The Components of Plant Tissue Culture Media II - Horticultural ...

Chapter 4 

The Components of Plant Tissue Culture Media ll: 

Organic Additions, Osmotic and pH Effects, 

and Support Systems 

Growth and morphogenesis of plant tissue 

cultures can be improved by small amounts of some 

organic nutrients. These are mainly vitamins 

(including some substances that are not strictly 

animal vitamins), amino acids and certain undefined 

supplements. The amount of these substances 

required for successful culture varies with the species 

and genotype, and is probably a reflection of the 

synthetic capacity of the explant. 

1.1. VITAMINS 

Vitamins are compounds required by animals in 

very small amounts as necessary ancillary food 

factors. Absence from the diet leads to abnormal 

growth and development and an unhealthy condition. 

Many of the same substances are also needed by plant 

cells as essential intermediates or metabolic catalysts, 

but intact plants, unlike animals, are able to produce 

their own requirements. Cultured plant cells and 

tissues can however become deficient in some 

factors; growth and survival is then improved by their 

addition to the culture medium. 

In early work, the requirements of tissue cultures 

for trace amounts of certain organic substances were 

satisfied by “undefined” supplements such as fruit 

juices, coconut milk, yeast or malt extracts and 

hydrolysed casein. These supplements can contribute 

vitamins, amino acids and growth regulants to a 

culture medium. The use of undefined supplements 

has declined as the need for specific organic 

compounds has been defined, and these have become 

listed in catalogues as pure chemicals. 

1.2. THE DEVELOPMENT OF VITAMIN MIXTURES 

The vitamins most frequently used in plant tissue 

culture media are thiamine (Vit. B1), nicotinic acid 

(niacin) and pyridoxine (Vit. B6) and apart from these 

three compounds, and myo-inositol, there is little 

common agreement about which other vitamins are 

really essential. 

The advantage of adding thiamine was discovered 

almost simultaneously by Bonner (1937, 1938), 

Robbins and Bartley (1937) and White (1937). 

Nicotinic acid and pyridoxine appear, in addition to 

1. ORGANIC SUPPLEMENTS 

E. F. George et al. (eds.), Plant Propagation by Tissue Culture 3rd Edition, 115–173. 

© 2008 Springer. 

115 

thiamine, in media published by Bonner (1940), 

Gautheret (1942) and White (1943b); this was 

following the findings of Bonner and Devirian (1939) 

that nicotinic acid improved the growth of isolated 

roots of tomato, pea and radish; and the papers of 

Robbins and Schmidt (1939a,b) which indicated that 

pyridoxine was also required for tomato root culture. 

These four vitamins; myo-inositol, thiamine, nicotinic 

acid, and pyridoxine are ingredients of Murashige 

and Skoog (1962) medium and have been used in 

varying proportions for the culture of tissues of many 

plant species (Chapter 3). However, unless there has 

been research on the requirements of a particular 

plant tissue or organ, it is not possible to conclude 

that all the vitamins which have been used in a 

particular experiment were essential. 

The requirements of cells for added vitamins vary 

according to the nature of the plant and the type of 

culture. Welander (1977) found that Nitsch and 

Nitsch (1965) vitamins were not necessary, or were 

even inhibitory to direct shoot formation on petiole 

explants of Begonia x hiemalis. Roest and 

Bokelmann (1975) on the other hand, obtained 

increased shoot formation on Chrysanthemum 

pedicels when MS vitamins were present. Callus of 

Pinus strobus grew best when the level of inositol in 

MS medium was reduced to 50 mg/l whereas that of 

P. echinata. proliferated most rapidly when no 

inositol was present (Kaul and Kochbar, 1985). 

Research workers often tend to adopt a ‘belt and 

braces’ attitude to minor media components, and add 

unusual supplements just to ensure that there is no 

missing factor which will limit the success of their 

experiment. Sometimes complex mixtures of as many 

as nine or ten vitamins have been employed. 

Experimentation often shows that some vitamins 

can be omitted from recommended media. Although 

four vitamins were used in MS medium, later work at 

Professor Skoog’s laboratory showed that the 

optimum rate of growth of tobacco callus tissue on 

MS salts required the addition of only myo-inositol 

and thiamine. The level of thiamine was increased 

four-fold over that used by Murashige and Skoog 

(1962), but nicotinic acid, pyridoxine and glycine

116 

(amino acid) were unnecessary (Linsmaier and 

Skoog, 1965). A similar simplification of the MS 

vitamins was made by Earle and Torrey (1965) for 

the culture of Convolvulus callus. 

Soczck and Hempel (1988) found that in the 

medium of Murashige et al. (1974) devised for the 

shoot culture of Gerbera jamesonii, thiamine, pyridoxine 

and inositol could be omitted without any 

reduction in the rate of shoot multiplication of their 

local cultivars. Ishihara and Katano (1982) found that 

Malus shoot cultures could be grown on MS salts 

alone, and that inositol and thiamine were largely 

unnecessary. 

1.3. SPECIFIC COMPOUNDS 

Myo-inositol. Myo-inositol (also sometimes 

described as meso-inositol or i-inositol) is the only 

one of the nine theoretical stereoisomers of inositol 

which has significant biological importance. 

Medically it has been classed as a member of the 

Vitamin B complex and is required for the growth of 

yeast and many mammalian cells in tissue culture. 

Rats and mice require it for hair growth and can 

develop dermatitis when it is not in the diet. Myoinositol 

has been classed as a plant ‘vitamin’, but note 

that some authors think that it should be regarded as a 

supplementary carbohydrate, although it does not 

contribute to carbohydrate utilization as an energy 

source or as an osmoticum. 

Historical use in tissue cultures. Myo-inositol 

was first shown by Jacquiot (1951) to favour bud 

formation by elm cambial tissue when supplied at 20- 

1000 mg/l. Necrosis was retarded, though the 

proliferation of the callus was not promoted. Myoinositol 

at 100 mg/1 was also used by Morel and 

Wetmore (1951) in combination with six other 

vitamins for the culture of callus from the 

monocotyledon Amorphophallus rivieri (Araceae). 

Bud initials appeared on some cultures and both roots 

and buds on others according to the concentration of 

auxin employed. The vitamin was adopted by both 

Wood and Braun (1961) and Murashige and Skoog 

(1962) in combination with thiamine, nicotinic acid 

and pyridoxine in their preferred media fur the 

culture of Catharanthus roseus and Nicotiana 

tabacum respectively. Many other workers have since 

included it in culture media with favourable results 

on the rate of callus growth or the induction of 

morphogenesis. Letham (1966) found that myoinositol 

interacted with cytokinin to promote cell 

division in carrot phloem explants. 

The Components of Plant Tissue Culture Media II 

Occurrence and biochemistry. Part of the 

growth promoting property of coconut milk is due to 

its myo-inositol content (Pollard et al., 1961). 

Coconut milk also contains scyllo-inositol (Table 

4.1). This can also promote growth but to a smaller 

extent than the myo-isomer (Pollard et al., 1961). 

Inositol is a constituent of yeast extract (Steiner et al., 

1969; Steiner and Lester, 1972) and small quantities 

may also be contained in commercial agar (Wolter 

and Skoog, 1966). Myo-inositol is a natural 

constituent of plants and much of it is often 

incorporated into phosphatidyl-inositol which may be 

an important factor in the functioning of membranes 

(Jung et al., 1972; Harran and Dickinson, 1978). The 

phosphatidylinositol cycle controls various cellular 

responses in animal cells and yeasts, but evidence of 

it playing a similar role in plants is only just being 

accumulated. Enzymes which are thought to be 

involved in the cycle have been observed to have 

activities in plants and lithium chloride (which 

inhibits myo-inositol-1-phosphatase and decreases the 

cycle) inhibits callus formation in Brassica oleracea 

(Bagga et al., 1987), and callus growth in 

Amaranthus paniculatus (Das et al., 1987). In both 

plants the inhibition is reversed by myo-inositol. 

As the myo-inositol molecule has six hydroxyl 

units, it can react with up to six acid molecules 

forming various esters. It appears that inositol 

phosphates act as second messengers to the primary 

action of auxin in plants: phytic acid (inositol hexaphosphate) 

is one of these. Added to culture media it 

can promote tissue growth if it can serve as a source 

of inositol (Watanabe et al., 1971). In some species, 

auxin can be stored and may be transported as IAAmyo-inositol 

ester (Chapter 5). o-Methyl-inositol is 

present in quite large quantities in legumes; inositol 

methyl ethers are known to occur in plants of several 

other families, although their function is unknown 

(Phillips and Smith, 1974). 

The stimulatory effect of myo-inositol in plant 

cultures probably arises partly from the participation 

of the compound in biosynthetic pathways leading to 

the formation of the pectin and hemicelluloses needed 

in cell walls (Loewus et al., 1962; Loewus, 1974; 

Loewus and Loewus, 1980; Harran and Dickinson, 

1978; Verma and Dougall, 1979; Loewus and 

Loewus, 1980) and may have a role in the uptake and 

utilization of ions (Wood and Braun, 1961). In the 

experiments of Staudt (1984) mentioned below, when 

the P04 3– content of the medium was raised to 4.41 

mM, the rate of callus growth of cv. ‘Aris’ was

progressively enhanced as the myo- inositol in the 

medium was put up to 4000 mg/l. This result seems 

Chapter 4 

to stress the importance of inositol-containing 

phospholipids for growth. 

Table 4.1. Substances identified as components of coconut milk (water) from mature green fruits and market-purchased fruits. 

SUBSTANCE QUANTITY/REFERENCE SUBSTANCE QUANTITY/REFERENCE 

Mature green 

fruits 

Mature fresh 

fruits 

Mature 

green 

fruits 

Mature fresh 

fruits 

Amino acids (mg/l) Sugars (g/l) 

Alanine 127.3 (14) 312 (13), 177.1 

(14) 

Sucrose 9.2 (14) 8.9 (14) 

Arginine 25.6 (14) 133 (13), 16.8 

(14) 

Glucose 7.3 (14) 2.5 (14) 

Aspartic acid 35.9 (14) 65 (13), 5.4 (14) Fructose 5.3 (14) 2.5 (14) 

Asparagine 10.1 (14) ca.60 (13), 10.1 

(14) 

Sugar alcohols (g/l) 

γ-Aminobutyric 34.6 (14) 820 (13), 168.8 Mannitol (1) 

acid 

(14) 

Glutamine acid 70.8 (14) 240 (13), 78.7 

(14) 

Sorbitol 15.0 (12), (17) 

Glutamine 45.4 (14) ca.60 (13), 13.4 

(14) 

myo-Inositol 0.1 (12), (17) 

Glycine 9.7 (14) 13.9 (14) scyllo-Inositol 0.5 (12), (17) 

Histidine 6.3 (14) Trace (13,14) Vitamins (mg/l) 

Homoserine -- (14) 5.2 (14) Nicotinic acid 0.64 (4) 

Hydroxyproline Trace (13,14) Pantolhenic acid 0.52 (4) 

Lysine 21.4 65.8 (14) Biotin, Riboflavin 0.02 (4) 

Methionine 16.9 (14) 8 (13), Trace (14) Riboflavin 0.01 (4) 

Phenylalanine -- (14) 12 (13), 10.2 (14) Folic acid 0.003 (4) 

Proline 31.9 97 (13), 21.6 (14) Thiamine, pyridoxine Trace (4) 

Serine 45.3 (14) Growth substances (mg/l) 

Typtophan 39 (13) Auxin 0.07 (7), (28) 

Threonine 16.2 (2) 44 (13), 26.3 (14) Gibberellin Yes (10,28) 

Tyrosine 6.4 (14) 16 (13), 3.1 (14) 1,3-Diphenylurea 5.8 (8), (6,17) 

Valine 20.6 (14) 27 (13), 15.1 (14) Zeatin (22,26) 

Other nitrogenous compounds Zeatin glucoside (26) 

Ammonium (19) Zeatin riboside (20), (24), (25) 

Ethanolamine (19) 6-Oxypurine growth 

promoter 

(27) 

Dihydroxyphenyl 

alanine 

(19) Unknown cytokinin/s 6, (18) (22) 

Inorganic elements (mg/100g dry wt.) Other (mg/l) 

Potassium 312.0 (3) RNA-polymerase (23) 

Sodium 105 (3) RNA-phosphorus 20.0 (14) 35.4 (14) 

Phosphorus 37.0 (3) DNA-phosphorus 0.1 (14) 3.5 (14) 

Magnesium 30.0 (3) Uracil, Adenine 21 

Organic acids (meq/ml) Leucoanthocyanins (11) (15,17) 

Malic acid 34.3 (14) 12.0 (14) Phyllococosine (16) 

Shikimic, Quinic 

and 2 unknowns 

0.6 (14) 0.41 (2) Acid Phosphatase (5,9) 

Pyrrolidone 

carboxylic acid 

0.4 (14) 0.2 (14) Diastase (2) 

Citric acid 0.4 (14) 0.3 (14) Dehydrogenase (5) 

Succinic acid -- (14) 0.3 (14) Peroxidase (5) 

Catalase (5) 

Numbered references (within brackets) in the above table are listed in Section 1.11 of this Chapter. 

117

118 The Components of Plant Tissue Culture Media II 

Activity in tissue cultures. Cultured plant tissues 

vary in their capacity for myo-inositol biosynthesis. 

Intact shoots are usually able to produce their own 

requirements, but although many unorganised tissues 

are able to grow slowly without the vitamin being 

added to the medium (Murashige, 1974) the addition 

of a small quantity is frequently found to stimulate 

cell division. The compound has been discovered to 

be essential to some plants. In the opinion of Kaul 

and Sabharwal (1975) this includes all monocotyledons, 

the media for which, if they do not contain 

inositol, need to be complemented with coconut milk, 

or yeast extract. 

Fraxinus pennsylvanica callus had an absolute 

requirement for 10 mg/1 myo-inositol to achieve 

maximum growth; higher levels, up to 250 mg/l had 

no further effect on fresh or dry weight yields (Wolter 

and Skoog, 1966). The formation of shoot buds on 

callus of Haworthia spp was shown to be dependent 

on the availability of myo-inositol (Kaul and 

Sabharwal, 1972, 1975). In a revised Linsmaier and 

Skoog (1965) medium [Staudt (1984) containing 1.84 

mM PO4 3– ], callus tissue of Vitis vinifera cv ‘Müller- 

Thurgau’ did not require myo-inositol for growth, but 

that of Vitis vinifera x V. riparia cv. ‘Aris’ was 

dependent on it and the rate of growth increased as 

the level of myo-inositol was increased up to 250 

mg/l (Staudt, 1984). 

Gupta et al. (1988) found that it was essential to 

add 5 g/l myo-inositol to Gupta and Durzan (1985) 

DCR-1 medium to induce embryogenesis (embryonal 

suspensor masses) from female gametophyte tissue of 

Pseudotsuga menziesii and Pinus taeda. The 

concentration necessary seems insufficient to have 

acted as an osmotic stimulus (see section 3). myo- 

Inositol reduced the rate of proliferation in shoot 

cultures of Euphorbia fulgens (Zhang et al., 1986). 

Thiamine. Thiamine (Vit. B1, aneurine) in the 

form of thiamine pyrophosphate, is an essential cofactor 

in carbohydrate metabolism and is directly 

involved in the biosynthesis of some amino acids. It 

has been added to plant culture media more 

frequently than any other vitamin. Tissues of most 

plants seem to require it for growth, the need 

becoming more apparent with consecutive passages, 

but some cultured cells are self sufficient. The maize 

suspension cultures of Polikarpochkina et al. (1979) 

showed much less growth in passage 2, and died in 

the third passage when thiamine was omitted from 

the medium. 

MS medium contains 0.3 μM thiamine. That this 

may not be sufficient to obtain optimum results from 

some cultures is illustrated by the results of Barwale 

et al. (1986): increasing the concentration of 

thiamine-HCI in MS medium to 5 μM, increased the 

frequency with which zygotic embryos of Glycine 

max formed somatic embryos from 33% to 58%. 

Adding 30 μM nicotinic acid (normally 4 μM) 

improved the occurrence of embryogenesis even 

further to 76%. Thiamine was found to be essential 

for stimulating embryogenic callus induction in 

Zoysia japonica, a warm season turf grass from Japan 

(Asano et al., 1996). It has also been shown to 

stimulate adventitious rooting of Taxus spp. (Chée, 

1995). 

There can be an interaction between thiamine and 

cytokinin growth regulators. Digby and Skoog (1966) 

discovered that normal callus cultures of tobacco 

produced an adequate level of thiamine to support 

growth providing a relatively high level of kinetin 

(ca. 1 mg/l) was added to the medium, but the tissue 

failed to grow when moved to a medium with less 

added kinetin unless thiamine was provided. 

Sometimes a change from a thiamine-requiring to 

a thiamine-sufficient state occurs during culture (see 

habituation – Chapter 7). In rice callus, thiamine 

influenced morphogenesis in a way that depended on 

which state the cells were in. Presence of the vitamin 

in a pre-culture (Stage I) medium caused thiaminesufficient 

callus to form root primordia on an 

induction (Stage II) medium, but suppressed the 

stimulating effect of kinetin on Stage II shoot 

formation in thiamine-requiring callus. It was 

essential to omit thiamine from the Stage I medium to 

induce thiamine-sufficient callus to produce shoots at 

Stage II (Inoue and Maeda, 1982). 

1.4. OTHER VITAMINS 

Pantothenic acid. Pantothenic acid plays an 

important role in the growth of certain tissues. It 

favoured callus production by hawthorn stem 

fragments (Morel, 1946) and stimulated tissue 

proliferation in willow and black henbane (Telle and 

Gautheret, 1947; Gautheret, 1948). However, 

pantothenic acid showed no effects with carrot, vine 

and Virginia creeper tissues which synthesize it in 

significant amounts (ca. 1 μg/ml). 

Vitamin C. The effect of Vitamin C (L-ascorbic 

acid) as a component of culture media will be 

discussed in Chapter 12. The compound is also used 

during explant isolation and to prevent blackening.

Besides, its role as an antioxidant, ascorbic acid is 

involved in cell division and elongation, e.g., in 

tobacco cells (de Pinto et al., 1999). Ascorbic acid 

(4-8 x 10 –4 M) also enhanced shoot formation in both 

young and old tobacco callus. (Joy et al., 1988). It 

speeded up the shoot-forming process, and 

completely reversed the inhibition of shoot formation 

by gibberellic acid in young callus, but was less 

effective in old callus. Clearly its action here was not 

as a vitamin. 

Vitamin D. Some vitamins in the D group, 

notably vitamin D2 and D3 can have a growth 

regulatory effect on plant tissue cultures. Their effect 

is discussed in Chapter 7. 

Vitamin E. The antioxidant activity of vitamin E 

(α-tocopherol) will be discussed in Chapter 12. 

Other vitamins. Evidence has been obtained that 

folic acid slows tissue proliferation in the dark, while 

enhancing it in the light. This is probably because it 

is hydrolysed in the light to p-aminobenzoic acid 

(PAB). In the presence of auxin, PAB has been 

shown to have a weak growth-stimulatory effect on 

cultured plant tissues (de Capite, 1952a,b). 

Riboflavin which is a component of some vitamin 

mixtures, has been found to inhibit callus formation 

but it may improve the growth and quality of shoots 

(Drew and Smith, 1986). Suppression of callus 

growth can mean that the vitamin may either inhibit 

or stimulate root formation on cuttings. Riboflavin 

has been shown to stimulate adventitious rooting on 

shoots of Carica papaya (Drew et al., 1993), apple 

shoots (van der Krieken et al., 1992) and Eucalyptus 

globulus (Trindade and Pais, 1997). It also enhances 

embryogenic callus induction in Zoysia japonica in 

association with cytokinins and thiamine (Asano 

et al., 1996). 

Glycine is occasionally described as a vitamin in 

plant tissue cultures: its use has been described in the 

section on amino acids. 

Adenine. Adenine (or adenine sulphate) has been 

widely used in tissue culture media, but because it 

mainly gives rise to effects which are similar to those 

produced by cytokinins, it is considered in the chapter 

on cytokinins (Chapter 6). 

Stability. Some vitamins are heat-labile; see the 

section on medium preparation in Volume 2. 

1.5. UNDEFINED SUPPLEMENTS 

Many undefined supplements were employed in 

early tissue culture media. Their use has slowly 

declined as the balance between inorganic salts has 

been improved, and as the effect of amino acids and 

Chapter 4 

119 

growth substances has become better understood. 

Nevertheless several supplements of uncertain and 

variable composition are still in common use. 

The first successful cultures of plant tissue 

involved the use of yeast extract (Robbins, 1922; 

White, 1934). Other undefined additions made to 

plant tissue culture media have been: 

• meat, malt and yeast extracts and fibrin digest; 

• juices, pulps and extracts from various fruits 

(Steward and Shantz, 1959; Ranga Swamy, 1963; 

Guha and Maheshwari, 1964, 1967), including those 

from bananas and tomatoes (La Rue, 1949); 

• the fluids which nourish immature zygotic 

embryos; 

• extracts of seedlings (Saalbach and Koblitz, 1978) 

or plant leaves (Borkird and Sink, 1983); 

• the extract of boiled potatoes and corn steep 

liquor (Fox and Miller, 1959); 

• plant sap or the extract of roots or rhizomes. Plant 

roots are thought to be the main site of cytokinin 

synthesis in plants (Chapter 6); 

• protein (usually casein) hydrolysates (containing a 

mixture of all the amino acids present in the original 

protein). Casein hydrolysates are sometimes termed 

casamino acids: they are discussed in Chapter 3). 

Many of these amendments can be a source of 

amino acids, peptides, fatty acids, carbohydrates, 

vitamins and plant growth substances in different 

concentrations. Those which have been most widely 

used are described below. 

1.6. YEAST EXTRACT. 

Yeast extract (YE) is used less as an ingredient of 

plant media nowadays than in former times, when it 

was added as a source of amino acids and vitamins, 

especially inositol and thiamine (Vitamin B1) (Bonner 

and Addicott, l937; Robbins and Bartley, 1937). In a 

medium consisting only of macro- and micronutrients, 

the provision of yeast extract was often 

found to be essential for tissue growth (White, 1934; 

Robbins and Bartley, 1937). The vitamin content of 

yeast extract distinguishes it from casein hydrolysate 

(CH) so that in such media CH or amino acids alone, 

could not be substituted for YE (Straus and La Rue, 

1954; Nickell and Maretzki, 1969). It was soon 

found that amino acids such as glycine, lysine and 

arginine, and vitamins such as thiamine and nicotinic 

acid, could serve as replacements for YE, for 

example in the growth of tomato roots (Skinner and 

Street, 1954), or sugar cane cell suspensions (Nickell 

and Maretzki, 1969).


The percentage of amino acids in a typical yeast 

extract is high (e.g. 7% amino nitrogen - Nickell and 

Maretzki, 1969; Bridson, 1978; Thom et al., 1981), 

but there is less glutamic acid than in casein or other 

protein hydrolysate. Malt extract contains little 

nitrogen (ca. 0.5% in total). 

Yeast extract has been typically added to media in 

concentrations of 0.1-1 g/l; occasionally 5, 10 and 

even 20 g/l (Morel and Muller, 1964) have been 

included. It normally only enhances growth in media 

containing relatively low concentrations of nitrogen, 

or where vitamins are lacking. Addition of 125-5000 

mg/l YE to MS medium completely inhibited the 

growth of green callus of 5 different plants whereas 

small quantities added to Vasil and Hildebrandt 

(1966) THS medium (which contained 0.6 times the 

quantity of NO3 – and NH4 + ions and unlike MS did 

not contain nicotinic acid or pyridoxine) gave more 

vigorous growth of carrot, endive and lettuce callus 

than occurred on MS. There was still no growth of 

parsley and tomato callus on THS medium: these 

tissues only grew well on unmodified MS (Vasil and 

Hildebrandt, 1966a,b,c). 

Stage I media are sometimes fortified with yeast 

extract to reveal the presence of micro-organisms 

which may have escaped decontamination 

procedures: it is then omitted at later stages of 

culture. 

Yeast extract has been shown to have some 

unusual properties which may relate to its amino acid 

content. It elicits phytoalexin accumulation in 

several plant species and in Glycyrrhiza echinata 

suspensions it stimulated chalcone synthase activity 

leading to the formation of narengin (Ayabe et al., 

1988). It also stimulated furomocoumarin production 

in Glehnia littoralis cell suspensions (Kitamura et al., 

1998). On Monnier (1976, 1978) medium 1 g/l yeast 

extract was found to inhibit the growth of immature 

zygotic embryos of Linum, an effect which, when 

0.05 mg/1 BAP and 400 mg/l glutamine were added, 

induced the direct formation of adventitious embryos 

(Pretova and Williams, 1986). 

Yeast extract is now purchased directly from 

chemical suppliers. In the 1930s and 1940s it was 

prepared in the laboratory. Brink et al. (1944) 

macerated yeast in water which was then boiled for 

30 minutes and, after cooling, the starchy material 

was removed by centrifugation. However, Robbins 

and Bartley (1937) found that the active components 

of yeast could be extracted with 80% ethanol. 

1.7. POTATO EXTRACT 

Workers in China found that there was a sharp 

increase in the number of pollen plants produced 

from wheat anthers when they were cultured on an 

agar solidified medium containing only an extract of 

boiled potatoes, 0.1 mM FeEDTA, 9% sucrose and 

growth regulators. Potato extract alone or potato 

extract combined with components of conventional 

culture media (Chuang et al., 1978) has since been 

found to provide a useful medium for the anther 

culture of wheat and some other cereal plants. For 

example, the potato medium was found to be better 

for the anther culture of spring wheat than the 

synthetic (N6) medium (McGregor and McHughen, 

1990). Sopory et al. (1978) obtained the initiation of 

embryogenesis from potato anthers on potato extract 

alone and Lichter (1981) found it beneficial to add 

2.5 g/l Difco potato extract to a medium for Brassica 

napus anther culture, but it was omitted by Chuong 

and Beversdorf (1985) when they repeated this work. 

We are not aware of potato extract being added to 

media for micropropagation, apart from occasional 

reports of its use for orchid propagation. Sagawa and 

Kunisaki (1982) supplemented 1 litre of Vacin and 

Went (1949) medium with the extract from 100g 

potatoes boiled for 5 minutes, and Harvais (1982) 

added 5% of an extract from 200g potatoes boiled in 

1 litre water to his orchid medium. Of interest was 

the finding that potato juice treatment enabled in vitro 

cultures of Doritaenopsis (Orchidaceae) to recover 

from hyperhydricity (Zou, 1995). 

1.8. MALT EXTRACT 

Although no longer commonly used, malt extract 

seems to play a specific role in cultures of Citrus. 

Malt extract, mainly a source of carbohydrates, was 

shown to initiate embryogenesis in nucellar explants 

(Rangan et al., 1968; Rangan, 1984). Several recent 

studies showed a role for the extract in the 

multiplication of Citrus sinensis somatic embryos 

(Das et al., 1995), and in other Citrus spp. (Jumin, 

1995), in the promotion of plantlet formation from 

somatic embryos derived from styles of different 

Citrus cultivars (De Pasquale et al., 1994), and in 

somatic embryogenesis and plantlet regeneration 

from pistil thin cell layers of Citrus (Carimi et al., 

1999). Malt extract also promoted germination of 

early cotyledonary stage embryos arising from the in 

vitro rescue of zygotic embryos of sour orange 

(Carimi et al., 1998). The extract is commercially 

available and used at a level of 0.5 – 1 g/l.

1.9. BANANA HOMOGENATE 

Homogenised banana fruit is sometimes added to 

media for the culture of orchids and is often reported 

to promote growth. The reason for its stimulatory 

effect has not been explained. One suggestion 

mentioned earlier is that it might help to stabilise the 

pH of the medium. Pierik et al. (1988) found that it 

was slightly inhibitory to the germination of 

Paphiopedilum ciliolare seedlings but promoted the 

growth of seedlings once germination had taken 

place. 

1.10. FLUIDS WHICH NOURISH EMBRYOS 

The liquid which is present in the embryo sac of 

immature fruits of Aesculus (e.g. A. woerlitzensis) 

(Shantz and Steward, 1956, 1964; Steward and 

Shantz, 1959; Steward and Rao, 1970) and Juglans 

regia (Steward and Caplin, 1952) has been found to 

have a strong growth-promoting effect on some plant 

tissues cultured on simple media, although growth 

inhibition has occasionally been reported 

(Fonnesbech, 1972). Fluid from the immature female 

gametophyte of Ginkgo biloba (Steward and Caplin, 

1952) and extracts from the female gametophyte of 

Pseudotsuga menziesii (Mapes and Zaerr, 1981) and 

immature Zea mays grains (less than two weeks after 

pollination) can have a similar effect. The most 

readily obtained fluid with this kind of activity is 

coconut milk (water). 

1.11. COCONUT MILK/WATER 

When added to a medium containing auxin, the 

liquid endosperm of Cocos nucifera fruits can induce 

plant cells to divide and grow rapidly. The fluid is 

most commonly referred to as coconut milk, although 

Tulecke et al. (1961) maintained that the correct 

English term is ‘coconut water’, because the term 

coconut milk also describes the white liquid obtained 

by grating the solid white coconut endosperm (the 

‘meat’) in water and this is not generally used in 

tissue culture media. However, in this section, both 

terms are used. 

Coconut milk was first used in tissue cultures by 

Van Overbeek et al. (1941, 1942) who found that its 

addition to a culture medium was necessary for the 

development of very young embryos of Datura 

stramonium. Gautheret (1942) found that coconut 

milk could be used to initiate and maintain growth in 

tissue cultures of several plants, and Caplin and 

Steward (1948) showed that callus derived from 

phloem tissue explants of Daucus carota roots grew 

much more rapidly when 15% coconut milk was 

Chapter 4 

121 

added to a medium containing IAA. Unlike other 

undefined supplements to culture media (such as 

yeast extract, malt extract and casein hydrolysate) 

coconut milk has proved harder to replace by fully 

defined media. The liquid has been found to be 

beneficial for inducing growth of both callus and 

suspension cultures and for the induction of 

morphogenesis. Although commercial plant tissue 

culture laboratories (particularly those in temperate 

countries) would endeavour not to use this ingredient 

on account of its cost, it is still frequently employed 

for special purposes in research. 

It is possible to get callus growth on coconut milk 

alone (Steward et al., 1952), but normally it is added 

to a recognised medium. Effective stimulation only 

occurs when relatively large quantities are added to a 

medium; the incorporation of 10-15 percent by 

volume is quite usual. For instance, Burnet and 

Ibrahim (1973) found that 20% coconut milk (i.e. 

one-fifth of the final volume of the medium) was 

required for the initiation and continued growth of 

callus tissue of various Citrus species in MS medium; 

Rangan (1974) has obtained improved growth of 

Panicum miliaceum in MS medium using 2,4-D in 

the presence of 15% coconut milk. By contrast, Vasil 

and co-workers (e.g. Vasil and Vasil, 1981a,b) 

needed to add only 5% coconut milk to MS medium 

to obtain somatic embryogenesis from cereal callus 

and suspension cultures. 

Many workers try to avoid having to use coconut 

milk in their protocols. It is an undefined supplement 

whose composition can vary considerably (Swedlund 

and Locy, 1988). However, adding coconut milk to 

media often provides a simple way to obtain 

satisfactory growth or morphogenesis without the 

need to work out a suitably defined formulation. 

Suggestions that coconut milk is essential for a 

particular purpose need to be treated with some 

caution. For instance, in the culture of embryogenic 

callus from root and petiole explants of Daucus 

carota, coconut milk could be replaced satisfactorily 

either by adenine or kinetin, showing that it did not 

contribute any unique substances required for 

embryogenesis (Halperin and Wetherell, 1964). 

Preparation. Ready prepared coconut water 

(milk) can be purchased from some chemical 

suppliers, but the liquid from fresh nuts (obtained 

from the greengrocer) is usually perfectly adequate. 

One nut will usually yield at least 100 ml. The water 

is most simply drained from dehusked coconuts by 

drilling holes through two of the micropyles. Only 

normal uncontaminated water should be used and so


nuts should be extracted one by one, and the liquid 

endosperm from each examined to ascertain that it is 

unfermented before addition to a bulk supply. Water 

from green but mature coconuts may contain slightly 

different quantities of substances to that in the nuts 

purchased in the local market (Table 4.1) and has 

been said to be a more effective stimulant in plant 

media than that from ripe fruits, but Morel and 

Wetmore (1951) found to the contrary. Tulecke et al. 

(1961) discovered that the water from highly 

immature coconuts contained smaller quantities of 

the substances normally present in mature nuts. 

References to composition of coconut water 

(numbers refer to citations in Table 4.1). (1) 

Dunstan (1906), (2) DeKruijff (1906), (3) McCance 

and Widdowson (1940), (4) Vandenbelt (1945), (5) 

Sadasivan (1951), (6) Shantz and Steward (1952), (7) 

Paris and Duhamet (1953), (8) Shantz and Steward 

(1955), (9) Wilson and Cutter (1955), (10) Radley 

and Dear (1958), (11) Steward and Shantz (1959), 

(12) Pollard et al. (1961), (13) Figures of Steward 

et al. (1961), given by Raghavan (1977), (14) 

Tulecke et al. (1961), (15) Steward and Mohan Ram 

(1961), (16) Kuraishi and Okumura (1961), (17) 

Steward (1963), (18) Zwar et al. (1963), (19) Steward 

et al. (1964), (20) Letham (1968), (21) Steward et al. 

(1969), (22) Zwar and Bruce (1970), (23) Mondal 

et al. (1972), (24) Letham (1974), (25) Van Staden 

and Drewes (1975), (26) Van Staden (1976), (27) 

Letham (1982), (28) Dix and Van Staden (1982). 

Use of Coconut water. Coconut water is usually 

strained through cloth and deproteinized by being 

heated to 80-100°C for about 10 minutes while being 

stirred. It is then allowed to settle and the supernatant 

is separated from the coagulated proteins by filtration 

through paper. The liquid is stored frozen at -20°C. 

Borkird and Sink (1983) did not boil the water from 

fresh ripe coconuts, but having filtered it through 

several layers of cheesecloth, adjusted the pH to 10 

with 2 N NaOH and then kept it overnight at 4°C. 

The following day the pH was re-adjusted to 7.0 with 

5 N HCl, and the preparation was refiltered before 

being stored frozen at –20°C. 

Some workers autoclave media containing 

coconut milk; others filter-sterilise coconut milk and 

add it to a medium after autoclaving has been carried 

out. Morel and Wetmore (1951) used filter 

sterilisation, but found that the milk lost its potency if 

stored sterile (but presumably unfrozen) for 3 

months. Street (1977) advocated autoclaving coconut 

milk after it had been boiled and filtered; it was then 

stored at -20°C until required. 

Active ingredients. The remarkable growth 

stimulating property of coconut milk has led to 

attempts to isolate and identify the active principles. 

This has proved to be difficult because the fractions 

into which coconut milk has been separated each 

possess only a small proportion of the total activity 

and the different components appear to act 

synergistically. Substances so far identified include 

amino acids, organic acids, nucleic acids, purines, 

sugars, sugar alcohols, vitamins, growth substances 

and minerals (Table 4.1). The variable nature of the 

product is illustrated in the table by the analytical 

results obtained by different authors. 

Auxin activity. The liquid has been found to have 

some auxin activity which is increased by 

autoclaving, probably because any such growth 

substances exist in a bound form and are released by 

hydrolysis. But although coconut milk can stimulate 

the growth of some in vitro cultures in the absence of 

exogenous auxin, it normally contains little of this 

kind of growth regulator and an additional exogenous 

supply is generally required. In modern media, 

where organic compounds are often added in defined 

amounts, the main benefit from using coconut milk is 

almost certainly due to its providing highly active 

natural cytokinin growth substances. 

Cytokinin activity. Coconut milk was shown to 

have cytokinin activity by Kuraishi and Okumura 

(1961) and recognised natural cytokinin substances 

have since been isolated [9-β-D-ribo-furanosyl zeatin 

(Letham, 1968); zeatin and several unidentified ones 

(Zwar and Bruce, 1970); N, N′-diphenyl urea (Shantz 

and Steward, 1955)] but the levels of these 

compounds in various samples of coconut milk have 

not been published. An unusual cytokinin-like 

growth promoter, 2-(3-methylbut-2-enylamino)-purin- 

6-one was isolated by Letham (1982). 

Because coconut milk contains natural cytokinins, 

adding it to media often has the same effect as adding 

a recognised cytokinin. This means that a beneficial 

effect on growth or morphogenesis is often dependent 

on the presence of an auxin. Steward and Caplin 

(1951) showed that there was a synergistic action 

between 2,4-D and coconut milk in stimulating the 

growth of potato tuber tissue. Lin and Staba (1961) 

similarly found that coconut milk gave significantly 

improved callus growth on seedling explants of 

peppermint and spearmint initiated by 2,4-D, but only 

slightly improved the growth initiated by the auxin 2- 

BTOA (2-benziothiazoleoxyacetic acid). The 

occurrence of gibberellin-like substances in coconut 

milk has also been reported (Radley and Dear, 1958).

Suboptimum stimulation and inhibition. In 

cases where optimal concentrations of growth 

adjuvants have been determined, it has been found 

that the level of the same or analogous substances in 

coconut milk may be suboptimal. La Motte (1960) 

noted that 150 mg/l of tyrosine most effectively 

induced morphogenesis in tobacco callus cultures, but 

coconut milk added at 15% would provide only 0.96 

mg/l of this substance (Tulecke et al., 1961). Fresh 

and autoclaved coconut milk from mature nuts has 

proved inhibitory to growth or morphogenesis (Noh 

et al., 1988) in some instances. It is not known which 

ingredients cause the inhibition but the growth of 

cultured embryos seems particularly liable to be 

prevented, suggesting that the compound responsible 

might be a natural dormancy-inducing factor such as 

abscisic acid. Van Overbeck et al. (1942, 1944) 

found that a factor was present in coconut milk which 

Organic acids can have three roles in plant culture 

media: 

• they may act as chelating agents, improving the 

availability of some micronutrients, 

• they can buffer the medium against pH change, 

• they may act as nutrients. 

A beneficial effect is largely restricted to the acids 

of the Krebs’ cycle. Dougall et al. (1979) found that 

20 mM succinate, malate or fumarate supported 

maximum growth of wild carrot cells when the 

medium was initially adjusted to pH 4.5. Although 1 

mM glutarate, adipate, pimelate, suberate, azelate or 

phthalate controlled the pH of the medium, little or 

no cell growth took place. 

2.1. USE AS BUFFERS 

The addition of organic acids to plant media is not 

a recent development. Various authors have found 

that some organic acids and their sodium or 

potassium salts stabilise the pH of hydroponic 

solutions (Trelease and Trelease, 1933) or in vitro 

media (Van Overbeek et al., 1941, 1942; Arnow 

et al., 1953), although it must be admitted that they 

are not as effective as synthetic biological buffers in 

this respect (see Section 5). Norstog and Smith 

(1963) discovered that 100 mg/l malic acid acted as 

an effective buffering agent in their medium for 

barley embryo culture and also appeared to enhance 

growth in the presence of glutamine and alanine. 

Malic acid, now at 1000 mg/l was retained in the 

improved Norstog (1973) Barley II medium. In the 

Chapter 4 

2. ORGANIC ACIDS 

123 

was essential for the growth of Datura stramonium 

embryos, but that heating the milk or allowing it to 

stand could lead to the release of toxic substances. 

These could be removed by shaking with alcohols or 

ether or lead acetate precipitation. Duhamet and 

Mentzer (1955) isolated nine fractions of coconut 

milk by chromatography, and found one of these to 

be inhibitory to cultured crown gall tissues of black 

salsify when more than 10-20% coconut milk was 

incorporated into the medium. Norstog (1965) 

showed that autoclaved coconut milk could inhibit 

the growth of barley embryos but that filter-sterilised 

milk was stimulatory. Coconut water inhibited 

somatic embryo induction in Pinus taeda (Li and 

Huang, 1996) and both autoclaved or filter-sterilized 

coconut milk inhibited the growth of wheat embryoshoot 

apices (Smith, 1967). 

experiments of Schenk and Hildebrandt (1972) low 

levels of citrate and succinate ions did not impede 

callus growth of a wide variety of plants and 

appeared to be stimulatory in some species. The 

acids were also effective buffers between pH 5 and 

pH 6, but autoclaving a medium containing sodium 

citrate or citric acid caused a substantial pH increase.. 

2.1.1. Complexing with metals 

Divalent organic acids such is citric, maleic, malic 

and malonic (depending on species) are found in the 

xylem sap of plants, where together with amino acids 

they can complex with metal ions and assist their 

transport (White et al., 1981). These acids can also 

be secreted from cultured cells and tissues into the 

growth medium and will contribute to the 

conditioning effect. Ojima and Ohira (1980) 

discovered that malic and citric acids, released into 

the medium by rice cells during the latter half of a 

passage, were able to make unchelated ferric iron 

available, so correcting an iron deficiency. 

2.1.2. Nutritional role 

As explained in Chapter 3, adding Krebs’ cycle 

organic acids to the medium can enhance the 

metabolism of NH4 + . Gamborg and Shyluk (1970) 

found that some organic acids could promote 

ammonium utilisation and the incorporation of small 

quantities of sodium pyruvate, citric, malic and 

fumaric acids into the medium, was one factor which 

enabled Kao and Michayluk (1975) to culture Vicia 

hajastana cells at low density. Their mixture of


organic acid ions has been copied into many other 

media designed for protoplast culture. Cultures may 

not tolerate the addition of a large quantity of a free 

acid which will acidify the medium. For example, 

Triticale anther callus grew well on Chu et al. (1975) 

N6 medium supplemented with 35 mg/l of a mixture 

of sodium pyruvate, malic acid, fumaric acid, citric 

acid, but not when 100 mg/l was added (Chien and 

Kao, 1983). When organic anions are added to the 

medium from the sodium or potassium salts of an 

acid there are metallic cations to counterbalance the 

organic anions, and it seems to be possible to add 

larger quantities without toxicity. Five mM (1240 

mg/l .3H2O) potassium succinate enhanced the 

growth of cultured peach embryos (Ramming, 1990), 

and adding 15 mM (4052 mg/1 .4H2O) sodium 

succinate to MS medium (while also increasing the 

sucrose content from 3% to 6%) increased the cell 

volume and dry weight of Brassica nigra suspensions 

by 2.7 times (Molnar, 1988). 

Some plants seem to derive nutritional benefit 

from the presence of one particular organic acid. 

Murashige and Tucker (1969) showed that orange 

juice added to a medium containing MS salts 

promoted the growth of Citrus albedo callus. Malic 

and other Krebs’ cycle acids also have a similar 

effect; of these, citric acid produces the most 

pronounced growth stimulation. A concentration of 

up to 10.4 mM can be effective (Goldschmidt, 1976; 

Einset, l978; Erner and Reuveni, 1981). Succulent 

plants, in particular those in the family Crassulaccae, 

such as Bryophyllum and Kalanchoe fix relatively 

large amounts of carbon dioxide during darkness, 

converting it into organic acids, of which malic acid 

is particularly important. The organic acids are 

metabolised during daylight hours. In such plants, 

malic acid might be expected to prove especially 

efficient in enhancing growth if added to a culture 

medium. Lassocinski (1985) has shown this to be the 

case in chlorophyll-deficient cacti of three genera. 

The addition of L-malic acid to the medium of 

Savage et al. (1979) markedly improved the rate of 

survival and vigour of small cacti or areoles. 

Organic acid (citrate, lactate, succinate, tartrate, 

and oxalate) pretreatment of alfalfa callus 

dramatically decreased the growth of callus, but 

increased the subsequent yield of somatic embryos 

and embryo development, as well as conversion to 

plantlets (Nichol et al., 1991). They suggested that 

the acids may act in the physiological selection for 

embryogenic callus, by inducing preferential growth 

of slower-growing-compact cell aggregates compared 

to the faster growing friable callus. 

3. SUGARS -NUTRITIONAL AND REGULATORY EFFECTS 

Carbohydrates play an important role in in vitro 

cultures as an energy and carbon source, as well as an 

osmotic agent. In addition, carbohydrate-modulated 

gene expression in plants is known (Koch, 1996). 

Plant gene responses to changing carbohydrate status 

can vary markedly. Some genes are induced, some 

are repressed, and others minimally affected. As in 

microorganisms, sugar-sensitive plant genes are part 

of an ancient system of cellular adjustment to critical 

nutrient availability. However, there is no evidence 

that this role of carbohydrate is important in normal 

growth and organized development in cell 

cultures.3.1. Sugars as energy sources 

3.1.1. Carbohydrate autotrophy. 

Only a limited number of plant cell lines have 

been isolated which are autotrophic when cultured in 

vitro. Autotrophic cells are capable of fully 

supplying their own carbohydrate needs by carbon 

dioxide assimilation during photosynthesis 

(Bergmann, 1967; Tandeau de Marsac and Peaud- 

Lenoel, 1972a,b; Chandler et al., 1972; Anon, 1980; 

Larosa et al., 1981). Many autotrophic cultures have 

only been capable of relatively slow growth (e.g. 

Fukami and Hildebrandt, 1967), especially in the 

ambient atmosphere where the concentration of 

carbon dioxide is low (see Chapter 12). However, 

since these early trials, very good progess is being 

made with photoautrophic shoot cultures and photoautotrophic 

micropropagation is now possible (Kozai, 

1991). Success is dependent on enriching the CO2 

concentrations in the vessels during the photoperiod, 

reducing or eliminating sugar from the medium, and 

optimising the in vitro environment. 

Nevertheless, for the normal culture of either 

cells, tissues or organs, it is necessary to incorporate a 

carbon source into the medium. Sucrose is almost 

universally used for micropropagation purposes as it 

is so generally utilisable by tissue cultures. Refined 

white domestic sugar is sufficiently pure for most 

practical purposes. The presence of sucrose in tissue 

culture media specifically inhibits chlorophyll 

formation and photosynthesis (see below) making 

autotrophic growth less feasible.

3.2 ALTERNATIVES TO SUCROSE 

3.2.1. Other Sugars. 

The selection of sucrose as the most suitable 

energy source for cultures follows many comparisons 

between possible alternatives. Some of the first work 

of this kind on the carbohydrate nutrition of plant 

tissue was done by Gautheret (1945) using normal 

carrot tissue. Sucrose was found to be the best source 

of carbon followed by glucose, maltose and raffinose; 

fructose was less effective and mannose and lactose 

were the least suitable. The findings of this and other 

work is summarized in Table 4.2. Sucrose has almost 

invariably been found to be the best carbohydrate; 

glucose is generally found to support growth equally 

well, and in a few plants it may result in better in 

Chapter 4 

vitro growth than sucrose, or promote organogenesis 

where sucrose will not; but being more expensive 

than sucrose, glucose will only be preferred for 

micropropagation where it produces clearly 

advantageous results. 

Multiplication of Alnus crispa, A. cordata and A. 

rubra shoot cultures was best on glucose, while that 

of A. glutinosa was best on sucrose (Tremblay and 

Lalonde, 1984; Tremblay et al., 1984; Barghchi, 

1988). Direct shoot formation from Capsicum annum 

leaf discs in a 16 h day required the presence of 

glucose (Phillips and Hubstenberger, 1985). Glucose 

is required for the culture of isolated roots of wheat 

(Furguson, 1967) and some other monocotyledons 

(Bhojwani and Razdan, 1983). 

Table 4.2. The main sugars which can utilized by plants. 

The value of as sugar for carbon nutrition is indicated by the size of the type. 

SUGAR Reducing Capacity Products of hydrolytic/enzymatic 

breakdown 

Monosaccharides 

Hexoses 

Glucose Reducing sugar None 

Fructose Reducing sugar None 

Galactose Reducing sugar None 

Mannose Reducing sugar None 

Pentoses 

Arabinose Slow reduction None 

Ribose Slow reduction None 

Xylose Slow reduction None 

Disaccharides 

Sucrose Non-reducing Glucose, fructose 

Maltose Reducing sugar Glucose 

Cellobiose Reducing sugar Glucose 

Trehalose Non-reducing Glucose 

Lactose Reducing sugar Glucose, fructose 

Trisaccharides 

Raffinose Non-reducing Glucose, galactose, fructose 

Some other monosaccharides such as arabinose 

and xylose; disaccharides such as cellobiose, maltose 

and trehalose; and some polysaccharides; all of which 

are capable of being broken down to glucose and 

fructose (Table 4.2), can also sometimes be used as 

partial replacements for sucrose (Straus and LaRue, 

1954; Sievert and Hildebrandt, 1965; Yatazawa et al., 

1967; Smith and Stone, 1973; Minocha and Halperin, 

1974; Zaghmout and Torres, 1985). In Phaseolus 

callus, Jeffs and Northcote (1967) found that sucrose 

could be replaced by maltose and trehalose (all three 

sugars have an alpha-glucosyl radical at the nonreducing 

end), but not by glucose or fructose alone or 

125 

in combination, or by several other different sugars. 

Galactose has been said to be toxic to most plant 

tissues; it inhibits the growth of orchids and other 

plants in concentrations as low as 0.01% (0.9 mM) 

(Ernst et al., 1971; Arditti and Ernst, 1984). 

However, cells can become adapted and grown on 

galactose, e.g., sugar cane cells (Maretzski and 

Thom, 1978). The key was the induction of the 

enzyme galactose kinase, which converts galactose to 

galactose-1-phosphate. More recently, other reports 

on galactose use have appeared. It promoted callus 

growth in rugosa rose, but inhibited somatic 

embryogenesis (Kunitake et al., 1993). Galactose


promoted early somatic embryo maturation stages in 

European silver fir (Schuller and Reuther, 1993). 

When used instead of sucrose, it improved rooting of 

Annona squamosa microshoots (Lemos and Blake, 

1996). In addition, galactose has been found to 

reduce or overcome hyperhydricity in shoot cultures 

(Druart, 1988; see Volume 2). Fructose has also been 

reported to be effective in preventing hyperhydricity 

(Rugini et al., 1987). 

There are some situations where fructose supports 

growth just as well as sucrose or glucose (Steffen 

et al., 1988) and occasionally it gives better results. 

Some orchid species have been reported to grow 

better on fructose than glucose (Ernst, 1967; Ernst 

et al., 1971; Arditti, 1979). Fructose was the best 

sugar for the production of adventitious shoots from 

Glycine max cotyledonary nodes, especially if the 

concentration of nutrient salts supplied was 

inadequate (Wright et al., 1986). Shoot and leaf 

growth and axillary shoot formation in Castanea 

shoot cultures was stimulated when sucrose was 

replaced by 30 g/l fructose. The growth of basal 

callus was reduced and it was possible to propagate 

from mature explants of C. crenata, although this was 

not possible on the same medium supplemented with 

sucrose (Chauvin and Salesses, 1988). However, 

fructose was reported to be toxic to carrot tissue if, as 

the sole source of carbon, it was autoclaved with 

White (1943a) A medium. When filter sterilized, 

fructose supported the growth of callus cultures 

which had a final weight 70% of those grown on 

sucrose (Pollard et al., 1961). 

Sucrose in culture media is usually hydrolysed 

totally, or partially, into the component 

monosaccharides glucose and fructose (see below) 

and so it is logical to compare the efficacy of 

combinations of these two sugars with that of 

sucrose. Kromer and Kukulczanka (1985) found that 

meristem tips of Canna indica survived better on a 

mixture of 25 g/l glucose plus 5 g/l fructose, than on 

30 g/l sucrose. Germination of Paphiopedilum orchid 

seeds was best on a medium containing 5g/l fructose 

plus 5 g/l glucose; a mixture of 7.5 g/l of each sugar 

was optimal for further growth of the seedlings 

(Pierik et al., 1988). In spite of its rapid hydrolysis to 

glucose and fructose, sucrose appears to have a 

specific stimulatory effect on embryo development in 

Douglas fir, that was not observed when it was 

replaced by the monosaccharides (Taber et al., 1998). 

The general superiority of sucrose over glucose 

for the culture of organised plant tissues such as 

isolated roots may be on account of the more 

effective translocation of sucrose to apical meristems 

(Butcher and Street, 1964). In addition, there could 

be an osmotic effect, because, from an equal weight 

of compound, a solution of glucose has almost twice 

the molarity of a sucrose solution, and will thus, in 

the absence of inversion of the disaccharide, induce a 

more negative water potential (see below). 

Maltose. Plant species vary in their ability to 

utilise unusual sugars. For instance, although 

Gautheret (1945) could grow carrot callus on 

maltose, Mathes et al. (1973) obtained only minimal 

growth of Acer tissue on media supplemented with 

this sugar. Similarly, growth of soybean tissue on 

maltose is normally very slow, but variant strains of 

cells have been selected which can utilise it (Limberg 

et al., 1979), perhaps because the new genotypes 

possessed an improved capacity for its active 

transport. Later studies have given a more prominent 

role to maltose as a component of tissue culture 

media. Maltose serves as both a carbon source and as 

an osmoticum. Compared to sucrose there is a slower 

rate of extracellular hydrolysis, it is taken up more 

slowly, and hydrolysed intracellularly more slowly. 

Maltose led to a substantial increase in somatic 

embryos from Petunia anthers (Raquin, 1983). It 

also led to an increase in callus induction and plantlet 

regeneration during in vitro androgenesis of 

hexaploid winter triticale and wheat (Karsai et al., 

1994). Maltose also increased callus induction in rice 

microspore culture, with an acceleration of initial cell 

divisions (Xie et al., 1995). For barley microspore 

culture, the inclusion of maltose led to a higher 

frequency of green plants (Finnie et al., 1989). 

Maltose has been reported to equal or surpass sucrose 

in supporting embryogenesis in a number of species, 

including carrot (Verma and Dougall, 1977; 

Kinnersley and Henderson, 1988), alfalfa (Strickland 

et al., 1987), wild cherry (Reidiboym-Talleux et al., 

1999), Malus (Daigny et al., 1996), Abies (Norgaard 

1997) and loblolly pine (Li et al., 1998). The number 

of plants regenerated from indica (Biswas and 

Zapata, 1993), and japonica (Jain et al., 1997) rice 

varieties was also greater when protoplasts were 

cultured with maltose rather than sucrose. Transfer 

from a medium containing sucrose or glucose to one 

supplemented with maltose has been used by Stuart 

et al. (1986) and Redenbaugh et al. (1987) to enhance 

the conversion of alfalfa embryos. Similarly, maltose 

led to a much higher germination rate from asparagus 

somatic embryos than sucrose (Kunitake et al., 1997). 

Lactose. The disaccharide lactose has been 

detected in only a few plants. When added to tissue

culture media it has been found to induce the activity 

of β-galactosidase enzyme which can be secreted into 

the medium. The hydrolysis of lactose to galactose 

and glucose then permits the growth of Nemesia 

strumosa and Petunia hybrida callus, cucumber 

suspensions (Hess et al., 1979; Callebaut and Motte, 

1988), cotton callus and cell suspensions (Mitchell 

et al., 1980), and Japanese morning glory callus 

(Hisajima and Thorpe, 1981). The key to lactose 

utilization in Japanese morning glory was not only 

the extracellular hydrolysis of this disaccharide, but 

the induction of galactose kinase, which prevented 

the accumulation of toxic galactose (Hisajima and 

Thorpe, 1985). Rodriguez and Lorenzo Martin 

(1987) found that adding 30 g/l lactose to MS 

medium instead of sucrose increased the number of 

shoots produced by a Musa accuminata shoot culture, 

but no new shoots were produced on subsequent 

subculture, although they were when sucrose was 

present. 

In addition to lactose, plant cells have been shown 

to become adapted and then to grow on other 

galactose-containing oligosaccharides, including 

melibiose (Nickell and Maretzki, 1970; Gross et al., 

1981), raffinose (Wright and Northcote, 1972; 

Thorpe and Laishley, 1974; Gross et al., 1981), and 

stachyose (Verma and Dougall, 1977; Gross et al., 

1981). 

Corn syrups. Kinnersley and Henderson (1988) 

have shown that certain corn syrups can be used as 

carbon sources in plant culture media and that they 

may induce morphogenesis which is not provoked by 

supplementing with sucrose. Embryogenesis was 

induced in a 10-year old non-embryogenic cell line of 

Daucus carota and plantlets were obtained from 

Nicotiana tabacum anthers by using syrups. Those 

used contained a mixture of glucose, maltose, 

maltotriose and higher polysaccharides. Their 

stimulatory effect was reproduced by mixtures of 

maltose and glucose. 

3.2.2. Sugar alcohols. 

Sugar alcohols were thought not usually to be 

metabolised by plant tissues and therefore 

unavailable as carbon sources. For this reason, 

mannitol and sorbitol have been frequently employed 

as osmotica to modify the water potential of a culture 

medium. In these circumstances, sufficient sucrose 

must also be present to supply the energy requirement 

of the tissues. Adding either mannitol or sorbitol to 

the medium may make boron unavailable (See 

Chapter 3). 

Chapter 4 

127 

Mannitol was found to be metabolised by 

Fraxinus tissues (Wolter and Skoog, 1966). Later, 

studies with carrot and tobacco suspensions and 

cotyledon cultures of radiata pine showed that 

although mannitol was taken up very slowly, it was 

readily metabolized (Thompson et al., 1986). Thus, 

this sugat alcohol is only of value as a short-term 

osmotic agent. In contrast, sorbitol is readily taken 

up and metabolized in some species. It has been 

found to support the growth of apple callus (Chong 

and Taper, 1972, 1974a,b) and that of other rosaceous 

plants (Coffin et al., 1976), occasionally giving rise 

to more vigorous growth than can be obtained on 

sucrose. The ability of Rosaceae to use sorbitol as a 

carbon source is reported to be variety dependent. 

Albrecht (1986) found that shoot cultures of one 

crabapple variety required sorbitol for growth and 

would not grow on sucrose; another benefited from 

being grown on a mixture of sorbitol and sucrose and 

the growth of a third suffered if any sucrose was 

replaced by sorbitol. The apple rootstock ‘Ottawa 3’ 

produced abnormal shoots on sorbitol (Chong and 

Pua, 1985). Evidence is accumulating to show that 

sugar alcohols generally exhibit non-osmotic roles in 

regulating morphogenesis and metabolism in plants 

that do not produce polyols as primary photosynthetic 

products (Steinitz, 1999). In addition to being 

metabolised to varying degrees in heterotrophic 

cultures, such as tobacco, maize, rice, citrus and 

chichory, sugar alcohols stimulate specific molecular 

and physiological responses, where they apparently 

act as chemical signals. 

The cyclic hexahydric alcohol myo-inositol does 

not seem to provide a source of energy (Smith and 

Stone, 1973) and its beneficial effect on the growth of 

cultured tissues when used as a supplementary 

nutrient must depend on its participation in 

biosynthetic pathways (see vitamins above). 

3.2.3. Starch. 

Cultured cells of a few plants are able to utilise 

starch in the growth medium and appear to do so by 

release of extracellular amylases (Nickell and 

Burkholder, 1950). Growth rates of these cultures are 

increased by the addition of gibberellic acid, probably 

because it increases the synthesis or secretion of 

amylase enzymes (Maretzki et al., 1971, 1974). 

3.3. HYDROLYSIS OF SUCROSE. 

The remainder of this section on sugars is devoted 

to the apparent effects of sucrose concentration on 

cell differentiation and morphogenesis. Reports on


the subject should be tempered with the knowledge 

that some or all of the sucrose in the medium is liable 

to be broken down into its constituent hexose sugars, 

and that such inversion will also occur within plant 

tissues, where reducing sugar levels of at least 0.5 per 

cent are likely to occur (Helgeson et al., 1972). 

3.3.1. Autoclaving. 

A partial hydrolysis of sucrose takes place during 

the autoclaving of media (Ball, 1953; Wolter and 

Skoog, 1966) the extent being greater when the 

compound is dissolved together with other medium 

constituents than when it is autoclaved in pure 

aqueous solution (Ferguson et al., 1958). In a fungal 

medium, not dissimilar to a plant culture medium, 

Bretzloff (1954) found that sucrose inversion during 

autoclaving (15 min at 15 lbs/in 2 ) was dependent on 

pH in the following way: 

pH 3.0 100% 

pH 3.4 75% 

pH 3.8 40% 

pH 4.2 25% 

pH 4.7 12.5% 

pH 5.0 10% 

pH 6.0 0% 

These results suggest that the proportion of 

sucrose hydrolysed by autoclaving media at 

conventional pH levels (5.5-5.8) should be negligible. 

Most evidence suggests that this is not the case and 

that 10-15% sucrose can be converted into glucose 

and fructose. Cultures of some plants grow better in 

media containing autoclaved (rather than filtersterilised) 

sucrose (Ball, 1953; Guha and Johri, 1966; 

Johri and Guha, 1963; Verma and Van Huystee, 

1971; White, 1932) suggesting that the cells benefit 

from the availability of glucose and/or fructose. 

However, Nitsch and Nitsch (1956) noted that 

glucose only supported the growth of Helianthus 

callus if it had been autoclaved, and Romberger and 

Tabor (1971) that the growth of Picea shoot apices 

was less when the medium contained sucrose 

autoclaved separately in water (or sucrose autoclaved 

with only the organic constituents of the medium) 

than when all the constituents had been autoclaved 

together. They suggested that a stimulatory 

substance might be released when sugars are 

autoclaved with agar. 

In other species there has been no difference 

between the growth of cultures supplied with 

autoclaved or filter sterilised sucrose (Mathes et al., 

1973) or growth has been less on a medium 

containing autoclaved instead of filter sterilised 

sucrose (Stehsel and Caplin, 1969). 

3.3.2. Enzymatic breakdown. 

Sucrose in the medium is also inverted into 

monosaccharides during the in vitro culture of plant 

material. This occurs by the action of invertase 

located in the plant cell walls (Burstrom, 1957; 

Yoshida et al., 1973) or by the release of extracellular 

enzyme (King and Street, 1977). In most cultures, 

inversion of sucrose into glucose and fructose takes 

place in the medium; but, because the secretion of 

invertase enzymes varies, the degree to which it 

occurs differs from one kind of plant to another. 

After 28 days on a medium containing either 3 or 4% 

sucrose, single explants of Hemerocallis and 

Delphinium had used 20-30% of the sugar. Of that 

which remained in the medium which had supported 

Hemerocallis, about 45% was sucrose, while only 5% 

was sucrose in the media in which Delphinium had 

been grown. In both cases the rest of the sucrose had 

been inverted (Lumsden et al., 1990). 

The sucrose-inverting capacity of tomato root 

cultures was greatest in media of pH 3.6-4.7. 

Activity sharply declined in less acid media (Weston 

and Street, 1968). Helgeson et al. (1972) found that 

omission of IAA auxin from the medium in which 

tobacco callus was cultured, caused there to be a 

marked rise in reducing sugar due to the progressive 

hydrolysis of sucrose, both in the medium and in the 

tissue. A temporary increase in reducing sugars also 

occurred at the end of the lag phase when newly 

transferred callus pieces started to grow rapidly. It is 

interesting to note that cell wall invertase possessed 

catalytic activity in situ, whether or not tobacco tissue 

was grown on sucrose (Obata-Sasamoto and Thorpe, 

1983). 

In cultures of some species, uptake of sugar may 

depend on the prior extracellular hydrolysis of sugar. 

This is the case in sugar cane (Komor et al., 1981); 

and possibly also in Dendrobium orchids (Hew et al., 

1988) and carrot (Kanabus et al., 1986). Nearly all 

the sucrose in suspension cultures of sugar cane and 

sugar beet was hydrolysed in 3 days (Zamski and 

Wyse, 1985) and Daucus carota suspensions have 

been reported to hydrolyse all of the sucrose in the 

medium (Thorpe, 1982) into the constituent hexoses 

within 3 days (Kanabus et al., 1986) or within 24 h 

(Dijkema et al., 1990). However, most species can 

take up sucrose directly as was shown through studies 

with asymetrically labeled 14 C-sucrose (Parr and 

Edelman, 1975), and metabolise it intracellularly. 

Within the cell, soluble invertase, sucrose phosphate 

synthetase and sucrose synthetase serve to hydrolyse 

sucrose (Thorpe, 1982). Thus, in Acer (Copping and

Street, 1972) the soluble invertase activity paralleled 

growth rate, while in tobacco (Thorpe and Meier, 

1973) and Japanese morning glory (Hisajima et al., 

1978) sucrose synthetase was more important. In the 

last species, the change in activity of sucrose 

synthetase was greater than that of sucrose phosphate 

synthetase, an enzyme not extensively examined in 

cultured cells. 

The growth of shoots from non-dormant buds of 

mulberry is not promoted by sucrose, only by 

maltose, glucose or fructose. Even though mulberry 

tissue hydrolysed sucrose into component 

monosaccharides, shoots did not develop. In the 

presence of 3% fructose, sucrose was actually 

inhibitory to shoot development at concentrations as 

low as 0.2% (Oka and Ohyama, 1982). 

3.4. UPTAKE. 

The uptake of sugar molecules into plant tissues 

appears to be partly through passive permeation and 

partly through active transport. The extent of the two 

mechanisms may vary. Active uptake is associated 

with the withdrawal of protons (H + ) from the 

medium. Charge compensation is effected by the 

excretion of a cation (H + or K + ) (Komor et al., 1977, 

1981). Glucose was taken up preferentially by carrot 

suspensions during the first 7 days of a 14 day 

passage; fructose uptake followed during days 7-9 

(Dijkema et al., 1990). At concentrations below 200 

mM, glucose was taken up more rapidly into 

strawberry fruit discs and protoplasts than either 

sucrose or fructose (Scott and Breen, 1988). 

3.5. EFFECTIVE CONCENTRATIONS. 

In most of the comparisons between the 

nutritional capabilities of sugars discussed above, the 

criterion of excellence has been the most rapid 

growth of unorganised callus or suspension-cultured 

cells. For this purpose 2-4% sucrose w/v is usually 

optimal. Similar concentrations are also used in 

media employed for micropropagation, but 

laboratories probably pay insufficient attention to the 

effects of sucrose on morphogenesis (see below) and 

plantlet development. Sucrose levels in culture 

media which result in good callus growth may not be 

optimal for morphogenesis, and either lower or 

higher levels may be more effective. 

The optimum concentration of sucrose to induce 

morphogenesis or growth differs between different 

genotypes, sometimes even between those which are 

closely related. For instance, Damiano et al. (1987) 

found that the concentration of sucrose necessary to 

Chapter 4 

129 

produce the best rate of shoot proliferation in 

Eucalyptus gunnii shoot cultures varied between 

clones. The influence of sucrose concentration on 

direct shoot formation from Chrysanthemum explants 

varied with plant cultivar (Fig 4.1). 

Experiments of Molnar (1988) have shown that 

the optimum level of sucrose may depend upon the 

other amendments added to a culture medium. The 

most rapid growth of Brassica nigra suspensions on 

one containing MS salts (but less iron and B5 

vitamins) occurred when 2% sucrose was added. 

However, when it was supplemented with 1-4 g/l 

casein hydrolysate or a mixture of 3 defined amino 

acids, growth was increased on up to 6% sucrose. A 

similar result was obtained if, instead of the amino 

acids, 15 mM sodium succinate was added. There 

was an extended growth period and the harvested dry 

weight of the culture was 2.8 times that on the 

original medium with 2% sucrose. 

The level of sucrose in the medium may have a 

direct effect on the type of morphogenesis. Thus, 

sucrose (87 mM) favored organogenesis, while a 

higher level (350 mM) favoured somatic 

embryogenesis from immature zygotic embryos of 

sunflower (Jeannin et al., 1995). In vitro minicrowns 

of asparagus developed short, thickened storage roots 

at high frequencies when the sucrose concentration in 

the medium was increased to 6% (Conner and 

Falloon, 1993). Lower sucrose concentrations, even 

with the addition of non- or poorly metabolised 

carbohydrates, such as cellobiose, maltose, mannose, 

melibiose and sorbitol produced thin fibrous roots, 

indicating that the additional sucrose was nutritional 

rather than osmotic. 

The respiration rate of cultured plant tissues rises 

as the concentration of added sucrose or glucose is 

increased. In wheat callus, it was found to reach a 

maximum when 90 g/1 (0.263 M) was added to the 

medium, even though 20 g/l produced the highest rate 

of growth and number of adventitious shoots (Galiba 

and Erdei, 1986). The uptake of inorganic ions can 

be dependent on sugar concentration and the benefit 

of adding increased quantities of nutrients to a 

medium may not be apparent unless the amount of 

sugar is increased at the same time (Gamborg et al., 

1974). 

3.5.1. Cell differentiation 

Formation of vascular elements. Although 

sugars are clearly involved in the differentiation of 

xylem and phloem elements in cultured cells, it is still 

uncertain whether they have a regulatory role apart 

from providing a carbon energy source necessary for


active cell metabolism. Sucrose is generally required 

to be present in addition to IAA before tracheid 

elements are differentiated in tissue cultures. The 

number of both sieve and xylem elements formed 

[and possibly the proportion of each kind - Wetmore 

and Rier (1963); Rier and Beslow (1967)] depends on 

sucrose concentration (Aloni, 1980). In Helianthus 

tuberosus tuber slices, although sucrose, glucose and 

trehalose were best for supporting cell division and 

tracheid formation, maltose was only a moderately 

effective carbon source (Minocha and Halperin, 

1974). Shininger (1979) has concluded that only 

carbohydrates which enable significant cell division 

are capable of promoting tracheary element 

formation. The occurrence of lignin in cultured cells 

is not invariably associated with thickened cell walls. 

Sycamore suspension cultures produced large 

amounts of lignin when grown on a medium with 

abnormally high sucrose (more than 6%) and 2,4-D 

levels. It was deposited within the cells and released 

into the medium (Carceller et al., 1971). 

Fig. 4.1 The effect of sucrose concentration on direct adventitious shoot formation from flower pedicels of two chrysanthemum cultivars 

[from data of Roest and Bokelmann, 1975]. 

Chlorophyll formation. Levels of sucrose 

normally used to support the growth of tissue cultures 

are often inhibitory to chlorophyll synthesis (Rier and 

Chen, 1964; Edelman and Hanson, 1972) but the 

degree of inhibition does vary according to the 

species of plant from which the tissue was derived. 

In experiments of Hildebrandt et al. (1963) and 

Fukami and Hildebrandt (1967) for example, carrot 

and rose callus had a high chlorophyll content on 

Hildebrandt et al. (1946) tobacco medium with 2-8% 

sucrose; but tissue of endive, lettuce and spinach only 

produced large amounts of the pigment on a medium 

with no added sucrose (although a small amount of 

sugar was probably supplied by 15-16% coconut 

milk). 

Cymbidium protocorms contain high chlorophyll 

levels only if they are cultured on media containing 

0.2-0.5% sucrose. Their degree of greening declines 

rapidly when they are grown on sucrose 

concentrations higher than this (Vanséveren-Van 

Espen, 1973). Likewise, orchid protocorm-like 

bodies will not become green and cannot develop into 

plantlets if sucrose is present in the medium beyond 

the stage of their differentiation from the explants. 

Where added sucrose does reduce chlorophyll 

formation, it is thought that the synthesis of 5aminolaevulinic 

acid (ALA - a precursor of the 

porphyrin molecules of which chlorophyll is 

composed) is reduced due to an inhibition of the 

activity of the enzyme ALA synthase (Pamplin and

Chapman, 1975). Sugars apart from sucrose are not 

inhibitory (Edelman and Hanson, 1972; El Hinnawiy, 

1974). It has been said that cells grown on sucrose 

for prolonged periods may permanently lose the 

ability to synthesise chlorophyll (Van Huystee, 

1977). Plastids become converted to amyloplasts 

packed with starch and this may change the 

expression of plastid DNA (Gunning and Steer, 1975) 

or result in a reduction of plastid RNA (Rosner et al., 

1977). 

A small amount of photosynthesis may be carried 

out by cultured shoots providing they are not 

maintained on media containing a high concentration 

of sucrose. Photosynthesis increased in Rosa shoot 

cultures when they were grown initially on 20 or 40 

g/l sucrose which was decreased to 10 g/l in 

successive subcultures (Langford and Wainwright, 

1987). An increase in photosynthesis occurs when 

sucrose is omitted from the medium in which rooted 

plantlets are growing (Short et al., 1987), but these 

treatments are not successful in ensuring a greater 

survival of plantlets when they are transferred extra 

vitrum. A more recent study also showed the relative 

contribution of autotrophic and heterotrophic carbon 

metabolism in cultured potato plants (Wolf et al., 

1998). With 8% sucrose in the medium 90% of the 

tissue carbon was of heterotrophic origin in lightgrown 

plants; while on 3% sucrose, only 50% was of 

heterotrophic origin. 

3.6 STARCH ACCUMULATION AND MORPHOGSNESIS 

3.6.1. Starch deposition preceeding morphogenesis. 

Cells of callus and suspension cultures commonly 

accumulate starch in their plastids and it is 

particularly prevalent in cells at the stationary phase. 

Starch in cells of rice suspensions had different 

chemical properties to that in the endosperm of seeds 

(Landry and Smyth, 1988). In searching for features 

which might be related to later morphogenetic events 

in Nicotiana callus, Murashige and co-workers 

(Murashige and Nakano, 1968; Thorpe and 

Murashige, 1968a, b) noticed that starch accumulated 

preferentially in cells sited where shoot primordia 

ultimately formed. The starch is produced from 

sucrose supplied in the culture medium (Thorpe et al., 

1986). As a result of this work, it was suggested that 

starch accumulation might be a prerequisite of 

morphogenesis. In tobacco, starch presumably acts 

as a direct cellular reserve of the energy required for 

morphogenesis, because it disappears rapidly as 

meristenoids and shoot primordia are formed (Thorpe 

and Meier, 1974, 1975). Morphogenesis is an 

Chapter 4 

131 

energy-demanding process and callus maintained on 

a shoot-inducing medium does have a greater 

respiration rate than similar tissue kept on a noninductive 

medium (Thorpe and Murashige, 1970; 

Thorpe and Meier, 1972). Organ-forming callus of 

tobacco has been found to accumulate starch prior to 

shoot or root formation, whereas callus not capable of 

morphogenesis did not do so (Kavi Kishor and 

Mehta, 1982). 

3.6.2. Morphogenesis without starch deposition 

Cells of other plants which become committed to 

initiate organs do not necessarily accumulate starch 

as a preliminary to morphogenesis and it seems likely 

that the occurrence of this phenomenon is speciesrelated. 

The deposition of starch was observed as an 

early manifestation of organogenesis in Pinus 

coulteri embryos (Patel and Berlyn, 1983), but not in 

those of Picea abies (Von Arnold, 1987). Although 

zygotic embryos of the latter species immediately 

began to accumulate starch (particularly in the 

chloroplasts of cells in the cortex) when they were 

placed on a medium containing sucrose. It was never 

observed in meristematic cells from which 

adventitious buds developed (Von Arnold, 1987). 

However, if a major role for the accumulation of 

starch prior to the initiation of organized development 

is for energy production, this role would be satisfied 

by the lipid reserves in zygotic embryos of conifers 

(Thorpe, 1982). Indeed, the rapid and nearly linear 

degradation of triglycerides during the period of high 

respiration during shoot initiation in excised 

cotyledons of radiata pine (Biondi and Thorpe, 1982; 

Douglas et al., 1982) would support this view. 

Meristematic centres in bulb scales of Nerine 

bowdenii can be detected as groups of cells from 

which starch is absent (Grootaarts et al., 1981). 

Starch was not accumulated in caulogenic callus of 

Rosa persica x R. xanthina initiated from recentlyinitiated 

shoot cultures, but cells did accumulate 

starch when the shoot forming capacity of the callus 

was lost after more than three passages (Lloyd et al., 

1988). Callus derived from barley embryos was 

noted to accumulate starch very rapidly and this was 

accompanied by a reduction in osmotic pressure 

within the cells (Granatek and Cockerline, 1978). 

Gibberellic acid, which in this plant could be used to 

induce shoot formation, brought about an increase in 

cell osmolarity. 

There are some further examples where a 

diminution of the amount of stored carbohydrate in 

cultured tissues has restored or improved their


organogenetic capacity. A salt-tolerant line of alfalfa 

cells which showed no ability for shoot regeneration 

after three and a half years in culture on 3% sucrose 

was induced to form shoots and plantlets by being 

cultured for one passage of 24 days on 1% sucrose, 

before being returned to a medium containing 3% 

sucrose and a high 2,4-D level (Rains et al., 1980, 

and personal communication). Cells which in 3% 

sucrose were full of starch became starch-depleted 

during culture on a lower sucrose level. The number 

of somatic embryos formed by embryogenic 

‘Shamouti’ orange callus, was increased when 

sucrose was omitted from the medium for one 

passage, before being returned to Murashige and 

Tucker (1969) medium with 5-6% sucrose (Kochba 

and Button, 1974). 

3.6.3. Unusual sugars 

In some plants, unusual sugars are able to regulate 

morphogenesis and differentiation. Galactose stimulates 

embryogenesis in Citrus cultures (Kochba et al., 

1978) and can enhance the maturation of alfalfa 

embryos. Callus of Cucumis sativus grew most 

rapidly on raffinose and was capable of forming roots 

when grown on this sugar; somatic embryos were 

only differentiated when the callus was cultured on 

sucrose (88-175 mM), but if a small amount of 

stachyose (0.3 mM) was added to 88 mM sucrose the 

callus produced adventitious shoots instead (Kim and 

Janick, 1989). Stachyose is the major translocated 

carbohydrate in cucurbits. 

4. OSMOTIC EFFECT OF MEDIA INGREDIENTS 

Besides having a purely nutritive effect, solutions 

of inorganic salts and sugars, which compose tissue 

culture media, influence plant cell growth through 

their osmotic properties. A discussion is most 

conveniently accommodated at this point, as many of 

the papers published on the subject stress the osmotic 

effects of added sugars. 

4.1. OSMOTIC AND WATER POTENTIALS: A GENERAL 

INTRODUCTION 

Water movement into and out of a plant cell is 

governed by the relative concentrations of dissolved 

substances in the external and internal solutions, and 

by the pressure exerted by its restraining cell wall. 

The manner of defining the respective forces has 

changed in recent years, and as both old and new 

terminology are found in the tissue culture literature, 

the following brief description may assist the reader. 

More detailed explanations can be found in many text 

books on plant physiology. 

In the older concept, cells were considered to take 

up water by suction (i.e. by exerting a negative 

pressure) induced by the osmotically active 

concentration of dissolved substances within the cell. 

The suction force or suction pressure (SP) was 

defined as that resulting from the osmotic pressure of 

the cell sap (OPcs) minus the osmotic pressure of the 

external solution (OPext), and the pressure exerted on, 

and stretching the cell wall, turgor pressure (TP) - so 

called because it is at a maximum when the cells are 

turgid. This may be represented by the equations: 

SP = (OPcs - OPext) – TP or 

SP = OPcs - (OPext + TP). 

These definitions devised by botanists, are not 

satisfactory thermodynamically because water should 

be considered to move down an energy gradient, 

losing energy as it does so. The term water potential 

(Ψ - Greek capital letter psi) is now used, and 

solutions of compounds in water are said to exert an 

osmotic potential. As the potential of pure water is 

defined as being zero, and dissolved substances cause 

it to be reduced, solutions have osmotic potentials, Ψs 

(or Ψπ - Greek small letter pi) which are negative in 

value. Water is said to move from a region of high 

potential (having a less negative value) to one that is 

lower (having a more negative value). Both osmotic 

pressure and osmotic potential are used as terms in 

tissue culture literature and are equivalent except that 

they are opposite in sign (i.e. an OP of +6 bar equals 

a Ψs of -6 bar). Thermodynamically, the cell’s turgor 

pressure is defined as a positive pressure potential 

(Ψp). The water potential of a cell (Ψcell ) is then 

equal to the sum of its osmotic and pressure 

potentials plus the force holding water in 

microcapillaries or bound to the cell wall matrix 

(Ψm): 

Ψ cell = Ψs + Ψp + Ψm 

Modern statements of the older suction pressure 

concept are therefore that the difference in water 

potential (i.e. the direction of water movement) 

between a cell and solution outside is given by: 

ΔΨ = (Ψsinside cell – Ψsoutside cell) – Ψp or 

ΔΨ = Ψ cell – Ψπoutside 

and when ΔΨ = 0 (at equilibrium) 

Ψπoutside = Ψ cell 

The force of water movement between two cells, `

‘A’ and ‘B’, of different water potential, is given 

by: 

ΔΨ = Ψ cellA – ΨcellB 

the direction of movement being towards the 

more negative water potential. 

The osmotic potential (pressure) of solutions is 

determined by their molar concentration and by 

temperature. The water potential of a plant tissue 

culture medium (Ψtcm) is equivalent to the osmotic 

potential of the dissolved compounds (Ψs). There is 

no pressure potential but, if they are added, 

substances such as agar and Gelrite contribute a 

matric potential (Ψm): 

Ψtcm = Ψs + Ψm 

Osmotic pressure and water potential are 

measured in standard pressure units thus: 

1 bar = 0.987 atm 

= 10 6 dynes cm –2 

= 10 5 Pa (0.1 MPa = 1 bar). 

Whereas molarity is defined as number of gram 

moles of a substance in one litre of a solution (i.e. one 

litre of solution requires less than one litre of 

solvent), molality is the number of gram moles of 

solute per kilogram of solvent, and thus, unlike 

osmotic potential (osmolality, measured in pressure 

units) is independent of temperature. It is therefore 

more convenient to give measurements of osmotic 

pressure in osmolality units. The osmole (Osm) is 

defined as: 

The unit of the osmolality of a solution exerting 

an osmotic pressure equal to that of an ideal nondissociating 

substance which has a concentration 

of one mole of solute per kilogram of solvent. 

The osmolality of a very dilute solution of a 

substance which does not dissociate into ions, will be 

the same as its molality (i.e. g moles per kilogram of 

solvent). The osmolality of a weak solution of a salt, 

Chapter 4 

or salts, which has completely dissociated into ions, 

will equal that of the total molality of the ions. 

The osmotic potential of dilute solutions 

approximates to Van’t Hoff’s equation: Ψ = -cRT; 

where 

c = concentration of solutes in mol/litre; 

R = the gas constant and 

T = temperature in °K 

From the above equation, at 0°C one litre of a 

solution containing 1 mole of an undissociated 

compound, or 1 mole of ions, could be expected to 

have an osmolality of 1 Osm/kg, and an osmotic 

(water) potential of: 

Ψ= -1 (mole) x 0.082054 (atm /mole/°C) 

x 273.16 (°K) = - 22.414 atm 

Thus, although in practice the Van’t Hoff 

equation must be corrected by the osmotic 

coefficient, Φ: 

Ψ = − Φ cRT (Lang, 1967), 

it is possible to give approximate figures for 

converting osmolality into osmotic potential pressure 

units. These are shown in Table 4.3. This table can 

also be used to estimate the osmotic potential of nondissociating 

molecules such as sugars or mannitol. 

Thus at 25°C, 30 g/l sucrose (molecular wt. 342.3) 

should exert an osmotic potential of: 

30 

-2.4789 x = −0.217 MPa 

342. 

3 

The observed potential is -0.223 Mpa (Table 4.4). 

A definition. The osmotic properties of solutions 

can be difficult to describe without confusion. In this 

book, the addition of solutes to a solvent (which 

makes the osmotic or water potential more negative, 

but makes the osmolality of the solution increase to a 

larger positive value), has been said to reduce 

Table 4.3 Factors by which osmolality (Osm/kg) should be multiplied to estimate an equivalent osmotic potential 

in pressure units. 

Multiply Osm/kg by the factor shown for an equivalent osmotic potential in 

pressure units 1 

For 

conversion to 

15 ºC 20 ºC 0 ºC 25 ºC 30 ºC 

Atm -22.414 -23.645 -24.055 -24.465 -24.875 

Bar -22.711 -23.958 -24.374 -24.789 -25.205 

Dyne/cm 2 

-22.711 

x 10 6 

-23.958 

x 10 6 

-24.374 

x 10 6 

-24.789 

x 10 6 

-25.205 

x 10 6 

Mpa -2.2711 -2.3958 -2.4374 -2.4789 -2.5205 

* Divide pressure units by the figures shown to find approximate osmolality, 

-223 

e.g. − 223 kPa = 

1000 

x 

1 

- 2. 

4789 

= 0.090 Osm/kg. 

133


Table 4.4 The osmolality and osmotic potential of sucrose solutions at different concentrations. 

Sucrose concentration Osmolality Osmotic potential at 

(% , w/v) (mM) (Osm/kg) 25 ºC (MPa) 

0.5 14.61 0.015 -0.037 

1.0 29.21 0.030 -0.074 

1.5 43.82 0.045 -0.112 

2.0 58.43 0.060 -0.149 

2.5 73.04 0.075 -0.186 

3.0 87.64 0.090 -0.223 

4.0 116.86 0.121 -0.300 

5.0 175.28 0.186 -0.461 

6.0 233.71 0.253 -0.627 

8.0 292.14 0.324 -0.803 

10.0 350.57 0.396 -0.982 

(decrease) the osmotic potential of solutions. MS 

medium containing 3% sucrose (osmolality, ca. 186 

mOsm/kg; ca. -461 kPa at 25°C) is thus described as 

having a lower water potential than the medium of 

White (1954) supplemented with 2% sucrose 

(osmolality, ca. 78 mOsm/kg; ca. −193 kPa at 25°C). 

4.2. THE OSMOTIC POTENTIAL OF TISSUE CULTURE 

MEDIA 

4.2.1. The total osmotic potential of solutes 

The approximate total osmotic potential of a 

medium due to dissolved substances, can be 

estimated from: Ψs = Ψsmacronutrients + Ψssugars 

When 3% w/v sucrose is added to Murashige and 

Skoog (1962) medium, the osmolality of a filter 

sterilised preparation rises from 0.096 to 0.186 

Osm/kg and at 25°C, the osmotic potential of the 

medium decreases from -0.237 to -0.460 MPa. 

Sugars are thus responsible for much of the osmotic 

potential of normal plant culture media. Even 

without any inversion to monosaccharides, the 

addition of 3% w/v sucrose is responsible for over four 

fifths of the total osmotic potential of White (1954) 

medium, 60% of that of Schenk and Hildebrandt 

(1972), and for just under one half that of MS. 

The contribution of the gelling agent. The water 

potential of media solidified with gels is more 

negative than that of a liquid medium, due to their 

matric potential, but this component is probably 

relatively small (Amador and Stewart, 1987). In the 

following sections, the matric potential of semi-solid 

media containing ca. 6 g/l agar has been assumed to 

be -0.01 MPa at 25°C, but as adding extra agar to 

media helps to prevent hyperhydricity, it is possible 

that this is an underestimate. 

The contribution of nutrient salts. Inorganic salts 

dissociate into ions when they are dissolved in water, 

so that the water potential of their solutions 

(especially weak solutions) does not depend on the 

molality (or molarity) of undissociated compounds, 

but on the molality (or molarity) of their ions. Thus a 

solution of KCl with a molality of 0.1, will have a 

theoretical osmolality of 0.2, because in solution it 

dissociates into 0.1 mole K + and 0.1 mole Cl – . 

Osmolality of a solution of mixed salts is dependent 

on the total molality of ions in solution. 

Dissociation may not be complete, especially 

when several different compounds are dissolved 

together as in plant culture media, which is a further 

reason why calculated predictions of water potential 

may be imprecise. In practice, osmotic potentials 

should be determined by actual measurement with an 

osmometer. Clearly though, osmotic potential of a 

culture medium is related to the concentration of 

solutes, particularly that of the macronutrients and 

sugar. 

Of the inorganic salts in nutrient media, the 

macronutrients contribute most to the final osmotic 

(water) potential because of their greater 

concentration. The osmolality of these relatively 

dilute solutions is very similar to the total osmolarity 

of the constituent ions at 0°C, and can therefore be 

estimated from the total molarity of the macronutrient 

ions. Thus based on its macronutrient composition, a 

liquid Murashige and Skoog (1962) medium (without 

sugar) with a total macronutrient ion concentration of 

95.75 mM, will have an osmolality of ca. 0.0958 

osmoles (Osm) per kilogram of water solvent (95.8 

mOsm/kg), at 25°C, an osmotic potential of ca. - 

0.237 MPa (237 kPa). Estimates of osmolality

derived in this way agree closely to actual 

measurements of osmolality or osmolarity for named 

media given in the papers of Yoshida et al. (1973), 

Kavi Kishor and Reddy (1986) and Lazzeri et al. 

(1988) (see Table 4.5). 

The contribution of sugars. The osmolality and 

osmotic potential of sucrose solutions can be read 

from Table 4.6. Those of mannitol and sorbitol 

solutions of equivalent molarities will be 

approximately comparable. At concentrations up to 

3% w/v, the osmolality of sucrose is close to 

molarity. It will be seen that the osmotic potential (in 

MPa) of sucrose solutions at 25°C can be roughly 

estimated by multiplying the % weight/volume 

concentration by -0.075: that of the monosaccharides 

fructose, glucose, mannitol and sorbitol (which have 

a molecular weight approximately 0.52 times that of 

sucrose), by multiplying by -0.14. 

If any of the sucrose in a medium becomes 

hydrolysed into monosaccharides, the osmotic 

potential of the combined sugar components (sucrose 

+ glucose + fructose), (Ψssugars), will be lower (more 

negative) than would be estimated from Table 4.5. 

The effect can be seen in Table 4.6. From this data it 

seems that 40-50% of the sucrose added to MS 

medium by Lazzeri et al. (1988) was broken down 

into monosaccharides during autoclaving. Hydrolysis 

of sucrose by plant-derived invertase enzymes will 

also have a similar effect on osmotic potential. In 

some suspension cultures, all sucrose remaining in 

the medium is inverted within 24 h. A fully inverted 

sucrose solution would have almost double the 

negative potential of the original solution, but as the 

appearance of glucose and fructose by enzymatic 

hydrolysis usually occurs concurrently with the 

uptake of sugars by the tissues, it will tend to have a 

stabilizing effect on osmotic potential during the 

passage of a culture. Reported measurements of the 

Chapter 4 

osmolality of MS medium containing 3% sucrose, 

after autoclaving, are: 

230 mOsm/kg (0.65% Phytagar; Lazzeri et al., 

1988) 

240 ± 20 mOsm/kg (0.6-0.925% agar; Scherer 

et al., 1988) 

230 ± 50 mOsm/kg (0.2-0.4% Gelrite; Scherer 

et al., 1988) 

4.2.2. Decreasing osmotic potential with other 

osmotica 

By adding soluble substances in place of some of 

the sugar in a medium, it can be shown that sugars 

not only act as a carbohydrate source, but also as 

osmoregulants. Osmotica employed for the 

deliberate modification of osmotic potential, should 

be largely lacking other biological effects. Those 

most frequently selected are the sugar alcohols 

mannitol and sorbitol. It is assumed that plants that 

do not have a native pathway for sugar alcohol 

biosynthesis are also deficient in pathways to 

assimilate them. Sugar alcohols, though, are usually 

translocated, and may be metabolised and utilized to 

various degrees (Steinitz, 1999; for mannitol 

Lipavska and Vreugedenhil, 1996 Tian and Russell, 

1999; for sorbitol Pua et al., 1984). Polyethylene 

glycol may be more helpful as an inert 

nonpenetrating osmolyte although it may contain 

toxic contaminants (Chazen et al., 1995). Mannitol 

can easily penetrate cell walls, but the plasmalemma 

is considered to be relatively impermeable to it 

(Rains, 1989), whereas high-molecular-weight 

polyethylene glycol 4000 is too large to penetrate cell 

walls (Carpita et al., 1979; Rains, 1989). Thus, a 

nonpenetrating osmolyte cannot penetrate into the 

plant cells, but inhibits water uptake. Sodium 

sulphate and sodium chloride have also been used in 

some experiments. 

Table 4.5. Predicted osmolality and the osmolality actually observed after autoclaving (data from Lazzeri et al., 1988). 

Predicted osmolality 

(no sucrose hydrolysis) 

Oberved total 

Salts Agar Sucrose Total osmolality 

(mOsm/kg) (mOsm/kg) (mOsm/kg) (mOsm/kg) (mOsm/kg) 

MS + agar 96 4 - 100 - 

MS + agar + 0.5% sucrose 96 4 15 115 115 

MS + agar + 1.0% sucrose 96 4 30 130 140 

MS + agar + 2.0% sucrose 96 4 60 160 184† 

MS + agar + 4.0% sucrose 

† 161 mOsm/kg by Brown et al. (1989) 

96 4 121 221 276 

135


Table 4.6. The osmolality and osmotic potential of sucrose solutions of different concentrations. 

Sucrose Concentration Osmolality Osmotic potential at 

25°C 

(% w/v) mM Osm/Kg MPa 

0.5 14.61 0.015 -0.037 

1.0 29.21 0.030 -0.074 

1.5 43.82 0.045 -0.112 

2.0 58.43 0.060 -0.149 

2.5 73.04 0.075 -0.186 

3.0 87.64 0.090 -0.223 

4.0 116.86 0.121 -0.300 

6.0 175.28 0.186 -0.461 

8.0 233.71 0.253 -0.627 

10.0 292.14 0.324 -0.803 

12.0 350.57 0.396 -0.982 

4.3. EFFECTS AND USES OF OSMOLYTES IN TISSUE 

CULTURE MEDIA 

4.3.1. Protoplast isolation and culture 

The osmotic potential of a plant cell is counterbalanced 

by the pressure potential exerted by the cell 

wall. To safely remove the cell wall during 

protoplast isolation without damaging the plasma 

membrane, it has been found necessary to plasmolyse 

cells before wall-degrading enzymes are used. This is 

done by placing the cells in a solution of lower water 

potential than that of the cell. 

Glucose, sucrose and especially mannitol and 

sorbitol, are usually added to protoplast isolation 

media for this purpose, either singly or in 

combination, at a total concentration of 0.35-0.7 M. 

These addenda are then retained in the subsequent 

protoplast culture medium, their concentration being 

progressively reduced as cell colonies start to grow. 

Tobacco cell suspensions take up only a very small 

amount of mannitol from solution (Thompson and 

Thorpe, 1981) and its effect as an osmotic agent 

appears to be exerted outside the cell (Thorpe, 1982). 

When protoplasts were isolated from Pseudotsuga 

and Pinus suspensions, which required a high 

concentration of inositol to induce embryogenesis , it 

was found to be essential to add 60 g/l (0.33 M) myoinositol 

(plus 30 g/l sucrose, 20 g/l glucose and 10 g/l 

sorbitol) to the isolation and culture media (Gupta 

et al., 1988). Mannitol and further amounts of 

sorbitol could not serve as substitutes. 

4.3.2. Osmotic effects on growth 

Solutions of different concentrations partly exert 

their effect on growth and morphogenesis by their 

nutritional value, and partly through their varying 

osmotic potential. Lapeña et al (1988) estimated that 

three quarters of the sucrose necessary to promote the 

optimum rate of direct adventitious shoot formation 

from Digitalis obscura hypocotyls, was required to 

supply energy, while the surplus regulated 

morphogenesis osmotically. 

How osmotic potential influences cellular 

processes is still far from clear. Cells maintained in 

an environment with low (highly negative) osmotic 

potential, lose water and in consequence the water 

potential of the cell decreases. This brings about 

changes in metabolism and cells accumulate high 

levels of proline (Rabe, 1990). The activity of the 

main respiratory pathway of cells (the cytochrome 

pathway) is reduced in conditions of osmotic stress, 

in favour of an alternative oxidase system (De Klerk- 

Kiebert and Van der Plas, 1985). The increase of the 

concentration of osmolytes, may also result in high 

levels of the plant hormone abscisic acid, both extra 

vitrum and in vitro (recent reviews Zhu, 2002; Riera 

et al., 2005). 

Equilibrium between the water potential of the 

medium and that of Echinopsis callus, only occurred 

when the callus was dead. Normally the water 

potential of the medium was greater so that water 

flowed into the callus (Kirkham and Holder, 1981). 

Clearly this situation could not occur in media which 

were too concentrated, and Cleland (1977) proposed 

that a critical water potential needs to be established 

within a cell before cell expansion and cell division, 

can occur. The osmotic concentration of culture 

media could therefore be expected to influence the 

rate of cell division or the success of morphogenesis 

of the cells or tissues they support. Both the 

inorganic and organic components will be

contributory. The cells of many plants which are 

natives of sea-shores or deserts (e.g. many cacti) 

characteristically have a low water potential (Ψcell) 

and in consequence may need to be cultured in media 

of relatively low (highly negative) osmotic potentials 

(Lassocinski, 1985). Sucrose concentrations of 4.5- 

6% have sometimes been found to be beneficial for 

such plants (Sachar and Iyer, 1959; Johnson and 

Emino, 1979; Mauseth, 1979; Lassocinski, 1985). 

Above normal sucrose concentrations can often be 

beneficial in media for anther culture [e.g 13% 

sucrose in Gamborg et al. (1968) B5 medium - 

Chuong and Beversdorf, 1985], and for the culture of 

immature embryos [e.g. 10% sucose in MS medium - 

Stafford and Davies (1979); 12.5% in Phillips and 

Collins (1979) L2. medium - Phillips et al. (1982)]. 

If the osmotic potential of the medium does 

indeed influence the growth of tissue cultures, one 

might expect the sucrose concentration, which is 

optimal for growth, to vary from one medium to 

another, more sucrose being required in dilute media 

than in more concentrated ones. Evans et al. (1976) 

found this was so with cultures of soybean tissue. 

Maximum rates of callus growth were obtained in 

media containing either: 

1. 50-75% of MS basal salts + 3-4% sucrose, or, 

2. 75-100% of MS basal salts + 2% sucrose. 

Similarly Yoshida et al. (1973) obtained equally 

good growth rates of Nicotiana glutinosa callus with 

nutrient media in which 

1. the salts exerted -0.274 MPa and sucrose -0.223 

MPa, or 

2. the salts exerted -0.365 MPa and sucrose -0.091 

MPa. 

It was essential to add 60 g/l sucrose (i.e. 

Ψssucrose= -0.461 MPa at 25°C) to the ‘MEDIUM’ salts 

of de Fossard et al. (1974) (Ψ = -0.135 MPa) to 

obtain germination and seedling growth from 

immature zygotic embryos of tomato. However, if 

the ‘HIGH’ salts (Ψsmedium = -0.260 MPa) were used, 

60 g/l sucrose gave only slightly better growth than 

2.1 g/l (Ψssucrose = -0.156 MPa) (Neal and Topoleski, 

1983). 

A detailed examination of osmotic effects of 

culture media on callus cultures was conducted by 

Kimball et al. (1975). Various organic substances 

were added to a modified Miller (1961) medium 

(which included 2% sucrose), to decrease the osmotic 

potential (Ψs) from -0.290 MPa. Surprisingly, the 

greatest callus growth was said to occur at -1.290 to 

-1.490 MPa (unusually low potentials which normally 

inhibit growth — see below) in the presence of 

Chapter 4 

137 

mannitol or sorbitol, and between -1.090 to -1.290 

MPa when extra sucrose or glucose were added. On 

the standard medium, many cells of the callus were 

irregularly shaped; as the osmotic potential of the 

solution was decreased there were fewer irregularities 

and at about Ψs = -1.090 MPa all the cells were 

spherical. The percentage dry matter of cultures also 

increased as Ψs was decreased. 

Doley and Leyton (1970) found that decreasing 

the water (osmotic) potential of half White (1963) 

medium by -0.100 or -0.200 MPa through adding 

more sucrose (and/or polyethylene glycol), caused the 

rate of callus growth from the cut ends of Fraxinus 

stem sections to be lower than on a standard medium. 

At the reduced water potential, callus had suberised 

surfaces and grew through the activity of a vascular 

cambium. It also contained more lignified xylem and 

sclereids. At each potential there was an optimal 

IAA concentration for xylem differentiation. 

When the concentration of sucrose in a high salt 

medium such as MS is increased above 4-5 per cent, 

there begins to be a progressive inhibition of cell 

growth in many types of culture. This appears to be 

an osmotic effect because addition of other 

osmotically-active substances (such as mannitol and 

polyethyleneglycol) to the medium causes a similar 

response (Maretzki et al., 1972). Usually, high 

concentrations of sucrose are not toxic, at least not in 

the short term, and cell growth resumes when tissues 

or organs are transferred to media containing normal 

levels of sugar. Increase of Ψs is one method of 

extending the shelf life of cultures. Pech and Romani 

(1979) found that the addition of 0.4 M mannitol to 

MS medium (modified organics), was able to prevent 

the rapid cell lysis and death which occurred when 

2,4-D was withdrawn from pear suspension cultures. 

Decreasing the osmotic potential (usually by 

adding mannitol) together with lowering of the 

temperature has been used to reduce the growth rate 

for preservation of valuable genotypes in vitro. This 

has been reported, among others, for potato (Gopal 

et al., 2002; Harding et al., 1997), Dioscorea alata 

(Borges et al., 2003) and enset (Negash et al., 2001) 

4.3.3. Tissue water content 

The water content of cultured tissues decreases as 

the level of sucrose in the medium is increased. 

Isolated embryos of barley were grown by Dunwell 

(1981) on MS medium in sucrose concentrations up 

to 12 per cent. Dry weight increased as the sucrose 

concentration was raised to 6 or 9 per cent and shoot 

length of some varieties was also greater than on 3 

per cent sucrose. Water content of the developing


plantlets was inversely proportional to the sucrose 

level. 

The dry weight of Lilium auratum bulbs and roots 

on MS medium increased as the sucrose 

concentration was augmented to 90 g/l but decreased 

dramatically with 150 g/l because growth was 

inhibited. The fresh weight/dry weight ratio of both 

kinds of tissue once again declined progressively as 

sucrose was added to the medium (0 - 150 g/l). In a 

medium containing 30 mg/l sucrose, the number and 

fresh weight of bulblets increased as MS salt strength 

was raised from one eighth to two times its normal 

concentration. An interaction between salt and 

sucrose concentrations was demonstrated in that 

optimum dry weight of bulbs could be obtained in 

either single strength MS + 120 g/l sucrose 

(osmolality, without sucrose inversion = ca. 492 

mOsm/kg; Ψs = ca. -1.22 MPa), or double strength 

MS + 60 g/l sucrose (osmolality, without sucrose 

inversion = ca. 378 mOsm/kg; Ψs = ca. -0.94 MPa) 

(Takayama and Misawa, 1979). 

4.3.4. Morphogenesis 

The osmotic effect of sucrose in culture solutions 

was well demonstrated by a series of experiments on 

tobacco callus by Brown et al., (1979): rates of callus 

growth and shoot regeneration which were optimal on 

culture media containing 3 per cent sucrose, could be 

maintained when the sugar was replaced partially by 

osmotically equivalent levels of mannitol. The 

optimal (medium plus sucrose) here was between 

-0.4 and -0.6 MPa, and increasing sucrose levels 

above 3 per cent brought a progressive decrease in 

shoot regeneration. Similar results were obtained by 

Barg and Umiel (1977), but when they kept the 

osmotic potential of the culture solution roughly 

constant by additions of mannitol, the sucrose 

concentrations optimal for tobacco callus growth or 

morphogenesis were not the same (Fig. 4.2) 

Brown and Thorpe (1980) subsequently found 

that callus of Nicotiana capable of forming shoots, 

had a water potential (Ψ) of -0.8 MPa, while nonshoot-forming 

callus had a Ψ of -0.4 MPa. The two 

relationships were: 

Shoot forming callus 

Ψcell = Ψs +Ψp + Ψm 

–0.8 =–1 + 0.4 + 0 MPa 

Non-shoot-forming callus 

Ψcell = Ψs +Ψp + Ψm 

–0.4 = –0 + 0.3 + 0 MPa 

Correct water potential. An optimum rate of 

growth and adventitious shoot formation of wheat 

callus occurred on MS medium containing 2% 

sucrose. Only a small number of shoots were 

produced on the medium supplemented with 1% 

sucrose, but if mannitol was added so that the total 

Ψcell = Ψs was the same as when 2% sucrose was 

present, the formation of adventitious shoots was 

stimulated (Galiba and Erdei, 1986). Very similar 

results were obtained by Lapeña et al. (1988). A 

small number of shoot buds were produced from 

Digitalis obscura hypocotyls on MS medium 

containing 1% sucrose: more than twice as many if 

the medium contained 2% sucrose (total Ψs given as 

-0.336 MPa), or 1% sucrose plus mannitol to again 

give a total Ψs equal to -0.336 MPa. 

Water potential can modify commitment. 

Morphogenesis can also be regulated by altering the 

water potential of media. Shepard and Totten (1977) 

found that very small (ca. 1-2 mm) calluses formed 

from potato mesophyll protoplasts were unable to 

survive in 1 or 2% sucrose, and the base of larger (5- 

10 mm) ones turned brown, while the upper portions 

turned green but formed roots and no shoots. The 

calli became fully green only on 0.2-0.5% sucrose. 

At these levels shoots were formed in the presence of 

0.2-0.3 M mannitol. When the level of mannitol was 

reduced to 0.05 M, the proportion of calli 

differentiating shoots fell from 61% to 2%. The 

possibility of an osmotic affect was suggested 

because equimolar concentrations of myo-inositol 

were just as effective in promoting shoot 

regeneration. 

Another way to modify morphogenesis is to 

increase the ionic concentration of the medium. Pith 

phloem callus of tobacco proliferates on Zapata et al. 

(1983) MY1 medium supplemented with 10 –5 M IAA 

and 2.5 x 10 –6 M kinetin, but forms shoots on 

Murashige et al. (1972) medium containing 10 –5 M 

IAA and 10 –5 M kinetin. These two media contain 

very similar macronutrients (total ionic concentrations, 

respectively 96 and 101mM), yet adding 0.5- 

1.0% sodium sulphate (additional osmolality 89-130 

mOsm/kg) decreased the shoot formation of callus 

grown on the shoot-forming medium, but increased it 

on the medium which previously only supported 

callus proliferation (Pua et al., 1985a). Callus 

cultured in the presence of sodium sulphate retained 

its shoot-producing capacity over a long period, 

although the effect was not permanent (Pua et al., 

1985b; Chandler et al., 1987). In these experiments 

shoot formation was also enhanced by sodium 

chloride and mannitol. 

Increasing the level of sucrose from 1 to 3 per 

cent in MS medium containing 0.3 mg/l IAA,

induced tobacco callus to form shoots, while further 

increasing it to 6 per cent resulted in root 

differentiation (Rawal and Mehta, 1982; Mehta, 

1982). The formation of adventitious shoots from 

Nicotiana tabacum pith callus is inhibited on a 

medium with MS salts if 10-15% sucrose is added. 

Preferential zones of cell division and meristemoids 

produced in 3% sucrose then become disorganised 

into parenchymatous tissue (Hammersley-Straw and 

Thorpe, 1988). 

Chapter 4 

It should be noted that auxin which has a very 

important influence on the growth and 

morphogenesis of cultured plant cells, causes their 

osmotic potential to be altered (Van Overbeek, 1942; 

Hackett, 1952; Ketellapper, 1953). When tobacco 

callus is grown on a medium which promotes shoot 

regeneration, the cells have a greater osmotic 

pressure (or more negative water potential) than 

callus grown on a non-inductive medium (Brown and 

Thorpe, 1980; Brown, 1982). 

Fig. 4.2 The effect of sucrose concentration on the growth and morphogenesis of tobacco callus. 

[Drawn from data for four lines of tobacco callus in Barg and Umiel, 1977]. Callus growth = solid line. Morphogenesis = line of dashes. 

Scale was, 1 = No differentiation, 2 = Dark green callus with meristemoids, 3 = leafy shoots 

Apogamous buds on ferns. Whittier and Steeves 

(1960) found a very clear effect of glucose 

concentration on the formation of apogamous buds on 

prothalli of the fern Pteridium. (Apogamous buds 

give rise to the leafy and spore-producing generation 

of the plant which has the haploid genetic 

constitution of the prothallus). Bud formation was 

greatest between 2-3% glucose (optimum 2.5%). 

Results which confirm this observation were obtained 

by Menon and Lal (1972) in the moss Physcomitrium 

pyriforme. Here apogamous sporophytes were 

formed most freely in low sucrose concentrations 

(0.5-2%) and low light conditions (50-100 lux), and 

were not produced at all when prothalli were cultured 

in 6% sucrose or high light (5000-6000 lux). Whittier 

and Steeves (loc. cit.) noted that they could not obtain 

the same rate of apogamous bud production by using 

0.25% glucose plus mannitol or polyethyleneglycol; 

on the other hand, adding these osmotica to 2.5% 

glucose (so that the osmotic potential of the solution 

139 

was equivalent to one with 8% sucrose) did reduce 

bud formation. It therefore appeared that the 

stimulatory effect of glucose on morphogenesis was 

mainly due to its action as a respiratory substrate, but 

that inhibition might be caused by an excessively 

depressed osmotic potential. 

Differentiation of floral buds. Pieces of coldstored 

chicory root were found by Margara and 

Rancillac (1966) to require more sucrose (up to 68 

g/l; 199 mM) to form floral shoots than to produce 

vegetative shoots (as little as 17 g/l; 50 mM). Tran 

Thanh Van and co-workers (Tran Thanh Van and 

Trinh, 1978) have similarly shown that the specific 

formation of vegetative buds, flower buds, callus or 

roots by thin cell layers excised from tobacco stems, 

could be controlled by selecting appropriate 

concentrations of sugars and of auxin and cytokinin 

growth regulators. 

Root formation and root growth. Media of small 

osmotic potential are usually employed for the


induction and growth of roots on micropropagated 

shoots. High salt levels are frequently inhibitory to 

root initiation. Where such levels have been used for 

Stage II of shoot cultures, it is common to select a 

low salts medium (e.g. ¼ or ½ MS), when detached 

shoots are required to be rooted at Stage III. By 

testing four concentrations of MS salts (quarter, half, 

three quarters and full strength) against four levels of 

sucrose (1, 2, 3 and 4%), Harris and Stevenson 

(1979) found that correct salt concentration (½ or ¼ 

MS) was more important than sucrose concentration 

for root induction on grapevine cuttings in vitro. The 

benefit of low salt levels for root initiation may be 

due more to the need for a low nitrogen level, than 

for an increased osmotic potential. Dunstan (1982) 

showed that microcuttings of several tree-fruit 

rootstocks rooted best on MS salts, but that in media 

of these concentrations, the amount of added sugar 

was not critical, although it was essential for there to 

be some present. For the best rooting of Castanea, it 

was important to place shoots in Lloyd and McCown 

(1981) WPM medium containing 4% sucrose (Serres, 

1988). 

There are reports that an excessive sugar 

concentration can inhibit root formation. Green 

cotyledons of Sinapis alba and Raphanus sativus 

were found by Lovell et al. (1972) to form roots in 

2% sucrose in the dark, but not in light of 5500 lux 

luminous intensity. In the light, rooting did occur if 

the explants were kept in water, or (to a lesser extent) 

if they were treated with DCMU (a chemical inhibitor 

of photosynthesis) before culture in 2% sucrose. The 

authors of this paper suggested that sugars were 

produced within the plant tissues during 

photosynthesis, which, added to the sucrose absorbed 

from the medium, provided too great a total sugar 

concentration for rooting. Rahman and Blake (1988) 

reached the same conclusion in experiments on 

Artocarpus heterophyllus. When shoots of this plant 

were kept on a rooting medium in the dark, the 

number and weight of roots formed on shoots, 

increased with the inclusion of up to 80 g/l sucrose. 

The optimum sucrose concentration was 40 g/l if the 

shoots were grown in the light. 

Root formation on avocado cuttings in 0.3 MS 

salts [plus Linsmaier and Skoog (1965) vitamins] was 

satisfactory with 1.5, 3 or 6% sucrose, and only 

reduced when 9% sucrose was added (Pliego-Alfaro, 

1988). 

Although 4% (and occasionally 8%) sucrose has 

been used in media for isolated root culture, 2% has 

been used in the great majority of cases (Butcher and 

Street, 1964). In an investigation into the effects of 

sucrose concentration on the growth of tomato roots, 

Street and McGregor (1952) found that although 

sucrose concentrations of between 1.5 and 2.5% 

caused the same rate of increase of root fresh weight, 

1.5% sucrose was optimal. It produced the best rate 

of growth of the main root axis, and the greatest 

number and total length of lateral roots. 

Somatic embryogenesis. The osmotic potential 

of a medium can influence whether somatic 

embryogenesis can occur and can regulate the proper 

development of embryos. As will be shown below, a 

low osmotic potential is often favourable, but this is 

not always the case. For instance immature 

cotyledons of Glycine max produced somatic 

embryos on Phillips and Collins (1979) L2 medium 

containing less than 2% sucrose, but not if the 

concentration of sugar was increased above this level 

(Lippmann and Lippmann, 1984). 

Placing tissues in solutions with high osmotic 

potential will cause cells to become plasmolysed, 

leading to the breaking of cytoplasmic 

interconnections between adjacent cells 

(plasmodesmata). Wetherell (1984) has suggested 

that when cells and cell groups of higher plants are 

isolated by this process, they become enabled to 

develop independently, and express their totipotency. 

He pointed out that the isolation of cells of lower 

plants induces regeneration, and plasmolysis has long 

been known to initiate regeneration in multicellular 

algae, the leaves of mosses, fern prothallia and the 

gemmae of liverworts (Narayanaswami and LaRue, 

1955; Miller, 1968). Carrot cell cultures preplasmolysed 

for 45 min in 0.5-1.0 M sucrose or 1.0 

M sorbitol gave rise to many more somatic embryos 

when incubated in Wetherell (1969) medium with 0.5 

mg/l 2,4-D than if they had not been pre-treated in 

this way. Moreover embryo formation was more 

closely synchronized. Ikeda-Iwai et al. (2003) found 

that in Arabidopsis a 6-12 hour treatment with 0.7 M 

sucrose, sorbitol or mannitol resulted in somatic 

embryogenesis. 

Callus derived from hypocotyls of Albizia 

richardiana, produced the greatest numbers of 

adventitious shoots on B5 medium containing 4% 

sucrose, but somatic embryos grew most readily 

when 2% sucrose was added. At least 1% sucrose 

was necessary for any kind of morphogenesis to take 

place (Tomar and Gupta, 1988). A similar result was 

obtained by Ćulafić et al. (1987) with callus from 

axillary buds of Rumex acetosella: adventitious 

shoots were produced on a medium containing MS

salts and 2% sucrose (Ψs = ca. -0.39 MPa at 25°C), 

but embryogenesis occurred when the sucrose 

concentration was increased to 6% (Ψssucrose = -0.46 

MPa, Ψs = ca. -0.70 MPa at 25°C) or if the medium 

was supplemented with 2% sucrose plus 21.3 g/l 

mannitol or sorbitol (which together have the same 

osmolality as 6% sucrose). 

A low (highly negative) osmotic potential helps to 

induce somatic embryogenesis in some other plants. 

Adding 10-30 g/l sorbitol to Kumar et al. (1988) L-6 

medium (total macronutient ions 64.26 mM; 20 g/l 

sucrose), caused there to be a high level of 

embryogenesis in Vigna aconitifolia suspensions and 

the capacity for embryogenesis to be retained in longterm 

cultures. The formation of somatic embryos in 

ovary callus of Fuchsia hybrida was accelerated by 

adding 5% sucrose to B5 medium (Dabin and Beguin, 

1987), and the induction of embryogenic callus of 

Euphorbia longan required the culture of young 

leaflets on B5 medium with 6% sucrose (Litz, 1988). 

There are exceptions, particularly with regard to 

embryo growth. The proportion of Ipomoea batatas 

somatic embryos forming shoots was greatest when a 

medium containing MS inorganics contained 1.6%, 

rather than 3% sucrose (Chée et al., 1990). Protocorm 

proliferation of orchids is most rapid when tissue is 

cultured in high concentrations of sucrose, but for 

plantlet growth, the level of sucrose must be reduced 

(Homès and Vanseveran-Van Espen, 1973). 

The induction of embryogenic callus from 

immature seed embryos of Zea mays was best on MS 

medium with 12% sucrose (Lu et al., 1982), and Ho 

and Vasil (1983) used 6-10% sucrose in MS medium 

to promote the formation of pro-embryoids from 

young leaves of Saccharum officinarum. However, 

in the experiments of Ahloowalia and Maretski 

(1983), somatic embryo formation from callus of this 

plant was best on MS medium with 3% sucrose, but 

growth of the embryos into complete plantlets 

required that the embryos should be cultured first on 

MS medium with 6% sucrose and then on MS with 

3% sucrose. 

Polyethylene glycol 4000 (PEG 4000) improves 

root and shoot emergence without limiting embryo 

histodifferentiation in soybean somatic embryos 

(Walker and Parrott, 2001). Likewise in spruce, it 

was reported that polyethylene glycol might improve 

the quality of somatic embryos by promoting normal 

differentiation of the embryonic shoot and root (e.g. 

Stasolla et al., 2003). Non-penetrating osmotica like 

polyethylene glycol cannot enter plant cells, but 

restrict water uptake and provide a simulated drought 

Chapter 4 

141 

stress during embryo development. A combination of 

ABA and an osmoticum prevents precocious 

germination in white spruce (Attree et al., 1991) 

and allows embryo development to proceed. 

Advantageous effects of polyethylene glycol and 

ABA have been reported in a number of species 

(Hevea brasiliensis, Linossier et al., 1997; Picia 

abies, Bozhkov and Von Arnold, 1998; white spruce, 

Stasolla et al., 2003, Corydalis yanhusuo, Sagare 

et al., 2000; and Panax ginseng, Langhansová et al., 

2004). 

When cultured on Sears and Deckard (1982) 

medium, embryogenesis in callus initiated from 

immature embryos of ‘Chinese Spring’ and some 

other varieties of wheat was incomplete, because 

shoot apices germinated and grew before embryos 

had properly formed. More typical somatic embryos 

could be obtained by adding 40 mM sodium or 

potassium chloride to the medium. The salts had to 

be removed to allow plantlets to develop normally 

(Galiba and Yamada, 1988). Transferring somatic 

embryos to a medium of lower (more negative) water 

potential is often necessary to ensure their further 

growth and/or germination. High sucrose levels are 

often required in media for the culture of zygotic 

embryos if they are isolated when immature. The use 

of 50-120 g/l sucrose in media is then reported, the 

higher concentrations usually being added to very 

weak salt mixtures. Embryos which are more fully 

developed when excised, grow satisfactorily in a 

medium with 10-30 g/l sugar. 

Storage organ formation. At high 

concentration, sucrose promotes the formation of 

tubers, bulbs and corms (e.g. Xu et al., 1998; 

Vreugdenhil et al., 1998; Ziv 2005, Gerrits and de 

Klerk 1992). This promotion might be mediated by 

ABA since osmotic stress induces ABA synthesis 

(Riera et al., 2005) and ABA promotes bulb (Kim 

et al., 1994) and tuber (Xu et al., 1998) formation. 

The situation is, however, more complex. Suttle and 

Hultstrand (1994) did not find a reduction of tuber 

formation in potato by adding fluridone, an ABAsynthesis 

inhibitor, and Xu et al. (1998) did not 

observe an increased ABA-level at high sucrose 

concentration. So in potato, the effect of sucrose is 

not likely to be mediated by ABA. Exogenous ABA 

does not promote Gladiolus corm formation (Dantu 

and Bhojwani, 1995) but bulb formation of lily was 

completely inhibited by fluridone and restored by 

simultaneous addition of ABA (Kim et al., 1994). 

To establish the effect of osmoticum directly, 

experiments have been carried out with addition of


mannitol instead of sucrose. Mannitol did not 

promote corm production of Gladiolus (De Bruyn 

and Ferreira, 1992) or bulb production in onion 

(Kahane and Rancillac, 1996), but data for lily 

suggested that, although it had a toxic effect, in this 

plant mannitol did stimulate bulb formation (Gerrits 

and De Klerk, 1992). 

Anther culture. The use of high concentrations 

of sucrose is commonly reported in papers on anther 

culture where the addition of 5-20% sucrose to the 

culture medium is found to assist the development of 

somatic embryos from pollen microspores. This 

appears to be due to an osmotic regulation of 

morphogenesis (Sunderland and Dunwell, 1977), for 

once embryoid development has commenced, such 

high levels of sucrose are no longer required, or may 

be inhibitory. A high concentration of mannitol has 

been used for pretreatment before the culture of 

barley anthers (Roberts-Oehlschlager and Dunwell 

1990) and pollen (Wei et al., 1986); tobacco anthers 

(Imamura and Harada 1980) and pollen (Imamura 

et al., 1982); and before wheat microspore culture 

(Hu et al., 1995). A high concentration of mannitol 

has also been used to induce osmotic stress in 

microspore derived embryos of Brassica napus 

(Huang et al., 1991) and before anther culture of 

Brassica campestris (Hamaoka et al., 1991). Isolated 

microspores of Brassica napus cultured on a high 

concentration of mannitol and at a low concentration 

of sucrose (0.08–0.1%) yield no embryos whereas on 

high polyethyleneglycol 4000 the embryo yield is 

comparable to that of the sucrose control (Ikeda-Iwai, 

2003). These results demonstrate that in microspore 

embryogenesis of Brassica napus the level of 

metabolizable carbohydrate required for microspore 

embryo induction and formation may be very low and 

that an appropriate osmoticum (polyethylene glycol 

4000 or sucrose) is required. 

The temporary presence of high sucrose 

concentrations is said to prevent the proliferation of 

callus from diploid cells of the anther that would 

otherwise swamp the growth of the pollen-derived 

embryoids. The concentration of macronutrient ions 

generally used in anther culture media is not 

especially low. In a sample of reports it was found to 

be 68.7 mM (George et al., 1987), and so the total 

osmotic potential, Ψs, (salts plus sucrose) of many 

anther culture media is in the range -0.55 to -1.15 

MPa. 

4.3.5. Relative humidity. 

The vapour pressure of water is reduced by 

dissolving substances in it. This means that the 

relative humidity of the air within closed culture 

vessels is dependent on the water potential of the 

medium according to the equation: 

1000RT 

⎛ p 

Ψ = lne ⎜ 

W ⎜ 

0 ⎝ p0 

(after Glasstone, 1947) 

⎞ ⎛ dp ⎞ 

⎟ . 

⎟ ⎜ p − c ⎟ 

⎠ ⎝ dc ⎠ 

where: Ψ, R and T are as in the Van’t Hoff equation, 

W0 is the molecular weight of water, c is the 

concentration of the solution in moles per litre, and p0 

and p are respectively the vapour pressures of water 

and the solution. 

If the change in vapour pressure dependent on the 

density of the solution, p – c (dp/dc), is treated as 

unity (legitimate perhaps for very dilute solutions, or 

when the equation is expressed in molality, rather 

than molarity), it is possible to estimate relative 

humidity (100 × p/p0) above tissue culture media of 

known water potentials, from: 

1000RT ⎛ 

Ψ = lne 

18. 

016 ⎜ 

⎝ 

p ⎞ 

⎟ , (Lang, 1967) 

⎠ 

p0 

The relative humidity above most plant tissue 

cultures in closed vessels is thus calculated to be in 

the range 99.25-99.75% (Table 4.7), the osmolality of 

some typical media being 

• White (1963), liquid, 2% sucrose, 106 mOsm/kg 

• MS, agar, 3% sucrose, 230 mOsm/kg 

• MS, agar, 6% sucrose, 359 mOsm/kg 

• MS, agar, 12% sucrose (unusual), 659 mOsm/kg 

(in these cases, 50% hydrolysis of sucrose into 

monosaccharides is assumed to have taken place 

during autoclaving) 

Relative humidity can be reduced below the levels 

indicated above by, for example, covering vessels 

with gas-permeable closures while using nongelatinous 

support systems (see Section 6.3.1).

Table 4.7 The relative humidity within culture vessels which would be expected to result from the use of 

plant culture media of various osmolalities or water potentials 

The relative acidity or alkalinity of a solution is 

assessed by its pH. This is a measure of the hydrogen 

ion concentration; the greater the concentration of H + 

ions (actually H3O + ions), the more acid the solution. 

As pH is defined as the negative logarithm of 

hydrogen ion concentration, acid solutions have low 

pH values (0-7) and alkaline solutions, high values 

(7-14). Solutions of pH 4 (the concentration of H + is 

10 –4 mol.l –1 ) are therefore more acid than those of pH 

5 (where the concentration of H + is 10 –5 mol.l –1 ); 

solutions of pH 9 are more alkaline than those of pH 

8. Pure water, without any dissolved gases such as 

CO2, has a neutral pH of 7. To judge the effect of 

medium pH, it is essential to discriminate between 

the various sites where the pH might have an effect: 

(1) in the explant, (2) in the medium and (3) at the 

interface between explant and medium. 

The pH of a culture medium must be such that it 

does not disrupt the plant tissue. Within the 

acceptable limits the pH also: 

• governs whether salts will remain in a soluble 

form; 

• influences the uptake of medium ingredients and 

plant growth regulator additives; 

• has an effect on chemical reactions (especially 

those catalysed by enzymes); and 

• affects the gelling efficiency of agar. 

This means that the effective range of pH for 

media is restricted. As will be explained, medium pH 

is altered during culture, but as a rule of thumb, the 

initial pH is set at 5.5 – 6.0. In culture media, 

detrimental effects of an adverse pH are generally 

related to ion availability and nutrient uptake rather 

than cell damage. 

5.1. THE pH OF MEDIA 

5.1.1. Buffering 

The components of common tissue culture media 

have only little buffering capacity. Vacin and Went 

(1949) investigated the effect on pH of each 

Chapter 4 143 

Relative humidity in vessel 

Water potential (kPa) at temperature: 

(%) 

Osmolality 

(mOsm/kg) 15 ºC 20 ºC 25 ºC 30 ºC 

98 1121 -2686 -2733 -2780 -2826 

99 558 -1337 -1360 -1383 -1406 

99.5 278 -667 -678 -690 -701 

99.75 139 -333 -339 -344 -350 

5. pH OF TISSUE CULTURE MEDIA 

compound in their medium. The chemicals which 

seemed to be most instrumental in changing pH were 

FeSO4.7H2O and Ca(NO3)2.4H2O. Replacing the 

former with ferric tartrate at a weight which 

maintained the original molar concentration of iron, 

and substituting Ca3(PO4)2 and KNO3 for 

Ca(NO3)2.4H2O, they found that the solution was 

more effectively buffered. While amino acids also 

showed promise as buffering agents, KH2PO4 was 

ineffective unless it was at high concentration. In 

some early experiments attempts were made to 

stabilise pH by incorporating a mixture of KH2PO4 

and K2HPO4 into a medium (see Kordan, 1959, for 

example), but Street and Henshaw (1966) found that 

significant buffering was only achieved by soluble 

phosphates at levels inhibitory to plant growth. For 

this reason Sheat et al. (1959) proposed the buffering 

of plant root culture media with sparingly-soluble 

calcium phosphates, but unfortunately if these 

compounds are autoclaved with other medium 

constituents, they absorb micronutrients which then 

become unavailable. 

A buffer is a compound which can poise the pH 

level at a selected level: effective buffers should 

maintain the pH with little change as culture 

proceeds. As noted before, plant tissue culture media 

are normally poorly buffered. However, pH is 

stabilised to a certain extent when tissues are cultured 

in media containing both nitrate and ammonium ions. 

Agar and Gelrite gelling agents may have a slight 

buffering capacity (Scherer, 1988). 

Organic acids: Many organic acids can act as 

buffers in plant culture media. By stabilizing pH at 

ca. pH 5.5, they can facilitate the uptake of NH4 + 

when this is the only source of nitrogen, and by their 

own metabolism, assist the conversion of NH4 + into 

amino acids. There can be improvement to growth 

from adding organic acids to media containing both 

NH4 + and NO3 – , but this is not always the case. 

Norstog and Smith (1963) noted that 0.75 mM malic


acid was an effective buffer and appeared to enhance 

the effect of the glutamine and alanine which they 

added to their medium. Vyskot and Bezdek (1984) 

found that the buffering capacity of MS medium was 

increased by adding either 1.25 mM sodium citrate or 

1.97 mM citric acid plus 6.07 mM dibasic sodium 

phosphate. Citric acid and some other organic acids 

have been noted to enhance the growth of Citrus 

callus when added to the medium (presumably that of 

Murashige and Tucker, 1969) (Erner and Reuveni, 

1981). For the propagation of various cacti from 

axillary buds, Vyskot and Jara (1984) added sodium 

citrate to MS medium to increase its buffering 

capacity. 

Recognized biological buffers: Unlike organic 

acids, conventional buffers are not metabolised by the 

plant, but can poise pH levels very effectively. 

Compounds which have been used in plant culture 

media for critical purposes such as protoplast 

isolation and culture, and culture of cells at very low 

inoculation densities, include: 

• TRIS, Tris(hydroxymethyl)aminomethane; 

• Tricine, N-tris(hydroxymethyl)methylglycine; 

• MES, 2-(N-morpholino)ethanesulphonic acid; 

• HEPES, 4-(2-hydroxyethyl)-1-piperazine(2ethanesulphonic 

acid); and 

• CAPS, 3-cyclohexylamino-1-propanesulphonic 

acid. 

Such compounds may have biological effects 

which are unrelated to their buffering capacity. 

Depending on the plant species, they have been 

known to kill protoplasts or greatly increase the rate 

of cell division and/or plant regeneration (Conrad 

et al., 1981). The ‘biological’ buffers MES and 

HEPES have been developed for biological research 

(Good et al., 1966). 

MES: MES is one of the few highly effective and 

commercially available buffers with significant 

buffering capacity in the pH range 5-6 to which plant 

culture media are usually adjusted and has only a low 

capacity to complex with micronutrients. It is not 

toxic to most plants, although there are some which 

are sensitive. Ramage and Williams (2002) report 

that shoot regeneration from tobacco leaf discs was 

not affected by MES when increasing the 

concentration up to 100 mM. De Klerk et al (2007), 

though, observed a decrease of rooting from apple 

stem slices with increasing MES concentration (see 

below; Fig. 4.4). This effect of MES was not 

understood. It only occurred during the first days of 

the rooting process and was not observed during the 

outgrowth phase after the meristems had been 

formed. 

During the culture of thin cell layers of Nicotiana, 

Tiburcio et al. (1989) found that the pH of LS 

medium could be kept close to 5.8 for 28 days by 

adding 50 mM MES, whereas without the buffer pH 

gradually decreased to 5.25. Regulating pH with 

MES alters the type of morphogenesis which occur in 

this (and other) tissues (see below). 

Parfitt et al. (1988) found 10 mM MES to be an 

effective buffer in four different media used for 

tobacco, carrot and tomato callus cultures, and peach 

and carnation shoot cultures, although stabilizing pH 

did not result in superior growth. The tobacco, peach 

and carnation cultures were damaged by 50 mM 

MES. Tris was toxic at all concentrations tested, 

although Klein and Manos (1960) had found that the 

addition of only 0.5 mM Tris effectively increased 

the fresh weight of callus which could be grown on 

White (1954) medium when iron was chelated with 

EDTA. 

MES has also been used successfully to buffer 

many cultures initiated from single protoplasts 

(Müller et al., 1983) and its inclusion in the culture 

medium can be essential for the survival of individual 

cells and their division to form callus colonies (e.g. 

those of Datura innoxia - Koop et al., 1983). MES 

was found to be somewhat toxic to single protoplasts 

of Brassica napus, but ‘Polybuffer 74’ (PB-74, a 

mixture of polyaminosulphonates), allowed excellent 

microcolony growth in the pH range 5.5-7.0 

(Spangenberg et al., 1986). Used at 1/100 of the 

commercially available solution, it has a buffering 

capacity of a 1.3 mM buffer at its pK value (Koop 

et al., 1983). 

Banana homogenate is widely used in orchid 

micropropagation media. Ernst (1974) noted that it 

appeared to buffer the medium in which slipper 

orchid seedlings were being grown. 

5.1.2. The uptake of ions and molecules 

The pH of the medium has an effect on the 

availability of many minerals (Scholten and Pierik, 

1998). In general, the uptake of negatively charged 

ions (anions) is favoured at acid pH, while that of 

cations (positively charged) is best when the pH is 

increased. As mentioned before, the relative uptake of 

nutrient cations and anions will alter the pH of the 

medium. The release of hydroxyl ions from the plant 

in exchange for nitrate ions results in media 

becoming more alkaline; when ammonium ions are

taken up in exchange for protons, media become 

more acid (Table 4.8). 

Nitrate and ammonium ions: The uptake of 

ammonium and nitrate ions is markedly affected by 

pH. Excised plant roots can be grown with NH4 + as 

the sole source of nitrogen providing the pH is 

maintained within the range 6.8 to 7.2 and iron is 

available in a chelated form. At pH levels below 6.4 

the roots grow slowly on ammonium alone and have 

an abnormal appearance (Sheat et al.,1959). This 

accords with experiments on intact plants where 

ammonium as the only source of nitrogen is found to 

be poorly taken up at low pH. It was most effectively 

utilised in Asparagus in a medium buffered at pH 5.5. 

At low pH (ca. 4 or less) the ability of the roots of 

most plants to take up ions of any kind may be 

impaired, there may be a loss of soluble cell 

constituents and growth of both the main axis and 

that of laterals is depressed (Asher, 1978). 

Nitrate on the other hand, is not readily absorbed 

by plant cells at neutral pH or above (Martin and 

Rose, 1976). Growth of tumour callus of Rumex 

acetosa on a nitrate-containing medium was greater 

at pH 3.5 than pH 5.0 (Nickell and Burkholder, 

1950), while Chevre et al. (1983) reported that 

axillary bud multiplication in shoot cultures of 

Castanea was most satisfactory when the pH of MS 

was reduced to 4 and the Ca 2+ and Mg 2+ 

concentrations were doubled. 

pH Stabilization: One of the chief advantages of 

having both NO3 – and NH4 + ions in the medium is 

that uptake of one provides a better pH environment 

for the uptake of the other. The pH of the medium is 

thereby stabilized. Uptake of nitrate ions by plant 

cells leads to a drift towards an alkaline pH, while 

NH4 + uptake results in a more rapid shift towards 

acidity (Street, 1969; Behrend and Mateles, 1975; 

Hyndman et al., 1982). For each equivalent of 

ammonium incorporated into organic matter, about 

0.8-1 H + (proton) equivalents are released into the 

external medium; for each equivalent of nitrate 

assimilated, 1-1.2 proton equivalents are removed 

from the medium (Fuggi et al., 1981). Raven (1986) 

calculated that there should be no change of pH 

Table 4.8 The uptake of ions and its consequences in plant culture media 

Uptake anion (e.g. NH4 + ) 

Rate Best in alkaline or weakly acid solutions 

Consequence Protons (H + ) extruded by plant 

Medium becomes more ACID 

Chapter 4 145 

Uptake cation (e.g. NO3 ⎯ ) 

Best in relatively acid solutions 

Hydroxyl (OH ⎯ ) ions extruded by plant 

Medium becomes more ALKALINE 

resulting from NO3 – or NH4 + uptake, when the ratio 

of the two is 2 to 1. 

The pH shifts caused by uptake of nitrate and 

ammonium during culture (see above) can lead to a 

situation of nitrogen deficiency if either is used as the 

sole nitrogen source without the addition of a buffer 

(see later) (Hyndman et al., 1982). The uptake of 

NH4 + , when this is the sole source of nitrogen is only 

efficient when a buffer or an organic acid is also 

present in the medium. In media containing both 

NO3 – and NH4 + with an initial pH of 5-6, preferential 

uptake of NH4 + causes the pH to drop during the early 

growth of the culture. This results in increased NO3 – 

utilisation (Martin and Rose, 1976) and a gradual pH 

rise. The final pH of the medium depends on the 

relative proportions of NO3 – and NH4 + which are 

provided (Gamborg et al., 1968). After 7 days of root 

culture, White (1943a) medium (containing only 

nitrate), adjusted to pH 4.8-4.9, had a pH of 5.8-6.0 

(Street et al., 1951, 1952), but Sheat et al. (1959) 

could stabilize the medium at pH 5.8 by having one 

fiftieth of the total amount of nitrogen as ammonium 

ion, the rest as nitrate. Changes in the pH of a 

medium do however vary from one kind of plant to 

another. Ramage and Williams (2002) observed a 

decrease in pH when tobacco leaf discs were cultured 

with only NH4 + nitrogen whereas no such decrease 

was observed on medium with both NH4 + and NO3 – . 

No organogenesis occurred when the medium with 

only NH4 + was unbuffered but the inclusion of MES 

resulted in the formation of meristems (but no 

shoots). 

Media differing in total nitrogen levels (but all 

having the same ratio of nitrate to ammonium as MS 

medium), had a final pH of ca. 4.5 after being used 

for Stage III root initiation on rose shoots, whereas 

those containing ammonium alone had a final pH of 

ca. 4.1 (Hyndman et al., 1982). A similar observation 

with MS medium itself was made by Delfel and 

Smith (1980). No matter what the starting value in 

the range 4.5-8.0, the final pH after culture of 

Cephalotaxus callus was always 4.2. The medium of 

De Jong et al. (1974) always had a pH of 4.8-5.0 after 

Begonia buds had been cultured, whatever the


starting pH in the range 4.0-6.5. This is not to say that 

the initial pH was unimportant, because there was an 

optimum for growth and development (see later) 

(Berghoef and Bruinsma, 1979). 

Other Compounds: The availability and uptake 

of other inorganic ions and organic molecules is also 

affected by pH. As explained above, the uptake of 

phosphate is most efficient from acid solutions. 

Petunia cells took up phosphate most rapidly at pH 4 

and its uptake declined as the pH was raised (Chin 

and Miller, 1982). Vacin and Went (1949) noted the 

formation of iron phosphate complexes in their 

medium by pH changes. Insoluble iron phosphates 

can also be formed in MS medium at pH 6.2 or above 

unless the proportion of EDTA to iron is increased 

(Dalton et al., 1983). 

The uptake of Cl – ions into barley roots is 

favoured by low pH, but Jacobson et al. (1971) 

noticed that it was only notably less at high pH in 

solutions strongly stirred by high aeration. They 

therefore suggested that H + ions secreted from plant 

roots as a result of the uptake of anions, can maintain 

a zone of reduced pH in the Nernst layer, a stationary 

film of water immediately adjacent to cultured plant 

tissue (Nernst, 1904). In plant tissue culture, uptake 

of ions and molecules may therefore more liable to be 

affected by adverse pH in agitated liquid media, than 

in media solidified by agar. 

Lysine uptake into tobacco cells was found by 

Harrington and Henke (1981) to be stimulated by low 

pH. The effect of pH on uptake is especially relevant 

for auxins (e.g., Edwards and Goldsmith 1980). 

Depending on the pH and their pKa, auxins are either 

present as an undissociated molecule or as an anion. 

For influx, the undissociated auxin molecule may 

pass through the membrane by diffusion whereas the 

anion is taken up by a carrier (Delbarre et al., 1996; 

Morris 2000). Dissociation dependens upon the pH. 

In the apoplast, the pH is low, ca. 5. When taken up, 

the auxin enters the cytoplasm with pH 7. At this pH, 

most auxin is present as anion and cannot diffuse out. 

Efflux of the anion is brought about by an efflux 

carrier system. Thus, the net uptake into cells of plant 

growth regulators which are weak lipophilic acids 

(such as IAA, NAA, 2,4-D and abscisic acid) will be 

greater, the more acid the medium and the greater the 

difference between its pH and that of the cell 

cytoplasm (Rubery, 1980). Shvetsov and Gamburg 

(1981) did in fact find that the rate of 2,4-D uptake 

into cultured corn cells increased as the pH of the 

medium fell from 5.5 to 4. Increased uptake of auxin 

at low pH was also found in apple microcuttings, 

both for IBA (Harbage et al., 1998) and IAA (De 

Klerk et al., 2007). As previously mentioned, auxins 

can themselves modify intra- and extra-cellular pH. 

Adding 2,4-D to a medium increased the uptake of 

nicotine into culture of Acer pseudoplatanus cells 

(Kurkdjian et al., 1982). 

5.1.3. Choosing the pH of culture media: Starting pH 

Many plant cells and tissues in vitro, will tolerate 

pH in the range of about 4.0-7.2; those inoculated 

Fig. 4.3 Development of pH during tissue culture. The pH was set before autoclaving as is usually done, and 

measured directly after autoclaving and after 5 days of culture with 1-mm stem-slices cut from apple microshoots. 

The medium was in Petri dishes with BBL agar and modified MS medium. Per Petri dish, 30 ml of medium was 

added and 30 slices were cultured. (Data from de Klerk et al., 2007).

into media adjusted to pH 2.5-3.0 or 8.0 will probably 

die (Butenko et al., 1984). Best results are usually 

obtained in slightly acid conditions. In a random 

sample of papers on micropropagation, the average 

initial pH adopted for several different media was 

found to be 5.6 (mode 5.7) but adjustments to as low 

as 3.5 and as high as 7.1 had been made. 

The pH for most plant cultures is thus lower than 

that which is optimum for hydroponic cultures, where 

intact plants with their roots in aerated solution 

usually grow most rapidly when the pH of the 

solution is in the range 6.0-7.3 (De Capite, 1948; 

Sholto Douglas, 1976; Cooper, 1979). 

Suspension cultured cells of Ipomoea, grew 

satisfactorily in Rose and Martin (1975) P2 medium 

adjusted initially to pH 5.6 or pH 6.3, but the yield of 

dry cells was less at two extremes (pH 4.8 and pH 

7.1). Martin and Rose (1976) supposed that a low 

yield of cells from a culture started at pH 7.1 was due 

to the inability of the culture to utilise NO3 – , but the 

cause of a reduction in growth at pH 4.8 was less 

obvious. It might have resulted from the plant having 

to expend energy to maintain an appropriate 

physiological pH internally. 

Kartha (1981) found that pH 5.6-5.8 supported the 

growth of most meristem tips in culture and that 

cassava meristems did not grow for a prolonged 

period on a medium adjusted to pH 4.8. The optimum 

pH (before autoclaving) for the growth of carnation 

shoot tips on Linsmaier and Skoog (1965) medium, 

was 5.5-6.5. When the medium was supplemented 

with 4 mg/l adenine sulphate and 2 g/l casein 

hydrolysate, the optimum pH was 5, and on media 

adjusted to 6.0 and 6.5, there was significantly less 

growth (Davis et al., 1977). Shoot proliferation in 

Camellia sasanqua shoot cultures was best when the 

pH of a medium with MS salts was adjusted to 5-5.5. 

Only in these flasks was the capacity of juvenile 

explants to produce more shoots than adult ones 

really pronounced (Torres and Carlisi, 1986). 

Norstog and Smith (1963) suggested that the pH 

of the medium used for the culture of isolated zygotic 

embryos, should not be greater than 5.2. 

5.1.4. pH adjustment 

Because there is then no need to take special 

aseptic precautions, and it is impractical to adjust pH 

once medium has been dispensed into small lots, the 

pH of a medium is usually adjusted with acid or 

alkali before autoclaving. According to Krieg and 

Gerhardt (1981), agar is partially hydrolysed if 

autoclaved at pH 6 or less and will not solidify so 

effectively when cooled. They recommend that agar 

Chapter 4 147 

media for bacteriological purposes should be 

sterilised at a pH greater than 6 and, if necessary, 

should be adjusted to acid conditions with sterile acid 

after autoclaving, when they have cooled to 45-50°C. 

The degree of hydrolysis in plant culture media 

autoclaved at pH 5.6-5.7, is presumably small. 

The effect of autoclaving: Autoclaving changes 

the pH of media (Fig. 4.3). In media without sugars, 

the change is usually small unless the phosphate 

concentration is low, when more significant 

fluctuations occur. Media autoclaved with sucrose 

generally have a slightly lower pH than those 

autoclaved without it, but if maltose, glucose, or 

fructose have been added instead of sucrose, the post- 

autoclave pH is significantly reduced (Owen et al., 

1991). 

The pH of liquid media containing MS salts [e.g. 

Linsmaier and Skoog (1965) or Skirvin and Chu 

(1979) media] containing 3-3.4% sucrose, has been 

found to fall during autoclaving from an adjusted 

level of 5.7, to pH 5.17 (Singha, 1982), to pH 5.5 

(Owen et al., 1991), or to pH 4.6 (Skirvin et al., 

1986). The drop in pH may vary according to the pH 

to which the medium was initially adjusted. In the 

experiments of Skirvin et al. (loc. cit.), the pH of a 

medium adjusted to 5.0, fell to 4.2; one adjusted to 

6.4, fell to 5.1; that set at pH 8.5, fell to 8.1. 

Most agars cause the pH of media to increase 

immediately they are dissolved. Knudson (1946) 

noticed that the pH of his medium shifted from 4.6- 

4.7 to 5.4-5.5 once agar had been added and 

dissolved; and Singha (1982) discovered that the 

unadjusted pH of MS medium rose from pH 4 to pH 

5.2, depending on the amount of agar added. 

However if a medium containing agar was adjusted to 

pH 5.7, and then autoclaved, the medium became 

more acid than if agar had not been added, the fall in 

pH being generally in proportion to the amount of 

agar present. 

The effect of storage. The pH of autoclaved plant 

media tends to fall if they are stored. Vacin and 

Went (1949) noted that autoclaving just accelerated a 

drop in the pH of Knudson (1946) C medium, as 

solutions left to stand showed similar changes. 

Sterilisation by filtration (see below) was not a 

satisfactory alternative, as it too effected pH changes. 

The compounds particularly responsible were thought 

to be unchelated ferrous sulphate (when the pH had 

initially been set between 3 and 6), and calcium 

nitrate (when the original pH was 6 to 9). Complex 

iron phosphates, unavailable to plants, were produced 

from the ferrous sulphate, but if iron was added as


ferric citrate or ferric tartrate (chelates), no significant 

pH changes resulted from autoclaving, and plants 

showed no iron-deficiency symptoms. 

Skirvin et al. (1986) found that both with and 

without agar, autoclaved MS medium tended to 

become more acid after 6 weeks storage, for example: 

MS medium 

Time Liquid With 0.6% Difco 

Bacto Agar 

Starting pH 5.7 5.7 

After autoclaving 4.6 4.6 

6 weeks later 4.1 4.4 

To minimise a change in the pH of stored media, 

it is suggested that they are kept in the dark: Owen 

et al., (1991) reported that the pH of MS containing 

0.1M sucrose or 0.8% Phytagar, remained relatively 

stable after autoclaving if kept in the dark at 4°C, but 

fell by up to 0.8 units if stored in the light at 25°C. De 

Klerk et al. (2007) using BBL agar, also observed a 

shift of pH, but this was negligible when 10 mM 

MES was added (Fig. 4.3). 

Hydrolysis: Some organic components of culture 

media are liable to be hydrolysed by autoclaving in 

acid media. The degree of hydrolysis of different 

brands of agar may be one factor influencing the 

incidence of hyperhydricity in plant cultures. Agar 

media may not solidify satisfactorily when the initial 

pH has been adjusted to 4-4.5. The reduction in pH 

which occurs in most media during autoclaving may 

also cause unsatisfactory gelling of agar which has 

been added in low concentrations. Part of the sucrose 

added to plant culture media adjusted to pH 5.5 is 

also hydrolysed during autoclaving: the proportion 

degraded increases if the pH of the medium is much 

less than this. Hydrolysis of sucrose is, however, not 

necessarily detrimental. 

Activated charcoal: The presence of activated 

charcoal can alter the pH of a medium. As in the 

production of activated charcoal it is, at one stage 

washed with HCl, the pH of a medium can be 

lowered by acid residues (Owen et al., 1995; Wann 

et al., 1997). 

4.1.5. pH changes during culture 

Due to the differential uptake of anions and 

cations into plant tissues, the pH of culture media 

does not usually stay constant, but changes as ions 

and compounds are absorbed by the plant. It is usual 

for media containing nitrate and ammonium ions to 

decline slowly in pH during a passage, after being 

adjusted initially to pH 5.4-5.7, Sometimes after a 

preliminary decrease, the pH may begin to rise and 

return to a value close to, or even well above that at 

which the culture was initiated. The pH of White 

(1954) medium, which contains only NO3 – nitrogen, 

drifted from an initial 5.0 to 5.5 towards neutrality as 

callus was cultured on it (Klein and Manos, 1960). 

With some cultures, the initial pH of the medium 

may have little effect. Cell suspension cultures of 

Dioscorea deltoides in the medium of Kaul and Staba 

(1968) [containing MS salts], adjusted to a pH either 

3.5, 4.3, 5.8 or 6.3, all had a pH of 4.6-4.7 within 10 

h of inoculation. There was a further fall to pH 4.0- 

4.2 in the next 2-3 days, but during the following 15- 

17 days the pH was 4.7-5.0, finally rising to pH 6.0- 

6.3 on about day 19 (Butenko et al., 1984). Similarly, 

Skirvin et al. (1986) found that MS medium adjusted 

to pH 3.33, 5.11, 6.63, or 7.98 before autoclaving, 

and then used for the culture of Cucumis melo callus, 

had a pH after 48 h in the range 4.6-5.0. Visseur 

(1987) also reported that although the pH of his 

medium (similar macronutrients to MS but more Ca 2+ 

and PO4 3– ) decreased if it was solidified with agar, 

but on a 2-phase medium, the final pH was 6.9 ± 0.4 

irrespective of whether it was initially 4.8, 5.5 or 6.2. 

Despite the above remarks, it should be noted that 

the nature of the pH drift which occurs in any one 

medium, differs widely, according to the species of 

plant grown upon it. The pH of the medium 

supporting shoot cultures of Disanthus cercidifolius 

changed from 5.5 to 6.5 over a 6 week period, 

necessitating frequent subculturing to prevent the 

onset of senescence, whereas in the medium in which 

shoots of the calcifuge Lapageria rosea were grown, 

the pH, initially set to 3.5-5.0, only changed to 3.8- 

4.1 (Howard and Marks, 1987). Note also that the 

reversion of media to a homeostatic pH, may be due 

to the presence of both NO3 – and NH4 + ions (Dougall, 

1980). Adjustment of media containing only one of 

these nitrogen sources to a range of pH levels, would 

be expected to result in a more variable set of final 

values. 

As the pH of media deviate from the original 

titration level, simple unmonitored cultures may not 

provide the most favourable pH for different phases 

of growth and differentiation. In Rosa ‘Paul’s Scarlet’ 

suspensions, the optimum pH for the cell division 

phase was 5.2-5.4: pH 5.5-6.0 was best for the cell 

expansion phase (Nesius and Fletcher, 1973). The 

maximum growth rate of Daucus carota habituated 

callus on White (1954) medium with iron as Fe- 

EDTA, occurred at pH 6.0 (Klein and Manos, 1960).

It has been suggested that acidification of media is 

partly due to the accumulation of carbon dioxide in 

tightly closed culture flasks (Leva et al., 1984), but 

the decrease in pH associated with incubating anther 

cultures with 5% CO2 was found by Johansson and 

Eriksson (1984) to be only ca. 0.1 units. Removal of 

CO2 from an aerated cell suspension culture of 

Poinsettia resulted in an increase of about 0.2 pHunits 

(Preil, 1991). 

Auxin plant growth regulators promote cell 

growth by inducing the efflux of H + ions through the 

cell wall. Hydrogen ion efflux from the cell is 

accompanied by potassium ion influx. When cultures 

are incubated in a medium containing an auxin, the 

medium will therefore become more acid while the 

pH of the cell sap will rise. The extent of these 

changes was found by Kurkdjian et al. (1982) to be 

proportional to auxin (2,4-D) concentration. 

5.2. pH CONTROL WITHIN THE PLANT 

The various compartments of cells have a 

different pH and this pH is maintained (Felle, 2001). 

In the symplasm, the pH of the cytoplasm is ca. 7 and 

of the vacuole ca. 5. The apoplasm has a pH of ca. 5. 

Plant cells typically generate an excess of acidic 

compounds during metabolism which have to be 

neutralised (Felle, 1998). One of the most important 

ways by which this is accomplished is for H + , or K + 

to be pumped out of the cell, in exchange for anions 

(e.g. OH – ), thereby decreasing the extracellular pH. 

Plant cells also compensate for an excess of H + by the 

degradation of organic acids. Synthesis of organic 

acids, such as malate, from neutral precursors is used 

to increase H + concentration when the cytoplasmic 

pH rises, for instance if plants are grown in alkaline 

soils (Raven and Smith, 1976; Findenegg et al., 

1986). In intact plants, there is usually a downwards 

gradient from the low pH external to the cell, to 

higher pH levels in more mature parts, and this 

enables the upwards transport of non-electrolytic 

compounds such as sugars and amino acids (Böttger 

and Luthen, 1986). 

Altering the pH of the external solution 

surrounding roots or cells can alter the pH of the cell 

(Smith and Raven, 1979). Because of necessary 

controls, the pH of the cytoplasm may be only 

slightly altered, that of vacuoles may show a more 

marked change. Changing the pH of the medium in 

which photo-autotrophic Chenopodium rubrum 

suspensions were cultured from 4.5 to 6.3, caused the 

pH of the cytoplasm to rise from 7.4 to 7.6 and that of 

the cell vacuoles to increase from 5.3 to 6.6. The 

increase in cytoplasmic pH caused there to be a 

marked diversion of carbon metabolism, away from 

sugar and starch, into the production of lipids, amino 

acids and proteins (Hüsemann et al., 1990). The 

maintenance of the pH is also illustrated in an 

experiment with detached leaves of Vicia faba (Felle 

and Hanstein, 2002). When the leaves were placed in 

a 10 mM MES-TRIS buffer and transferred to buffer 

with another pH, changes in the pH of the apoplasm 

were small: with an initial buffer pH = 4.1 and 

transfer to buffer pH = 6.8, the apoplastic pH of 

substomatal cavities increased from 4.71 to 5.13 and 

in the reverse transfer decreased from 5.13 to 4.70. 

This indicates that the pH of the apoplasm is not 

strongly influenced by the medium but stays close to 

the ‘natural’ pH of ca. 5.0. No exact details are given 

but in this experiment the distance between the site of 

the pH measurements and the MES-TRIS solution in 

which the leaves had been placed was probably large. 

The symplasm has a much larger capacity to buffer 

(Felle, 2001) so that its pH will be even less 

influenced by the medium pH. Thus, within the 

explant the pH of both apoplasm and symplasm will 

be affected only little by the medium pH. The 

situation may be different at the interface between 

explant and medium. The influence of medium pH 

will extend towards inner tissues of the explant as the 

buffering capacity of the medium is increased (and 

thus overcomes buffering by the tissue). Inside the 

explant, the pH will also greatly influence movement 

Table 4.9. The influence of the initial pH of Linsmaier Skoog (1965) medium on morphogenesis in thin cell layers of Nicotiana 

(data of Mutaftschiev et al., 1987) 

Initial pH 

of 

the medium 

Mean number of 

organs per 

explant 

Chapter 4 149 

Mean percentage of explants forming: 

Callus Roots Vegetative buds Flowers 

3.8 4 ± 2 60 ± 10 40 ± 10 0 0 

5.0 2 ± 1 90 ± 10 10 ± 10 0 0 

6.1 20 ± 10 0 0 100 0 

6.8 6 ± 2 0 0 20 ± 10 80 ± 10


through membranes, i.e. uptake in cells, as this often 

depends on the dissociation of compounds which is 

pH dependent. 

Changing the pH of the medium can thus have a 

regulatory role on plant cultures which is similar to 

that of plant growth regulating chemicals, one of the 

actions of which is to modify intracellular pH and the 

quantity of free calcium ions. Auxins can modify 

cytoplasmic pH by triggering the release of H + from 

cells. In plant tissue culture these ions can acidify the 

medium (Kurkdjian et al., 1982). Proton release is 

thought to be the first step in acid-triggered and 

turgor-triggered growth (Schubert and Matzke, 1985). 

It should be noted that pH changes themselves may 

act as a signal (Felle, 2001). 

5.3. THE EFFECT OF pH ON CULTURES 

5.3.1. Initiating cultures at low pH 

Plants of the family Ericaceae which only grow 

well on acid soils (e.g. rhododendrons and 

blueberries), have been said to grow best on media 

such as Anderson (1975), Anderson (1978; 1980) and 

Lloyd and McCown (1981) WPM, when the pH is 

first set to ca. 4.5 (Anderson, 1975; Skirvin, 1981), 

but for highly calcifuge species such as Magnolia 

soulangiana, a starting pH of 3.5 can result in the 

highest rate of shoot proliferation in shoot cultures 

(Howard and Marks, 1987). Chevre et al. (1983) state 

that chestnut shoot cultures grew and proliferated best 

at pH 4 provided MS medium was modified by 

doubling the usual levels of calcium and magnesium. 

De Jong et al. (1974), using a specially developed 

medium, found that a low pH value favoured the 

growth of floral organs. A similar result was seen by 

Berghoef and Bruinsma (1979c) with Begonia buds. 

Growth was greatest when the pH of the medium was 

initially adjusted to acid, 4.5-5.0 being optimal. At 

pH 4.0 the buds became glassy. 

Before the discovery of effective chelating agents 

for plant cultures, root cultures were grown on media 

with a low pH. Tomato roots were, for instance, 

unable to grow on media similar to those of White 

(1943a), when the pH rose to 5.2 (Street et al., 1951). 

Boll and Street (1951) were able to show that the 

depression of growth at high pH was due to the loss 

of Fe from the medium and that it could be overcome 

by adding a chelated form of iron (see chapter 3). 

Using FeEDTA, Torrey (1956) discovered that 

isolated pea roots [grown on a medium containing 

Bonner and Devirian (1939) A macronutrients, which 

do not contain NH4 + ], grew optimally at pH 6.0-6.4 

but were clearly inhibited at pH 7.0 or greater; and 

Street (1969) reported that growth of tomato roots 

could be obtained between pH 4.0 and pH 7.2, if 

EDTA was present in the medium. Because agar does 

not gel properly when the initial pH of the medium is 

adjusted to 4, it is necessary to use liquid media for 

low pH cultures; or employ another gelling agent, or 

a mechanical support. 

Fig. 4.4 Effect of medium pH on adventitious rooting from apple stem disks. The pH at the x-axis is the pH as measured at the 

start of culture after autoclaving (cf. Fig. 4.3). (from de Klerk et al., 2007).

Some other cultures may also be beneficially 

started at low pH, which may indicate that the tissues 

have an initial requirement for NO3 – . Embryogenesis 

of Pelargonium was induced more effectively if MS, 

or other media, were adjusted to pH 4.5-5.0 before 

autoclaving (rather than pH 5.5 and above) 

(Marsolais et al., 1991). 

5.3.2. Differentiation and Morphogenesis 

Differentiation and morphogenesis are frequently 

found to be pH-dependent. Xylogenesis depends on 

the medium pH (Khan et al., 1986). The growth of 

callus and the formation of adventitious organs from 

thin cell layers excised from superficial tissues of the 

inflorescence rachis of Nicotiana, depended on the 

initial pH of Linsmaier and Skoog (1965) medium 

containing 0.5 μM IBA and 3 μM kinetin (Table 4.9) 

(Mutaftschiev et al., 1987). Pasqua et al. (2002) 

reported many quantitative effects of pH during 

regeneration from tobacco thin cell layers. The types 

of callus produced from the plumules of Hevea 

seedlings differed according to the pH of the medium 

devised by Chua (1966). Soft and spongy callus 

formed at acid (5.4) or alkaline (8.0) pH. A compact 

callus was obtained between pH 6.2 and 6.8. 

5.3.3. Adventitious root formation 

There are several reports in the literature which 

show that the pH of the medium can influence root 

formation of some plants in vitro. A slightly acid pH 

seems to be preferred by most species. Zatkyo and 

Molnar (1986), who showed a close correlation 

between the acidity of the medium (pH 7.0 to 3.0) 

and the rooting of Vitis, Ribes nigrum and Aronia 

melancarpa shoots, suggested that this was because 

acidity is necessary for auxin action. 

Sharma et al. (1981) found it advantageous to 

reduce the pH of the medium to 4.5 to induce rooting 

of Bougainvillea shoots and a reduction of the pH of 

MS medium to 4.0 (accompanied by incubation in the 

dark) was required for reliable root formation of two 

Santalum species (Barlass et al., 1980) and Correa 

decumbens and Prostanthera striatifolia (Williams 

et al., 1984; 1985). Other Australian woody species 

rooted satisfactorily at pH 5.5 and pH 4 was 

inhibitory (Williams et al., loc. cit.). Shoots from 

carnation meristem tips rooted more readily at pH 5.5 

than pH 6.0 (Stone, 1963), and rooting of excised 

potato buds was best at pH 5.7, root formation being 

inhibited at pH 4.8 and at pH 6.2 or above (Mellor 

and Stace-Smith, 1969). Direct root formation on 

Nautilocalyx leaf segments was retarded if a modified 

MS medium containing IAA was adjusted initially to 

Chapter 4 151 

an acid pH (3.5 or 4.0) or a neutral pH (6.5). Good 

and rapid root formation occurred when the medium 

was adjusted to between pH 5.0 to 6.3 (Venverloo, 

1976). De Klerk et al (2007), working with apple 

stem slices, found only a small effect of pH on 

rooting (Fig. 4.4): when the pH was set before 

autoclaving at 4.5 (after autoclaving the pH was 

4.54), the number of roots was 4.5, and with the pH 

set at 8.0 (after autoclaving the pH had dropped to 

5.65), the number of roots per slice increased to 7. In 

medium buffered with MES, the maximum number 

of roots was formed at pH 4.4 (measured after autoclaving). 

In these experiments, the dose-response 

curve for root number did not correspond with the 

effect of pH on IAA uptake. Such discrepancy 

between the effects of the pH on uptake and root 

number, was also reported by Harbage, Stimart and 

Auer (1998). 

Direct formation of roots from Lilium auratum 

bulb scales occurred when MS medium was adjusted 

within the range 4-7 but was optimal at pH 6. The pH 

range for adventitious bulblet formation in this plant 

was from 4 to 8, but most bulblets were produced 

when the initial pH was between 5 and 7 (Takayama 

and Misawa, 1979). 

Substrates which are to acid or too alkaline can 

adversely affect rooting ex vitro. 

5.3.4. Embryogenesis 

Smith and Krikorian (1989) discovered that preglobular 

stage pro-embryos (PGSP) of carrot could be 

made to proliferate from tissues capable of direct 

embryo formation, with no auxin, on a medium 

containing 1-5 mM NH4 + (and no nitrate). Somatic 

embryos were formed when this tissue was moved to 

MS medium. The pH of the ‘ammonium-nitrogen’ 

medium fell from 5.5 to 4.0 in each subculture 

period, and it was later found (Smith and Krikorian, 

1990a,b) that culture on a medium of low pH was 

essential for the maintenance of PGSP cultures. 

Sustained culture at a pH equal or greater than 5.7, 

with no auxin, allowed somatic embryo development. 

A similar observation to that of Smith and Krikorian 

had been made by (Stuart et al., 1987). Although the 

pH of a suspension culture of alfalfa ‘Regen-S’ cells 

in a modified Schenk and Hildebrandt (1972) 

medium with 15 mM NH4 + was adjusted to 5.8, it 

quickly fell back to pH 4.4-5.0 in a few hours. The 

pH then gradually increased as somatic embryos were 

produced, until at day 14 it was 5.0. In certain 

suspension cultures, the pH was titrated daily to 5.5, 

but on each occasion soon returned to nearly the same 

pH as that in flasks which were untouched. Even so,


the pH-adjusted suspensions produced more embryos 

than the controls. 

The ammonium ion has been found to be essential 

for embryogenesis. Is one of its functions to reduce 

the pH of the medium through rapid uptake and 

metabolism, thereby facilitating the uptake of nitrate, 

upon which embryogenesis is really dependent? 

Embryogenesis from leaf explants of Ostericum 

koreanum, was found to depend strongly on pH (Cho 

et al 2003). As the explants were cultured continuously 

with NAA, it is possible that the observed relationship 

was caused by differential NAA uptake. This is also 

suggested by the slower rate of embryo development 

seen at low pH, because this would be expected where 

there is a high internal NAA concentration. 

6. LIQUID MEDIA AND SUPPORT SYSTEMS 

The nutritional requirements of plant cultures can 

be supplied by liquid media but growth in liquid 

medium may be retarded and development affected 

by oxygen deprivation and hyperhydration. The 

oxygen concentration of liquid media is often 

insufficient to meet the respiratory requirements of 

submerged cells and tissues. It can be increased 

either by raising the oxygen concentration of the 

medium or placing cells or tissues in direct contact 

with air. If the water potential of the medium is 

greater (less negative) than that of a cell, water flows 

into the cell and the vacuole becomes distended. 

Cells and tissues affected in this way are described as 

hyperhydric. Shoots often show physiological disturbances 

with symptoms that can be recognised 

visually (Chapter 13) (Debergh et al., 1992; Gaspar 

et al., 1987; Preece and Sutter, 1991; Ziv, 1991). The 

term hyperhydric is preferable to the previously used 

term ‘vitrified’ for reasons explained by Debergh 

et al. (1992). Water potential is determined by 

osmotic potential of the solutes in the medium and, in 

the case of a gelled medium, by the matric potential 

of the gel (Section 4.1) (Beruto et al., 1995; Fujiwara 

and Kozai, 1995; Owens and Wozniak, 1991). 

Reductions in hyperhydricity can be achieved by 

increasing the concentrations of the solutes and the 

gel. Hyperhydricity may also be reduced through 

evaporation of water from tissues if they are placed in 

contact with air. 

Contact of cultured tissues with air, to alleviate 

problems of hyperhydricity and hypoxia, can be 

achieved by the use of either porous or semi-solid 

(gelled) supports. The advantage of supports, as 

opposed to thin layers of liquid medium, is that 

tissues can be placed in a sufficient volume of 

medium to prevent depletion of nutrients and allow 

for the dispersion of any toxins that might be 

produced by the plant tissues. The relative 

advantages of liquid medium, solid supports or gelled 

media, varies with the type of material being 

cultured, the purpose of the culture and the scale of 

culture, as discussed below. 

6.1. LIQUID MEDIA 

Liquid medium, without supporting structures, is 

used for the culture of protoplasts, cells or root 

systems for the production of secondary metabolites, 

and the propagation of somatic embryos, 

meristematic nodules, microtubers and shoot clusters. 

In liquid medium, these cultures often give faster 

growth rates than on agar-solidifed medium. 

Cultures may be fully or only partially immersed in 

the medium. 

Aeration of liquid medium in stationary Petri 

dishes is sometimes adequate for the culture of 

protoplasts and cells because of the shallow depth of 

the medium, but may still be suboptimal. Anthony 

et al. (1995) cultured protoplasts of cassava, in liquid 

medium in Petri dishes with an underlying layer of 

agarose in which glass rods were embedded 

vertically. Sustained protoplast division was 

observed in the cultures with glass rods but not in the 

controls without glass rods. The authors suggested 

that the glass rods extended the liquid meniscus, 

where the cell colonies were clustered thus causing 

gaseous exchange between the liquid and the 

atmosphere above to be facilitated. 

Anthony et al. (1997) cultured protoplasts of 

Passiflora and Petunia in 30 ml glass bottles 

containing protoplast suspensions in 2 ml aliquots, 

either alone or in the presence of the oxygen carriers 

Erythrogen or oxygenated Perfluorodecalin. Cell 

division in each of the two species was stimulated by 

both oxygen carriers. 

Laboratory-scale experimentation on immersed 

cultures of cells, tissues and organs, may be carried 

out in 125 ml or 250 ml Erlenmeyer flasks. Largescale 

cultures are usually carried out in bioreactors 

with a capacity of 1 litre or more. The concentration 

of oxygen in the medium is raised by oxygen in the 

gas phase above and air bubbles inside the liquid. 

Increasing the oxygen concentration and circulation 

of the medium is facilitated in flasks by the use of 

gyratory shakers and in bioreactors by stirring and/or 

bubbling air through the medium (Chapter 1). The

use of bioreactors often involves the automated 

adjustment of the culture medium. The design of 

bioreactors for plant cells and organs was reviewed 

by Doran (1993) and the use of shake-flasks and 

bioreactors for the scale-up of embryogenic plant 

suspension cultures has been reviewed by Tautorus 

and Dunstan (1995). The importance of oxygen 

concentration in bioreactors can be illustrated by an 

investigation into the growth of hairy roots of Atropa 

belladonna (Yu and Doran, 1994). They found that 

no growth occurred at oxygen tensions of 50% air 

saturation but between 70% and 100% air saturation, 

total root length and the number of root tips increased 

exponentially. Hyperhydricity in liquid cultures may 

be avoided by adding to the medium osmoregulators, 

such as mannitol, maltose and sorbitol, and inhibitors 

of gibberellin biosynthesis including ancymidol and 

paclobutrazol (Ziv, 1989). 

Plantlets and microtubers can be cultured by 

partial submersion in liquid medium. One method of 

aerating tissues is by the automated flooding and 

evacuation of tissues by liquid medium. This method 

has been used to produce microtubers of potato from 

single node cuttings (Teisson and Alvard, 1999). An 

alternative approach to aeration is to apply the liquid 

medium over the plant tissues as a nutrient mist. For 

example, Kurata et al. (1991) found that nodes of 

potato grew better in nutrient mist than on agar-based 

cultures. 

6.2. SUPPORT BY SEMI-SOLID MATRICES 

Gelled media provide semi-solid, supporting 

matrices that are widely used for protoplast, cell, 

tissue and organ culture. Agar, agarose, gellan gums 

and various other products have been used as gelling 

agents. 

6.2.1. Agar 

Agar is very widely employed for the preparation 

of semi-solid culture media. It has the advantages 

that have made it so widely used for the culture of 

bacteria, namely :- 

• it forms gels with water that melt at approx. 

100°C and solidify at approx. 45°C, and are thus 

stable at all feasible incubation temperatures; 

• gels are not digested by plant enzymes; 

• agar does not strongly react with media 

constituents. 

To ensure adequate contact between tissue and 

medium, a lower concentration of agar is generally 

used for plant cultures than for the culture of bacteria. 

Plant media are not firmly gelled, but only rendered 

Chapter 4 153 

semi-solid. Depending on brand, concentrations of 

between 0.5-1.0% agar are generally used for this 

purpose. Agar is thought to be composed of a 

complex mixture of related polysaccharides built up 

from galactose. These range from an uncharged 

neutral polymer fraction, agarose, that has the 

capacity to form strong gels, to highly charged 

anionic polysaccharides, sometimes called 

agaropectins, which give agar its viscosity. Agar is 

extracted from species of Gelidium and other red 

algae, collected from the sea in several different 

countries. It varies in nature according to country of 

origin, the year of collection and the way in which it 

has been extracted and processed. The proportion of 

agarose to total polysaccharides can vary from 50 to 

90% (Adrian and Assoumani, 1983). Agars contain 

small amounts of macro- and micro-elements; 

particularly calcium, sodium, potassium, and 

phosphate (Beruto et al., 1995; Debergh, 1983; 

Scherer et al., 1988), carbohydrates, traces of amino 

acids and vitamins (Day, 1942) that affect the 

osmotic and nutrient characteristics of a gel. They 

also contain phenolic substances (Scherer et al., 

1988) and less pure grades may contain long chain 

fatty acids, inhibitory to the growth of some bacteria. 

As agar can be the most expensive component of 

plant media, there is interest in minimising its 

concentration. Concentrations of agar can be 

considered inadequate if they do not support explants 

or lead to hyperhydricity. Hyperhydricity decreases 

as the agar concentration is raised but there may be 

an accompanying reduction in the rate of growth. For 

example, Debergh et al. (1981) found that shoot 

cultures of Cynara scolymus were hyperhydric on 

medium containing 0.6% Difco ‘Bacto’ agar. No 

hyperhydricity occurred on medium containing 1.1% 

agar but shoot proliferation was reduced. Likewise, 

Hakkaart and Versluijs (1983) found that shoots of 

carnation were hyperhydric on medium containing 

0.6% agar whereas growth was severely reduced on 

medium containing 1.2%. During studies to optimise 

the production of morphogenic callus from leaf discs 

of sugarbeet, Owens and Wozniak (1991) found large 

differences in the numbers of somatic embryos and 

shoots according to the gelling agent employed. 

They found that water availability, determined by gel 

matric potential, was the dominant factor involved. 

When they adjusted the concentrations of the gelling 

agents to give media of equal gel matric potential, 

somatic embryos and shoots were found in similar 

numbers on Bacto agar (0.7%), HGT agarose 

(0.46%), Phytagar (0.62%) and Gelrite (0.12%).


Various brands and grades of agar are available 

commercially. These differ in the amounts of 

impurities they contain and their gelling capabilities. 

The gelling capacity of Difco brands of agar 

increased with increasing purity i.e. ‘Noble’ > 

‘Purified’ > ‘Bacto’ (Debergh, 1983). After 

impurities of agar were removed by sealing agar in a 

semi-permeable bags and washing in deionized water, 

the water potentials of gels of three brands were 

substantially lower than unwashed gels (Beruto et al., 

1995). A ten-fold difference in the regeneration rate 

of sugar cane was observed by Anders et al. (1988) 

on media gelled with the best and least effective of 

seven brands of agar. Scholten and Pierik (1998) 

investigated the different growth characteristics of 

seven different agar brands on the growth of axillary 

shoots, adventitious shoots and adventitious roots of 

rose, lily and cactus. They concluded that no single 

bioassay could identify ‘good or bad’ agars for a 

large group of plant species but Merk 1614, Daishin, 

MC29, and BD Purified gave the best results in most 

experiments. ‘Daishin’ showed no batch-to-batch 

variations. They found no relationship between price 

and quality of the brands of agar. 

Agarose. Agarose is the high gel strength moiety 

of agar. It consists of β-D(1→3) galactopyranose and 

3,6-anhydro-α-L(1 → 4) galactopyranose polymer 

chains of 20-160 monosaccharide units alternatively 

linked to form double helices. There are also several 

different agaro-pectin products available, which have 

been extracted from agar and treated to remove most 

of the residual sulphate side groupings (Shillito et al., 

1983). Because preparation involves additional 

processes, agarose is much more expensive than agar 

and its use is only warranted for valuable cultures, 

including protoplast and anther culture. Brands may 

differ widely in their suitability for these applications. 

Concentrations of 0.4-1.0% are used. Agarose 

derivatives are available which melt and gel at 

temperatures below 30°C, making them especially 

suitable for testing media ingredients that are heatlabile, 

or for embedding protoplasts. Low meltingpoint 

agarose is prepared by introducing 

hydroxyethyl groups into the agarose molecules 

(Shillito et al., 1983). Another advantage of agarose 

over agar lies in the removal of toxic components of 

agar during its preparation. Bolandi et al. (1999) 

preferred to embed protoplasts in agarose, rather than 

use liquid medium, because in agarose the semi-solid 

matrix applies a direct pressure on the plasma 

membrane of the protoplasts. They mixed protoplasts 

of sunflower with 0.5% agarose, pipetted 50 μl 

droplets of the mixture into Petri dishes and covered 

them with a thin layer of culture medium. A similar 

method was used to culture protoplasts of Dioscorea 

by Tor et al. (1999). The use of droplets has the 

advantage that a high plating density can be achieved 

in the droplets, while exposing the protoplasts to a 

larger reservoir of culture medium in the liquid phase. 

Bishoi et al. (2000) initially cultured anthers of 

Basmati rice on liquid medium, then used 1.0% 

agarose to culture calli derived from microspores. 

6.2.2. Gellan gum 

Gellan gum is a widely used gelling agent in plant 

tissue culture, that is marketed under various trade 

names including Gelrite, Phytagel and Kelcogel. It 

is an exopolysaccharide that encapsulates cells of the 

bacterium Sphingomonas paucimobilis (= Auromonas 

elodea = Pseudomonas elodea), from which it is 

obtained by industrial fermentation. The structure, 

physico-chemical properties and the rheology of 

solutions of gellan gum and related polysaccharides 

has been reviewed by Banik et al. (2000). Gellan 

gum consists of a linear repeating tetrasaccharide of 

D-glucose, D-glucuronic acid, D-glucose and Lrhamnose. 

Heating solutions of gellan gum in 

solutions that contain cations, such as K + , Ca 2+ , Mg 2+ , 

causes the polysaccharide to form a gel in which the 

polymers form a half-staggered parallel double helix. 

The commercial deacetylated and purified 

polysaccharide forms a firm non-elastic gel. The gel 

sets rapidly at a temperature, determined by the 

concentrations of the polysaccharide and the cation, 

which varies between 35-50 °C (Kang et al., 1982). 

The commercial product contains significant 

quantities of potassium, sodium, calcium and 

magnesium (Pasqualetto et al., 1988a,b; Scherer 

et al., 1988) but is said to be free of the organic 

impurities found in agar. It is unclear whether or not 

these cations remain fully available to plant cultures. 

Some researchers (Gawel et al., 1986; 1990; 

Trolinder and Goodin, 1987; Umbeck et al., 1987) 

add an extra 750 mg l –1 MgCl2 to a medium 

containing MS salts to aid the gelling of 1.6% Gelrite. 

In most other reports on the use of Gelrite, cations in 

the medium have been sufficient to produce a gel. 

Beruto et al. (1995) found that 0.12 % Gelrite and 

0.7% Bacto agar have equivalent matric potential 

and support equivalent adventitious regeneration in 

leaf discs of sugarbeet. As gellan gum is used in 

lower concentration than agar, the cost per litre of 

medium is less. It produces a clear gel in which plant 

tissues can be more easily seen and microbial

contamination more easily detected than in agar gels. 

It has proved to be a suitable gelling agent for tissue 

cultures of many herbaceous plants and there are 

reports of its successful use for callus culture, the 

direct and indirect formation of adventitious organs 

and somatic embryos, shoot culture of herbaceous 

and semi-woody species and the rooting of plantlets. 

In most cases the results have been as good as, and 

sometimes superior to, those obtainable on agarsolidified 

media. Anders et al. (1988) described the 

regeneration of greater numbers of plants from of 

sugar cane on Gelrite than on the most productive 

brand of agar, and Koetje et al. (1989) obtained more 

somatic embryos from callus cultures of rice on 

media solidified with Gelrite than with Bacto-agar. 

Shoot cultures, particularly of some woody species, 

are liable to become hyperhydric if Gelrite, like agar, 

is used at too low a concentration. There are sharp 

differences in the response of different species to 

concentration of Gelrite. Turner and Singha (1990) 

found the highest rate of shoot proliferation in Geum 

occurred on 0.2% and in Malus on 0.4%. Garin et al. 

(2000) obtained more mature somatic embryos of 

Pinus strobus on gellan gum at 1% concentration of 

than at 0.6%. Pasqualetto et al. (1986a,b) used 

mixtures of Gelrite (0.1-0.15%) and Sigma @ agar 

(0.2-0.3%) to prevent the hyperhydricity that 

occurred in shoot cultures of Malus domestica ‘Gala’ 

on media solidified with Gelrite alone. Nairn (1988) 

used a mixture of Gelrite (0.194%) and Difco 

‘Bacto’ agar (0.024%) to prevent the hyperhydricity 

that occurred in shoot cultures of Pinus radiata on 

medium gelled with 0.2% Gelrite alone. 

6.2.3. Alginates 

Alginic acid is a binary linear heteropolymer 1,4β-D-mannuronic 

acid and 1,4-α-L-guluronic acid 

(Larkin et al., 1988). It is extracted from various 

species of brown algae. When the sodium salt is 

exposed to calcium ions, gelation occurs. Alginates 

are widely used for protoplast culture and to 

encapsulate artificial seed. 

Protoplasts embedded in beads or thin films of 

alginate can plated densely while yet exposed to a 

large pool of medium that dilutes inhibitors and toxic 

substances. Embedded protoplasts can be surrounded 

by nurse cells, either free in the surrounding medium 

or separated by filters or membranes. Alginate has 

the advantages over agarose that protoplasts do not 

have to be exposed to elevated temperatures when 

they are mixed with the gelling agent and the gel can 

be liquified by adding sodium citrate, releasing 

Chapter 4 155 

protoplasts or cell colonies for transfer to other 

media. The method of embedding protoplasts in 

beads as employed by Larkin et al. (1988) involved 

mixing the protoplast suspensions with an aqueous 

solution of sodium alginate and dropping it, through a 

needle, into a solution of calcium chloride. Beads 

containing the protoplasts were formed when the 

alginate made contact with the calcium ions. Beads 

with a final concentration of 1.5% sodium alginate 

were washed and cultured in liquid medium on an 

orbital shaker. Protoplasts may also be captured in 

thin layers of alginate (Dovzhenko et al., 1998). 

Synthetic seeds (synseeds, somatic seeds) 

encapsulated in alginate (Fig. 4.4), can be prepared 

from somatic embryos (Timbert et al., 1995), shoot 

tips (Maruyama et al., 1998), apical and axillary buds 

(Piccioni and Standardi, 1995), single nodes 

(Piccioni, 1997), and cell aggregates from hairy roots 

(Repunte et al.,1995). The uses of sythetic seeds 

include direct planting into soil, storage of tissues and 

transfer of materials between laboratories under 

sterile conditions. The methods of encapsulation of 

somatic embryos of carrot were described by Timbert 

et al. (1995). Torpedo-shaped embryos were mixed 

with a 1% sodium alginate solution. The mixture was 

dropped into a solution 100 mM calcium chloride in 

10% sucrose. The beads (3-3.5 mm in diameter) were 

then rinsed in a 10% sucrose solution. 

6.2.4. Starch 

Sorvari (1986a,c) found that plantlets formed in 

higher frequencies in anther cultures of barley on a 

medium solidified with 5% corn starch or barley 

starch rather than with agar. Corn starch only formed 

a weak gel and it was necessary to place a polyester 

net on its surface to prevent the explants from 

sinking. Sorvari (1986b) found that it took 5-14 

weeks for adventitious shoots to form on potato discs 

on agar-solidified medium but only 3 weeks on 

medium containing barley starch. Henderson and 

Kinnersley (1988) found that embryogenic carrot 

callus cultures grew slightly better on media gelled 

with 12% corn starch than on 0.9% Difco `Bacto’ 

agar. 

6.2.5. ‘Kappa’--carrageenan 

Carrageenan is a product of sea weeds of the 

genus Euchema and the kappa form has strong 

gelling properties. Like gellan gum, kappacarrageenan 

requires the presence of cations for 

gelation. In Linsmaier and Skoog (1965) medium at 

0.6% w/v, the gel strength was slightly less than that 

of 0.2% Gelrite or 0.8% of extra pure agar (Ichi et al.,


1986). Chauvin et al. (1999) found that regeneration 

from cultures of tulip, gladiolus and tobacco shoots 

was possible in the presence of 200 mg l –1 

kanamycin, whereas in several other gelling agents a 

concentration of 100 mg l –1 inhibited regeneration. 

6.2.6. Pectins 

A mixture of pectin and agar can be a less 

expensive substitute for agar. For example, a semisolid 

medium consisting of 0.2% agar plus 0.8-1.0% 

pectin, was employed for shoot culture of strawberry 

and some other plants (Zimmerman, 1979). 

6.2.7. Other gelling agents 

Battachary et al. (1994) found sago (from 

Metroxylon sagu) and isubgol (from Plantago ovata) 

were satisfactory substitutes for agar at, respectively, 

one eighth and one tenth of the cost of Sigma purified 

agar A7921. 

6.3. POROUS SUPPORTS 

Aeration of the tissues on a porous substrate is 

usually better than it would be in agar or static liquid. 

Chin et al. (1988) used a buoyant polypropylene 

membrane floated on top of a liquid medium to 

culture cells and protoplasts of Asparagus. The 

membrane (Celgard 3500, Questar Corp., Charlotte, 

N.C.) has a pore size of 0.04 mm and is autoclavable. 

Conner and Meredith (1984) found that cells grew 

more rapidly on filter papers laid over polyurethane 

foam pads saturated with medium than on agar. 

Young et al. (1991) supported shoots of tomato over 

liquid culture medium on a floating microporous 

polypropylene membrane and entrained the growing 

shoots through polypropylene netting. They reported 

opportunities for the development of this method for 

mechanisation by mass handling. Membrane rafts 

were also used by Teng (1997) and Watad et al. 

(1995,1996). 

Cheng and Voqui (1977) and Cheng (1978) used 

polyester fleece to support cultures of Douglas fir that 

were irrigated with liquid media in Petri dishes. 

Plantlets that were regenerated from cotyledon 

explants were cultured on 3 mm-thick fabric. When a 

protoplast suspension was dispersed over 0.5 mmthick 

fabric, numerous colonies were produced in 12 

days, whereas in the absence of the support, cell 

colonies failed to proliferate beyond the 20 cell stage. 

A major advantage in using this type of fabric support 

is that media can be changed simply and quickly 

without disturbing the tissues. The system has also 

been used for protoplast culture of other plants (e.g. 

by Russell and McCown, 1986). 

Heller and Gautheret (1949) found that tissues 

could be satisfactorily cultured on pieces of ashless 

filter paper moistened by contact with liquid medium. 

Very small explants, such as meristem tips, that might 

be lost if placed in a rotated or agitated liquid medium, 

can be successfully cultured if placed on an M-shaped 

strip of filter paper (sometimes called a ‘Heller’ 

support). When the folded paper is placed in a tube of 

liquid medium, the side arms act as wicks (Goodwin, 

1966). This method of support ensures excellent tissue 

aeration but the extra time required for preparation and 

insertion has meant that paper wicks are only used for 

special purposes such as the initial cultural stages of 

single small explants which are otherwise difficult to 

establish. Whether explants grow best on agar or on 

filter paper supports, varies from one species of plant 

to another. Davis et al. (1977) found that carnation 

shoot tips grew less well on filter paper bridges than on 

0.6% agar but axillary bud explants of Leucospermum 

survived on bridges but not on agar. 

Paper was also used in the construction of plugs 

(marketed by Ilacon Ltd, Tonbridge, UK TN9 1NR) 

known as Sorbarods (Roberts and Smith, 1990). These 

are cylindrical (20 mm in length and 18 mm in 

diameter) and consist of cold-crimped cellulose, 

longitudinally folded, wrapped in cellulose paper. The 

plug has a porosity (total volume – volume of 

cellulose) of 94.2% and high capillarity, so that the 

culture medium is efficiently drawn up into the plug, 

leaving the sides of the plug in direct contact with air. 

Roots permeate the plugs and are protected by the 

cellulose during transfer to soil. Plantlets of 

chrysanthemum in Sorbarods formed longer stems, 

larger leaves, more roots, and developed greater fresh 

mass, dry weights and fresh to dry mass ratios than 

plantlets in agar-solidified medium (Roberts and 

Smith, 1990). The greater fresh to dry mass ratio 

indicates that contact with liquid medium led to greater 

hydration of tissues. This was subsequently controlled 

by the inclusion of a growth retardant, paclobutrazol (1 

mg l –1 ), in the culture medium (Smith et al., 1990a). 

Other porous materials that have been used to support 

plant growth include rockwool (Woodward et al., 

1991; Tanaka et al., 1991), polyurethane foam 

(Gutman and Shiryaeva, 1980; Scherer et al., 1988), 

vermiculite (Kirdmanee et al., 1995), a mixture of 

vermiculite and Gelrite (Jay-Allemand et al., 1992). 

Afreen-Zobayed et al. (2000) cultured sweet 

potato, on sugar-free medium in autotrophic 

conditions, on mixtures of paper pulp and vermiculite 

in various proportions. Optimal growth was obtained 

on a mixture containing 70% paper pulp. On this

mixture, the fresh mass of plantlets was greater by a 

factor of 2.7 than on agar-solidified medium. 

Mixtures of paper pulp and vermiculite, in 

unspecified proportions, are prepared in a commercial 

product known as Florialite (Nisshinbo Industries, 

Inc., Tokyo). Afreen-Zobayed et al. (1999) found 

that growth rates of plantlets of sweet potato grown 

autotrophically on Florialite were greater, in 

ascending order, on agar, gellan gum, vermiculite, 

Sorbarods and Florialite (best). The dry mass of 

leaves and roots were greater by factors of 2.9 and 

2.8, respectively, on Florialite than on an agar matrix. 

These authors observed that roots spread better in 

Florialite than in Sorbarods. They attributed this to 

the net-like orientation of fibers in Florialite that 

contrasted with the vertical orientation of fibres in 

Sorbarods. Ichimura and Oda (1995) found three 

substances that stimulated plant growth in extracts of 

paper pulp. Each was characterised by low molecular 

weight and high polarity. It is possible that these 

substances contribute to the superior growth observed 

on substrates containing paper pulp. 

6.3.1. Opportunities for improved ventilation and 

photoautotrophy 

When plantlets are cultured in vessels containing 

air at a relative humidity (RH) of less than 100%, 

transpiration occurs which is an important factor in 

reducing hyperhydricity (Gribble, 1999). Relative 

humidity in culture vessels can be reduced through 

ventilation, but gelled substrates are then unsuitable 

because the absorbance of water by the roots of a 

transpiring plant is impeded by the gel’s low hydraulic 

conductivity (Fujiwara and Kozai, 1995) and this 

increases as the gel dries. Thus a common feature of 

studies using ventilated vessels has been the use of 

liquid medium supported by porous materials. 

For example, when plantlets of chrysanthemum 

were grown in Sorbarods in a culture vessel with air 

94% RH, a reduction in the tissue hydration was 

indicated by a significantly lower fresh to dry mass 

ratio than at 100% RH (Smith et al., 1990b) and 

increases in stem length and leaf area. In this 

ADRIAN J. & ASSOUMANI M. 1983 Gums and hydrocolloids in 

nutrition. pp. 301-333 in Rechcigl M. (ed.) 1983 CRC Handbook 

of Nutritional Supplements Vol. II. Agricultural Use. CRC Press 

Inc. Baton Rouge. 

AFREEN-ZOBAYED F., ZOBAYED S.M.A., KUBOTA C., 

KOZAI T. & HASEGAWA O. 2000 A combination of vermiculite 

and paper pulp supporting material for the photoautotrophic 

micropropagation of sweet potato. Plant Sci. 157, 225-231. 

Chapter 4 157 

REFERENCES 

investigation, the RH was reduced to 94% by gaseous 

diffusion through a gas-permeable membrane that 

covered holes drilled in the lid of the culture vessel. 

The use of such ventilated culture vessels can 

significantly improve plant growth by reducing 

hyperhydration and facilitating the movement of 

solutes to the leaves in the transpiration stream. It 

also provides opportunities for photoautotrophic 

growth in sugar-free media. When plantlets are 

cultured in closed vessels, carbon dioxide 

concentrations fall to low levels in the light period, as 

Kozai and Sekimoto (1988) demonstrated in cultures 

of strawberry plants. Photosynthesis requires an 

adequate supply of carbon dioxide and suitable 

lighting. Adequate concentrations of carbon dioxide 

for photoautotrophy can be maintained in ventilated 

culture vessels with (Afreen-Zobayed et al., 1999, 

2000) or without (Horan et al., 1995) elevated levels 

of carbon dioxide in the atmosphere outside the 

culture vessel. Adequate lighting can be achieved 

under lights delivering a photosynthetic photon flux 

of 150 μmol m –2 s –1 in a culture room (Afreen- 

Zobayed et al., 1999, 2000) or in day-light in a 

greenhouse (Horan et al., 1995). The environmental 

requirements of photoautotrophy in vitro have been 

reviewed by Jeong et al. (1995) and its advantages 

have been described by Kozai et al. (1995). 

6.4. IMMOBILISED CELLS 

Yields of secondary metabolites are usually greater 

in differentiated, slow-growing cells than in fast 

growing, undifferentiated cells. By immobilising cells 

in a suitable matrix, their rate of growth can be slowed 

and the production of secondary products enhanced. 

Several ingenious methods of immobilisation have 

been employed. Examples include immobilization in 

spirally wound cotton (Choi et al. (1995), glass fibre 

fabric reinforced with a gelling solution of hybrid SiO2 

precursors (Campostrini et al., 1996), loofa sponge and 

polyurethane foam (Liu et al., 1999) and alginate 

beads (Serp et al., 2000). 

AFREEN-ZOBAYED F., ZOBAYED S.M.A., KUBOTA C., 

KOZAI T. & HASEGAWA O. 1999 Supporting material 

affects the growth and development of in vitro sweet potato 

plantlets cultured photoautotrophically. In Vitro Cell. Dev. – 

Pl. 35, 470-474. 

AHLOOWALIA B.S. & MARETZKI A. 1983 Plant regeneration via 

somatic embryogenesis in sugarcane. Plant Cell Rep. 2, 21-25. 

ALBRECHT C. 1986 Optimization of tissue culture media. Comb. 

Proc. Int. Plant Prop. Soc. 1985, 35, 196-199.


ALONI R. 1980 Role of auxin and sucrose in the differentiation of 

seive and tracheary elements in plant tissue cultures. Planta 150, 

255-263. 

AMADOR A.M. & STEWART K.A. 1987 Osmotic potential and 

pH of fluid drilling gels as influenced by moisture loss and 

incorporation of growth regulators. J. Am. Soc. Hortic. Sci. 112, 

26-28. 

ANDERS J., LARRABEE P.L. & FAHEY J.W. 1988 Evaluation 

of gelrite and numerous agar sources for in vitro regeneration of 

sugarcane. HortScience 23, 755. 

ANDERSON W.C. 1975 Propagation of rhododendrons by tissue 

culture. I. Development of a culture medium for multiplication of 

shoots. Comb. Proc. Int. Plant Prop. Soc. 25, 129-135. 

ANDERSON W.C. 1978 Tissue culture propagation of rhododendrons. 

In Vitro 14, 334. 

ANDERSON W.C. 1980 Tissue culture of red raspberries. pp. 27- 

34 in Proceedings of the Conference on Nursery Production of 

Fruit Plants –Applications and Feasibility. U.S.D.A., A.R.S., 

ARR-NE-11. 

ANON (RESEARCH GROUP 301) 1976 A sharp increase of the 

frequency of pollen plant induction in wheat with potato medium. 

(Chinese) Acta Genet. Sin. 3, 25-31. 

ANON 1980 Ann. Rep. and Accounts, British Petroleum Co. 

Ltd. 1979. 

ANTHONY P., DAVEY M.R., POWER J.B. & LOWE K.C., 1995 

An improved protocol for the culture of cassava leaf protoplasts. 

Plant Cell Tissue Organ Cult. 42, 299-302. 

ANTHONY P., DAVEY M.R., POWER J.B. & LOWE K.C. 1997 

Enhanced mitotic division of cultured Passiflora and Petunia 

protoplasts by oxygenated perfluorocarbon and haemoglobin. 

Biotechnol. Tech. 11, 581-584. 

ARDITTI J. & ERNST R. 1984 Physiology of germinating orchid 

seeds. pp. 177-222 in Arditti J (ed.) 1984 Orchid Biology - 

Reviews and Perspectives III. Comstock Publishing, Cornell 

Univ. Press, Ithaca, London. 

ARDITTI J. 1979 Aspects of the physiology of orchids. Adv. Bot. 

Res. 7, 421-655. 

ARNOW P., OLESON J.J. & WILLIAMS J.H. 1953 The effect of 

arginine in the nutrition of Chlorella vulgaris. Am. J. Bot. 40, 

100-104. 

ASANO Y., KATSUMOTO H., INOKUMA C., KANEKO S., 

ITO Y. & FUJIIE A. 1996 Cytokinin and thiamine requirements 

and stimulative effects of riboflavin and α-ketoglutaric acid on 

embryogenic callus induction from the seeds of Zoysia japonica 

Steud. J. Plant Physiol. 149, 413-417. 

ASHER C.J. 1978 Natural and synthetic culture media for 

spermatophytes. pp. 575-609 in Recheige M. Jr. (ed.). CRC 

Handbook Series in Nutrition and Food. Section G., Culture 

Media and Food Supplements. Diets Vol 3. 

ATTREE S.M., MOORE D., SAWHNEY V.R. & FOWKE L.C. 

1991 Enhanced maturation and desiccation tolerance of white 

spruce [Picea glauca (Moench.) Voss] somatic embryos: effects 

of a non-plasmolysing water stress and abscisic acid. Ann. Bot. 

68, 519-525. 

AYABE S., UDAGAWA A. & FURUYA T. 1988 Stimulation of 

chalcone synthase activity by yeast extract in cultured 

Glycorrhiza echinata cells and 5-deoxyflavone formation by 

isolated protoplasts. Plant Cell Rep. 7, 35-38. 

BAGGA S., DAS R. & SOPORY S.K. 1987 Inhibition of cell 

proliferation and glyoxalase-1 activity by calmodulin inhibitors 

and lithium in Brassica oleracea. J. Plant Physiol. 129, 149-153. 

BALL E. 1953 Hydrolysis of sucrose by autoclaving media, a 

neglected aspect in the culture of plant tissues. B. Torrey Bot. 

Club 80, 409-411. 

BANIK R.M., KANARI B. & UPADHYAY S.N., 2000. 

Exopolysaccharide of the gellan family: prospects and potential. 

World J. Microb. Biot. 16, 407-414. 

BARGHCHI M. 1988 Micropropagation of Alnus cordata (Loisel.) 

Loisel. Plant Cell Tissue Organ Cult. 15, 233-244. 

BARG R. & UMIEL N. 1977 Effects of sugar concentration on 

growth, greenery and shoot formation in callus cultures of four 

genetic lines of tobacco. Z. Pflanzenphysiol. 81, 161-166. 

BARLASS M., GRANT W.J.R. & SKENE K.G.M. 1980 Shoot 

regeneration in vitro from native Australian fruit-bearing trees – 

Quandong and Plum bush. Aust. J. Bot. 28, 405-409. 

BARWALE U.B., KERNS H.R. & WIDHOLM J.M. 1986 Plant 

regeneration from callus cultures of several soybean genotypes 

via embryogenesis and organogenesis. Planta 167, 473-481. 

BEHREND J. & MATELES R.I. 1975 Nitrogen metabolism in 

plant cell suspension cultures. I. Effect of amino acids on growth. 

Plant Physiol. 56, 584-589. 

BERGHOEF J. & BRUINSMA J. 1979 Flower development of 

Begonia franconis Liebm. IV. Adventitious flower bud formation 

in excised inflorescence pedicels in vitro. Z. Pflanzenphysiol. 94, 

407- 416. 

BERGMANN L. 1967 Wachstum gruner Suspensionskulturen von 

Nicotiana tabacum Var. ‘Samsun’ mit CO2 als Kohlenstoffquelle. 

Planta 74, 243-249. 

BERUTO D., BERUTO M., CICCARELLI C. & DEBERGH P. 

1995 Matric potential evaluations and measurements for gelled 

substrates. Physiol. Plant. 94, 151-157. 

BHATTACHARYA P., DEY S. & BHATTACHARYA B.C. 1994 

Use of low-cost gelling agents and support matrices for 

industrial-scale plant tissue culture. Plant Cell Tissue Organ Cult. 

37, 15-23. 

BHOJWANI S.S. & RAZDAN M.K. 1983 Plant Tissue Culture: 

Theory and Practice. Elsevier Science Publishers B.V., 

Amsterdam. 

BIONDI S. & THORPE T.A. 1982 Growth regulator effects, 

metabolite changes and respiration during shoot initiation in 

cultured cotyledon explants of Pinus radiata. Bot. Gaz. 143, 

20-25. 

BISHNOI U.S., JAIN R.K., GUPTA K.R., CHOWDHURY V.K. 

& CHOWDHURY J.B. 2000 High frequency androgenesis in 

indica x Basmati rice hybrids using liquid culture media. Plant 

Cell Tissue Organ Cult. 61, 153-159. 

BISWAS G.C.G. & ZAPATA F.J. 1993 High-frequency plant 

regeneration from protoplasts of indica rice (Oryza sativa L.) 

using maltose. J. Plant Physiol. 141, 470-475. 

BOLANDI A.R., BRANCHARD M., ALIBERT G., SERIEYS H. 

& SARRAFI A. 1999 Cytoplasmic-nuclear interaction and 

medium effect for protoplast culture in sunflower. Plant Cell 

Tissue Organ Cult. 57, 189-193. 

BOLL W.G. & STREET H.E. 1951 Studies on the growth of excised 

roots. 1. The stimulatory effect of molybdenum and copper on the 

growth of excised tomato roots. New Phytol. 50, 52-75. 

BONNER J. & ADDICOTT F. 1937 Cultivation in vitro of excised 

pea roots. Bot. Gaz. 99, 144-170. 

BONNER J. & DEVIRIAN P.S. 1939 Growth factor requirements 

of four species of isolated roots. Am. J. Bot. 26, 661-665. 

BONNER J. & DEVIRIAN P.S. 1939 Growth factor requirements 

of four species of isolated roots. Am. J. Bot. 26, 661-665. 

BONNER J. 1937 Vitamin B1, a growth factor for higher plants. 

Science 85, 183-184. 

BONNER J. 1938a Thiamin (Vitamin B1) and the growth of roots: 

the relation of chemical structure to physiological activity. Am. J. 

Bot. 25, 543-549. 

BONNER J. 1940a Specificity of nicotinic acid as a growth factor 

for isolated pea roots. Plant Physiol. 15, 553-557.

BORGES M., MENESES S., VAZQUEZ J., GARCIA M., 

AGUILERA N., INFANTE Z., RODRIGUEZ A. & FONSECA 

M. 2003 In vitro conservation of Dioscorea alata L. germplasm 

by slow growth. Plant Gen. Res. Newsl. 133, 8-12. 

BORKIRD C. & SINK K.C. 1983 Medium components for shoot 

cultures of chlorophyll-deficient mutants of Petunia inflata. Plant 

Cell Rep. 2, 1-4. 

BÖTTGER M. & LUTHEN H. 1986 Possible linkage between 

NADH-oxidation and proton secretion in Zea mays L. roots. J. 

Exp. Bot. 37, 666-675. 

BOZHKOV P.V. & VON ARNOLD S. 1998 Polyethylene glycol 

promotes maturation but inhibits further development of Picea 

abies somatic embryos. Physiol. Plant. 104, 211-224. 

BRETZLOFF C.W. Jr. 1954 The growth and fruiting of Sordaria 

fimicola. Am. J. Bot. 41, 58-67. 

BRIDSON E.Y. 1978 Diets, culture media and food supplements. 

pp. 91-281 in Rechcigl M. Jr. (ed.) CRC Handbook Series in 

Nutrition and Food. Section G. Vol.3. 

BRINK R.A., COOPER D.C. & AUSHERMAN L.E. 1944 A 

hybrid between Hordeum jubatum and Secale cereale. J. Hered. 

35, 67-75. 

BROWN D.C.W. & THORPE T.A. 1982 Mitochondrial activity 

during shoot formation and growth in tobacco callus. Physiol. 

Plant. 54, 125-130. 

BROWN D.C.W. & THORPE T.A. 1980 Changes in water 

potential and its components during shoot formation in tobacco 

callus. Physiol. Plant. 49, 83-87. 

BROWN D.C.W., LEUNG D.W.M. & THORPE T.A. 1979 

Osmotic requirement for shoot formation in tobacco callus. 

Physiol. Plant. 46, 36-41. 

BROWN C., BROOKS E.J., PEARSON D. & MATHIAS R.J. 

1989 Control of embryogenesis and organogenesis in immature 

wheat embryo callus using increased medium osmolarity and 

abscisic acid. J. Plant Physiol. 133, 727-733. 

BURNET G. & IBRAHIM R.K. 1973 Tissue culture of Citrus peel 

and its potential for flavonoid synthesis. Z. Pflanzenphysiol. 69, 

152-162. 

BURSTROM H. 1957 Root surface development, sucrose 

inversion and free space. Physiol. Plant. 10, 741-751. 

BUTCHER D.N. & STREET H.E. 1964 Excised root culture. Bot. 

Rev. 30, 513-586. 

BUTENKO R.G., LIPSKY A.K.H., CHERNYAK N.D. & ARYA 

H.C. 1984 Changes in culture medium pH by cell suspension 

cultures of Dioscorea deltoidea. Plant Sci. Lett. 35, 207-212. 

CALLEBAUT A. & MOTTE J.-C. 1988 Growth of cucumber cells 

in media with lactose or milk whey as carbon source. Plant Cell 

Rep. 7, 162- 165. 

CAMPOSTRINI R., CARTURAN G., CANIATO R., PIOVAN 

A., FILIPPINI R., INNOCENTI G. & CAPPELLETTI E.M. 

1996 Immobilization of plant cells in hybrid sol-gel materials. J. 

Sol-Gel Sci. Techn. 7, 87-97. 

CANHOTO J.M. & CRUZ G.S. 1994 Improvement of somatic 

embryogenesis in Feijoa sellowiana Berg (Myrtaceae) by 

manipulation of culture media composition. In Vitro Cell. Dev. – 

Pl. 30, 21-25. 

CAPLIN S.M. & STEWARD F.C. 1948 Effects of coconut milk on 

the growth of explants from carrot root. Science 108, 655-657. 

CARCELLER M., DAVEY M.R., FOWLER M.W. & STREET 

H.E. 1971 The influence of sucrose, 2,4-D and kinetin on the 

growth, fine structure and lignin content of cultured sycamore 

cells. Protoplasma 73, 367-385. 

CARIMI F., DE PASQUALE F. & CRESCIMANNO F.G. 1999 

Somatic embryogenesis and plant regeneration from pistil thin 

cell layers of Citrus. Plant Cell Rep. 18, 935-940. 

CARIMI F., DE PASQUALE F. & PUGLIA A.M. 1998 In vitro 

rescue of zygotic embryos of sour orange, Citrus aurantium L., 

Chapter 4 159 

and their detection based on RFLP analysis. Plant Breeding 117, 

261-266. 

CARPITA N., SABULARSE D., MONTEZINOS D. & DELMER 

D.P. (1979) Determination of the pore size of cell walls of living 

plant cells. Science 205,1144-1147. 

CHANDLER M.T., TANDEAU DE MARSAC N. & 

KOUCHKOVSKY Y. 1972 Photosynthetic growth of tobacco 

cells in liquid suspensions. Can. J. Bot. 50, 2265-2270. 

CHANDLER S.F., RAGOLSKY E., PUA E.-C. & THORPE T.A. 

1987 Some morphogenic effects of sodium sulphate on tobacco 

callus. Plant Cell Tissue Organ Cult. 11, 141-150. 

CHAUVIN J.E. & SALESSES G. 1988 Advances in chestnut 

micropropagation (Castanea sp.). Acta Hortic. 227, 340-345. 

CHAUVIN J.E., MARHADOUR S., COHAT J. & LE NARD M. 

1999 Effects of gelling agents on in vitro regeneration and 

kanamycin efficiency as a selective agent in plant transformation 

procedures. Plant Cell Tissue Organ Cult. 58, 213-217. 

CHAZEN O., HARTUNG W. & NEUMANN P.M. 1995 The 

different effects of PEG 6000 and NaCl on leaf development are 

associated with differential inhibition of root water transport. 

Plant Cell Environ. 18, 727-735. 

CHEE P.P. 1995 Stimulation of adventitious rooting of Taxus 

species by thiamine. Plant Cell Rep. 14, 753-757. 

CHEE R.P., SCHULTHEIS J.R. & CANTCLIFFE D.J. 1990 Plant 

recovery from sweet potato somatic embryos. HortScience 25, 

795-797. 

CHENG T.-Y. & VOQUI T.H. 1977 Regeneration of Douglas fir 

plantlets through tissue culture. Science 198, 306-307. 

CHENG T.-Y. 1978 Clonal propagation of woody species through 

tissue culture techniques. Comb. Proc. Int. Plant Prop. Soc. 28, 

139-155. 

CHEVRE A.-M., GILL S.S., MOURAS A. & SALESSES G. 1983 

In vitro multiplication of chestnut. J. Hortic. Sci. 58, 23-29. 

CHIEN Y.C. & KAO K.N. 1983 Effects of osmolality, cytokinin 

and organic acids on pollen callus formation in Triticale anthers. 

Can. J. Bot. 61, 639-641. 

CHIN C. & MILLER D. 1982 Some characteristics of the 

phosphate uptake by Petunia cells. HortScience 17, 488. 

CHIN C.-K., KONG Y. & PEDERSEN H. 1988 Culture of 

droplets containing asparagus cells and protoplasts on polypropylene 

membrane. Plant Cell Tissue Organ Cult. 15, 59-65. 

CHOI H.J., TAO B.Y. & OKOS M.R. 1995 Enhancement of 

secondary metabolite production by immobilized Gossypium 

arboreum cells. Biotechnol. Progr. 11, 306-311. 

CHONG C. & PUA E.-C. 1985 Carbon nutrition of Ottawa 3 apple 

rootstock during stages of in vitro propagation. J. Hortic. Sci. 60, 

285-290. 

CHONG C. & TAPER C.D. 1972 Malus tissue culture. I. Sorbitol 

(D-glucitol) as carbon source for callus initiation and growth. 

Can. J. Bot. 50, 1399-1404. 

CHONG C. & TAPER C.D. 1974a Malus tissue cultures. II. Sorbitol 

metabolism and carbon nutrition. Can. J. Bot. 52, 2361-2364. 

CHONG C. & TAPER C.D. 1974b Influence of light intensity on 

sorbitol metabolism, growth and chlorophyll content of Malus 

tissue cultures. Ann. Bot. 38, 359-362. 

CHU C.-C., WANG C.-C., SUN C.-S., HSU C., YIN K.-C., CHU 

C.-Y. & BI F.-Y. 1975 Establishment of an efficient medium for 

anther culture of rice, through comparative experiments on the 

nitrogen sources. Sci.Sinica 18, 659-668. 

CHUA S.E. 1966 Studies on tissue culture of Hevea brasiliensis. I. 

Role of osmotic concentration, carbohydrate and pH value in 

induction of callus growth in plumule tissue from Hevea 

seedling. J. Rubber Res. Inst. Malaya 19, 272-276. 

CHUANG C.-C., OUYANG T.-W., CHIA H., CHOU S.-M. & 

CHING C.-K. 1978 A set of potato media for wheat anther


culture. pp. 51-56 in Plant Tissue Culture. Proceedings of the 

Peking Symposium 1978. Pitman, Boston, London, Melbourne. 

CHUONG P.V. & BEVERSDORF W.D. 1985 High frequency 

embryogenesis through isolated microspore culture in Brassica 

napus L and B. carinata Braun. Plant Sci. 39, 219-226. 

CLELAND R. 1977 The control of cell enlargement. pp. 101-115 

in Jennings D.H. (ed.) S.E.B. Symp. 31. University Press, 

Cambridge. 

COFFIN R., TAPER C.D. & CHONG C. 1976 Sorbitol and 

sucrose as carbon source for callus culture of some species of the 

Rosaceae. Can. J. Bot. 54, 547-551. 

CONNER A.J. & FALLOON P.G. 1993 Osmotic versus 

nutritional effects when rooting in vitro asparagus minicrowns on 

high sucrose. Plant Sci. 89, 101-106. 

CONNER A.J. & MEREDITH C.P. 1984 An improved 

polyurethane support system for monitoring growth in plant cell 

cultures. Plant Cell Tissue Organ Cult. 3, 59-68. 

CONRAD P.L., CONRAD J.M., DURBIN R.D. & HELGESON J.P. 

1981 Effects of some organic buffers on division of protoplastderived 

cells and plant regeneration. Plant Physiol. 67, Suppl., 116. 

COOPER A. 1979 The ABC of NFT. Grower Books, London. 

ISBN 0 901 361224. 

COPPING L.G. & STREET H.E. 1972 Properties of the 

invertases of cultured sycamore cells and changes in their activity 

during culture growth. Physiol. Plant. 26, 346-354. 

CULAFIC L., BUDIMIR S., VUJICIC R. & NESKOVIC M. 1987 

Induction of somatic embryogenesis and embryo development in 

Rumex acetosella L. Plant Cell Tissue Organ Cult. 11, 133-139. 

DABIN P. & BEGUIN F. 1987 Somatic embryogenesis in 

Fuchsia. Acta Hortic. 212, 725-726. 

DAIGNY G., PAUL H. & SANGWAN-NORRELL B.S. 1996 

Factors influencing secondaru somatic embryogenesis in Malus x 

domestica Borkh. (cv ‘Gloster 69’). Plant Cell Rep. 16, 153-157. 

DALTON C.C., IQBAL K. & TURNER D.A. 1983 Iron phosphate 

precipitation in Murashige and Skoog media. Physiol. Plant. 57, 

472-476. 

DAMIANO C., CURIR P. & COSMI T. 1987 Short note on the 

effects of sugar on the growth of Eucalyptus gunnii in vitro. Acta 

Hortic. 212, 553-556. 

DANTU P.K. & BHOJWANI S.S. 1995 In vitro corm formation 

and field evaluation of corm-derived plants of Gladiolus. Sci. 

Hortic. 61, 115-129. 

DAS R., BAGGA S. & SOPORY S.K. 1987 Involvement of 

phosphoinositides, calmodulin and glyoxidase-1 in cell 

proliferation in callus cultures of Amaranthus paniculatus. Plant 

Sci. 53, 45-51. 

DAS T., MITRA G.C. & CHATTERJEE A. 1995 Micropropagation 

of Citrus sinensis var. mosambi: an important scion. 

Phytomorph. 45, 57-64. 

DAVIS M.J., BAKER R. & HANAN J.J. 1977 Clonal multiplication 

of carnation by micropropagation. J. Am. Soc. Hortic. 

Sci. 102, 48-53. 

DAY D. 1942 Thiamin content of agar. B. Torrey Bot. Club 69, 

11-20. 

DE BRUYN M.H. AND FERREIRA D.I. 1992 In vitro corm 

production of Gladiolus dalenii and G. tristis. Plant Cell Tissue 

Organ Cult. 31, 123-128. 

DE CAPITE L. 1948 pH of nutritive solutions and its influence on 

the development of plants. Ann. Fac. Agrar. Univ. Perugia 5, 

135-145. 

DE CAPITE L. 1952a Combined action of p-aminobenzoic acid and 

indoleacetic acid on the vascular parenchyma of Jerusalem 

artichoke cultured in vitro. Compt. Rend. Soc. Biol. 146, 863-865. 

DE CAPITE L. 1952b Action of p-aminophenylsulphonamide and 

p-amino-benzoic acid on the vascular parenchyma of Jerusalem 

artichoke and of crown-gall tissue of salsify. Compt. Rend. Acad. 

Sci. Paris 234, 2478-2480. 

DE FOSSARD R.A., MYINT A. & LEE E.C.M. 1974 A broad 

spectrum tissue culture experiment with tobacco (Nicotiana 

tabacum) pith tissue callus. Physiol. Plant. 31, 125-130. 

DE JONG A.W. & BRUINSMA J. 1974 Pistil development in 

Cleome flowers III. Effects of growth-regulating substances on 

flower buds of Cleome iberidella Welv. ex Oliv. grown in vitro. 

Z. Pflanzenphysiol. 73, 142-151. 

DE JONG A.W., SMIT A.L. & BRUINSMA J. 1974 Pistil 

development in Cleome flowers II. Effects of nutrients on flower 

buds of Cleome iberidella Welv. ex Oliv. grown in vitro. Z. 

Pflanzenphysiol. 72, 227-236. 

DE KLERK G.J., HANECAKOVA J. & JASIK J. 2007 Effect 

of medium-pH on adventitious root formation from thin stem 

disks cut from apple microshoots. Plant Cell Tissue Organ 

Cult. (in press) 

DE KLERK-KIEBERT Y.M. & VAN DER PLAS L.H.W. 1985 

Relationship of respiratory pathways in soybean cell suspensions 

to growth of the cells at various glucose concentrations. Plant 


DE KRUIJFF E. 1906 Composition of coconut water and presence 

of diastase in coconuts. B. Dep. Agric. Indes Neerland. 4, 1-8. 

DE PASQUALE F., CARIMI F. & CRESCIMANNO F.G. 1994 

Somatic embryogenesis from styles of different cultivars of 

Citrus limon (L.) Burm. Aust. J. Bot, 42, 587-594. 

DE PINTO M.C., FRANCIS D. & DE GARA D. 1999 The redox 

state of the ascorbate-dehydroascrobate pair as a specific sensor 

of cell division in tobacco BY-2 cells. Protoplasma 209, 90-97. 

DEBERGH P.C. 1983 Effects of agar brand and concentration on 

the tissue culture medium. Physiol. Plant. 59, 270-276. 

DEBERGH P., AITKEN-CHRISTIE J., COHEN D., GROUT B., 

VON ARNOLD S., ZIMMERMAN R. & ZIV M. 1992 Reconsideration 

of the term ‘vitrification’ as used in micropropagation. 


DEBERGH P., HARBAOUI Y. & LEMEUR R. 1981. Mass 

propagation of globe artichoke (Cyanara scolymus): evaluation 

of different hypotheses to overcome vitrification with special 

reference to water potential. Physiol. Plant. 53, 181-187 

DELBARRE A., MULLER P., IMHOFF V. & GUERN J. 1996 

Comparison of mechanisms controlling uptake and accumulation 

of 2,4-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, 

and indole-3-acetic acid in suspension-cultured tobacco cells. 


DELFEL N.E. & SMITH L.J. 1980 The importance of culture conditions 

and medium component interactions on the growth of 

Cephalotaxus harringtonia tissue cultures. Planta Medica 40, 

237-244. 

DIGBY J. & SKOOG F. 1966 Cytokinin activation of thiamine 

biosynthesis in tobacco callus cultures. Plant Physiol. 41, 647-652. 

DIJKEMA C., BOOIJ H., DE JAGER P.A., DE VRIES S.C., 

SCHAAFSMA T.J. & VAN KAMMEN A. 1990 Aerobic hexose 

metabolism in embryogenic cell lines of Daucus carota studied 

by 13 C NMR using 13 C-enriched substrates. pp. 343-348 in 

Nijkamp et al. (eds.) 1990 (q.v.). 

DIX L. & VAN STADEN J. 1982 Auxin and gibberellin-like 

substances in coconut milk and malt extract. Plant Cell Tissue 

Organ Cult. 1, 239-245. 

DOLEY D. & LEYTON L. 1970 Effects of growth regulating 

substances and water potential on the development of wound 

callus in Fraxinus. New Phytol. 69, 87-102. 

DORAN P.M. 1993 Design of bioreactors for plant cells and 

organs. pp 116-169 in Fiechter A. (ed.) Bioprocess design and 

control. Vol. 48 Springer-Verlag, Berlin. pp 116-169.

DOUGALL D.K. 1980 Nutrition and Metabolism. Plant Tissue 

Culture as a Source of Biochemicals. C.R.C. Press, Boca Raton, 

Florida. 

DOUGALL D.K., WEYRAUCH K.W. & ALTON W. 1979 The 

effects of organic acids on growth and anthocyanin production by 

wild carrot cells growing on ammonia as a sole nitrogen source. 

In Vitro 15, 189-190 (Abst. 103). 

DOUGLAS T.J., VILLALOBOS V.M., THOMPSON M.R. & 

THORPE T.A. 1982 Lipid and pigment changes during shootinititaion 

in cultured explants of Pinus radiata. Physiol. Plant. 

55, 470-477. 

DOVZHENKO A., BERGEN U. & KOOP H.U. 1998 Thinalginate-layer 

technique for protoplast culture of tobacco leaf 

protoplasts: shoot formation in less than two weeks. Protoplasma 

204, 114-118. 

DREW R.A. & SMITH N.G. 1986 Growth of apical and lateral 

buds of pawpaw (Carica papaya L.) as affected by nutritional 

and hormonal factors. J. Hortic. Sci. 61, 535-543. 

DREW R.A., MCCOMB J.A. & CONSIDINE J.A. 1993 

Rhizogenesis and root growth of Carica papaya L. in vitro in 

relation to auxin sensitive phases and use of riboflavin. Plant 


DRUART PH. 1988 Regulation of axillary branching in 

micropropagation of woody fruit species. Acta Hortic. 227, 

369-380. 

DUHAMET L. & MENTZER C. 1955 Essais d’isolement des 

substances excito-formatrices du lait de coco. Compt. Rend. 

Acad. Sci. Paris 241, 86-88. 

DUNSTAN D.I. 1982 Transplantation and post-transplantation of 

micropropagated tree-fruit rootstocks. Comb. Proc. Int. Plant 

Prop. Soc., 1981 31, 39-44. 

DUNSTAN W.R. 1906 Report on a sample of coconut “water” 

from Ceylon. Trop. Agric. (Ceylon) 26, 377-378. 

DUNWELL J.M. 1981 Influence of genotype and environment on 

growth of barley Hordeum vulgare embryos in vitro. Ann. Bot. 

48, 535-542. 

EMONS A.M.C., SAMALLO-DROPPERS A. & VAN DER 

TOORN C. 1993 The influence of sucrose, mannitol, L-proline, 

abscisic acid and gibberellic acid on the maturation of somatic 

embryos of Zea mays L. from suspension cultures. J. Plant 

Physiol. 142, 597-604. 

EARLE E.D. & TORREY J.G. 1965 Colony formation by isolated 

Convolvulus cells plated on defined media. Plant Physiol. 40, 

520-528. 

EDELMAN J. & HANSON A.D. 1972 Sucrose suppression of 

chlorophyll synthesis in carrot-tissue cultures. J. Exp. Bot. 23, 

469-478. 

EDWARDS K.J. & GOLDSMITH M.H.M. 1980 pH-dependent 

accumulation of indoleacetic acid by corn coleoptile sections. 


EINSET J.W. 1978 Citrus tissue culture - stimulation of fruit 

explant cultures with orange juice. Plant Physiol. 62, 885-888. 

EL HINNAWIY E. 1974 Effect of some growth regulating 

substances and carbohydrates on chlorophyll production in 

Melilotus alba (Desr.) callus tissue cultures. Z. Pflanzenphysiol. 

74, 95-105. 

ERNER Y. & REUVENI O. 1981 Promotion of citrus tissue 

culture by citric acid. Plant Physiol. 67, Suppl., 27. 

ERNER Y. & REUVENI O. 1981 Promotion of citrus tissue 

culture by citric acid. Plant Physiol. 67, Suppl., 27. 

ERNST R. 1967 Effects of carbohydrate selection on the growth 

rate of freshly germinated Phalaenopsis and Dendrobium seed. 

Am. Orchid Soc. Bull. 36, 1068-1073. 

ERNST R. 1974 The use of activated charcoal in asymbiotic 

seedling culture of Paphiopedilum. Am. Orchid Soc. Bull. 43, 

35-38. 

Chapter 4 161 

ERNST R., ARDITTI J. & HEALEY P.L. 1971 Carbohydrate 

physiology of orchid seedlings. II. Hydrolysis and effects of 

oligosaccharides. Am. J. Bot. 58, 827-835. 

EVANS D.A., SHARP W.R. & PADDOCK E.F. 1976 Variation in 

callus proliferation and root morphogenesis in leaf tissue cultures 

of Glycine max strain T 219. Phytomorph. 26, 379-384. 

FELLE H.H. 1998. The apoplastic pH of the Zea mays root cortex 

as measured with pH-sensitive microelectrodes: aspects of 

regulation. J. Exp. Bot. 49, 987-995. 

FELLE H.H. 2001 pH: Signal and messenger in plant cells. Plant 

Biol. 3, 577-591. 

FELLE H.H. & HANSTEIN S. 2002 The apoplastic pH of the 

substomatal cavity of Vicia faba leaves and its regulation 

responding to different stress factors. J. Exp. Bot. 53, 73-82. 

FERGUSON J.D., STREET H.E. & DAVID S.B. 1958 The carbohydrate 

nutrition of tomato roots. V. The promotion and 

inhibition of excised root growth by various sugars and sugar 

alcohols. Ann. Bot. 22, 513-524. 

FINDENEGG G.R., VAN BEUSICHEM M.L. & KELTJENS 

W.G. 1986 Proton balance of plants: physiological, agronomical 

and economical implications. Neth. J. Agric. Sci. 34, 371-379. 

FINNIE S.J., POWELL W. & DYER A.F. 1989 The effect of 

carbohydrate composition and concentration on anther culture 

response in barley. Plant Breeding 103, 110-118. 

FONNESBECH M. 1972 Organic nutrients in the media for the 

propagation of Cymbidium in vitro. Physiol. Plant. 27, 360-364. 

FOX J.E. & MILLER C. 1959 Factors in corn steep water 

promoting growth of plant tissues. Plant Physiol. 34, 577-579. 

FUGGI A., RUGANO V.M., VONA V. & RIGANO C. 1981 

Nitrate and ammonium assimilation in algae cell-suspensions and 

related pH variations in the external medium, monitored by 

electrodes. Plant Sci. Lett. 23, 129-138. 

FUJIWARA A. (ed.) 1982 Plant Tissue Culture 1982. Proc. 5th. 

Int. Cong. Plant Tiss. Cell Cult., Japan. Assoc. Plant Tissue 

Culture, Japan. 

FUJIWARA K. & KOZAI T. 1995 Physical microenvironment and 

its effects. pp. 319-369 in Aitken-Christie J., Kozai T. and Smith 

M.L. (eds.) Automation and Environmental Control in Plant 

Tissue Culture. Kluwer Academic Publishers, The Netherlands. 

FUKAMI T. & HILDEBRANDT A.C. 1967 Growth and chlorophyll 

formation in edible green plant callus tissues in vitro on 

media with limited sugar supplements. Bot. Mag. Tokyo 80, 

199-212. 

FURGUSON J.D. 1967 The nutrition of excised wheat roots. 


GALIBA G. & ERDEI L. 1986 Dependence of wheat callus 

growth, differentiation and mineral content on carbohydrate 

supply. Plant Sci. 45, 65-70. 

GALIBA G. & YAMADA Y. 1988 A novel method for increasing 

the frequency of somatic embryogenesis in wheat tissue culture 

by NaCl and KCl supplementation. Plant Cell Rep. 7, 55-58. 

GAMBORG O.L. & SHYLUK J.P. 1970 The culture of plant cells 

with ammonium salts as a sole nitrogen source. Plant Physiol. 45, 

598-600. 

GAMBORG O.L., CONSTABEL F. & SHYLUK J.P. 1974 

Organogenesis in callus from shoot apices of Pisum sativum. 


GAMBORG O.L., MILLER R.A. & OJIMA K. 1968 Nutrient 

requirements of suspension cultures of soybean root cells. Exp. 

Cell Res. 50, 151-158. 

GAMBORG O.L., MILLER R.A. & OJIMA K. 1968 Nutrient 

requirements of suspension cultures of soybean root cells. Exp. 

Cell Res. 50, 151-158. 

GARIN E., BERNIER-CARDOU M. , ISABEL N., 

KLIMASZEWSKA K. & PLOURDE A. 2000 Effect of sugars, 

amino acids and culture technique on maturation of somatic


embryos of Pinus strobus on medium with two gellan gum 

concentrations. Plant Cell Tissue Organ Cult. 62, 27-37. 

GASPAR T., KEVERS C., DEBERGH P., MAENE L., PAQUES 

M. & BOXUS P. 1987 Vitrification: morphological, physiological 

and ecological aspects. in Bonga J.M. and Durzan D.J. 

(eds.) Cell and Tissue Culture in Forestry Vol. 1 Kluwer 

Academic Press Publ., Dordrecht. 

GAUTHERET R.J. 1942 Manuel Technique de Culture des Tissue 

Végétaux. Masson et Cie, Paris. 

GAUTHERET R.J. 1945 Une voie nouvelle en biologie végétale: 

la culture des tissus. Gallimard, Paris. 

GAUTHERET R.J. 1948 Sur la culture indéfinie des tissus de Salix 

caprea. Compt. Rend. Soc. Biol. 142, 807. 

GAWEL N.J., RAO A.P. & ROBACKER C.D. 1986 Somatic 

embryogenesis from leaf and petiole callus cultures of 

Gossypium hirsutum L. Plant Cell Rep. 5, 457-459. 

GAWEL N.J. & ROBACKER C.D. 1990 Somatic embryogenesis 

in two Gossypium hirsutum genotypes on semi-solid versus 

liquid proliferation media. Plant Cell Tissue Organ Cult.23, 

201-204. 

GEORGE E.F., PUTTOCK D.J.M. & GEORGE H.J. 1987 Plant 

Culture Media Vol.1. Exegetics Ltd., Westbury, England. 

GERRITS M. & DE KLERK G.J. 1992 Dry-matter partitioning 

between bulbs and leaves in plantlets of Lilium speciosum 

regenerated in vitro. Acta Bot. Neerl. 41, 461-468. 

GLASSTONE S. 1946 Textbook of Physical Chemistry. Second 

Edition. MacMillan, London. 

GOLDSCHMIDT E.E. 1976 Edogenous growth substances of 

citrus tissues. HortScience 11, 95-99. 

GOOD N.E., WINGET G.D., WINTER W., CONNOLLY T.N., 

IZAWA S. & SINGH R.M. 1966 Hydrogen ion buffers for 

biological research. Biochemistry 5, 467-477. 

GOODWIN P.B. 1966 An improved medium for the rapid growth 

of isolated potato buds. J. Exp. Bot. 17, 590-595. 

GOPAL J., CHAMAIL A. & SARKAR D. 2002 Slow-growth in 

vitro conservation of potato germplasm at normal propagation 

temperature. Pot. Res. 45, 203-213. 

GRANATEK C.H. & COCKERLINE A.W. 1978 Callus formation 

versus differentiation of cultured barley embryos: hormonal 

osmotic interactions. In Vitro 14, 212-217. 

GRAY D.J., CONGER B.V. & SONGSTAD D.D. 1987 

Desiccated quiescent somatic embryos of orchard grass for use as 

synthetic seeds. In Vitro Cell. Dev. 23, 29-32. 

GRIBBLE K. 1999 The influence of relative humidity on 

vitrification, growth and morphology of Gypsophila paniculata 

L. Plant Growth Regul. 27, 179-188. 

GROOTAARTS H., SCHEL J.H.N. & PIERIK R.L.M. 1981 The 

origin of bulblets formed on excised twin scales of Nerine 

bowdenii W. Watts. Plant Cell Tissue Organ Cult. 1, 39-46. 

GROSS K.C., PHARR D.M. & LOCY R.D. 1981 Growth of callus 

initiated from cucumber hypocotyls on galactose and galactosecontaining 

oligosaccharides. Plant Sci. Lett. 20, 333-341. 

GUHA S. & JOHRI B.M. 1966 In vitro development of overy and 

ovule of Allium cepa L. Phytomorph. 16, 353-364. 

GUHA S. & MAHESHWARI S.C. 1964 In vitro production of 

embryos from anthers of Datura. Nature 204, 497. 

GUHA S. & MAHESHWARI S.C. 1967 Development of 

embryoids from pollen grains of Datura in vitro. Phytomorph. 

17, 454-461. 

GUNNING B.E.S. & STEER M.W. 1975 Ultrastructure and the 

Biology of Plant Cells. Arnold, London. 

GUPTA P.K. & DURZAN D.J. 1985 Shoot multiplication from 

mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar 

pine (Pinus lambertiana). Plant Cell Rep. 4, 177-179. 

GUPTA P.K., DANDEKAR A.M. & DURZAN D.J. 1988 Somatic 

proembryo formation and transient expression of a luciferase 

gene in Douglas fir and loblolly pine protoplasts. Plant Sci. 58, 

85-92. 

GUTMAN T.S. & SHIRYAEVA G.A. 1980 New method for 

growing cultures of isolated tissues of higher plants. Rastit. 

Resur. 16, 601-606. 

HACKETT D.P. 1952 The osmotic change during auxin-induced 

water uptake by potato tissue. Plant Physiol. 27, 279-284. 

HAKKAART F.A. & VERSLUIJS J.M.A. 1983 Some factors 

affecting glassiness in carnation meristem tip cultures. Neth. J. 

Plant Path. 89, 47-53. 

HALPERIN W. & WETHERELL D.F. 1964 Adventive embryony 

in tissue cultures of the wild carrot, Daucus carota. Am. J. Bot. 

51, 274-283. 

HAMAOKA Y., FUJITA Y. & IWAI S. 1991 Effects of 

temperature on the mode of pollen development in anther culture 

of Brassica campestris. Physiol. Plant. 82, 67-72. 

HAMMERSLEY-STRAW D.R.H. & THORPE T.A. 1988 Use of 

osmotic inhibition in studies of shoot formation in tobacco callus 

cultures. Bot. Gaz. 149, 303-310. 

HARBAGE J.F., STIMART D.P. & AUER C. 1998 pH affects 

1H-indole-3-butyric acid uptake but not metabolism during the 

initiation phase of adventitious root induction in apple 

microcuttings. J. Am. Soc. Hortic. Sci. 123, 6-10. 

HARDING K., BENSON E.E. & CLACHER K. 1997 Plant 

conservation biotechnology: an overview. Agro Food Ind. Hi 

Tech. 8, 24-29. 

HARRAN S. & DICKINSON D.B. 1978 Metabolism of myoinositol 

and growth in various sugars of suspension cultured 

cells. Planta 141, 77-82. 

HARRINGTON H.M. & HENKE R.R. 1981 Amino acid transport 

into cultured tobacco cells. I. Lysine transport. Plant Physiol. 67, 

373-378. 

HARRIS R.E. & STEVENSON J.H. 1979 Virus elimination and 

rapid propagation of grapes in vitro. Comb. Proc. Int. Plant Prop. 

Soc. 29, 95-108. 

HARVAIS G. 1982 An improved medium for growing the orchid 

Cypripedium reginae axenically. Can. J. Bot. 60, 2547-2555. 

HELGESON J.P., UPPER C.D. & HABERLACH G.T. 1972 

Medium and tissue sugar concentrations during cytokinincontrolled 

growth of tobacco callus tissues. pp. 484-492 in Carr 

D.J. (ed.) Plant Growth Substances 1970. Springer Verlag, 

Berlin, Heidelberg, New York. 

HELLER R. & GAUTHERET R.J. 1949 Sur l’emploi de papier 

filtre sans cendres commes support pour les cultures de tissues 

végétaux. Compt. Rend. Seance Soc. Biol., Paris 143, 335-337. 

HENDERSON W.E. & KINNERSLEY A.M. 1988 Corn starch as 

an alternative gelling agent for plant tissue culture. Plant Cell 


HESS D., LEIPOLDT G. & ILLG R.D. 1979 Investigations on the 

lactose induction of β-galactosides activity in callus tissue 

cultures of Nemesia strumosa and Petunia hybrida. Z. 


HEW C.S., TING S.K. & CHIA T.F. 1988 Substrate utilization by 

Dendrobium tissues. Bot. Gaz. 149, 153-157. 

HILDEBRANDT A.C., RIKER A.J. & DUGGAR B.M. 1946 The 

influence of the composition of the medium on growth in vitro of 

excised tobacco and sunflower tissue cultures. Am. J. Bot. 33, 

591-597. 

HILDEBRANDT A.C., WILMAR J.C., JOHNS H. & RIKER A.J. 

1963 Growth of edible chlorophyllous plant tissues in vitro. Am. 

J. Bot. 50, 248-254. 

HISAJIMA S. & THORPE T.A. 1981 Lactose-adapted cultured 

cells of Japanese morning-glory. Acta Physiol. Plant. 3, 187-191. 

HISAJIMA S. & THORPE T.A. 1985 Lactose metabolism in 

lactose-adapted cells of Japanese morning-glory. J. Plant 

Physiol. 118, 145-151.

HISAJIMA S., ARAI Y. & ITO T. 1978 Changes in sugar 

contents and some enzyme activities during growth of morningglory 

callus (In Japanese). J. Jpn. Soc. Starch Sci. 25, 223-228. 

HO W.-J. & VASIL I.K. 1983 Somatic embryogenesis in 

sugarcane (Saccharum officinarum L.): Growth and plant 

regeneration from embryogenic cell suspension cultures. Ann. 

Bot. 51, 719-726. 

HOMÈS J. & VANSVEREN-VAN ESPEN N. 1973 Effets du 

saccharose et de la lumière sur le développement et la 

morphologie de protocormes d’Orchides cultivés in vitro. Bull. 

Soc. Roy. Bot. Belg. 106, 89-106. 

HOOKER T.S. & THORPE T.A. 1997 Effects of water deficit 

stress on the developmental growth of excised tomato roots 

cultured in vitro. In Vitro Cell. Dev.-Pl. 33, 245-251. 

HORAN I., WALKER S., ROBERTS A.V., MOTTLEY J. & 

SIMPKINS I. 1995 Micropropagation of roses: the benefits of 

pruned mother-plantlets at Stage II and a greenhouse 

environment at stage III. J. Hortic. Sci., 70, 799-806. 

HOWARD B.H. & MARKS T.R. 1987 The in vitro - in vivo 

interface. pp. 101-111 in Jackson M.B., Mantell S.H. & Blake J. 

(eds.) Advances in the Chemical Manipulation of Plant Tissue 

Cultures. Monograph 16, British Plant Growth Regulator Group, 

Bristol. 

HU T.C., ZIAUDDIN A., SIMION E. & KASHA K.J. 1995 Isolated 

microscopore culture of wheat (Triticum aestivum L.) in a 

defined media. I: Effects of pretreatment, isolation methods, and 

hormones. In Vitro Cell. Dev. Biol., Plant. 31, 79-83. 

HUANG B., BIRD S., KEMBLE R., MIKI B. & KELLER W. 

1991 Plant regeneration from microspore-derived embryos of 

Brassica napus: effect of embryo age, culture temperature, 

osmotic pressure and abscisic acid. In Vitro Cell. Dev. Plant 27, 

28-31. 

HUSEMANN W., AMINO S., FISCHER K., HERZBECK H. & 

CALLIS R. 1990 Light dependent growth and differentiation 

processes in photoautotrophic cell cultures. pp. 373-378 in 

Nijkamp et al. (eds.) 1990 (q.v.). 

HYNDMAN S.E., HASEGAWA P.M. & BRESSAN R.A. 1982 The 

role of sucrose and nitrogen in adventitious root formation on 

cultured rose shoots. Plant Cell Tissue Organ Cult. 1, 229-238. 

ICHI T., KODA T., ASAI I., HATANAKA A. & SEKIYA J. 1986 

Effects of gelling agents on in vitro culture of plant tissues. 

Agric. Biol. Chem. 50, 2397-2399. 

ICHIMURA K. & ODA M. 1995 Stimulation of root growth in 

several vegetables by wood pulp extract. J. Jpn Soc. Hortic. Sci. 

63 (Suppl. 4), 797-803. 

IKEDA-IWAI, M., UMEHARA M., SATOH S. & KAMADA H. 

2003 Stress-induced somatic embryogenesis in vegetative tissues 

of Arabidopsis thaliana. Plant J. 34, 107-114. 

ILIĆ-GRUBOR K., ATTREE S. M. & FOWKE L. C. 1998 

Induction of microspore-derived embryos of Brassica napus L. 

with polyethylene glycol (PEG) as osmoticum in a low sucrose 

medium Plant Cell Rep. 17, 329-333. 

IMAMURA J. &. HARADA H. 1980. Effects of abscisic acid and 

water stress on the embryo and plantlet formation in anther 

culture of Nicotiana tabacum cv. Samsun. Z. Pflanzenphysiol. 

100, 285-289. 

IMAMURA J., OKABE E., KYO M. & HARADA H. 1982 

Embryogenesis and plantlet formation through direct culture of 

isolated pollen of Nicotiana tabacum cv. Samsum and Nicotiana 

rustica cv. rustica. Plant Cell Physiol. 23, 713-716. 

INOUE M. & MAEDA E. 1982 Control of organ formation in rice 

callus using two-step culture method. pp. 183-184 in Fujiwara A. 

(ed.) 1982 (q.v.). 

ISHIHARA A. & KATANO M. 1982 Propagation of apple 

cultivars and rootstocks by shoot-tip culture. pp. 733-734 in 

Fujiwara A. (ed.) 1982 (q.v.). 

Chapter 4 163 

JACOBSON L., COOPER B.R. & VOLZ M.G. 1971 The 

interaction of pH and aeration in Cl uptake by barley roots. 


JACQUIOT C. 1951 Action of meso-inositol and of adenine on 

bud formation in the cambium tissue of Ulmus campestris 

cultivated in vitro. Compt. Rend. Acad. Sci. Paris 233, 815-817. 

JAIN R.K., DAVEY M.R. COCKING E.C. & WU R. 1997 

Carbohydrate and osmotic requirements for high-frequency plant 

regeneration from protoplast-derived colonies of indica and 

japonica rice varieties. J. Exp. Bot. 48, 751-758. 

JAY-ALLEMAND C., CAPELLI P. & CORNU D. 1992 Root 

development of in vitro hybrid walnut microcuttings in a 

vermiculite-containing gelrite medium. Sci. Hortic. 51, 335-342. 

JEANNIN G., BRONNER R. & HAHNE G. 1995 Somatic 

embryogenesis and organogenesis induced on the immature 

zygotic embryo of sunflower (Helianthus annus L.) cultivated in 

vitro: Role of Sugar. Plant Cell Rep. 15, 200-204. 

JEFFS R.A. & NORTHCOTE D.H. 1967 The influence of IAA 

and sugar on the pattern of induced differentiation in plant tissue 

culture. J. Cell Sci. 2, 77-88. 

JEONG B.R., FUJIWARA K. & KOZAI T. 1995 Environmental 

control and photoautotrophic micropropagation. Hortic. Rev. 17, 

123-170. 

JOHANSSON L. & ERIKSSON T. 1984 Effects of carbon dioxide 

in anther cultures. Physiol. Plant 60, 26-30. 

JOHNSON J.L. & EMINO E.R. 1979 In vitro propagation of 

Mammillaria elongata. HortScience 14, 605-606. 

JOHRI B.M. & GUHA S. 1963 In vitro development of onion 

plants from flowers. pp. 215-223 in Maheshwari and Ranga 

Swamy (eds.) 1963 (q.v.). 

JOY IV R.W., PATEL K.R. & THORPE T.A. 1988 Ascorbic acid 

enhancement of organogenesis in tobacco. Plant Cell Tissue 

Organ Cult. 13, 219-228. 

JOY IV R.W., PATEL K.R. & THORPE T.A. 1988 Ascorbic acid 

enhancement of organogenesis in tobacco callus. Plant Cell 


JUMIN H.B. 1995 Plant regeneration via somatic embryogenesis 

in Citrus and its relatives. Phytomorphology 45, 1-8. 

JUNG P., TANNER W. & WOLTER K. 1972 The fate of myoinositol 

in Fraximus tissue cultures. Phytochemistry 11, 1655-1659. 

KAHANE R. & RANCILLAC M. 1996 Carbohydrates in onion 

cultured in vitro. Acta Bot. Gall. 143, 117-123 

KANABUS J., BRESSAN R.A. & CARPITA N.C. 1986 Carbon 

assimilation in carrot cells in liquid culture. Plant Physiol. 82, 

363-368. 

KANG K.S., VEEDER G.T., MIRRASOUL P.J., KANEKO T. & 

COTTRELL W. 1982 Agar-like polysaccharide produced by a 

Pseudomonas species: Production and basic properties. Appl. 

Environ. Microbiol. 43, 1086-1091. 

KAO K.N. & MICHAYLUK M.R. 1975 Nutritional requirements 

for growth of Vicia hajastana cells and protoplasts at a very low 

population density in liquid media. Planta 126, 105-110. 

KARSAI I., BEDO Z. & HAYES P.M. 1994 Effect of induction 

medium pH and maltose concentration on in vitro androgenesis 

of hexaploid winter triticale and wheat. Plant Cell Tissue Organ 

Cult. 39, 49-53. 

KARTHA K.K. 1981 Meristem culture and cryopreservation – 

Methods and applications. pp. 181-211 in Thorpe T.A. (ed.) 

Plant Tissue Culture: Methods and Applications in Agriculture. 

Academic Press, New York, London, Toronto, Sydney. 

KAUL B. & STABA E.J. 1968 Dioscorea tissue cultures. 1. 

Biosynthesis and isolation of diesgenin from Dioscorea deltoidea 

callus and suspension cells. Lloydia 31, 171-179. 

KAUL K. & KOCHHAR T.S. 1985 Growth and differentiation of 

callus cultures of Pinus. Plant Cell Rep. 4, 180-183.


KAUL K. & SABHARWAL P.S. 1972 Morphogenetic studies on 

Haworthia: Establishment of tissue culture and control of 

differentiation. Am. J. Bot. 59, 377-385. 

KAUL K. & SABHARWAL P.S. 1975 Morphogenetic studies on 

Haworthia: Effects of inositol on growth and differentiation. Am. 

J. Bot. 62, 655-659. 

KAVI KISHOR P.B. & MEHTA A.R. 1982 Some aspects of 

carbohydrate metabolism in organ-differentiating tobacco and 

non-differentiating cotton callus cultures. pp. 143-144 in 

Fujiwara A. (ed.) 1982 (q.v.). 

KAVI KISHOR P.B. & REDDY G.M. 1986 Regeneration of 

plants from long-term cultures of Oryza sativa L. Plant Cell Rep. 

5, 391-393. 

KETELLAPPER H.J. 1953 The mechanism of the action of indole- 

3-acetic acid on the water absorption by Avena coleoptile 

sections. Acta Bot. Neerl. 2, 387-444. 

KIM K.S., DAVELAAR E. & DE KLERK G.J. (1994) Abscisic 

acid controls dormancy development and bulb formation in lily 

plantlets regenerated in vitro. Physiol. Plant. 90, 59-64. 

KHAN A., CHAUHAN Y.S. & ROBERTS L.W. 1986 In vitro 

studies on xylogenesis in citrus-fruit vesicles. 2. Effect of pH of 

the nutrient medium on the induction of cytodifferentiation. Plant 

Sci. 46, 213-216. 

KIM Y.-H. & JANICK J. 1989b Somatic embryogenesis and 

organogenesis in cucumber. HortScience 24, 702. 

KIMBALL S.L., BEVERSDORF W.D. & BINGHAM E.T. 1975 

Influence of osmotic potential on the growth and development of 

soybean tissue cultures. Crop Sci. 15, 750-752. 

KING P.J. & STREET H.E. 1977 Growth patterns in cell cultures. 

pp. 307-387 in Street H.E. (ed.) Plant Tissue and Cell Culture. 

Bot. Monographs Vol.11, Blackwell Scientific Public-ations. 

Oxford, London. 

KINNERSLEY A.M. & HENDERSON W.E. 1988 Alternative 

carbohydrates promote differentiation of plant cells. Plant Cell 


KINNERSLEY A.M. & HENDERSON W.E. 1988 Alternative 

carbohydrates promote differentiation of plant cells. Plant Cell 


KIRDMANEE C., KITAYA Y. & KOZAI T. 1995. Effects of CO2 

enrichment and supporting material in-vitro on photoautotrophic 

growth of eucalyptus plantlets in-vitro and ex-vitro In Vitro Cell. 

Dev. –Pl. 31, 144-149 

KIRKHAM M.B. & HOLDER P.L. 1981 Water osmotic and turgor 

potentials of kinetin-treated callus. HortScience 16, 306-307. 

KITAMURA Y., IKENAGA T., OOE Y., HIRAOKA N. & 

MIZUKAMI H. 1998 Induction of furanocoumarin biosynthesis 

in Glehnia littoralis cell suspension cultures by elicitor treatment. 

Phytochemistry 48, 113-117. 

KLEIN R.M. & MANOS G.E. 1960 Use of metal chelates for plant 

tissue cultures. Ann. NY. Acad. Sci. 88, 416-425. 

KNUDSON L. 1946 A new nutrient solution for the germination of 

orchid seed. Am. Orchid Soc. Bull. 15, 214-217. 

KOCH K.E. 1996 Carbohydrate-modulated gene expression in 

plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 509-540. 

KOCHBA J. & BUTTON J. 1974 The stimulation of 

embryogenesis and embryoid development in habituated ovular 

callus from the ‘Shamouti’ orange (Citrus sinensis) as affected by 

tissue age and sucrose concentration. Z. Pflanzenphysiol. 73, 

415-421. 

KOCHBA J., SPIEGEL-ROY P., SAAD S. & NEUMANN H. 

1978 Stimulation of embryogenesis in Citrus tissue culture by 

galactose. Naturwissenschaften 65, 261-262. 

KOETJE D.S., GRIMES H.D., WANG Y.-C. & HODGES T.K. 

1989 Regeneration of indica rice (Oryza sativa L.) from primary 

callus from immature embryos. J. Plant Physiol. 13, 184-190. 

KOMOR E., ROTTER M. & TANNER W. 1977 A protoncotransport 

system in a higher plant: sucrose transport in Ricinus 

communis. Plant Sci. Lett. 9, 153-162. 

KOMOR E., THOM M. & MARETZKI A. 1981 The mechanism of 

sugar uptake by sugarcane suspension cells. Planta 153, 181-192. 

KOOP H.U., WEBER G. & SCHWEIGER H.G. 1983 Individual 

culture of selected single cells and protoplasts of higher plants in 

microdroplets of defined media. Z. Pflanzenphysiol. 112, 21-34. 

KORDAN H.A. 1959 Proliferation of excised juice vesicles of 

lemon in vitro. Science 129, 779-780. 

KOTT L.S. & BEVERSDORF W.D. 1990 Enhanced plant 

regeneration from microspore-derived embryos of Brassica 

napus by chilling, partial desiccation and age selection. Plant 


KOZAI T. & SEIKIMOTO K. 1988. Effects of the number of air 

changes per hour of the closed vessel and the photosynthetic 

photon flux on the carbon dioxide concentration inside the vessel 

and the growth of strawberry plantlets in vitro. Environ. Contr. 

Biol. 26, 21-29. 

KOZAI T., FUJIWARA K. & KITAYA Y. 1995 Modelling, 

measurement and control in plant tissue culture. Acta Hortic. 

393, 63-73. 

KOZAI T. 1991a Photoautotrophic micropropagation. In Vitro 

Cell. Dev. – Pl. 27, 47-51. 

KOZAI T. 1991b Micropropagation under photoautotrophic 

conditions. pp. 447-469 in Debergh P.C. and Zimmerman R.H. 

(eds.) Micropropagation. Technology and Application. Kluwer 

Academic Publishers, Dordrecht, Boston, London. ISBN 0-7923- 

0818-2. 

KRIEG N.R. & GERHARDT P. 1981 pp. 143-156 in Gerhardt P. 

Murray R.G.E., Costilow R.N., Nester E.W., Wood W.A., Krieg 

N.R. and Briggs Phillips G. Manual of Methods for General 

Bacteriology. Am. Soc. Microbiol. Washington D.C. 

KROMER K. & KUKULCZANKA K. 1985 In vitro cultures of 

meristem tips of Canna indica L. Acta Hortic. 167, 279-285. 

KUMAR A.S., GAMBORG O.L. & NABORS M.W. 1988 Plant 

regeneration from suspension cultures of Vigna aconitifolia. 

Plant Cell Rep. 7, 138-141. 

KUNITAKE H., IMAMIZO H. & MII M. 1993 Somatic 

embryogenesis and plant regeneration from immature seedderived 

calli of rugosa rose (Rosa rugosa Thumb). Plant Sci. 90, 

187-194. 

KUNITAKE H., NAKASHIMA T., MORI, K. & TANAKA, M. 

1997 Normalization of asparagus somatic embryogenesis using a 

maltose-containing medium. J. Plant Physiol. 150, 458-461. 

KURAISHI S. & OKUMURA F.S. 1961 A new green-leaf growth 

stimulating factor, phyllococosine, from coconut milk. Nature 

189, 148-149. 

KURATA K., IBARAKI Y. & GOTO E. 1991 System for micropropagation 

by nutrient mist supply. Trans. ASAE 34, 621-624. 

KURITAKE H., NAKASHIMA T., MORI K. & TANAKA M. 

1997 Normalization of asparagus somatic embryogenesis using a 

maltose-containing medium. J. Plant Physiol. 150, 458-461. 

KURKDJIAN A., MATHIEU Y. & GUERN J. 1982 Evidence for 

an action of 2,4-dichlorophenoxyacetic acid on the vacuolar pH 

of Acer pseudoplatanus cells in suspension culture. Plant Sci. 

Lett. 27, 77-86. 

LA RUE C.D. 1949 Cultures of the endosperm of maize. Am. J. 

Bot. 36, 798. 

LAMOTTE C.E. 1960 The effects of tyrosine and other amino 

acids on the formation of buds in tobacco callus. Dissertation 

Abstracts 21, 31-32. 

LANDRY L.G. & SMYTH D.A. 1988 Characterization of starch 

produced by suspension cultures of Indica rice (Oryza sativa L.). 

Plant Cell Tissue Organ Cult. 15, 23-32.

LANG A.R.G. 1967 Osmotic coefficients and water potentials of 

sodium chloride solutions from 0 to 40ºC. Austr. J. Chem. 20, 

2017-2023. 

LANGFORD P.J. & WAINWRIGHT H. 1987 Effects of sucrose 

concentration on the photosynthetic ability of rose shoots in vitro. 

Ann. Bot. 60, 633-640. 

LANGHANSOVÁ L., KONRÁDOVÁ H. & VANĔK T. 2004 

Polyethylene glycol and abscisic acid improve maturation and 

regeneration of Panax ginseng somatic embryos. Plant Cell Rep. 

22, 725-730. 

LAPEÑA L., PÉREZ-BERMÚDEZ P. & SEGURA J. 1988 

Morphogenesis in hypocotyl cultures of Digitalis obscura: 

influence of carbohydrate levels and sources. Plant Science 57, 

247-252. 

LARKIN P.J., DAVIES P.A. & TANNER G.J. 1988 Nurse culture 

of low numbers of Medicago and Nicotiana protoplasts using 

calcium alginate beads. Plant Sci. 58, 203-210. 

LARKIN P.J., DAVIES P.A. & TANNER G.J. 1988. Nurse 

cultures of low numbers of Medicago and Nicotiana protoplasts 

using calcium alginate beads. Plant Sci. 58, 203-210. 

LAROSA P.C., HASEGAWA P.M. & BRESSAN R.A. 1981 

Initiation of photoautotrophic potato cells. HortScience 16, 433. 

LASSOCINSKI W. 1985 Chlorophyll-deficient cacti in tissue 

cultures. Acta Hortic. 167, 287-293. 

LAZZERI P.A., HILDEBRAND D.F., SUNEGA J., WILLIAMS 

E.G. & COLLINS G.B. 1988 Soybean somatic embryogenesis: 

interactions between sucrose and auxin. Plant Cell Rep. 7, 517-520. 

LEMOS E.E.P. & BLAKE J. 1996 Micropropagation of juvenile 

and adult Annona squamosa. Plant Cell Tissue Organ Cult. 46, 

77-79. 

LETHAM D.S. 1966 Regulation of cell division in plant tissues. II. 

A cytokinin in plant extracts: isolation and interaction with other 

growth regulators. Phytochemistry 5, 269-286. 

LETHAM D.S. 1968 A new cytokinin bioassay and the naturally 

occurring cytokinin complex. pp. 19-31 in Wightman and 

Setterfield (eds.) Biochemistry and Physiology of Plant Growth 

Substances. Runge Press, Ottawa. 

LETHAM D.S. 1974 Regulators of cell division in plant tissues. 

XX. The cytokinins of coconut milk. Physiol. Plant. 32, 66-70. 

LETHAM D.S. 1982 A compound in coconut milk which actively 

promoted radish cotyledon expansion, but exhibited negligible 

activity in tissue culture bioassays for cytokinins, was identified 

as the 6 oxypurine, 2-(3-methylbut-2-enylamino)-purin-6-one. 

Plant Sci. Lett. 26, 241-249. 

LEVA A.R., BARROSO M. & MURILLO J.M. 1984 La 

moltiplicazione del melo con la tecnica della micropropagazione. 

Vriazione del pH in substrati diversi durante la fase di 

multiplicazione. Riv. Ortoflorofrutti. Ital. 68, 483-492. 

LI X.Y. & HUANG F.H.. 1996 Induction of somatic 

embryogenesis in Loblolly pine (Pinus taeda L.). In Vitro Cell. 

Dev. – Pl. 32, 129-135. 

LI X.Y., HUANG F.H., MURPHY J.B. & GBUR E.R. JR. 1998 

Polyethylene glycol and maltose enhance somatic embryo 

maturation in Loblolly pine (Pinus taeda L.). In Vitro Cell. Dev. 

-Pl. 34, 22-26. 

LICHTER R. 1981 Anther culture of Brassica napus in a liquid 

culture medium. Z. Pflanzenphysiol. 103, 229-237. 

LIMBERG M., CRESS D. & LARK K.G. 1979 Variants of 

soybean cells which can grow in suspension with maltose as a 

carbon-energy source. Plant Physiol. 63, 718-721. 

LIN M.-L. & STABA E.J. 1961 Peppermint and spearmint tissue 

cultures. I. Callus formation and submerged culture. Lloydia 24, 

139-145. 

LINOSSIER L., VEISSEIRE P., CAILLOUX F. & COUDRET A. 

1997 Effects of abscisic acid and high concentrations of PEG on 

Chapter 4 165 

Hevea brasiliensis somatic embryos development. Plant Sci. 124, 

183-191. 

LINSMAIER E.M. & SKOOG F. 1965 Organic growth factor 

requirements of tobacco tissue cultures. Physiol. Plant. 18, 100- 

127. 

LIPAVSKA H. & VREUGDENHIL D. 1996 Uptake of mannitol 

from the media by in vitro grown plants. Plant Cell Tissue Organ 

Cult.45, 103-107. 

LIPPMANN B. & LIPPMANN G. 1984 Induction of somatic 

embryos in cotyledonary tissue of soybean, Glycine max L. Merr. 


LITZ R.E. 1988 Somatic embryogenesis from cultured leaf 

explants of the tropical tree Euphoria longan Stend. J. Plant 

Physiol. 132, 190-193. 

LIU Y.K., SEKI M. & FURUSAKI S. 1999 Plant cell 

immobilization in loofa sponge using two-way bubble circular 

system. J. Chem. Eng. Jpn. 32, 8-14. 

LLOYD D., ROBERTS A.V. & SHORT K.C. 1988 The induction 

in vitro of adventitious shoots in Rosa. Euphytica 37, 31-36. 

LLOYD G. & McCOWN B. 1981 Commercially-feasible 

micropropagation of Mountain laurel, Kalmia latifolia, by use of 

shoot tip culture. Int. Plant Prop. Soc. Proc. 30, 421-427. 

LLOYD G. & McCOWN B. 1981 Commercially-feasible 

micropropagation of Mountain laurel, Kalmia latifolia, by use of 

shoot tip culture. Int. Plant Prop. Soc. Proc. 30, 421-427. 

LOEWUS F.A. & LOEWUS M.W. 1980 Myo-inositol: 

Biosynthesis and metabolism. pp. 43-76 in Stumpf and Conn 

(eds.): The Biochemistry of Plants 3. Academic Press N.Y. 43-76. 

LOEWUS F.A. 1974 The biochemistry of myo-inositol in plants. 

Recent Adv. Phytochem. 8, 179-207. 

LOEWUS F.A., KELLY S. & NEUFELD E.F. 1962 Metabolism 

of myo-inositol in plants: Conversion to pectin hemicellulose, Dxylose 

and sugar acids. Proc. Natl. Acad. Sci. USA 48, 421-425. 

LOU H. & KAKO S. 1995 Role of high sugar concentrations in 

inducing somatic embryogenesis from cucumber cotyledons. Sci. 

Hortic. 64, 11-20. 

LOVELL P.H., ILLSLEY A. & MOORE K.G. 1972 The effects of 

light intensity and sucrose on root formation, photosynthetic 

ability, and senescence in detached cotyledons of Sinapis alba L. 

and Raphanus sativus L. Ann. Bot. 36, 123-134. 

LU C., VASIL I.K. & OZIAS-AKINS P. 1982 Somatic embryogenesis 

in Zea mays. Theor. Appl. Genet. 62, 109-112. 

LUMSDEN P.J., PRYCE S. & LEIFERT C. 1990 Effect of 

mineral nutrition on the growth and multiplication of in vitro 

cultured plants. pp. 108-113 in Nijkamp et al. (eds.) 1990 (q.v.). 

MADHUSUDHAN R., RAS S.R. & RAVISHANKAR G.A. 1995 

Osmolarity as a measure of growth of plant cells in suspension 

cultures. Enz. Microb. Tech. 17, 989-991. 

MAPES M.O. & ZAERR J.B. 1981 The effect of the female 

gametophyte on the growth of cultured Douglas fir embryos. 

Ann. Bot. 48, 577-582. 

MAHESHAWARI P. AND RANGASWAMY N.S. 1963 (eds.). 

Plant Tissue and Organ Culture. Int. Soc. Plant Morphologists, 

Univ. Delhi, India. 

MARETZKI A. & THOM, M. 1978 Characteristics of a galactose-adapted 

sugarcane cell line grown in suspension culture. 


MARETZKI A., DELA-CRUZ A. & NICKELL L.G. 1971 

Extracellular hydrolysis of starch in sugarcane cell suspensions. 


MARETZKI A., THOM A. & NICKELL L.G. 1972 Influence of 

osmotic potentials on the growth and chemical composition of 

sugarcane cell cultures. Hawaii Plant Res. 58, 183-199. 

MARETZKI A., THOM M. & NICKELL L.G. 1974 Utilisation 

and metabolism of carbohydrates in cell and callus cultures. pp.


329-361 in Street H.E. (ed.) Tissue Culture and Plant Science. 

Academic Press, London, New York, San Francisco. 

MARGARA J. & RANCILLAC M. 1966 Observations 

préliminaires sur le rôle du milieu nutritif dans l’initiation florale 

des bourgeons néoformés in vitro chez Cichorium intybus L. 

Compt. Rend. Acad. Sci. Paris 263D, 1455-1458. 

MARSOLAIS A.A., WILSON D.P.M., TSUJITA M.J. & 

SENARATNA T. 1991 Somatic embryogenesis and artificial seed 

production in Zonal (Pelargonium horterum) and Regal 

(Pelargonium domesticum) geranium. Can. J. Bot. 69, 1188-1193. 

MARTIN S.M. & ROSE D. 1976 Growth of plant cell (Ipomoea) 

suspension cultures at controlled pH levels. Can. J. Bot. 54, 

1264-1270. 

MARUYAMA E., ISHII K. & KINOSHITA I. 1998 Alginate 

encapsulation technique and cryogenic procedures for long-term 

storage of the tropical forest tree Guazuma crinita Mart. in vitro 

cultures. Jarq-Jpn. Agr. Res. Q. 32, 301-309. 

MATHES M.C., MORSELLI M. & MARVIN J.W. 1973 Use of 

various carbon sources by isolated maple callus cultures. Plant 

Cell Physiol. 14, 797-801. 

MAUSETH J.D. 1979 Cytokinin-elicited formation of the pith-rib 

meristem and other effects of growth regulators on the 

morphogenesis of Echino cereus (Cactaceae) seedling shoot 

apical meristems. Am. J. Bot. 66, 446-451. 

McCANCE R.A. & WIDDOWSON E.M. 1940 The Chemical 

Composition of Foods. British M.R.C. Spec. Rep. Serv. No. 235. 

MCGREGOR L.J. & MCHUGHEN A. 1990 The influence of 

various cultural factors on anther culture of four cultivars of spring 

wheat (Triticum aestivum L.). Can. J. Plant Sci. 70, 183-192. 

MEHTA A.R. 1982 Comparative studies on some physiological 

aspects in organ forming (diploid as well as haploid) tobacco and 

non-organ forming cotton callus tissues. pp. 125-126 in Fujiwara 

A. (ed.) 1982 (q.v.). 

MELLOR F.C. & STACE-SMITH R. 1969 Development of excised 

potato buds in nutrient medium. Can. J. Bot. 47, 1617-1621. 

MENON M.K.C. & LAL M. 1972 Influence of sucrose on the 

differentiation of cells with zygote-like potentialities in a moss. 

Naturwissenschaften 59, 514. 

MERKLE S.A., PARROTT W.A. & FLINN B.S. 1995 Morphogenetic 

aspects of somatic embryogenesis. pp. 155-205 in Thorpe 

T.A. (ed.) In Vitro Embryogenesis in Plants. Kluwer Acad. Publ., 

Dordrecht. 

MERYL SMITH M. & STONE B.A. 1973 Studies on Lolium 

multiflorum endosperm in tissue culture. 1. Nutrition. Aust. J. 

Biol. Sci. 26, 123-133. 

MILLER C.O. 1961 A kinetin-like compound in maize. Proc. Natl. 

Acad. Sci. USA 47, 170-174. 

MILLER J.H. 1968 Fern gametophytes as experimental material. 

Bot. Rev. 34, 361-426. 

MINOCHA S.C. & HALPERIN W. 1974 Hormones and 

metabolites which control tracheid differentiation with or without 

concomitant effects on growth in cultured tuber tissue of 

Helianthus tuberosus L. Planta 116, 319-331. 

MITCHELL E.D., JOHNSON B.B. & WHITTLE T. 1980 βgalactosidase 

activity in cultured cotton cells (Gossypium 

hirsutum L.). A comparison between cells growing on sucrose 

and lactose. In Vitro 16, 907-912. 

MOLNAR S.J. 1988 Nutrient modifications for improved growth 

of Brassica nigra cell suspension cultures. Plant Cell Tissue 

Organ Cult. 15, 257-267. 

MONDAL H., MANDAL R.K. & BISWAS B.B. 1972 RNA 

polymerase from eukaryotic cells. Isolation and purification of 

enzymes and factors from chromatin of coconut nuclei. Eur. J. 

Biochem. 25, 463-470. 

MONNIER M. 1976 Culture in vitro de l’embryon immature de 

Capsella bursa-pastoris Moench (L.). Rev. Cytol. Biol. Vég. 39, 

1-120. 

MONNIER M. 1978 Culture of zygotic embryos. pp. 277-286 in 

Thorpe T. A. (ed.) 1978 Frontiers of Plant Tissue Culture. Int. 

Assoc. for Plant Tissue Culture. Distrib. by The Bookstore, 

Univ. Calgary, Alberta, T2N 1N4, Canada. 

MOREL G. & MULLER J.-F. 1964 In vitro culture of the apical 

meristem of the potato. Compt. Rend. Acad. Sci., Paris 258, 

5250-5252. 

MOREL G. & WETMORE R.H. 1951 Tissue culture of 

monocotyledons. Am. J. Bot. 38, 138-140. 

MOREL G. 1946 Action de l’acid pantothénique sur la croissance 

de tissus d’Aubépine cultivés in vitro. Compt. Rend. Acad. Sci., 

Paris 223, 166-168. 

MORRIS D.A. 2000 Transmembrane auxin carrier systems – 

Dynamic regulators of polar auxin transport. Plant Growth Reg. 

32, 161-172. 

MÜLLER J.F., MISSIONIER C. & CABOCHE M. 1983 Low 

density growth of cells derived from Nicotiana and Petunia 

protoplasts: Influence of the source of protoplasts and 

comparison of the growth- promoting activity of various auxins. 


MURASHIGE T. & NAKANO R.T. 1968 The light requirement 

for shoot initiation in tobacco callus culture. Am. J. Bot. 55, 710. 

MURASHIGE T. & SKOOG F. 1962 A revised medium for rapid 

growth and bio-assays with tobacco tissue cultures. Physiol. 


MURASHIGE T. & TUCKER D.P.H. 1969 Growth factor 

requirements of citrus tissue culture. pp. 1155-1161 in Chapman 

H. D. (ed.) Proc. 1st Int. Citrus Symp. Vol. 3, Univ. Calif., 

Riverside Publication. 

MURASHIGE T. & TUCKER D.P.H. 1969 Growth factor 

requirements of citrus tissue culture. pp. 1155-1161 in Chapman 

H. D. (ed.) Proc. 1st Int. Citrus Symp. Vol. 3, Univ. Calif., 

Riverside Publication. 

MURASHIGE T. 1974 Plant propagation through tissue cultures. 

Annu. Rev. Plant Physiol. 25, 135-166. 

MURASHIGE T., SHABDE M.N., HASEGAWA P.N., 

TAKATORI F.H. & JONES J.B. 1972 Propagation of asparagus 

through shoot apex culture. I. Nutrient media for formation of 

plantlets. J. Am. Soc. Hort. Sci. 97, 158-161. 

MURASHIGE T., SERPA M. & JONES J.B. 1974 Clonal 

multiplication of Gerbera through tissue culture. HortScience 9, 

175-180. 

MUTAFTSCHIEV S., COUSSON A. & TRAN THANH VAN K. 

1987 Modulation of cell growth and differentiation by pH and 

oligosaccharides. pp. 29-42 in Jackson M.B., Mantell S.H. & 

Blake J. (eds.) Advances in the Chemical Manipulation of Plant 

Tissue Cultures. Monograph 16, British Plant Growth Regulator 

Group, Bristol. 

NAIRN B.J. 1988 Significance of gelling agents in a production 

tissue culture laboratory. Comb. Proc. Int. Plant Prop. Soc. 1987 

37, 200-205. 

NARAYANASWAMI S. & LARUE C.D. 1955 The morphogenic 

effects of various physical factors on the gemmae of Lunaria. 

Phytomorph. 5, 99-109. 

NEAL C.A. & TOPOLESKI L.D. 1983 Effects of the basal 

medium on growth of immature tomato embryos in vitro. J. Am. 

Soc. Hortic. Sci. 108, 434-438. 

NEGASH A., KRENS F., SCHAART J. & VISSER B. 2001 In 

vitro conservation of enset under slow-growth conditions. Plant 


NERNST W. 1904 Theorie der Reaktiongeschwindigkeit in heterogenen 

Systemen. Phys. Chem. 47, 52-55.

NESIUS K.K. & FLETCHER J.S. 1973 Carbon dioxide and pH 

requirement of non-photosynthetic tissue culture cells. Physiol. 


NICHOL J.W., SLADE D., VISS P. & STUARD D.A. 1991 Effect 

of organic acid pretreatment on the regeneration and 

development (conversion) of whole plants from callus cultures of 

alfalfa, Medicago sativa L. Plant Sci. 79, 181-192. 

NICHOL J.W., SLADE D., VISS P. & STUART D.A. 1991 

Effect of organic acid pretreatment on the regeneration and 

development (conversion) of whole plants from callus cultures of 

alfalfa, Medicago sativa L. Plant Sci. 79, 181-192. 

NICKELL L.G. & BURKHOLDER P.R. 1950 Atypical growth of 

plants. II. Growth in vitro of virus tumors of Rumex in relation to 

temperature, pH and various sources of nitrogen, carbon and 

sulfur. Am. J. Bot. 37, 538-547. 

NICKELL L.G. & BURKHOLDER P.R. 1950 Atypical growth of 

plants. II. Growth in vitro of virus tumors of Rumex in relation to 

temperature, pH and various sources of nitrogen, carbon and 

sulfur. Am. J. Bot. 37, 538-547. 

NICKELL L.G. & MARETZKI A. 1969 Growth of suspension 

cultures of sugarcane cells in chemically defined media. Physiol. 


NICKELL L.G. & MARETZKI A. 1970 The utilization of sugars 

and starch as carbon sources by sugarcane cell suspension 

cultures. Plant Cell Physiol. 11, 183-185. 

NIJKAMP H.I.J., VAN DER PLAS I.H.W. & VAN AARTRIJK J. 

(eds.) Progress in Plant Cellular and Molecular Biology. Proc. 

VIIth Int. Cong. on Plant Tissue and Cell Culture. Amsterdam, 

The Netherlands. 24-29 June 1990. Kluwer Academic Publishers, 

Dortrecht, Netherlands. 

NITSCH J.P. & NITSCH C. 1956 Auxin-dependent growth of 

excised Helianthus tissues. Am. J. Bot. 43, 839-851. 

NITSCH J.P. & NITSCH C. 1965 Néoformation de fleurs in vitro 

chez une espèce de jours courts: Plumbaga indica L. Ann. Phys. 

Vég. 7, 251-256. 

NITZSCHE W. & WENZEL G. 1977 Haploids in Plant Breeding. 

Suppl. 8 to Z. Pflanzenzucht. Parey. Berlin and Hamburg. 

NOH E.W., MINOCHA S.C. & RIEMENSCHNEIDER D.E. 1988 

Adventitious shoot formation from embryonic explants of red 

pine (Pinus resinosa). Physiol. Plant. 74, 119-124. 

NORGAARD J.V. 1997 Somatic embryo maturation and plant regeneration 

in Abies nordmanniana LK. Plant Sci. 124, 211-221. 

NORSTOG K. & SMITH J. 1963 Culture of small barley embryos 

on defined media. Science 142, 1655-1656. 

NORSTOG K. & SMITH J. 1963 Culture of small barley embryos 

on defined media. Science 142, 1655-1656. 

NORSTOG K. 1965b Induction of apogamy in megagametophytes 

of Zamia integrifolia. Am. J. Bot. 52, 993-999. 

NORSTOG K. 1973 New synthetic medium for the culture of 

premature barley embryos. In Vitro 8, 307-308. 

OBATA-SASAMOTO H. & THORPE T.A. 1983 Cell wall 

invertase activity in cultured tobacco tissues. Plant Cell Tissue 

Organ Cult. 2, 3-9. 

OBATA-SASAMOTO H., BEAUDOIN-EAGAN L. & THORPE 

T.A. 1984 C-metabolism during callus induction in tobacco. 


OJIMA K. & OHIRA K. 1980 The exudation and characterization 

of iron-solubilizing compounds in a rice cell suspension culture. 

Plant Cell Physiol. 21, 1151-1161. 

OKA S. & OHYAMA K. 1982 Sugar utilization in mulberry 

(Morus alba L.) bud culture. pp. 67-68 in Fujiwara A. (ed.) 1982 

(q.v.). 

OWEN H.R., WENGERD D. & MILLER A.R. 1991 Culture 

medium pH influenced by basal medium, carbohydrate source, 

gelling agent, activated charcoal, and medium storage method. 


Chapter 4 167 

OWENS L.D. & WOZNIAK C.A. 1991 Measurement and effects 

of gel matric potential and expressibility on production of 

morphogenic callus by cultured sugarbeet leaf discs. Plant Cell 


PAMPLIN E.J. & CHAPMAN J.M. 1975 Sucrose suppression of 

chlorophyll synthesis in tissue cultures. J. Exp. Bot. 26, 212-220. 

PARFITT D.E., ALMEHDI A.A. & BLOKSBERG L.N. 1988 Use 

of organic buffers in plant tissue-culture systems. Sci. Hortic. 36, 

157-163. 

PARIS D. & DUHAMET L. 1953 Action d’un mélange d’acides 

aminés et vitamines sur la proliferation des cultures de croun-gall 

de Scorsonère; comparison avec l’action du lait de coco. Compt. 

Rend. Acad. Sci. Paris 236, 1690-1692. 

PARR D.R. & EDELMAN J. 1975 Release of hydrolytic 

enzymes from the cell walls of intact and disrupted carrot callus 

tissue. Planta 127, 111-119. 

PASQUA G., MANES F., MONACELLI B., NATALE L. & 

ANSELMI S. 2002 Effects of the culture medium pH and ion 

uptake in in vitro vegetative organogenesis in thin cell layers of 

tobacco. Plant Sci. 162, 947-955. 

PASQUALETTO P.-L., WERGIN W.P. & ZIMMERMAN R.H. 

1988 Changes in structure and elemental composition of vitrified 

leaves of `Gala’ apple in vitro. Acta Hortic. 227, 352-357. 

PASQUALETTO P.-L., ZIMMERMAN R.H. & FORDHAM I. 

1986a Gelling agent and growth regulator effects on shoot 

vitrification of `Gala’ apple in vitro. J. Am. Soc. Hortic. Sci. 111, 

976-980 & 112, 407. 

PASQUALETTO P.-L., ZIMMERMAN R.H. & FORDHAM I. 

1988b The influence of cation and gelling agent concentrations 

on vitrification of apple cultivars in vitro. Plant Cell Tissue 

Organ Cult. 14, 31-40. 

PATEL K.R. & BERLYN G.P. 1983 Cytochemical investigations 

on multiple bud formation in tissue cultures of Pinus coulteri. 

Can. J. Bot. 61, 575-585. 

PECH J.C. & ROMANI R.J. 1979 Senescence of pear fruit cells 

cultured in a continuously renewed auxin-deprived medium. 


PHILLIPS D.V. & SMITH A.E. 1974 Soluble carbohydrates in 

soybean. Can. J. Bot. 52, 2447-2452. 

PHILLIPS G.C. & COLLINS G.B. 1979 In vitro tissue culture of 

selected legumes and plant regeneration from callus cultures of 

red clover. Crop Sci. 19, 59-64. 

PHILLIPS G.C. & COLLINS G.B. 1984 Chapter 7 – Red Clover 

and other forage legumes. pp. 169-210 in Sharp W.R., Evans 

D.A., Ammirato P.V. & Yamada Y. (eds.) Handbook of Plant 

Cell Culture. Vol. 2. Crop Species. Macmillan Publishing Co., 

New York, London. 

PHILLIPS G.C. & HUBSTENBERGER J.F. 1985 Organogenesis in 

pepper tissue cultures. Plant Cell Tissue Organ Cult. 4, 261-269. 

PICCIONI E. & STANDARDI A. 1995 Encapsulation of micropropagated 

buds of six woody species. Plant Cell Tissue Organ 

Cult. 42, 221-226. 

PICCIONI E. 1997 Plantlets from encapsulated micropropagated 

buds of M.26 apple rootstock. Plant Cell Tissue Organ Cult. 47, 

255-260. 

PIERIK R.L.M. 1988 In vitro culture of higher plants as a tool in 

the propagation of horticultural crops. Acta Hortic. 226, 25-40. 

PIERIK R.L.M., SPRENKELS P.A., VAN DER HARST B. & 

VAN DER MEYS Q.G. 1988 Seed germination and further 

development of plantlets of Paphiopedilum ciliolare Pfitz. in 

vitro. Sci. Hortic. 34, 139-153. 

PLIEGO-ALFARO F. 1988 Development of an in-vitro rooting 

bioassay using juvenile-stage stem cuttings of Persea americana 

Mill. J. Hort. Sci. 63,295-301.


POLIKARPOCHKINA R.T., GAMBURG K.Z. & KHAVIN E.E. 

1979 Cell-suspension culture of maize (Zea mays L.). Z. 


POLLARD J.K., SHANTZ E.M. & STEWARD F.C. 1961 

Hexitols in coconut milk: their role in the nurture of dividing 

cells. Plant Physiol. 36, 492-501. 

PREECE J.E. & SUTTER E.G. 1991 Acclimatization of micropropagated 

plants to the greenhouse and field. pp. 71-93 in 

Debergh P.C. and Zimmerman R.H. (eds.) Micropropagation. 

Technology and Application. Kluwer Academic Publishers, 

Dordrecht, Boston, London. ISBN 0-7923-0818-2. 

PREIL W. 1991 Application of bioreactors in plant propagation. 

pp. 425-445 in Debergh P.C. and Zimmerman R.H. (eds.) Micropropagation. 

Technology and Application. Kluwer Academic 

Publishers, Dordrecht, Boston, London. ISBN 0-7923-0818-2. 

PRETOVA A. & WILLIAMS E.G. 1986 Direct somatic 

embryogenesis from immature zygotic embryos of flax (Linum 

usitatissimum L.). J. Plant Physiol. 126, 155-161. 

PUA E.C. & CHONG C. 1984 Requirement for sorbitol (Dglucitol) 

as carbon source for in vitro propagation of Malus 

robusta No. 5. Can. J.Bot. 62, 1545-1549. 

PUA E.-C., RAGOLSKY E., CHANDLER S.F. & THORPE T.A. 

1985a Effect of sodium sulfate on in vitro organogenesis of 

tobacco callus. Plant Cell Tissue Organ Cult. 5, 55-62. 

PUA E.-C., RAGOLSKY E. & THORPE T.A. 1985b Retention of 

shoot regeneration capacity of tobacco callus by Na2SO4. Plant 

Cell Rep. 4, 225-228. 

RABE E. 1990 Stress physiology: the functional significance of the 

accumulation of nitrogen-containing compounds. J. Hortic. Sci. 

65, 231-243 

RADLEY M. & DEAR E. 1958 Occurrence of gibberellin-like 

substances in coconut. Nature 182, 1098. 

RAGHAVAN V. 1977 Diets and culture media for plant embryos. 

pp. 361-413 in Recheigl M. Jr. (ed.) 1977 CRC Handbook Series 

in Nutrition & Food Vol. 4. CRC Press, Baton Rouge, Florida. 

RAHMAN M.A. & BLAKE J. 1988 The effects of medium 

composition and culture conditions on in vitro rooting and ex 

vitro establishment of jackfruit (Artocarpas heterophyllus Lam.). 


RAINS D.W. 1989 Plant tissue and protoplast culture: application 

to stress physiology and biochemistry. In: Jones H.G., Flowers 

T.J. & Jones M.B. (eds) Plants under stress. Cambridge 

University Press, London, pp 181-196. 

RAINS D.W., VALENTINE R.C. & HOLLAENDER A. (eds.) 

1980 Genetic Engineering of Osmoregulation. Plenum Press, 

New York, London. 

RAMAGE C.M. & WILLIAMS R.R. 2002 Inorganic nitrogen 

requirements during shoot organogenesis in tobacco leaf discs. J. 

Exp. Bot 53, 1437-1443. 

RAMMING D.W. 1990 The use of embryo culture in fruit 

breeding. HortScience 25, 393-398. 

RANGA SWAMY N.S. 1963 Studies on culturing seeds of 

Orobanche aegyptiaca. pp. 345-354 in Maheshwari and Ranga 

Swamy (eds.) 1963 (q.v.). 

RANGAN T.S. 1984 Clonal propagation. pp. 68-73 in Vasil I.K. 

(ed.) Cell Culture and Somatic Cell Genetics of Plants Vol. 1. 

Acad. Press, New York. 

RANGAN T.S. 1974 Morphogenic investigations on tissue 

cultures of Panicum miliaceum. Z. Pflanzenphysiol. 72, 456-459. 

RANGAN T.S., MURASHIGE T. & BITTERS W.P. 1968 In 

vitro initiation of nucellar embryos in monoembryonic Citrus. 

HortScience 3, 226-227. 

RAQUIN C. 1983 Utilization of different sugars as carbon sources 

for in vitro anther culture of petunia. Z. Pflanzenphysiol. 111, 

Suppl., 453-457. 

RAVEN J.A. & SMITH F.A. 1976 Cytoplasmic pH regulation and 

electrogenic H + extrusion. Curr. Adv. Plant Sci. 8, 649-660. 

RAVEN J.A. 1986 Biochemical disposal of excess H + in growing 

plants? New Phytol. 104, 175-206. 

RAWAL S.K. & MEHTA A.R. 1982 Changes in enzyme activity 

and isoperoxidases in haploid tobacco callus during 

organogenesis. Plant Sci. Lett. 24, 67-77. 

REDENBAUGH K., VISS P., SLADE D. & FUJII J.A. 1987 

Scale-up: artificial seeds. pp. 473-493 in Green C.E, Sommers 

D.A., Hackett W.P. and Biesboer D.D. (eds.) Plant Tissue and 

Cell Culture. Alan R. Liss, Inc., New York 

REIDIBOYM-TALLEUX L., DIEMER F., SOURDIOUX M., 

CHAPELAIN K. & DE-MARCH G.G. 1999 Improvement of 

somatic embryogenesis in wild cherry (Prunus avium), effect of 

maltose and ABA supplements. Plant Cell Tissue Organ Cult. 

55, 199-209. 

REPUNTE V.P., TAYA M. & TONE S. 1995 Preparation of 

artificial seeds using cell aggregates from horseradish hairy roots 

encapsulated in alginate gel with paraffin coat. J. Ferment. 

Bioeng. 79, 83-86. 

RIER J.P. & BESLOW D.T. 1967 Sucrose concentration and the 

differentiation of xylem in callus. Bot. Gaz. 128, 73-77. 

RIER J.P. & CHEN P.K. 1964 Pigment induction in plant tissue 

cultures. Plant Physiol. 39, Suppl. 

RIERA M., VALON C., FENZI F., GIRAUDAT J. & LEUNG J. 

2005 The genetics of adaptive responses to drought stress: 

abscisic acid-dependent and abscisic acid-independent signalling 

components. Physiol. Plant. 123, 111-119. 

ROBBINS W.J. & BARTLEY M.A. 1937 Vitamin B, and the 

growth of excised tomato roots. Science 85, 246-247. 

ROBBINS W.J. & SCHMIDT M.B. 1939a Vitamin B6, a growth 

substance for isolated tomato roots. Proc. Nat. Acad. Sci. Wash. 

25, 1-3. 

ROBBINS W.J. & SCHMIDT M.B. 1939b Further experiments on 

excised tomato roots. Am. J. Bot. 26, 149-159. 

ROBBINS W.J. 1922 Cultivation of excised root tips and stem tips 

under sterile conditions. Bot. Gaz. 73, 376-390. 

ROBERTS A.V. & SMITH E.F. 1990 The preparation in vitro of 

chrysanthemum for transplantation to soil. (1). Protection of roots 

by cellulose plugs. Plant Cell Tissue Organ Cult. 21, 129-32. 

ROBERTS D.R., SUTTON B.C.S. & FLINN B.S. 1990 

Synchronous and high frequency germination of interior spruce 

somatic embryos following partial drying at high relative 

humidity. Can. J. Bot. 68, 1086-1090. 

ROBERTS-OEHLSCHLAGER, S.L. & DUNWELL J.M. 1990 

Barley anther culture – pretreatment on mannitol stimulates 

production of microspore-derived embryos. Plant Cell Tissue 

Organ Cult. 20, 235-240. 

RODRIGUEZ G. & LORENZO MARTIN J.R. 1987 In vitro 

propagation of Canary Island banana (Musa acuminata Colla 

AAA var. Dwarf Cavendish). Studies of factors affecting culture 

obtention, preservation and conformity of the plants. Acta Hortic. 

212, 577-583. 

ROEST S. & BOKELMANN G.S. 1975 Vegetative propagation of 

Chrysanthemum morifolium Ram. in vitro. Sci. Hortic. 3, 317-330. 

ROMBERGER J.A. & TABOR C.A. 1971 The Picea abies shoot 

apical meristem in culture. I. Agar and autoclaving effects. Am. 

J. Bot. 58, 131-140. 

ROSE D. & MARTIN S.M. 1975 Effect of ammonium on growth 

of plant cells (Ipomoea sp.) in suspension cultures. Can. J. Bot. 

53, 1942-1949. 

ROSNER A., GRESSEL J. & JAKOB K.M. 1977 Discoordination 

of ribosomal RNA metabolism during metabolic shifts of 

Spirodela plants. Biochim. Biophys. Acta 474, 386-397. 

RUBERY P.H. 1980 The mechanism of transmembrane auxin 

transport and its relation to the chemiosmotic hypothesis of the

polar transport of auxin. pp. 50-60 in Skoog F. (ed.) 1980 Plant 

Growth Substances. Proc. 10 th Int. Cong. Plant Growth 

Substances, Madison, Wisconsin 1979. Springer Verlag, Berlin, 

Heidelberg. 

RUGINI E., TARINI P. & ROSSODIVITA M.E. 1987 Control of 

shoot vitrification of almond and olive grown in vitro. Acta 

Hortic. 212, 177-183. 

RUSSELL J.A. & McCOWN B.H. 1986 Culture and regeneration 

of Populus leaf protoplasts isolated from non-seedling tissue. 

Plant Sci. 46, 133-142. 

SAALBACH G. & KOBLITZ H. 1978 Attempts to initiate callus 

formation from barley leaves. Plant Sci. Lett. 13, 165-169. 

SACHAR R.C. & IYER R.D. 1959 Effect of auxin, kinetin and 

gibberellin on the placental tissue of Opuntia dillenii Haw. 

cultured in vitro. Phytomorph. 9, 1-3. 

SADASIVAN V. 1951 The phosphatases in coconut (Cocos 

nucifera). Arch. Biochem. 30, 159-164. 

SAGARE A.P., LEE Y.L., LIN T.C., CHEN C.C. & TSAY H.S. 

2000 Cytokinin-induced somatic embryogenesis and plant 

regeneration in Corydalis yanhusuo (Fumariaceae) -a medicinal 

plant. Plant Sci. 160,139-147. 

SAGAWA Y. & KUNISAKI J.T. 1982 Clonal propagation of 

orchids by tissue culture. pp. 683-684 in Fujiwara A. (ed.) 1982 

(q.v.). 

SAVAGE A.D., KING J. & GAMBORG O.L. 1979 Recovery of a 

pantothenate auxotroph from a cell suspension culture of Datura 

innoxia Mill. Plant Sci. Lett. 16, 367-376. 

SCHENK R.U. & HILDEBRANDT A.C. 1972 Medium and 

techniques for induction and growth of monocotyledonous and 

dicotyledonous plant cell cultures. Can. J. Bot. 50, 199-204. 

SCHERER P.A. 1988 Standardization of plant micropropagation 

by usage of a liquid medium with polyurethane foam plugs or a 

solidified medium with the gellan gum gelrite instead of agar. 

Acta Hortic. 226, 107-114. 

SCHERER P.A., MULLER E., LIPPERT H. & WOLFF G. 1988 

Multielement analysis of agar and gelrite impurities investigated 

by inductively coupled plasma emission spectrometry as well as 

physical properties of tissue culture media prepared with agar or 

gellan gum gelrite. Acta Hortic. 226, 655-658. 

SCHOLTEN H.J. & PIERIK R.L.M. 1998 Agar as a gelling agent: 

differential biological effects in vitro. Sci. Hortic. 77, 109-116. 

SCHUBERT S. & MATZKE H. 1985 Influence of phytohormones 

and other effectors on proton extrusion by isolated protoplasts 

from rape leaves. Physiol. Plant. 64, 285-289. 

SCHULLER A. & REUTHER G. 1993 Response of Abies alba 

embryonal-suspensor mass to various carbohydrate treatments. 


SCOTT J. & BREEN P. 1988 Sugar uptake by strawberry fruit 

discs and protoplasts. HortScience 23, 756. 

SEARS R.G. & DECKARD E.L. 1982 Tissue culture variability 

in wheat: callus induction and plant regeneration. Crop Sci. 22, 

546-550. 

SENARATNA T., MCKERSEE B.D. & BOWLEY S.R. 1989a 

Desiccation tolerance of alfalfa (Medicago sativa L.) somatic 

embryos. Influence of abscisic acid, stress pretreatments and 

drying rates. Plant Sci. 65, 253-259. 

SENARATNA T., McKERSIE B. & ECCLESTONE S. 1989b 

Germination of desiccated somatic embryos of alfalfa (Medicago 

sativa L.). Plant Physiol. 89, Suppl., 135. 

SERP D., CANTANA E., HEINZEN C., VON STOCKAR U. & 

MARISON I.W. 2000 Characterization of an encapsulation 

device for the production of monodisperse alginate beads for cell 

immobilization. Biotechnol. Bioeng. 70, 41-53. 

SERRES R.A. 1988 Effects of sucrose and basal medium 

concentration on in vitro rooting of American chestnut. 

HortScience 23, 739. 

Chapter 4 169 

SHANTZ E.M. & STEWARD F.C. 1952 Coconut milk factor: the 

growth-promoting substances in coconut milk. J. Am. Chem. 

Soc. 74, 6133. 

SHANTZ E.M. & STEWARD F.C. 1955 The identification of 

compound A from coconut milk as 1,3-diphenylurea. J. Am. 

Chem. Soc. 77, 6351-6353. 

SHANTZ E.M. & STEWARD F.C. 1956 The general nature of 

some nitrogen free growth-promoting substances from Aesculus 

and Cocos. Plant Physiol. 30, Suppl., XXXV. 

SHANTZ E.M. & STEWARD F.C. 1964 Growth-promoting 

substances from the environment of the embryo. II. The growthstimulating 

complexes of coconut milk, corn and Aesculus. pp. 

59-75 in Actes du Coll. Int. C.N.R.S. No. 123. 

SHARMA A.K., PRASAD R.N. & CHATURVEDI H.C. 1981 

Clonal propagation of Bougainvillea glabra `Magnifica’ through 

shoot apex culture. Plant Cell Tissue Organ Cult. 1, 33-38. 

SHEAT D.E.G., FLETCHER B.H. & STREET H.E. 1959 Studies 

on the growth of excised roots. VIII. The growth of excised 

tomato roots supplied with various inorganic sources of nitrogen. 

New Phytol. 58, 128-141. 

SHEPARD J.F. & TOTTEN R.E. 1977 Mesophyll cell protoplasts 

of potato: isolation, proliferation and plant regeneration. Plant 

Physiol. 60, 313-316. 

SHILLITO R.D., PASZKOWSKI J. & POTRYKUS I. 1983 

Agarose plating and a bead type culture technique enable and 

stimulate development of protoplast-derived colonies in a number 

of plant species. Plant Cell Rep. 2, 244-247. 

SHININGER T.L. 1979 The control of vascular development. 

Annu. Rev. Plant Physiol. 30, 313-337. 

SHOLTO-DOUGLAS J. 1976 Advanced Guide to Hydroponics. 

Pelham Books, London. ISBN 0-7207-08303. 

SHORT K.C., WARBURTON J. & ROBERT A.V. 1987 In vitro 

hardening of cultured cauliflower and chrysanthemum plantlets 

to humidity. Acta Hortic. 212, 329-334. 

SHVETSOV S.G. & GAMBURG K.Z. 1981 Uptake of 2,4-D by 

corn cells in a suspension culture. Dokl. Akad. Nauk S.S.S.R. 

257, 765-768. 

SIEVERT R.C. & HILDEBRANDT A.C. 1965 Variations within 

single cell clones of tobacco tissue cultures. Am. J. Bot. 52, 

742-750. 

SINGHA S. 1982 Influence of agar concentration on in vitro shoot 

proliferation of Malus sp. `Almey’ and Pyrus communis `Seckel’. 

J. Am. Soc. Hortic. Sci. 107, 657-660. 

SKINNER J.C. & STREET H.E. 1954 Studies on the growth of 

excised roots. II. Observations on the growth of excised 

groundsel roots. New Phytol. 53, 44-67. 

SKIRVIN R.M. 1981 Fruit crops. pp. 51-139 in Conger B.V. (ed.) 

Cloning Agricultural Plants via In Vitro Techniques. CRC Press, 

Inc., Boca Raton, Florida. 

SKIRVIN R.M. & CHU M.C 1979 In vitro propagation of 

‘Forever Yours’ rose (Rosa hybrida) HortScience 14, 608-610. 

SKIRVIN R.M., CHU M.C., MANN M.L., YOUNG H., 

SULLIVAN J. & FERMANIAN T. 1986 Stability of tissue 

culture medium pH as a function of autoclaving, time, and 

cultured plant material. Plant Cell Rep. 5, 292-294. 

SMITH C.W. 1967 A study of the growth of excised embryo shoot 

apices of wheat in vitro. Ann. Bot. 31, 593-605. 

SMITH D.L. & KRIKORIAN A.D. 1989 Release of somatic 

embryogenic potential from excised zygotic embryos of carrot 

and maintenance of polyembryonic cultures in hormone-free 

medium. Am. J. Bot. 76, 1832-1843. 

SMITH D.L. & KRIKORIAN A.D. 1990a Somatic proembryo 

production from excised wounded zygotic carrot embryos on 

hormone-free medium; evaluation of the effects of pH, ethylene 

and activated charcoal. Plant Cell Rep. 9, 34-37.


SMITH D.L. & KRIKORIAN A.D. 1990b pH control of carrot 

somatic embryogenesis. pp. 449-453 in Nijkamp et al. (eds.) 

1990 (q.v.). 

SMITH E.F., ROBERTS A.V. & MOTTLEY J. 1990a The 

preparation in vitro of chrysanthemum for transplantation to soil. 

(2) Improved resistance to desiccation conferred by 

paclobutrazol. Plant Cell Tissue Organ Cult., 21, 133-40. 

SMITH E.F., ROBERTS A.V. & MOTTLEY J. 1990b The 

preparation in vitro of chrysanthemum for transplantation to soil. 

(3) Improved resistance to desiccation conferred by reduced 

humidity. Plant Cell Tissue Organ Cult. , 21, 141-5. 

SMITH F.A. & RAVEN J.A. 1979 Intercellular pH and its 

regulation. Annu. Rev. Plant Physiol. 30, 289-311. 

SMITH M.M. & STONE B.A. 1973 Studies on Lolium multiflorum 

endosperm in tissue culture. I. Nutrition. Aust. J. Biol. 26, 123-133. 

SOCZEK U. & HEMPEL M. 1988 The influence of some organic 

medium compounds on multiplication of gerbera in vitro. Acta 

Hortic. 226, 643-646. 

SOPORY S.K., JACOBSEN E. & WENZEL G. 1978 Production 

of monohaploid embryoids and plantlets in cultured anthers of 

Solanum tuberosum. Plant Sci. Lett. 12, 47-54. 

SORVARI S. 1986a The effect of starch gelatinized nutrient media 

in barley anther cultures. Ann. Agric. Fenn. 25, 127-133. 

SORVARI S. 1986b Differentiation of potato discs in barley starch 

gelatinized nutrient media. Ann. Agric. Fenn. 25, 135-138. 

SORVARI S. 1986c Comparison of anther cultures of barley 

cultivars in barley-starch and agar gelatinized media. Ann. Agric. 

Fenn. 25, 249-254. 

SPANGENBERG G., KOOP H.-U., LICHTER R. & SCHWEI- 

GER H.G. 1986 Microculture of single protoplasts of Brassica 

napus. Physiol. Plant. 66, 1-8. 

STAFFORD A. & DAVIES D.R. 1979 The culture of immature 

pea embryos. Ann. Bot. 44, 315-321. 

STASOLLA C. & YEUNG E.C. 1999 Ascorbic acid improves 

conversion of white spruce somatic embryos. In Vitro Cell. 

Dev.-Pl. 35, 316-319. 

STASOLLA C., KONG L., YEUNG E.C. & THORPE T.A. 2003 

Maturation of somatic embryos in conifers: morphogenesis, 

physiology, biochemistry and molecular biology. In Vitro Cell. 

Dev. – Pl. 38, 93-105. 

STAUDT G. 1984 The effect of myo-inositol on the growth of 

callus tissue in Vitis. J. Plant Physiol. 116, 161-166. 

STEFFEN J.D., SACHS R.M. & HACKETT W.P. 1988 Growth 

and development of reproductive and vegetative tissues of 

Bougainvillea cultured in vitro as a function of carbohydrate. 

Am. J. Bot. 75, 1219-1224. 

STEHSEL M.L. & CAPLIN S.M. 1969 Sugars: autoclaving versus 

sterile filtration and the growth of carrot root tissue. Life Sci. 8, 

1255-1259. 

STEINER S. & LESTER R.L. 1972 Studies on the diversity of 

inositol-containing yeast phospholipids: incorporation of 2deoxyglucose 

into lipid. J. Bacteriol. 109, 81-88. 

STEINER S., SMITH S., WAECHTER C.J. & LESTER R.L. 1969 

Isolation and partial characterization of a major inositolcontaining 

yeast phospholipid in baker’s yeast, mannosyldiinositol, 

diphosphorylceramide. Proc. Natl. Acad. Sci. USA 64, 

1042-1048. 

STEINITZ B. (1999) Sugar alcohols display nonosmotic roles in 

regulating morphogenesis and metabolism in plants that do not 

produce polyols as primary photosynthetic products. J. Plant 

Physiol. 155, 1-8. 

STEWARD F.C. & CAPLIN S.M. 1951 A tissue culture from 

potato tuber: the synergistic action of 2,4-D and of coconut milk. 

Science 113, 518-520. 

STEWARD F.C. & CAPLIN S.M. 1952 Investigations on growth 

and metabolism of plant cells. IV. Evidence on the role of the 

coconut milk factor. Ann. Bot. 16, 491-504. 

STEWARD F.C. & MOHAN RAM H.Y. 1961 Determining 

factors in cell growth: some implications for morphogenesis in 

plants. Adv. Morph. 1, 189-265. 

STEWARD F.C. & RAO K.V.N. 1970 Investigations on the growth 

and metabolism of cultured explants of Daucus carota. III. The 

range of responses induced in carrot explants by exogenous growth 

factors and by trace elements. Planta 91, 129-145. 

STEWARD F.C. & SHANTZ E.M. 1956 The chemical induction 

of growth in plant tissue cultures. pp. 165-186 in Wain and 

Wightman (eds.) 1956 The Chemistry and Mode of Action of 

Plant Growth Substances. Butterworth Scientific Publications 

Ltd., London. 

STEWARD F.C. & SHANTZ E.M. 1959 The chemical regulation 

of growth: Some substances and extracts which induce growth 

and morphogenesis. Annu. Rev. Plant Physiol. 10, 379-404. 

STEWARD F.C. 1963 Carrots and coconuts: Some investigations 

on growth. pp. 178-197 in Maheshwari and Ranga Swamy (eds.) 

1963 (q.v.). 

STEWARD F.C., CAPLIN S.M. & MILLAR F.K. 1952 

Investigations on growth and metabolism of plant cells. I. New 

techniques for the investigation of metabolism, nutrition, and 

growth in undifferentiated cells. Ann. Bot. 16, 57-77. 

STEWARD F.C., MAPES M.O. & AMMIRATO P.V. 1969 

Growth and morphogenesis in tissue and free cell cultures. pp. 

329-376 in Steward F.C. (ed.) 1969 Plant Physiology - A Treatise 

Vol. VB. Academic Press, New York, London. 

STEWARD F.C., MAPES M.O., KENT A.E. & HOLSTEN R.D. 

1964 Growth and development of cultured plant cells. Science 

143, 20-27. 

STEWARD F.C., SHANTZ E.M., POLLARD J.K., MAPES M.O. 

& MITRA J. 1961 Growth induction in explanted cells and 

tissues: Metabolic and morphogenetic manifestations. pp. 193- 

246 in Rudnick D. (ed.) Synthesis of Molecular and Cellular 

Structure. Ronald Press, New York. 

STONE O.M. 1963 Factors affecting the growth of carnation 

plants from shoot apices. Ann. Appl. Biol. 52, 199-209. 

STRAUS J. & LA RUE C.D. 1954 Maize endosperm tissue grown 

in vitro. 1. Culture requirements. Am. J. Bot. 41, 687-694. 

STREET H.E. & HENSHAW G.G. 1966 Introduction and methods 

employed in plant tissue culture. pp. 459-532 in Willmer E.N. 

Cells and Tissues in Culture – Methods and Physiology. Vol 3. 

Academic Press, London, New York]. 

STREET H.E. & McGREGOR S.M. 1952 The carbohydrate 

nutrition of tomato roots. III. The effects of external sucrose 

concentration on the growth and anatomy of excised roots. Ann. 

Bot. 16, 185-205. 

STREET H.E. 1969 Growth in organised and unorganised systems 

- knowledge gained by culture of organs and tissue explants. pp. 

3-224 in Steward F. C. (ed.) 1969. Plant Physiology - a Treatise. 

5B. Academic Press, New York. 

STREET H.E. 1977 Laboratory organization. pp. 11-30 in Street 

H.E. (ed.) Plant Tissue and Cell Culture. Bot. Monographs 

Vol.11, Blackwell Scientific Public-ations. Oxford, London. 

STREET H.E., McGONAGLE M.P. & LOWE J.S. 1951 

Observations on the `staling’ of White’s medium by excised 

tomato roots. Physiol. Plant. 4, 592-616. 

STREET H.E., McGONAGLE M.P. & McGREGOR S.M. 1952 

Observations on the ‘staling’ of White’s medium by excised 

tomato roots. II. Iron availability. Physiol. Plant. 5, 248-276. 

STRICKLAND S.G., NICHOL J.W., McCALL C.M. & STUART 

D.A. 1987 Effect of carbohydrate source on alfalfa 

embryogenesis. Plant Sci. 48, 113-121.

STUART D.A., STRICKLAND S.G. & NICHOL J.W. 1986 

Enhanced somatic embryogenesis using maltose. U.S. Patent No. 

4801545 , 

STUART D.A., STRICKLAND S.G. & WALKER K.A. 1987 

Bioreactor production of alfalfa somatic embryos. HortScience 

22, 800-803. 

SUNDERLAND N. & DUNWELL J.M. 1977 Anther and pollen 

culture. pp. 223-265 in Street H.E. (ed.) Plant Tissue and Cell 

Culture. Bot. Monographs Vol.11, Blackwell Scientific Publications. 

Oxford, London. 

SUTTLE J.C. & HULTSTRAND J.F. 1994 Role of endogenous 

abscisic acid in potato microtuber dormancy. Plant Physiol. 105, 

891-896. 

SWEDLUND B. & LOCY R.D. 1988 Somatic embryogenesis and 

plant regeneration in two-year old cultures of Zea diploperennis. 


TABER R.P., ZHANG C. & HU W.S. 1998 Kinetics of Douglasfir 

(Pseudotsuga menziesii) somatic embryo development. Can. 

J. Bot. 76, 863-871. 

TAKAYAMA S. & MISAWA M. 1979 Differentiation in Lilium 

bulb scales grown in vitro - effect of various cultural conditions. 


TAKAYAMA S. & MISAWA M. 1979 Differentiation in Lilium 

bulb scales grown in vitro - effect of various cultural conditions. 


TANAKA M., HIRANO T., GOI M., HIGASHIURA T., 

SASAHARA H. & MURASAKI K. 1991 Practical application of 

a novel disposable film culture vessel in micropropagation. Acta 

Hortic. 300, 77-84. 

TANDEAU DE MARSAC N. & PEAUD-LENOEL C. 1972a 

Cultures photosynthtique de lignes clonales de cellules de tabac. 

Compt. Rend. Acad. Sci. Paris 274D, 1800-1802. 

TANDEAU DE MARSAC N. & PEAUD-LENOEL C. 1972b 

Exchanges d’oxygène et assimilation de gaz carbonique de tissus 

de tabac en culture photosynthétique. Compt. Rend. Acad. Sci. 

Paris 274D, 2310-2313. 

TAUTORUS T.E. & DUNSTAN D.I. 1995 Scale-up of embryonic 

plant suspension cultures in bioreactors. In: S Jain, P Gupta, and 

R Newton (Eds) Somatic embryogenesis in woody plants Vol. 1. 

Kluwer Academic Publishers, The Netherlands pp 265-292. 

TEISSON C. & ALVARD D. 1999 In vitro production of potato 

microtubers in liquid medium using temporary immersion. Potato 

Res. 42, 499-504. 

TELLE J. & GAUTHERET R.J. 1947 Sur la culture indéfinie des 

tissus de la racine de jusquiame (Hyascyamus niger). Compt. 

Rend. Acad. Sci. Paris 224, 1653-1654. 

TENG W.L. 1997 Regeneration of Anthurium adventitious shoots 

using liquid or raft culture. Plant Cell Tissue Organ Cult. 49, 

153-156. 

THOM M., MARETZKI A., KOMOR E. & SAKAI W.S. 1981 

Nutrient uptake and accumulation by sugarcane cell cultures in 

relation to the growth cycle. Plant Cell Tissue Organ Cult. 1, 3-14. 

THOMPSON M.R. & THORPE T.A. 1981 Mannitol metabolism 

in cell cultures. Plant Physiol. 67, Suppl., 27. 

THOMPSON M.R., DOUGLAS T.J., OBATA-SASAMOTO H. & 

THORPE T.A. 1986 Mannitol metabolism in cultured plant 

cells. Physiol. Plant. 67, 365-369. 

THORPE T.A & MEIER D.D. 1973 Sucrose metabolism during 

tobacco callus growth. Phytochemistry 12, 493-497. 

THORPE T.A. 1982 Carbohydrate utilization and metabolism. 

pp. 325-368 in Bonga J.M. & Durzan D.J. (eds.) Tissue Culture 

in Forestry. Martinus Nijhoff/Dr. W. Junk Publishers, The 

Hague. 

THORPE T.A. & LAISHLEY E.J. 1974 Carbohydrate oxidation 

during Nicotiana tabacum callus growth. Phytochemistry 13, 

1323-1328. 

Chapter 4 171 

THORPE T.A. & LAISHLEY E.J. 1973 Glucose oxidation 

during shoot initiation in tobacco callus cultures. J. Exp. Bot. 

24, 1082-1089. 

THORPE T.A. & MEIER D.D. 1972 Starch metabolism, repiration 

and shoot formation in tobacco callus cultures. Physiol. Plant. 27, 

365-369. 

THORPE T.A. & MEIER D.D. 1973 Effects of gibberellic acid 

and abscisic acid on shoot formation in tobacco callus cultures. 


THORPE T.A. & MEIER D.D. 1974 Starch metabolism in shootforming 

tobacco callus. J. Exp. Bot. 25, 288-294. 

THORPE T.A. & MEIER D.D. 1975 Effect of gibberellic acid on 

starch metabolism in tobacco callus cultured under shoot-forming 

conditions. Phytomorph. 25, 238-245. 

THORPE T.A. & MURASHIGE T. 1968a Some histochemical 

changes underlying shoot initiation in tobacco callus culture. Am. 

J. Bot. 55, 710. 

THORPE T.A. & MURASHIGE T. 1968b Starch accumulation in 

shoot-forming tobacco callus cultures. Science 160, 421-422. 

THORPE T.A. & MURASHIGE T. 1970 Some histochemical 

changes underlying shoot initiation in tobacco callus cultures. 

Can. J. Bot. 48, 277-285. 

THORPE T.A. 1982 Physiological and biochemical aspects of 

organogenesis in vitro. pp. 121-124 in Fujiwara A. (ed.) 1982 

(q.v.). 

THORPE T.A., JOY R.W. IV & LEUNG W.M. 1986 Starch turnover 

in shoot-forming tobacco callus. Physiol. Plant. 66, 58-62. 

TIAN H.Q. & RUSSELL S.D. 1998 Culture-induced changes in 

osmolality of tobacco cell suspensions using four exogenous 

sugars. Plant Cell Tiss Organ Cult. 55, 9-13 

TIBURCIO A.F., GENDY C.A. & TRAN THANH VAN K. 1989 

Morphogenesis in tobacco subepidermal cells: putrescine as a 

marker of root differentiation. Plant Cell Tissue Organ Cult. 19, 

43-54. 

TIMBERT R., BARBOTIN J.-N., KERSULEC A., BAZINET C. 

& THOMAS D. 1995 Physico-chemical properties of the 

encapsulation matrix and germination of carrot somatic embryos. 

Biotechnol. Bioeng. 46, 573-578. 

TOMAR U.K. & GUPTA S.C. 1988 Somatic embryogenesis and 

organogenesis in callus cultures of a tree legume - Albizia 

richardiana King. Plant Cell Rep. 7, 70-73. 

TOR M., TWYFORD C.T., FUNES I., BOCCON-GIBOD J., 

AINSWORTH C.C. & MANTELL S.H. 1998 Isolation and 

culture of protoplasts from immature leaves and embryogenic 

cell suspensions of Dioscorea yams: tools for transient gene 

expression studies. Plant Cell Tissue Organ Cult. 53, 113-125. 

TORRES K. & CARLISI J.A. 1986 Shoot and root organogenesis 

of Camellia sasanqua. Plant Cell Rep. 5, 381-384. 

TORREY J.G. 1956 Chemical factors limiting lateral root 

formation in isolated pea roots. Physiol. Plant. 9, 370-388 

TRAN THANH VAN K. & TRINH H. 1978 Plant propagation: 

non-identical and identical copies. pp. 134-158 in Hughes K.W., 

Henke R. & Constantin M. (eds.) Propagation of Higher Plants 

through Tissue Culture. Technical Inf. Center, U.S. Dept. 

Energy. Spingfield, Va. CONF-7804111. 

TRELEASE S.F. & TRELEASE H.M. 1933 Physiologically 

balanced culture solutions with stable hydrogen-ion concentration. 

Science 78, 438-439. 

TREMBLAY F.M. & LALONDE M. 1984 Requirements for in 

vitro propagation of seven nitrogen-fixing Alnus species. Plant 


TREMBLAY F.M., NESME X. & LALONDE M. 1984 Selection 

and micropropagation of nodulating and non-nodulating clones of 

Alnus crispa (Ait.) Pursh. Plant Soil 78, 171-179. 

TRINDADE H. & PAIS M.S. 1997 In vitro studies on Eucalyptus 

globulus rooting ability. In Vitro Cell. Dev.-Pl. 33, 1-5.


TROLINDER N.L. & GOODIN J.R. 1987 Somatic embryogenesis 

and plant regeneration in cotton (Gossypium hirsutum L.). Plant 

Cell Rep. 6, 231-234. 

TULECKE W., WEINSTEIN L.H., RUTNER A. & 

LAURENCOT H.J. 1961 The biochemical composition of 

coconut water as related to its use in plant tissue culture. Contrib. 

Boyce Thomps. 21, 115-128. 

TURNER S.R. & SINGHA S. 1990 Vitrification of crabapple, pear 

and geum on gellan gum-solidified culture medium. HortScience 

25, 1648-1650. 

UMBECK P., JOHNSON G., BARTON K. & SWAIN W. 1987 

Genetically transformed cotton (Gossypium hirsutum L.) plants. 

Nat. Biotechnol. 5, 263-266. 

VACIN E.F. & WENT F.W. 1949 Some pH changes in nutrient 

solutions. Bot. Gaz. 110, 605-613. 

VAIN P., MCMULLEN M.D. & FINER J.J. 1993 Osmotic 

treatment enhances particle bombardment-mediated transient and 

stable transformation of maize. Plant Cell Rep. 12, 84-88. 

VAN DER KRIEKEN W.M., BRETELER H., VISSER M.H.M. & 

JORDI W. 1992 Effect of light and riboflavin on indolebutyric 

acid-induced root formation on apply in vitro. Physiol. Plant. 85, 

589-594. 

VAN HUYSTEE R.B. 1977 Porphyrin metabolism in peanut cells 

cultured in sucrose containing medium. Acta Hortic. 78, 83-87. 

VAN OVERBEEK J. 1942 Water uptake by excised root systems of 

the tomato due to non-osmotic forces. Am. J. Bot. 29, 677-683. 

VAN OVERBEEK J., CONKLIN M.E. & BLAKESLEE A.F. 1941 

Factors in coconut milk essential for growth and development of 

very young Datura embryos. Science 94, 350-351. 

VAN OVERBEEK J., CONKLIN M.E. & BLAKESLEE A.F. 

1942 Cultivation in vitro of small Datura embryos. Am. J. Bot. 

29, 472-477. 

VAN OVERBEEK J., SIU R. & HAAGEN-SMIT A.J. 1944 

Factors affecting the growth of Datura embryos in vitro. Am. J. 

Bot. 31, 219-224. 

VAN STADEN J. & DREWES S.E. 1975 Identification of zeatin 

and zeatin riboside in coconut milk. Physiol. Plant. 34, 106-109. 

VAN STADEN J. 1976 The identification of zeatin glucoside from 

coconut milk. Physiol. Plant. 36, 123-126. 

VANDENBELT J.M. 1945 Nutritive value of coconut. Nature 156, 

174-175. 

VANSVEREN-VAN ESPEN N. 1973 Effets du saccharose sur le 

contenu en chlorophylles de protocormes de Cymbidium Sw. 

(Orchidaceae) cultivés in vitro. Bull. Soc. Roy. Bot. Belg. 106, 

107-115. 

VASIL I.K. & HILDEBRANDT A.C. 1966a Variations of 

morphogenetic behaviour in plant tissue cultures. I. Cichorium 

endivia. Am. J. Bot. 53, 860-869. 

VASIL I.K. & HILDEBRANDT A.C. 1966b Variations of 

morphogenetic behaviour in plant tissue cultures. II. 

Petroselinum hortense. Am. J. Bot. 53, 869-874. 

VASIL I.K. & HILDEBRANDT A.C. 1966c Growth and 

chlorophyll production in plant callus tissues grown in vitro. 


VASIL V. & VASIL I.K. 1981a Somatic embryogenesis and plant 

regeneration from tissue cultures of Pennisetum americanum, and 

P. americanum x P. purpureum hybrid. Am. J. Bot. 68, 864-872. 

VASIL V. & VASIL I.K. 1981b Somatic embryogenesis and plant 

regeneration from suspension cultures of pearl millet 

(Pennisetum americanum). Ann. Bot. 47, 669-678. 

VENVERLOO C. 1976 The formation of adventitious organs. III. 

A comparison of root and shoot formation on Nautilocalyx 

explants. Z. Pflanzenphysiol. 80, 310-322. 

VERMA D.C. & DOUGALL D.K. 1977 Influence of carbohydrates 

on quantitative aspects of growth and embryo formation in wild 

carrot suspension cultures. Plant Physiol. 53, 81-85. 

VERMA D.C. & DOUGALL D.K. 1979 Biosynthesis of myo- 

inositol and its role as a precursor of cell-wall polysaccharides in 

suspension cultures of wild-carrot cells. Planta 146, 55-62. 

VERMA D.P.S. & VAN HUYSTEE R.B. 1971 Derivation, 

characteristics and large scale culture of a cell line from Arachis 

hypogaea L. cotyledons. Exp. Cell Res. 69, 402-408. 

VISEUR J. 1987 Micropropagation of pear, Pyrus communis L., in 

a double-phase culture medium. Acta Hortic. 212, 117-124. 

VON ARNOLD S. 1987 Effect of sucrose on starch accumulation 

in, and adventitous bud formation on, embryos of Picea abies. 

Ann. Bot. 59, 15-22. 

VREUGDENHIL D., BOOGAARD Y., VISSER, R.G.F. & DE 

BRUIJN S.M. 1998 Comparison of tuber and shoot formation 

from in vitro cultured potato explants. Plant Cell Tiss Organ Cult. 

53, 197-204. 

VYSKOT B. & BEZDEK M. 1984 Stabilization of synthetic media 

for plant tissue culture and cell cultures. Biol. Plant. 26, 132-143. 

VYSKOT B. & JARA Z. 1984 Clonal propagation of cacti through 

axillary buds in vitro. J. Hortic. Sci. 59, 449-452. 

WALKER D.R. & PARROTT W.A. 2001 Effect of polyethylene 

glycol and sugar alcohols on soybean somatic embryo 

germination and conversion. Plant Cell Tissue Organ Cult. 64, 

55-62. 

WATAD A.A., AHRONI A., ZUKER A., SHEJTMAN H., 

NISSIM A. & VAINSTEIN A. 1996 Adventitious shoot formation 

from carnation stem segments: A comparison of different 

culture procedures. Sci. Hortic. 65, 313-320 

WANN S.R., VEAZEY R. L., KAPHAMMER J. 1997 Activated 

charcoal does not catalyze sucrose hydrolysis in tissue culture 

media during autoclaving. Plant Cell Tissue Organ Cult. 50, 

221-224. 

WATAD A.A., KOCHBA M., NISSIM A. & GABA V. 1995 

Improvement of Aconitum napellus micropropagation by liquid 

culture on floating membrane rafts. Plant Cell Rep. 14, 345-348. 

WATANABE K., TANAKA K., ASADA K. & KASAL Z. 1971 

The growth promoting effect of phytic acid on callus tissues of 

rice seed. Plant Cell Physiol. 12, 161-164. 

WEI Z.M., KYO M. & HARADA H. 1986 Callus formation and 

plant-regeneration through direct culture of isolated pollen of 

Hordeum vulgare cv. Sabarlis. Theor. Appl. Genet.72, 252-255. 

WELANDER T. 1977 In vitro organogensis in explants from 

different cultivars of Begonia hiemalis. Physiol. Plant. 41, 

142-145. 

WESTON G.D. & STREET H.E. 1968 Sugar absorption and sucrose 

inversion by excised tomato roots. Ann. Bot. 32, 521-529. 

WETHERELL D.F. 1969 Phytochrome in cultured wild carrot 

tissue. I. Synthesis. Plant Physiol. 44, 1734-1737. 

WETHERELL D.F. 1984 Enhanced adventive embryogenesis 

resulting from plasmolysis of cultured wild carrot cells. Plant 


WETMORE R.H. & RIER J.P. 1963 Experimental induction of 

vascular tissues in callus of angiosperms. Am. J. Bot. 50, 418-430. 

WHITE M.C., DECKER A.M. & CHANEY R.L. 1981 Metal 

complexation in xylem fluid. I. Chemical composition of tomato 

and soybean stem exudate. Plant Physiol. 67, 292-300. 

WHITE P.R. 1932 Influence of some environmental conditions on 

the growth of excised root-tips of wheat seedlings in liquid 

media. Plant Physiol. 7, 613-628. 

WHITE P.R. 1934 Potentially unlimited growth of excised tomato 

root tips in a liquid medium. Plant Physiol. 9, 585-600. 

WHITE P.R. 1937 Survival of isolated tomato roots at sub-optimal 

and supra-optimal temperatures. Plant Physiol. 12, 771-776. 

WHITE P.R. 1943a A Handbook of Plant Tissue Culture. The 

Jacques Catlell Press, Lancaster, Pa.

WHITE P.R. 1943b Nutrient deficiency studies and an improved 

inorganic nutrient for cultivation of excised tomato roots. Growth 

7, 53-65. 

WHITE P.R. 1954 The Cultivation of Animal and Plant Cells. 1st. 

edition. Ronald Press, New York. 

WHITE P.R. 1963 The Cultivation of Animal and Plant Cells. 2nd 

edition. Ronald Press, New York. 

WHITTIER D.P. & STEEVES T.A. 1960 The induction of 

apogamy in the bracken fern. Can. J. Bot. 38, 925-930. 

WILLIAMS R.R., TAJI A.A. & BOLTON J.A. 1984 In vitro 

propagation of Dampiera diversifolia and Prostanthera 

rotundifolia. Plant Cell Tissue Organ Cult. 3, 273-281. 

WILLIAMS R.R., TAJI A.M. & BOLTON J.A. 1985 Specificity 

and interaction among auxins, light and pH in rooting of 

Australian woody species in vitro. HortScience 20, 1052-1053. 

WILSON K.S. & CUTTER V.M. 1955 Localization of acid 

phosphatase in the embryo sac and endosperm of Cocos nucifera. 

Am. J. Bot. 42, 116-119. 

WOLF S., KALMAN-ROTEM N., YAKIR D. & ZIV M. 1998 

Autotrophic and heterotrophic carbon assimilation of in vitro 

grown potato (Solanum tuberosum L.) J. Plant Physiol. 135, 

574-580. 

WOLTER K.E. & SKOOG F. 1966 Nutritional requirements of 

Fraxinus callus cultures. Am. J. Bot. 53, 263-269. 

WOOD H.N. & BRAUN A.C. 1961 Studies on the regulation of 

certain essential biosynthetic systems in normal and crown-gall 

tumor cells. Proc. Natl. Acad. Sci. USA 47, 1907-1913. 

WOODWARD S., THOMSON R.J. & NEALE W. 1991 

Observations on the propagation of carnivorous plants by in vitro 

culture. Newsl. Int. Plant Prop. Soc. Spring 1991. 

WRIGHT K. & NORTHCOTE D.H. 1972 Induced root differentiation 

in sycamore callus. J. Cell Sci. 11, 319-337. 

WRIGHT M.S., KOEHLER S.M., HINCHEE M.A. & CARNES 

M.G. 1986 Plant regeneration by organogenesis in Glycine max. 


XIE J., GAO M., CAI Q., CHENG X., SHEN Y. & LIANG Z.. 

1995 Improved isolated microspore culture efficiency in medium 

with maltose and optimized growth regulator combination in 

japonica rice (Oryza sativa). Plant Cell Tissue Organ Cult. 42, 

245-250. 

XU X., VAN LAMMEREN A.A.M., VERMEER E. & 

VREUGDENHIL D. 1998 The role of gibberellin, abscisic acid, 

and sucrose in the regulation of potato tuber formation in vitro. 


YATAZAWA M., FURUHASHI K. & SHIMIZU M. 1967 

Growth of callus tissue from rice root in vitro. Plant Cell 

Physiol. 8, 363-373. 

Chapter 4 173 

YOSHIDA F., KOBAYASHI T. & YOSHIDA T. 1973 The 

mineral nutrition of cultured chlorophyllous cells of tobacco. I. 

Effects of p salts, p sucrose, Ca, Cl and B in the medium on the 

yield, friability, chlorophyll contents and mineral absorption of 

cells. Plant Cell Physiol. 14, 329-339. 

YOUNG R.E., HALE A., CAMPER N.D., KEESE R.J. & 

ADELBERG J.W. 1991 Approaching mechanization of plant 

micropropagation. Trans. ASAE 34, 328-333 

YU S.X. & DORAN P.M. 1994 Oxygen requirements and masstransfer 

in hairy-root culture. Biotechnol. Bioeng. 44, 880-887. 

ZAGHMOUT O. & TORRES K.C. 1985 The effects of various 

carbohydrate sources and concentrations on the growth of Lilium 

longiflorum ‘Harson’ grown in vitro. HortScience 20, 660. 

ZAMSKI E. & WYSE R.E. 1985 Stereospecificity of glucose 

carrier in sugar beet suspension cells. Plant Physiol. 78, 291-295. 

ZAPATA F.J, KHUSH G.S., CRILL J.P., NEU M.R., ROMERO 

R.O., TORRIZO L.R. & ALEJAR M. 1983 Rice anther culture at 

IRRI. pp. 27-86 in Cell and Tissue Culture Techniques for Cereal 

Crop Improvement. Science Press Beijing, China. Gordon and 

Breach Science Publications, Inc., New York. 

ZATYKO J.M. & MOLNAR I. 1986 Adventitious root formation 

of different fruit species influenced by the pH of medium. p. 29 

in Abstracts VI Intl. Cong. Plant Tissue & Cell Culture. 

Minneapolis, Minn. 

ZHANG B., STOLTZ L.P. & SNYDER J.C. 1986 In vitro 

proliferation and in vivo establishment of Euphorbia fulgens. 

HortScience 21, 859. 

ZHOU T.S. 1995 In vitro culture of Doritaenopsis: comparison 

between formation of the hyperhydric protocorm-like-body 

(PLB) and the normal PLB. Plant Cell Rep. 15, 181-185. 

ZHU J.K. (2002) Salt and drought stress signal transduction in 

plants. Annu. Rev. Plant Biol. 53, 247-273. 

ZIMMERMAN R.H. 1979 The laboratory of micropropagation at 

Cesena, Italy. Comb. Proc. Int. Plant Prop. Soc. 29, 398-400. 

ZIV M, 1991. Quality of micropropagated plants - vitrification. In 

Vitro Cell. Dev. – Pl. 27, 64-69. 

ZIV, M. 1989. Enhanced shoot and cormlet proliferation in liquid 

cultured gladiolus buds by growth retardants. Plant Cell Tissue 

Organ Cult. 17, 101-110. 

ZIV M. 2005 Simple bioreactors for mass propagation of plants. 

Plant Cell Tissue Organ Cult. 81, 277-285 

ZWAR J.A. & BRUCE M.I. 1970 Cytokinins from apple extract 

and coconut milk. Aust. J. Biol. Sci. 23, 289-297. 

ZWAR J.A., KEFFORD N.P., BOTTOMLEY W. & BRUCE M.I. 

1963 A comparison of plant cell division inducers from coconut 

milk and apple fruitlets. Nature 200, 679-680.

The Components of Plant Tissue Culture Media II - Horticultural ...

Create successful ePaper yourself

Delete template?

Save as template?