SCIENTIA HORTICULTUR~ ELSEVIER Scientia Horticulturae69 (1997) 239-257 Recent trends in the biotechnology of Chrysanthemum: a critical review G.R. Rout, P. Das Plant Bioteehnologv Dit,ision Plant Tissue Culture Laboratory, Regional Plant Resource Centre, Bhubaneswar 751 015, India Received 5 March 1996 Abstract Various techniques have been developed which could help breeders to meet the demand of the cut flower industry in the next century. Available methods for the transfer of genes could significantly shorten the breeding procedures and overcome some of the agronomic and environmental problems which would otherwise not be possible through conventional methods. On the other hand, nutritional requirements (mineral nutrients), carbohydrates and other organic compounds (vitamins, amino acids, etc), environmental factors (e.g. light, gaseous environment, temperature and humidity) and treatments with growth regulators have helped in achieving high proliferation rates to allow commercially viable micropropagation. An overview of the regeneration of chrysanthemum by direct and indirect organogenesis, embryogenesis from explants and embryo rescue is presented in this article. In addition, the use of these techniques in association with several biotechnological methods to enrich the genome of chrysanthemum, such as selection of somaclonal variants, screening for various usetul characteristics and genetic transformation, is reviewed. © 1997 Elsevier Science B.V. Keywords: Plant biotechnology; Genetic transformation; Regeneration; Chrysanthemum (Dendranthema grandiflora) 1. Introduction Chrysanthemum ( D e n d r a n t h e m a grandiflora) is one of the most important cut flowers and pot plants grown in many parts of the world. The commercial cultivars are usually propagated vegetatively through cuttings and suckers. Breeding programmes Abbreviations: BAP: 6-benzylaminopurine;Kn: kinetin; IAA: indole-3-aceticacid; NAA: Inaphthyleneacetic acid; IBA: indole-3-butyricacid; 2,4-D: 2,4-dichlorophenoxyacetic acid; Ads: adenine sulphate Corresponding author. 0304-4238/97/$t7.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0304-4238(97)00008-3 240 G.R. Rout, P. Das / Scientia Horticulturae 69 (1997) 239-257 have focussed on improving various characteristics to enhance the ornamental value, including the colour, size and form of the flower, production quality and reaction to the environment (Broertjes et al., 1980). Although desirable traits have been introduced by classical breeding, there are limitations to this technique. Firstly, there is a limited gene pool. Secondly, distant crosses may be limited by incompatibility or differences in ploidy level between putative parents. Thirdly, characteristics such as uniform growth and synchronous flowering are polygenic. Hence, sexual crossing may alter the delicate balance of factors determining plant growth and development (Mol et al., 1989). As an alternative approach to classical breeding methods, attempts are being made to develop transformation systems using agrobacterium-based gene vectors (Hutchinson et al., 1989; Lu et al., 1990 and Van Wordragen et al., 1989). Biotechnology involving modern tissue culture, cell biology and molecular biology offers the opportunity to develop new germplasms that are better adapted to changing demands. In this way, extensive studies have been carried out with chrysanthemum on various aspects ofits biotechnology, such as micropropagation, adventitious shoot bud regeneration from various explants and somatic embryogenesis. In this communication we provide an overview of the different biotechnological applications available for the micropropagation, regeneration and improvement of chrysanthemum. 2. Establishment of aseptic cultures It is relatively easy to establish in vitro cultures from most of the organs of chrysanthemum. The best source is a virus-indexed parent plant grown in the greenhouse. The general procedures for virus elimination have been discussed (Brierley and Lorentz, 1960; Haakkart and Quak, 1964; Paludan, 1973). In general, most explants were washed with 5-10% detergent solution 'Teepol' for 20 min. The explants were disinfected with 0.1% HgC12 for 20-25 min followed by several rinses (2-5 times) with sterile distilled water. The explants were cultured on either basal MS (Murashige and Skoog, 1962) or B 5 (Gamborg et al., 1968) medium supplemented with different growth regulators at concentrations needed to achiev the target response. Lu et al. (1990). Surface sterilized initial explants with 70% ethanol for 40 s followed by 1.25% sodium hypochlorite solution with Tween-20 for 2 to 3 min and then rinsed them three times in sterile water. The petals were surface sterilized for 5 min in 5% Chlorox and a drop of Tween-20 and then rinsed twice in sterile distilled water before being used as explants (Bush et al., 1976). 3. Type of culture 3.1. Meristem culture Axillary and terminal buds are used for mass multiplication in vitro (Rout et al., 1996). A single bud produces a single shoot or multiple shoots depending on the species and composition of the medium. Numerous factors have been found to influence the induction of morphogenesis in plant cell and tissue cultures. Several culture media are in G.R. Rout, P. Das / Scientia Horticulturae 69 (1997) 239-257 241 common use including formulations derived by Murashige and Skoog (1962) and Gamborg et al. (1968). Micropropagation of chrysanthemum has been reported by various researchers using cultures of shoot apex and axillary bud explants (Ben-Jaacov and Langhans, 1972; Roest and Bokelmann, 1973; Earle and Langhans, 1974a; Bush et al., 1976; Grewal and Sharma, 1978; Wang and Ma, 1978; l e e et al., 1979; Lazar et al., 1981; Lazar and Cachita, 1982; de Donato and Peruceo, 1984; Gertsson and Andersson, 1985; Ahmed, 1986; Ahmed and Andrea, 1987; Sun and Li, 1987; Widiastoety, 1987; Prasad and Chaturvedi, 1988), callus derived from stem and leaf explants (Hill, 1968; Earle and Langhans, 1974b) and floral parts (Bush et al., 1976; Roest and Bokelmann, 1975; Lee et al., 1979; Sutter and Langhans, 1981; Lazar et al., 1981; Lazar and Cachita, 1983; Chen et al., 1985; Prasad and Chaturvedi, 1988; Ohishi and Sakurai, 1988; Lu et al., 1990; Kaul et al., 1990; Bhattacharya et al., 1990 Corneanu and Corneanu, 1992; Kumar and Kumar, 1995). Prasad et al. (1983) reported that the rates of multiplication were less than those obtained for four important cultivars of Chrysanthemum morifolium cvs. Otome Zakura, Pandhari, Revdi and Turbulent, on a modified Murashige and Skoog's (1962) medium supplemented with 1.5 mg 1-~ BAP and 0.5 mg 1-1 IAA. A maximum number of 31 shoots were formed per explant of 'Otome Zakura' in 60 days of culture. It was estimated that about nine million plants could be produced from a single explant in one year using this method. The growth ofthe shoot tips differed depending on the growth regulator composition of the medium. Three main types of growth were observed: (a) elongation of the apex to form only one or a few shoots; (b) an undifferentiated, fast growing white callus; and (c) an intermediate type of growth composed of light green callus with many small, narrow leaf-like structures. The intermediate type of growth was found to be the best for further proliferation and formation of plantlets when the medium was supplemented with 10% coconut milk or, to a lesser degree, with 25 ppm inositol, 0.8 ppm kinetin and 0.5 ppm IAA. Growth of the shoot was more pronounced on semi-solid medium compared with liquid medium (Ben-Jaacov and Langhans, 1972). Earle and Langhans, 1974a,b) reported that multiple shoots were obtained from a single shoot tip of Chrysanthemum sp. In most cases, an average of five shoots per culture was obtained within 4 weeks on Skoog medium (Linsmaier and Skoog, 1965) containing 2 ppm kinetin and 0.02 ppm anaphthaleneacetic acid (NAA). Success in culturing the explants depended on a number of factors inducing the size of the explant, its physiological age and the source of the plant. Wang and Ma (1978) reported that shoot tips between 0.2-0.5 mm and shoot meristems between 0.1-0.2 mm produced only a single shoot (Fig. 1). Larger explants (0.5 to 1.55 mm) formed multiple shoots (Fig. 2 and Fig. 3). 3.2. Callus culture and plant regeneration Callus cultures of chrysanthemum have been established in many laboratories. Appropriate auxin and cytokinin levels are required for callus production from each species or variety. Hill (1968) established callus from shoot tip explants of chrysanthemum. He succeeded in stimulating the growth and proliferation of shoot-like structures on a medium containing 2 mg I 1 NAA and 0.8 mg 1 ~ kinetin. Roest and Bokelmann (1975) investigated the role of growth regulators, explant length, sugar, vitamins and 242 G.R, Rout, P. Das / Scientia Horticulturae 69 (1997) 239-257 Fig. 1. Proliferation of shoots derived from leaf explants after 4 weeks of culture (Bar = 0.5 mm). Fig. 2. Proliferation and elongation of shoots of Chr3,'santhemum morifolium cv. Deep Pink cultured on B 5 +2.5 mg I -~ B A P + 1.0 mg 1-I I A A + 3 0 g I I D-glucose after 8 weeks of culture (Bar = 20 mm). Fig. 3. Root formation in the excised shoots in liquid MS medium supplemented with 0,25 mg I i I B A + 2 % ( w / v ) sucrose after 15 days of culture (Bar = 50 ram). Fig. 4, Plants bearing flowers in the greenhouse (Bar = 0.2 cm). Fig. 5. Shoot-tips encapsulated as beads in 2.5% sodium alginate matrix (Bar = 10 ram), Fig. 6. Emerging shoot and roots from the encapsulated shoot tip (Bar = 5 ram). G.R. Rout, P. Das / Scientia Horticulturae 69 (1997) 239-257 243 minerals on adventitious shoot formation via callus from pedicel explants. They also confirmed that a combination of BAP and IAA was most favourable for shoot bud regeneration. In the absence of vitamins and minerals, or in the presence of the macro and micro elements (devoid of KNO 3 and NH 4 NO3), adventitious shoot formation was completely suppressed. Bhattacharya et al. (1990) reported the influence of auxins on callus production and obtained good green calli from both leaf and stem segments on MS basal salts supplemented with 2 mg 1-1 2,4-D within 2 weeks of culture. Furthermore, the calli regenerated into shoot buds after transfer to solid MS medium supplemented with 0.1 mg 1-~ IAA and 0.2 mg 1-i BAP. Kaul et al. (1990) regenerated adventitious shoots from leaf and stem explants of 11 chrysanthemum cultivars. MS basal medium supplemented with 5 I-tM NAA was optimum for both types of explants. They also reported that stem explants were superior to leaf explants. The morphogenic development was noted 4 - 6 days after the initiation of cultures depending upon the explant type. Similar results were found by Rout et al. (1996) for shoot bud regeneration from leaf and stem explants of C. morifolium cv. Deep pink using different basal nutrient media, growth regulators and carbohydrates. B 5 basal salts were more effective than MS basal salts within 2 weeks of culture; small adventitious shoot primordia were visible around the cut end of the stem and leaf segments when cultured on B 5 basal salts supplemented with 2.5 mg 1-J BAP, 1.0 mg 1 i IAA and 3% ( w / v ) D-glucose. The differentiated shoot primordia subsequently developed into dark green shoots. The majority of the explants became yellow and died within 3 - 4 weeks on media without growth regulators. They also reported that callusing of stem and leaf segments was noted only in cultures devoid of BAP. The rate of callus growth was faster with an increase in the concentration of either NAA or IAA in the medium. Kinetin was less effective in inducing the regeneration of shoot buds compared with BAP and adenine sulphate (ads). In general, a higher regeneration frequency was observed with leaf and stem segments derived from semi-mature segments compared with immature segments on media containing 2.5-3.0 mg 1-~ BAP, !.0-1.5 mg l-t IAA and 30 g 1-~ D-glucose. There was a difference in the regeneration frequency between the stem and the leaf explants grown on B 5 basal media supplemented with 2.5-3.0 mg 1-~ BAP + 1.0-1.5 mg 1-~ IAA. Regeneration of shoots was better with leaf than stem explants, as reported earlier in chrysanthemum (Miyazaki et al., 1976). The frequency of the regenerated shoots/explant varied from 12.4-36.4 with leaf and 8.4-36.0 with stem explants derived from immature sources. The explants derived from semi-mature sources produced 20.6-44.6 shoot buds from leaf and 16.4-37.4 from stem explants. The differential response could be due to the varying concentrations of growth regulators and the type of explants used (Miyazaki and Tashiro, 1978). Semi-mature leaf segments showed a higher potential for shoot bud regeneration than the immature segments, which perhaps resulted from a hormonal imbalance caused by accumulation of endogenous cytokinin in the leaf tissues. Shoot bud regeneration from leaf explants increased linearly with a decrease in the concentration of IAA, as reported earlier (Rout et al., 1996). In contrast, the number of regenerated shoots per explant was low at 1.0 mg 1-~ and high at 1.5 mg 1-~ of IAA. The role of BAP and IAA, along with 3% D-glucose, in the medium was turther studied by the authors using both stem and leaf explants. They reported that the addition of BAP was essential for the development of 244 G.R. Rout, P. Das / Seientia Horticulturae 69 (1997) 239-257 transferable shoots. All other treatments except BAP (1.5-3.0 mg 1- l ) and IAA (1.0-1.5 mg 1- J), did not show any significant effect on stem or leaf explants for shoot bud regeneration. The media containing 2.5 mg 1 i BAP + 1.0 mg 1- ~ IAA and 2.5 mg 1 t BAP + 1.5 mg 1- ~ IAA helped in the maximum regeneration of shoots both in the immature and the semi-mature leaf and stem exptants. Bhattacharya et al. (1990) reported that a combination of 0.1 mg 1-J IAA and 0.2 mg I-~ BAP was most appropriate for callus formation from nodal segments, shoot apices and leaf and for the regeneration of shoots t¥om callus. An average shoot height of 3 - 4 cm (after 4 weeks of culture) with 2 - 4 leaves per cm height of the stem and production of at least 3 - 4 shoots from each explant was achieved by the authors. Preliminary observations indicate that regeneration frequency could be improved by manipulating the light intensity and the composition of hormones in the culture medium. The regeneration frequency was higher in stem and leaf derived cultures under a 14 h photoperiod compared with those under continuous light (Rout et al., 1996). The rate of shoot bud regeneration was, however, affected in both a 14 h photoperiod and continuous light when the medium was supplemented either with a high dose of IAA ( < 2.5 mg 1- ~) alone or in combination with a number of cytokinins. Stem and leaf explants derived from both mature and semi-mature sources also showed a differential rate of shoot bud regeneration on different media at 55 p~Em-2 s ~ light intensity. The reason that juvenility played an important role in regeneration is not clear, but the number of regenerated shoot buds/culture depended on the composition of the culture medium, especially on the level of PGRs and the light regime. The media containing 2.5 mg 1 J BAP and 1.0-1.5 mg 1-~ IAA induced a high rate of regeneration at a 14 h photoperiod, whereas the other media with IAA (2.0-3.0 mg 1 ~) recorded a higher regeneration rate at low light intensity. There have been several reports concerning shoot bud regeneration from calli derived from various explants (Kohno et al., 1978; Schum and Preil, 1981; Amaqasa and Kameya, 1989; Bhattacharya et al., 1990; Zito and Teo, 1990 and Lu et al., 1990). Bannier and Stepenkus (1976) reported that callus was only able to survive down to a temperature of -6.6°C, whereas callus acclimatized for 6 weeks at 4.5°C survived at a temperature of - 16.1°C. Callus age was an important factor for shoot bud regeneration. The regeneration ability decreased when calli were stored at low temperatures for long periods. Eight cultivars were tested for their regeneration ability using different types of explants on the same media. Cuitivar and explant type had a greater effect on regeneration than the type of medium (Rademaker et al., 1990). Bhattacharya et al. (1990) reported rapid mass propagation of C. morifolium through callus cultures derived from stem and leaf explants. Callus (I cm 2) regenerated 2 - 3 shoots after 3 weeks on MS solid medium supplemented with 0.1 mg 1-1 IAA and 0.2 mg 1-J BAP. Each of the regenerated shoots, when transferred to the same medium without agar, yielded about 150 new shoots. He also reported that about 1014 plantlets could be produced in a year from one explant alone. A high concentration (5.0 mg 1-~) of BAP and NAA promoted callus induction and formation of shoots from achenes and petals of C. coccineum, but a high concentration (5.0 mg 1 ~) of 2,4-D inhibited shoot formation (Fujii and Shimzu, 1990). Direct plant regeneration was achieved using stem segments of fresh C. morifolium cv. Royal Purple on MS basal media supplemented with 6-benzyladenine at 0.5-2.0 mg 1 G.R. Rout, P. Das / Scientia Horticulturae 69 (1997)239-257 245 NAA (l-naphthaleneacetic acid). The morphogenic potential varied with the stage of maturity of the stem. The highest percentage of shoot formation (100%) and the greatest average number of shoots per explant (14.6) were observed on segments taken from the apical portion of the stem. Organogenic potential declined in the relatively mature explants (Lu et al., 1990). Sutter and Langhans (1981) compared 9 year old leaf callus of C. morifolium with those of 1 month old callus derived from leaf explants to assess the regeneration ability of long term cultures. Aberrant forms, variable leaf shapes and stunted growth were observed in 15% of them while the remaining 85% were characterised by exessive growth oflateral shoots. Plants regenerated from ray florets of 16 cultivars of Chrysanthemum morifolium were evaluated for somaclonal variations with regard to floral characters and inflorescence morphology. Ohishi and Sakurai (1988) reported that petal tissues of 21 cut-flower cultivars developed callus with ahigher ability to regenerate of adventitious shoots on MS medium supplemented with 10 mg 1 ~ IAA, 10 mg 1-~ BAP and 0. 1 mg 1-~ kinetin. Kaul et al. (1990) reported that adventitious shoots were regenerated from leaf and stem explants of 11 chrysanthemum cultivars on MS basal medium supplemented with 5 mM BAP and 5 mM NAA. They also noted that stem explants were superior to leaf explants and there were wide variations between the cultivars in their shoot regeneration frequency. Shoots on stem explants originated mainly from cortical cells which rapidly divided and ruptured the epidermis. Miyazaki and Tashiro (1978) reported that stem segments of Chrysanthemum morifolium cv. Kayono-sakura derived from young plants (9 week old) produced adventitious shoots more readily than those from older plants (19 weeks). They also observed that segments taken during the winter regenerated more shoots than those taken during spring or summer. The response of segments from different internodes varied with the concentrations of the growth substances used. The number of explants that formed shoots declined as the length of the stem section increased. The leaf explants produced higher pyrethrin content than stem and other explants (Paul et al., 1988) but pyrethrin synthesis decreased when the culture was kept for a longer period (Ravishankar et al., 1989; Zieg et al., 1983). Staba et al. (1984) recorded the highest pyrethrin content in liquid cultures kept for one week in the dark and 2 weeks in the light. Pyrethrin synthesis was also high between the mid and late exponential growth phases. The maximum accumulation occurred at the end of the exponential phase (about 21 days after incubation). A time-course study on tissue growth and pyrethrin synthesis showed that a combination of 0.5 mg 1 ~ each of 2,4-D and BAP resulted in the best callus growth and the highest pyrethrin yield (Krishna et al., 1993). Histological examination of regenerating explants has shown that adventitious shoots arose from different tissues, depending on the initial explant. On stem explants, shoots originated from cortical cells (Miyazaki et al., 1976; Kaul et al., 1990), whereas with flower pedicels individual epidermal cells were identified as the sole progenitors of shoots (Broertjes et al., 1976), 3.3. OL,ule culture The ovule culture technique provides an effective means for the production of hybrids between diploids and polyploids in chrysanthemum. Watanabe (1977) compared shoot 246 G.R. Rout. P. Das / Scientia Horticulturae 69 (1997) 239 257 bud regeneration from ovules with that from other culture systems. He also reported that ovary culture has been employed to overcome hybrid sterility and success was achieved in obtaining F l hybrids and androgenic polyhaploids. 3.4. Protoplast culture Chrysanthemum protoplasts were isolated from a white spider cultivar and other two breeding lines by Schum and Preil (1981) and they succeeded in developing green calli from these protoplasts. Sauvadet et al. (1990) produced callus colonies from protoplasts. They observed colony formation in 16, and division in 18, out of 29 of the clones studied. Callus was obtained from colonies of five clones but buds were produced only in one case. The regeneration potential was retained over a prolonged culture period and the regenerated plants were successfully grown in the the greenhouse. 4. Somatic embryogenesis The greatest potential for clonal multiplication is through somatic embryogenesis where a single isolated cell can be induced to produce first an embryo, then a complete plant. Somatic embryogenesis in chrysanthemum (Dendranthema grandiflora cv. Iridon, Fortune, Goldmine, Helka and Zest) has been obtained by using leaf mid-rib explants cultured on modified MS basal medium supplemented with 1.0 mg I 1 2,4-D and 0.2 mg 1 ~ BAP (May and Trigiano, 1991). These authors also noted that the induction of somatic embryogenesis depended on the photoperiod and sucrose concentration. The maximum production of somatic embryos was achieved on medium containing 9-18% sucrose when the cultures were incubated first in the dark for 28 days, followed by a period of 10 days in the light. Twelve of the 23 cultivars evaluated produced somatic embryos, but complete plants were recovered only from five of these. The regenerated plants were phenotypically similar to parent plants in growth habit, leaf morphology and flower colour. Pavingerova et al. (1994) reported somatic embryogenesis and regeneration plants after of agrobacterium-mediated transformation of callus of C. rnoriolium cvs. Yellow Spider, White Snowdon, Orange Westland and Mistletoe. 5. Root development The induction of roots in regenerated shoots depends on the composition mineral nutrients and growth regulators in the medium. Hill (1968) reported rooting of shoots on filter-paper bridges in liquid medium without sugar and growth substances. Ben-Jaacov and Langhans (1972) noted that the development of roots only occurred after some elongation of the shoots on MS medium containing BAP and IAA. They also reported that roots were easily induced when the cultures were still in the rotating flask by increasing the concentration of 1AA to 1.0 ppm in the medium. Rooting, however, was not desirable at this stage since it was easier to transfer without roots. Roest and Bokelmann (1975) successfully induced roots in the adventitious shoots of 'Super G.R. Rout, P. Das / Scientia Horticulturae 69 (I 997) 239 257 247 Yellow' and 'Bravo' on liquid MS nutrient media supplemented with 10 7 g ml ~ IAA. In most cases, the shoots and roots developed on a single medium containing 4.4 mM BAP and 5.7 mM IAA. At higher concentrations of auxin (17.1 mM IAA), the shoots failed to regenerate roots (Kaul et al., 1990). Rooting depended on the age of the explant; better rooting was achieved when the shoots (1.5-2.0 cm) were transferred to sterile pots containing 1/2 MS medium without growth regulators (Bush et al., 1976). Tian et al. (1993) reported that 88% of rooting was obtained on media containing 0.01-1.00 mg 1-~ of paclobutrazol. Good rooting of 'Deep Pink' was achieved on half strength basal MS salts supplemented with 0.25 mg I 1 of IBA or IAA (Rout et al., 1996). The maximum percentage of rooting was obtained on 1 / 2 strength basal MS salts supplemented with 0.25 mg 1 i IBA and 2% ( w / v ) sucrose (Fig. 3). The performance of rooting was better in the liquid medium than on solid medium after 7 - 8 days of culture. Rooting was delayed in MS media supplemented with IAA (0.1-0.5 mg 1-1 ) with the formation of callus at the basal end. Chlorosis of the leaves occurred when the shoots were maintained for a longer period on the rooting medium (Rout et al., 1996). Bhattacharya et al. (1990) reported that 90% of explants formed roots with more than 15 roots per shoot of 'Birbal Sahni' in 4 weeks on half strength MS medium supplemented with 0.1 mg 1 ~ IAA 7 days after the transfer (Lu et al., 1990; Kumar and Kumar, 1995). Twelve different chrysanthemum cultivars were rooted within 10 days on 1 / 2 strength basal medium without growth regulators (Kaul et al., 1990). The effects of different levels of salt and sucrose on root development in chrysanthemum were reported (Earle and Langhans, 1974a,b; Roest and Bokelmann, 1975; Sun and Li, 1987; Tian et ai., 1993). There have been very few reports with regard to the role of the culture environment, such as temperature and light, on rooting. Rooting was achieved in 90% of the cultures of 'Deep Pink' rooted with about 2.0 Klux of light, whereas higher light intensities (3.0 Klux) gave a lower rooting percentage (Rout et al., 1996). 6. Transfer of plants to soil Smith et al. (1990) reported a very simple and usetul in vitro procedure to prepare plants for transfer to soil. They concluded that the rooting potential of chrysanthemum shoots could be increased by keeping young shoots in liquid rooting medium containing 1 mg 1-t paclobutrazol in culture vessels that maintained a RH of 100%, 96% or 94% for 4 weeks. Roberts and Smith (1990) successfully and impressively tackled the transplantation problem of in vitro grown plantlets and reported up to 100% rooting using either an agar-solidified MS medium or cellulose plugs which were saturated with liquid culture medium. After transplantation and exposure to an atmosphere of reduced RH, plantlets in sorbarods wilted less but transpired more water than plantlets taken bare rooted from agar. Wardle et al. (1983) reported that chrysanthemum plantlets cultured under low humidity exhibited a high mortality rate and those that survived were smaller and had few roots. Smith (1992) reported that paclobutrazol in the medium reduced the relative humidity of the culture vessel to 94-96%. Paclobutrazol reduced vitrification, increased wax and improved stomatal physiology in chrysanthemum. Smith et al. (1990) reported that the plantlets grown on liquid medium containing 0-3.0 mg 1-~ of various 248 G.R. Rout. P. Das / Scientia Horticulturae 69 (1997) 239 257 growth retardants induced resistance to wilting and inhibited synthesis of gibberellins. Rout et al. (1996) reported high percentage of survival when shoots were rooted in liquid medium (Fig. 4). 7. Clonai stability through tissue culture Skirvin and Janick (1976) were among the first to emphasize the importance of cional variation in genotype improvement of horticultural crops. Sutter and Langhans (1981) reported that the plants derived from long-term cultures showed delayed flowering and the addition of GA 3 (gibberellic acid) or IAA to the culture medium had no effect in restoring normal flowering. Somaclonal variation was more common among in vitro cultured adventitious shoots of many species including chrysanthemum (Skirvin, 1978). Malaure et al. (1991) recently reported that shoots derived from ray florets of 16 cultivars of chrysanthemum showed more variation than plants regenerated from vegetative parts. However, in vitro selection and somaclonal variation are random processes and have yet to be used to achieve specific goals in chrysanthemum improvement. 8. Induction of variability in tissue culture The variability which might be a curse to a propagator could be of much interest to the chrysanthemum breeder. Variability of any type is always welcomed by the breeder (Skirvin, 1978), and with tissue culture it might be possible to isolate improved forms of standard cultivars which have resistance to lodging, pests and diseases. Variability has been reported in different chrysanthemum cultivars through physical or chemical mutagenesis (Jang et al., 1987; Matsumoto and Onozawa, 1989) or low temperature tolerant mutants (Huitema et al., 1986). Dalsou and Short (1987) isolated 16 tolerant clones that were resistant to sodium chloride. Nikaido and Onogawa (1989) were also able to develop mutants that contained higher levels of flavonoids and carotenoids. Malaure et al. (1991) found that somaclonal variation was exhibited in plants regenerated from ray-florets of C h r y s a n t h e m u m m o r i f o l i u m . Morphological changes were also observed in plants derived from petal segments (Ohishi and Sakurai, 1988). Subsequently, Ahloowalia (1992) developed 20 new variants which differed in height, leaf, flower shape and petal size and curvature. 9. Cryopreservation studies All over the world many new cultivars come onto the commercial market annually. However, for breeders and nurseries with a restricted supply of labour and land, it is difficult to maintain many cultivars. An effective method of in vitro storage for chrysanthemum would allow breeders and nurseries to maintain a greater number of cultivars. There are two ways to store plants in vitro; slow growth techniques for embryos and the use of cryopreservation (International Board of Plant Genetic Re- G.R. Rout, P. Das / Scientia Horticulturae 69 (1997) 239-257 249 sources, 1983). Techniques for the induction of slow growth of embryos in chrysanthemum are known (Preil and Hoffmann, 1985; Bajaj, 1986; Fukai et al., 1988). The cryopreservation of callus culture has been reported, but a protocol for easy and reproducible cryopreservation of shoot tips of the chrysanthemum has not yet been standardized (Bannier and Stepenkus, 1976). Fukai and Oe (1990) reported a method for the cryopreservation of chrysanthemum (C. morifolium) shoot tips. Shoot tips were placed into 0.1 mg 1 ~ BAP, 1.0 mg 1-~ NAA, 2% sucrose and 5% dimethyl sulphoxide (DMSO) for 2 days, slowly cooled with a cryoprotectant solution (10% DMSO and 3% sucrose) at a rate of 0.2°C per minute from 0 to 40°C and then immersed and stored in liquid nitrogen. After thawing in warm water, more than 87% of these shoot tips regenerated shoots. Later Fukai et al. (1988) reported the application of cryopreservation techniques and the survival rates of 12 species and two interspecific hybrids of chrysanthemum. The shoot regeneration rates of the frozen shoot tips varied from 94 to 100% depending on the species. They also reported that the shoot tips of chrysanthemum showed high viability even after 8 months of storage in liquid nitrogen. The thawed chrysanthemum shoot tips grew and flowered normally in a greenhouse under natural conditions. 10. Seed development and embryo rescue The technique of embryo rescue has been used successfully in many crops to reduce the generation time (Sanders and Ziebur, 1963) and circumvent post fertilization barriers to recovering interspecific hybrids (Davies, 1960; Tanaka and Watanabe, 1972; Stimart and Ascher, 1974; Watanabe, 1977; Chandler and Beard, 1978; Haghighi and Ascher, 1988). It has also been a useful means for culturing seeds without well developed endosperms (Anderson et al., 1990). Globular and heart shaped embryos could be rescued using Norstog (1973) or Murashige and Skoog (1962) media, respectively. Embryogenic stages have been documented for several chrysanthemum species (Harling, 1951; Nagami, 1961; Tanaka and Watanabe, 1972). Anderson et al. (1990) reported a system for embryo rescue and seed set. The embryos exhibited higher percentages of seed set, germination, and progeny reaching anthesis compared with normal development. The progeny of garden parents developed through embryo rescue were significantly earlier in total generation time than the corresponding non-rescued seedlings. These two techniques have great potential for breeding chrysanthemum and other perennial crops. 11. In vitro development of encapsulated organs Application of synthetic seed technology in the field of micropropagation, storage and transport has been well recognized in several crops (Ganapathi et al., 1994). The shoot tips were encapsulated in different concentrations of sodium alginate to determine the optimum concentration for encapsulation. Among the different gel matrixes tested, MS + 0.1 mg 1 t IBA showed better response for germination in synthetic media. 250 G.R. Rout, P. Das / Scientia Horticulturae 69 (1997) 239-257 About 95% of cultures germinated on media containing MS + 0.1 mg 1 1 IBA within a week. The developing shoots and roots emerged through the alginate matrix (2%) and grew into plantlets in 2 weeks (Fig. 5 and Fig. 6) (Rout and Das, unpublished data). This technique has additional advantages for handling and transportation. Encapsulation may be useful in the conservation of chrysanthemum germplasm. 12. Agrobacterium mediated transformation Plant genetic transformation has already led to a better understanding of mechanisms involved in plant gene regulation (Wising et al., 1988). Gene transfer enables the introduction of foreign genes, or specifically designed hybrid genes, into host plant genomes, thus creating novel varieties with specifically designed characters including resistance to environmental stress, pests and disease (Ahmed and Sagi, 1993). As an alternative to traditional approach to introducing genetic changes, attempts have been made to develop transformation systems using agrobacterium based gene vectors (Hutchinson et al., 1989; Lu et al., 1990; Van Wordragen et al., 1989 and de Jong et al., 1993). Chrysanthemum transformation involved inoculation of leaf pieces of the related species C. indicum (Ledger et al., 1991) and stem explants of C. morifolium (Lemieux et al., 1990; Firoozababy et al., 1991). Susceptibility of chrysanthemum to wild type Agrobacterium strains has been reported (Van Wordragen et al., 1992). However, transformation efficiencies were mostly cultivar-dependent (Van Wordragen et al., 1992). Use of GUS intron reporter gene revealed that low efficiency gene transfer and transient gene expression took place. Lowe et al. (1993) developed a system for producing transformed plants from explants of C. morifolium. The susceptibility of the cultivar Super White to various wild-type strains of Agrobacterium tumefaciens and A. rhizogenesis. Urban et al. (1992) attempted to transform three cultivars of D. grandi.flora. They reported success with 'Iridon' using Agrobacterium EHA 105 (pB 1121) on leaf explants, de Jong et al. (1993) reported enhanced regeneration ability of the leaf explants after co-cultivation with Agrobacterium tumefaciens. Subsequently, Urban et al. (1994) reported an efficient, high-frequency transformation protocol for cv. Iridon and regeneration protocols for cultivars Hekla and Polaris. They evaluated three wild-type strains of A. tumefaciens (Ach5, A281 and Chry5) for tumour production in three cultivars. Chry5 and A281 were significantly more virulent on all three cultivars than was Ach5. Transformed plants of 'Iridon' were obtained using Agrobacterium strain EHA 105, a disarmed version of A281. Transformed shoots regenerated and rooted on medium containing 50 I~g ml-I kanamycin. Vegetatively propagated progeny of transformed plants were identified which expressed GUS activity and contained multiple copies ofthe TSWV (Tomato Spotted Wilt Virus) nucleocapsid gene. 13. Conclusion Here an attempt has been made to summarize the pertinent literature on the application of biotechnology to chrysanthemum (see Table l). A wide range of G.R. Rout. P. Das / Scientia Horticulturae 69 (1997) 239-257 251 Table I In vitro culture of Chr)'santhemum Species/cultivars Explant source Morphogenic response Reference C. morifi*lium C. moriJolium s st Hill, 1968 Ben-Jaacov and Langhans, 1972 C. cinerariaefolium ca C. mori/~)lium C. morifolium C. mori[ollum C. ntori[oliurn cv. Indianapolis C. mor{folium cvs. Blue Bird, Montana, Meladion. Delaware C. mor~)lium cv. Shin Dong C. morifolium C. morifolium cvs. Super Yellow Blanche poitevine C. mor(fblium cv. Bronze Bornh C. morS?~lium cv. Super Yellow C. mori~)lium cv. White spider C. mor(f~lium st s s p, pe st st, if Shoot bud regeneration Organogenesis, plantlet formation Shoot bud regeneration and rooting Plantlet production Organogenesis Root and shoot formation Regeneration of shoot Bud and plantlet production Shoot multiplication, rooting, plantlet formation St differentiation Shoot and root Lee et al., 1979 pt fp Callus formation Shoot and rootregeneration Schum and Preil, 1981 Lazar et al., 1981 sm I Organogenesis Organogenesis Lazar and Cachita, 1982 Slusarkiewick et al., 1982 c Lazar and Cachita. 1983 C. mort/hilum C. hortorum cvs. Pink Camino, Super Yellow, Spider C. mor~lolium cvs. Winter westland, Yellow westland, Dark west[and. Snowdon, Yellow Snowdon, Altis and Blanche Chrysanthemum W C. moril)~lium C. mori]))lium 1 am A plantlet formation organogenesis Multiplication, rooting, transfer to soil Shoot multiplication, plantlet formation Organogenesis Shoot elongation, rooting C. moril)~lium cv. Birbal sahni C. morifolium, C. coccineurn C. mori[olium ab Im Roest and Bokelmann, 1973 Earle and Langhans, 1974a Roest and Bokelmann, 1976 Miyazaki et al., t976 Bush el al., 1976 Wang and Ma, 1978 Dabin and Choiseg, 1983 de Donato and Peruceo, 1984 Chen et al., 1985 Gertsson and Andersson. 1985 St rooting Shoot multiplication and Ahmed. 1986 st st, ab, 1 St Sun and Li. 1987 Widiastoety, 1987 Ahmed and Andrea, 1987 1, s. r Plantlet formation Callus formation Multiple shoots, plantlet formation Callus formation p Callus formation Amaqasa and Kameya, 1989 1 Callus formation, shoot bud regeneration Rademaker et al., 1990 Prasad and Chaturvedi, 1988 252 G.R. Rout. P. Das / Scientia Horticulturae 69 (1997) 239-257 Table 1 (continued) Species/cultivars Explant source Morphogenic response Reference C. coccineum p Fujii and Shimzu, 1990 C. moriJblium s cv. Royal Purple C. morifolium cvs. I. s C. cinerariaefolium I C. moriJblium I, s C. morifi)lium pt C. mor~)lium I Callus formation, shoot bud regeneration, plantlet Iormation and transfer to soil Organogenesis, plantlet and establishment in soil Adventitious shoot formation and rooting Callus proliferation production of pyrethrins Organogenesis, plantlet formation Callus. shoot bud regeneration and rooting Somatic embryogenesis mb Somatic embryogenesis May and Trigiano, 1991 C. hortorum C. mor~)lium St St Corneanu and Corneanu, 1992 Tian et al., 1993 C. morifolium 1 Organogenesis Rooting and field establishment Somatic embryogenesis Shoot bud regeneration, rooting and establishment in soil Organogenesis, rooting and establishment in soil Kumar and Kumar, t995 Lu et al., 1990 Kaul et al., 1990 Zito and Teo. 1990 Bhattacharya et al., 1990 Sauvadet et al., 1990 Sauvadet et al., 1990 cv. Yellow Spider C. mor~)lium cv. lridon Pavingerova et al., 1994 cv. Yellow Spider C. maximum s C. morifi)lium I. s cv. Deep Pink Rout et al., 1996 ab: axillary bud; fp: floral peduncle; sm: shoot meristem; s: stem; st: shoot tip: 1: leaf; r: root; ca: capitulum; pd: pedicel; tin: lateral meristem; if: inflorescences; pt: protoplast; p: petal; pe: petal epidermis; mb: mid-rib. c h r y s a n t h e m u m cultivars were m i c r o p r o p a g a t e d using various explants. High rates of shoot regeneration could be a c h i e v e d from vegetative explants already held in tissue culture. C r y o p r e s e r v a t i o n and e m b r y o rescue are n e w e r techniques which have potential for c o m m e r c i a l exploitation. Protoplast manipulation and transformation techniques are well a d v a n c e d and m o l e c u l a r b i o l o g y indicate genes showed promise for i m p r o v i n g c h r y s a n t h e m u m . Field trials of transgenic plants are under way to d e v e l o p strategies for g r o w i n g such plants c o m m e r c i a l l y . Studies o f foreign gene expression in chrysanthem u m , h o w e v e r , have been h a m p e r e d to s o m e extent by the p o o r level of expression o f the inserted genes. Studies to find non-constitutive promoters specific to c h r y s a n t h e m u m are therefore very important. Integration o f foreign genes appears to o c c u r at r a n d o m sites, via n o n - h o m o l o g o u s recombination. A l t h o u g h this has not yet caused any serious p r o b l e m in the transgenic plants, studies on site-specific integration should continue. Such specific integration w o u l d allow the targeting o f genes to aparticular site on the g e n o m e and allelic r e p l a c e m e n t of an existing gene with an e n g i n e e r e d alternative. Such targeting w o u l d G.R. Rout, P. Das / Scientia Horticulturae 69 (1997) 239-257 253 result in a more reproducible quantitative and qualitative expression from a particular transfer (De Block, 1993). Regardless of the approach used, a clear understanding of the processes leading to differentiation and morphogenesis in higher plants is essential, both in terms of general knowledge and for practical purposes of chrysanthemum improvement. Acknowledgements The authors wish to acknowlege the Department of Biotechnology, Government of India, for financial assistance. The authors wish to thank to John Mottley, Department of Life Sciences, University of East London, UK, for editing the manuscript. References Ahloowalia, B.S., 1992. In vitro radiation induced mutants in Chrysanthemum. Mutation Breeding News Lett., 39: 6. Ahmed, H.A., 1986. In vitro regeneration and propagation of meristem apices of chrysanthemum. Kerteszetiegyetem-Kozlemenyei.. 50: 199-214. Ahmed, H.A. and Andrea, M., 1987. Effect of Chrysanthemum multiplication by meristem-tip culture. Acta Hort., 212: 99-98. Ahmed, K.Z. and Sagi, F., 1993. 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