In Vitro Cellular & Developmental Biology - Plant (2018) 54:637–641
https://doi.org/10.1007/s11627-018-9935-9
PLANT TISSUE CULTURE
Micropropagation of Glossonema varians (Stocks) Benth. ex Hook.f.—a
rare Asclepiadeae of Indian Thar Desert
Aarti Paliwal 1 & Narpat S. Shekhawat 1 & Harchand R. Dagla 1
Received: 18 February 2018 / Accepted: 30 August 2018 / Published online: 3 October 2018 / Editor: Wenhao Dai
# The Society for In Vitro Biology 2018
Abstract
In the present study, an in vitro regeneration protocol for Glossonema varians (Stocks) Benth. ex Hook.f. of family
Asclepiadaceae was optimized. Cotyledonary nodes of in vitro cultured seedlings were used as explants for activation of axillary
shoot buds. Axillary shoot buds were initially activated on 0.1 mg L−1 6-benzylaminopurine (BAP) and then multiplied on
0.05 mg L−1 BAP. Shoots were rooted in vitro on 1/4 strength Murashige and Skoog medium containing 0.1 mg L−1 2naphthoxyacetic acid and 100 mg L−1 activated charcoal. The cultures were maintained in a 12 h photoperiod at 40–
50 μmol m−2 s−1 spectral flux photon, 25–30 ± 2°C, and 60% relative humidity (RH). Up to 80% of in vitro regenerated plantlets
were acclimatized on soilrite in cotton-plugged culture tubes in the greenhouse. This protocol can be a useful method to mass
propagate and conserve this rare plant to balance biodiversity in the desert ecosystem.
Keywords Glossonema varians . Rare plant . Dodha . Cotyledonary node . In vitro regeneration
Glossonema varians (Stocks) Benth. ex Hook.f. of the family
Asclepiadaceae is a rare plant of Indian Thar Desert and is
locally known as ‘Dodha’ (Fig. 1A). The plant is also distributed in Bahrain, Iran, Pakistan, and Saudi Arabia. Propagation
and conservation of this rare plant are needed to balance the
biodiversity in the desert ecosystem. Plant tissue culture techniques facilitate the understanding of the physical and chemical requirements and the growth, development, and regeneration of plantlets from this species (Dagla 2012). It also plays
an important role in the conservation and vegetative mass
propagation of plants with poor seed setting and viability.
Micropropagation and molecular characterization of plants
of the Indian Thar Desert have been reported by Dagla and
Shekhawat (2005a, b), Dagla et al. (2007, 2012, 2014), Gehlot
et al. (2014), Nair and Dagla (2016), Upendra and Dagla
(2016), Goswami and Dagla (2017), Jukanti et al. (2017),
Upendra et al. (2017), and Nair et al. (2018). Plantlet regeneration through callus differentiation of Glossonema edule, a
* Harchand R. Dagla
hrdagla@gmail.com
1
Plant Biotechnology and Molecular Biology Laboratory,
Biotechnology Unit Department of Botany, Jai Narain Vyas
University, Jodhpur, India
critically endangered desert plant of Qatar, has also been reported by Al Hadidi et al. (2017). Plants regenerated through
axillary shoot bud activation are more reliable clones than the
plants regenerated through callus differentiation. The chances
of somaclonal variations are more in plants regenerated
through callus differentiation than axillary shoots. The present
investigation aimed to develop a reliable and reproducible
method for axillary shoot multiplication and in vitro plant
regeneration of Glossonema varians. Establishment of in vitro
cultures and regeneration of plantlets through mature shoot
segments were not possible due to the rare availability of this
plant in natural habitats. In vitro cultured seedlings were used
as a source of cotyledonary nodes for shoot multiplication and
plantlet regeneration. In vitro developmental biology of
G. varians may be a useful method for characterization, mass
propagation, and conservation of this rare plant.
Murashige and Skoog (MS) medium (Murashige and
Skoog 1962) containing 3% (w/v) sucrose and 0.8% (w/v)
bacteriological agar type I (HiMedia®, Mumbai, India) was
used for in vitro culture of G. varians. The pH of the medium
was adjusted to 5.8 with 0.5 N NaOH and 0.1 N NaCl. The
medium was autoclaved (Nat Steel Equipment Pvt. Ltd.,
Mumbai, India) at 121°C and 1.06 kg cm−2 for 15 min. The
chemicals used in MS medium were procured from
HiMedia®, and plant growth regulators were procured from
Loba Chemie Pvt. Ltd., Mumbai, India.
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PALIWAL ET AL.
Figure 1. Glossonema varians (Stocks) Benth. ex Hook.f. A In natural habitat. B Flower twig. C Follicles. D Seeds.
Mature follicles (fruits) of G. varians were collected from
Jaisalmer of western Rajasthan, India. These were pretreated
with 70% (w/v) ethanol for 30 s and surface-sterilized with
0.1% (w/v) aqueous solution of HgCl2 for 3–4 min, followed
by 4–5 washes with sterile water. Surface-sterilized follicles
were incised longitudinally, and the seeds were removed. The
excised seeds were inoculated onto 15 mL of hormone-free
semi-solid MS medium in 25 × 150 mm cotton-plugged culture tubes (Borosil Hindustan National Glass Industries Ltd.,
Haryana, India) for germination. The cultures were incubated
under a 12-h photoperiod at 40–50 μmol m−2 s−1 SFP (spectral flux photon) from white fluorescent tube (Philips India
Ltd., Mumbai, India) at 25–30 ± 2°C and 60% relative humidity (RH).
Cotyledonary nodes of in vitro cultured seedlings were
inoculated onto MS medium containing 0.1 mg L −1 6benzylaminopurine (BAP), 3% (w/v) sucrose, and 0.8%
(w/v) agar for activation of shoot buds. In vitro-produced axillary shoots were cultured further on MS medium containing
0.0, 0.05, 0.1, 0.2, or 0.5 mg L−1 BAP in either 400 mL
MICROPROPAGATION OF GLOSSONEMA VARIANSIN VITRO CELL.DEV.BIOL.—PLANT (2018) 54:637–641
polypropylene caped glass culture bottles (Kasablanka,
Bengaluru, India) containing 50 mL of medium or cottonplugged 100 mL Erlenmeyer flasks (Borosil Hindustan
National Glass Industries Ltd.) containing 30 mL of medium
for optimization of shoot amplification.
Shoots were separated from clumps and inoculated onto 1/4,
1/2, and full-strength MS medium containing
100 mg L −1activated charcoal, and 0.0, 0.01, 0.02, or
0.5 mg L−1 indole-3-butyric acid (IBA) or 2-naphthoxyacetic
acid (NOA) for induction of roots.
In vitro regenerated plantlets were removed from culture
vessels and washed with running tap water to remove adhered
nutrient media from the roots. The plantlets were then transferred to polypropylene caped culture bottles or cottonplugged tubes (25 × 150 mm) containing autoclaved soilritemix (perlite, peat moss, and vermiculite in equal ratio)
(Keltech Energies Ltd., Bangaluru, India) and moistened with
1/4 strength MS salts. The culture bottles and tubes were
initially placed at 70–80% relative humidity and 25–30
± 2°C near the water-cooled pad end of the greenhouse,
and MS salts were supplied to the plantlets regularly
after 8–10 d. Caps of the culture bottles were loosened
15 d after transfer of the plantlets to soilrite. Plantlets
Figure 2. Glossonema varians
(Stocks) Benth. ex Hook.f.: A
Seedling on hormone-free MS
medium. B Shoot multiplication
on MS medium (Murashige and
Skoog 1962) containing
0.1 mg L−1 6-benzylaminopurine
(BAP). C Bunch of shoots
cultured on MS medium
containing 0.05 mg L−1 BAP. D
In vitro root induction from
shoots on 1/4 strength MS
medium containing 0.1 mg L−1 2naphthoxyacetic acid and
100 mg L−1 activated charcoal. E
Acclimatized plantlets.
639
were gradually shifted (1.2–1.5 m) towards the fan side
of the greenhouse. Hardened plantlets were transferred
to polybags containing sand, garden soil, and organic
manure in a 3:1:1 ratio for field transfer.
The experiments were set up in a randomized block design
and repeated three times. Each value for mass multiplication
of shoots and root induction containing IBA, NOA, and various strengths of MS medium represented an average number
of five replicates using 25, 30, and 15 explants, respectively.
The data were analyzed statistically using SPSS v.17 (SPSS,
Chicago, IL). The significance of differences among the
means was carried out using Duncan’s multiple range test at
p < 0.05 (Duncan 1955).
Seeds were germinated after 8–10 d of inoculation on hormone free MS medium (Fig. 2A). Axillary shoots buds were
activated from cotyledonary nodes after 1 wk of culture on
MS medium containing 0.1 mg L−1 BAP. Shoots multiplied
on 0.1 mg L−1 BAP were hyperhydrated, thin, and pale yellow
with small leaves (Fig. 2B). Optimum growth of shoots was
obtained on MS medium supplemented with 0.05 mg L−1
BAP at a temperature of 25–30 ± 2°C in Erlenmeyer flask
(Fig. 2C and Fig. 3). In the present experiment, cottonplugged Erlenmeyer flasks were found to be more suitable
640
PALIWAL ET AL.
Shoot number and Shoot length (cm)
7
Shoot number
6
Shoot length
5
4
3.8 a
3.8 a
3 ab
3.6 a
3
2.4 b
2.6 b
2
2.2 bc
1.6 cd
1.1 c
1
0.8 d
0
0
0.05
0.1
BAP (mgl-1)
0.2
0.5
Figure 3. Glossonema varians (Stocks) Benth. ex Hook.f. Mass multiplication of shoots on MS medium (Murashige and Skoog 1962) containing various concentrations of 6-benzylaminopurine (BAP). The observed
data were analyzed by one-way ANOVA and Duncan’s multiple range
test (p < 0.05), and each value represents an average number of five replicates using 25 explants. Different letters represent statistically significant differences with standard deviation error bars.
for optimum growth than culture bottles. Shoots cultured in
bottles were hyperhydrated and yellowish.
Among various concentrations of IBA and NOA used
for induction of roots, 0.1 mg L−1 NOA was found to
be most suitable (Table 1, Fig. 2D). One-fourth strength
MS medium proved to be optimal for induction of roots
(Fig. 4). About 53% of shoots were rooted after 4 wk
of inoculation (Table 1). It is a common practice to
transfer shoots from a higher strength to less concentrated solutions to induce rooting (Bopana and Saxena
2008). In Lavandula vera (Andrade et al. 1999) and
Dendrocalamus longispathus (Saxena and Bhojwani
Table 1 In vitro root induction
from shoots of Glossonema
varians (Stocks) Benth. ex
Hook.f. on 1/4 MS medium
(Murashige and Skoog 1962)
containing various concentrations
of auxins and 100 mg L−1
activated charcoal
1993), the rooting frequency was higher when shoots
were rooted on low-strength MS medium. The positive
effect of a reduced strength of MS salts on root induction is that the concentration of nitrogen ions needed for
root formation is much lower than for shoot formation
and growth (Driver and Suttle, 1987).
Higher moisture content was found to be detrimental
for the survival of plantlets in culture bottles. Cottonplugged culture tubes were found to be more suitable
for hardening of in vitro regenerated plantlets than bottles. In Thapsia garganica, improved ventilation of culture vessels by using lids with holes plugged with cotton wool significantly improved acclimatization
(Makunga et al. 2006). In the present study, about 70–
80% of in vitro regenerated plantlets were hardened
successfully when grown on soilrite in culture tubes
(Fig. 2E ). The present in vitro regeneration protocol
of G. varians will be an efficient method for mass
propagation and maintenance of this species in the desert ecosystem.
Conclusions
Axillary shoot buds of cotyledonary nodes were activated
successfully on 0.1 mg L−1 BAP and then multiplied on
0.05 mg L−1 BAP. One-fourth strength MS medium containing 0.1 mg L−1 NOA and 100 mg L−1 activated charcoal was
suitable for induction of roots. In vitro regenerated shoots
were hardened efficiently on soilrite in cotton-plugged culture
tubes.
IBA (mg L−1)
NOA (mg L−1)
Shoot
response (%)
Roots per node
Mean ± SD
Root length (cm)
Mean ± SD
0.0
0.01
0.05
0.1
0.2
0.5
–
–
–
–
–
–
–
–
–
–
–
0.01
0.05
0.1
0.2
0.5
0
20
40
46.66
46.66
40
20
46.66
53.33
40
20
0.00 ± 0.00d
1.07 ± 0.26c
1.53 ± 0.52bc
1.73 ± 0.46bc
1.73 ± 0.46bc
1.07 ± 0.46c
1.20 ± 0.41bc
1.80 ± 1.08b
2.53 ± 1.81a
1.80 ± 1.21b
1.60 ± 0.83bc
0.00 ± 0.00g
0.37 ± 0.12de
0.41 ± 0.15bc
0.44 ± 0.12bc
0.29 ± 0.10e
0.19 ± 0.08f
0.41 ± 0.22bcd
0.49 ± 0.12b
0.65 ± 0.15a
0.31 ± 0.18de
0.29 ± 0.12e
Means followed by the same letter within columns are not significantly different (p < 0.05) using Duncan’s
multiple range test, and each value represents an average number of five replicates using 30 explants
IBA, indole-3-butyric acid; NOA, 2-naphthoxyacetic acid
MICROPROPAGATION OF GLOSSONEMA VARIANSIN VITRO CELL.DEV.BIOL.—PLANT (2018) 54:637–641
3.50
Root number and Root length (cm)
Root number
3.00
Root length
2.53 a
2.50
2.00
1.50
1.20 b
1.40 b
1.00
0.50
0.65 a
0.28 b
0.33 b
Full
Half
Strength of MS medium
0.00
One-fourth
Figure 4. Glossonema varians (Stocks) Benth. ex Hook.f. In vitro root
induction on various strengths of MS medium containing 0.1 mg L−1 2naphthoxyacetic acid and 100 mg L−1 activated charcoal. The observed
data were analyzed by one-way ANOVA and Duncan’s multiple range
test (p < 0.05), and each value represents an average number of five replicates using 15 explants. Different letters represent statistically significant differences with standard deviation error bars.
Acknowledgments The authors acknowledge the Department of
Biotechnology (DBT), Government of India, for establishment of regional facilities for Micropropagation and Hardening of plants of arid regions
in the Department of Botany, Jai Narain Vyas University, Jodhpur, India.
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Narpat Shekhawat