Indian Journal of Biotechnology Vol 10, April 2011, pp 228-231
Establishment of a rapid multiplication protocol of Coleus forskohlii Briq. and in vitro conservation by reduced growth Pratibha Dube1, Moumita Gangopadhyay1, Saikat Dewanjee2 and M Nasim Ali1* 1
IRDM Faculty Centre, Ramkrishna Mission Vivekananda University, Ramkrishna Mission Ashrama Narendrapur, Kolkata 700 103, India 2 Advanced Pharmacognosy Research Laboratory, Department of Pharmaceutical Technology Jadavpur University, Kolkata 700 032, India Received 14 January 2010; revised 2 August 2010; accepted 5 October 2010
Coleus forskohlli Briq., an endangered medicinal plant, has been successfully micropropagated under in vitro conditions. Best adventitious shoots were developed in solid Murashige and Skoog’s medium supplemented with 1.5 mg L-l 6-benzylaminopurine, while best rhizogenesis was achieved without growth regulator. The second event of success was in vitro conservation of the species by slow growth technique employing osmotic regulators (sorbitol and mannitol). 3 M mannitol showed best performance for reducing growth. This study elucidated an efficient method of ex situ conservation of germplasm and sustainable management of this medicinally important plant. Keywords: Coleus forskohlii, in vitro conservation, mannitol, micropropagation, slow growth
Introduction Coleus forskohlii Briq. (Family: Lamiaceae), commonly known as ‘Medicinal Coleus, is a small, perennial, and aromatic herb. Since long back, it is used in traditional Indian medicines for digestive remedy. Roots of this plant are the only source of forskolin, a labdane diterpene1. Forskolin possesses cardiotonic, hypotensive and anti-inflammatory activities2. It has been reported to stimulate the production of cAMP by activating adenyle cyclase3. Due to a wide range of medicinal activities, this plant has increased market demand in both domestic and international level. Increasing market demand coupled with non-restricted harvesting from natural flora, the population of this herb is decreasing alarmingly from the natural habitat, which has necessitated the use of biotechnology in conservation and sustainable management of this endangered plant species4,5. Micropropagation and slow or reduced growth method are the two commonly used in vitro conservation methods. Micropropagation is one of the powerful tools used to conserve elite, rare and endangered species. It is an effective mean for rapid _______________ *Author for correspondence: Mobile: 09475944498 E-mail:
[email protected]
multiplication of species and ex situ conservation. Therefore, rapid and large scale production of medicinal and aromatic plants employing micropropagation has gained significant interest5. The main drawback of plant cell culture is the danger of spontaneous changes. Therefore, continuous culture of plant cells is often undesirable. In this situation, slow or reduced growth technique would be a potential tool for a more efficient and cost-effective rapid propagation system for medium term storage. The principle of reduced-growth storage is based on the manipulation of culture condition by adding growth retarding agents to allow the culture to remain viable but with almost negligible growth rate, which could be targeted for ex situ conservation of plants. The present Study has been designed to develop a rapid multiplication protocol via micropropagation and a novel protocol for in vitro conservation via slow or reduced growth. Materials and Methods Collection of Plant Material
Young branches of C. forskohlii were excised (explants) from Medicinal Plant Garden, Ramkrishna Mission Vivekananda University (RMVU), Narendrapur, West Bengal and was identified by the Taxonomist of Botanical Survey of India, Shibpur, Howrah. The voucher specimen was deposited in the Herbarium of RMVU.
DUBE et al: RAPID MULTIPLICATION AND IN VITRO CONSERVATION OF C. FORSKOHLII
Preparation and Surface Sterilization of Explants
The explants containing apical buds with two axially placed leaves were used as a source for this experiment. They were washed with running tap water to remove adhering dust particles. The explants were then surface sterilized with 1% bavistin and 0.5% streptomycine solution for about 5-7 min. In the end, they were treated with 0.1% murcuric chloride solution for 1-3 min, followed by 3-4 washings with sterile distilled water.
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growth retardants in the culture medium. Cultures were maintained at 22±2°C and 16 h photoperiod. At the end 30 d, the culture was tested for re-growth ability by transferring to growth and regeneration medium. The plantlets resuming normal growth by shoot elongation as well as by sprouting of new shoot buds were considered as viable. Percentage viability, shoot elongation (cm), number of leaves per plant and fresh wt (mg) were recorded. Statistical Analysis
Media and Culture Conditions
After surface sterilization, sterile explants were inoculated on sterilized solid Murashige and Skoog’s (MS) media6 fortified with 3% sucrose. All cultures were maintained at 22±2°C and 16 h photoperiod (light intensity 40-80 µmol m-2 s-1 provided by coolwhite fluorescent tubes, Philips India). After 7 d, the cultures showing axillary bud breaking were transferred to media containing different growth hormones. Organogenesis of Explants
To test the effect of different growth regulators on shoot multiplication, mainly two types of cytokinin were taken into consideration. The MS medium was supplemented with different concentrations (1.0, 1.5 & 2.0 mg L-1) of 6-benzylaminopurine (BAP) and kinetin (Kn). Shoot regeneration (caulogenesis) response to growth hormones at different concentrations were expressed as percentage of responding explants and number of regenerated shoots per explant. For root generation (rhizogenesis), the in vitro regenerated shoots were transferred into MS medium supplemented with different -1 concentrations (0.5 & 1.0 mg L ) of indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). Explants in MS media without phytohormones served as control in both caulogenesis and rhizogenesis studies. The best resulting media for rooting was identified on the basis of first appearance of root (d) and number of roots per shoot.
Each set of experiment consisted of 10 replicates were repeated thrice. The results were calculated as mean±SD. Data were examined by analysis of variance to detect differences (P ≤ 0.05) between the means and compared using Tukey’s Test at the same (5%) probability level using GraphPad (GraphPad Software, San Diego, CA). Results and Discussion Response of Explants to Growth Hormones for Shoot Regeneration
Prior to treatment with plant growth regulators, sterilized C. forskohlii were inoculated in solid MS media. After 7 d, it was noticed that adventitious shoots started originating directly (Fig. 1a) from inoculated explants. Only those cultures showing healthy and non-contaminated plantlets were transferred to media containing different growth hormones and cultured for 30 d. Shoots were originated directly at the axillary position of the nodal explants in presence of cytokinins in the medium. Among two cytokinins (BAP & Kn), BAP at 1.5 mg L-l was found to be superior (Fig. 1b) for shoot regeneration in terms of percentage of response, plant height and number of shoots per explants (Table 1). The beneficial effect of BAP further became evident when large number of shoots gradually formed a compact colony comprising of shoots at different developmental stages (Fig. 1c). Amongst different
Slow or Reduced Growth Technique
Nodal segments were dissected from in vitro regenerated C. forskohlii used as the source material for slow growth experiment. Effect of mannitol and sorbitol at different concentrations (1, 2, 3 & 4 M) on reduced growth was studied. Two set of experiment, one in MS media and another in MS media supplemented with BAP (1.5 mg L-1) were taken for comparing morphological changes due to addition of
Fig. 1—Different developmental stages of micropropagation of C. forskohlii: (a) Shoot regeneration from sprouting shoot buds, (b) emergence of multiple shoots in BAP 1.5 mg L-1, and (c) well established in vitro culture showing root on the medium.
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selected concentrations of BAP, 1.5 mg L-1 was found to be optimum in term of % response (96.7±5.8), shoot length (5.1±0.1 cm) and number of shoots per explants (4.7±0.5). Kn did not produce significant effect on shoot generation as compared with control culture. Response of Explants to Growth Hormones for Root Regeneration
In course of culture, roots appeared from the base of shoots. Best rhizogenesis was achieved in the absence of selected growth regulators in term of first appearance of root (7.7±0.5 d) and number of roots per shoot (5.7±0.5). It is evident from Table 2 that Table 1—Effect of cytokinins on shoot regeneration from nodal explants of C. forskohlli after 30 d culture period Treatment
Conc. (mg L-1)
% response
Shoot height (cm)
No. of shoots per explant
4.6±0.2a, b 2.2±0.4a
Control
0.0
56.7±5.8a
BAP
1.0 1.5 2.0
80.0±10.0b, c, d 4.7±0.1a, b 3.1±0.3b 96.7±5.8b, e 5.1±0.1a, b 4.7±0.5c 86.7±5.8b, e, d 5.0±0.2a 4.5 ± 0.5c
Kn
1.0 1.5 2.0
63.3±5.8a, c 70.0±10.0a, 73.3±5.8a, d
d
3.7±0.3c 3.2±0.4b, d 4.3±0.3a, c 3.8±0.4d 4.1±0.1b, c 3.7±0.5b
The data were represented as mean±SD; means with the same letters are not significantly different at P ≤ 0.05. Table 2—Effect of auxins on rooting of C. forskohlli after 20 d culture period Treatment
Concentration (mg L-1)
First appearance of root (d)
No. of roots per shoot
0.0 0.5 1.0 0.5 1.0
7.7 ± 0.5a 10.6 ± 0.8b 12.2 ± 0.4c 10.1 ± 1.0b 11.6 ± 0.5c
5.7 ± 0.5a 3.9 ± 0.3b 2.3 ± 0.5c 4.1 ± 0.6b 3.5 ± 0.8b
Control IBA IAA
The data were represented as mean±SD; means with the same letters are not significantly different at P ≤ 0.05.
20-d-old culture in MS medium without hormone gave rise to maximum number of roots per shoot within minimum time period. The incidence of highly efficient root formation on auxin-free medium may be due to the availability of higher quantity of endogenous auxin. Direct regeneration, as described in this report is more desirable because this mode of regeneration gives better chance of producing genetically uniform plants. The micropropagation protocol reported herein could be useful for mass multiplication from limited donor plants within a short time. As the mode of regeneration is direct, this protocol was beneficial over callus mediated regeneration7,8. Slow Growth or Reduced Growth Technique
The principle of reduced-growth storage is based on the manipulation of culture condition or culture media to allow the culture to remain viable at a very slow growth rate. In this technique, the growth of explants is controlled by adding growth retarding agents in the medium9. This makes least necessity for sub-culturing explants. Table 3 shows the overall performance of the explants under reduced growth condition for 30 d with two growth retardants (mannitol & sorbitol) at different concentrations. From this study, it was observed that 3 M mannitol in media showed best performance for reducing growth of this plant species in term of height (2.0±0.3 cm), number of leaves (3.3±1.6) and fresh wt (324.6±20.3 mg). The use of mannitol for reducing growth was also reported in cultures of Ipomea batatus10. In test for post-storage viability, 90±10% recovery was observed regardless of storage duration, provided culture medium contained both mannitol and sorbitol. Figs 2a and b present a comparative morphological view of treated and non-treated explants in the same culture period. Prior to resume normal growth, the post-stored shoots were found to pass through a lag phase and then explants responded to regeneration by
Table 3—Effect of various growth retardants on in vitro culture of C. forskohlli after 30 d culture Mannitol Concentrations
1M
Sorbitol 2M
a
3M a
2.2±0.2 5.3±0.6 b, c
4M a
2.0±0.3 3.3±1.6 a
MS
1M a
2.2±0.3 3.7±0.6 a
2M a
3M a
2.3±0.3 2.4±0.2 5.3±0.6c 5.7±0.6c
MS+ BAP (1.5 mg L-1)
4M a
2.2±0.2 4.3±0.6 a, b
a
5.1±0.2 8.7±0.6d
5.2±0.3b 19.0±0.6e
Height (cm) No. of leaves
2.3±0.4 5.0±1.0a, b, c
Fresh wt of plantlets (mg)
385.5±18.6a, c 379.6±12.3a, c 324.6±20.3b 366.4±9.1c 402.6±14.5c 391.2±18.1a 336.8±10.5b 376.7±21.1c 456.1±16.1d 510.8±15.7e
The data were represented as mean ± SD; means with the same letters are not significantly different at P ≤ 0.05.
2.3±0.1 4.0±1.0 a
b
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Acknowledgement The authors thank Swami Viswamayananda and Professor S K Gupta, Chief Scientific Advisor of Medicinal Plant Garden, RMVU, Narendrapur, West Bengal for their generous help and scientific advice. References
Fig. 2—A comparative view of C. forskohlii reduced growth culture: (a) Reduced growth culture in MS media containing 3 M mannitol, (b) control set, grown in basal MS media containing BAP 1.5 mg L-1, and (c) different developmental stages of C. forskohlii after transferring growth retardant treated medium to regeneration medium.
developing axillary shoots at nodal zones in regeneration medium supplemented with BAP 1.5 mg L-1 (Fig. 2c). Present experiment showed that C. forskohlli could be conserved by reduced growth method using osmoticum (mannitol, sorbitol) in the medium. Presence of osmoticum in culture medium, somehow, prevents explants from normal uptake of nutrients, resulting in slow growth. Thus, in vitro slow growth method is a better option for conserving germplasm of plants10. The present protocol of in vitro mass multiplication and slow growth technique holds a huge potential for large scale exploitation of C. forskohlli as well as for the ex situ conservation of this medicinally important plant.
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