In Vitro Propagation of Desmodium gangeticum (L.) DC. from Cotyledonary Nodal Explants
UR Vishwakarma, AM Gurav, PC Sharma
Regional Research Institute (Ay.), Kothrud, Pune - 4110 38 (MH), India
|Date of Submission||09-Dec-2008|
|Date of Decision||29-Jan-2009|
|Date of Acceptance||05-Feb-2009|
|Date of Web Publication||30-Dec-2009|
A M Gurav
Regional Research Institute (Ay.), Kothrud, Pune - 4110 38 (MH)
Source of Support: None, Conflict of Interest: None
| Abstract|| |
An in vitro procedure for rapid multiplication of medicinally important plant Desmodium gangeticum (L.) DC. (Fabaceae), has been developed using cotyledonary nodal explant. An average of 9.2 shoots per explant were obtained by culturing cotyledonary nodal explaint on Murashige and Skoog's medium containing 8.8 μM BAP and 21.2 μM NAA, in combination, within 28 days. These shoots were rooted on half strength MS medium supplemented with IAA 17.1 μM. Rooted plantlets were hardened using 1:1:1 mixture of soil, river sand and vermiculite under green house conditions.
Keywords: Desmodium gangeticum, cotyledonary nodes, regeneration, growth regulators
|How to cite this article:|
Vishwakarma U R, Gurav A M, Sharma P C. In Vitro Propagation of Desmodium gangeticum (L.) DC. from Cotyledonary Nodal Explants. Phcog Mag 2009;5:145-50
|How to cite this URL:|
Vishwakarma U R, Gurav A M, Sharma P C. In Vitro Propagation of Desmodium gangeticum (L.) DC. from Cotyledonary Nodal Explants. Phcog Mag [serial online] 2009 [cited 2021 Dec 4];5:145-50. Available from: http://www.phcog.com/text.asp?2009/5/18/145/57973
| Introduction|| |
Desmodium gangeticum (L.) DC. is an important Ayurvedic medicinal plant, belonging to family fabaceae. It is an erect woody under shrub, 2-4 feet high, grows wild in tower Himalayan regions and throughout India. It is also found in Ceylon, Burma, Malay peninsula and Islands, China, Philippines and Tropical Africa ,,.
Whole plant, mainly roots are used in medicine. The plant is a bitter tonic, digestive, antidysentric, alterative, aphrodisiac, antipyretic, anticatarrhal, febrifuge. It is used to cure typhoid and other fevers, asthma, bronchitis, vomiting, dysentery, piles, biliousness, chorela, scorpion sting and snake bite. The roots of Desmodium gangeticum (L.) DC. are used as one of the ingredients of two very important Ayurvedic preparations, 'Dashmoola Kwatha' and `Dashamootarishta' , .
It is identified as promising plant, which is in great demand and of high commercial potential. Estimated domestic demand for Desmodium gangeticum (L.) DC. is about 678.4 tonnes/year  . The drug, Desmodium gangeticum (L.) DC. is mostly collected from wild sources to meet the requirement of pharmaceutical industries, as such no efforts have yet been made towards its cultivation except Dhan prakash et at  . Department of Indian Systems of Medicine and Homeopathy, Ministry of Health and Family Welfare, Government of India, has formulated a Central Scheme for Cultivation and Development of Medicinal Plants. Desmodium gangeticum (L.) DC. is one of the species identified for promoting cultivation in order to reduce pressure on their natural habitat and to meet the shortage against the demand of the industry  . Efforts on propagation of Desmodium gangeticum (L.) DC. by seed and stem cuttings have been successfully made , . There is no report on tissue culture studies of D. gangeticum (L.) DC., though such studies on other species of Desmodium have been reported such as D. Heterocarpon (L.) DC. and D. ovatifotium Wall.  . Therefore, efforts were made to evolve the techniques for rapid multiplication through tissue culture.
| Material and Methods|| |
Fresh mature seeds of Desmodium gangeticum (L.)DC., were collected from healthy plants growing in garden of Regional Research Institute, Pune, India. The plant was identified with the help of Flora of British India and confirmed by comparing the authenticated herbarium specimen available in Botanical Survey of India, Western Circle, Pune. Herbarium was prepared, deposited in the herbarium section of the Institute with voucher specimen number 215. Seeds were treated with concentrated H 2 SO 4 for 15 minutes to facilitate germination and thoroughly washed under tap water. The seeds were then soaked in 5% Teepol solution for 5 minutes and washed again under tap water. Later, the seeds were treated with 0.1% HgCl 2 solution for I minute followed by several times washing with sterile distilled water under aseptic condition and inoculated on MS plain medium containing 3 percent sucrose The pH of medium was adjusted to 5.7 with 1N NaOH/1N HCl, before addition of 0.8 percent agar and autoclaved at 15 tb/Inch 2 pressure and 121° C temperature for 20 minutes. In the initial stage of seed germination, cultures were kept in dark at 25° C and 92% humidity, in Environmental test chamber, for 4-5 days. The cultures were, then transferred to culture room, where they were maintained at 25° C+ 2° C and 8/16- h (light/dark) photoperiod provided through white fluorescent tubes with light intensity of 3000 lux.
After germination of seeds, cotyledonary nodes of 11.5 cm length with both cotyledons were excised from 14 days old seedlings and cultured on MS medium  supplemented with BAP and Kn, singly or in combination with IAA or NAA, in different concentrations, by embedding 2-3 mm of the epicotyle end of the embryonic axis in the medium.
Well developed shoots regenerated from cotyledonary nodal explants were excised and rooted on half and full strength MS plain medium supplemented with or without IAA and I BA, singly, as well as in different concentrations.
Observations were made every day. Final observation was taken on 28 th day, with respect to number of cultures responded for callusing, shooting and rooting, average number of shoots per explaint, average height of the shoot, average number and length of roots per shoot. All the experiments were repeated thrice with 24 replicates. Data obtained from the experiments were analyzed for mean and standard error using software SYSTAT 11 (Stat Soft Inc.,).
Plantlets with well developed roots were removed carefully from culture tubes and washed under running tap water to remove medium sticking to the surface. Then the plantlets were dipped in 1 aqueous solution of Bavistin for 10 minutes and washed with water. The treated plantlets were transferred to small plastic pots containing sterile soil: river sand: vermiculite (1:1:1). The pots were covered with plastic bags to maintain high humidity, for 14-15 days. The plants were sprayed with half strength MS salt solution twice a week for a period of 2 weeks. Then the hardened plants were transferred to green house. Well grown plants were shifted to bigger earthen pots containing garden soil and farm yard manure, after 2-3 months and watered on alternate days.
| Results and Discussion|| |
Effect of phytohormones on shoot regeneration from cotyledonary nodes
Cotyledonary nodal explants with both cotyledons were excised from in vitro grown seedlings were cultured on MS medium supplemented with BAP and Kn, singly or in combination with IAA or NAA, in different concentrations, to find out their influence on shoot regeneration[Figure 1]A. Cotyledonary nodal explants inoculated on MS plain medium showed callus formation, root initiation at cut end dipped in the medium and regeneration of 1-2 shoots, directly from axillary buds, at the end of 28 days. Cotyledonary nodes, which were inoculated on MS medium supplemented with different concentrations (4.6-18.4 µM) of kn also showed response towards shooting and callusing but failed to improve the number of proliferating shoots and on an average 2.7 shoots per explant were regenerated from cotyledonary nodal explants directly on MS+ Kn (9.2 µM) [Table 1].
However incorporation of BAP in the medium improved shoots proliferation. The number of shoots per explant was highest on MS medium containing BAP (8.8µM), singly, where over 3 shoots of 0.5 cm were regenerated from cotyledonary explants after 28 days. The number of shoots per explant increased with increase in concentration of BAP up to optimal level (8.8 µM). However increase in concentration of BAP resulted in decrease of shoot length. The number of shoots per explant declined when the concentration of BAP was increased beyond 8.8 µM [Table 2]. This supports the findings of Gulati and Jaiwal  and Cheng et at  . Effectiveness of BAP in shoot regeneration from cotyledonary nodes has been reported in several other species of leguminaceae e.g. Datbergia tatifotia Roxb.  , Arachis hypogea L.  , Vigna mungo (L.) Hepper  , Acacia nitotica subsp. indica Brenan  and Stercutia urens Roxb.  . BAP was found to be more effective than Kn when added in MS medium, either in combination with IAA or NAA, in different concentrations. Addition of IAA or NAA in combination with Kn did not improve the number of shoots per explant except that the length of shoot was increased [Table 3] and [Table 4]. Highest number of shoots i.e., 2.3 was observed in explants inoculated on MS+ Kn (9.2µM) + IAA (5.7µM), whereas when BAP in combination with IAA or NM was incorporated in MS medium the number of shoots per explant was considerably increased. However, the number of shoots per explant was relatively lower on the medium supplemented with BAP + IAA as compared to BAP + NM [Table 5] and [Table 6]. Highest number of shoots (9.2) per explant was obtained on MS+BAP (8.8 µM) + NM (21.2µM), but these shoots failed to elongate, average shoot length being 0.8 cm only [Figure 1]B. Duhoux and Davies  reported regeneration of multiple shoots in Acacia albida Define. from cotyledonary nodes inoculated on MS medium containing BAP and NM in combination.
Effect of phytohormones on in vitro rooting
Root initiation was observed in shoots of Desmodium gangeticum (L.) DC. inoculated on both full strength and half strength MS medium supplemented with or without IAA or IBA [Table 7] and [Table 8]. IAA was found to be the best for rooting.100 percent rooting with maximum average number (10.3) of roots were obtained in shoots inoculated either on half strength or full strength MS medium supplemented with IAA (17.1µM) [Figure 1]C. Root initiation was found earlier in shoots inoculated on half strength MS medium either supplemented with IAA or IBA.
The plantlets were transferred to small plastic pots containing sterile soil: river sand: vermiculite (1:1:1) for hardening [Figure 1]D. Fully developed rooted plantlets were transferred to field after acclimatization (hardening), where they showed 80% survival rate. This efficient and simple protocol reported herein could be useful for rapid multiplication of this medicinally important plant.
| Acknowledgements|| |
The authors thank Central Council for Research in Ayurveda and Siddha, New Delhi, India for providing working facilities and encouragement for the present work.
| References|| |
|1.||Anonymous, The Wealth of India (Raw materials),Vol (D-E), (CSIR, New Delhi, 1952) 41-43. |
|2.||J.D.Hooker, Flora of British India,Vol.2, Reprinted edition, (Periodical Experts, Delhi, 1973) 168. |
|3.||T. Cook, The Flora of Presidency of Bombay,Vol.13,(Botanical Survey of India, Calcutta,1967)3:79-380. |
|4.||K.R. Kirtikar and B.D. Basu , Indian Medicinal Plants,Vol.1 ,(Lalit Mohan Basu and Co., India,1935) 204-212,758-760. |
|5.||R. N. Chops, S. L. Nayar and L.C. Chops. Glossary of indian Medicinl plants,(CSIR, New Delhi, 1956) 94. |
|6.||Anonymous, Agenda for the meeting of medicinal plants board, (Annexures), Sep.2001, Vol.2(Dept. of ISM and H, Ministry of Health and Family Welfare, Govt. of India, New Delhi,2001) 910,38,44, 48-50, 55,65. |
|7.||P.Dhan, A. Niranjan and S.K.Tewari. Chemistry of D. gangeticum cultivated on sodic soil. Journal of Medicinal and Aromatic Plants Science 22/4A and 23/1A: 21-25(2000). |
|8.||G.S. Rawat and A.K. Sharma, Stratrergies for the conservation medicinal plants in the Himalayas, In Prosects of Medicinal Plants, (Indian Society of Plant Genetic Resources, N. Delhi, 1998) 29-36. |
|9.||U.R. Vishwakarma, M.B.Yelne and P.C.Shanna. Effect of various treatments on seed germination of Desmodium gangeticum (L.) DC.(Shalapami). BMBER.20(1-4):85-91(1999). |
|10.||U.R. Vishwakanna, M.B. Yelne and P.C. Sharma. Vegetative propagation of Desmodium gangeticum (L.) DC.(Shalapanri) by stem cuttings. BMBER.24(1-4):110-120 (2003). |
|11.||D.S.Wofford, K.H. Quesenbeny and D.D. Balensperger. Tissue culture regeneration of Desmodium. Crop. Sci. 32: 266-268 (1992). |
|12.||T. Murashige and F. Skoog. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiology Plant. 15 473-497 (1962) |
|13.||A.Gulati and P.K.Jaiwal . Plant regeneration from cotyledonary node explant of mungbean (Vigna radiata(L.) Wiiczek.). Plant Cell Reports13:523-527 (1994). |
|14.||T.Y.Cheng, H. Saka and H. V. Thank. Plant regeneration from soyabean cotyledonary node segments in culture. Plant Science Letters 19: 91-99 (1980). |
|15.||C. Pradhan, S. Pattnaik and P.K Chand. Rapid in vitro propagation of East Indian Rosewood (Dalbergia latifolia Roxb.) through high frequency shoot proliferation from cotyledonary nodes. J. Plant Biochemistry and Biotechnology 1: 61-64(1998). |
|16.||P. Venkatachalam, N. Geetha, K.S Rao, N. Jayabalan and S. Saravanababu. BAP - regulated direct shoot organogenesis from cultured seedling exlants of ground nut (Arachis hypogea L.). Indian Journal of Experimental Biology 37: 807-812 (1999). |
|17.||S. Ignacimuthu, G. Franklin and G Melchias. Multiple shoot formation and in vitro fruiting from cotyledonary nodes of Vigna mango (L.) Hepper. Current Science 73 (9): 733-735(1997). |
|18.||Dewan, K. Nanda and S.C. Gupta. In vitro micropropagation Acacia nilotim subsp. indica Brenan via cotyledonary nodes. Plant Cell Reports 12:18-21(1992) |
|19.||S.D. Purohit, A Dave and G kukda. In vitro conservation strategies for a rare medical herb safed musli (Chlorophytum. borivilianum Sant. et. Femand.). Indian Journal of Plant Genetic Resources 7(2): 201-204 (1994). |
|20.||E. Duhox and D. Davis. Shoot production from cotyledonary buda Acacia albida (synonym Faidherbia albida) and influence of sucrose on rhizogenesis. J. P1. Physiol. 121:175-180 (1985). |
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]