Micropropagation of Vegetable Rennet (Withania coagulans[Stocks] Dunal)—A Critically Endangered Medicinal Plant

Abstract

Withania coagulans (Stocks) Dunal (Solanaceae), popularly called vegetable rennet, is a critically endangered and highly valued medicinal plant. Overexploitation and reproductive failure forced the plant species toward the verge of complete extinction. We describe here the development of a simple, rapid, and cost effective in vitro micropropagation system for W. coagulans for mass-scale production of true-to-type plantlets using nodal shoot segments. Exactly 95.5 ± 0.34% explants responded within 8-10 days (d) and produced multiple shoot buds (4.1 ± 0.10 shoots of 2.95 ± 0.15 cm length) on 0.8% agar-gelled Murashige and Skoog's (MS) basal medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), and additives (100 mg L −1 L-ascorbic acid, 25 mg L −1 each citric acid, adenine sulphate, and L-arginine). The shoots in cultures were multiplied by repeated transfer on MS medium with 4.44 μM BAP, 0.57 μM IAA, and additives. Further cultures were multiplied on a large-scale through the subculturing The authors are grateful to 727 Downloaded by [McMaster University] at 09:37 05 November 2014 728 M. S. Rathore et al. of shoot clumps differentiated in vitro, on MS medium supplemented with 1.11 μM BAP, 0.57 μM IAA, and additives. Maximum number (19.1 ± 0.28) of healthy (6.15 ± 0.25 cm) and viable shoots differentiated on this medium. The microshoots were rooted both in vitro and ex vitro. Exactly 67.3 ± 1.01% microshoots rooted in vitro within 25-30 d on agar-gelled half-strength MS salts supplemented with 29.52 μM indole-3-butyric acid (IBA) and 200 mg L −1 of activated charcoal (AC). Alternatively, 73.8 ± 0.65% cloned shoots rooted on sterile soilrite (soilless compost and soil conditioner) under ex vitro conditions after pulse treatment with 2.46 mM IBA for 300 s. The clones of W. coagulans were hardened in a greenhouse within 40-45 d by slow and gradual exposure of plantlets from high relative humidity (RH; 70-80%) and low (26 ± 2 • C) temperature to low and high (34 ± 2 • C) temperature. The hardened plantlets were transferred to soil and stored in agro-net house with more than 90% survival rate. Replacement of pure and laboratory grade sucrose with commercial grade sugar, use of less expensive commercial grade agar-agar in culture medium, higher rate of shoot proliferation, single step ex vitro rooting, and hardening of plantlets in the greenhouse are advantageous features of the protocol. The micropropagation protocol defined here is reproducible, easy to follow, and would be helpful in large-scale restoration programs through true-to-type mass-multiplication of W. coagulans.

Shoot regeneration from leaf explants of Withania coagulans (Stocks) Dunal and genetic stability evaluation of regenerates with RAPD and ISSR markers

“…Poly-gamo-dioecious nature of flowers and self-incompatibility (Gilani et al, 2009) limits the chances of seed setting and the plant species is found in vegetative state under arid conditions (Bhandari, 1990). The natural regeneration rate does not sustain to keep up with the exploitation rate and the plant species is threatened in its habitat and declared critically endangered (Jain et al, 2010;Rathore et al, 2012).…”

“…A number of tissue culture studies have been carried out on W. somnifera, but W. coagulans received little attention so far for biotechnological studies (Mirjalili et al, 2011;Mishra et al, 2012;Kushwaha et al, 2013;Rathore and Kheni, 2015). A few attempts were made on in vitro propagation of W. coagulans using different explants and mode of regeneration (Jain et al, 2009(Jain et al, , 2010Valizadeh, 2009, 2011;Rathore et al, 2012). Plant regeneration techniques are attractive and potential ways for utilization of plant genetic resources as well as their conservation.…”

“…Overexploitation, hostile environmental factors, habitat disturbances and reproductive failure threaten the survival and render the plant species vulnerable to complete extinction (Rathore et al, 2012). Hence harvest of plant material from wild is not a viable option to meet the commercial demand and some other strategies must be devised to meet the requirements.…”

CNS depressant and analgesic effects of Withania coagulans (Stocks) dunal fruits collected from Khuzdar district of Balochistan, Pakistan

“…Withania coagulans (Stocks) Dunal, a shrub from Solanacea family, locally known as local inhabitants [5,6]. The berries from this plant due to the presence of enzyme in husk and pulp are used as milk coagulating agent.…”

In vitro Shoot Multiplication of Withania coagulans (Stocks) Dunal

“…Micropropagation studies on W. cogulans is reviewed recently (Teixeira da Silva et al 2015). Its natural propagation occurs through seeds, which is a slow process and resultant seeds may not germinate in the absence of favourable germination conditions (Rathore et al 2012). …”