An efficient protocol for in vitro propagation of the wild legume Cicer microphyllum Benth., a crop wild relative of chickpea (Cicer arietinum L.)
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Cicer microphyllum Benth. is a wild legume that is adapted to the extremely adverse climatic conditions of the cold Himalayan deserts. This rare species is a crop wild relative (CWR) that could be used for chickpea (Cicer arietinum L.) breeding programs to improve abiotic stress tolerance. However, the availability of C. microphyllum biological material is very limited due to its high altitude and remote location. These factors make in vitro plant tissue culture an excellent alternative for the conservation and propagation of this species. In this study, an efficient and reproducible in vitro regeneration protocol was established for C. microphyllum. This process enabled the regeneration of multiple shoots through indirect organogenesis from excised embryos. In addition, the effects of combinations of auxin and cytokinin concentrations in Murashige and Skoog (MS) medium were tested. The highest percentage of callus induction was observed on MS with α-Naphthaleneacetic acid (NAA) and 6-Benzylaminopurine (BAP), and the addition of AgNO3 to MS with NAA and BAP significantly improved shoot morphogenesis. The highest root percentage was observed on half-strength MS with Indole-3-butyric acid (IBA). Regenerated plantlets had a 65% survival rate 25 d after transplanting to garden soil, vermiculite, and vermicompost (1:1:1) potting mix. This protocol can be routinely used in large-scale propagation and possibly in germplasm conservation of this rare and important CWR species for additional use in chickpea breeding programs.
KeywordsShoot bud Micropropagation Plant tissue culture
The authors are grateful to the DRDO HQ, Ministry of Defense, Government of India, for providing the necessary facilities and financial assistance. The DIHAR (Leh, India) is duly acknowledged for providing logistic support during the collection of C. microphyllum seeds.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
- Ballabh B, Chaurasia OP, Pande PC, Ahmed Z (2007) Raw edible plants of cold desert Ladakh. Indian J Tradit Knowl 6:182–184Google Scholar
- Ghauri EG, Afridi MS, Marwat GA, Inayatur-Rahman AM (2013) Micro-propagation of Stevia Rebaudiana Bertoni through root explants. Pak J Bot 45:1411–1416Google Scholar
- Ignacimuthu S, Franklin G, Melchias G (1997) Multiple shoot formation and in vitro fruiting from cotyledonary nodes of Vigna mungo (L.) Hepper. Curr Sci 73:733–735Google Scholar
- Kadri A, Chalak L, El Bitar A, Nicolas N, Mrou‚ S, De March GG (2014) In vitro plant regeneration system for two Middle East cultivars of chickpea (Cicer arietinum). Adv Crop Sci Technol 2:1–4Google Scholar
- Khoury CK, Castaneda-Alvarez NP, Achicanoy HA, Sosa CC, Bernau V, Kassa MT, Norton SL, van der Maesen LJG, Upadhyaya HD, Ramirez-Villegas J, Jarvis A, Struik PC (2015) Crop wild relatives of pigeonpea [Cajanus cajan (L.) Millsp.]: distributions, ex situ conservation status, and potential genetic resources for abiotic stress tolerance. Biol Conserv 184:259–270CrossRefGoogle Scholar
- Kumar V, Parvatam G, Ravishankar GA (2009) AgNO3 - a potential regulator of ethylene activity and plant growth modulator. eJ Biotechnol 12:0. https://doi.org/10.2225/vol12-issue2-fulltext-1
- McCouch S, Baute GJ, Bradeen J, Bramel P, Bretting PK, Buckler E, Burke JM, Charest D, Cloutier S, Cole G, Dempewolf H, Dingkuhn M, Feuillet C, Gepts P, Grattapaglia D, Guarino L, Jackson S, Knapp S, Langridge P, Lawton-Rauh A, Lijua Q, Lusty C, Michael T, Myles S, Naito K, Nelson RL, Pontarollo R, Richards CM, Rieseberg L, Ross-Ibarra J, Rounsley S, Hamilton RS, Schurr U, Stein N, Tomooka N, van der Knaap E, van TD, Toll J, Valls J, Varshney RK, Ward J, Waugh R, Wenzl P, Zamir D (2013) Agriculture: feeding the future. Nature 499:23–24CrossRefGoogle Scholar
- Pandey P, Irulappan V, Bagavathiannan MV, Senthil-Kumar M (2017) Impact of combined abiotic and biotic stresses on plant growth and avenues for crop improvement by exploiting physio-morphological traits. Front Plant Sci 8:1–15Google Scholar
- Polisetty R, Patil P, Deveshwar JJ, Khetarpal S, Chandra R (1996) Rooting and establishment of in vitro grown shoot tip explants of chickpea (Cicer arietinum L.). Indian J Exp Biol 34:806–809Google Scholar
- Singh RK, Singh S, Anandhan S, Shannon LM, Quiroz-Figueroa FR, Ruiz-May E (2017) First insights into the biochemical and molecular response to cold stress in Cicer microphyllum, a crop wild relative of chickpea (Cicer arietinum). Russ J Plant Physiol 64:758–765Google Scholar
- Singh RK, Singh S, Pandey P, Anandhan S, Goyary D, Pande V, Ahmed Z (2013) Construction of cold induced subtracted cDNA library from Cicer microphyllum and transcript characterization of identified novel wound induced gene. Protoplasma 250:459–469Google Scholar
- Singh S, Anandham S, Arum Kumar MB, Goyary D, Singh NK, Das SC (2007) In vitro establishment of Cicer microphyllum , a wild relative of cultivated chickpea from cold desert of Ladakh. J Arid Legume 4:66–68Google Scholar
- Singh S, Singh I, Kapoor K, Gaur PM, Chaturvedi SK, Singh, N. P, Sandhu JS (2014) Chickpea. In Singh M, Bisht IS , Dutta M (eds) Broadening the genetic base of grain legumes. Springer, New Delhi, 51–73Google Scholar