An efficient protocol for in vitro propagation of the wild legume Cicer microphyllum Benth., a crop wild relative of chickpea (Cicer arietinum L.)

  • Rupesh Kumar Singh
  • Sivalingam Anandhan
  • Luz María García-Pérez
  • Eliel Ruiz-May
  • Eusebio Nava Pérez
  • Francisco Roberto Quiroz-FigueroaEmail author
Plant Tissue Culture


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.


Shoot 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.

Supplementary material

11627_2018_9958_MOESM1_ESM.docx (30 kb)
ESM 1 (DOCX 30 kb)


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Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  1. 1.Centro de Química de Vila Real (CQ-VR)Universidade de Trás-os-Montes e Alto DouroVila RealPortugal
  2. 2.Directorate of Onion and Garlic ResearchPuneIndia
  3. 3.Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional Unidad Sinaloa (CIIDIR-IPN Unidad Sinaloa), Laboratorio de Fitomejoramiento MolecularInstituto Politécnico NacionalGuasaveMexico
  4. 4.Red de Estudios Moleculares Avanzados,Cluster BioMimic®Instituto de Ecología A. CXalapaMexico

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