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

Abstract

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 AgNO 3 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.