Plumular meristem transformation system for chickpea: an efficient method to overcome recalcitrant tissue culture responses

Abstract

Major biotic and abiotic stresses have led to the reduction of chickpea productivity, creating a strong barrier for its utilization as major food legume. Genetic transformation has revolutionized the chickpea improvement programs for biotic stress tolerance. Recent knowledge on genomic resources offers a range of approaches for genetic improvement of chickpea with a greater resolution, but relies on the transgenic establishment which is the major bottleneck in this process. Poor tissue culture responses along with insensitivity to in vitro rooting hindered the transgenic chickpea development through tissue culture based transformation methods. In the present report, Agrobacterium-mediated plumular meristem transformation was adapted in chickpea to bypass the recalcitrancy of regenerable tissue. Embryonic axis of immature seedlings of chickpea cultivar was decapitated followed by pricking of plumular meristem present in shoot apex and cotyledonary nodes. Pricking is followed by infection by Agrobacterium tumefaciens carrying binary vector pBI121. Three days co-cultivation was performed with infected explants in 6-benzylaminopurine and α-naphthaleneacetic acid supplemented modified Murashige and Skoog medium. Transient GUS expression was observed in co-cultivated explants and PCR based screening strategy allowed the establishment of primary transgenic events with a frequency of 60%. Kanamycin mediated stringent selection of T1 events helped to eliminate the chimeric plants and 44% of T1 progenies were confirmed through PCR. Further, Southern hybridization was performed to identify transgene integration in T1 events. Additionally, prominent GUS activity in T1 events confirmed the expression of transgene. Plumular transformation method, based on culture dependent Agrobacterium-infection and culture independent plant selection as well as establishment, was reported for the first time in chickpea. This transformation method will boost the recovery of novel genotypes with improved agronomic traits and mutant development for functional genomics in chickpea. Agrobacterium-mediated transformation of plumular meristem in chickpea is established, following in vitro co-cultivation of explants and culture independent selection of transgenic plants, at higher frequency.