An improved protocol for efficient regeneration, transformation and gene-editing using CRISPR/Cas9 in pigeonpea (Cajanus cajan(L.) Millsp.)

Abstract Background: Pigeonpea (Cajanus cajan (L.) Millsp.; 2n = 2× = 22) is a drought-tolerant perennial grain legume commonly cultivated in India's rain-fed and dry land zones, and it is a remarkable natural source of minerals and protein worldwide. Despite decades of research, the constraints linked to tissue culture continues to hinder the genetic improvement of the crop for instances; the explants inability to produce an embryogenic calli, direct shoot formation, and limited regeneration potential, as well as a lack of an effective transformation system. Thereby, efficient calli production, in-vitro recovery, and a systematic technique to co-delivery of Cas9 and sgRNA into the plant remain pre-requisites for efficient genome alteration. Results: To establish a CRISPR/Cas9 based genome editing platform in pigeonpea, a CRISPR/Cas9 mediated binary vector for coherent gene modification (for example, knockout, Homology Donor Repair) was designed. Calli production was achieved at a rate of over 70% by combining with a standardized robust genetic transformation method of pigeonpea using embryonic axis and cotyledonary node explants. The addition of tailored growth regulators and silver nitrate to shoot induction media boosted in-vitro regeneration by up to 89 %. Finally, as a hallmark, for biolistic-mediated transformation, an improved regeneration strategy was used, resulting in the successful incorporation of the transforming binary vector into the embryogenic calli with a transformation efficiency of 46%. PCR and Southern blot analyses were used to validate the results in the end.Conclusion: The standardized shoot regeneration method and successful transformation method of Cas9 in this research, made pioneer steps for facilitating the procedures for the improvement of this crop, which is as yet considered as recalcitrant. Furthering the protocol uncovers significant contributions in both effectiveness and convenience over existing pigeonpea transformation techniques, thereby enabling genetic research on other legumes/dicot crops achievable, as well.