POOJA MALIK, MUKTA PRAJAPATI, SAPNA BHORIA, RANJANA JAIWAL, PAWAN K. JAIWAL, MINAKSHI PRASAD AND DARSHNA CHAUDHARY*
Centre for Biotechnology, M. D. University, Rohtak-124 001 (Haryana), India
*(e-mail: darshnarajan.cbt@mdurohtak.ac.in; Mobile : 98137 75909)
(Received: November 4, 2022; Accepted: December 8, 2022)
ABSTRACT
Trifolium alexandrinum is a significant annual, winter, multi-cut (about 4 to 6), protein-rich forage legume
crop whose genetic modification for the improvement of biomass yield and quality requires an effective in vitro plant regeneration protocol. In this study, a simple, efficient and reproducible regeneration system based on direct shoot organogenesis was developed using cotyledon explants. The various factors affecting in vitro regeneration e.g. type and concentration of cytokinin, age of donor seedling, size and explant type and genotype were optimized. Cotyledons with petiole excised from three-day old seedlings (pre-conditioned on 5 µM BAP) of T. alexandrinum cv. Mescavi on MSB5 basal medium with 10 µM BAP directly produced 25.88 shoots per explant in most of the cultures (94%) without the formation of intermediate callus within four weeks. The shoots were successfully rooted on NAA containing medium and the resulted plants were fertile and morphologically identical to seed raised plants. This regeneration system can be used further for transfer of agronomical important genes via Agrobacterium tumefaciens for genetic improvement of this crop.
Key words : In vitro regeneration, berseem, type and concentration of cytokinin, age and size of explants, genotype, Trifolium alexandrinum