Overcoming in vitro regeneration bottlenecks: a high-frequency regeneration protocol for cucumber (Cucumis sativus L.) via cotyledonary nodes

Cucumber is an economically important crop; nonetheless, genotype-dependent and inefficient in vitro regeneration technologies limit its genetic transformation and genome editing potential. The goal of this study was to develop an effective and reproducible cucumber regeneration protocol by comparing explant types, plant growth regulator combinations, and in vitro culture conditions under various auxin-cytokinin regimes. In this study, three types of explants were evaluated viz., cotyledonary nodes, cotyledons, and hypocotyls, with cotyledonary nodes demonstrating the highest regenerative capacity. All of these explants exhibited considerable callus induction on MS medium supplemented with BAP (1.5 mg/L), NAA (0.5 mg/L), and AgNO 3 (0.5 mg/L), demonstrating that cytokinin and auxin work together to promote cell division and organogenesis. However, shoot regeneration was only obtained from nodal explants, with callus produced on MS media containing BAP, IAA, and AgNO 3 . These were subsequently moved to shoot induction medium containing MS supplemented with BAP (1.0 mg/L) and Kinetin (0.5 mg/L), where several shoot buds emerged from each cotyledonary node. These were then moved to a rooting medium containing IBA (2.0 mg/L) and Kinetin (0.5 mg/L), resulting in increased root number, length, and induction efficiency (98–99%). The regenerated plantlets were then successfully acclimatized and moved into the greenhouse. To investigate the genetic fidelity of the regenerated plantlets, a total of 13 ISSR markers as well as 46 SSR markers distributed across all seven chromosomes of the crop were used, revealing consistent monomorphic banding patterns across the regenerants, confirming their genetic stability and lack of somatic variation. The methodology was also validated in another cucumber cultivar, Kalyanpur Green, which demonstrated excellent regeneration. An efficient and highly reproducible tissue culture methodology for cucumber has been developed, which can be effectively employed to address limitations in practical applications of functional genomics and genome-editing technologies in the crop. This procedure examines the influence of various phytohormone dosages and other growing conditions on high-frequency tissue regeneration from cotyledonary nodes in cucumber, presenting a possible platform for cucumber improvement via transformation and genome editing.