Establishment of an Efficient Tissue Culture‐Based Regeneration Protocol of Cucumber ( Cucumis sativus L.)

Cucumber ( Cucumis sativus L.) is an important vegetable crop of global significance with high nutritional and commercial values. Its recalcitrance to in vitro manipulation, however, limits biotechnological progress. Strong genotype‐dependency and low morphogenic competence are the main reasons that reliable cultivar‐specific regeneration systems are essential for the successful application of genetic transformation and genome editing technologies. In the present study, we developed an efficient and reproducible regeneration protocol for C. sativus cv. “Kalyanpur Green” by evaluating morphogenic responses, i.e., callus induction, shoot organogenesis and rooting of cotyledon, and hypocotyl and nodal explants with different combinations of plant growth regulators (PGRs). Analysis of variance indicated highly significant differences ( p < 0.001) for 13 of 14 in vitro traits evaluated, except for shoot length. The highest callus induction (100%) was obtained on MS medium supplemented with BAP (1.0–2.0 mg/L) in combination with NAA (0.3–0.5 mg/L) or IAA (0.2–0.5 mg/L). Among the tested explants only cotyledonary nodes were found to be responsive for shoot induction with 100% regeneration and maximum proliferation on BAP (1.5–2.0 mg/L) alone or in combination with AgNO3 (4.5 mg/L) after optimized callus induction. The best rooting percentage (100% induction, 5.33 roots/explant, 7.47 cm mean length) was obtained on IBA (2.0 mg/L) and kinetin (0.5 mg/L). Thirteen ISSR primers were used to confirm the genetic fidelity of the regenerated plantlets, of which UBC‐809 and UBC‐810 had the maximum amplification efficiency (150–1500 bp amplicons). Monomorphic banding patterns confirmed true‐to‐type nature of the regenerants. The optimized protocol will provide a strong base for micropropagation, genetic transformation, and genome editing based improvement of C. sativus cv. “Kalyanpur Green”.