Micropropagation, micromorphological evaluation and genetic homogeneity validation of Cucumis collosus (Rottl.) Cogn.: A wild progenitor of melon

Cucumis collosus (Rottl.) Cogn., a wild ancestor of melon cultivars, is an underutilized cucurbit of arid horticulture having nutraceutical and therapeutic significance. C. collosus has great potential for enhancing the genetic base of cultivated melon and other cucurbits. This investigation is the first report on a robust and reliable micropropagation system for C. collosus (var. AHK-119) using nodal explants, followed by micromorphological and genetic stability assessment. More than 95% explants showed axillary shoot-bud induction on MS (Murashige and Skoog, 1962) medium containing 0.5 mg L⁻¹ BAP and developed a maximum of 3.2 ± 0.6 shoots measuring 3.2 ± 0.1 cm length. After evaluating the various parameters, the best shoot multiplication (Shoot Number 34.9 ± 1.7, Shoot Length 6.7 ± 0.9 cm) was achieved on MS medium having half-strength of nitrates (825 mg L⁻¹ NH₄NO₃ and 950 mg L⁻¹ KNO₃) + 0.25 mg L⁻¹ 6-benzylaminopurine (BAP) + 0.1 mg L⁻¹ Kinetin (Kin) + 0.1 mg L⁻¹ Indole-3-acetic acid (IAA) + additives (50 mg L⁻¹ ascorbic acid and 25 mg L⁻¹ each of adenine sulfate, L-arginine and citric acid), after 4 weeks. Micro-shoots were rooted using three approaches: In Vitro Continuous Treatment (IVCT), In Vitro Pulse-Treatment (IVPT) and Ex Vitro Pulse Treatment (EVPT). Of these, EVPT approach proved to be most successful in terms of rooting response (80%), root number (6.4 ± 0.9) and root length (5.7 ± 0.7 cm). The foliar micro-morphology analysis of in vitro leaves (IVL), ex vitro leaves (EVL) and mother plant leaves (MPL) demonstrated notable adaptive alterations during the transitional stages and produced structurally stable plants similar to the mother plant. Furthermore, the validation of genetic homogeneity using Start Codon Targeted (SCoT) and Inter Simple Sequence Repeats (ISSR) markers revealed monomorphic banding patterns among the micropropagated and the mother plant. The discussed method can be applied for large-scale commercial production of C. collosus for arid horticulture, urban/vertical farming, breeding purposes and improvement of other cucurbits.