Meta-topolin mediated enhanced micropropagation, foliar-micromorphological evaluation and genetic homogeneity validation in African pumpkin (Momordica balsamina L.)

Abstract

An efficient micropropagation method has been defined utilizing meta-topolin (mT) for the first time in Momordica balsamina L., an underutilized cucurbit with high nutraceutical and therapeutic potential. Cent-percent nodal explants exhibited bud-breaking on Murashige and Skoog's (MS, 1962) medium containing 1.0 mg L⁻¹ mT and 0.1 mg L⁻¹ Indole-3-acetic acid (IAA) and produced 4.65 ± 0.30 shoots measuring 4.60 ± 0.37 cm in length. During multiplication, MS medium supplemented with 0.5 mg L⁻¹ mT, 0.1 mg L⁻¹ IAA, and additives resulted in the maximum shoot number (44.20 ± 1.21) with 6.54 ± 0.13 cm shoot length per culture vessel, after 4 weeks of the fourth subculture. The types of cytokinins [mT or 6-benzylaminopurine (BAP)] influenced the development of foliar micromorphological characteristics. The leaves developed on mT and IAA containing medium exhibited superior foliar micromorphological parameters such as stomatal index, stomatal density, druse density, well-developed veins/veinlets in comparison to leaves developed on BAP and IAA containing medium. Ex vitro rooting and concurrent acclimatization (EVRCA) were achieved in greenhouse by pulse-treating the shoot-base with 250 mg L⁻¹ indole-3-butyric acid (IBA) for 5 min. About 81 % shoots rooted ex vitro and developed 7.85 ± 0.32 roots per shoot measuring 6.31 ± 0.34 cm root length, after 4 weeks. Around 86 % of ex vitro rooted plantlets survived in the nursery and sets fruits. To ensure genetic homogeneity of the protocol, Start Codon Targeted (SCoT) and Inter Simple Sequence Repeats (ISSR) markers were utilized and the monomorphic banding profiles confirmed the clonal nature of the regenerants. Therefore, the defined protocol appears to be simple, robust and consistent for the large-scale production of genetically identical plants of M. balsamina.