Development of an efficient regeneration protocol for sesame (Sesamum indicum L. var. BINATIL-02) through optimized in vitro culture techniques

Abstract

Sesame (Sesamum indicum L.), one of the earliest cultivated oilseed crops in human civilization, is an important agricultural and economic crop in Bangladesh as the second largest oilseed crop contributing to food security and income for the rural people. While, it has an excellent biochemical profile, with up to 57% oil rich in unsaturated fatty acids and naturally occurring antioxidant lignans, its productivity is still below its potential owing to various biotic stresses, abiotic constraints, and recalcitrance of sesame in conventional breeding and in vitro regeneration. The main objective of this study is to develop an efficient in vitro regeneration system for Sesamum indicum var. of BINATIL-02 which is high-oil yielding gamma-ray mutant produced at the Bangladesh Institute of Nuclear Agriculture (BINA). It will create a platform for subsequent micropropagation and genetic engineering. 16 sterilization combinations of 70% ethanol, 0.1% mercuric chloride and sodium hypochlorite (Clorox) with Tween-20 to sterilize the seed surface were evaluated. Among them, one of the combinations, 50% Clorox and Tween-20 for 30 minutes, yielded the best result with 87% germination and 0% contamination as confirmed with a one-way ANOVA test. The best germination medium was Water Agar supplemented with 3% sucrose with 90% germination and 95.7% seedling survival rate. Cotyledonary leaves of 14-day old seedlings had 25 times more regeneration potential than hypocotyl explants. Explants were cultured on MS media containing BAP, IAA, AgNO3 and GA3. The optimal shoot regeneration frequency (71.1%) was achieved with the hormone combination of 2.5 mg/l BAP and 0.75 mg/l IAA, which was further increased to 82% with AgNO₃ supplementation, at the same time reducing necrosis and vitrification. The use of activated charcoal caused of total collapse of shoot survival and was excluded, while GA₃ resolved cytokinin-induced shoot stunting, resulting in 89% shoot elongation frequency. Rooting was induced on a ½ strength MS medium supplemented with 1 mg/l IAA and 1 mg/l AgNO3. After 30 days, 80% of shoots formed healthy roots and plants were successfully transferred to sterilized soils. The development of an efficient, rapid and reproducible regeneration system is an important step towards the improvement of sesame crop in Bangladesh.