Efficient invitro plant regeneration from cotyledon-derived callus cultures of sesame (Sesamum indicum L.) and genetic analysis of True-to-Type regenerants using RAPD and SSR markers

Sesame (Sesamum indicum L.) is an important oilseed crop which is rich in unsaturated fatty acids, proteins and natural antioxidants. Reports on tissue culture methods involving regeneration through callus phase are scanty, genotype dependent and have been reported with low regeneration frequency. This recalcitrant nature will impede the application of modern biotechnological tools for genetic advancement studies in sesame. An efficient protocol for invitro shoot organogenesis from callus cultures has been established using cotyledon explants excised from 1-week-old seedlings of sesame cv. TMV 3. Maximum frequency of greenish white friable compact (GWFC) callus (72.0%) was obtained on Murashige and Skoog (MS) medium supplemented with 4.54 μM of 2, 4-dichlorophenoxyacetic acid (2, 4-D). Organogenic GWFC callus cultured on MS medium containing 4.4 μM of 6-benzylaminopurine (BAP) and 10 μM of silver nitrate (AgNO3) showed the optimum frequency of shoot regeneration (66%) and produced 4.6 shoots per 150 mg callus. Optimum rooting of 70.2%, an average of 6.02 ± 0.8 roots per shoot was achieved on MS medium fortified with 5.6 μM of indole-3-acetic acid (IAA). The rooted plantlets were acclimatized successfully in a greenhouse with a 65.8% survival rate. Non significant phenotypic aberrations were observed among the ex vitro transferred plantlets. The genetic stability of regenerants was assessed by using Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeat (SSR) markers. PCR amplification using 10 RAPD and 10 SSR primers generated 71 distinct and scorable DNA bands. DNA banding patterns reveal the absence of somaclonal variation among the plantlets regenerated through indirect organogenesis compared to that of mother plant and confirms the genetic purity of the invitro raised plants. The developed protocol could be effectively employed for obtaining genetic traits invitro through genetic transformation studies.