Optimisation of explant-specific isolation, culture, and micro-callus induction of sesame (Sesamum indicum L.) protoplasts

The low yield of sesame (Sesamum indicum L.) compared to other oilseed crops hinders its successful commercialisation process. Sesame yield improvement using molecular-biotechnological tools involving gene manipulation is a better alternative than traditional breeding because it requires less time, effort, and labour. Protoplasts are plant cells devoid of the cell wall. Protoplast systems are deployed as versatile cell-based tools for tissue culture, genomics, transcriptomics, proteomics, metabolomics, and epigenetics studies leading to crop improvement. Inadequate reports of sesame protoplasts restrict its potential use in this crop. In the present study, we are reporting the successful isolation and purification of sesame protoplasts from four different sesame explants: hypocotyl, internode, leaf, and hypocotyl-derived callus using the one-step enzymatic digestion method. We have carefully optimised enzyme combinations, digestion durations, temperatures, and shaking speeds for every tested explant to obtain the highest protoplast yield and viability. Maximum yield (9.9 x 106 protoplasts/gm fresh weight) and viability (92.1%) were achieved from callus explants. We purified isolated protoplasts by floating them over a 20% (w/v) sucrose solution. Among tested media, callus-derived protoplasts divided rapidly in Murashige and Skoog broth medium supplemented with 17.77 M 6-benzylaminopurine (BAP) and 0.54 M -naphthalene acetic acid (NAA). In the same medium, protoplasts divided into the 2-cell stage after 2-3 days of culture and progressed to the micro-callus stage at 0.24 {+/-} 0.05% frequency within 16-20 days of culture. Sesame protoplasts could be an excellent system for investigating potential avenues for biotechnological improvements of sesame including transient gene expression and CRISPR-based studies.