Protoplast isolation and plant regeneration in two cultivated coriander varieties, Co-1 and RS

An efficient and reproducible protocol for plant regeneration from protoplasts has been developed in two Coriandrum sativum varieties – Co-1 and Rajendraswathi (RS). The RS coriander is rich in oil and shows resistance to diseases whereas Co-1 is known for better yield. In the present study, protoplast isolation – the first step of fusion was undertaken in cotyledon-derived embryogenic cell suspensions by using enzymatic solutions of different concentrations. Digestion with 2% cellulase Onozuka R-10, 1% pectinase and 0.2% macerozyme R-10 showed the maximum yield of protoplasts (4.60 × 10⁶/g of callus) when mannitol was added as the osmoticum. The cell division and plating efficiency, however, varied with the different levels of plant growth regulators (PGRs) used. In a liquid medium amended with 0.2 mg/l α-naphthalene acetic acid (NAA) and 0.2 mg/l 6-benzyladenine (BA), protoplasts showed a higher plating efficiency (6.2% in Co-1 and 5.4% in RS) compared to other PGR treatments. On a 2,4-dichlorophenoxy acetic acid (2,4-D)-supplemented medium, cells divided fast, produced micro-colonies, and formed the callus on which a heterogeneous mixture of somatic embryos was produced in both the varieties tested. The maximum number of somatic embryos was produced (47/callus mass) in a medium to which 1 mg/l 2,4-D was added. On a BA and gibberellic acid (GA₃)-supplemented medium,somatic embryos germinated into plantlets, and the maximum germination frequency (81%)was noted in a medium to which1 mg/l BA and 0.5 mg/l GA₃was added. A successful somatic embryo-mediated plant regeneration was achieved from protoplasts in both coriander varieties, and the regenerated plants were morphologically similar to mother plants. The whole process of “protoplast to plant recovery”, enzymatic mixtures, osmotic condition, and role of PGRs are discussed in this article.