Protoplast Isolation, Culture and Plant Regeneration in Catharanthus roseus (L.) G. Don via Somatic Embryogenesis

Mehpara      Maqsood; A.      Mujib; Dipti      Tonk; M.      Z. Abdin

Abstract

An efficient protocol for plant regeneration from protoplasts in Catharanthus roseus is reported. Hypocotylderived embryogenic callus was used as a source of protoplasts. The embryogenic suspension was established in liquid MS, added with 1.0 mg l-1 NAA and 1.0 mg l-1 BAP; and then suspension was treated with various enzymatic solutions either alone or in combinations. The cocktail of cellulase (2.0%), pectinase (1.0%), macerozyme (0.02%) and driselase (0.50%) showed maximum yield of protoplasts (37.25 ± 1.86a X 10⁵) with highest viability (65.25 ± 3.26a). The yield (8.50 ± 0.42a X 10⁶) and viable protoplasts number (70.18 ± 3.51a) was even more when sorbitol was added with above enzyme mixtures as osmoticum. In 0.50 mg l-1 NAA +0.50 mg l-1 2, 4-D containing medium, protoplasts divided well and maximum number of micro colony formation was noticed (13.33 ± 1.53a/Petriplate). The callus biomass (fresh weight) was, however low in protoplast derived embryogenic callus (PDEC) than in normal embryogenic callus (NEC). Biochemically, the protein, proline, sugar and enzyme activity level (CAT, SOD, GR and APX) were also higher in PDEC than in NEC. The protoplast derived somatic embryos latter germinated and regenerated into plantlets. The recovery period from ‘protoplast to plantlet’ was around 40 weeks in contrast to 16 weeks in normal somatic embryogenesis pathway.

Keywords: Alkaloids, antioxidative enymes, Catharanthus roseus, embryogenic callus, enzymatic digestion, fluorecein diacetate, hypocotyl, micro-colony, osmoticum, somatic embryogenesis, regeneration, suspension culture, plant growth regulators, protoplast, protoplast isolation