Establishment of in vitro regeneration systems from callus and protoplast in capsicum frutescens L.

Abstract

The present study entitled “Establishment of in vitro regeneration systems from callus and protoplast in Capsicum frutescens L. was carried out in the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2015-2017. The objective of the study was to establish callus culture from different explants in C. frutescens, to establish protocol for protoplast isolation from callus/leaf mesophyll and to culture protoplast. The study was carried out in two phases viz., establishment of callus culture and organogenesis; and standardization of protocol for protoplast culture. Callus was induced from leaves and internodal segments from in vitro raised seedlings. Among the 44 treatments in MS medium with different combinations of auxins (NAA, IAA, IBA, 2,4-D and picloram) and cytokinins (BA and Kn), 100 per cent callus induction was obtained in MS media supplemented with picloram 0.50, 1.00, 1.50 and 2.00 mg L-1 (C41, C42, C43, and C44), NAA 1.50 mg L-1, NAA 2.00 mg L-1(C3, C4) and BA 3.00 mg L-1 + NAA 1.00 mg L-1 (C29). Among the 78 treatments tried for organogenesis, calli obtained from C29 treatment showed organogenesis in MS + BA 3.00 mg L-1 + IBA 1.00 mg L-1 (R37) and (MS + BA 5.00 mg L-1 + IAA 2.00 mg L-1 (R61) in 41 and 90 days, respectively. The microshoots obtained recorded 83.33 per cent rooting in MS medium supplemented with IAA 1.50 mg L-1(Rt7) in 12.83 days. Leaves excised from in vitro seedlings, and calli produced in MS + BA 3.00 mg L-1 + NAA 1.00 mg L-1 (C29), were used as explants for protoplast isolation. Leaf bits incubated in cell protoplast washing (CPW) solution containing cellulase 2.00 per cent, macerozyme 0.50 per cent and mannitol 0.50 M (maintained at pH 5.8) for 6 h (DM28) in dark at 27oC, yielded (124 x 105) protoplast per g, with a viability of 95.16 per cent. The callus yielded maximum protoplast (36 x 105 protoplasts per g) in an enzyme combination of cellulase 2.00 per cent + macerozyme 0.50 per cent + mannitol 0.60 M (maintained at pH 5.8) after 4 h 91 (DM47) of incubation under same conditions. In protoplast purification, floatation medium with 21 per cent sucrose recorded maximum protoplast yield (30 x 105 protoplasts per g tissue and 10 x 105 protoplasts per g callus) and maximum viability (90.91 per cent and 100 per cent), from leaf derived and callus derived protoplast, respectively. The purified protoplasts with 10 x105 plating density initiated microcalli in liquid MS medium supplemented with mannitol 0.50 M, 2,4- D 0.50 mg L-1 and sucrose 30g L-1 (PCM5) in 45 days. Further development to visual colony formation of microcalli was obtained on addition of liquid MS medium supplemented with mannitol 0.40 M and sucrose 5g L-1, in 60 days from callus derived protoplast and in 70 days from leaf derived protoplast. In the study, maximum callusing response was obtained in MS medium with picloram 1.50 mg L-1. Organogenesis was obtained from the calli derived in MS medium with BA 3.00 mg L-1 and NAA 1.00 mg L-1. The shoot initiated from the calli in MS medium with BA 3.00 mg L-1 and IBA 1.00 mg L-1. The rooting of microshoots could be obtained in MS medium with IAA 1.50 mg L-1. In protoplast isolation, leaf gave higher protoplast yield and viability in CPW solution with cellulase 2.00 per cent, macerozyme 0.50 per cent and mannitol 0.50 M, incubated in dark for 6 h and callus, in CPW solution with cellulase 2.00 per cent, macerozyme 0.50 per cent and mannitol 0.60 M, incubated in dark for 4 h. The protoplasts purified in 21 per cent sucrose supplemented floatation medium and adjusted to a plating density of 10 x 105, initiated microcalli in liquid MS medium supplemented with mannitol 0.50 M, 2,4- D 0.50 mg L-1 and sucrose 30g L-1. The visual colony formation of microcalli was obtained on addition of liquid MS medium supplemented with mannitol 0.40 M and sucrose 5g L-1. In this study, a callus mediated in vitro regeneration system has been established in C. frutescens. A protocol has also been developed for protoplast isolation from leaf and calli, and its culture resulting in microcalli formation.