Isolation and culture of leaf protoplasts of Indian mulberry (Morus indica var. K-2)

Abstract

Mesophyll protoplasts of Indian mulberry leaf (Morus indica var. K2) were isolated enzymatically from shoots obtained in vitro in a mixture of cellulase enzyme Onozuka RS 2% to 0.1% Pectolyase Macerozyme and 1% in the mannitol at 0, 6 M for 14 to 16 hours. This enzyme mix has also allowed the release of protoplasts from mature leaf tissue. We regularly observed a regeneration of the cell wall for the two sources of protoplasts, but the divisions are rare. Protoplasts cultured in liquid medium regenerate a cell wall (60%) but fail to divide and grow. Protoplasts cultured in agarose beads / layers of Ca-alginate present the first divisions after 7 to 10 days of culture when grown at a density of 5 × 10 plates / ml. Structures up to four cells are seen in 5-7% of the cultures on MS medium supplemented with glucose at 0, 6 M, 1 mg / l TDZ / BAP, 1 mg / l 2,4-D and 15% coconut milk. The use of glucose is essential and we obtain a formation of micro-colonies after one month of culture.