PROTOPLAST AND CELL CULTURE OF CERTAIN ORCHIDS

Abstract

More Protoplasts per gramme fresh weight were isolated from older leaves (1 - 3cm lenf!;thl of Oncidium Gower Ramsey than younger leaves ( < 1cm length). Aranda Christine. However, preparations obtained using older leaves were prone to larger amounts of contamination by raphides. Much less protoplasts could be obtained from protocorms of both orchids. Average protoplast yield was about 2.9 x 104 /gfw. Protoplasts cultured in basal 8p medium remained viable for an average of 8 weeks after isolation. Various factors found to affect the viability of Oncidium protoplasts in culture include pre-culture of protocorms prior to isolation, concentration of coconut water, arginine, putrescine and glucose. Plant growth regulators were required for continued viability of protoplasts. Cultures grown in medium without plant growth regulators were not viable after 4 days in culture. Protoplasts cultured in agarose-solidified 8p medium could be induced to regenerate cell walls and then divide. The highest colony formation frequency (9.2%) obtained when Oncidium protoplasts were cultured at a density of 3 - 3.5 x 10 power 5 . A small proportion of protoplasts proceeded to divide normally (path 1). The other mode of division appears to be peculiar to orchid protoplasts and has not been reported elsewhere. Coenocytic budding of protoplasts was also observed. Percentage of protoplasts undergoing coenocytic budding was as high as 86% in certain experiments. Larger colonies were obtained from protocorm-derived protoplasts than from leaf mesophyll-derived protoplasts. Micro-colonies failed to develop further upon reaching the 40 - 60 cell stage, and browning of the medium occurred after 10 weeks in culture. Individual cells were successfully isolated leaves (1 - 3cm length) of Aranda Christine and Aranda Tay Swee Eng. The recommended enzyme mixture for isolation is 1% pectolyase Y23, 2% macerozyme R10, 0 .3% Aranda PDS, 5mM MES and 0.06M sucrose (1-step incubation method). Conditions for obtaining clean preparations of cells were studied. Cell divisions were stimulated by culturing in conditioned medium, more than in VW medium, both without any plant growth regulators. Isolated cells began division within 1 or 2 days after culture. The colonies formed were much larger than protoplast-derived colonies and were the result of normal cell division. They had a distinct observed shape and organisation. Protrusions to develop from them. Sub-culture were also of colonies onto fresh medium was necessary to obtain further growth of the colonies. Average size of colonies sub-cultured to 0.5-macro MS medium was 0.5mm with 85% of them having developed protrusions. In time, fine chains of cells grew out from superficial cells, giving the colonies a 'hairy' appearance when viewed under a binocular microscope.