Dendrobium Officinale Leaf Protoplast Culture And In Vitro Apical Somatic Embryo Research

Effects of isolated and cultured on protoplasts derived from young leaves of Dendrobium officinale Kimura et Migo aspectic tube plantlet were discussed in the paper. In our study, the appropriate condition in isolate proptoplast is basic solution contained 0.5% Pectinase and 1.0% Cellulase Onozuka R-10. The time of enzyme treatment is 6h. Osmotic medium concen- tration is 0.5M. The sugar is superior to mannitol and glucose. However, in the osmotic medium the yield is decreased within 28.7-38.1%. If the basic solution contained 150mg.L-1 ascorbic acid and 20 mg.L-1 CaCl2 will increased the activity of protoplast 9.2% and 15.7%. Moreover,if the young leaf pieces were plasmolyzed before incubated in enzyme solution will increased the yield and activity of protoplast 3.23% and 4.2%.In the culture of the protoplast, the first division occurred within 3 to 5 days, and the embryogenic cell cluster could be observed 30 days after culture. The appropriate condition in the culture is MS1 supplement- ed with 0.5mg.L-1'NAAand 1.0mg.L-16-BA. For protoplast survival in the initial culture relatively low temperature (2±0.5 ℃ ) and low light intensity (150 1x) are preferable. Culture density is 5×105 cell . mL-1. The liquid thin culture and agrose-liquid doublelayer are superior to agrose bead methed and agrose island culture in the time of division and division frequency. However,cell colony frequency is weakest in the liquid thin culture than others.If keep young leaves in the dark 24h before incubated in enzyme solution will enhance the stability of protoplast and increase the division frequency 2.4%. 50mg.L-1 ascorbic acid and 5mg.L-1 CaCl2 and 100 mg.L-1 Arg also increased division frequency. But, keep leaves under low temperature prior to isolateion of protoplasts neither increased the percentages of protoplasts in total cells after enzyme treatment,nor increased the percentages of division.The histological processes of plant regeneration from root-tip of Dendrobium officinale Kimura et Migo were studied. Segments produced clusters of somatic embryos from embryogenic calli or directly from root apical meristems , and that also can convert into bud directly in the different tissue culture conditions. Modified media B5 with 1.0 mg.L-1 2,4 - D and 3.0mg.L~1 6-BA is suit to induce calli, and 0.5 mg.L-1 NAA is fit for somatic embryos inducement from calli. 6-BA encreased direct somatic embryogenesis on root tip distinctly,but NAA is retard it. NAA also prolong the time of root-tip convert into bud directly. With histological observation on these processes suggests protocorm like bodies(PLBs) of Dendrobium officinale Kimura et Migo can be considered a ture somatic embryos that origin from a single-cell.