Protocols of Protoplast Isolation and Cell Fusion in Dendrobium nobile

Abstract: To construct the protocols of protoplast isolation and cell fusion in Dendrobium nobile， the tender leaves of tissue cultured seedling were used as the material. The survival rate was applied for evaluating the quality of newly harvested protoplast， four significant factors of the cellulase concentration， pectinase concentration，macerozyme concentration and enzymolysis time were screened for their optimum levels during protoplast isolation through orthogonal experiment design. It found that the survival rate could reach the highest value of 94.42% with 9.5 mg/mL， 5.0 mg/mL， 6.0 mg/mL and 3.0 h， respectively. Then the PEG 4000 was applied for inducing the cell fusion with the method of high pH and high calcium. The heterokaryon fusion rate was selected as the assessment criteria， the main four factors of the PEG concentration， CaCl2 concentration， pH value and fusion time were screened for their optimum levels by using the experimental design of response surface method (RSM). Through this method， the fusion rate could reach the highest value of 0.924% with four factors of 38.41%， 0.44 mol/L， 9.26 and 28.87 min respectively. Meanwhile， the RSM detected that the synergistic effects reached to the significant level of 0.05 between the PEG concentration and the CaCl2 concentration， PEG concentration and pH， CaCl2 concentration and fusion time. Furthermore， RSM also identified that the heterokaryon fusion rate was maintained around 0.94% when the pH value was 9.26 and the PEG concentration， CaCl2 concentration， fusion time were varied within narrow ranges. This study improved the protocols of protoplast isolation and cell fusion in D. nobile， which is meaningful for germplasms’ protection and development， and it also supplied a solid bases in experimental material for breeding.

CSTR: 32200.14.cjcb.2016.08.0005