Studies On Isolation And Culture Of Microspore Protoplasts Of Solanum Tuberosum

Microspore protoplasts, as a special experimental system in the study of plant cell engineering, have both advantages of haploid and protoplasts. And they can be served as an important segment in potato nalytic-synthetic breeding?project. The present investigation first studies the condition and some effectional factors on isolation and culture of haploid protoplasts isolated from the microspores at the stage of meiosis tetrad , uninucleate and mature pollen grains of tetraploid cultivas Gannongshu No.1, Gannongshu No.2, Gannongshu No.3 and dihaploid 81- 15 of potato. The result from microspore protoplast isolation showed: 1. 1% snailase?.2% cellulase?.8% Ilemicellulase is the best enzyme combination dissolved in K3 medium to isolate protoplasts from potato tetrad after cold pretreatment. the highest yield of protoplasts of Gannongshu No.1, Gannongshu No.2, Gannongshu No.3 and dihaploid 81-15 up to 75.1 7%, 71.43%, 78.05%, and 54.19% respectively. 2. Protoplasts can not be obtained from uninucleate of microspore. 3. Protoplasts having been obtained from mature pollen of Gannongshu No.1, Gannongshu No.2 and Gannongshu No.3 via old treatment- germination-enzyme?method . with highest yield up to 10.12%, 9.70%and 17.71%; and 1% Cellulase (R-10) ?.5% Hemicellulase (Sigma)+1% Pectinlase (Serva)+0.5% Potassium dextran sulfate?% PVP is the suitable enzyme combination. 4. Sucrose is the best osmotic pressure regulator in the process of isolation tetrad protoplasts , and 10.3% is optimum ; while rnannitol is better in isolation mature pollen protoplasts , and 18.2% is optimum. 5. 30 mm Germination duration is suitable for isolation mature pollen protoplasts. 6. The protoplast vigor can be improved 8.4% or so through reducing enzyme concentration. 3 7. Adding protectants to enzyme solution has very significant effect on decreasing the crushing and browning of protoplasts and improving their vigor. The result from microspore protoplasts culture showed: 1. Adding 2,4-D 1.OmgIL, KT 0.5mgIL, 6-BA 0.4 mgIL, demising NH4~ and NAA can keep tetrad protoplasts integrity, decrease budding in future culture and improve division frequency. 2. Tetrad protoplasts in low density of 1 X 103/ml can be induced to divided in K3 media added nther wall factor?(L-serine, L-glutamin et al). 3. 5-7d cold pretreatment has very important significance in inducing tetrad protoplasts to divide. The highest division frequency up to 12.6% and finally format small cell clumps, while tetrad could not divide without cold pretreatment. 4. During the tetrad protoplast culture, it will be better using 揟hin liquid layer culturend ouble layer culture?method. 5.The protoplasts from mature pollen can not be induced to divide, only modified its shape. 6. The nearer bloodship between anther wall tissue and tetrad protoplasts, the more apparent nurse function of anther wall tissue to tetrad protoplasts seemed to have.