Isolation And Culture Of Pollen Protoplasts Of Solanum Tuberosum

The paper reported here the isolation and culture of pollen protoplasts of different stage from potato pollen grains. The present study selected six good quality of tetraploidy cultivas and three dihaploidy varieties as donors, the technique system of isolation and culture of pollen protoplasts were established. Using sucrose as osmotic pressure regulator, the highest isolation percentage of tetrad protoplasts was up to 84.6% in the mixed enzymatic solution including 1.2% snailase. The protoplasts from the material after 7days of cold treatment regenerated cell wall after 24h , and began its first cell division after 48h culture in K3 basic medium supplemented with I OOmg/L L-serine, 800mgIL L-glutamin, 0.1% calf serum and Img/L 2,4-D, 0.5mg/L KT, 0.4mgIL 6-BA with thin liquid culture. Cell division frequency was 15.4% and after further culture of 1 Sdays?, some cells continued division and formation of small cell clumps. Anther floating culture for pollen shedding into the culture medium I day, then followed by the treatment of high osmotic pressure enzymatic solution including 0.8mol/L mannitol, young pollen protoplasts at uninucleate were isolated , with percentage about 12.8%. The factors of influence germination were also studied. Supplemented with 0.1 mmol/L K4 and I .OmmolIL Mg2~+ can increase the percentage of germination, and 0.1 Smmol/L Na4 speed the germination. After a procedure of optimization. Over 70% of pollen germination rate was obtained with liquid the medium composed of 10% Sucrose, 0.02%CaC12, 0.02%H3B03, 0.02%MgSO4. 7H20, 0.01%KNO3 for a wide range. The procedure of isolation potato mature pollen protoplasts by 揷old treatment- germination-enzymeethod was established. Germinated the grains after 7 days of cold treatment for 30mm, then transferred them into the enzymatic solution supplemented with 1 .2mol/L mannitol, the yield of pollen protoplasts was up to 21.04%. While the isolation percentage declined to 16.05% and the yield of sub-protoplasts was up to 5.35% when the germination duration was 2 hours. Culture experiments showed: Mature pollen protoplast is difficult to be induced to dedifferentiate; Tube-subprotoplast has strong ability of regeneration the cell wall and the trend of self-fusion. 3 The factors of affecting pollen cryopreservation in Solanum tuberosum and protoplasts isolation were also investigated. The viability of cryopreservation pollen grains of I 8hours?desiccation duration before thrown into LN was higher than that of l2hours?or 6hours? The method of step by step defrozen at -2OC棗4C?5~C is best. Using pollen grains after cryopreservation for 90d as the materials to isolate protoplasts, the isolation percent was 16.21%. Although the viability of cryopreservation pollen is declined than that of fresh pollen, protoplasts could also be isolated from them and the difference of isolation percentage were not significant. In conclusion, this is the first report, as far as we know, on the establishment of the procedures of isolation and culture of pollen protoplasts in detail. The result showed the procedure was general suitable .This isolated cells are potentially useful for further study in genetic engineering.