Isolation And Culture Of Pollen Protoplasts In Solanum Tuberosum L.

Pollen protoplasts,as a special experiment system in the study of plant cell engineering,have both advantanges of haploid and protoplasts,they can be served as an impotrant segment in Solanum tuberosum L. breeding project.The present investigation studing the condition and some effectional factors on isolation and culture of pollen protoplasts from microspores at the stage of tetrad,monokaryotic,mature pollen grains and pollen tube of potato.The pollen grains of different stage were culture in vitro after cold temperature and mannitol pretreatment,the results showed that the pollen grains of monokaryotic stage had gigantic potential of sporophyte development,and the percentage of dedifferentiation was up to 4.1%.The pollen grains of tetrad stage didn't promoted division,most of mature pollen grains trended to gametophyte development after culture.The highest isolation percentage of tertade protoplasts was up to 74.2% after 5~7 days of cold pretreatment in the enzyme solution containing 1% snailase,0.5%driselase,1.2%cellulase,0.3mol/lsucrose and K3 medium salts.The protoplasts regenerated cell wall after 1day,and begain first division after 2~3d days of culture in K3 basic medium supplemented with 1mg/l 2,4-D,0.2mg/l 6-BA,1mg/l NAA,800mg/l L-glutamin, 100mg/l L-serine,0.3mol/l sucrose and 0.1% calf serum,the division percentage was up to 14.2%.After 10~15days of culture some cells continued division and formation of small cell clumps.For isolation monokaryotic young pollen protoplasts in Solanum tuberosum L.,the technique of preparation de-exined pollen was established.The procedure included four steps:(1)stratvation treatment, (2)cold hydrated(at4℃), (3)heat-shock(at34℃), (4)osmotic-shock. The highest percentage of de-exined pollen was up to 34.5%,and the de-exined pollen was germination in vitro with a rate of 38.3%.Using mannitol and sorbitol as osmotic pressure regulator,the highest isolation percentage of young pollen protoplasts was up to 24.3% in the mixed enzymatic solution containing 1% cellulase,0.5%pectinase,0.5%macerozyme,0.5%PDS and K3'macroelement and microelement,after 2days of culture regenerated cell wall. The factors of influence germination were also studied.The highest germination rate was up to 83.4% with the liquid medium containing 10%sucrose,10%PEG(8000),0.02%Ca(NO3)2,0.02%H3BO3,0.5mmol/l KNO3 and supplemented with 0.5mmol/lMg2+,0.1mmol/l Na+and 1mg/l 6-BA can increase the germination rate.The study established the procedure of isolation potato mature pollen protoplasts"cold treatment-germination-enzyme".The pollen grains was germinated 30min after 5~7days of cold pretreatment ,then transferred them into the enzymatic solution (component of enzymatic as above) supplemented with 0.8mol/l mannitol,0.2mol/l sorbitol and K3'macroelement and microelement or 13mol/lCPW ,the yield of mature pollen protoplast was up to 24.7%.when the pollen grains germinated after 1~2h,then trasfeered them into enzymatic solution (component of enzymatic as above)about 4~5h pollen tube subprotoplasts were released.The subprotoplasts trended to spontaneous fusion at the first stage of isolation, after 1 day of culture regenerated cell wall.