【作者】 吴旺泽；

【导师】 王蒂； 王清； 张金文；

【作者基本信息】 甘肃农业大学 ， 作物遗传育种， 2004， 硕士

【摘要】 实验采用6个优良四倍体栽培种和3个双单倍体不同发育时期的花粉为供试材料，研究花粉原生质体分离与培养的技术体系，取得如下结果：1.对不同发育时期的花粉经低温和甘露醇处理后，进行培养，其中单核期幼嫩花粉培养后脱分化频率达4.1%，具有孢子体发育的巨大潜力。四分体时期的花粉培养后不能启动分裂；成熟花粉培养后易趋向于配子体发育方向。2.四分体时期的花粉经低温处理5~7d后，在0.3mol/l的蔗糖做渗透压调节剂，1%蜗牛酶+0.5%崩溃酶+1.2%纤维素酶的混合酶液中，四分体原生质体分离率最高达74.2%，在K3改良培养基+1mg/l 2,4-D+0.2mg/l 6-BA+1mg/l NAA+800 mg/l谷氨酰胺+100mg/l丝氨酸+0.1%小牛血清的培养基中培养1d后再生细胞壁，2~3d后再生细胞壁的原生质体启动第一次分裂，分裂频率最高达14.2%，培养10~15d后，无组织生长的细胞最后增殖生成细胞团。3.单核期幼嫩花粉先经饥饿处理，再经低温水合、高温热激、渗激多步冲击的方法制备了马铃薯脱外壁花粉，Favorita最高脱壁率达34.5%；对脱外壁花粉进行了离体人工萌发，最高萌发率达38.3%。脱外壁花粉在0.6mol/l甘露醇+0.2mol/l山梨醇做渗透压的酶液中，花粉原生质体最高分离率达24.3%，质体培养１~2d后再生细胞壁，3d后再生细胞壁加厚，个别原生质体膨大后表现出孢子体发育迹象。4.马铃薯花粉离体萌发研究结果表明，在10%蔗糖+10%PEG（8000）+0.02%Ca(NO3)2+0.02%H3BO3+0.5mmol/l K+的培养基上马铃薯花粉萌发率最高达83.4%，添加低浓度的Na+,6-BA能促进花粉萌发和花粉管伸长。5.针对马铃薯成熟花粉的特点建立了“低温—萌发—酶解”的花粉原生质体分离方法。低温处理5~7d的材料，在萌发液中萌发30min后在0.8mol/l甘露醇+0.2mol/l山梨醇渗透压调节剂下，在添加有K3培养基大量和微量元素或13mol/l的CPW盐离子的酶液中，原生质体分离率达24.7%。6.当花粉管长到约2~3mm后，转入酶液后1~2h就可观察到花粉粒发生质壁分离，4~5h后花粉管亚原生质体被释放。花粉管亚原生质体大小差别较大，大的直径约50~60μm，小的约10μm，刚分离的亚原生质体易自发融合，培养1d后再生细胞壁。更多还原

【Abstract】 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. <WP=8>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.更多还原