【作者】 张淑红；

【导师】 王蒂；

【作者基本信息】 甘肃农业大学 ， 作物遗传育种， 2004， 硕士

【摘要】 马铃薯是唯一的粮菜兼用作物，其原生质体培养及体细胞杂交是“分解－综合”育种的重要环节，也是将具有抗逆、抗病等优良性状的野生种资源引入栽培种中的重要途径。试验以3个双单倍体品系和3个野生二倍体为材料，进行了马铃薯叶肉原生质体的培养及融合的研究，同时也探讨了一些外界条件如真空抽气、低温预处理、褐化抑制剂等对原生质体培养的影响，获得了如下结果：真空抽气能够有效地提高原生质体的数量，降低原生质体的破碎比例，使原生质体在培养过程中的杂质大大减少。材料的种类和基因型与原生质体的产量和质量密切相关。双单倍体品系无论在原生质体的产量、活力，还是在原生质体的植板率上均优于野生二倍体，尤其以2－10品系最好，其产量可达5.83×106/g鲜叶，活力为61.02％，植板率为32％；CW－2－3最差，其产量为0.31×106g鲜叶，活力为16.40％，植板率为0％。基因型不同，所获得的原生质体的褐化指数也不同，野生二倍体的褐化指数均比双单倍体的高。野生二倍体中以CW－2－3的褐化指数最高，为18.33％；双单倍体中以2－10的褐化指数最低，为9％。但在培养基中添加0.6mg/L的AgNO3，可有效抑制原生质体在培养中的褐化现象，从而提高细胞分裂频率。NAA或6－BA会引起马铃薯叶肉原生质体的褐化，但二者互作时却又抑制了原生质体的褐化。1.2mg/L的NAA引起的褐化指数比NAA0.8，1.0mg/L的高，褐化率为14％,0.6，0.8mg/L的NAA引起的褐化指数差异性不显著；6－BA在0.2，0.4，0.6mg/L三个水平上差异性均显著，0.6mg/L6－BA褐化率最高，为17.33％。对CW－2－7的试管苗进行2d的低温预处理，能够有效提高原生质体的植板率。以4℃的处理效果最好，原生质体植板率与15℃和室温的相比，差异性显著。对CW－2－7和2－10进行PEG高Ca2+高pH诱导融合时，最适宜的温度为30℃，最适宜的pH值为9.0；进行电融合时最适宜的电气参数为：交流电场强度125V/cm、直流脉冲场强0.75KV/cm、脉冲宽度40μs，在此参数下的电场处理不影响原生质体的活力。通过马铃薯叶肉原生质体的分离及培养，获得了3个双单倍体品系和1个野生二倍体的再生植株。更多还原

【Abstract】 Potato (Solanum tuberosum L.) is the only crop used both as food and vegetable.Protoplast culture and somatic hybridization is the important procedure of analytical-synthetic breeding of potato and the important way to transfer resistant properties of wild species into cultivar of potato.In this reseach,three anther derived dihaploid strains and three diploid wild species were used as the donor tissues to investigate the protoplast culture and somatic hybridization of potato.The effect of some outside factors such as vaccum infiltration,cold pretreament,browning inhibitor were also discussed.The results showed as follows:The treatment of vaccum infiltration on donor tissues could improve the yield and reduce the broken ratio of proptoplasts.There were less impurities in the protoplasts when they were cultured.There was an intimate relationship between the yield, viability, plating effeciency of protoplast and the donor tissues’ variety and genotypes.The protoplast’yield,quality and plating effeciency of dihaploid strains were all better than diploid wild species’.2-10 was the best,its protoplast yield was 5.83×106/g fresh leaves,viability was 61.02% and plating effeciency was 32%.CW-2-3 was the worst,its protoplast yield was 0.31×106/g fresh leaves,viability was 16.40% and plating effeciency was 0%.The cold 2d-pretreatment with the tube plantlets of CW-2-7 could improve its plating efficiency of protoplast.With 4℃ pretreament,the plating effeciency of protoplast was the highest compared with 15℃ and Room Temperature.The browning rates were very different with different genotypes,and diploid wild species’browning rates were all higher than the dihaploid strains’.The browning rates of CW-2-3 was the highest,18.33%;2-10’s browning rate was the lowest,9%.But the addition of AgNO3 into the culture medium of protoplast could prevent its browning,and the best concentration of AgNO3 was 0.6mg/L. <WP=8>NAA or 6-BA could cause the browning of mesoplyll protoplasts of potato.But when they were cooperated,they could prevent the browning of protoplasts.The browning rates of NAA1.2mg/L were the highest among the three NAA concentration levels. The variances of protoplast browning rates were all siginificant in the three 6-BA concentration levels.The protoplasts browning rates of 6-BA0.6mg/L were the highest,17.33%.The chemical fusion by PEG method and electro-fusion were between CW-2-7 and 2-10.The results showed that the best temperature was 30℃ and the best pH was 9.0 in the chemical fusion.The best parameters of electro-fusion was:AC125V/cm,DC0.75KV/cm,PW40μs.With those parameters of electro-fusion,no side-effect was found on the protoplast viability.Regenerated plantlets from three dihaploid strains and one diploid wild species were got by the isolation and culture of mesophyll protoplast of potato.更多还原