【作者】 田振东；

【导师】 戴朝曦；

【作者基本信息】 甘肃农业大学 ， 作物遗传育种， 2000， 硕士

【摘要】 体细胞融合技术在马铃薯这一无性繁殖作物的生物技术育种中具有重要的理论与实践意义。电融合是一项新兴的融合技术，在马铃薯体细胞杂交中已显示出良好的应用前景。但是目前在马铃薯的体细胞电融合具体操作中，普遍存在成对融合频率低的问题。因为单倍体细胞的成对融合可以使融合产物保持原有物种种性。为了提高在电融合中成对细胞融合的频率，本实验以普通栽培种（S.tuberosum L.）花药诱导而来的双单倍体品系“81-15”实生苗下胚轴原生质体和“84-3”叶肉原生质体为材料，进行了提高马铃薯原生质体自体融合和异体融合的成对融合频率的技术探讨。结果表明：在自然光和室温（25±2℃）条件下培养游离材料，结合游离结束前半小时将摇床转速由40r/min增至50-55r/mim的处理，可以得到活力旺盛、质膜较为稳定一致的高质量原生质体，融合时破碎少，融合同步性好。自体融合试验表明：采用较低的原生质体密度（5×10~4个/mL），较低的成串电压（"81-15"：200v/cm，"84-3"：230v/cm）和适中的成串时间（40s），两材料的理想细胞串（含二个原生质体）频率均可达36％左右。成串过程中对交流电压作适当调整可将理想细胞串频率提高到45％左右，在施以150μs，0.8kv/cm的适中高压直流脉冲后，两材料自体成对融合频率均可高达42％左右。异体融合试验表明：采用较低的原生质体密度（5×10~4个/mL），调整具有不同融合特性的双亲原生质体比率可以得到较高的成对异体融合频率。本实验在“81-15”下胚轴原生质体与“84-3”叶肉原生质体比率为1:2的条件下，成对异体融合频率高达26％。成串过程中通过调整交流电场强度，可以明显提高成对异体融合频率。在最理想条件下，成对异体融合频率最高可达32％，比一般条件下的平均频率高约7％。此外，实验结果还表明：融合物在培养过程中，应适时加入新鲜等渗培养基，从分裂到分化阶段不宜降低培养基的渗透压。更多还原

【Abstract】 Somatic electrofusion techniques are of great importance in the potato biotechnology breeding theoretically and practically. Electric-field induced protoplasts fusion as an newly developed method. has recently showed bright practical prospect ,but the low frequency of two cell fusion is the main problem in the practice of the potato somatic electrofusion because the two haploid cell fusion can keep the same chromosome number of a species. In order to obtain the high two cell fusion frequency in mass somatic fusion experiments, the optimum electric paramete and other effective factors in potato electrofusion were studied .The mesophyll cells from an anther derived dihaploid strain 4-3 and the hypocotyls from true potato seeds of S.tuberosum L. dihaploid strain 1 -.15were used as the donor tissues .The results showed that the high vigor ,stable membrane protoplasts could be obtained when the materials cultured under sunlight and 25C combined with accelerating shalker rotation speed from 40r/min to 50-55r/min ahead of protoplasts purification 30 mm. Homologous electrofusion experiments reveled that at the intermediate protoplasts densities(5 X I 04/ml),using low AC field( 1-15. 200Vpp/cm.4-3 23OVpp/cm)and moderate duration(40s),the desirable ell chain with two protoplasts of the two materials could reach about 36%.lt was found that adjustment of Vpp intensity during cell alignment could increase desirable ell chain frequency from about 36% up to 45%,after treatment with 0.8kv/cm DC square pulse for 150 ,42% optimal frequency of two cell fusion were obtained. The results of experiments on the heterofusion proved that ,at low densities(5>( I 04/ml),by adjusting the ratio of two materials protoplasts which exists inherent difference in fusability could get higher heterofusion frequency . In the present experiments, at the ratio of 1:2( 1-15 hypocotyl protoplasts: 4-3 mesophyll protoplasts), the binary heterofusion frequency reached 26%. It has been noted that with adjusting AC intensity could obviously increase binary heterofusion frequecies during the course of cell alignment in both homologous and heterologous electrofusion experiments. At the present studies, under the optimum conditions, the frequecies of binary heterofusion reached 32% which increased 7% at the average nevel compared with unadjusted conditions. In addition the studies also indicated that it is very important to add fresh , unchanged osmolarity medium timely during culturing fusion products, it is unadvisable to reduce medium osmolarity from protoplasts division stage to callus differentiation stage.更多还原