摘要
搜集和创造新的种质资源，扩大变异来源是所有作物育种研究的重要课题。马铃薯栽培种大多为同源四倍体，遗传分离复杂，后代筛选繁琐，使得常规育种效率低。近年来发展起来的体细胞融合、基因导入、体细胞无性系变异等育种新技术在某种程度上可克服以上缺点，为育种开辟了一条新途径。通过原生质体培养再生的植株常常发生高频率变异，利用这一特性作为获得突变体的手段进而筛选新品种(系)可成为马铃薯品种改良的途径之一。但同时也应看到变异对体细胞融合，基因导入造成的不利影响。因此，深入全面地研究其遗传变异是这一培养体系应用中十分重要的工作。
本研究对马铃薯叶肉原生质体再生植株的54个株系及其母本进行分子水平(RAPD分析)、细胞水平(染色体数目)、表型水平(11个性状)的鉴定，全面深入地研究了再生植株的遗传变异；同时对小量法提取DNA及RAPD稳定性进行了研究，主要结果如下：
对双单倍体3#原生质体再生植株39份和双单倍体8#再生植株15份材料的表型分析表明再生植株的表型除8个株系变化较大，7个株系没变化，其它39个株系变异较小。源于同一愈伤组织的植株表型相近，发生明显变异的性状有结薯状况，株高和小叶数。
在54个再生植株的染色体检测中，有7个株系是双单倍体，2n=24；40个株系是四倍体，2n=48；少数为非整倍体和六倍体，变异丰富。同时也观察到有丝分裂过程中染色体联会分离的环状构型，染色体核内复制、胞质不分离等现象。
比较表型与染色体检测结果发现，原生质体再生植株的表型变异往往来源于其有丝分裂变异造成的染色体数目的变异。
用23个随机引物对3#母本及其39个再生植株，用23个随机引物对8#母本及其15个再生植株进行RAPD分析结果显示，再生株系与母本在所检测的位点范围内均有差异。3#再生株系变异率小，介于5.6％-18.2％之间，较集中，8#变异率大介于4.3％-32.6％较分散。原生质体再生植株在分子水平上的变异更为普遍，但各株系变异率低(个别株系除外)。在表型没有或很少变异的植株中，RAPD分析也显示出普遍变异，这说明再生植株的分子变化主要发生在不影响基因表达的DNA非编码区。
改良的小量法提取DNA，可快速、经济的提取高质量的DNA。同时通过优化选择反应参数(本实验结果显示DNA浓度在150-300ug／L之间可获得良好的扩增结果；MgCl_2浓度在1.5—4mmol／L范围内扩增效果最好；dNTP在反应中的最终起始浓度在100—300μmol／L之间为最佳；引物在0.3—3.0μmol／L之间为最佳浓度)获得了稳定的RAPD结果。
本研究首次从三个不同水平鉴定马铃薯原生质体再生植株的遗传变异，对马铃薯双
单倍体原生质体再生植株遗传变异得到全面详尽的认识，从而为以原生质体培养为载体
技术的育种工作提供理论依据。同时对小量法提取DNA作了改进，增加了RAPD稳定
性，对提高RAPD分析结果的可靠性具有十分重要的意义。
Collecting,creating new germplasm resources and enlarging the orign of variation are the important works in crop breeding research . Almost all potato cultivars are homogeneous tetraploid .Its complex genetic segregation and troublesome selecting work which make the conventional breeding very difficult .New breeding technology -protoplast fusion , genetic trasformation and somaclonal variation can overcome these shortcomings in some degree and make a new way for breeding .Protoplast-derived plants often generate a high somaclonal variation ,using the specific traits to get variants and selecting new variety could be a way of potato improvement. At the same time,however, disadvantage of this variation to protoplast fusion and genetic trasformation deserve attention . Therefore studying the genetic variation of protoplast-derived plants has very significance .
The morphology, chromosome variation and RAPD polymorphism of 54 plants regenerated from mesophyll cell protoplasts were analyzed ; and also DNA extraction and the stability of RAPD were studied.
Total 39 clones derived from the protoplasts of dihaploid 3#and 15 clones derived from the protoplasts of dihaploid 8# were analyzed.
The results showed that the morphological variations were universal. Including tuber> height N and leaflets.
Except 15 regenerated plants occurred much or no variation, 39 had only little variation . The plants derived from the same callus were similar in morphology.
Total 54 clones were examined in chromosomal level ,7 clones had normal chromosome number ( 2n= 24 ) ; 40 clones were tetraploid ( 2n= 48 ) ; a few aneuploid and hexploid ( 2n= 72 ) were found .Endomitosis and the ring -like chromosome were also observed during mitosis .
Contrasting morphology results with chromosome results, it was obviously that the chromosomal variation often led to morphological variation .
Total 23 and 23 random primers were used to analyze RAPD polymorphism of plants from 3# and 8#. respectively. The results showed that all regenerated plants were different from their parents in the site examined. The genetic variation frequency of 3# was from 5.6% to 18.2%, and 8# was from 4.3%-32.6%. The variation of molecular level was also universal .The molecular variation,however, was not always leading to morphological
variation.
The method of little amount material DNA extraction was improved in some degree. By optimizing RAPD reaction parameter ( The concentration of DNA templates is from 150 ug/.L to 300ug/.L, The concentration of MgCl2 is from 1.5mmol/L to 4mmol/L, The concentration of dNTP is from 100umol/L to 300umol/L, The concentration of primer is from 0.3umol/L to 3.0umol/L.) , the more stable RAPD result was obtained.
The genetic variations were first syntheticly analyzed at three different levels for the protoplast-derived plants in potato, which give us an overall knowledge of these variations. The results could provide the theoretical base for potato breeding using protoplast culture system .In addition,little material DNA extraction method was improved and optimized reaction parameter of RAPD could improve its stability in some degree.
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