【作者】 郝玉金；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2000， 博士

【摘要】 本研究以柑桔、苹果、草莓、梨和猕猴桃5种有代表性的果树为试验材料，对离体保存方法及其遗传变异进行了研究，主要研究结果如下： 1建立了5个基因型的柑桔单细胞姊妹系以及苹果和草莓各2个基因型的单芽姊妹系，为遗传变异研究提供了材料。 2建立了根据细胞大小计算有丝分裂指数以及判断细胞倍性的方法，利用该方法在活体条件下对柑桔胚性愈伤组织中不同倍性细胞群体的有丝分裂指数进行了计算，表明四倍体变异细胞在常规培养条件下的有丝分裂受到抑制，并通过程序性死亡被淘汰。 3完善或改进了5种果树组培材料的离体保存方法，各种材料用缓慢生长法保存1a的生长量相当于正常条件下约3周的生长量，转入正常培养条件后可以恢复生长；超低温保存后柑桔胚性愈伤组织获得了90％以上的存活率，苹果利草莓茎尖的存活率相对较低，为72.7％-89.3％，表明柑桔胚性愈伤组织更适于用超低温保存，苹果和草莓茎尖用缓慢生长法更好。 4柑桔胚性愈伤组织的染色体观察表明，染色体变异普遍存在并在愈伤组织形成早期就已发生，异常有丝分裂是造成染色体变异的细胞遗传学机制；继代培养和离体保存1a后愈伤组织的倍性组成保持了相对稳定性，这是由于多倍体变异细胞在不断产生的同时也通过程序性死亡途径不断被淘汰。 5用RAPD分子标记对继代培养和离体保存1a后的遗传变异进行了检测，继代培养前后的8个纽荷尔脐橙单细胞姊妹系用102个随机引物共扩增出9 796条带，除引物OPH-15检测到4条变异带外，其余相同，变异率约为0.04％；上述细胞系和引物也用于超低温保存后的变异检测，共扩增出9792条带，保存前后未发现变异；缓慢生长法保存的8个红马叙葡萄柚单细胞姊妹系用98个随机引物共扩增出8624条带，也没有检测到变异。 6用AFLP分子标记对离体保存后的遗传变异进行了检测，苹果DNA用MseⅠ／Eco RⅠ双酶切，20个引物对扩增，缓慢生长法保存前后的8个嘎拉单芽姊妹系共获得29760条带，超低温保存前后的8个M₂₆单芽姊妹系共获得28480条带，都 fP中农业人学2000 M博卜论义没有发现变异：草萄＊＊A用人力el／凡JI双酶切（凡JI对甲基化敏感），16个引物对扩增，缓慢生长法保存前后的 8个黑龙江 1号单芽姊妹系共获得 20 992条带，保存前后未发现变异，超低温保存前后的8个女峰单芽姊妹系共获得ZI 512条带，各单芽系在引物对M－CAG／PCCA的扩增产物中都增加了一条带，是DNA甲基化导致的，其它引物对的扩增产物没有差异。 7对转化的柑桔愈伤组织、梨和猕猴桃转化组培苗（或茎尖）成功地进行了离体保存，通过GUS基因的化学染色、PCR扩增、SSCP标记和 Southern杂交验证了GUS基因在离体保存过程中的稳定存在和表达。 8利用MSAP标记对各种材料的DNA甲基化状况进行了研究，发现具有胚状体发生能力的愈伤组织与丧失胚状体发生能力的愈伤组织相比，其DNA甲基化程度要低：经低温和超低温保存后，柑桔愈伤组织胚状体发生能力增强，同时伴随着＊＊A甲基化程度降低；超低温保存的苹果和草萄茎尖恢复生长中生根能力增强，也伴随着DN A甲基化程度降低：表明离体保存过程中的各种胁迫能够诱导＊＊A甲基化变化，其中的脱甲基化可能与愈伤组织或茎尖的再生或分化能力增强有关。更多还原

【Abstract】 In the present paper,five kinds of representative fruit crops including citrus,apple,strawberry,pear and kiwi fruit were used to investigate the approaches of in vitro conservation and the genetic variations,The main results were as follows:1 Single-cell sibling lines of 5 citrus genotypes and single-bud sibling lines of 2 apple and 2 strawberry genotypes were established,which provided materials for the studies on somaclonal variation.2 A method to calculate mitosis index and to estimate cell ploidy was put forward and experimentally verified,and then was used to calculate in vivo the mitosis index of different ploid cells in the same citrus callus. The results indicated that quantitatively predominant diploid cells inhibited the mitosis of tetraploid cells in diploid genotype. Later,these tetraploid cells disappeared via programmed cell death under the normal subculture condition. However,this inhibition by the diploid cells was neutralized by low-density culture condition.3 Approach for in vitro conservation of 5 kinds of fruit crops were established and optimized. The average growth mass of material conserved for one year without subculture by slow-growth was just equal to that of 3-week culture under routine subculture condition. The conserved materials survived after being transferred into routine culture condition. With regard to cryopreservation,viability of citrus calli was above 90% whereas shoot-tips of apples and strawberries were 72.7% to 89.3% respectively,as indicated that cryopreservation was more suitable than slow-growth for citrus callus conservation,and slow-growth method was suitable for the conservation of shoot-tips of apple and strawberry.4 Chromosome variation in citrus calli occurred at the early stage of the callus formation was observed. However,the relative genetic stability of chromosome ploidy was maintained during subculture and in vitro conservation,which resulted from the disappearing of variant cells by programmed cell death.5 RAPD markers were adopted to detect the DNA-level variations of citrus callus during subculture and in vitro conservation. As for 8 single-cell sibling lines of Newhall navel,a total of 9 796 bands obtained from the subculture calli with 102 primers. Most of those bands were the same only except 4 bands obtained from the primer OPH-15,as showed a variation frequency of about 0.04%. While no aberration was observed in 9 792 bands obtained from cryopreserved callus. Similarly,8 624 bands without diversity were obtained from 8 Red Marsh single-cell sibling lines conserved in vitro by slow-growth method.6 AFLP markers were also used to detect the DNA-level variations of shoot-tips from single-bud sibling lines of apple and strawberry. After the DNAs of apples were digested by Mse I and EcoR I and amplified with 20 pairs of specific primers,a total of 29 760 bands were observed from Malus pumila var. Gala conserved by slow-growth and 28 480 bands from cryopreserved M. pumila var. M26,no variant band was found. After the DNAs of strawberries were digested by Mse I and Pst I,the latter one sensitive to DNA methylation,and amplified with 16 pairs of specific primers,a total of 20 992 and 21512 bands from Fragaria vesca var Heilongjiang No.l conserved by slow-growth and cryopreserved F. xananassa var. Joho was obtained respectively. As for the former,no difference in band profile was found. But there was a new band scored from every single-bud sibling lines of the latter,which resulted from DNA methylation.7 The transformants of citrus callus,pear and kiwifruit shoot-tips with GUS gene were successfully conserved in vitro by slow-growth and cryopreservation. Chemical stain,RAPD,SSCP marker and Southern blotting confirmed the stable expression and integrity of GUS gene in host genome.8 MSAP markers were used to investigate the DNA methylation status of tested materials. DNA methylation level of Newhall navel callus incapable to regenerate embryoid was higher than that of callus with embryogenesis capacity. Conservation in vitro enh更多还原