【作者】 李林光；

【导师】 张志宏；

【作者基本信息】 沈阳农业大学 ， 果树学， 2008， 博士

【摘要】 苹果是世界性果树,世界上相当多的国家都将其列为主要消费果品。由于果树多倍体一般具有生长旺盛、果实大且少籽或无籽、产量高、适应性和抗逆性强等特点,且能够利用无性繁殖的方式固定其优良性状,使之保持稳定而不分离,所以多倍体育种一直被认为是创造苹果新品种的重要途径。本研究以金冠、嘎拉、富士苹果的成熟胚和寒富、烟嘎1号的离体叶片为试材,通过秋水仙素诱变技术和二倍体与多倍体杂交的方法,获得苹果多倍体新种质。同时在分子水平上探究苹果四倍体与二倍体之间的差异,为阐明苹果多倍体基因组进化机理奠定基础。主要研究结果如下:1.建立了苹果成熟胚离体诱导四倍体的技术体系。金冠的成熟胚在0.5%的秋水仙素+1%二甲基亚砜的混合溶液中浸泡48h后接种在MS+TDZ 1.0 mg·L^-1+IBA 0.5 mg·L^-1培养基上进行再生培养,诱变率达10.6%。2.建立了以寒富、烟嘎苹果的试管苗叶片离体诱导四倍体的技术体系。寒富苹果叶片在附加15、30、60、120 mg·L^-1秋水仙素的液体再生培养基(MS+1.0 mg·L^-1 TDZ+0.5 mg·L^-1 NAA+200 mg·L^-1 LH)中静置暗培养5 d,各个浓度处理均诱导出四倍体植株,诱变率在5.3%～22.2%之间。寒富、烟嘎1号苹果叶片在附加50 mg·L^-1秋水仙碱固体再生培养基上处理5 d亦获得了四倍体植株,诱变率分别为20.0%和15.0%。3.将诱变获得的倍性嵌合体植株的叶片接种到MS+TDZ 1.0 mg·L^-1+IBA 0.5 mg·L^-1+蔗糖30 g·L^-1+琼脂6 g L^-1培养基上进行再生,从金冠、嘎拉、富士的倍性嵌合体中均分离出了同质突变体。4.比较了3种获得苹果三倍体的方案:（1）通过四倍体与二倍体杂交获得三倍体;（2）通过三倍体与二倍体杂交获得三倍体;（3）在三倍体实生后代中筛选三倍体。表明最好的方法是二倍体与四倍体间的杂交,出现三倍体比率明显高于其他两种方法。5.苹果多倍体植株生长健壮,枝条粗壮,叶片大而圆,粗糙,叶厚,叶边锯齿较多,节间较短。多倍体的气孔比二倍体和非整倍体的气孔大得多,在气孔密度上,多倍体小于二倍体和非整倍体。苹果三倍体实生后代中不同倍性植株在形态上差异显著,根据植株形态和叶形指数,可以容易地将多倍体（三倍体、四倍体）植株与二倍体植株和非整倍体植株区分开来。6.二倍体苹果品种富士和烟嘎1号叶绿素a、叶绿素b、叶绿素总含量和类胡萝卜素分别低于其同源四倍体天星和四倍体烟嘎1号品种,但叶绿素a/b比值与倍性没有表现出一致的变化规律。对二倍体品种富士和烟嘎1号及其各自同源四倍体品种的叶绿素荧光参数日变化规律进行了测定,结果显示二倍体品种富士和烟嘎1号Fo日平均值分别为214.6和208.0,分别较天星和四倍体烟嘎1号高出9.4和8.3。二倍体品种富士和烟嘎1号的PI值分别小于其同源四倍体品种天星和四倍体烟嘎1号。7.通过电镜观察,二、四倍体苹果花粉的大部分侧面观均为长椭圆形,极面观为三角形,具3纵沟孔;花粉表面为纵向条状纹饰,有穿孔。四倍体花粉量明显比二倍体少,非椭圆形花粉粒特别多。四倍体P/E值为1.8,二倍体为2.0,差异显著。四倍体胚囊存在不同程度的异常结构。8.采用ISSR分子标记技术对富士、烟嘎1号、寒富的二倍体及其同源四倍体进行PCR扩增。8条引物共扩增出65条180～2500bp谱带,其中24条带具有多态性,占36.9%,每个引物可扩增出0～5条多态性带,平均为3条。三组苹果（富士和天星、寒富和四倍体寒富、烟嘎1号和四倍体烟嘎1号）之间有所差异,但富士、烟嘎1号、寒富的二倍体及其同源四倍体间的遗传相似系数全部为1,不能区分。9.利用高效液相色谱仪测定了苹果二倍体品种及其同源四倍体叶片DNA水解样中胞嘧啶和5-甲基胞嘧啶的含量。四倍体天星与二倍体富士相比,天星的DNA甲基化程度降低,为29.33%,富士为35.34%。四倍体寒富和二倍体寒富甲基化程度分别是14.37%和14.49%,差异不大。10.本研究共获得金冠、嘎拉、富士成熟胚诱导的同质四倍体251株,寒富叶片诱导的四倍体20株,烟嘎1号叶片诱导的四倍体13株。从四倍体与二倍体杂交后代、三倍体与二倍体杂交后代及三倍体实生后代中共筛选出三倍体46株、四倍体5株。上述多倍体种质的获得为选育苹果多倍体品种奠定了重要基础。更多还原

【Abstract】 The apple is the world fruit,and most countries in the world rank it with the main consuming fruits. Because polyploidy fruit trees generally have the characteristics of vigorous growing,big fruit size, fewer seeds or seedless,high production,strong adaptability and resistance,and could keep excellent characteristics with asexual propagation,polyploidy breeding is considered as the importance approach of create new apple cultivars.This study obtained apple novel polyploidy germplasm with the mature embryos of ’Golden Delicious’,’Gala’,’Fuji’ and the in vitro leaf of ’Yan 1 Gala’,’Hanfu’ as materials by colchicine induction and polypolid hybridization.Besides on that,we analyzed the difference between apple tetraploid and diploid on molecule level,which will lay a foundation for the work on elucidating the evolution mechanism of apple polyploidy.The main results are as follows:1.The system of induction of tetraploid from apple mature embryos in vitro was successfully established.The induction rates was 10.6%when the mature embryos of ’Golden Delicious’ apple were treated with 0.5%colchicines and 1%DMSO for 48h and then cultured on MS medium supplemented with TDZ 1.0mg·L^-1,IBA 0.5 mg·L^-1.2.In vitro tetraploid induction of ’Hanfu’ and ’Yan 1 gala’ apple were studied by treating leaf with colchicines solution.The inducing percentage was from 5.3%to 22.2%when the leavess of ’Hanfu’ were treated in liquid medium(MS+TDZ 1.0 mg·L^-1+NAA 0.5 mg·L^-1+LH 200 mg·L^-1) supplemented with colchicines 15 mg·L^-1,30 mg·L^-1,60 mg·L^-1 or 120 mg·L^-1 for five days at dark.And tetraploid plantlets were also obtained from ’Hanfu’ and ’Yan 1 gala’ when the leaves were treated on solid medium supplemented with colchicines for five days at dark.The induction rates were 20.0%and 15.0% for ’Hanfu’ and ’Yan 1 gala’ respectively.3.The leaves of ploidy chimeras were inoculate to culture medium which contained MS+TDZ 1.0 mg·L^-1+IBA 0.5 mg·L^-1+sucrose30 g·L^-1+ Agar 30g·L^-1 for adventitious shoots regeneration.The homogeneity mutants could be separated form the ’Golden delicious’,’Gala’ and ’Fuji’ ploidy chimeras.4.Comparing the three schemes for obtaining apple triploid plants:（1） hybridization between diploid and tetraploid plant;（2） crossing between diploid and triploid plant;（3） selecting triploid plants from triploid seedling progenies.Result show that the best methed was the hybridization between diploid and tetraploid plant,as the percentage of triploid plants obviously higher than that using other schemes.5.Apple polyploid plants were vigorous with strong shoots and short internodes,and its leaves were big,round,coarse,and thick with more teeth,.Stomas of triploid seedling progenies were bigger than that of diploid and aneuploid plant,but the density of stomas was less than that in diploid and aneuploid plant. Among the polyploid seedling progenies of apple,different ploidy plants had marked discrimination in plant shape.Based on plant shape and index of leaf shape,polyploid（tetraploid and triploid） plants could easily be distinguished from the diploid plants and the aneuploid plants. 6.The chlorophyll a,chlorophyll b,the total amount of chlorophyll and carotenoid of diploid apple cultivars ’Fuji’ and ’Yan 1 Gala’ were below their autotetraploid ’Tensei’ and ’tetraploid Yan 1 Gala’, respectively.However,chlorophyll a/b ratio have not showed consistent changes.The chlorophyll fluorescence parameters changes in the law of diploid varieties ’Fuji’ and ’Yan 1 Gala’ and their respective autotetraploid species were measured and the results showed that FO averages of diploid varieties ’Fuji’ and ’Yan 1 Gala’ were 214.6 and 208.0,which are higher than the ’Tensei’ and ’tetraploid Yan 1 Gala’-9.4 and 8.3.The ’PI’ on diploid varieties Fuji and ’Yan I Gala’ were lower than their autotetraploid ’Tensei’ and ’tetraploid Yan 1 Gala’ respecitively.7.The diploidy and tetraploidy pollens of apples were observed by electron microscope,the pollens have three longthways channels,most of their profiles were oblong and polars were triangle.The pollen surfaces have longways striations and perforations.The pollen quantity of tetraploids were less than that of diploids,and nonablong pollens were only few.P/E value of tetraploids was 1.8,and that of diploids was 2.0,which have significant difference.8.The ’Fuji’,’Yan 1 Gala’ and ’Hanfu’ of diploid and autotetraploid were analyzed with ISSR markers.A total of 65 bands,ranged from 180 to 2500 bp,were amplified with eight primers. Twenty-four bands were polymorphic,accounting for 36.9%,and each primer can amplify from 0 to 5 polymorphic bands,with 3 bands at average.Three groups of apples（’Fuji’ and ’Tensei’,’Hanfu’ and ’tetraploid Hanfu’,’Yan 1 Gala’ and ’tetraploid Yan 1 Gala’） have difference,but the genetic similarity coefficients between diploidys and its autotetraploidys of ’Fuji’,’Yan 1 Gala’ and ’Hanfu’ all were 1,and they could not distinguish each other.9.The content of cytosine and 5-methyl cytosine in hydrolysis DNA samples extracted from leaves of the diploid of apple and its autotetraploid was identified used HPLC.The ’Tensei’ had low DNA methylation which was 29.33%and ’Fuji’ was 35.34%.The methylation degree of ’Hanfu’ diploid and tetraploid were 14.37%and 14.49%respectively,with no significant difference.10.Two hundred and fifty-one homogeneity tetraploid trees were induced from embryos of apple cultivars ’Golden Delicious’,’Gala’ and ’Fuji’.Twenty and thirteen tetraploid plants were induced from leaves of ’Hanfu’ and ’Yan Gala 1’ respectively.Forty-six triploid tress and 5 teraploid trees were obtained from the offsprings of teraploid crossing diploid,the offsprings of triploid crossing diploid,and the self-progenies of triploid.These polyploidy germplasms laid the important foundation for breeding polyploid apple cultivars.更多还原