【作者】 张艳梅；

【导师】 于锡宏；

【作者基本信息】 东北农业大学 ， 蔬菜学， 2012， 硕士

【摘要】 本试验以4个榨菜(Brassica juneea Czern.et Coss.var.tumida)株系B2-1014,B2-1020, BZ(?)-1005,BZ(?)-1003为材料,利用榨菜离体培养及实生苗处理诱导多倍体的方法,对影响榨菜离体植株再生及秋水仙素诱导多倍体影响因素进行了研究,初步建立了榨菜的多倍体诱导方法。同时对游离小孢子培养诱导胚状体进行了初步的研究,为下一步研究诱导单倍体植株,并进行加倍得到榨菜二倍体植株打下了基础。研究结果如下：通过对榨菜4个株系的茎尖及下胚轴进行离体培养,均能成功诱导出愈伤组织,其中以榨菜株系B2-1014诱导率最高,为49.0%,利用愈伤组织诱导率高的株系B2-1014进行离体培养,筛选出其最佳再生培养基组合：愈伤组织诱导培养基(MS+0.5mg/L6-BA+0.8mg/LNAA)；不定芽诱导培养基(MS+2.0mg/L6-BA+0.2mg/LNAA)；不定根诱导的培养基(MS+0.2mg/LNAA).下胚轴与茎尖最佳再生培养基组合相同,但其诱导效果不同,茎尖离体培养：愈伤组织的诱导率达95.6%,不定芽再生率达95.6%,生根率达100%;下胚轴离体培养：愈伤组织的诱导率达90.0%,不定芽再生率达92.2%,生根率达97.8%。不同苗龄对榨菜再生频率呈显著性影响,以茎尖做外植体,其苗龄为5d时分化能力最强,下胚轴则是4d。通过组织培养和实生苗处理诱导榨菜多倍体均能获得四倍体植株,不同的处理方式、处理条件可以产生不同的诱导效果。在组织培养中,以株系B2-1014进行诱导,茎尖为外植体、秋水仙素浓度0.4%、培养48h时四倍体诱导效果最好,为30%；在相同条件下,下胚轴为外植体,四倍体诱变率为15.6%。利用实生苗处理进行榨菜多倍体诱导,以点滴生长点(2.0g/L)和浸根(40mg/L、处理时间3d)同时处理四倍体诱变率最高,为14.4%,灌根处理(40mg/L,处理时间3d)四倍体诱变率最低,为7.8%;榨菜不同株系多倍体的诱变率不同,4种材料中以榨菜株系B2-1014四倍体诱导效果最好,显著高于其他株系。不同处理方法的最适苗龄相同,3片叶的四倍体诱变率最高。对榨菜4个株系进行游离小孢子培养中,不同株系、不同基本培养基其胚状体诱导率不同。在不同株系与基本培养基的组合中,B2-1014在1/2NLN基本培养基中的胚状体诱导效果最好,诱导率为31.1%。而同样采用1/2NLN基本培养基,B2-1020,BZ(?)-1005,BZ(?)-1003的胚状体诱导率分别为8.9%,5.6%和4.4%。更多还原

【Abstract】 In this experiment, we studied some important influencing factors in the progress of tuber mustard (Brassica juneea Czern.et Coss.var.tumida) vitro plantlet regeneration by the method of tuber mustard lines in vitro culture and seedling polyploid induction. The material used in the experiment are4tuber mustard strains named B2-1014, B2-1020, BZ(?)-1005, BZ(?)-1003respectively. We established the system of tuber mustard polyploid induction, in order to get diploid plant and did preliminary study on embryoids induction from isolated microspore culture. The results as follows:We induced successful callus result in tuber mustard in vitro culture using the shoot tips and hypocotyls of four strains. One of them that is B2-1014had the highest rate of induction.It was49.0%. We used the strain B2-1014, which had the highest rate of induction strain in vitro culture. The optimum regeneration medium were screened callus induction medium (MS+0.5mg/L6-BA+0.8mg/LNAA),Adventitiousbud induction medium(MS+2.0mg/L6-BA+0.2mg/LNAA); Adventitious root induction medium(MS+0.2mg/LNAA). Hypocotyls and shoot tips had the same optimum regeneration medium, but had different induction results. Isolated culture of shoot tips: the inductivity of the callus was95.6%, the regeneration rate of the adventitious bud was95.6%and the rooting rate was100%. Isolated culture of hypocotyls:the inductivity of the callus was up to90.0%, the regeneration rate of the adventitious bud was92.2%and the rooting rate was97.8%. There was a significant difference in the regeneration frequency in different seedling ages in vitro. The explantation was the stem apex which has the strongest differentiation capacity with the seedling age5d and that of the hypocotyls is4d.We could both get the tetraploid plants, through tissue culture and seedling inducted the polyploidy tuber mustard. And there were different conditions and different approaches may lead to different induction results. In tissue culture, when the B2-1014was used, the stem apex explants showed the best induction effects, and the best induction condition is (concentrations of colchicine0.4%, culture time48h), in which we obtained the highest doubling rate30%for tetraploid. The induction of other treatment showed that:the hypocotyls of isolated culture hypocotyls is concentrations of colchicines0.4%, culture time48h, the doubling rate of tetraploid is15.6%. Seedling induced the polyploidy of tuber mustard, then drip growing point(2.0g/L)and dip the root (40mg/L, culture time3d), simultaneous processing the highest doubling rate of tetraploid is14.4%; Dip the root, the concentrations is40mg/L, the doubling rate of tetraploid is7.8%. Polyploid of different lines of the tuber mustard had different the rate of induction. The best treatment was the lines of the tuber mustard B2-1014. It was significantly higher than other lines. Different methods had the same the optimum seeding age,3leaves of tetraploid time had the higest rate of induction.Four tuber mustards were used in the study of isolated microspore culture, from which we can know the influence of different lines of tuber mustard and different basic medium for embryoid induction. The results showed that, in different combinations of tuber mustard line and basic medium, the combination of tuber mustard line B2-1014and basic medium1/2NLN showed the best induction effect, the mutation rate was31.1%. But the mutation rate of tuber mustard lines B2-1020, BZ(?)-1005and BZ(?)-1003in basic medium1/2NLN were8.9%,5.6%and4.4%respectively.更多还原