白鹤芋体胚高效快繁体系的建立及多倍体诱导研究

发布时间：2018-08-27 17:06

【摘要】：本研究以白鹤芋‘Mojo'品种的花序及试管苗根茎为试验材料,通过对激素种类、激素浓度、AgN03浓度及营养水平等方面,开展白鹤芋体胚发生及器官发生的研究。利用秋水仙素、氨磺灵及氟乐灵对白鹤芋进行多倍体诱导,为今后白鹤芋多倍体育种及新品种的选育提供参考。主要研究结果如下:(1)白鹤芋‘Mojo’品种花序胚性愈伤诱导最适配方为MS+TDZ 5.0 mg·L-1 + NAA 0.2 mg·L-1,诱导率达64.97%。根茎胚性愈伤诱导最佳配方为MS+2,4-D 4 mg·L-1 +TDZ 0.5 mg·L-1,诱导率为87.44%;根茎器官发生型愈伤诱导最佳配方为MS+2,4-D 1mg·L-1+TDZ 0.5 mg·L-1,诱导率为92.33%。根茎是胚性愈伤诱导的最佳外植体。(2)愈伤组织增殖阶段,2mg·L-1 AgNO3作用时,愈伤组织增殖效果及生长状态最佳,增殖系数达3.08。(3)胚状体诱导最佳激素配方为 MS+6-BA 1 mg·L-1 + TDZ 0.2 mg·L-1 +NAA 0.2 mg·L-1,诱导率为68.32%;体胚萌发最佳营养和激素水平为1/2MS+TDZ 0.1 mg·L-1 + IBA 1.0 mg·L-1,萌发率为70.54%;植株再生的最佳配方为MS+6-BA 0.8 mg·L-1 +IBA 0.2 mg·L-1,再生率为89.34%。植株最佳生根配方为 MS+AC 1 g·L-1+NAA0.2 mg·L-1。(4)白鹤芋体胚形态形成过程与单子叶植物胚的形态形成过程类似,主要分为以下几个阶段:胚性细胞、球形胚、椭圆形胚、盾形胚,子叶型胚、成熟体细胞胚。(5)染色体制片技术优化,早上8:45-9:15取材,用0.002 mol·L-18-羟基喹啉或0.07 mmol·L-1放线菌酮预处理4 h所得的染色体制片效果最佳。白鹤芋‘Mojo'品种的染色体数目为2n=2x=30,为二倍体。(6)以白鹤芋‘Mojo'品种的体胚及幼芽为试验材料,研究不同浓度的秋水仙素、氨磺灵及氟乐灵溶液对体胚及幼芽四倍体的诱导情况。得出白鹤芋体胚四倍体诱导的最佳方法为0.08%秋水仙素处理15 d。幼芽四倍体诱导的最佳方法为100 mg·L-1氟乐灵处理5 d。幼芽的诱导率要比体胚的高,幼芽的多倍体诱导较体胚的更容易些。(7)对白鹤芋的四倍体在形态学、染色体数目和气孔进行鉴定。发现四倍体植株较二倍体植株生长健壮,叶片宽大厚实,叶色深绿,叶型指数变小,叶型更加匀称,株型紧凑。白鹤芋二倍体的染色体数目为2n=2x=30,四倍体的染色体数目为2n=4x=60,染色体数目增加了一倍。四倍体气孔明显比二倍体的大,而且同等面积上的气孔数减少。
[Abstract]:In this study, the inflorescence and root stem of Mojo' variety were used as experimental materials. The somatic embryogenesis and organogenesis were studied in the aspects of hormone type, hormone concentration and AgN03 concentration and nutrition level. Colchicine, ammosulfurin and fluralin were used to induce polyploidy of white crane taro, which provided references for polyploid breeding and breeding of new varieties of white crane taro. The main results were as follows: (1) the best formula for embryogenic callus induction was MS TDZ 5.0 mg L ~ (-1) NAA 0.2 mg L ~ (-1), and the induction rate was 64.97%. The best formula of rhizome embryogenic callus induction was MS 24-D 4 mg L -1 TDZ 0.5 mg L -1, the inducing rate was 87.440.The best formula of rhizome embryogenic callus induction was MS 2N 4-D 1mg L -1 TDZ 0.5 mg L -1, and the induction rate was 92.33%. Rhizome was the best explant for embryogenic callus induction. (2) the callus proliferation and growth state were the best when the callus was treated with 2 mg L ~ (-1) AgNO3. (3) the best hormone formula for embryoid induction was MS 6-BA 1 mg L-1 TDZ 0.2 mg L -1 NAA 0.2 mg L -1, the best somatic embryo germination nutrition and hormone level was 1/2MS TDZ 0.1 mg L -1 IBA 1.0 mg L -1, and the germination rate was 70.54%. The optimum formula was MS 6-BA 0.8 mg L -1 IBA 0.2 mg L -1 and regeneration rate 89.34 4. The best rooting formula was MS AC 1 g L-1 NAA0.2 mg L -1. (4) the somatic embryogenesis process was similar to that of monocotyledonous embryos, and was mainly divided into the following stages: embryogenic cells, globular embryos, elliptical embryos, pellet embryos, and peltate embryos. Cotyledonous embryo, mature somatic embryo. (5) the chromosome slice technique was optimized. The chromosome was prepared with 0.002 mol L-18-hydroxyquinoline or 0.07 mmol L-1 actinomycin for 4 h at 8: 45-9: 15 in the morning. The chromosome number of Mojo' was 2nnnt2xan30, which was diploid. (6) the somatic embryos and buds of Mojo' were used as experimental materials to study the induction of tetraploid of somatic embryos and buds with different concentrations of colchicine, amsulfurin and trifluridine solution. The best method of tetraploid induction was 0.08% colchicine for 15 days. The best method for inducing tetraploid was 100 mg L-1 trifluridine for 5 days. The induction rate of young bud was higher than that of somatic embryo, and the polyploid induction of young bud was easier than that of somatic embryo. (7) the tetraploid was identified in morphology, chromosome number and stomata. It was found that tetraploid plants grew stronger than diploid plants, their leaves were thicker and thicker, leaf color was dark green, leaf shape index was smaller, leaf shape was more symmetrical, and plant type was compact. The chromosome number of diploid and tetraploid was 2nnm2xc30 and 2nm4xm60respectively, and the number of chromosome doubled. The stomata of tetraploid were larger than that of diploid, and the number of stomata in the same area was decreased.
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S682.36

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