花生再生体系的优化及AhSAD基因启动子的转化

发布时间：2018-03-26 13:00

本文选题：花生　切入点：愈伤组织　出处：《郑州大学》2016年硕士论文

【摘要】：本研究选用花生品种远杂9307、冀甜1号、罗汉果、花37、白沙1016的上胚轴为外植体,对愈伤组织诱导和再生体系进行研究,通过比较不同预培养时间、不同激素配比、不同基因型的诱导愈伤差异确立适宜花生上胚轴愈伤组织诱导的最佳培养基。分别从体细胞胚发生途径和器官间接发生途径探索花生上胚轴离体再生的最佳途径。确立最适宜的培养条件进行19种不同花生品种筛选。在优化的再生体系基础上进行农杆菌介导的遗传转化。遗传转化方面,对农杆菌介导的遗传转化条件进行优化,分别从Km筛选浓度,受体材料、侵染时间、共培养时间完善转化流程,将花生Δ9-硬脂酰-ACP脱氢酶基因启动子AhSAD转化不同花生品种的受体材料,获得阳性植株,主要研究结果如下:(1)预培养时间不同,愈伤组织状态存在明显差异,预培养0d的上胚轴易形成胚性愈伤,预培养7d的上胚轴状态优于3d和10d。(2)确定MSB5+Pic3mg/L为最适宜诱导培养基,胚性愈伤诱导率最高可达60%以上,该类培养基适宜本研究中多数花生品种的愈伤诱导,并可进行多次继代培养得到重复性体胚发生培养物。MSB5+Pic3mg/L+Gln1g/L(+)AgNO3(2mg/L)为最佳继代培养基。(3)愈伤组织进一步诱导体细胞胚发生的最佳培养基为MS+0.4mg/L6-BA+0.1mg/LNAA,成苗率最高为46.77%。继代过程先在6-BA0.4mg/L+NAA0.1mg/L再生,交替使用MS+6-BA3.0mg/L+NAA 0.5mg/L成苗状况较好;愈伤组织诱导不定芽的最佳培养基为MS+TDZ0.2mg/L+6-BA3.0mg/L+NAA0.5mg/L,可诱导出不定芽且分化率最高为42%。(4)不同基因型的花生品种在相同的培养基中出现诱导和再生差异,9048,9102出愈率在70%-80%之间,9020,9092,9110出愈率为60%-70%,9059出愈率最低仅为16%。9099,9097再生率在40%-50%之间,9036,9093次之。(5)最终确定胚性愈伤组织和无菌苗上胚轴作为受体材料时,卡那霉素筛选浓度为80mg/L和100mg/L,两者侵染时间在10min时褐化率最低,转化率最高。胚性愈伤共培养2d时,抗卡那霉素的抗性芽数最多;无菌苗的上胚轴共培养3d时,抗性愈伤诱导率最高。(6)经过卡那霉素筛选,获得36抗性植株,其中7株经GUS组织活性检测和PCR检测鉴定为阳性植株。
[Abstract]:In this study, the epicotyl of peanut varieties Yuanza 9307, Ji Tian 1, Luohanguo, Flower 37 and Baisha 1016 were used as explants to study callus induction and regeneration system. The optimum medium for callus induction of peanut epicotyl was established by different genotypic callus induction. The best way of regeneration of peanut epicotyl in vitro was explored from somatic embryogenesis pathway and organ indirect pathway respectively. To establish the most suitable culture conditions for screening 19 different peanut varieties, and to carry out Agrobacterium-mediated genetic transformation on the basis of optimized regeneration system. The conditions of genetic transformation mediated by Agrobacterium tumefaciens were optimized, and the transformation process was improved by screening concentration, receptor material, infection time and co-culture time from km, respectively. Peanut 螖 9-stearyl ACP dehydrogenase gene promoter AhSAD was transformed into different peanut varieties to obtain positive plants. The main results were as follows: 1) the callus status was significantly different with different pre-culture time. The embryogenic callus was easily formed on the epicotyl of precultured for 0 d, and the state of the epicotyl of precultured 7 days was better than that of 3 d and 10 d). MSB5 Pic3mg/L was the most suitable medium for induction, and the highest rate of embryogenic callus induction could be more than 60%. The medium was suitable for callus induction of most peanut varieties in this study. The best medium for further induction of embryogenesis from callus was MS 0.4mg/L6-BA 0.1 mg / L LNAA, and the highest seedling rate was 46.77%, and the optimum medium for inducing embryogenesis was MS 0.4mg/L6-BA 0.1 mg / L LNAA, and the reproducible somatic embryogenesis culture medium .MSB5 Pic3mg/L Gln1g-1 / L (Agno _ 3 / L _ 2 mg / L) was the best subculture medium for further induction of embryogenesis of somatic cells by MS 0.4mg/L6-BA 0.1 mg 路L ~ (NAA), and the highest plantlet rate was 46.77%. Cheng first regenerates in 6-BA0.4mg/L NAA0.1mg/L, Ms 6-BA3.0mg/L NAA 0.5mg/L was used alternately in good condition. The best medium for inducing adventitious buds was MS TDZ0.2mg/L 6-BA3.0mg/L NAA 0.5 mg / L, which could induce adventitious buds and the highest differentiation rate was 42%.) the difference of induction and regeneration between different genotypes of peanut cultivars in the same medium was between 70% and 80%. The recovery rate of 9020 / 9092 / 9110 was 60-70 / 9059 and the lowest was 160.909/ 9097 / 9097. The regeneration rate was between 40- 50% and 9036,9093 / 9093, respectively.) finally, when the embryogenic callus and the aseptocotyl of the sterile seedlings were used as the recipient materials, The screening concentration of kanamycin was 80mg/L and 100mg / L, the infection time was the lowest and the transformation rate was the highest in 10min. The number of resistant buds of kanamycin was the most when the embryogenic callus co-cultured for 2 days, and the number of resistant buds was the highest when the epicotyl of sterile plantlets was co-cultured for 3 days. The resistant callus induction rate was the highest, and 36 resistant plants were obtained by kanamycin screening. Seven of them were identified as positive plants by GUS tissue activity test and PCR assay.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S565.2

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