小麦茎尖再生体系的优化与CL5227基因的遗传转化

发布时间：2018-04-29 18:06

本文选题：茎尖 + 愈伤组织　；参考：《甘肃农业大学》2017年硕士论文

【摘要】：本研究选取三个小麦品种,甘春20号、甘春24号、中麦175为试验材料,进行茎尖愈伤组织培养及植株再生的研究,探讨了基因型、茎尖生长天数及切取部位、培养基、激素(2,4-D,KT与6-BA)对愈伤组织诱导、分化及植株再生的影响。初步建立了中麦175茎尖愈伤组织培养及植株再生体系,并对其优化。对中麦175茎尖愈伤组织进行遗传转化,探讨了影响农杆菌介导的遗传转化因素,初步建立了农杆菌介导的中麦175茎尖遗传转化体系,成功的将Cl5227基因转入小麦中。研究结果如下:(1)通过对甘春20号,甘春24号,中麦175进行茎尖愈伤组织培养,经分析得出,不同基因型对茎尖愈伤组织诱导及分化有显著的影响;筛选出再生能力好的品种-中麦175,用于下一步的试验;生长3d的茎尖基部第一切段为愈伤组织培养最佳部位;确定了最佳愈伤组织诱导培养基:MS基本成分+2,4-D 2mg/L+CH0.5g/L+Pro 0.5g/L+Gln 0.25g/L+蔗糖30g/L+琼脂6g/L,p H=5.8;2,4-D浓度显著影响茎尖愈伤组织的诱导,随着2,4-D浓度的升高,诱导率呈先升高后降低的趋势,浓度为2mg/L的2,4-D最适宜愈伤的诱导;最佳分化培养基为:MS基本成分+CH0.5g/L+Pro 0.5 g/L+Gln 0.25 g/L+蔗糖30g/L+琼脂6g/L+KT 2mg/L+6-BA 2mg/L,pH=5.8。其中,6-BA与K T浓度均达到2mg/L时分化率达到最大。(2)通过对农杆菌介导的中麦175茎尖进行遗传转化,研究发现,侵染时间、菌液浓度、共培养时间对愈伤组织遗传转化有很大影响;愈伤组织的存活率随侵染时间的延长表现出降低的趋势,菌液浓度OD600=0.6,菌液时间为30min时分化率达到最大;茎尖愈伤组织共培养时间对转化的影响中,侵染后的愈伤组织在共培养3d时,分化率达到最大,为共培养适宜天数。500 mg/L头孢霉素(Cef)加入诱导筛选培养基和分化筛选培养基中,可以有效抑制农杆菌生长,且对小麦茎尖愈伤组织的生长影响较小。经诱导筛选21d后的愈伤组织,放置在加入5mg/L卡那霉素的分化筛选培养基上,得到的分化率较高,可有效的进行抗性愈伤的筛选。(3)经培养基抗性筛选后得到2株抗性植株,但经PCR检测,只有1株抗性植株呈阳性。
[Abstract]:In this study, three wheat varieties, Ganchun 20, Ganchun 24 and Zhongmai 175, were selected as experimental materials to study the callus culture and plant regeneration of stem tip. The genotypes, days of shoot tip growth, cutting position and culture medium were discussed. The effects of KT and 6-BA) on callus induction, differentiation and plant regeneration. The callus culture and plant regeneration system of Zhongmai 175 were established and optimized. The genetic transformation of the stem tip callus of Zhongmai 175 was carried out, and the factors affecting the genetic transformation mediated by Agrobacterium tumefaciens were discussed. The Agrobacterium tumefaciens mediated genetic transformation system of the stem tip of Zhongmai 175 was established, and the Cl5227 gene was successfully transferred into wheat. The results were as follows: 1) Callus culture of stem tip of Ganchun 20, Ganchun 24 and Zhongmai 175 was carried out. The results showed that different genotypes had significant effects on callus induction and differentiation. Zhongmai 175, a variety with good regeneration ability, was selected for further experiment, and the best part of callus culture was at the base of stem tip for 3 days. The optimum medium for callus induction was determined as follows: the concentration of 2n4-D 2mg/L CH0.5g/L Pro 0.5g/L Gln 0.25g/L sucrose 30g/L Agar (6g / L) significantly affected the callus induction at stem tip. With the increase of 24-D concentration, the induction rate increased first and then decreased. The optimal medium for callus induction was 2mg/L, and the optimal medium for differentiation was CH0.5g/L Pro 0.5 g / L Gln 0.25 g / L sucrose 30g/L Agar 6g/L KT 2mg/L 6-BA 2mg / L 6g/L KT 2mg/L 6-BA 2mg / L, pH = 5.8. The optimum medium for differentiation was CH0.5g/L Pro 0.5 g / L Gln 0.25 g / L sucrose 30g/L. Among them, the concentration of 6-BA and KT reached the maximum rate of 2mg/L time division. (2) the genetic transformation of Zhongmai 175 stem tip mediated by Agrobacterium tumefaciens was carried out. The results showed that infection time, concentration of bacterial solution and co-culture time had great influence on genetic transformation of callus. The survival rate of callus decreased with the increase of infection time. The concentration of OD600,0.6 and the time of 30min were the highest, and the effect of co-culture time of callus on the transformation was observed. The differentiation rate of the infected callus reached the maximum when co-cultured for 3 days. For the suitable days of co-culture, mg/L cefs were added to the induction and screening medium, which could effectively inhibit the growth of Agrobacterium tumefaciens. And the effect on the growth of wheat stem tip callus was small. The callus after 21 days of induction and screening was placed on the differentiation medium with 5mg/L kanamycin, and the rate of differentiation was higher, which could be used to screen the resistant callus effectively, and two resistant plants were obtained after the selection of resistant callus in the medium. But only one resistant plant was positive by PCR.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S512.1

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