农杆菌介导高粱遗传转化的相关因素优化

doi:10.16035/j.issn.1001-7283.2018.01.008

摘要/Abstract

摘要：

为建立一个稳定的高粱遗传转化体系,以高粱RHMC品种为试验材料,采用内含有P ^CAMBIA3301质粒载体的根癌农杆菌EHA105介导的方法研究了高粱遗传转化的相关因素。结果表明,外植体预培养2d,GUS表达效果最好;在侵染液和共培养基中加入200μmol/L乙酰丁香酮时,GUS表达效果最好;侵染液浓度OD₆₀₀值的适宜范围为0.8~1.0;农杆菌侵染愈伤组织的适宜时间为5~10min;共培养的适宜时间为2d。初步建立了一套高粱遗传转化体系,为今后高粱转化提供一些参考。

关键词: 高粱, 农杆菌, 遗传转化, 愈伤组织

Abstract:

Using sorghum variety RHMC and the agrobacterium strain EHA105 containing P ^CAMBIA3301 plasmid as the materials, we studied the factors which influence agrobacterium-mediated transformation efficiency of the sorghum so as to establish a stable transformation system. The results showed that when the explants were pre-cultured for 2d, the GUS staining was the best. When 200μmol/L acetosyringone was added to infecting fluid and co-culture media, the GUS staining was the best. The suitable concentration range of the OD₆₀₀ was 0.8-1.0. The suitable time of the infection with agrobacterium was 5-10min. The suitable time of the co-culture was 2d. In this study, a stable transformation system was preliminary established. This provided some references for the transformation of sorghum in the future.

Key words: Sorghum, Agrobacterium, Genetic transformation, Callus

张微,王良群,刘勇,郝艳芳,杨伟,白鸿雁,武擘. 农杆菌介导高粱遗传转化的相关因素优化[J]. 作物杂志, 2018 (1): 56–61

Wei Zhang,Liangqun Wang,Yong Liu,Yanfang Hao,Wei Yang,Hongyan Bai,Bo Wu. Optimization of the Factors Related to the Efficiency of Agrobacterium-Mediated Transformation of Sorghum[J]. Crops, 2018(1): 56–61

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预培养天数(d) Preculture time	供试愈伤组织数 Total number of callus	GUS基因表达的愈伤组织数 Number of callus with GUS expression	愈伤组织染色区 Callus staining region	抗性愈伤诱导率(%) Ratio of resistant callus
0	50	0	0	0
1	50	0	0	0
2	50	1.27±0.25aA	约为1.2%,浅蓝色About 1.2%, light blue	2.54±0.56aA
3	50	1.03±0.48bB	约为1.0%,浅蓝色About 1.0%, light blue	2.06±0.71bB
4	50	0	0	0

乙酰丁香酮的浓度(μmol/L) AS concentration	供试愈伤组织数 Total number of callus	GUS基因表达的愈伤组织数 Number of callus with GUS expression	愈伤组织染色区 Callus staining region	抗性愈伤诱导率(%) Ratio of resistant callus
0	50	0	0	0
100	50	1.33±0.34bB	约为0.8%,浅蓝色About 0.8%, light blue	2.66±0.33cC
200	50	1.65±0.15aA	约为2.6%,深蓝色About 2.6%, deep blue	3.30±0.52aA
300	50	1.52±0.86aA	约为2.5%,深蓝色About 2.5%, deep blue	3.04±0.64bB
400	50	1.14±1.25cC	约为2.1%,深蓝色About 2.1%, deep blue	2.28±0.23dD

侵染液浓度 Infecting fluid concentration	供试愈伤组织数 Total number of callus	GUS基因表达的愈伤组织数 Number of callus with GUS expression	愈伤组织染色区 Callus staining region	抗性愈伤诱导率(%) Ratio of resistant callus
0	50	0	0	0
0.2	50	0	0	0
0.4	50	0	0	0
0.6	50	1.14±0.15dD	约1.0%,浅蓝色About 1.0%, light blue	2.28±1.26dD
0.8	50	1.66±0.36bB	约2.3%,浅蓝色About 2.3%, light blue	3.32±0.58bB
1.0	50	1.85±0.43aA	约2.5%,浅蓝色About 2.5%, light blue	3.70±0.46aA
1.2	50	1.52±0.22cCB	约0.7%,深蓝色About 0.7%, deep blue	3.04±0.31cC

侵染时间(min) Infection time	供试愈伤组织数 Total number of callus	GUS基因表达的愈伤组织数 Number of callus with GUS expression	愈伤组织染色区 Callus staining region	抗性愈伤诱导率(%) Ratio of resistant callus
0	50	0	0	0
5	50	1.57±0.84bBA	约2.5%,浅蓝色About 2.5%, light blue	3.14±0.22bBA
10	50	1.62±0.23aA	约2.5%,浅蓝色About 2.5%, light blue	3.24±0.51aA
15	50	1.59±0.15bBA	约2.6%,浅蓝色About 2.6%, light blue	3.18±0.64bBA

共培养时间(d) Co-culture time	供试愈伤组织数 Total number of callus	GUS基因表达的愈伤组织数 Number of callus with GUS expression	愈伤组织染色区 Callus staining region	抗性愈伤诱导率(%) Ratio of resistant callus
0	50	0	0	0
1	50	0	0	0
2	50	1.65±0.72aA	约2.5%,浅蓝色About 2.5%, light blue	3.30±1.20aA
3	50	1.53±0.36aA	约2.7%,浅蓝色About 2.7%, light blue	3.06±0.33aA
4	50	0.48±0.15bB	约1.2%,浅蓝色About 1.2%, light blue	0.96±0.58bB
5	50	0	0	0