Crops ›› 2018, Vol. 34 ›› Issue (1): 56-61.doi: 10.16035/j.issn.1001-7283.2018.01.008

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Optimization of the Factors Related to the Efficiency of Agrobacterium-Mediated Transformation of Sorghum

Zhang Wei1,2,Wang Liangqun1,2,Liu Yong1,Hao Yanfang1,2,Yang Wei1,Bai Hongyan1,Wu Bo1   

  1. 1 Shanxi Key Laboratory of Sorghum Genetic and Germplasm Innovation, Jinzhong 030600, Shanxi, China
    2 Sorghum Institute, Shanxi Academy of Agricultural Sciences, Jinzhong 030600, Shanxi, China
  • Received:2017-11-01 Revised:2017-11-29 Online:2018-02-20 Published:2018-08-24

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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

Fig.1

The structure of the PCAMBIA3301 plasmid"

Fig.2

The PCAMBIA3301 plasmid was leading in agrobacterium||| A, The agrobacterium strain EHA105 containing PCAMBIA3301 plasmid; B, PCR detection of colony"

Table 1

Effect of preculture time on transformation of the sorghum"

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

Table 2

Effect of different AS concentration on transformation of the sorghum"

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

Table 3

Effect of different infecting fluid concentration on transformation of the sorghum"

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

Table 4

Effect of different infection time on transformation of the sorghum"

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

Table 5

Effect of different co-culture time on transformation of the sorghum"

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
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