农杆菌介导大麦无筛选标记转基因植株的获得

doi:10.3864/j.issn.0578-1752.2020.18.002

Abstract

Abstract:

【Objective】Genotype dependence is a very serious problem in barley genetic transformation in which Golden Promise has been mainly used. In addition, the transformation efficiency of barley is still very low in China. Moreover, the generation of marker-free transgenic barley plants is very important to the future commercialization of genetically modified barley due to the considerably increased public concerns. It is necessary to establish an efficient genetic transformation system for generating marker-free transgenic barley plants, expand available barley genotypes for genetic transformation, and provide technical support for functional genomics study and breeding and potential commercialization of genetically modified varieties of barley. 【Method】The composition and auxin adding ratio in the medium used for barley transformation as well as culture regime was optimized to establish an efficient Agrobacterium-mediated genetic transformation system for barley using the immature embryos approximately 14 days post anthesis of a good agronomic trait variety Vlamingh. A double T-DNA vector pWMB123 containing the Bar gene and the GUS gene cassette was introduced into barley by Agrobacterium-mediated transformation to obtain marker-free transgenic barley plants in T₁ generation. 【Result】 It is indicated that the shoot induction medium supplemented with 1.0 mg·L^-1 KT, 0.5 mg·L^-1 6-BA and 0.05 mg·L^-1 NAA significantly promoted the differentiation ability of barley transformed calli. The adding of 2.5 mg·L^-1 copper sulfate in shoot induction medium lightened the albinism of transgenic barley plantlets. Through the exploration of different hormone ratios and medium components, it was found that the SM1 supplemented with 1.0 mg·L^-1 IBA without other auxin had the best rooting effect. A total of 138 immature embryos were transformed with Agrobacterium containing pWMB123 vector, and fourteen transgenic barley plants were obtained with a transformation efficiency of 10.14%. Detection by PCR, Quick Stix strips and histochemical staining assays confirmed that all T₀ plants contained Bar gene, among which four plants did not contain GUS gene. The co-transformation efficiency of the two T-DNAs is 71.43%. In the T₁ populations derived from the four T₀ transgenic lines containing Bar and GUS genes, two transgenic plants with GUS gene and without Bar gene were screened out in line BL8, and the efficiency for marker-free transgenic plants was 6.9%. The two foreign genes of Bar and GUS were identified to be integrated in most T₁transgenic barley plants be multiple copies using Southern blot. It was further confirmed that BL8-15 and BL8-19 were marker-free transgenic plants. 【Conclusion】An efficient genetic transformation system mediated by Agrobacterium-mediated for generating marker-free transgenic plants with healthy roots and less albinism were successfully established in barley.

Key words: barley, transformation, marker-free transgenic, agrobacterium-mediated

GONG Qiang,WANG Ke,YE XingGuo,DU LiPu,XU YanHao. Generation of Marker-Free Transgenic Barley Plants by Agrobacterium-Mediated Transformation[J].Scientia Agricultura Sinica, 2020, 53(18): 3638-3649.

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References 35

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组分 Composition	侵染液 IM	共培养培养基 CM	第一次筛选培养基 SM1	第二次筛选培养基 SM2	过渡培养基 TM	分化培养基 DM
MS	0.434	0.434	4.340	4.340	2.700	2.170
麦芽糖 Maltose			30	30	20	20
葡萄糖 Glucose	10	10
乙酰丁香酮Acetosyringone	0.02745	0.03924
谷氨酰胺 Glutamine					0.75
酪蛋白水解物Casein hydrolysate			1	1
肌醇 Myoinositol			0.35	0.35
脯氨酸 Proline			0.69	0.69
盐酸硫胺Thiamine HCl			0.001	0.001	0.004
麦草畏Dicamba			0.0025	0.0025
二氯苯氧乙酸2, 4-D					0.0025
硫酸铜CuSO₄			0.00125	0.00125	0.00125	0.00125
6-苄氨基嘌呤6-BA					0.0001
琼脂糖Agarose		8
植物凝胶Phytagel			3.5	3.5	3.5	3.0
草铵膦Phosphinothricin^a			0.005	0.010	0.010	0.005
羧苄青霉素Carbenicillin^a			0.25	0.25	0.25	0.25
头孢噻肟Cefotaxime^a			0.25

培养基编号 Medium ID	IAA	IBA	NAA
SR1	1	1	1
SR2	1	1	2
SR3	1	2	1
SR4	2	1	1
SR5	1	1	0
SR6	1	0	1
SR7	0	1	1
SR8	1	0	0
SR9	2	0	0
SR10	0	0	1
SR11	0	0	1.5
SR12	0	0	2
SR13	0	1	0
SR14	0	1.5	0
SR15	0	2	0

株系 Lines	Bar⁺GUS⁺	Bar⁺GUS^—	Bar^—GUS⁺
BL1	19	1	0
BL6	23	0	0
BL-7	24	2	0
BL-8	27	0	2

Generation of Marker-Free Transgenic Barley Plants by Agrobacterium-Mediated Transformation

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