Analysis of Agrobacterium-Mediated Genetic Transformation System of indica Rice 9311 and Huazhan

doi:10.16819/j.1001-7216.2023.220305

Abstract

Abstract:

【Objective】 The rice transgenic technology is an important tool to promote rice molecular biology research and precision breeding. Agrobacterium-mediated transgenic technology has become the mainstream method for rice genetic transformation. Nevertheless, due to its genotype, the genetic transformation efficiency of typical indica rice is still quite low. It is urgent to improve its transformation efficiency. 【Method】 The effects of medium composition and photoperiod on genetic transformation efficiency of recalcitrated indica rice varieties 9311 and Huazhan were investigated. 【Result】 The results showed that MS macro element, B5 micro element, N6 organic matter and maltose were the best combination for 9311 tissue culture. MS macro element, MS micro element, MS organic matter and maltose were the best combination for Huazhan tissue culture. Adding 0.5 mg/L BAP or 1.5 mg/L KT into the induction medium could significantly improve the regeneration rate of 9311 and Huazhan tissue culture, up to more than 70.0%. The transformation efficiency with 100 μmol/L AS in the infection solution was higher than that with 200 μmol/L AS. Photoperiod experiments suggested that different photoperiod strategies should be adopted for 9311 and Huazhan transformation. 9311 transformation efficiency peaked, 6.0%?6.4%, under continuous darkness at the induction and selection stages and continuous light at the differentiation stage. Huazhan transformation efficiency reached the highest, 5.0%?7.5%, under 12 h photoperiod at all stages. 【Conclusion】 Collectively, the present study optimized tissue culture and genetic transformation system of recalcitrant indica rice varieties 9311 and Huazhan. Our methods could also be applied to the genetic transformation of other related indica rice genotypes.

Key words: recalcitrated indica rice, Agrobacterium tumefaciens, tissue culture, genetic transformation

摘要：

【目的】水稻转基因技术是推动水稻分子生物学研究和精准育种的重要工具。农杆菌介导法是水稻遗传转化的主流方法。然而受基因型限制，典型籼稻的转化效率仍然偏低，需提高其转化效率。【方法】以顽拗型籼稻品种9311和华占为受体材料，研究了不同大量元素、微量元素、有机物和碳源的组合，植物激素浓度，侵染培养基，乙酰丁香酮（AS）浓度和光周期对遗传转化效率的影响。【结果】在成熟种子组织培养试验中，MS大量元素、B5微量元素、N6有机物和麦芽糖为9311最优组合，华占最优组合为MS大量元素、MS微量元素、MS有机物和麦芽糖。诱导培养基中添加0.5 mg/L的BAP或1.5 mg/L的KT可显著提高9311和华占的组织培养再生率，可达70.0%以上。在侵染过程中，添加100 μmol/L AS的转化效率高于200 μmol/L AS。光周期实验显示9311和华占宜采用不同的光周期策略，9311在全黑暗条件下进行愈伤诱导和筛选，在全光照条件下进行分化，转化效率最高，达6.0%~6.4%，而华占在12 h光照/12 h黑暗条件下进行诱导、筛选和分化，获得的转化效率最高（5.0%~7.5%）。【结论】本研究优化了顽拗型籼稻9311和华占的组织培养和遗传转化体系，该体系也可以应用于其他相关籼稻基因型的遗传转化。

关键词: 顽拗型籼稻, 根癌农杆菌, 组织培养, 遗传转化

ZHANG Jia, WANG Huijie, HE Zhengquan, LIU Wenzhen. Analysis of Agrobacterium-Mediated Genetic Transformation System of indica Rice 9311 and Huazhan[J]. Chinese Journal OF Rice Science, 2023, 37(2): 213-224.

张佳, 王慧杰, 何正权, 刘文真. 农杆菌介导的籼稻9311和华占遗传转化体系的研究[J]. 中国水稻科学, 2023, 37(2): 213-224.

Figures/Tables 11

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培养基 Medium	成分 Composition
愈伤诱导培养基 Callus induction medium	MS大量+B5微量+N6有机+麦芽糖+铁盐+0.3 g/L酸水解酪蛋白+2.87 g/L脯氨酸+2.5 mg/L 2, 4-D+0.5 mg/L BAP+1.5 mg/L ABA +3.9 g/L植物凝胶 MS Macro+B5 Micro+N6 Vitamins+Maltose+Fe-Na-EDTA+0.3 g/L acid hydrolyzed casein+2.87 g/L Proline+2.5 mg/L 2, 4-D+0.5 mg/L BAP+1.5 mg/L ABA +3.9 g/L Phytagel
侵染培养基 Infection medium	AA大量+AA微量+AA有机+20 g/L蔗糖+10 g/L葡萄糖+铁盐+0.5 g/L酸水解酪蛋白+876 mg/L L-谷氨酰胺+174 mg/L 精氨酸+260 mg/L天冬氨酸 AA Macro+AA Micro+AA Vitamins+20 g/L sucrose+10 g/L glucose+Fe-Na-EDTA+0.5 g/L acid hydrolyzed casein +876 mg/L L-glutamine +174 mg/L arginine +260 mg/L aspartic acid
选择培养基 Selection medium	MS大量+B5微量+N6有机+麦芽糖+铁盐+0.3 g/L酸水解酪蛋白+2.87 g/L脯氨酸+30 g/L甘露醇+2.5 mg/L 2,4-D+0.5 mg/L BAP +5 g/L植物凝胶 MS Macro+B5 Micro+N6 Vitamins+Maltose+Fe-Na-EDTA+0.3 g/L acid hydrolyzed casein+2.87 g/L Proline+30 g/L mannitol+2.5 mg/L 2,4-D+0.5 mg/L BAP+5 g/L Phytagel
分化培养基 Regeneration medium	MS大量+MS微量+B5有机+麦芽糖+铁盐+2 g/L酸水解酪蛋白+30 g/L山梨醇+0.2 mg/L NAA +4 mg/L BAP+5 g/L植物凝胶 MS Macro+MS Micro+B5 Vitamins+Maltose+Fe-Na-EDTA+2 g/L acid hydrolyzed casein+30 g/L sorbitol +0.2 mg/L NAA+4 mg/L BAP+5 g/L Phytagel MS大量+MS微量+B5有机+铁盐+30 g/L葡萄糖+0.5 mg/L IBA+4 g/L植物凝胶 MS Macro+MS Micro+B5 Vitamins+Fe-Na-EDTA+30 g/L glucose+0.5 mg/L IBA+4 g/L Phytagel
生根培养基 Rooting medium
YEM培养基 YEM medium	10 g/L甘露醇+2 g/L C-谷氨酰胺+0.5 g/L磷酸二氢钾+0.2 g/L氯化钠+0.2 g/L硫酸镁+0.3 g/L酵母提取物+15 g/L Agar 10 g/L mannitol +2 g/L C-glutamine +0.5 g/L monopotassium phosphate+0.2 g/L sodium chloride+0.2 g/L magnesium sulfate +0.3 g/L yeast extract +15 g/L Agar

培养基 Medium	成分 Composition
愈伤诱导培养基 Callus induction medium	MS大量+B5微量+N6有机+麦芽糖+铁盐+0.3 g/L酸水解酪蛋白+2.87 g/L脯氨酸+2.5 mg/L 2, 4-D+0.5 mg/L BAP+1.5 mg/L ABA +3.9 g/L植物凝胶 MS Macro+B5 Micro+N6 Vitamins+Maltose+Fe-Na-EDTA+0.3 g/L acid hydrolyzed casein+2.87 g/L Proline+2.5 mg/L 2, 4-D+0.5 mg/L BAP+1.5 mg/L ABA +3.9 g/L Phytagel
侵染培养基 Infection medium	AA大量+AA微量+AA有机+20 g/L蔗糖+10 g/L葡萄糖+铁盐+0.5 g/L酸水解酪蛋白+876 mg/L L-谷氨酰胺+174 mg/L 精氨酸+260 mg/L天冬氨酸 AA Macro+AA Micro+AA Vitamins+20 g/L sucrose+10 g/L glucose+Fe-Na-EDTA+0.5 g/L acid hydrolyzed casein +876 mg/L L-glutamine +174 mg/L arginine +260 mg/L aspartic acid
选择培养基 Selection medium	MS大量+B5微量+N6有机+麦芽糖+铁盐+0.3 g/L酸水解酪蛋白+2.87 g/L脯氨酸+30 g/L甘露醇+2.5 mg/L 2,4-D+0.5 mg/L BAP +5 g/L植物凝胶 MS Macro+B5 Micro+N6 Vitamins+Maltose+Fe-Na-EDTA+0.3 g/L acid hydrolyzed casein+2.87 g/L Proline+30 g/L mannitol+2.5 mg/L 2,4-D+0.5 mg/L BAP+5 g/L Phytagel
分化培养基 Regeneration medium	MS大量+MS微量+B5有机+麦芽糖+铁盐+2 g/L酸水解酪蛋白+30 g/L山梨醇+0.2 mg/L NAA +4 mg/L BAP+5 g/L植物凝胶 MS Macro+MS Micro+B5 Vitamins+Maltose+Fe-Na-EDTA+2 g/L acid hydrolyzed casein+30 g/L sorbitol +0.2 mg/L NAA+4 mg/L BAP+5 g/L Phytagel MS大量+MS微量+B5有机+铁盐+30 g/L葡萄糖+0.5 mg/L IBA+4 g/L植物凝胶 MS Macro+MS Micro+B5 Vitamins+Fe-Na-EDTA+30 g/L glucose+0.5 mg/L IBA+4 g/L Phytagel
生根培养基 Rooting medium
YEM培养基 YEM medium	10 g/L甘露醇+2 g/L C-谷氨酰胺+0.5 g/L磷酸二氢钾+0.2 g/L氯化钠+0.2 g/L硫酸镁+0.3 g/L酵母提取物+15 g/L Agar 10 g/L mannitol +2 g/L C-glutamine +0.5 g/L monopotassium phosphate+0.2 g/L sodium chloride+0.2 g/L magnesium sulfate +0.3 g/L yeast extract +15 g/L Agar

培养基名称 Media	基本培养基 Basal media（BM）			碳源 Carbon source	平均愈伤诱导率 Average frequency of callus induction / %		组织培养再生率 Regeneration rate of tissue culture / %
培养基名称 Media	大量 Macro	微量 Micro	有机 Vitamin	碳源 Carbon source	9311	华占Huazhan	9311	华占 Huazhan
RBM₁	N6	N6	N6	蔗糖Sucrose	76.0±4.0	81.3±1.2	14.6±2.1 ab	2.7±2.7 bc
RBM₂	N6	MS×5	MS	麦芽糖Maltose	78.3±0.7	75.7±1.7	2.7±2.7 c	7.8±2.2 ab
RBM₃	N6	B5	B5	混合 Mixed	69.7±3.5	79.5±5.5	7.2±2.9 abc	2.4±2.4 bc
RBM₄	MS	N6	MS	混合 Mixed	69.3±4.8	77.0±1.0	15.9±4.1 ab	13.0±2.4 a
RBM₅	MS	MS×5	B5	蔗糖Sucrose	82.7±2.0	71.3±2.6	5.2±0.4 bc	8.9±2.2 ab
RBM₆	MS	B5	N6	麦芽糖Maltose	78.0±5.3	68.0±3.0	20.0±5.5 a	8.5±2.6 ab
RBM₇	DKN	N6	B5	麦芽糖Maltose	48.7±3.5	56.7±3.4	0.0±0.0 c	0.0±0.0 c
RBM₈	DKN	MS×5	N6	混合 Mixed	49.3±3.2	53.7±2.3	0.0±0.0 c	0.0±0.0 c
RBM₉	DKN	B5	MS	蔗糖Sucrose	49.0±1.5	57.0±1.0	0.0±0.0 c	0.0±0.0 c

培养基名称 Media	基本培养基 Basal media（BM）			碳源 Carbon source	平均愈伤诱导率 Average frequency of callus induction / %		组织培养再生率 Regeneration rate of tissue culture / %
培养基名称 Media	大量 Macro	微量 Micro	有机 Vitamin	碳源 Carbon source	9311	华占Huazhan	9311	华占 Huazhan
RBM₁	N6	N6	N6	蔗糖Sucrose	76.0±4.0	81.3±1.2	14.6±2.1 ab	2.7±2.7 bc
RBM₂	N6	MS×5	MS	麦芽糖Maltose	78.3±0.7	75.7±1.7	2.7±2.7 c	7.8±2.2 ab
RBM₃	N6	B5	B5	混合 Mixed	69.7±3.5	79.5±5.5	7.2±2.9 abc	2.4±2.4 bc
RBM₄	MS	N6	MS	混合 Mixed	69.3±4.8	77.0±1.0	15.9±4.1 ab	13.0±2.4 a
RBM₅	MS	MS×5	B5	蔗糖Sucrose	82.7±2.0	71.3±2.6	5.2±0.4 bc	8.9±2.2 ab
RBM₆	MS	B5	N6	麦芽糖Maltose	78.0±5.3	68.0±3.0	20.0±5.5 a	8.5±2.6 ab
RBM₇	DKN	N6	B5	麦芽糖Maltose	48.7±3.5	56.7±3.4	0.0±0.0 c	0.0±0.0 c
RBM₈	DKN	MS×5	N6	混合 Mixed	49.3±3.2	53.7±2.3	0.0±0.0 c	0.0±0.0 c
RBM₉	DKN	B5	MS	蔗糖Sucrose	49.0±1.5	57.0±1.0	0.0±0.0 c	0.0±0.0 c

基本培养基 Basal medium	处理 Treatment	植物激素浓度 Phytohormone concentration / (mg L⁻¹)				组织培养再生率 Regeneration rate of tissue culture / %
基本培养基 Basal medium	处理 Treatment	BAP	KT	TDZ	ABA	9311	华占 Huazhan
BM₂	T₁	0.25	0.00	0.10	0.00	11.3±3.0 bc	6.0±1.7 d
	T₂	0.25	0.75	0.00	1.50	12.6±3.5 bc	18.5±3.5 bc
	T₃	0.25	1.50	0.20	3.00	39.0±8.2 a	31.9±2.7 a
	T₄	0.00	0.00	0.00	3.00	1.7±1.7 c	0.0±0.0 e
	T₅	0.00	0.75	0.20	0.00	2.4±2.4 c	8.9±1.8 cd
	T₆	0.00	1.50	0.10	1.50	34.6±5.4 a	24.4±4.8 ab
	T₇	0.50	0.00	0.20	1.50	22.2±4.1 b	14.9±3.6 cd
	T₈	0.50	0.75	0.10	3.00	9.2±1.5 c	9.4±3.0 cd
	T₉	0.50	1.50	0.00	0.00	1.5±1.5 c	26.2±0.4 ab