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作物杂志,2019, 第3期: 73–79 doi: 10.16035/j.issn.1001-7283.2019.03.012

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

农杆菌介导谷子成熟胚遗传转化体系的建立与优化

李颜方,杜艳伟,张正,王高鸿,赵根有,赵晋锋,余爱丽   

  1. 山西省农业科学院谷子研究所,046011,山西长治
  • 收稿日期:2018-11-15 修回日期:2018-12-21 出版日期:2019-06-15 发布日期:2019-06-12
  • 通讯作者: 赵晋锋
  • 作者简介:李颜方,助理研究员,研究方向为遗传育种;
  • 基金资助:
    国家现代农业技术体系项目(CARS-06-13.5-A23);山西省农业科学院科技自主创新能力提升工程项目(2017ZZCX-09);山西省农业科学院特色农业技术攻关(YGG17021);山西省农业科学院农业科技创新项目(YCX2018206)

Establishment and Optimization of Agrobacterium Mediated Transformation System for Mature Embryo of Foxtail Millet

Li Yanfang,Du Yanwei,Zhang Zheng,Wang Gaohong,Zhao Genyou,Zhao Jinfeng,Yu Aili   

  1. Millet Research Institute, Shanxi Academy of Agricultural Sciences, Changzhi 046011, Shanxi, China
  • Received:2018-11-15 Revised:2018-12-21 Online:2019-06-15 Published:2019-06-12
  • Contact: Jinfeng Zhao

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摘要：

为建立一个稳定的谷子遗传转化体系,对216份谷子种质资源进行筛选,确定材料178成熟胚为受体材料,从植物激素浓度、防褐化剂、农杆菌菌液浓度、侵染时间、乙酰丁香酮（AS）浓度等方面对谷子再生及转化体系进行优化。结果表明,在MS培养基中加入生长素2,4-D 9μmol/L、细胞分裂素Kinetin 4μmol/L、硝酸银8mg/L,愈伤组织诱导率最高且能有效防止褐化。在侵染液和共培养基中加入100μmol/L AS,gus表达效果最好。农杆菌溶液OD600为0.3~0.5,侵染15min,共培养3d时,抗性愈伤组织率最高。获得的转基因植株,经PCR检测及gus染色分析,确定外源bar基因已成功整合到谷子基因组中。初步建立了一套谷子遗传转化体系,为今后谷子遗传转化提供一些参考。

关键词: 谷子, 农杆菌, 成熟胚, 遗传转化, 愈伤组织

Abstract:

In this study, we developed the foxtail millet in vitro transformation system by using Agrobacterium mediated based on screening 216 foxtail millet varieties to obtain high quality embryo callus. Calli induced from mature seeds of variety 178 as explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA 3301 which contained β-glucuronidase (gus) as the reporter gene, transformed into embryogenic callus for optimizing transformation conditions. For infection with Agrobacterium, calluses derived from shoot apex explants were prepared and submerged in bacterial suspension for different durations, meanwhile the concentration of AS in co-cultivation medium was optimized. Resistant calli were gained by selecting them on a medium containing phosphinothricin and kanamycin. Transformed plants were obtained from the resistant calli after differentiation and regeneration. The target gene was detected by gus activity assay and PCR analysis of bar gene. These results indicated the foreign gene has been integrated into the genome of millet and can be effectively expressed.

Key words: Foxtail millet, Agrobacterium, Mature embryos, Genetic transformation, Callus

表1

试验所用的培养基"

培养基Media 培养基组分Medium composition
愈伤组织诱导培养基（A）
Calli inducing medium		 MS培养基+2.0mg/L 2,4-D+0.5mg/L Kinetin+40g/L麦芽糖+35mg/L ZnSO4·7H2O+0.6mg/L CuSO4·5H2O+
8mg/L AgNO3+4g/L植物凝胶,pH 5.8
分化培养基（B）
Differential medium		 MS培养基+2.0mg/L Kinetin+20g/L麦芽糖+7g/L Phytoblend（植物凝胶类似物）,pH 5.8
农杆菌悬浮侵染培养基（C）
Suspending medium		 MS培养基+40g/L麦芽糖+35mg/L ZnSO4·7H2O+0.6mg/L CuSO4·5H2O+100μmol/L乙酰丁香酮（AS）+1g/L泊洛沙姆,pH 5.8
共培养基（D）
Co-cultural medium		 MS培养基+2.0mg/L 2,4-D+0.5mg/L Kinetin+40g/L麦芽糖+35mg/L ZnSO4·7H2O+0.6mg/L CuSO4·5H2O+
100μmol/L AS+1g/L泊洛沙姆+4g/L植物凝胶,pH 5.8
筛选分化培养基（E）
Screening and differentiation medium		 分化培养基+150mg/L特美汀+5mg/L草丁膦,pH 5.8
筛选再生培养基（F）
Screening and regeneration medium		 MS培养基+2.0mg/L 2,4-D+0.5mg/L Kinetin+40g/L麦芽糖+35mg/L ZnSO4·7H2O+0.6mg/L CuSO4·5H2O+
4g/L植物凝胶+150mg/L特美汀+10mg/L草丁膦,pH 5.8
生根培养基（G）
Rooting medium		 1/2 MS培养基+30g/L蔗糖+7g/L Phytoblend,pH 5.7

表2

适宜组织培养的谷子基因型"

材料编号
Material code		 基因型
Genotype		 发芽率
Rate of germination
 愈伤组织诱导率
Rate of callus induction
 抗性愈伤组织率
Rate of resistance callus		 gus表达率
Rate of gusexpression
19 豫谷1号Yugu 1 83.5 84.2 32.6 11.4
50 长农35 Changnong 35 80.5 83.1 35.8 25.3
63 长生06 Changsheng 06 68.5 85.3 18.9 7.5
125 长生07 Changsheng 07 80.5 81.7 24.6 19.2
129 沁州黄Qinzhouhuang 96.6 87.2 12.6 3.9
164 冀谷11 Jigu 11 78.4 72.6 48.7 35.3
168 金谷1号Jingu 1 91.5 86.8 34.2 19.5
178 晋谷21 Jingu 21 82.6 79.6 42.5 38.9

图1

AgNO3浓度对愈伤组织生长状态的影响"

图2

2,4-D浓度对愈伤组织生长状态的影响"

图3

Kinetin浓度对愈伤组织生长状态的影响"

图4

菌液浓度对转化结果的影响"

图5

侵染时间对转化结果的影响"

图6

AS浓度对转化结果的影响"

图7

谷子转化植株bar基因检测结果 性对照;2：阴性对照;3~11：不同转化株系"

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