Crops ›› 2019, Vol. 35 ›› Issue (3): 73-79.doi: 10.16035/j.issn.1001-7283.2019.03.012

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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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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

Table 1

Media used in experiment"

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

Table 2

Genotype of millet suitable for tissue culture %"

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

Fig.1

Effects of AgNO3 concentration on callus growth status"

Fig.2

Effects of 2,4-D concentration on callus growth status"

Fig.3

Effects of Kinetin concentration on callus growth status"

Fig.4

Effects of Agrobacterium concentration on transformation results"

Fig.5

Effects of infection time on transformation results"

Fig.6

Effects of AS concentration on transformation results"

Fig.7

The test results of transgenic Setaria italic Positive control; 2, Negative control; 3-11, Different transformed lines; Marker, DL 5000"

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