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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (10): 1423-1432.doi: 10.3724/SP.J.1006.2018.01423

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS •     Next Articles

Use of Bar Gene for the Stable Transformation of Herbicide-resistant Foxtail Millet Plants

Qian-Nan CHEN1,2,Ke WANG1,Sha TANG1,Li-Pu DU1,Hui ZHI1,Guan-Qing JIA1,Bao-Hua ZHAO2,Xing-Guo YE1,Xian-Min DIAO1,*()   

  1. 1 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    2 College of Life Science, Hebei Normal University, Shijiazhuang 050024, Hebei, China
  • Received:2017-11-23 Accepted:2018-07-20 Online:2018-10-10 Published:2018-08-01
  • Contact: Xian-Min DIAO E-mail:diaoxianmin@caas.cn
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31501324);This study was supported by the National Natural Science Foundation of China(31522040);the Fundamental Research Funds of CAAS(Y2017JC15);the Fundamental Research Funds of CAAS(CAAS-XTCX2016001-5);the Fundamental Research Funds of CAAS(CAAS-XTCX2016002);the China Agriculture Research System(CARS-06-13.5-A04);the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences

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Abstract:

Efficiency of Agrobacterium-mediated genetic transformation has been the main factor that restricts the study of functional genes and transgenic breeding in foxtail millet. In this study, foxtail millet variety Jigu 11 panicle primordia were used as explants. Young panicles of 0.5-1.0 cm in length were picked and cut into small pieces. The young embryos were cultured in modified MS medium for inducing embryogenic calli, totally forming 3120 embryogenic calli in 15-20 days. Soaking the calli in the infection suspension prior to transformation, and the heat treatment at 45°C for 3 min could effectively improve the transient genetic transformation efficiency by 26.1%. The transformed calli were screened with phosphinothricin (PPT), in which 513 were resistant calli, with the resistant calli rate of 16.4%. Seven herbicide-resistant plants were obtained after resistant calli differentiation and seedling culture. Six T0 transgenic positive plants were identified by PCR and Southern blot. PPT resistance analysis was carried out on leaves in T3 generation of transformed plants, and combined with Bar protein antibody test strip identification, the results confirmed that the Bar gene stably incorporated into the genome of foxtail millet seedlings. This study established a stable genetic transformation system in foxtail millet, which is of great significance in improving the efficiency of molecular breeding, and prompting foxtail millet as a new model plants.

Key words: foxtail millet, Agrobacterium, genetic transformation, heat shock, herbicide-resistant

Table 1

Media composition of Agrobacterium-mediated foxtail millet calli transformation (g L-1)"

组分
Composition		 诱导培养基
IM		 侵染液
IL		 共培养基
CM		 恢复培养基RM 筛选培养基
SM		 分化培养基DM 壮苗培养基
SSM
MS 4.43 0.443 0.443 4.43 4.43 4.43 2.215
L-Glu 0.5 — — 0.5 0.5 — —
CEH 0.8 0.8 0.8 0.8 0.8 — —
MES 1.95 0.5 0.5 1.95 1.95 — —
inositol 0.1 — — 0.1 0.1 0.1 —
VCa — — — — — 0.01 0.01
Glucose — 36 10 — — — —
Sucrose — 68.5 — — — 20 30
Maltose 40 — — 40 40 — —
CuSO4a — 0.0006 — — — — —
AgNO3a 0.005 — 0.005 0.005 0.005 — —
2,4-Da 0.002 — 0.002 0.002 0.002 — —
KTa 0.0005 — 0.005 0.0005 0.0005 0.002 —
NAAa — — — — — — 0.0005
ASa — — 0.1962, L-1 — — — —
Cefa — — — 0.1 — — —
Carba — — — 0.25 0.25 — —
Tima — — — — — 0.15 0.15
PPTa — — — — 0.005/0.01 — 0.001
Agar 5 — 8 5 5 — —
Phytagel — — — — — 2.5 2

Table 2

Genetic transformation heat treatment"

热激温度
Heat temperature (°C)		 热激时间
Heat time (min)		 冰浴时间
Ice bath time (min)
25(CK) — —
37 3 1
45 3 1
47 3 1

Fig. 1

Panicle and calli status at different induction periods (bar = 0.5 cm) A: calli after induction for 7 d; B: calli after induction for 20 d; C: calli after induction for 40 d; D: young panicles in different sizes."

Table 3

Comparison of calli induction efficiency after 15 days of young panicle induction"

幼穗大小
Panicle size (cm)		 愈伤组织数量
Calli number		 幼穗数量
Panicle number		 愈伤组织诱导率
Calli induction rate (%)a
≤ 0.5 23 30 74.35±2.35
18 25
20 27
0.5-1.0 181 198 92.74±1.13
190 204
207 221
1.1-1.5 104 137 75.14±4.11
97 123
77 109
1.6-2.0 43 96 39.16±4.98
39 103
31 89
≥ 2.0 17 75 21.54±1.85
13 68
21 92

Fig. 2

Effect of calli induction time on transformation efficiency A: calli after induction for 7 d; B: calli after induction for 15 d; C: calli after induction for 30 d; D: GUS transient conversion efficiency; E: GUS staining of calli after induction for 15 d; F: GUS staining of calli after induction for 30 d. * and ** indicates a significant difference at P ≤ 0.05 and P ≤ 0.01 levels, respectively."

Fig. 3

Transient expression of GUS after heat shock treatment of foxtail millet calli A: GUS transient expression rate at different temperatures of 3 min; B: GUS staining under CK (25°C); C: GUS staining at 45°C heat shock; D: GUS transient expression rate at different time under 45°C heat shock; E: 45°C heat shock 1.5 min GUS staining; F: 45°C heat shock 3 min GUS staining. * and ** indicates a significant difference at P ≤ 0.05 and P ≤ 0.01 levels, respectively."

Fig. 4

Identification of T0 generation transgenic lines A: calli differentiation; B: regenerated plant; C: strong seedling; D: transplanting of regenerated plants; E: bar PCR detection, 1-7 are T0 generation transformed plants; F: Southern blot of T0 generation of transgenic lines, 1-5: digested with Sac I, 7-11: digested with Hind III, 6: wild type. M: marker DL2000; H: water control; WT: wild type; P: plasmid."

Fig. 5

Bar gene resistance test of T3 generation transgenic lines A: 2.5 g L-1 PPT solution coated leaves; B: 2.5 g L-1 PPT solution sprayed leaves in transgenic lines (left) and in CK (right); C: Bar protein test results. CK: wild type control; Negative: negative transgenic lines; Positive: positive transgenic line."

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