Welcome to Acta Agronomica Sinica，

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

RichHTML337

83

PDF

706

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."

[1] Li H W, Li C H, Pao W K . Cytogenetical and genetical studies of the interspecific cross between the cultivated foxtail millet, Setaria italica(L.) Beauv., and the green foxtail millet S. viridis L. J Am Soc Agron, 1945,37:32-54
[2] Zhang C, Zhang H, Li J X . Advances of millet research on nutrition and application. J Chin Cereals Oils Assoc, 2009,22:51-55
[3] Doust A N, Kellogg E A, Devos K M, Bennetzen J L . Foxtail millet: A sequence-driven grass model system. Plant Physiol, 2009,149:137-141
doi: 10.1104/pp.108.129627 pmid: 19126705
[4] Brutnell T P, Wang L, Swartwood K, Goldschmidt A, Jackson D, Zhu X G, Kellogg E, Eck J V . Setaria viridis: a model for C4 photosynthesi. Plant Cell, 2010,22:2537-2544
[5] 董云洲, 段胜军, 赵连元, 杨秀海, 贾士荣 . 用基因枪转化花粉获得转基因谷子与玉米. 中国农业科学, 1999,32(2):112-115
Dong Y Z, Duan S J, Zhao L Y, Yang X H, Jia S R . Production of transgenic millet and maize plants by particle bombardment. Sci Agric Sin, 1999, 32(2):112-115 (in Chinese with English abstract)
[6] Dong Y Z, Duan S J . Production of transgenic millet plants via particle bombardment. Acta Bot Boreali-Occident Sin, 2000,20:175-178
[7] Diao X M, Chen Z L, Duan S J, Liu Y L, Zhao L Y, Sun J S . Factors influencing foxtail millet embryogenic calli transformation by particle bombardments. Acta Agric Boreali-Sin, 1999,14:31-36
doi: 10.3321/j.issn:1000-7091.1999.03.007
[8] Wang Y F, Li W, Diao X M . Genetic transformation of foxtail millet mediated by Agrobacterium tumefaciens. J Hebei Agric Sci, 2003,7:1-6
[9] Liu Y H, Feng X Y, Xu Y G, Yu J L, Ao G M, Peng Z M, Zhao Q . Overexpression of millet ZIPlike gene (SiPf40) affects lateral bud outgrowth in tobacco and millet. Plant Physiol Biochem, 2009,47:1051-1060
[10] Qin F F, Zhao Q, Ao G M, Yu J J . Co-suppression of Si401, a maize pollen specific Zm401 homologous gene, results in aberrant anther development in foxtail millet. Euphytica, 2008,163:103-111
[11] 李丛 . 谷子DREB类转录因子SiARDP功能及其表达调控研究. 中国农业大学博士学位论文, 北京, 2014
Li C . The Function and Regulation of the Foxtail Millet DREB-type Transcriptional Factor SiARDP. PhD Dissertation of China Agricultural University, Beijing, China, 2014 ( in Chinese with English abstract)
[12] Martins P K, Ribeiro A P, Cunha B A D B, Kobayashi A K, Molinari H B C . A simple and highly efficientAgrobacterium- mediated transformation protocol for Setaria viridis. Biotechnol Rep, 2015,6:41-44
[13] Martins P K, Nakayama T J, Ribeiro A P, Cunha B A D B D, Nepomuceno A L, Harmon F G, Kobayashi A K, Molinari H B C . Setaria viridis floral-dip: a simple and rapid Agrobacterium- mediated transformation method. Biotechnol Rep, 2015, 6:61-63
[14] Saha P, Blumwald E . Spike-dip transformation of Setaria viridis. Plant J, 2016,86:89-101
[15] Ortiz J P A, Reggiardo M I, Ravizzini R A, Ahabe S G, Cervigni G D L, Spitteler M A, Morata M M, Elias F E, Valleios R H . Hygromycin resistance as an eficient selectable marker for wheat stable transformation. Plant Cell Rep, 1996,15:877-881
doi: 10.1007/BF00231579 pmid: 24178266
[16] Bevan M W, Flavell R B, Chihon M D . A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature, 1983,304:184-187
doi: 10.1038/304184a0
[17] Thompson C J, Mowa N R, Tizard R, Crameri R, Davies J E, Lauwcreys M, Botterman J . Characterization of the herbicide- resistance gene bar from Streptomyees hygroseopicus. EMBO J, 1987,6:2519-2523
[18] Howe A R, Gasser C S, Brown S M, Padgette S R, Hart J, Parker G B, Fromm M E, Armstrong C L . Glyphosate as a selective agent for the production of fertile transgenie maize (Zea mays L.) plants. Mol Breed, 2002,10:153-164
[19] 孙敬三, 陈维伦 . 植物生物技术和作物改良. 北京: 中国科学技术出版杜, 1990. pp 5-14
Sun J S, Chen W L. Plant Biotechnology and Crop Improvement. Beijing: China Science and Technology Press, 1990. pp 5-14(in Chinese)
[20] 王天宇 . 抗除草剂基因在作物杂种优势中的利用及进展. 作物杂志, 1998, ( 5):33-34
Wang T Y . Utilization and progress of herbicide resistant genes in crop heterosis. Crops, 1998, ( 5):33-34 (in Chinese)
[21] 张晓东, 李冬梅, 徐文英, 蒋有绎, 胡道芬, 韩立新 . 利用基因枪将HMW谷蛋白亚基基因与除草剂Basta抗性基因导入小麦不同外植体获得转基因植株. 遗传, 1998, ( 增刊1):5-10
Zhang X D, Li D M, Xu W Y, Jiang Y Y, Hu D F, Han L X . Transgenic plants were obtained by introducing the HMW glutenin subunit gene and the herbicide Basta resistance gene into different explants of wheat using gene gun. Hereditas, 1998, ( suppl-1):5-10 (in Chinese)
[22] 傅荣昭, 曹光诚, 马江生, 李文彬, 孙勇加, 陈占宽, 易明林, 郅玉宝, 林作辑 . 用基因枪法将人工雄性不育基因导入小麦的研究初报. 遗传学报, 1997,24:358-361
doi: 10.1007/BF02951625
Fu R Z, Cao G C, Ma J S, Li W B, Sun Y J, Chen Z K, Yi M L, Zhi Y B, Lin Z Y . Preliminary study of transferring engineered male sterile gene into wheat via microgrojectile bombardment. J Genet Genomics, 1997,24:358-361 (in Chinese with English abstract)
doi: 10.1007/BF02951625
[23] Huang D N, Zhang S Q, Xue R, Hua Z H, Xie X B, Wang X L . A new method to identify and improve the purity of hybrid rice with herbicide resistant gene. Chin Rice Res Newsl, 1998,6:1
[24] 王天宇, 石云素, 辛志勇, Darmency H . 抗除草剂谷子新种质的创制、鉴定与利用. 中国农业科技导报, 2000, ( 5):62-66
Wang T Y, Shi Y S, Xin Z Y, Darmency H . Establishment, identification and utilization of new herbicide-resistant millet germplasm. J Agric Sci Tech China, 2000, ( 5):62-66 (in Chinese)
[25] Naciri Y, Darmency H, Belliard J, Dessaint F, Pernès J . Breeding strategy in foxtail millet, Setaria italica L.Beauv. following interspecific hybridization. Euphytica, 1992,60:97-103
[26] Wang T Y, Darmency H . Dinitroaniline herbicide cross -resistance in resistant Setaria italica lines selected from interspecific cross with S. viridis. Pest Manage Sci, 1997,49:277-283
[27] Heap I M, Morrison I N . Resistance to aryloxyphenoxpropionate and cyclohexanedione herbicides in green foxtail (Setaria viridis). Weed Sci, 1996,44:25-30
[28] Wang T Y, Darmency H . Cross -resistance to aryloxyphenoxypropionate and cyclohexanedione herbicides in foxtail millet(Setaria italica). Pestic Biochem Physiol, 1997,59:81-88
[29] Maniatis T, Fritsch E F, Sambrook J F E F . Molecular Cloning: A Laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory Press, 1989. pp 304-331
[30] 柳琳 . 根癌农杆菌介导的谷子遗传转化体系的构建. 河北师范大学硕士学位论文, 河北石家庄, 2010
Liu L . Agrobacterium-tumefaciens Mediated Transformation of Millet Genetic System. MS Thesis of Hebei Normal University, Shijiazhuang, China, 2010 ( in Chinese with English abstract)
[31] Hiei Y, Ishida Y, Kasaoka K, Komari T . Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection withAgrobacterium tumefaciens. Plant Cell, 2006,87:233-243
[32] 徐游 . 农杆菌介导Hi-II玉米幼胚遗传转化效率及高效再生体系的研究. 江西农业大学硕士学位论文, 江西南昌, 2013
Xu Y . Agrobacterium-mediated Maize Immature Embryo Hi-II Genetic Transformation Efficiency and Efficient Regeneration System. MS Thesis of Jiangxi Agricultural University, Nanchang, China, 2013 ( in Chinese with English abstract)
[33] Liu Y H, Yu J J, Zhao Q, Zhu D Y, Ao G M . Genetic transformation of millet (Setaria italica) by Agrobacterium-mediated. J Agric Biotechnol, 2005,13:32-37
[34] Lambe P, Dinant M, Matagne R . Differential long term expression and methylation of the hygromycin phosphotrans pherase (hph) and(β-glucuronidase) calli. Plant Sci, 1995,108:51-62
[35] 牛玉红 . 谷子抗除草剂基因Srf的分子标记. 河北农业大学硕士学位论文, 河北保定, 2001
doi: 10.7666/d.y385779
Niu Y H . Molecular Tagging of Srf, A Gene Conferring Sethoxydim Resistance in Foxtail Millet (Setaria italica (L.) Beauv.). MS Thesis of Hebei Agricultural University, Baoding, Hebei, China, 2001 ( in Chinese with English abstract)
doi: 10.7666/d.y385779
[1] JIN Min-Shan, QU Rui-Fang, LI Hong-Ying, HAN Yan-Qing, MA Fang-Fang, HAN Yuan-Huai, XING Guo-Fang. Identification of sugar transporter gene family SiSTPs in foxtail millet and its participation in stress response [J]. Acta Agronomica Sinica, 2022, 48(4): 825-839.
[2] DU Xiao-Fen, WANG Zhi-Lan, HAN Kang-Ni, LIAN Shi-Chao, LI Yu-Xin, ZHANG Lin-Yi, WANG Jun. Identification and analysis of RNA editing sites of chloroplast genes in foxtail millet [Setaria italica (L.) P. Beauv.] [J]. Acta Agronomica Sinica, 2022, 48(4): 873-885.
[3] FENG Ya, ZHU Xi, LUO Hong-Yu, LI Shi-Gui, ZHANG Ning, SI Huai-Jun. Functional analysis of StMAPK4 in response to low temperature stress in potato [J]. Acta Agronomica Sinica, 2022, 48(4): 896-907.
[4] ZHAO Mei-Cheng, DIAO Xian-Min. Phylogeny of wild Setaria species and their utilization in foxtail millet breeding [J]. Acta Agronomica Sinica, 2022, 48(2): 267-279.
[5] MA Gui-Fang, MAN Xia-Xia, ZHANG Yi-Juan, GAO Hao, SUN Zhao-Xia, LI Hong-Ying, HAN Yuan-Huai, HOU Si-Yu. Integrated analysis between folate metabolites profiles and transcriptome of panicle in foxtail millet [J]. Acta Agronomica Sinica, 2021, 47(5): 837-846.
[6] TANG Rui-Min, JIA Xiao-Yun, ZHU Wen-Jiao, YIN Jing-Ming, YANG Qing. Cloning of potato heat shock transcription factor StHsfA3 gene and its functional analysis in heat tolerance [J]. Acta Agronomica Sinica, 2021, 47(4): 672-683.
[7] JIA Xiao-Ping, LI Jian-Feng, ZHANG Bo, QUAN Jian-Zhang, WANG Yong-Fang, ZHAO Yuan, ZHANG Xiao-Mei, WANG Zhen-Shan, SANG Lu-Man, DONG Zhi-Ping. Responsive features of SiPRR37 to photoperiod and temperature, abiotic stress and identification of its favourable allelic variations in foxtail millet (Setaria italica L.) [J]. Acta Agronomica Sinica, 2021, 47(4): 638-649.
[8] JIA Xiao-Ping,YUAN Xi-Lei,LI Jian-Feng,WANG Yong-Fang,ZHANG Xiao-Mei,ZHANG Bo,QUAN Jian-Zhang,DONG Zhi-Ping. Photo-thermal interaction model under different photoperiod-temperature conditions and expression analysis of SiCCT gene in foxtail millet (Setaria italica L.) [J]. Acta Agronomica Sinica, 2020, 46(7): 1052-1062.
[9] HAN Le,DU Ping-Ping,XIAO Kai. Functional characteristics of TaPYR1, an abscisic acid receptor family gene in mediating wheat tolerance to drought stress [J]. Acta Agronomica Sinica, 2020, 46(6): 809-818.
[10] Jin-Feng ZHAO,Yan-Wei DU,Gao-Hong WANG,Yan-Fang LI,Gen-You ZHAO,Zhen-Hua WANG,Yu-Wen WANG,Ai-Li YU. Identification of PEPC genes from foxtail millet and its response to abiotic stress [J]. Acta Agronomica Sinica, 2020, 46(5): 700-711.
[11] CHEN Er-Ying, WANG Run-Feng, QIN Ling, YANG Yan-Bing, LI Fei-Fei, ZHANG Hua-Wen, WANG Hai-Lian, LIU Bin, KONG Qing-Hua, GUAN Yan-An. Comprehensive identification and evaluation of foxtail millet for saline-alkaline tolerance during germination [J]. Acta Agronomica Sinica, 2020, 46(10): 1591-1604.
[12] JIA Xiao-Ping,QUAN Jian-Zhang,WANG Yong-Fang,DONG Zhi-Ping,YUAN Xi-Lei,ZHANG Bo,LI Jian-Feng. Effects of different photoperiod conditions on agronomic traits of foxtail millet [J]. Acta Agronomica Sinica, 2019, 45(7): 1119-1127.
[13] YUAN Yi-Chuan, CHEN Xiao-Yu, LI Ming-Ming, LI Ping, JIA Ya-Tao, HAN Yuan-Huai, XING Guo-Fang. Screening of germplasm tolerant to low phosphorus of seedling stage and response of root protective enzymes to low phosphorus in foxtail millet [J]. Acta Agronomica Sinica, 2019, 45(4): 601-612.
[14] Xue-Jiao CHEN,Xu-Dong ZHANG,Zhi-Zhong HAN,Peng ZHANG,Zhi-Kuan JIA,Yan-Hao LIAN,Qing-Fang HAN. Fertilizer Response and Its Contribution to Yield of Foxtail Millet under Ridge-furrow Rainfall Harvesting Planting Model in Semi-arid Areas [J]. Acta Agronomica Sinica, 2018, 44(7): 1055-1066.
[15] Yu-Jie ZHANG,Yuan-Yuan ZHANG,Hua-Ning ZHANG,Ning QIN,Guo-Liang LI,Xiu-Lin GUO. Characterization and Regulatory Roles in Thermotolerance of Wheat Heat Shock Transcription Factor Gene TaHsfA2e [J]. Acta Agronomica Sinica, 2018, 44(12): 1818-1828.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No.12 Zhongguancun South Street, Beijing 100081,China Email:xbzw@chinajournal.net.cn
Supported by Beijing Magtech Co.Ltd