Establishment and Optimization of an Efficient Peanut
Genetic Transformation System

doi:10.11983/CBB25010

Abstract

Abstract: INTRODUCTION: To establish an efficient Agrobacterium-mediated genetic transformation system for peanuts and lay a foundation for the study of peanut gene functions and variety breeding.
RATIONALE: In this study, 11 peanut varieties were selected, and the cotyledonary leaflets from the variety with the highest bud cluster induction rate were screened out as experimental materials. By screening and optimizing influencing factors such as Agrobacterium strains, the optical density (OD) value of the bacterial suspension, the concentration of acetosyringone (AS), the concentration of surfactants, the infection method and duration, and the co-culture time, transgenic plants of peanut cotyledonary leaflets were obtained.
RESULTS: The results showed that using the embryo leaflet of Huayu 9133 as the receptor, the recombinant Agrobacterium containing eGFP (green fluorescent protein) and GUS (β-glucosidase) protein was used to infect and transform. It was found that when the infection solution was MS liquid+LBA4404 strain+100 μmol∙L^-1 AS+150 mg∙L^-1surfactant Silwet-77 + bacterial solution OD600 was 0.7, the infection method was vacuuming for 15 min + soaking for 20 min + co-culture for 4 d, the peanut conversion rate was the highest. The positive rates of CaMV 35S:eGFP and AhUBQ4:GUS were 52.67% and 57.67%, respectively.The transgenic plants were induced by tissue culture method. The transgenic plants containing eGFP protein were identified as transgenic positive plants by eGFP green fluorescence and PCR detection, and the transgenic plants containing GUS protein were identified as transgenic positive plants by GUS staining and PCR detection.
CONCLUSION: This experiment successfully established and optimized the peanut genetic transformation system, which provided a reference for the study of peanut gene function, the cultivation of resistant varieties, quality improvement and biotechnology research.

CaMV 35S:eGFP侵染材料荧光示意图(a: 再生芽丛明场, b: 再生芽丛荧光, c: 再生苗明场, d: 再生苗荧光)

Key words: Embryo lobule leaflet, Genetic transformation system, eGFP, GUS

Tingting Li, Zhiwen Yan, Yuanyuan Cui, Haosong Guo, Fangjun Chen, Qianqian Zhang, Xiaoqin Liu. Establishment and Optimization of an Efficient Peanut Genetic Transformation System[J]. Chinese Bulletin of Botany, DOI: 10.11983/CBB25010.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB25010

References

Bian HH,Zhao WC,Wei JW,Yu YM,Xue XR,Liu Z,Lü WX,Wang SH,Zhao FK (2020). Effects of Different Agrobacterium rhizogenes Strains on the Induction Rate and Positive Rate of Tomato Hairy Roots. Molecular Plant Breeding 20(24), 8237-8244.
边慧慧,赵文超,魏婧薇,余雨玫,薛晓汝,刘紫傲,吕伟兴,王绍辉,赵福宽(2020).不同发根农杆菌菌株对番茄毛状根诱导率及阳性率的影响.分子植物育种 20(24),8237-8244.
Chen D, Wang YR (2013). Analysis of the Influencing Factors of the International Competitiveness of China's Peanut Industry—An Empirical Analysis Based on the Indicators of the Production Link. Agricultural Technical Economics (11), 112-119.
陈迪,王一茹(2013).中国花生产业国际竞争力影响因素分析——基于生产环节指标的实证分析.农业技术经济 (11),112-119.
Chen XP, Lu Q, Hong YB, LI SX,LIANG XQ(2021).Research Progress of Peanut Genomics in Genetic Breeding . Guangdong Agricultural Sciences 48(12), 33-43.
陈小平,鲁清,洪彦彬,李少雄,梁炫强(2021).花生基因组学在遗传育种中的研究进展.广东农业科学48(12),33-43.
Chen MN,Yang QL,WangT,Chen N,Pan LJ,Chi XY,Yang Z,Wang M,Yu SL(2015). Agrobacterium-mediated genetic transformation of peanut and the efficient recovery of transgenic plants.Canadian Journal of Plant Science 95(4):735-744.
Deng HT, Yang YW, Feng HY, Li XK, Cui SL, Hou MY, Liu YR, Liu LF (2024). Optimization of Agrobacterium-Mediated Genetic Transformation System for Peanut. Molecular Plant Breeding,1-21.
邓洪涛,杨艳雯,冯鹤言,李秀坤,崔顺立,侯名语,刘盈茹,刘立峰(2024).农杆菌介导花生遗传转化体系的优化.分子植物育种,1-21.
Dong JJ (2019). Establishment of Peanut Regeneration System and Analysis of AhRR Gene Family. Master's Thesis. Zhengzhou: Henan Agricultural University, pp.1-55.
董建军(2019).花生再生体系的建立及AhRR基因家族分析.硕士论文.郑州：河南农业大学,pp1-55.
Eapen S,George L(1994). Agrobacterium tumefaciens-mediatedgene transfer in peanut ( Arachis hypogaea L.).Plant Cell Reportsc13,582－586.
Fang XP,Xu ZY, Zhang ZY, Yan LY, Chen KR,Luo LX,Chen JX,R.G. Birch, R.G. Dietzgen (1999). Genetic Studies on GUS Gene and NPTII Gene in the Progeny of Transgenic Peanuts.Peanut Science and Technology (Supplementary Issue 1),241-245.
方小平,许泽永,张宗义,晏立英,陈坤荣,罗莉霞,陈金香,R.G.Birch,R.G.Dietzgen(1999).GUS基因和NPTII基因在转基因花生后代的遗传研究.花生科技(S1),241-245.
GRABIELE M,CHALUP L,ROBLEDO G,SEIJO G(2012).Genetic and geographic origin of domesticated peanut as evidenced by 5S rDNA and chloroplast DNA sequences[J].Plant Systematics and Evolution 298,1151-1165.
HAMMONS R O(1994). The origin and history of the groundnut//The groundnut crop:a scientific basis for improvement.Chapman and Hall,New York,24-42.
He HW(2002). Establishment of the Peanut Regeneration System.Master's Thesis.Guangzhou:South China Normal University,pp.1-68.
何红卫(2002).花生再生系统的建立.硕士论文.广州:华南师范大学,pp1-68.
Hsieh, YF., Jain, M., Wang, J(2017).Direct organogenesis from cotyledonary node explants suitable for Agrobacterium-mediated transformation in peanut (Arachis hypogaea L.). Plant Cell Tiss Organ Cult 128,161–175.
Iqbal MM, Nazir F, Ali S, Asif MA, Zafar Y, Iqbal J, Ali GM (2012). Over expression of rice chitinase gene in transgenic peanut (Arachis hypogaea L.) improves resistance against leaf spot. Mol Biotechnol 50,129–136.
Jia Yuchen, Wang Li, Chen Qi, Li Lianguo, Li Qiaoling, Liu Dehu (2011).Establishment of the Genetic Transformation System for Peanut Cotyledonary Node Explants.Science Technology and Engineering,11(14),3142-3146.
贾宇臣,王利,陈琦,李连国,李巧玲,刘德虎(2011).花生子叶节外植体遗传转化体系的建立.科学技术与工程11(14),3142-3146.
Jia YC,Zhao K,Xue X,Li W,Li SW,Liu DH(2012).Research on the Genetic Transformation of the Rotavirus Antigen Protein G3VP7 Gene in Peanut.Journal of Biomedical Engineering,29(02),328-331.
贾宇臣,赵凯,薛昕,李薇,李少伟,刘德虎(2012).轮状病毒抗原蛋白G3VP7基因在花生中遗传转化的研究.生物医学工程学杂志 29(02),328-331.
Jiang Jing(2012).Construction of Regeneration Systems for Four Excellent Peanut Varieties.Master's Thesis.Guangxi:Guangxi University,pp.1-57.
蒋菁(2012).四个花生优良品种再生体系的构建.硕士论文,广西:广西大学,pp1-57.
Karthik,S.,Pavan, G., Sathish, S(2018). Genotype-independent and enhanced in planta Agrobacterium tumefaciens-mediated genetic transformation of peanut [Arachis hypogaea (L.)]. 3 Biotech 8, 202.
Liang XL, Zheng YX (2006). Research Progress in Transgenic Peanuts. Seed (01), 46-50.
梁雪莲,郑奕雄(2006).花生转基因研究进展.种子(01),46-50.
Liu F,Shan SH,Yan CX,Bi YP,Li GM,Lun WZ(2007). Research on the Influencing Factors of the Genetic Transformation and Regeneration System of Peanut Cotyledons. Journal of Peanut Science (01), 13-19.
刘峰,单世华,闫彩霞,毕玉平,李光敏,伦伟志(2007).花生子叶遗传转化再生体系影响因素的研究.花生学报(01),13-19.
Li HL,Wang D,Zhang SW,Lei J,Wu X,Wang XS,Ma YB(2020).Screening of Regeneration Systems for Different Soybean Varieties in Shanxi.Journal of Shanxi Agricultural Sciences 48(02),161-166.
李换丽,王丹,张树伟,雷佳,吴霞,王新胜,马燕斌(2020).山西不同大豆品种再生体系的筛选.山西农业科学 48 (02),161-166.
Liu FZ (2004). Research on the Transformation of Rs-afp1 Gene and γ-tmt Gene into Peanut and the Establishment of an Efficient Genetic Transformation System.Doctoral Dissertation.Tai'an:Shandong Agricultural University,pp.1-102.
刘风珍(2004).Rs-afp1基因和γ-tmt基因转化花生及高效遗传转化体系的研究.博士论文.泰安:山东农业大学,pp1-102.
Miao LJ,Zhang XiY,Huang BiY,Dong WZ,Tang FS,Qin L,Gao W,Han SY(2012).Study on the Induction of Multiple Buds from Young Leaves of Different Peanut Varieties. Henan Agricultural Sciences 41(11),45-48.
苗利娟,张新友,黄冰艳,董文召,汤丰收,秦利,高伟,韩锁义(2012).不同花生品种幼叶丛生芽诱导研究. 河南农业科学 41(11),45-48.
Mayavan S, Subramanyam K, Arun M, Rajesh M, Dev GK, Sivanandhan G, Jaganath B, Manickavasagam M, Selvaraj N, Ganapathi A (2013). Agrobacterium tumefaciens-mediated in planta seed transformation strategy in sugarcane. Plant Cell Rep 32,1557–1574
Qiu JM, Wen SJ, Liu HY, Liang XQ (2010). Optimization of the High-Efficiency Genetic Transformation System of Peanut Mediated by Agrobacterium tumefaciens. Chinese Journal of Oil Crop Sciences,32(02), 208-211.
邱金梅,温世杰,刘海燕,梁炫强(2010).根癌农杆菌介导花生高效遗传转化体系的优化.中国油料作物学报 32 (02), 208-211.
Qiao LX, Yu XL, Sui JM, Fan QC, Sun SM, Wang JS (2012).Optimization of the Agrobacterium-Mediated Genetic Transformation System of Peanut. Journal of Nuclear Agricultural Sciences 26(09),1244-1248+1358.
乔利仙,于新玲,隋炯明,范乾程,孙世孟,王晶珊(2012).农杆菌介导的花生遗传转化体系的优化.核农学报 26 (09),1244-1248+1358.
Shan SH,Zhuang WJ,Guan DY,Li CJ, Liu SH (2003). Research on Agrobacterium-Mediated Genetic Transformation of Peanut I. Molecular Identification of Plasmid Vectors and Conversion of Agrobacterium Strains. Journal of Peanut Science,(02),1-6.
单世华,庄伟建,官德义,李春娟,刘思衡(2003).以农杆菌为介导花生的遗传转化研究I.质粒载体的分子鉴定及农杆菌菌株的转换.花生学报 (02),1-6.
Shen HY, Xiong HC, Guo XT, Zuo YM (2012). A New Method of Peanut Genetic Transformation Mediated by Agrobacterium rhizogenes. Journal of Plant Nutrition and Fertilizer 18(02), 518-522.
申红芸,熊宏春,郭笑彤,左元梅(2012).一种发根农杆菌介导的花生遗传转化新方法.植物营养与肥料学报 18(02),518-522.
Wang FH, Feng Y, Ma XC, Yang XX, Zhang L (2019). Research on the Optimization of Agrobacterium-Mediated Genetic Transformation System of Peanut Embryonic Leaflets. Henan Agriculture (17), 23-25.
王凤欢,冯滢,马旭策,杨晓欣,张乐(2019).农杆菌介导花生胚小叶遗传转化体系的优化研究.河南农业 (17),23-25.
Wei LQ, Shao FX, Shan L, Bi YP (2008). Research Progress in Peanut Transgenics. China Biotechnology Journal (10), 124-129.
魏丽奇,邵凤霞,单雷,毕玉平(2008).花生转基因研究进展.中国生物工程杂志 (10),124-129.
Wang SY (2020). Creation, Identification and Genetic Analysis of Alien Chromosome Lines between Cultivated Peanut and Diploid Wild Species Arachis duranensis. Master's Thesis. Zhengzhou: Zhengzhou University, pp.1-80.
王思雨(2020).花生栽培种与二倍体野生种Arachis duranensis异染色体系创制、鉴定及遗传分析.硕士论文.郑州:郑州大学,pp1-80.
Wang FH,Feng Y,Ma XC,Yang XX,Zhang L (2019). Optimization Research on the Agrobacterium-Mediated Genetic Transformation System of Peanut Embryonic Leaflets. Henan Agriculture (17), 23-25.
王凤欢,冯滢,马旭策,杨晓欣,张乐(2019).农杆菌介导花生胚小叶遗传转化体系的优化研究.河南农业(17),23-25.
Wang Y(2012). Establishment and Optimization of the Peanut Cotyledon Regeneration System and Transformation System.Master's Thesis.Zhengzhou:Henan Agricultural University,pp.1-49.
王允(2012).花生子叶再生体系及转化体系的建立与优化.硕士论文.郑州:河南农业大学,pp1-49.
Wan XR,Yang LX,Li L(2007).Study on Plant Regeneration and Transformation Using Epicotyls of Peanut as Explants.Journal of Huazhong Agricultural University (01),15-19.
万小荣,杨丽霞,李玲(2007).花生上胚轴为外植体的植株再生及转化研究.华中农业大学学报(01),15-19.
Xu J,Zhang TtZhang JJ,Wang ZJ,Li L,Shan SH(2014). Establishment of Regeneration Systems for Different Peanut Varieties. Shandong Agricultural Sciences 46(06),23-26.
徐娟,张廷婷,张甲佳,王昭静,李林,单世华(2014).不同花生品种再生体系的建立.山东农业科学46(06),23-26.
Xu QQ, Liu FZ, Wan YS (2008). Study on the Main Factors Affecting the Genetic Transformation Rate of Peanut Mediated by Agrobacterium. Journal of Shandong Agricultural University (Natural Science Edition) (02), 161-165.
徐勤青,刘风珍,万勇善(2008).影响农杆菌介导花生遗传转化率主要因素的研究.山东农业大学学报(自然科学版) (02),161-165.
Xu ZY, Liao BS, Yan LY, Chen KR (2007). Research Progress in Transgenic Peanuts. Chinese Journal of Oil Crop Sciences (04), 489-496.
许泽永,廖伯寿,晏立英,陈坤荣(2007).转基因花生研究进展.中国油料作物学报 (04),489-496.
Yang KX,Tang R,Lü JW,Cheng LQ,Hu TH,Wang J (2018). Research Progress in Peanut Tissue Culture. Agricultural Technology Service 35(01), 69-70 + 72.
杨克相,唐容,吕建伟,成良强,胡廷会,王军(2018).花生组织培养研究进展.农技服务 35(01),69-70+72.
Yin DM (2020). A New Peanut Variety ‘Nongdahua 108’ with High Yield and High Resistance to Bacterial Wilt. China Seed Industry (11), 117-118.
殷冬梅(2020).高产高抗青枯病花生新品种农大花108.中国种业 (11),117-118.
Yang YW(2020). Optimization of Agrobacterium-Mediated Genetic Transformation System in Peanut . Master's Thesis. Hebei: Agricultural University of Hebei. pp.1-40.
杨艳雯（2020）.农杆菌介导花生遗传转化体系的优化.硕士论文.河北：河北农业大学.pp.1-40.
Zhang XQ, Liu M (2008). An Empirical Study on the International Competitiveness of China's Peanut Industry. Henan Agricultural Sciences (11), 42-46.
张秀青,刘珉(2008).中国花生产业国际竞争力实证研究.河南农业科学 (11),42-46.
Zhang RD, He LF (2015). Research Progress in Peanut Tissue Culture and Genetic Transformation. Journal of Anhui Agricultural Sciences (11), 27-29.
张铙丹,何龙飞(2015).花生组织培养及遗传转化研究进展.安徽农业科学 (11),27-29.
Zhu J,Han SY, Yuan M, He LQ, He GH, Huang JQ (2018). Optimization of the Conditions for Agrobacterium-Mediated Peanut Genetic Transformation. Chinese Journal of Oil Crop Sciences 40(02),191-198.
朱军,韩锁义,袁美,贺梁琼,何国浩,黄家权(2018).农杆菌介导的花生遗传转化条件优化.中国油料作物学报 40(02),191-198.
Zhai Q,Chen RQ,Liang XH,Zeng CC,Hu B,Li L,Li XY(2022).Establishment and Application of a Rapid Genetic Transformation Method for Peanut.Chinese Bulletin of Botany 57(03),327-339.?
翟琼,陈容钦,梁晓华,曾楚淳,胡博,李玲,李晓云(2022).一种花生快速遗传转化方法的建立与应用.植物学报57(03),327-339.

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Establishment and Optimization of an Efficient Peanut Genetic Transformation System

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