TECHNIQUE AND METHOD

Agrobacterium rhizogenes-mediated Transformation System of Pueraria lobata Hairy Roots

  • Zeng Wendan ,
  • Yan Huabing ,
  • Wu Zhengdan ,
  • Shang Xiaohong ,
  • Cao Sheng ,
  • Lu Liuying ,
  • Xiao Liang ,
  • Shi Pingli ,
  • Cheng Dong ,
  • Long Ziyuan ,
  • Li Jieyu
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  • 1Cash Crops Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China
    2Guangxi University, Nanning 530004, China

Received date: 2024-06-11

  Accepted date: 2024-12-26

  Online published: 2024-12-27

Abstract

INTRODUCTION:An efficient Agrobacterium rhizogenes-mediated transformation system for Pueraria lobata was established.

RATIONALE: In this study, tissue-cultured plantlets of P. lobata were used as explants to investigate the effects of different genotypes, A. rhizogenes strains, explants, precultivation times, infection times, culture days, subculture times, and culture methods on the efficiency of hairy root genetic transformation in P. lobata.

RESULTS: The results indicated that the induction rate of hairy root formation was the highest when the immature leaves of YG-19 were used as the explant material, reaching 10.2%. A. rhizogenes K599 was identified as the most suitable strain. The optimal explant material was immature leaves that had just unfolded from the first to second nodes of the 5th to 13th generation tissue culture plantlets subcultured for 8 days. After 3 days of pre-culture and 15 minutes of bacterial infection, the highest induction rate of hairy roots reached 22.4%. The optimal type of culture medium for the proliferation of hairy roots in P. lobatawas solid medium culture, and the fresh weight of hairy roots grown on solid medium was 75 times greater than that of hairy roots grown in liquid medium. PCR detection and fluorescence microscopy assays revealed that the expression of GFP and rolB genes in the hairy roots of P. lobata was stable, and the rate of cotransformation was 80%.

CONCLUSION: Genotype, A. rhizogenes strain, and culture duration were the most critical factors for the efficient genetic transformation of hairy roots in P. lobata.

Cite this article

Zeng Wendan , Yan Huabing , Wu Zhengdan , Shang Xiaohong , Cao Sheng , Lu Liuying , Xiao Liang , Shi Pingli , Cheng Dong , Long Ziyuan , Li Jieyu . Agrobacterium rhizogenes-mediated Transformation System of Pueraria lobata Hairy Roots[J]. Chinese Bulletin of Botany, 2025 , 60(3) : 425 -434 . DOI: 10.11983/CBB24092

References

[1] Aggarwal PR, Nag P, Choudhary P, Chakraborty N, Chakraborty S (2018). Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies. Plant Methods 14, 55.
[2] Cao XS, Xie HT, Song ML, Lu JH, Ma P, Huang BY, Wang MG, Tian YF, Chen F, Peng J, Lang ZB, Li GF, Zhu JK (2023). Cut-dip-budding delivery system enables genetic modifications in plants without tissue culture. Innovation (Camb) 4, 100345.
[3] Chen YM, Liu JY, Zhang T, Lu QY, Yan Q (2017). Induction of hairy roots in heavy metal hyperaccumulator Sedum alfredii. J Trop Subtrop Bot 25, 136-140. (in Chinese)
  陈友明, 刘静轶, 张腾, 卢倩云, 晏琼 (2017). 重金属超富集植物东南景天毛状根的诱导. 热带亚热带植物学报 25, 136-140.
[4] Cheng Y, Ma PT, Wu J (2022). Study on comparison of regeneration capacity of different varieties of common bean. J Sichuan Agric Univ 40, 465-471, 535. (in Chinese)
  程媛, 马朋涛, 武晶 (2022). 不同基因型普通菜豆再生能力研究. 四川农业大学学报 40, 465-471, 535.
[5] Crane C, Wright E, Dixon RA, Wang ZY (2016). Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens-transformed roots and Agrobacterium rhizogenes-transformed hairy roots. Planta 223, 1344-1354.
[6] Cui ML, Liu C, Piao CL, Liu CL (2020). A stable Agrobacterium rhizogenes-mediated transformation of cotton (Gos-sypium hirsutum L.) and plant regeneration from transformed hairy root via embryogenesis. Front Plant Sci 11, 604255.
[7] Du JY, Chen KY, Pu J, Zhou HY, Zhu GT, Zhang CZ, Du H (2023). The modification of gene editing vector for efficient GFPuv fluorescence screening and its application in potato genetic transformation. Sci Agric Sin 56, 2223-2236. (in Chinese)
  杜静雅, 陈凯园, 普金, 周会英, 祝光涛, 张春芝, 杜慧 (2023). 高效GFPuv荧光筛选基因编辑载体的改造及其在马铃薯遗传转化中的应用. 中国农业科学 56, 2223-2236.
[8] Fu CX, Jin ZP, Yang R, Wu FY, Zhao DX (2004). Establishment of Saussurea involucrata hairy roots culture and plantlet regeneration. Chin J Biotechnol 20, 366-371. (in Chinese)
  付春祥, 金治平, 杨睿, 吴风燕, 赵德修 (2004). 新疆雪莲毛状根的诱导及其植株再生体系的建立. 生物工程学报 20, 366-371.
[9] Geng LL, Niu LH, Gresshoff PM, Shu CL, Song FP, Huang DF, Zhang J (2012). Efficient production of Agrobacterium rhizogenes-transformed roots and composite plants in peanut (Arachis hypogaea L.). Plant Cell Tissue Organ Cult 109, 491-500.
[10] Hu JQ (2021). Study on Genetic Transformation System of Mulberry Mediated by Agrobacterium. Master's thesis. Chongqing: Southwest University. pp. 1-62. (in Chinese)
  胡建琼 (2021). 农杆菌介导的桑树遗传转化体系研究. 硕士论文. 重庆: 西南大学. pp. 1-62.
[11] Kiryushkin AS, Ilina EL, Guseva ED, Pawlowski K, Demchenko KN (2022). Hairy CRISPR: genome editing in plants using hairy root transformation. Plants (Basel) 11, 51.
[12] Liang P, Shi HP, Qi Y (2004). Effect of sucrose concentration on the growth and production of secondary metabolites in Pueraria phaseoloides hairy roots. Acta Biol Exp Sin 37, 384-390. (in Chinese)
  梁朋, 施和平, 齐莹 (2004). 蔗糖浓度对三裂叶野葛毛状根生长及其次生物质产生的影响. 实验生物学报 37, 384-390.
[13] Liu CF, Yu SH, Li L, Shi HP, Pan RZ (2000). The genetic transformation of Pueraria as medicinal plant by Agrobacterium rhizogenes. J Integr Plant Biol 42, 936-939. (in Chinese)
  刘传飞, 于树宏, 李玲, 施和平, 潘瑞炽 (2000). 发根土壤杆菌对葛属药用植物的遗传转化. 植物学报 42, 936-939.
[14] Mei GG, Chen A, Wang YR, Li SQ, Wu MY, Hu YL, Liu X, Hou XL (2024). A simple and efficient in planta transformation method based on the active regeneration capacity of plants. Plant Commun 5, 100822.
[15] Park SU, Facchini PJ (2000). Agrobacterium rhizogenes-mediated transformation of opium poppy, Papaver somniferum L., and California poppy, Eschscholzia californica Cham., root cultures. J Exp Bot 347, 1005-1016.
[16] Ren RY, Xue JK, Guo HY, Wei JC (2017). Induction of hairy roots of Scrophularia buergeriana and its plant regeneration. Chin Bull Bot 52, 783-787. (in Chinese)
  任如意, 薛巨坤, 国会艳, 魏继承 (2017). 北玄参毛状根诱导及其植株再生. 植物学报 52, 783-787.
[17] Shi HP, Kintzios S (2003). Genetic transformation of Pueraria phaseoloides with Agrobacterium rhizogenes and puerarin production in hairy roots. Plant Cell Rep 21, 1103-1107.
[18] Shi HP, Quan H, Kintzios S (2003). Induction of hairy roots of Pueraria phaseoloides and its culture in liquid and solid medium. Chin J Biotechnol 19, 307-311. (in Chinese)
  施和平, 权宏 Kintzios S (2003). 三裂叶野葛毛状根的诱导及其固体培养和液体培养. 生物工程学报 19, 307-311.
[19] Shi HP, Wang P, Yang SN, Guo YP (2016). Induction of hairy roots of Dianthus chinensis and its plant regeneration. Chin Bull Bot 51, 363-368. (in Chinese)
  施和平, 王蓓, 杨树楠, 郭亚鹏 (2016). 五寸石竹毛状根诱导及其植株再生. 植物学报 51, 363-368.
[20] Shi HP, Zhu YF, Zeng BQ, Zhou ZH, Yu ZA, Huang SQ (2017). Factors influencing induction and in vitro culture of hairy roots in Phytolacca americana L. Chin J Biotechnol 33, 272-283. (in Chinese)
  施和平, 朱远锋, 曾宝强, 周卓辉, 余震傲, 黄胜琴 (2017). 美洲商陆毛状根诱导及其离体培养的影响因素. 生物工程学报 33, 272-283.
[21] Sun H, Zhang JY, Luo LJ, Liu PD (2024). Development of transgenic hairy root induction and protoplast preparation systems for Stylosanthes leiocarpa. Acta Agrestia Sin 32, 1583-1591. (in Chinese)
  孙昊, 张建禹, 罗丽娟, 刘攀道 (2024). 光果柱花草转基因毛状根及其原生质体制备体系的建立. 草地学报 32, 1583-1591.
[22] Wan YM, Xiao SH, Bai YC, Fan JY, Wang Y, Wu CA (2023). Establishment and optimization of a high-efficient hairy-root system in foxtail millet (Setaria italica L.). Acta Agron Sin 49, 1758-1768. (in Chinese)
  万夷曼, 肖圣慧, 白依超, 范佳音, 王琰, 吴长艾 (2023). 谷子毛状根诱导方法的建立与优化. 作物学报 49, 1758-1768.
[23] Wang Y, Huang LJ, Li YW, Feng ZS, Mu ZH, Wang J, Wu XY, Wang BG, Lu ZF, Li GJ, Wu XH (2022). Checking transformation efficiency for different Lagenaria siceraria genotypes by using seed germination pouches as a growth carrier. Plant Cell Tissue Organ Cult 151, 199-206.
[24] Wang YH (2006). Study on establishment of system with high frequency for genetic transformation of Phellodendron chinese and plant regeneration. J Chin Med Mater 29, 641-644. (in Chinese)
  王跃华 (2006). 川黄柏高效遗传转化系统建立和植株再生研究. 中药材 29, 641-644.
[25] Xie XT, Huang QY, Wen GC, Yuan HW, He Y, Yan DL, Huang JQ, Wang XF, Zheng BS (2022). Construction of Agrobacterium rhizogenes-mediated transformation system of Carya illinoinensis without dependence on tissue culture. J Fruit Sci 39, 131-140. (in Chinese)
  谢晓婷, 黄巧宇, 温广超, 袁虎威, 何漪, 闫道良, 黄坚钦, 王晓飞, 郑炳松 (2022). 非组培依赖的发根农杆菌介导的薄壳山核桃转化体系构建. 果树学报 39, 131-140.
[26] Xu Y, Cao YP, Wang Y, Fu CX, Dai SJ (2019). Agrobacterium rhizogenes-mediated transformation system of Spinacia oleracea. Chin Bull Bot 54, 515-521. (in Chinese)
  徐悦, 曹英萍, 王玉, 付春祥, 戴绍军 (2019). 发根农杆菌介导的菠菜毛状根遗传转化体系的建立. 植物学报 54, 515-521.
[27] Yu SH, Liu CF, Li L, Pan RC (2001). Factors affecting genetic transformation of Pueraria lobata by Agrobacterium rhizogene. Chin J Appl Environ Biol 7, 474-477. (in Chinese)
  于树宏, 刘传飞, 李玲, 潘瑞炽 (2001). 影响发根农杆菌对结野葛遗传转化效率的因素. 应用与环境生物学报 7, 474-477.
[28] Yu SH, Liu CF, Li L, Pan RC (2002). Pueraria lobata hairy root culture in vitro and isoflavone production. J Plant Physiol Mol Biol 28, 281-286. (in Chinese)
  于树宏, 刘传飞, 李玲, 潘瑞炽 (2002). 野葛毛状根离体培养与异黄酮生产. 植物生理与分子生物学学报 28, 281-286.
[29] Zhang CC (2011). Research on Agrobacterium rhizogenes-mediated Genetic Transformation in Cotton. Master's thesis. Beijing: Chinese Academy of Agricultural Sciences. pp. 1-73. (in Chinese)
  张程程 (2011). 发根农杆菌介导的棉花遗传转化的研究. 硕士论文. 北京: 中国农业科学院. pp. 1-73.
[30] Zhang LL, Zhang J, Lü HL, Tan B, Wang W, Cheng J, Feng JC (2022). Establishment of leaf regeneration system for European plum. J Fruit Sci 39, 1945-1953. (in Chinese)
  张郎郎, 张洁, 吕虹霖, 谭彬, 王伟, 程钧, 冯建灿 (2022). 欧洲李叶片再生体系的建立. 果树学报 39, 1945-1953.
[31] Zhao XK, Sun H, Yang LY, Luo LJ, Liu PD (2023). Establishment of the genetic transformation system of Stylosanthes gracilis mediated with Agrobacterium rhizogenes. Acta Agrestia Sin 31, 581-586. (in Chinese)
  赵兴坤, 孙昊, 杨丽云, 罗丽娟, 刘攀道 (2023). 发根农杆菌介导的细茎柱花草毛状根转化体系的建立. 草地学报 31, 581-586.
[32] Zhu HY (2005). Study on Establishment of Regeneration System In Vitro and Transgenic Receptor System in Gerbera (Gerbera jamesonii Bolus). Master's thesis. Yangling: Northwest A&F University. pp. 1-64. (in Chinese)
  祝红艺 (2005). 非洲菊(Gerbera jamesonii Bolus)再生体系及遗传转化受体系统建立的研究. 硕士论文. 杨凌: 西北农林科技大学. pp. 1-64.
[33] Zhu WF, Li JL, Meng XW, Zhang PZ, Wu WT, Liu RH (2021). Research advances in chemical constituents and pharmacological activities of Pueraria genus. China J Chin Mater Med 46, 1311-1325. (in Chinese)
  朱卫丰, 李佳莉, 孟晓伟, 张普照, 吴文婷, 刘荣华 (2021). 葛属植物的化学成分及药理活性研究进展. 中国中药杂志 46, 1311-1325.
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