Establishment of an Agrobacterium rhizogenes-mediated Genetic Transformation System in Crossostephium chinense

doi:10.11983/CBB25165

Abstract

Abstract: INTRODUCTION: Plant genetic transformation systems serve as fundamental technical platforms for functional genomics research and molecular breeding. Crossostephium chinense, a valuable medicinal-ornamental species within the Asteraceae family, is characterized by its distinctive silvery-white foliage and exhibits multiple stress-resistant traits, including salt tolerance, drought resistance, and insect resistance. However, the absence of an efficient genetic transformation system has significantly impeded progress in functional gene studies and molecular breeding of this species. This study reports the first establishment of an Agrobacterium rhizogenes-mediated genetic transformation system for C. chinense, aiming to overcome this technical barrier. RATIONALE: Agrobacterium rhizogenes-mediated transformation has been widely adopted in diverse plant species due to its operational simplicity and short experimental cycle. Nevertheless, its application in Asteraceae plants, particularly in C. chinense, remains largely unexplored. In this study, we innovatively integrated the visual reporter system RUBY with developmental regulators Wus2 and IPT. A systematic comparison was conducted between tissue culture and non-tissue culture approaches, and the transformation efficiencies of different explant types—including rootless seedlings with attached cotyledons and stem segments—were evaluated. This work provides a comprehensive technical framework for establishing an efficient and stable genetic transformation system in C. chinense. RESULTS: Under tissue culture conditions, rootless seedlings with cotyledons attached exhibited superior transformation efficiency as explants, achieving a positive hairy root induction rate of 38.95% and successfully yielding transgenic regenerated plants. This efficiency was significantly higher than that of stem segment explants (17.45%). In non-tissue culture settings, the cut-dip-budding (CDB) method resulted in a (18.8±5.46)% hairy root induction rate using stem segments. Notably, the application of Wus2-IPT developmental regulators effectively promoted direct shoot organogenesis independent of plant growth regulators, thereby streamlining the regeneration process. CONCLUSION: This study successfully establishes the first efficient Agrobacterium rhizogenes-mediated genetic transformation system for C. chinense, identifying tissue-cultured rootless seedlings with attached cotyledons as the optimal explant type. The developed system not only provides a critical tool for gene functional analysis and molecular breeding in C. chinense but also offers a valuable reference for genetic transformation studies in other Asteraceae species. This breakthrough is expected to significantly accelerate the exploitation of elite gene resources and enhance germplasm innovation in C. chinense.

Key words: Crossostephium chinense, Agrobacterium rhizogenes, hairy roots, genetic transformation

Mengran Hao, Zhiyong Liu, Yanyan Sun, Mingxiu Sun, Jing Zhang, Aiping Song, Haibin Wang, Fadi Chen, Zhenxing Wang. Establishment of an Agrobacterium rhizogenes-mediated Genetic Transformation System in Crossostephium chinense[J]. Chinese Bulletin of Botany, DOI: 10.11983/CBB25165.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB25165

References

[1]陈雪鹃, 吴珏, 李雪珂, 孙明, 张启翔(2012).芙蓉菊组培快繁技术的研究..中南林业科技大学报, 32:100-104+127+151. [2]黄有军, 夏国华, 郑炳松, 黄坚钦, 邵香君(2007).芙蓉菊盐胁迫下的生长表现和生理响应..江西农业大学学报, /:389-392+408. [3]马曦, 张金睿, 庄红梅, 陈巍, 张蜜, 袁晓琪, 易小芳, 王聪聪, 王海云, 王燕(2025).发根农杆菌介导的芜菁高效遗传转化体系建立.园艺学报, 52:1389-1398. [4]吴琦, 王亚君, 赵琳, 杨秀伟, 马爱进(2017).芙蓉菊化学成分及其生物活性研究进展..药物评价研究, 40:1821-1827. [5]向太和, 王琳, 蒋欢, 田璟鸾(2011).发根农杆菌K599对菊花活体转化及其高效再生.园艺学报, 38:1365-1370. [6]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. [7]亓帅, 付建新, 王翊, 杨立文, 戴思兰(2014).甘菊下胚轴遗传转化体系的建立..分子植物育种, 12:356-362. [8]Bahramnejad B, Naji M, Bose R, Jha S(2019).A critical review on use of Agrobacterium rhizogenes and their associated binary vectors for plant transformation.Biotechnology Advances, 37:107405--. [9]Cao X, Xie H, Song M, Zhao L, Liu H, Li G, Zhu JK(2024).Simple method for transformation and gene editing in medicinal plants.Journal of Integrative Plant Biology, 66:17-19. [10]Clough SJ, Bent AF(1998).Floral dip: a simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana.The plant journal, 16:735-743. [11]Fan Y, Xu F, Zhou H, Liu X, Yang X, Weng K, Lyu S(2020).A fast, simple, high efficient and one-step generation of composite cucumber plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation.Plant Cell, Tissue and Organ Culture (PCTOC), 141:207-216. [12]Gu Y, Qin Y, Hua S, Shi J, Yang C, Peng Y, Dong W(2025).Novel methods for genetic transformation of watermelon (Citrullus lanatus) without tissue culture via Agrobacterium rhizogenes.Molecular Breeding, 45:22--. [13]Ikeuchi M, Shibata M, Rymen B, Iwase A, Bagman AM, Watt L(2018).A gene regulatory network for cellular reprogramming in plant regeneration.Plant and Cell Physiology, 59:770-782. [14]Kiryushkin AS, Ilina EL, Guseva ED, Pawlowski K, Demchenko KN(2021).Hairy CRISPR: genome editing in plants using hairy root transformation.Plants, 11:51--. [15]Krenek P, Samajova O, Luptovciak I, Doskocilova A, Komis G, Samaj, J(2015).Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications.Biotechnology Advances, 33:1024-1042. [16]Kumar S, Prakash S, Kumari P, Sanan-Mishra N(2025).A robust in-vitro and ex-vitro Agrobacterium rhizogenes-mediated hairy root transformation system in mungbean for efficient visual screening of transformants using the RUBY reporter.BMC Plant Biology, 25:724--. [17]Liu L, Qu JH, Wang CY, Liu M, Zhang CM, Zhang XY, GuO C, Wu CG, Yang GD, Huang JG, Yan K, Shu HR, Zheng CC, Zhang SZ(2024).An efficient genetic transformation system mediated by Rhizobium rhizogenes in fruit trees based on the transgenic hairy root to shoot conversion.Plant Biotechnology Journal, 22:2093-2103. [18]Liu YH, Cai XQ, Ning K, Xu P(2024).An Efficient Hairy Root Transformation Method for Common Bean based on Petiole Explants.Legume Research, 47:8-13. [19]Li XX, Gao YH, Ma HW, Wang YQ, Bu T, Yin W, Xia XL, Wang HL(2025).Non-tissue culture genetic modifications for plant improvement.Plant Molecular Biology, 115:67--. [20]Li X, Bu F, Zhang M, Li Z, Zhang Y, Chen H, Zhang Y(2025).Enhancing nature' s palette through the epigenetic breeding of flower color in chrysanthemum.New Phytologist, 245:2117-2132. [21]Lowe K, Wu E, Wang N, Hoerster G, Hastings C, Cho MJ, Gordon-Kamm W(2016).Morphogenic regulators Baby boom and Wuschel improve monocot transformation.The Plant Cell, 28:1998-2015. [22]Maher MF, Nasti RA, Vollbrecht M, Starker CG, Clark MD, Voytas DF(2020).Plant gene editing through de novo induction of meristems.Nature biotechnology, 38:84-89. [23]Mayo KJ, Gonzales BJ, Mason HS(2006).Genetic transformation of tobacco NT1 cells with Agrobacterium tumefaciens.Nature protocols, 1:1105-1111. [24]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 Communications, 5:/-/. [25]Sarkar S, Ghosh I, Roychowdhury D, Jha S(2018).The effects of rol genes of Agrobacterium rhizogenes on morphogenesis and secondary metabolite accumulation in medicinal plants. In Biotechnological approaches for medicinal and aromatic plants: Conservation, genetic improvement and utilization.Singapore: Springer Singapore, /:27-51. [26]Taak P, Tiwari S, Koul B(2020).Optimization of regeneration and Agrobacterium-mediated transformation of Stevia (Stevia rebaudiana Bertoni): a commercially important natural sweetener plant.Scientific reports, 10:16224--. [27]Tepfer D(1984).Genetic transformation of several species of higher plants by Agrobacterium rhizogenes: phenotypic consequences and sexual transmission of the transformed genotype and phenotype.Cell, 37:959-967. [28]Wang H, Cheng K, Li T, Lan X, Shen L, Zhao H, Lü S(2024).A Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation method of Idesia polycarpa and the generation of transgenic plants.Plants, 13:1791--. [29]Wang J, Liu S, Ma H, Tao Y, Feng S, Gong S, Zhang J, Zhou A(2020).Reliable and efficient Agrobacterium tumefaciens-mediated genetic transformation of Dianthus spiculifolius.Horticultural Plant Journal, 66:1998-204. [30]Wang Y, Yang X, Wang W, Wang Y, Chen X, Wu H, Gao ZY, Xu HH, Liu TK, Li Y, Xiao D, Liu WS, Hou XL, Wu H, Zhang CW(2025).Efficient genetic transformation and gene editing of Chinese cabbage using Agrobacterium rhizogenes.Plant Physiology, 1972:kiae543--. [31]Xin T, Tian H, Ma Y, Wang S, Yang L, LiX, Yang X(2022).Targeted creation of new mutants with compact plant architecture using CRISPR/Cas9 genome editing by an optimized genetic transformation procedure in cucurbit plants.Horticulture research, 9:uhab086--. [32]Yi X, Wang C, Yuan X, Zhang M, Zhang C, Qin T, Wang Y(2024).Exploring an economic and highly efficient genetic transformation and genome‐editing system for radish through developmental regulators and visible reporter.The Plant Journal, 120:1682-1692. [33].., :-. [34]Yi XF, Wang CC, Yuan XQ, Zhang M, Zhang CW, Qin TJ, Wang HY, Xu L, Liu LW, Wang Y(2024).Exploring an economic and highly efficient genetic transformation and genome‐editing system for radish through developmental regulators and visible reporter.The Plant Journal120(4), 1682-1692., :-. [35]Yu J, Deng S, Huang H, Mo J, Xu ZF, Wang Y(2023).Exploring the potential applications of the noninvasive reporter gene RUBY in plant genetic transformation.Forests, 141:637--. [36]Wang D, Zhong Y, Feng B, Qi X, Yan T, Liu J, Chen S(2023).The RUBY reporter enables efficient haploid identification in maize and tomato.Plant Biotechnology Journal, 21:1707-1715. [37]Zhao X, Meng Z, Wang Y, Chen W, Sun C, Cui B, Cui J, Yu M, Zeng Z, Guo S, Luo D, Cheng JQ, Zhang R, Cui H(2017).Pollen magnetofection for genetic modification with magnetic nanoparticles as gene carriers.Nature plants, 3:956-964. [38]Zhong J, Guo YH, Chen JT, Cao H, Liu M, Guo ZY, Zhang QX, Sun M(2021).Volatiles inheriting from Crossostephium chinense act as repellent weapons against aphids in Chrysanthemum lavandulifolium cultivars.Industrial Crops and Products, 166:--.