A Rapid In Planta Genetic Transformation System for MicroTom Tomato

doi:10.11983/CBB25061

Abstract

Abstract: MicroTom tomato (Solanum lycopersicum cv. ‘MicroTom’) is widely used for functional characterization due to its short life cycle and clear genetic background. However, the traditional genetic transformation system of MicroTom based on tissue culture is constrained by low efficiency, long transformation period and complex operation. Based on the somatic cell reprogramming mechanism triggered by wound signaling, this study established an efficient and rapid in planta genetic transformation system for MicroTom tomato. Wound hypocotyl were created by removing apical cotyledons and true leaves from two-week-old seedlings, followed by direct inoculation with Agrobacterium tumefaciens carrying binary vector pCY-H05251-VcDAD2-EGFP (enhanced green fluorescent protein) to induce shoot regeneration. Results showed that a 28.6% PCR-based positive efficiency of regenerated shoots in the T0 generation, with seeds derived within 4–5 months post inoculation. Antibiotic and fluorescence screening revealed approximate 73.5% lines in the T1-generation expressed the fused EGFP protein. Compared to conventional tissue culture-dependent transformation systems, this protocol enhanced transformation efficiency, shortened transformation period, and simplified sterile operational procedures. The in planta genetic transformation system provides a robust platform for functional genomics studies, and significantly lowers technical barriers in tomato genetic breeding.

Key words: MicroTom tomato, genetic transformation, in planta, gene function, molecular breeding

Weikun Wang, Wenqian Fu, Qiumei Hong, Yang Zheng, Jiahui Yu, Yan Wang, Liangmiao Liu, Li Yang, Changchun Wang, Weidong Guo. A Rapid In Planta Genetic Transformation System for MicroTom Tomato[J]. Chinese Bulletin of Botany, DOI: 10.11983/CBB25061.

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.chinbullbotany.com/EN/10.11983/CBB25061

References

[1]Ahmed S, Wan Azizan WAS, Akhond MAY, Juraimi AS, Ismail SI, Ahmed R, Md Hatta MA(2023).Optimization of in vitro regeneration protocol of tomato cv.MT1 for genetic transformation..Horticulturae 9, 800., 9:800-. [2]Anjanappa RB, Gruissem W(2021).Current progress and challenges in crop genetic transformation.J Plant Physiol, 261:153411-. [3]Bailey-Serres J, Parker JE, Ainsworth EA, Oldroyd GED, Schroeder JI(2019).Genetic strategies for improving crop yields.Nature, 575:109-118. [4]Barton KA, Binns AN, Matzke AJM, Chilton MDl(1983).Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA,and transmission of T-DNA to R1 progeny.Cell, 32:1033-1043. [5]Belanger JG, Copley TR, Hoyos-Villegas V, Charron JB, O’Donoughue L(2024).A comprehensive review of in planta stable transformation strategies.Plant Methods, 20:79-. [6]Chu P, Agapito-Tenfen SZ(2022).Unintended genomic outcomes in current and next generation GM techniques: a systematic review.Plants-basel, 11:2997-. [7]Cruz-Mendivil A, Rivera-Lopez J, German-Baez LJ, Lopez-Meyer M, Hernandez-Verdugo S, Lopez-Valenzuela JA, Reyes-Moreno C, Valdez-Ortiz A(2011).A simple and efficient protocol for plant regeneration and genetic transformation of tomato cv MicroTom for leaf explants.Hortic Sci, 46:1655-1660. [8]Dan YH, Fei ZJ, Rothan C(2007).MicroTom–a new model system for plant genomics.Genes Genom, 1:167-179. [9]Dan YH, Yan H, Munyikwa T, Dong J, Zhang YL, Armstrong CL(2006).Micro-Tom–a high-throughput model transformation system for functional genomics.Plant Cell Rep, 25:432-441. [10]Guo M, Zhang YL, Meng ZJ, Jiang J(2012).Optimization of factors affecting Agrobacterium-mediated transformation of MicroTom tomatoes.Genet Mol Res, 11:661-671. [11]胡丹玲, 孙永伟(2024).病毒介导的植物基因组编辑技术研究进展.植物学报, 59:452-462. [12]Khuong TTH, Crete P, Robaglia C, Caffarri S(2013).Optimisation of tomato Micro-Tom regeneration and selection on glufosinateBasta and dependency of gene silencing on transgene copy number.Plant Cell Rep, 32:1441-1454. [13]李君, 李岩, 刘德虎(2011).植物遗传转化的替代方法及研究进展.生物技术通报, 27:31-36. [14]Li Y, Chen Y, Zhou L, You SJ, Deng H, Chen Y, Alseekh S, Yuan Y, Fu R, Zhang ZX, Su D, Fernie AR, Bouzayen M, Ma T, Liu MC, Zhang Y(2020).Micro-Tom metabolic network: rewiring tomato metabolic regulatory network throughout the growth cycle.Mol Plant, 13:1203-1218. [15]刘小花, 张岚岚, 朱长青, 陈昆松, 徐昌杰(2008).番茄矮化微型机制及其在植物功能基因组学研究中的应用.遗传, 30:1257-1264. [16]Livak KJ, Schmittgen TD(2001).Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods, 25:402-408. [17]Lu JH, Li SS, Deng S, Wang MG, Wu YH, Li M, Dong JS, Lu SH, Su CL, Li GF, Lang ZB, Zhu JK(2024).A method of genetic transformation and gene editing of succulents without tissue culture.Plant Biotechnol J, 22:1981-1988. [18]Lu Y, Zhu HL(2022).The regulation of nutrient and flavor metabolism in tomato fruit.Vegetable Res, 2:5-. [19]McCormick S, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley R(1986).Leaf disc transformation of cultivated tomato (L.esculentum) using Agrobacterium tumefaciens. Plant Cell Rep, 5:81-84. [20]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-. [21]Phillips RL, Kaeppler SM, Olhoft P(1994).Genetic instability of plant tissue cultures: breakdown of normal controls.Proc Natl Acad Sci USA, 91:5222-5226. [22]Toth M, Toth ZG, Fekete S, Szabo Z, Toth Z(2020).Improved and highly efficient Agrobacterium rhizogenes-mediated genetic transformation protocol: efficient tools for functional analysis of root-specific resistance genes for Solanum lycopersicum cv. Micro-Tom..Sustainability, 14:6525-. [23]Wu XY, Zhang Y, Jiang X, Ma TS, Guo YT, Wu XX, Guo GS, Cheng XF(2024).Considerations in engineering viral vectors for genome editing in plants.Virology, 589:109922-. [24]熊发前, 刘俊仙, 刘菁, 贺梁琼, 蒋菁, 唐秀梅, 黄志鹏, 吴海宁, 钟瑞春, 韩柱强, 唐荣华(2019).花生的五种改良提取方法的比较分析及其应用.分子植物育种, 17:2207-2216. [25]Yang WT, Zhai HW, Wu FM, Deng L, Chao Y, Meng XW, Chen Q, Liu CH, Bie XM, Sun CL, Yu Y, Zhang XF, Zhang XY, Chang ZQ, Xue M, Zhao YJ, Meng XB, Li BS, Zhang XS, Zhang DJ, Zhao XY, Gao CX, Li JY, Li CY(2024).Peptide REF1 is a local wound signal promoting.Cell, 187:3024-3038. [26]Yasmeen A, Mirza B, Inayatullah S(2009).In planta transformation of tomato.Plant Mol Biol Rep, 27:20-28. [27]余柯达, 叶美娟, 陈文荣, 朱凯丽, 张常晶, 郭卫东(2016).蓝莓组织提取方法的研究.浙江师范大学学报自然科学版, 39:60-64. [28]张庆祝, 韩天富(2004).植物非组培遗传转化方法研究的进展.分子植物育种, 2:85-91. [29]郑立峰(2020).基于番茄高效再生体系的柚、基因转化体系的建立.硕士论文. 福州, 福建农林大学.,:32-33.