Characterization of Agrobacterium-mediated co-transformation events in rice using green and red fluorescent proteins

文献类型： 外文期刊

第一作者： Li, Lihua

作者： Li, Lihua;Dai, Liangying;Tian, Xudan;Wang, Lanlan;Zhao, Jianhua;Zhou, Jie;He, Haiyan;Qu, Shaohong;Tian, Xudan;Zhao, Jianhua

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关键词： Agrobacterium; Co-transformation; GFP; RFP; Selectable marker; Rice

期刊名称：MOLECULAR BIOLOGY REPORTS （ 影响因子：2.8； 五年影响因子：2.7 ）

ISSN： 0301-4851

年卷期： 2022 年 49 卷 10 期

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收录情况： SCI

摘要： Background Biotechnologists seeking to develop marker-free transgenic plants have established co-transformation methods. For co-transformation using mixed Agrobacterium strains, the mix ratio of Agrobacterium strains and selection scheme may influence co-transformation frequency. This study used fluorescent GFP and RFP markers to compose different selection schemes for observation of the selective dynamics of transformed rice cells and to investigate the factors affecting co-transformation efficiency. Methods and results We utilized GFP and RFP markers in co-transformation and tested the combinations of an antibiotic-selectable vector (pGFP-HPT) and a single RFP vector (pRFP) and of two antibiotic-selectable vectors (pGFP-HPT and pRFP-HPT) in rice. The pGFP-HPT/pRFP combination resulted in 70.9% to 81.2% of co-transformation frequencies while lower frequencies (56.6% on average) were obtained with the pGFP-HPT/pRFP-HPT combination. Based on GFP/RFP segregation patterns, 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny, which simulated the selection process of marker-free transgenic plants that carry an actual gene of interest. Transgene expression levels in the rice lines varied as revealed by RT-PCR, and tandem-linked T-DNAs were detected in co-transformants, suggesting that transgene expression might be affected by duplicated T-DNA structures. Conclusion Co-transformation via mixed Agrobacterium strains is feasible, and approximately 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny. The pGFP-HPT/pRFP and the pGFP-HPT/pRFP-HPT vector combinations showed distinctive selective dynamics of transformed rice cells, suggesting that co-transformation efficiency depends on both vector system and selection scheme.

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