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Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis

文献类型： 外文期刊

作者： Udayabhanu, Jinu 1 ; Huang, Tiandai 1 ; Xin, Shichao 1 ; Cheng, Jing 1 ; Hua, Yuwei 1 ; Huang, Huasun 1 ;

作者机构： 1.Chinese Acad Trop Agr Sci, State Key Lab Incubat Base Cultivat & Physiol Tro, Key Lab Biol & Genet Resources Rubber Tree, Rubber Res Inst,Minist Agr & Rural Affairs, Haikou 571101, Hainan, Peoples R China

2.Haikou Key Lab Trop Plant Seedling Innovat, Haikou 571101, Hainan, Peoples R China

关键词： Hevea brasiliensis; cotyledonary somatic embryos; Agrobacterium-mediated transformation; GUS staining; transmission electron microscopy

期刊名称：PLANTS-BASEL （ 影响因子：4.658； 五年影响因子：4.827 ）

ISSN：

年卷期： 2022 年 11 卷 8 期

页码：

收录情况： SCI

摘要： The recurring growth of bacterium in newly developed resistant cells and a minimal level of bacterial infection rate are the main limiting factors of Agrobacterium-mediated transformation experiments in Hevea brasiliensis. The current study aimed to optimize crucial factors of the transformation protocol in order to obtain an efficient transformation experimental model for Hevea using cotyledonary somatic embryos as explants. Transformation conditions such as antibiotic concentration, preculture duration, Agrobacterium concentration, sonication and cocultivation conditions were analyzed using the binary vector pCAMBIA2301. Transient transformation was confirmed by GUS histochemical staining. The best transformation efficiency was observed when the explants were not cultured on a preculture medium that contained acetosyringone at a level of 100 mu M. The best results were obtained using a bacterial density of 0.45 at OD 600 nm, 50 s of sonication of explants in a bacterial liquid culture and a total incubation time of 18 min in the same bacterial suspension. Transmission electron microscopical analysis confirmed the impacts of sonication on bacterial infection efficiency. Cocultivation conditions of 22 degrees C and 84 h of darkness were optimal for the transfer of T-DNA. Agrobacterium was eliminated with 500 mg/L of timentin, and the selection of transformants was performed using 100 mg/L of kanamycin in the selection medium. The presence of transgene was confirmed in the resistant embryos by polymerase chain reaction (PCR). The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest into the Hevea genome for the breeding of this economically important plant species in the future.

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