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An optimized CRISPRCas9-based gene editing system for efficiently generating homozygous edited plants in rubber tree ( Hevea brasiliensis) )

文献类型： 外文期刊

作者： Yang, Xianfeng 1 ; Lin, Qiufei 1 ; Udayabhanu, Jinu 1 ; Hua, Yuwei 1 ; Dai, Xuemei 1 ; Xin, Shichao 1 ; Wang, Xiaoyi 1 ; Huang, Huasun 1 ; Huang, Tiandai 1 ;

作者机构： 1.Chinese Acad Trop Agr Sci, Rubber Res Inst, Natl Key Lab Trop Crop Breeding, Key Lab Biol & Genet Resources Rubber Tree, Hainan 571101, Peoples R China

2.Chinese Acad Trop Agr Sci, Sanya Res Inst, Sanya 572025, Hainan, Peoples R China

3.Haikou Key Lab Innovat Seedlings Trop Plants, Haikou 571101, Peoples R China

关键词： Rubber tree; CRISPR/Cas9; Genetic transformation; Gene editing; Embryo; Homozygous

期刊名称：INDUSTRIAL CROPS AND PRODUCTS （ 影响因子：6.2； 五年影响因子：6.2 ）

ISSN： 0926-6690

年卷期： 2024 年 222 卷

页码：

收录情况： SCI

摘要： Previously, we have realized CRISPR/Cas9-RNP and plasmid-mediated genome editing in the rubber tree protoplast by transient transformation, but it is a challenge to generate editing plants hindered by low stable genetic transformation efficiency. In the present study, we firstly reported genome-edited rubber trees with expected phenotype, related works including demonstrated that 10 mg/L hygromycin is suitable for embryo screening, based on this foundation, CRISPR vector targeting the Phytoene desaturase gene ( HbPDS ) was transformed into embryos, the results showed that more than 90% of T0 edited embryos are chimeric, and the editing efficiency is 3.2%. In order to improve the homozygous embryo ratio, a T0 embryo with 9.8% edited cells was sliced into 33 small pieces for one more cycle embryogenesis to produce T1 generation embryos, it turns out the editing efficiency has been improved to nearly 90%, and 50% of T1 embryos were homozygous. At last, 8 plantlets were regenerated from the 29 T1 generation edited embryos, 4 chimeric and 4 homozygous albino plantlets, respectively. Next-generation sequencing demonstrated that, the threshold for the proportion of edited cells displaying expected albino phenotype is between 70 % and 85 %. Additionally, Tail-PCR' results, combined the different genotypes considered, the four homozygous plantlets can be confirmed as independently derived from different single transformed cells. Overall, this study presents a significant advancement in transgenic and gene editing for rubber trees, will be conducive to gene functional validation and accelerate the molecular breeding process of rubber tree.

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