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

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. • We found the optimal antibiotic for rubber tree embryo transformation is hygromycin. • We demonstrated that a constant 10 mg/L hygromycin is the best selection strategy. • To our knowledge, this study produced the highest genetic transformation efficiency in rubber tree. • It has been proven that most of the transformed T0 embryos are chimeric, and we overcome this problem. • This is the first gene editing rubber tree with expected phenotype reported publicly.