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Previously, we achieved gene editing using CRISPR/Cas9-RNP and plasmid in the PEG-mediated protoplasts transient transformation of rubber tree, and by targeting the HbPDS gene, callus with albino phenotype were obtained, but no edited plants were regenerated because the technology of embryogenesis from callus is not yet mature. In order to obtain gene edited seedlings, we used the same HbPDS target as previous in the callus editing, but used somatic embryos as the transformation receptor instead of callus. After hygromycin resistance screening, 116 positive T0 generation embryos were selected through Cas9 gene PCR detection, following by next generation sequencing, five embryos were found to be edited at the target, accounting for 4.3% of PCR positive embryos. At last, two regenerated plants were obtained, both were chimeric because only partial albino leaves appeared in the plantlet. Sequencing of both albino and green parts revealed that gene editing had occurred in all samples, besides a homozygous biallelic mutation in one albino leaf, all other leaves exhibited chimeric mutations, with mutant sequences in albino parts accounting for 86% to 100% ratio, while green parts accounting for 66% to 69% ratio. This indicates that the mutation threshold inducing the expected phenotype in rubber tree CRISPR/Cas9 editing plants is higher than 69%, ranging from 70% to 85%, providing theoretical guidance for obtaining gene editing seedlings with expected phenotype in the future. Meanwhile, it is proven that nearly all the regenerated plantlets obtained from T0 generation somatic embryos are chimeric, thus T0 generation embryos are not suitable as regenerated materials, but also providing insights to improve the regeneration procedure by using T1 embryo to get homozygous seedlings in the future. This is the first report about gene editing plants in rubber tree, although they are chimeric, it still enhances the understanding of the function of CRISPR/Cas9 in rubber tree, laying the foundation for improving and applying gene editing technology in rubber tree.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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