Establishment Of Transformation System For Diploid Potato And The Study Of Transgenic With S-RNase Gene Editing

Potato is the most important tuber crop.Cultivated potatoes are mainly tetraploid,of which the improvement is hampered by tetrasomic genetics and clonal propagation.Thus,more and more scientists are appealing to re-domesticate potato into an inbred line-based crop propagated by seeds at the diploid level.However,there are self-incompatibility of diploid potatoes,so that some excellent genes can not communicate,it is a major problem in diploid breeding.And plant genetic engineering technology can be through direct gene mutation to break the self-crossing disorder to achieve self-affinity.Thus providing raw materials for diploid breeding.In this study,we using the RNA-seq data generated from the style RNA of diploid clone S.phureja CIP 703541,We carried out de-novo assembly of RNA-seq data using Trinity?v2.2.0?.we Taking the S-RNase sequence from potato reference genome DM as the query,and aquired two new S-RNase.Base-on these sequence,we constructed a CRISPR/cas9 expression vector with NPT II gene and GFP gene.Use this gene editing system,we established the agrobacterium-mediated diploid potato transformation system,and knockout of S-RNase.Including the pre-culture time,the concentration of plant hormones in the regeneration stage,the Kan screening concentration of callus regeneration and the Kan screening concentration of the resistant shoots.The results showed that:?1?Pre-culture time was the most suitable for infection.?2?The plant hormone concentration in the regeneration stage was ZT:2.0 mg·L^-1+NAA 0.01 mg·L^-1 until buding.?3?Kan suitable screening pressure of100 mg·L^-1,rooting anti-sieve concentration of 50 mg·L^-1.?4?obtained by PCR amplification of the target gene mutation site Sanger sequencing,confirmed that the mutation has occurred.The genetic transformation system of diploid potato with CRISPR/cas 9 gene knockout was established,and the regenerated plants with S-RNase knockdown were successfully obtained,and the editing efficiency was13.5%.?5?obtained self-compatible diploid potato.