Development of an agrobacterium-delivered crispr/cas9 for phalaenopsis amabilis (L.) blume genome editing system

CRISPR/Cas9-genome editing system has been developed and started to be used in plants. Phalaenopsis amabilis is one of the popular orchids with beautiful flowers, but flowering time is relatively long, therefore other unique characters need to be created, such as variegated pattern leaves due to chloroplast mutations. VARIEGATA2 (VAR2) gene is the responsible gene for maintaining a chloroplast activity that the mutation will produce a variegated pattern of leaf. The objectives of this research are to get the best method for Agrobacterium-delivered T-DNA containing CRISPR/Cas9 into P.amabilis protocorm and to analyze the mutant phenotype in transformant plants. The genetic transformation was carried out by using A.tumefaciens EHA105 carried T-DNA with UBI::Cas9::U3::VAR2 in pRGEB32, and transformant selected in NP+10 ppm hygromycin medium. T-DNA integration in transformant genomes was confirmed by PCR. The RHS color chart was used for recording plant color of transformant. The results showed transformation efficiency was 1.2. A color change from green to pale yellowish was detected in the transformants. PCR results showed 545 bp HPT, 402 bp Cas9, 723 bp VAR, and 1200 bp trnL-F were amplified from transformants. In conclusion, Agrobacterium-delivered CRISPR/Cas9 can be applied for genome editing in orchid, that is simple, inexpensive and innovative. © 2020 Elsevier B.V., All rights reserved.