Agrobacterium-mediated transformation facillitates the CRISPR/Cas9 genome editing system in Dendrobium macrophyllum A. Rich orchid

As an ornamental plant, orchid has high economic value for its appearance. Some methods can be used to improve the orchid performance, for instance creating variegated plants that have green- and white-sectored leaves by mutating the VARIEGATED 2 (VAR2) gene using CRISPR/Cas9 Genome editing system. The objective of this study was to determine whether CRISPR/Cas9 could be introduced to the Dendrobium macrophyllum orchid protocorm by using Agrobacterium tumefaciens-mediated transformation method. Genetic transformation using A.tumefaciens is one of the inexpensive and reproducible molecular breeding approaches. The construct of T-DNA Ubi :: Cas9 :: U3 :: VAR2/ pRGEB32 in the A. tumefaciens strain EHA 105 was infected into 8-weeks old orchid protocorms, then the protocorms were maintained in Vacin and Went culture medium with addition of 6 mg/L-1 Hygromycin for antibiotic selection of transformants for 4 weeks. The results showed that the highest transformation efficiency (0.66%) was obtained in the 15- minute infection period although 0.43% in 30 minutes and 0.23% in 45 minutes. This result was confirmed by PCR analysis that several genes were amplified in the genome of D. macrophyllum such as, Cas9 (402 bp), HPT (545 bp), VAR2 (723 bp) and trnL-F (1200 bp) as internal control. Sequence analysis showed that there was a mutation in the form of substitution at the target site.