CRISPR-Cas9 Mediated Gene Editing Platform Through Callus-to-Plant Regeneration and Functional Analysis of DoALA4─DoALA6 in Dendrobium officinale.

Dendrobium orchids are well known for their great horticultural and medicinal values; however, the CRISPR/Cas9 gene editing system for Dendrobium species still needs to be improved. Therefore, this study aims to establish a CRISPR/Cas9-based functional validation system using Dendrobium officinale as a model species for the Dendrobium genus and to validate the DoALA4─DoALA6 genes, which may relate to growth and disease resistance. We first conducted a bioinformatics analysis of the P-type ATPase gene family in D. officinale, revealing the evolutionary diversity of P-type ATPase genes in orchids. Second, we inserted the GFP gene into the vector of CRISPR/Cas9 gene editing system to enhance the selection efficiency of genome-edited plants. Comparative analysis showed that different explants exhibited varying transformation efficiencies, ranging from 5% to 46.2%. Considering the regeneration capability, survival rate and gene editing efficiency, we selected callus as the transformation explant. Third, we used this editing system to generate DoALA4─DoALA6 mutants. Phenotypic observations of the mutants and inoculation of D. officinale with Sclerotium rolfsii indicated that DoALA4─DoALA6 are crucial for the growth of D. officinale and its resistance to southern blight disease. This efficient and stable CRISPR/Cas9 platform offers a foundation for further gene studies and Dendrobium breeding.