Genetic transformation of Cymbidium orchid by particle bombardment

Abstract

 A protocol is presented for genetically engineering Cymbidium orchid using particle bombardment. This protocol enabled the routine transformation of orchid plants that were previously difficult to transform. Liquid culture was used to generate a large number of protocorm-like bodies (PLBs) to be bombarded and to promote continued development of the bombarded meristematic tissue. Plasmid DNA (pKH200) carrying the GUS-INT and NPTII genes flanked by tobacco matrix attachment regions was introduced into the meristematic cells of PLBs by particle acceleration. The transformed PLBs were proliferated and selected for kanamycin resistance conferred by the introduced NPTII gene. Shoot regeneration was then induced from the kanamycin-resistant PLBs, and transgenic plantlets were produced. Both the kanamycin-resistant PLBs and regenerated shoots expressed the GUS-INT gene. The presence of the introduced gene in the transformed orchid plants was confirmed by PCR analysis, sequencing and Southern blot analysis of the PCR product. The recovered transgenic plants were established in soil and acclimatized in the greenhouse.