Gene Transfer Systems of Dendrobium (Orchidaceae)

Abstract

To enable future molecular breeding in orchids, gene transfer systems suitable for Dendrobhim orchids were studied. Five gene transfer methods explored with Dendrnhhim were microprojectile bombardment, Agrobacterhim-mediated transformation, protocorm electroinjection, seed imbibition, and pollen tube-mediated DNA delivery. Results indicate bombardment to be an effective method. Using a Dupont gunpowder-driven apparatus, transient e?q)ression averaged 38.3% for Dendrobhim UH800 protocorm-like bodies (PLB) and 15% for Dendrobhim UH44 etiolated shoots (ES); stable transformation rates averaged 11.7% for UH800 PLB and 0.17% for UH44 ES, bombarded with pBI426 and pBI121, respectively. Molecular data suggest plasmid DNA fragmentation in 5 of 8 UH800 transgenic plants, for three had both neo and gusA and the other five only had the neo fragment. Six parameters in micro projectile bombardments with a Bio-Rad helium-driven apparatus were tested: helium gas pressures, plasmid constructs, size and sources of gold particles, target plant genotype and type of tissue. Transient GUS expression rates of up to 80% on Dendrobium M61 and 100% on UH44 PLB were obtained when bombarded with pBI426 on 1.6-pm Bio-Rad gold particles under 1100 psi helium gas pressure. Gene transfer mediated by Agrobacterium tumefaciens has potential for Dendrobhim Southern hybridization analysis confirmed the presence of the introduced hygromycin phosphotransferase gene in the genomic DNA pooled from green proliferating PLB after 14.5 months of selection following cocultivation experiments with Agrobacterium LBA4301 (pUCD2614 + pUCD2716). LUX expression studies using LBA4301 (pTiC58 + pUCDil87) demonstrated a strong vir-inducing ability of Dendrobium PLB exudates and extracts. Electroinjection, giving transient expression rate of 10 to 16% and one transformant per approximately 50 protocorms, is also suitable for Dendrohhim Delivery of transgenes by seed imbibition or by the poUen-tube pathway was less effective, with some transient GUS expression and visual differences but no recovery of stable transformants. Tissue culture responses for Dendrobium ES and green leaf sections were evaluated for utility in transformation protocols.