Factors affecting gene delivery by particle bombardment ofDendrobium orchids

Abstract

Five parameters were examined for their effect on transformation ofDendrobium tissues by microprojectile bombardment. The superpromoter in pBI426 produced at least 1.5 times as many transient transformants as the single cauliflower mosaic virus 35S promoter in pBI121 (37 to 69% vs. 0 to 44%) with dark and frequent GUS (β-glucuronidase) staining. Tissue, genotype, and type of microparticle significantly affected transient GUS activity. Higher expression was seen in protocormlike bodies and in hybrid UH44 compared to etiolated shoots and protocorms and to hybrids M61 and K1329-39. Microparticles of 1.6-µm Bio-Rad gold were more effective than 1.0-µm ASI gold. Transient GUS activity did not differ among protocormlike bodies bombarded using helium propellant pressures of 650, 900, or 1100 psi. Transgenic plants were recovered fromDendrobium UH800 protocormlike bodies bombarded with pBI426-coated, 1.1-µm tungsten particles using an early-model gunpowder-driven apparatus with an estimated stable transformation rate of 11.7%. One transgenic plant ofDendrobium UH44 was recovered from etiolated shoot explants bombarded with pBI121-coated, 1.1-µm tungsten particles using the Dupont PDS-1000 with a stable transformation rate of 0.17%. Positive selection results showed 100 to 200 mg·liter−1 kanamycin to be appropriate for regeneration of transgenic plants from protocormlike bodies, protocorms, and etiolated shoot explants over a 3- to 9.5-mo. period.