Factors affecting gene delivery by particle bombardment ofDendrobium orchids |
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Authors: | Guo-Ling Nan Adelheid R Kuehnle |
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Institution: | (1) Department of Horticulture, University of Hawaii, 3190 Maile Way, 96822-2279 Honolulu, Hawaii |
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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. |
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Keywords: | genetic engineering Biolistic? process stable transformation etiolated shoot protocorm protocormlike body |
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