Title: Effect of Agrobacterium culture and inoculation density on transformation efficiency of a citrange (Citrus reticulata x Poncirus trifoliata). Authors
Submitted to: Journal of Forestry and Horticulture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 16, 2009
Publication Date: March 1, 2010
Citation: Niedz, R.P., Evens, T.J. 2010. Effect of Agrobacterium culture and inoculation density on transformation efficiency of a citrange (Citrus reticulata x Poncirus trifoliata). Journal of Forestry and Horticulture. 2(3):30-37. Interpretive Summary: Genetic engineering of plants results in the changing of one trait (e.g., resistance to a particular disease or pest) in an otherwise acceptable variety. However, plant species vary widely in how easy it is to produce a genetically engineered plant. Some important citrus types such as sweet oranges, grapefruit, and tangerines are not easy to genetically engineer. Understanding how to improve the efficiency of engineering these citrus types is important for producing the relatively large numbers of genetically engineered citrus trees required when testing the effects of a gene. The first step in genetic engineering of plants is to inoculate plant pieces with Agrobacterium, a bacterium that transfers the gene of interest into the plant. This study examined the effects of the stage of the Agrobacterium growth cycle and the concentration of the Agrobacterium in the solution used to treat the citrus plants on transformation. Transformation was determined on an important commercial citrus rootstock variety using the GUS gene, a gene that is easily scored in the laboratory.
Technical Abstract: The effect of Agrobacterium growth phase and density on transformation of citrus rootstock US-812 (Citrus reticulata x Poncirus trifoliata) epicotyl explants was determined. In the first experiment, Agrobacterium EHA105 containing pBINGUSint was grown in YEP medium to an OD600 of 1 and glycerol stocks made and stored at -80C. The three factors incubation time (18 to 24 hours), stock dilution (1/10, 1/100, 1/1000), and temperature (26C or 28C) were simultaneously varied and a response surface for OD600 generated for growth in baffled and unbaffled flasks. The resulting polynomial models for both flask types were highly significant with R2 values 0.98 and were suitable for predicting growth throughout the design space. In a second set of experiments, transformation efficiency of juvenile epicotyl explants was assessed from treatments in a 2x2 factorial arrangement of Agrobacterium culture OD600 (0.1 or 1) and inoculation density OD600 (0.1 or 1) using the GUS reporter gene. No significant differences were detected.