Effect of Agrobacterium culture and inoculation density on transformation efficiency of a citrange (Citrus reticulata x Poncirus trifoliata)

Randall P. Niedz^* and Terence J. Evens

Agricultural Research Service, U. S. Horticultural Research Laboratory,2001 South Rock Road, Ft. Pierce, FL 34945-3030

*Corresponding author. E-mail: randall.niedz@ars.usda.gov. Tel: (772) 462-5919.

Fax: (772) 462-5961

Accepted 16 November, 2009

   Abstract

This study focused on the effect of two factors, initial culture density and subsequent inoculation density on the transformation efficiency of citrus rootstock US-812 (Citrus reticulata x Poncirus trifoliata) epicotyl explants.  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 -80°C.  The three factors incubation time (18 to 24 h), glycerol stock dilution (1/10, 1/100, 1/1000), and temperature (26 or 28°C) were simultaneously varied and a response surface for OD₆₀₀ was generated for culture density in baffled and unbaffled flasks.  The resulting polynomial models for both flask types were highly significant with R² values of 0.98; this means that the polynomial model can be used to specify the stock dilution, temperature, and flask type to achieve a target density in a given amount of time (accurate from 18 to 24 h).  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.  The polynomial model developed in the first experiment was used to determine the conditions to achieve each factorial treatment combination.  No significant differences in main or interactive effects were detected.

Keywords:  Citrus, genetic transformation, rootstocks, Agrobacterium, GUS.