Oliveira, M.L.,Thomson, J.G. ,Stover, E.
Maria.oliveira@ars.usda.gov
U.S. Horticultural Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, 2001 South Rock Road, Fort Pierce, FL 34945, USA
Conventional breeding methods to incorporate resistance in citrus are very slow, due to extended juvenility from seedling trees and multiple generations needed to incorporate resistance from distant relatives. Use of transgenic methods may provide disease resistance in less time. Published protocols have relied on somatic embryogenesis from nucellar calli or from protoplast-derived cultures, or more commonly, shoot organogenesis from juvenile epicotyl or internodal stem segments. However, these A. tumefaciens-based systems typically have low transformation efficiencies and low transgenic plant recovery. A number of physiological factors associated with source tissues influence in vitro development and we are exploring alternative explants and treatments, in comparison to conventional methods using juvenile epicotyl stem segments. In Mexican lime, cotyledons provided an improvement in regeneration capacity, longer shoots and more robust rooting, with a very simple transformation procedure, reducing the time required for transgenic plant-recovery. Greenhouse internode explants treated with macerating enzymes prior to Agrobacterium co-cultivation are showing promise in transformation of citrus. In order to assess the effects of macerating enzymes, we examined the effect of different macerating enzymes on the transformation efficiency of Carrizo citrange and Tango mandarin. PCR analyses further confirmed the stable integration of the transgene. Transformation of citrus greenhouse internodes is therefore facilitated when explants are subjected to enzyme treatments prior to Agrobacterium infection.This procedure may be broadly applicable, permitting efficient recovery of other citrus genotypes recalcitrant to in vitro transformation.