Transformation of mature citrus

摘要

The invention relates to methods and compositions for genetic transformation of both juvenile and mature citrus. In some embodiments, the invention provides methods and compositions for genetic transformation of citrus using Rhizobia-mediated DNA delivery, and also methods of enhancing the frequency of genetic transformation of mature citrus by any DNA transfer method, including Sinorhizobium. Internodal stem sections prepared from epicotyls of citrus seedlings or freshly emerging shoots of mature citrus plants (e.g., first shoots from buds of mature plants following grafting onto rootstock or very young shoots of mature plants) are preconditioned for transformation by inducing callus formation on an artificial medium. All callus and any developing meristematic regions in immediately adjacent tissue are substantially or completely removed and the preconditioned explants are then transformed by Sinorhizobium or other known methods. Whole plants or shoots to be grafted onto rootstocks are subsequently regenerated from the transformed cells.

主权项

1. A method for transforming a mature citrus plant cell, comprising:
(a) placing stem sections comprising mature citrus plant cells on a tissue culture medium suitable for formation of callus tissue; (b) selecting only those stem sections forming callus; (c) removing resulting callus from said selected stem sections; (d) contacting one or more of the mature citrus plant cells from said selected stem sections having had the callus removed with an Agrobacterium spp. or Sinorhizobium spp. bacterium comprising:
(i) a first nucleic acid comprising a vir gene region of a Ti plasmid wherein the vir gene region acts to introduce a nucleic acid of interest into the plant cell in a VirD2-dependent manner; and(ii) a second nucleic acid comprising one or more T-DNA border sequence(s) operably linked to a nucleic acid of interest; and (e) selecting at least one transformed citrus plant cell comprising the nucleic acid of interest.