Increased Transgenic Plant Regeneration in Carrizo Citrange by the Combination of Agrobacterium Tumefaciens and Plasmolysis Treatment

Abstract

ABSTRACTProcedures for high efficiency production of transgenic citrus plants using an Agrobacterium tumefaciens system with plasmolysis treatment were developed. Longitudinally cut epicotyl segments of Carrizo citrange (Citrus sinensis L. Osbeck x Poncirus trifoliate L. Raf.)] were plasmolysed in different concentrations of sucrose and maltose [0, 3, 6, 8, 9, 10, 12 % (w/v)] prior to Agrobacterium inoculation. Plasmolysed epicotyl expiants were co-cultivated with either the hypervirulent Agrobacterium tumefaciens strain, the EHA-101 (harboring a binary vector pGA482GG) or Agll (carrying pCAMBIAl303 vector). Both binary vectors contained neomycin phosphotransferase II (NPT II) and b—glucuronidase (GUS) genes. The binary vector pCAMBIA1303 also contained a fused mGFP5 gene at the 3¢ end of GUS gene as a reporter. Epicotyl expiants of Carrizo citrange plasmolysed in 6–10% sucrose and 3% maltose showed transient GUS gene expression comprising up to 80–90% of the cut surface of expiants. Stable transformation frequencies were 120% when explant was treated with 6–10% sucrose. Plazmolysis treatment with 9–12% maltose eliminated the escapes from stable transgenic shoots. Regenerated putative transgenic shoots were harvested from the cut surface of epicotyl expiants within 2–3 months. Shoots were divided into basal and apical portions. Basal portions were assayed for GUS and apical portions were shoot tip grafted in vivo for the production of whole plants. The presence and expression of transgenes in the whole plants were verified by GUS assay, PCR and Southern analyses. The transformation efficiency in Carrizo citrange obtained is the highest so far reported for citrus.