Improved transformation efficiency in citrus by plasmolysis treatment

Kayim M., Koc N.

JOURNAL OF PLANT BIOCHEMISTRY AND BIOTECHNOLOGY, vol.14, no.1, pp.15-20, 2005 (SCI-Expanded) identifier identifier


Procedures for high efficiency production of transgenic citrus plants using an Agrobacterium tumefaciens system with plasmolysis treatment were developed. Longitudinally cut epicotyl segments of eight different citrus species ['Milam' Rough lemon (Citrus jambhiri Lush), 'Volkamer' lemon (Citrus volkameriana L), Rangpur lime (Citrus limonia L), 'Hamlin' sweet orange (Citrus sinensis L Osbeck), 'Duncan' grapefruit (Citrus paradisi Macf), Sour orange (Citrus aurantium L), 'Cleopatra' mandarin (Citrus reticulata Blanco) and Carrizo citrange (Citrus sinensis L Osbeck x Poncirus trifoliata L Raf)] were plasmolyzed in different concentrations of sucrose and maltose [0, 3, 6, 8, 9, 10, 12 % (w/v)] prior to Agrobacterium inoculation. Plasmolyzed epicotyl explants were co-cultivated with either the hypervirulent Agrobacterium tumefaciens strain, the EHA-101 (harboring a binary vector pGA482GG) or Agl-1 (carrying pCAMBIA1303 vector). Both binary vectors contained neomycin phosphotransferase II (NPT II) and beta-glucuronidase (GUS) genes. The binary vector, pCAMBIA1303 also contained a fused mGFP5 gene at the 3' end of GUS gene as a reporter. Epicotyl explants of Rangpur lime, Rough and 'Volkamer' lemons plasmolyzed in 9-12 % maltose showed transient GUS gene expression comprising up to 95 % of the cut surface of explants, while Carrizo citrange showed 80 % expression when they were plasmolyzed in 6-10 % sucrose. On the other hand, epicotyl explants of 'Hamlin' sweet orange, Grapefruit, Sour orange and 'Cleopatra' mandarin showed transient GUS expession in 80-90 % of explants with 6-10 % sucrose. Basal portions of the regenerated putative transgenic shoots harvested from the cut surface of epicotyl explants within 2-3 months, were assayed for GUS, and apical portions were shoot-tip grafted in vivo for the production of whole plants. The transformation efficiencies in different species obtained are the highest so far reported for citrus.