Journal Article

Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration

Magdalena Cervera, Antonio Navarro, Luis Navarro and Leandro Peña

in Tree Physiology

Volume 28, issue 1, pages 55-66
Published in print January 2008 | ISSN: 0829-318X
Published online January 2008 | e-ISSN: 1758-4469 | DOI: http://dx.doi.org/10.1093/treephys/28.1.55
Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration

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Genetic transformation of mature trees is difficult because adult tissues are recalcitrant to Agrobacterium tumefaciens infection and transformation and because transgenic mature events are less competent for regeneration. We have shown that reinvigoration allows manipulation of the vegetative phase to increase the potential for transformation and regeneration without loss of competence for flowering and fruiting. To produce transgenic plants from clementine mandarin (Citrus clementina hort. ex Tanaka), we optimized the conditions of the source material both ex vitro and in vitro. Grafting of mature buds on juvenile rootstocks in the spring and preventing multiple bud sprouting by removing all but one bud permitted selection of vigorous first flushes for in vitro culture. Use of additional virulence genes from A. tumefaciens to increase transformation frequency and optimization of culture media and conditions to enhance explant cell competence for T-DNA integration and organogenesis resulted in efficient and reliable transgenic plant production. Transformed regenerants from explants, cultured in media without antibiotics, were identified by a screenable marker (either β-glucuronidase or green fluorescent protein (GFP)), creating the possibility of generating transgenic clementine plants without antibiotic resistance marker genes. Stable integration of foreign genes was demonstrated by Southern blot analysis, and expression of these foreign genes was confirmed by detection of GFP fluorescence in leaves, floral organs and fruits of the transgenic plants.

Keywords: citrus; green fluorescent protein; reinvigoration; selectable marker-free plants; tree transformation

Journal Article.  0 words. 

Subjects: Plant Sciences and Forestry

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