Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration
Magdalena Cervera (1), Antonio Navarro (1), Luis Navarro (1) and Leandro Peña (1, 2)
1. Center of Plant Protection and Biotechnology, Instituto Valenciano de Investigaciones Agrarias, Ctra. Moncada-Náquera, km.
5, Moncada, Valencia, Spain / Corresponding author () / Received April 30, 2007; accepted June 14, 2007; published online October 15, 2007
Summary
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.
