ISHS Acta Horticulturae 738: International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species
TRANSFORMATION OF CHERRY: PRUNUS CERASUS L. 'MONTMORENCY' AND PRUNUS CERASUS × P. CANESCEN 'GISELA 6' MEDIATED BY AGROBACTERIUM TUMEFACIENS AND A TWO-STEP SELECTION SYSTEM |
| Authors: | G.-Q. Song, K.C. Sink |
| Keywords: | β-glucuronidase, gene transfer, regeneration, selection, woody plant |
| Abstract:
A two-step selection method was established to improve Agrobacterium tumefaciens-mediated stable transformation of sour cherry Prunus cerasus L. ‘Montmorency’ and rootstock P. cerasus × P. canescens ‘Gisela 6’. Whole leaf explants, each with four cuts perpendicular through the midrib, were co-cultivated with EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ß-glucuronidase (GUS) reporter gene (gusA). Selection and regeneration of transformed cells and shoots of both cultivars was carried out on selection media starting with 25 mg L-1 kanamycin (Km) for 4 weeks followed by 50 mg L-1 Km for 8 weeks. Compared with a one-step selection using 50 mg L-1 Km, the two-step selection method minimized necrosis of inoculated leaf explants, and the transformation frequency was increased from 3.1% to 7.6% for ‘Montmorency’ and from 3.3% to 9.7% for ‘Gisela 6’, respectively. Southern analyses of the GUS- and PCR-positive transformants, 3 for ‘Montmorency’ and 2 for ‘Gisela 6’, confirmed stable integration of the transgenes. | |
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