ISHS Acta Horticulturae 738: International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species
AGROBACTERIUM-MEDIATED TRANSFORMATION OF EMBRYOGENIC CULTURES IN 'MIYAGAWA WASE' SATSUMA MANDARIN (CITRUS UNSHIU MARC.) |
| Authors: | K.J. Song, S.B. Jin, K.Z. Riu |
| Keywords: | citrus breeding, gene transfer, GUS expression |
| Abstract:
To establish an Agrobacterium-mediated transformation system of ‘Miyagawa Wase’ satsuma mandarin (Citrus unshiu Marc.), we investigated some factors such as bacterial density, inoculation time, co-cultivation period, and acetosyringone concentration affecting transformation efficiency. Embryogenic cultures were induced and proliferated from undeveloped seeds. For genetic transformation, embryogenic cultures were infected with A. tumefaciens strain LBA4404 carrying pIG121 vector harboring hpt and gus genes. After co-cultivation, cultures were transferred on selective medium (MT basal medium supplemented with 500 mg/L malt extract, 7% lactose, 25 mg/L hygromycin, and 250 mg/L cefotaxime). After 4 weeks, hygromycin-resistant embryos were obtained and transformation efficiency was evaluated by transient GUS expression. The highest transformation efficiency was obtained at 0.6 OD600 of bacterial density, 20 min of inoculation time, 5 days of co-cultivation period, and 100 µM of acetosyringone concentration. Shoots of these embryos were grafted on rootstocks of trifoliate orange. Gene transfer was confirmed by PCR and southern blot analysis. | |
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