ISHS Acta Horticulturae 610: V International Symposium on Kiwifruit
GENE TRANSFER STRATEGY FOR KIWIFRUIT TO OBTAIN PURE TRANSGENIC PLANT THROUGH INDUCING ADVENTITIOUS ROOTS IN LEAF EXPLANTS WITH AGROBACTERIUM TUMEFACIENS |
| Authors: | M. Li, Z. Huang, L. Han, G. Zhao, Y. Li |
| Keywords: | Actinidia deliciosa, Actinidia chinensis, transformation, chimera, gus gene |
| Abstract:
Owing to regeneration usually occurred via an intermediate callus phase and no adventitious buds directly regenerated from leaves in vitro in kiwifruit genetic transformation, kanamycin-resistant calli selected from leaf explants were usually chimeras with transformed and untransformed tissues. Reports have shown that adventitious roots were usually originated from a single cell in leaf explants. Based on these results, we attempted to obtain pure kiwifruit transgenic plants via inducing adventitious roots in leaf explants with Agrobacterium tumefaciens. The best regeneration of adventitious roots from leaf explants of kiwifruit was observed on MS medium with 2.0 mg/L IBA in continuous dark. Large adventitious roots were regenerated at the cut surface of each leaf explants in three weeks. In addition, rooting was observed in medium with 500 mg/L carbenicillin better than 500 mg/L cefotaxime. No adventitious roots were induced in medium with over 6 mg/L kanamycin. The leaf explants were co-cultivated with Agrobacterium tumefaciens strain A281 containing gus gene in medium with 2 mg/L IBA and 20 mg/L acetosyringone for 3 days in dark, then transferred to selective medium containing 2 mg/L IBA, 500 mg/L carbenicillin and 10 mg/L kanamycin, cultured in continuous dark. Kanamycin-resistant adventitious roots formed on leaf explants were excised and transferred to medium containing 2 mg/L zeatin, 0.1 mg/L IBA, 500 mg/L carbenicillin and 50 mg/L kanamycin to form calli and shoots, cultured under a 14h photoperiod. From 96 leaf explants we obtained 12 kanamycin-resistant calli. GUS histochemical staining assay indicated that these calli were pure transgenic tissues. | |
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