ISHS Acta Horticulturae 652: I International Symposium on Grapevine Growing, Commerce and Research
RESULTS AND CONSTRAINTS IN TRANSFORMATION AND ADVENTITIOUS REGENERATION IN VITIS VINIFERA L. 'TINTA RORIZ' |
| Author: | A.L. Pinto-Sintra |
| Keywords: | A. tumefaciens, particle bombardment, β-glucuronidase, polymerase chain reaction, light microscopy, scanning electron microscopy |
| Abstract:
An adventitious regeneration protocol was established with the aim to obtain transgenic plants from an important grapevine variety in the Douro region (Tinta Roriz or Aragonez). Adult leaves showed enough degree of coincidence between competent cells for regeneration and for transformation by Agrobacterium to follow this path. Wounded leaves were assayed with A. tumefaciens strains with p35SGUSINT or were submitted to particle bombardment with p35SGUSINT or pJIC 100 (with 35S promoter and the gus, bar and hpt genes). Following transformation leaves were organogenically induced on medium without selective pressure and exposed to selection pressure 21 days after the gene transfer process. The shoots able to grow on media with selective pressure were selected as putative transgenic shoots. Positive amplification for the nptII gene was obtained in three shoots resistant to kanamycin, originated from transformations that had involved about 3200 cocultivated leaves. Bombarding about 750 leaves with the p35SGUSINT, positive amplification for nptII gene was obtained in three kanamycin resistant shoots. Bombarding about 1100 leaves with the pJIC100, positive amplification for bar gene was obtained in one phosphinotricin resistant shoot. Based on the data, we discuss some of the aspects that could be the cause of the very low regeneration rates of transformants. | |
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