ISHS Acta Horticulturae 738: International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species
IMPROVING REGENERATION AND TRANSFORMATION FOR RESISTANCE TO SHARKA IN PRUNUS |
| Authors: | C. Damiano, A. Gentile, S. Monticelli, V. Kondakova, E. Todorovska, I. Kamenova, I. Badjiakov, A. Atanassov, R. Scorza |
| Keywords: | DAS-ELISA, GUS expression, hypocotyl, kanamycin resistance, plant inoculation, PPV, stone fruit, transgenic plants |
| Abstract:
The biotechnological approach is a promising strategy to overcome some serious diseases which affect stone fruits such as Sharka. However, members of the genus Prunus are among the most challenging with respect to in vitro organogenesis: regeneration and transformation are highly dependent on species, genotype, and source of explants. We focused our activity on the development of adventitious shoot regeneration protocols using leaves of micropropagated Prunus varieties, and on the optimisation of regeneration ratios based on the hypocotyl slices strategy from mature peach seeds (‘Nemaguard’, ‘Missour’, ‘Montclar’, ‘Romestar’, ‘Percoco di Tursi’), apricot (‘Sancastrese’, ‘Hatif Hatif’, ‘Vitillo’, Boccuccia spinosa, ‘Palummella’), and plum (‘Stanley’, ‘St. Julien’). The infection of hypocotyl slices of ‘Stanley’ and ‘St. Julien’ with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPV-CP-33 carrying the Plum Pox Virus (PPV) coat protein, the GUS and NPTII genes, allowed the creation of 8 transgenic clones of ‘Stanley’ and 3 transgenic clones of ‘St. Julien’. GUS activity and molecular analysis of regenerated shoots confirmed the insertion of PPV-CP and NPTII genes in the plant genome and the absence of bacterial contamination. Challenging of transgenic ‘St. Julien’ clones with sap prepared from leaves of Nicotiana benthamiana infected with PPV produced no symptoms on inoculated and on post-inoculation generated leaves. DAS-ELISA results confirmed the absence of virus infection in transgenic plants. | |
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