Regeneração e transformação genética do portaenxerto de macieira, Marubakaido (Malus prunifolia, Willd Borhk)

Abstract

The first apple crops in Brazil began in late 60s and early 70s. The quality of available planting materials was low, since main producers of this fruit, such as Europe and United States, just initiated clonal cleaning programs, and thus were issues of the infected material by viruses and similar. In recent years apple tree culture took great socio-economic importance in Brazil, especially in Rio Grande do Sul and Santa Catarina. However the quality of seedlings imported and produced in the country is still not satisfactory. Recent introductions of propagative materials from United States, Europe and Japan have shown to partially infected with latent viruses, as analyzes of Virology Laboratory of Embrapa Grape and Wine. Viruses are responsible for damage at all stages of development of apple tree, they interfere in the necessary physiological processes for metabolism and normal growth of the plant. The production of virus resistant plants is critical to achieve a desired level of productivity. Therefore, the development of regeneration and transformation protocols is relevant for obtaining such plants. The objectives of this work were to develop protocols for regeneration and transformation to obtain apple plants, Marubakaido cultivar, with resistance to latent viruses (ASGV and ACLSV). For experiments leaf explants were subjected to different regeneration media with benzylaminopurine (BAP) and Thidiazuron (TDZ) at concentrations of 2, 3 and 5 mg L-1 and indolebutyricacid (IBA) at concentrations of 0.2 and 0.3 mg L-1. We also tested explant sources, as well as the effect of exposure of leaf explants to dark, the percentage of regenerated explants and number of shoots per explant. Leaves derived from newly rooted shoots in vitro have high regeneration rates and dark exposure for 20 days at the beginning of process, increase the regeneration rate and shoots number, respectively. In transformation experiments evaluated the resistance of Marubakaido tissues to hygromycin antibiotic, pre-culture of explants on the regeneration medium liquid and the effect of dark for two weeks with regard to the transformation efficiency. There was a high sensitivity to hygromycin (5 mg L-1), and the pre-cultivation of leaf explants prior to infection, affect their survival after the transformation process; exposure to a dark period after the transformation process improves the response of the explants. The absence of plasmid in Agrobacterium tumefaciens was the determining factor for failure of tested protocols in this apple rootstock.