Cultura de tecidos e transformação genética da palma forrageira Opuntia ficus-indica (L.) Mill

• Ano de defesa: 1999
• Autor(a) principal: Zárate, Rómulo Marino Llamoca
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Palavras-chave em Português: Bioquímica
• Link de acesso: http://www.repositorio.ufc.br/handle/riufc/44633

The prickly-pear (Opuntia ficus-indica) "palma forrageira" is widely used in the Brazilian North-East as a forage crop. One ofthe main constraint in the wide utilisation ofthe "palma forrageira" as a forage crop, is the low level of proteins in its phylloclads. The long term goal of the research here described, is to improve the nutritional quality of the pricklypear to farm animals, by gene transfer techniques. The main focus of this research was to establish protocols for the in vitro cultivation of this species, as well as to establish protocol for its transformation through particle bombardment. We have established the following protocols: (1) protocol for micropropagation through axillary bud proliferation; (2) protocol for whole plant regeneration from the shoot apical meristem; (3) protocol for induction and establishment of callus culture and cell suspension and (4) protocol for protoplasts isolation and cultivation. The studies towards the induction of somatic embryogenesis in cotiledonary explants achieved only partial success, as although pre-embryogenic structures were obtained, these were unable to give rise to mature embryos. Protocols for the transformation of isolated shoot apical meristems, young phylloclads and friable callus through particle bombardment were established. Integrative transformation of friable callus was demonstrated through genetic (amplification by PCR ofthe u i d A , n p t I l and t e t genes) and phenotypic (GUS activity and kanamycin resistance in the transformed calli) criteria. The analysis of GUS activity in elected lines of transformed calli, demonstrated that the enzyme B-glucuronidase represents 0.3% of the total soluble protein; this value is comparable to that obtained in model system such as maize and tobacco. The results described demonstrate that the cells ofthe prickly-pear are capable of withstanding the expression at high levels of a transgene and that the goal and that the production of transgenic varieties of the prickly-pear that express high levels of a gene for a protein of good nutritional quality is within reach, provided that protocols for plant regeneration are available. This is the first report on the expression of a transgene in the Cactaceae.