Subsidy for genetic transformation of Catasetum pileatum (Orchidaceae) plants using root and shoot meristematic tissues / Subsídios à transformação genética de plantas de Catasetum pileatum (Orchidaceae) por meio de tecidos merismáticos radiculares e caulinares

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

Research on in vitro conversion of root apical meristems into buds in Catasetum has contributed to a better understanding of competence, induction and cellular determination processes during plant development. Nowadays It demands advances in molecular studies. To achieve this, the use of transgenic plants is an important working tool. Furthermore, genetic transformation methods seem to be an efficient alternative for improvement of commercial plants with longlife cycles, such as orchids. Therefore, the aim of these studies was to establish a protocol for genetic transformation of Catasetum pileatum, using meristematic tissues as target explants. The potential of some gene promoters to induce the expression of uidA was evaluated in C. pileatum tissues. CaMV 35S, Pthi and the PTE027 showed the best results among all tested. The CaMV 35S and the Pthi1 were used in the permanent genetic transformation experiments. Lateral buds of shoot explants (CEs), root apices (ARs) and root segments (SRs) were tested as target explants for transformation. In order to obtain material containing higher quantity of proliferating cells, a tissue culture protocol was established using root segments (SRs). The formation of a globular structure on the proximal region of the explants was observed in the presence of 0.5mg.L-1 of BA. Histological sections of these structures showed the presence of a huge quantity of cells in intense cellular division. In advanced stages, it was observed superficial bud formation on the structures. These buds have the capacity to produce whole plants within one month, in appropriated culture medium. Explants were exposed to a range of hygromycin concentrations and thereupon concentrations of 25mg.L-1 and 10mg.L-1 were chosen for selecting CEs and ARs, and for selection of SRs, respectively. CEs and ARs were bombarded with tungsten microparticules containing adsorbed plasmidial DNA (P35S:uidA or Pthi1:uidA) and it was transferred to selective medium after one, two or three weeks. Nevertheless, these explants were not capable to survive in selective medium after two months. SRs were also bombarded with P35S:uidA or Pthi1:uidA. These explants expressed uidA between 48 hours and four weeks. After three months of selection, it was possible to see a blue stained bud transformed with Pthi1. The best conditions for genetic transformation of C. pileatum were followed: bombardment of newly isolated SRs, maintenance for two weeks in non-selective medium followed of transference to hygromicin medium up to three months. Although additional experiments will still be necessary to refine transformation methods and conduct molecular analysis, the results from the present study are the first reference about genetic transformation of Catasetum pileatum plants.

ASSUNTO(S)

catasetum pileatum meristemas transformação genética meristems catasetum pileatum genetic transformation

ACESSO AO ARTIGO

Documentos Relacionados