Applicazioni biotecnologiche per l'induzione di nuova variabilità genetica nelle orchidee
Author(s)
Grosso, Valentina
Date Issued
June 7, 2013
Type
Doctoral Thesis
Abstract
Orchidaceae family is one of the most developed monocotyledonous group; their pot plant and cut flower production have a high economical value in the international flower market. At first, in vitro culture protocols have been developed in this family and it is now usual to propagate orchids via protocorm-like body (PLB) formation, shoot multiplication or callus culture. One of the aims of the genetic improvement in orchids is to obtain new varieties and commercially attractive hybrid combinations. So far, the traditional breeding programs have led to very good results, but consumers desires are changeable and the need exists to generate always new stocks and to include some desirable combinations not allowed due to genetic incompatibility problems.
In our research work we have focused our efforts on the introduction of new genetic variability in Dendrobium and Phalaenopsis spp by means of a biotechnological approach which combines cellular and molecular manipulations to increase genetic variability. This achievement in turn should provide a clue to address these incompatibilities and to rise new genetic variability. With this in mind, we have developed in Dendrobium and Phalaenopsisspp a protocol for leaf and PLBs protoplasts isolation and somatic hybridization via PEG (Polyethylene glycol) or electrofusion. In order to obtain polyploid mutants we used cell cycle blocking agents in vitro. To make this technological approach more effective and efficient, flow cytometry has been largely applied to recognize and sort single hybridized cells from normal ones immediately after the fusion process and for a rapid screening and identification of the new polyploid mutants. We were successful in obtaining polyploidy mutants from in vitro explants; interestingly, some of them showed a downsizing genome situation exhibiting a partial polyploidy. The flow cytometry screening proved to be an usefull tool to select polyploids and to reduce cost and time consumption for the entire process. Molecular marker (RAPD and SSR) and cytogenetic approachs were also applied to differentiate orchid mutants allowing a genetic identification of controls from tetraploids and near-tetraploids,thus reducing the general efforts to grow up very large plant population of unknown ploidy.
Additional information
Dottorato di ricerca in Ortoflorofrutticoltura
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