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| Author(s): |
Eveline Carla da Rocha Tavano
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Piracicaba. |
| Institution: | Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC) |
| Defense date: | 2008-10-02 |
| Examining board members: |
Beatriz Madalena Januzzi Mendes;
Ricardo Harakava;
Francisco de Assis Alves Mourão Filho
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| Advisor: | Beatriz Madalena Januzzi Mendes |
| Abstract | |
Genetic transformation permits the introduction of agronomically important genes in plant genome and can be utilized in order to produce disease resistant plants. However for the recovery of transgenic plants is required to establish an efficient in vitro plant regeneration protocol. In this work the aim was to study an in vitro organogenesis and the genetic transformation of Volkamer lemon and sour orange with a sequence of the CTV coat protein gene. For in vitro organogenesis explant internodal segments collected from plants cultivated in greenhouse, epicotyl segments obtained from in vitro cultivated seedlings and cotyledon fragment with hypocotyl attached obtained from in vitro germinated seed were used as explant. These explants were cultured in EME medium supplemented with benzilaminopurine (BAP 0,0; 0,5; 1,0; 1,5; 2,0 mg L-1). Cultures were maintained under a 16 h photoperiod or in the dark for 10 d and then transferred to a 16 h photoperiod. The evaluation was performed 45 d after the incubation determining the number of responsive explant and the number of buds per explant. The anatomical characterization of in vitro regeneration process was carried out through histological analyses. The in vitro organogenesis occurred in the three types of explant tested, however cotyledon fragment with hypocotyl attached showed higher morphogenetic potential in both species. The best responses of regeneration were obtained when the medium was supplementation with 1,0 e 1,5 mg L-1 BAP for the Volkamer lemon and 0,5 e 1,0 mg L-1 BAP for the sour orange. The incubation in darkness favored the in vitro regeneration. The histological analyses showed that the regeneration process occurred through indirect organogenesis in the three types of explants tested. The developed protocol was use for genetic transformation of Volkamer lemon and sour orange with Agrobacterium, containing pCAMBIA 2201 plasmid with a sequence of CTV coat protein gene, in a hairpin construction. Volkamer lemon and sour orange shoots identified as transgenic by histochemical test GUS were micrografted into Carrizo citrange. PCR analysis were performed after micrografted showing the presence of the 671 pb fragment of the transgene. (AU) | |
| FAPESP's process: | 06/05454-6 - Organogenesis and genetic transformation of Volkamer lemon (Citrus volkameriana) |
| Grantee: | Eveline Carla da Rocha Tavano |
| Support Opportunities: | Scholarships in Brazil - Master |