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Doctoral Thesis
DOI
10.11606/T.11.2006.tde-12012007-160405
Document
Author
Full name
Dayse Cristina de Carvalho
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2006
Supervisor
Committee
Mourão Filho, Francisco de Assis Alves (President)
Jacomino, Angelo Pedro
Leonel, Sarita
Scarpare Filho, João Alexio
Stuchi, Eduardo Sanches
Title in Portuguese
Fusão de protoplastos visando a reconstrução da laranja azeda
Keywords in Portuguese
Citricultura
Cultura de tecidos vegetais
Hibridação vegetal
Melhoramento genético vegetal
Porta-enxertos
Protoplastos vegetais
Abstract in Portuguese
O objetivo deste trabalho foi aplicar a técnica de fusão química de protoplastos para desenvolver híbridos somáticos interespecíficos entre tangerinas (Citrus reticulata) e toranjas (Citrus grandis), visando a obtenção de porta-enxertos semelhantes à laranja azeda Citrus aurantium). Como fonte de protoplastos foram utilizadas suspensões celulares embriogênicas de tangelo 'Page' (C. reticulata x C. paradisi) e tangor 'Murcote' (C. reticulata x C. sinensis) e folhas jovens de seedlings de toranjas 'Lau Tau' e 'Ogami' (Citrus grandis). Após a fusão os protoplastos foram cultivados sob ausência de luz, até a formação de microcolônias, que foram então cultivadas em meio de cultura EME em dupla-fase, suplementado com 13,33 g/L de maltose para a indução da embriogênese. Os embriões globulares formados foram transferidos para meio EME com 25 g/L de sacarose e quando em estádio cotiledonar foram transferidos para meio de cultura suplementado com 1,5 g/L de extrato de malte. As brotações obtidas foram enxertadas in vitro sobre laranja 'Hamlin' e 'Valência'. As plantas obtidas foram levadas para casa de vegetação e cultivadas em substrato comercial. As 17 plantas regeneradas de tangelo 'Page' + toranja 'Lau Tau' apresentaram conformação fenotípica adversa aos genitores, com folhas de tamanho reduzido, ápice arredondado, coloração verde escura, mesófilo enrugado e ausência de pecíolo alado. A análise de citometria de fluxo confirmou o caráter diplóide das plantas regeneradas. Marcadores moleculares RAPD apresentaram padrão de bandas similar entre a planta regenerada e o genitor tangelo 'Page'. O protocolo utilizado para isolamento, fusão e cultura de protoplastos, bem como para a regeneração e aclimatização de plantas permitiram a obtenção 17 plantas da combinação entre tangelo 'Page' e toranja 'Lau Tau' com conformação fenotípica diferente dos genitores, duas plantas da combinação entre tangor 'Murcote' e toranja 'Ogami' e uma planta da combinação entre tangor 'Murcote' e toranja 'Lau Tau'.
Title in English
Protoplast fusion aiming the reconstruction of sour orange
Keywords in English
Citriculture
Genetic improvement
Plant protoplasts
Plant tissue culture
Rootstocks
Somatic hybridization
Abstract in English
The aim of this work was to apply the technique of chemical fusion of protoplasts, in order to develop interspecific somatic hybrids between mandarins (Citrus reticulata) and pummelos (Citrus grandis), in order to produce similar to sour orange (Citrus aurantium). The sources for protoplasts were embryogenic suspension cultures of 'Page' tangelo (C. reticulata x C. paradisi) and 'Murcott' tangor (C. reticulata x C. sinensis) and young leaves from seedlings of 'Lau Tau' and 'Ogami' pummelos (Citrus grandis). After the fusion, the protoplasts were cultivated in the absence of light, until the formation of microcolonies, and were then cultivated in double-phase EME medium, supplemented with 13.33 g/L of maltose for embryogenesis induction. The globular embryos thus formed were transferred to EME medium with 25 g/L of sucrose and, when in cotyledonal stage, were transferred to a culture medium supplemented with 1.5 g/L of malt extract. The shoots obtained were grafted in vitro onto 'Hamlin' and 'Valencia' sweet oranges. The regenerated plants were cultivated in a greenhouse, over commercial substrate. In this process, 17 plants were obtained. These plants presented phenotypic conformation different from the genitors, with leaves with reduced size, round apex, dark green coloration, rough leaf blade and absence of developed petiole. The analysis by flow cytometry confirmed the diploid character of the regenerated plants. RAPD molecular markers presented a similar band pattern between the regenerated specimen and the genitor 'Page' tangelo. The protocol used for isolation, hybridization and cultivation of protoplasts, as well as for the regeneration and acclimatization of the plants allowed the obtainment of 17 plants from the combination of 'Page' tangelo + 'Lau Tau' pummelo, with phenotypic conformation different from the genitors, two plants from the combination of 'Murcott' tangor + 'Ogami' pummelo and one plant from the combination of Murcote' tangor + 'Lau Tau' pummelo.
 
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Publishing Date
2007-01-18
 
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