Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange

Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange

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The development and optimization of efficient transformation protocols is essential in new citrus breeding programs, not only for rootstock, but also for scion improvement. Transgenic 'Hamlin' sweet orange (Citrus sinensis (L.) Osbeck) plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated in vitro. Factors influencing genetic transformation efficiency were evaluated including seedling incubation conditions, time of inoculation with Agrobacterium and co-culture conditions. Epicotyl segments were adequate explants for transformation, regenerating plants by direct organogenesis. Higher percentage of transformation was obtained with explants collected from seedlings germinated in darkness, transferred to 16 hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45 min. The best co-culture condition was the incubation of the explants in darkness, for three days in culture medium supplemented with 100 mM of acetosyringone. Genetic transformation was confirmed by performing ß-glucoronidase (GUS) assays and, subsequently, by PCR amplification for the nptII and GUS genes.

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Main Authors: Mendes, Beatriz Madalena Januzzi, Boscariol, Raquel Luciana, Filho, Francisco de Assis Alves Mourão, Almeida, Weliton Antonio Bastos de
Format: Digital revista
Language:eng
Published: Pesquisa Agropecuaria Brasileira 2002
Online Access:https://seer.sct.embrapa.br/index.php/pab/article/view/6425
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spelling rev-pab-br-article-64252014-07-18T19:20:10Z Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange Transformação genética de laranja 'Hamlin' via Agrobacterium Mendes, Beatriz Madalena Januzzi Boscariol, Raquel Luciana Filho, Francisco de Assis Alves Mourão Almeida, Weliton Antonio Bastos de Citrus sinensis; epicotyls; seedlings; transgenics; breeding methods Citrus sinensis; epicótilo; plântulas; transgênicos; métodos de melhoramento The development and optimization of efficient transformation protocols is essential in new citrus breeding programs, not only for rootstock, but also for scion improvement. Transgenic 'Hamlin' sweet orange (Citrus sinensis (L.) Osbeck) plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated in vitro. Factors influencing genetic transformation efficiency were evaluated including seedling incubation conditions, time of inoculation with Agrobacterium and co-culture conditions. Epicotyl segments were adequate explants for transformation, regenerating plants by direct organogenesis. Higher percentage of transformation was obtained with explants collected from seedlings germinated in darkness, transferred to 16 hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45 min. The best co-culture condition was the incubation of the explants in darkness, for three days in culture medium supplemented with 100 mM of acetosyringone. Genetic transformation was confirmed by performing ß-glucoronidase (GUS) assays and, subsequently, by PCR amplification for the nptII and GUS genes. O desenvolvimento e otimização de protocolos eficientes de transformação genética é essencial nos programas atuais de melhoramento de citros, tanto para porta-enxertos, como para copas de valor comercial. Plantas transgênicas de laranja 'Hamlin' (Citrus sinensis (L.) Osbeck) foram obtidas pela transformação genética de segmentos de epicótilo, coletados de plântulas germinadas in vitro, com Agrobacterium tumefaciens. Foram avaliados fatores que influenciam a eficiência da transformação genética, como: condições de incubação das plântulas utilizadas para coleta de explantes, tempo de inoculação com Agrobacterium e condições de co-cultivo. A regeneração de plantas a partir de segmentos de epicótilo ocorreu em alta freqüência, por organogênese direta. A maior porcentagem de plantas transgênicas foi obtida utilizando-se explantes coletados de plântulas germinadas no escuro e posteriormente transferidas, por 2-3 semanas, para condições de 16 horas de fotoperíodo, e infectados com Agrobacterium por um período de 15-45 minutos. As melhores condições de co-cultivo foram a incubação dos explantes no escuro, por três dias, em meio de cultura suplementado com 100 mM de acetoseringona. A transformação genética foi confirmada pelo teste histoquímico para ß-glucoronidase (GUS) e, posteriormente, pela amplificação de DNA, por PCR, para detecção dos genes nptII e GUS. Pesquisa Agropecuaria Brasileira Pesquisa Agropecuária Brasileira 2002-07-01 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion application/pdf https://seer.sct.embrapa.br/index.php/pab/article/view/6425 Pesquisa Agropecuaria Brasileira; v.37, n.7, jul. 2002; 955-961 Pesquisa Agropecuária Brasileira; v.37, n.7, jul. 2002; 955-961 1678-3921 0100-104x eng https://seer.sct.embrapa.br/index.php/pab/article/view/6425/3482
institution EMBRAPA
collection OJS
country Brasil
countrycode BR
component Revista
access En linea
databasecode rev-pab-br
tag revista
region America del Sur
libraryname Sistema de bibliotecas de EMBRAPA
language eng
format Digital
author Mendes, Beatriz Madalena Januzzi
Boscariol, Raquel Luciana
Filho, Francisco de Assis Alves Mourão
Almeida, Weliton Antonio Bastos de
spellingShingle Mendes, Beatriz Madalena Januzzi
Boscariol, Raquel Luciana
Filho, Francisco de Assis Alves Mourão
Almeida, Weliton Antonio Bastos de
Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange
author_facet Mendes, Beatriz Madalena Januzzi
Boscariol, Raquel Luciana
Filho, Francisco de Assis Alves Mourão
Almeida, Weliton Antonio Bastos de
author_sort Mendes, Beatriz Madalena Januzzi
title Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange
title_short Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange
title_full Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange
title_fullStr Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange
title_full_unstemmed Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange
title_sort agrobacterium-mediated genetic transformation of 'hamlin' sweet orange
description The development and optimization of efficient transformation protocols is essential in new citrus breeding programs, not only for rootstock, but also for scion improvement. Transgenic 'Hamlin' sweet orange (Citrus sinensis (L.) Osbeck) plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated in vitro. Factors influencing genetic transformation efficiency were evaluated including seedling incubation conditions, time of inoculation with Agrobacterium and co-culture conditions. Epicotyl segments were adequate explants for transformation, regenerating plants by direct organogenesis. Higher percentage of transformation was obtained with explants collected from seedlings germinated in darkness, transferred to 16 hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45 min. The best co-culture condition was the incubation of the explants in darkness, for three days in culture medium supplemented with 100 mM of acetosyringone. Genetic transformation was confirmed by performing ß-glucoronidase (GUS) assays and, subsequently, by PCR amplification for the nptII and GUS genes.
publisher Pesquisa Agropecuaria Brasileira
publishDate 2002
url https://seer.sct.embrapa.br/index.php/pab/article/view/6425
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