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Master's Dissertation
DOI
10.11606/D.11.2005.tde-29092005-134710
Document
Author
Full name
Flavio Trevisan
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2005
Supervisor
Committee
Mendes, Beatriz Madalena Januzzi (President)
Harakava, Ricardo
Mourão Filho, Francisco de Assis Alves
Title in Portuguese
Transformação genética de maracujazeiro (Passiflora edulis f. flavicarpa) para resistência ao vírus do endurecimento dos frutos
Keywords in Portuguese
maracujá
mosaico (doenças de planta)
planta transgênica
potyvirus
transformação genética
vírus de plantas
Abstract in Portuguese
O objetivo do trabalho foi estudar uma forma alternativa para o controle do endurecimento dos frutos do maracujazeiro, pela produção de plantas transgênicas contendo o gene da proteína capsidial do Passionfruit woodness virus - PWV. O vetor de expressão foi construído utilizando-se os plasmídeos pCambia 2300 e pCambia 2301, que contêm o gene de seleção nptII, para resistência ao antibiótico canamicina. O plasmídeo pCambia 2301 contém também o gene repórter uidA (GUS). Os plasmídeos foram introduzidos em Agrobacterium tumefaciens, estirpes EHA 105 e LBA 4404, pelo método do choque térmico. Os explantes para transformação genética constituíram-se de discos de folhas jovens (6 mm de diâmetro), das variedades IAC 275 e IAC 277, coletados de plantas mantidas em sob fotoperíodo de 16 h luz, a 27 °C. Os explantes foram inoculados com suspensão bacteriana (5x108 UFC/mL) por 20 min e transferidos para placa de Petri contendo o meio de cultura MS + thidiazuron (TDZ - 0,25 mg/L) + nitrato de prata (AgNO3 - 4 mg/L) + acetoseringona (1 µM/L). O co-cultivo foi realizado à temperatura de 24 °C, em ausência de luz, por um período de 3 dias. Para seleção e regeneração de plantas os explantes foram transferidos para meio de cultura de seleção MS + TDZ (0,25 mg/L) + AgNO3 (4 mg/L) + canamicina (100 mg/L) + cefotaxime (500 mg/L). A incubação foi realizada a 27 °C, em ausência de luz, por um período de 4 - 6 semanas. As gemas adventícias desenvolvidas foram transferidas para o meio de cultura MSM + 10% de água de coco e incubadas sob fotoperíodo de 16 h de luz. A transformação genética foi identificada pelo teste histoquímico GUS e por PCR. Obteve-se um total de 22 plantas PCR positivas. Destas, 8 foram analisadas por Southern blot para confirmação da integração do transgene. A transcrição e expressão do transgene foram analisadas por Northern e Western blot, respectivamente. As plantas transgênicas avaliadas foram multiplicadas e inoculadas com 3 diferentes estirpes do PWV. A linhagem T2 apresentou resistência a infecção dos três isolados utilizados.
Title in English
Passionfruit genetic transformation (Passiflora edulis f. flavicarpa) for resistance to woodiness virus
Keywords in English
genetic transformation
mosaic (plant diseases)
passionfruit
potyvirus
transgenic plants
Abstract in English
The main purpose of this work was to study an alternative way to control the Passionfruit woodiness virus - PWV through the production of transgenic plants which contained the Passionfruit woodness virus coat protein gene. The binary vector was built by using pCambia 2300 and pCambia 2301 plasmids, which contain the selection gene nptII. The pCambia 2301 plasmid also contains the reporter gene uidA (GUS). The plasmids were introduced into Agrobacterium tumefaciens, EHA 105 and LBA 4404 strains, via thermal shock method. The explants for the genetic transformation were young leaf disks (6 mm of diameter) of IAC 275 and IAC 277 varietys, extracted from plants kept under 16 h photoperiod, at 27 °C. The explants were inoculated with a bacterial suspension (5x108 UFC/mL) for 20 min and then transferred to Petri dishes containing cocolture medium MS + thidiazuron (TDZ - 0,25 mg/L) + silver nitrate (AgNO3 - 4 mg/L) + acetosyringone (1 µM/L). The co-culture was performed at 24 °C t, in the dark, for a three-day period. For the selection and regeneration of plants, the explants were transferred to the selection culture medium MS + TDZ (0,25 mg/L) + AgNO3 (4 mg/L) + kanamycin (100 mg/L) + cefotaxime (500 mg/L). The incubation was performed at 27 °C, in dark, for 4 - 6 weeks. The adventitious buds developed were then transferred to the culture medium MSM + 10% coconut water and kept incubated under 16 h photoperiod. The genetic transformation was identified through GUS and PCR tests. There were 22 PCR positive plants. Out of those, 8 were Southern blot analyzed for the confirmation of transgenc integration. The transgene transcription and expression were determined by Northern and Western blot respectively. The transgenic plants were then multiplied and inoculated with 3 different strains of PWV, and the line 2 showed resistance to the three strains used.
 
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Publishing Date
2006-09-27
 
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
  • MONTEIRO-HARA, Alessandra C. B. A., et al. Genetic Transformation of Passionflower and Evaluation of R1 and R2 generations for resistance to Cowpea aphid borne mosaic virus [doi:10.1094/PDIS-12-10-0873]. Plant Disease [online], 2011, vol. 95, n. 8, p. 1021-1025.
  • Monteiro-Hara, Alessandra Cristina Boffino de Almeida, et al. Genetic transformation of passionflower and evaluation of R1 and R2 generations for resistance to Cowpea aphid borne mosaic virus [doi:10.1094/PDIS-12-10-0873]. Plant Disease [online], 2011, vol. 95, p. 1021-1025.
  • TREVISAN, F., et al. Resistance to Passion fruit woodiness virus in transgenic passionflower expressing the virus coat protein gene [doi:10.1094/PD-90-1026]. Plant Disease [online], 2006, vol. 90, n. 8, p. 1026-1030.
  • TREVISAN, Flavio, et al. Resistance to Passion Fruit Woodiness Virus in Transgenic Passionflower Expressing the Virus Coat Protein Gene [doi:10.1094/PD-90-1026]. Plant Disease [online], 2006, vol. 90, nº 8, p. 1026-1030.
  • TREVISAN, Flavio, et al. Resistance to Passion fruit woodiness virus in transgenic plants of the yellow passion fruit expressing the viral coat protein gene. Acta Horticulturae, 2007, vol. 738, p. 495-499.
  • TREVISAN, Flavio, and MENDES, B. M. J. Optimization of passionfruit (Passiflora edulis f. flavicarpa) in vitro organogenesis. Scientia Agricola, 2005, vol. 62, nº 4, p. 346-350.
  • TREVISAN, Flavio, et al. Resistance of passion fruit woodiness virus in transgenic plants of the yellow passion fruit expressing the viral coat protein gene. In International Symposium on Biotechnology of Temperate Fruit crops adn tropical Species, Daytona Beach, Florida, EUA, 2005. ISHS Program and Abstract Book.Florida, EUA : University of Florida - IFAS, 2005. Resumo.
  • TREVISAN, Flavio, et al. Transfomação genética de maracujazeiros IAC-275 e IAC-277 via Agrobacterium para resistência ao vírus do endurecimento dos frutos (PWV). In 10º Encontro Científico dos Pós-Graduandos no CENA/USP, Piracicaba, SP, 2004. Anais do 10º Encontro Científico dos Pós-Graduandos no CENA/USP.Piracicba, SP : CENA/USP, 2004. Resumo.
  • TREVISAN, Flavio, et al. Transformação genética de maracujazeiro IAC-275 e IAC-277, via Agrobacterium, para resistência ao vírus do endurecimento dos frutos (PWV). In XXXVII Congresso Brasileiro de Fitopatologia, Gramado, 2004. Fitopatologia Brasileira.Brasilia : Sociedade Brasileira de Fitopatologia, 2004. Resumo.
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