Passionfruit genetic transformation (Passiflora edulis f. flavicarpa) for resistance to woodiness virus

Author(s)

Flavio Trevisan

• Document type: Master's Dissertation
• Press: Piracicaba.
• Institution: Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC)
• Defense date: 2005-08-29

Examining board members

Beatriz Madalena Januzzi Mendes; Ricardo Harakava; Francisco de Assis Alves Mourão Filho

• Advisor: Beatriz Madalena Januzzi Mendes

Abstract

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. (AU)