biologia plantarum

Biologia plantarum 51:333-339, 2007 | DOI: 10.1007/s10535-007-0065-1

Resistance of transgenic papaya plants to Papaya ringspot virus

S. Kertbundit¹, N. Pongtanom², P. Ruanjan², D. Chantasingh², A. Tanwanchai², S. Panyim¹, M. Juříček^3,*

¹ Department of Plant Science, Faculty of Science, Mahidol University, Payathai, Bangkok, Thailand

² Institute of Molecular Biology and Genetics, Mahidol University, Nakhon Pathom, Thailand

³ Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Prague, Czech Republic

The coat protein gene isolated from Papaya ringspot virus, Thai isolate, was used to generate transgenic papayas. A binary vector containing the coat protein gene under the control of a 35S promoter, was constructed and transformed into somatic embryos of papaya cultivar Khak Dum by microprojectile bombardment. Eight transgenic lines were identified from 1980 bombarded calli of papaya somatic embryos under kanamycin selection. Integration of the transferred genes into kanamycin resistant papaya calli was verified by PCR amplification of the coat protein gene, GUS assays and Southern blot hybridization. Although the coat protein gene was detected in all transgenic lines, only line G2 was found to be highly resistant to virus. This resistant line showed high degree of rearrangement of the inserted coat protein expression cassette while the coat protein gene itself had a deletion of 166 bp on the 3' end of its sequence. Although the transcription of the coat protein gene was detected in all transgenic lines by RT-PCR, only two transgenic papayas expressed the intact coat protein. Moreover, in the resistant line G2 the amount of the truncated coat protein mRNA was significantly decreased. These results point to an RNA mediated mechanism of coat protein mediated resistance in papaya, probably based on post-transcriptional gene silencing.

Keywords: biolistic transformation; coat protein; plant regeneration

Subjects: chlorophyll a,b; coat protein; fatty acid desaturase; heat stress, high temperature; in vitro culture, regeneration, proliferation, differentiation; papaya; papaya ringspot virus; photochemical efficiency, quenching; photosystems 1,2; polymerase chain reaction (PCR); Southern blot analysis; temperature, high; transgenic plants; Western blot analysis

Received: April 14, 2005; Accepted: February 24, 2006; Published: June 1, 2007  Show citation