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Development of Transgenic Tomato

M. A. Y. Akhond
Senior Scientific Officer
Biotechnology Division, BARI

M. R. Kabir
Scientific Officer
Biotechnology Division, BARI

M. S. Biswas
Scientific Officer
Biotechnology Division, BARI

M. Al-Amin
Principal Scientific Officer
Biotechnology Division, BARI

Abstract/ Executive Summary:

Cotyledon and hypocotyl explants of in vitro germinated tomato seedlings were cultured in MS media supplemented with different growth regulators along with a control treatment without growth regulators. The promoter, coding sequence and the terminator sequence for the GUS reporter gene were assembled and inserted into the T-DNA region of the binary vector harbouring kanamycin resistance. Regenerated shoots were subcultured every 4 weeks onto fresh medium. The developed protocol was used for transgenic plant regeneration following co-cultivation with Agrobacterium. An overall shoot regenaration frequency of 25% on the selection medium (100 mg/L kanamycin) was observed from hypocotyle explants. Well developed shoots were transferred to rooting medium under selection and all the shoots produced well developed root system. Four samples were tested which showed positive bands for both nptII and GUS genes supporting the presence of the transgenes in the plants. Plants showed normal growth pattern and were grown up to maturity. Fruits were harvested and the seeds were collected for further study.

Keywords: Tomato, Transformation, GUS, Plasmid

Location: Biotechnology Laboratory, BARI, Gazipur

Start Date: 01-10-2006

End Date: 31-07-2009

Program Area: Variety and Species

Commodity: Tomato

Objectives:

Development of an efficient in vitro plant regeneration and genetic transformation method for tomato.

Methodology:

Vector construction: The promoter, coding sequence and the terminator sequence for the GUS reporter gene and the kanamycin resistance (nptII) were collected in three different plasmid cloning vectors. Both chemical and electro competent E.coli cells were prepared in house and stored at -80° C. All the three plasmid vectors were transformed into competent E. coli cells and multiplied. The promoter and the reporter gene cassette was cut out from the cloning vector and inserted into the T-DNA region of the binary vector harbouring kanamycin resistance. Electrocompetent Agrobacterium cells were prepared and the assembled binary vector was transformed into the cells using electroporation.

Genetic transformation: The Agrobacterium cells carrying the binary plasmid vector was used for transformation. Tomato cotyledon and hypocotyl explants from three different BARI tomato varieties were co-cultivated with the Agrobacterium cells and cultured on selection medium containing kanamycin and carbenicillin. Regenerated shoots were subcultured every 4 weeks onto fresh medium. Transformed plants from previous year were transferred to soil and grown to maturity.

Source of Information: Annual report, Biotechnology Division 2009-2010

Article Link (Online Journal):

Funding Source:

1. Government

Budget: 2,00,000.00

Total Budget (Tk.) : 2,00,000.00

Achievement/Findings:

The newly constructed vector with the GUS reporter gene driven by potato promoter and terminator sequences are being tested with BARI tomato varieties. Shoot growth under kanamycin selection observed in some of the explants indicate that the vector construction was successful and the selectable marker gene is being expressed.

Knowledge Management: Report/Proceedings