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Development of Tomato Transformation Protocol

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

M. R. Kabir
Scientific Officer
Biotechnology Division, BARI

Abstract/ Executive Summary:

Development of an efficient tomato transformation protocol was attempted in this experiment. Transformation was carried out using a binary plasmid containing the kanamycin resistant selectable marker gene and the GUS reporter gene. The assembled binary vector was transformed into the electrocompetent Agrobacterium cells using electroporation. Explants from a promising BARI variety were co-cultivated with Agrobacterium cells and cultured on selection medium containing kanamycin and carbenicillin. The regenerated shoots were transferred on rooting media and insertion of the transgenes was confirmed in 12 transformed tomato plants by PCR using transgene-specific primers for nptII and gus, respectively. GUS testing was also performed for confirmation of the transgenic status. Seeds and DNA from the transgenic plants were extracted and preserved for further analyses.

Keywords: Tomato, transformation, Agrobacterium

Location: Biotechnology Laboratory, BARI Gazipur

Start Date: 01-07-2010

End Date: 30-06-2014

Program Area: Variety and Species

Commodity: Tomato

Objectives:

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

2. Confirmation of inserted genes in transgenic plants.

Methodology:

Regeneration

:

Explants from different varieties of tomato will be cultured in tissue culture media supplemented with growth regulators.

Genes to be used

:

Different marker and reporter genes e.g. nptII, GUS etc.

Transformation

:

Agrobacterium-mediated method

Source of Information: Annual report, Biotechnology Division 2014-2015

Article Link (Online Journal):

Funding Source:

1. World Bank	Budget: 45,00,000.00 approx.
2. Asian Development Bank	Budget: 45,00,000.00 approx.
3. Government	Budget: 45,00,000.00 approx.

Total Budget (Tk.) : 45,00,000.00 approx.

Achievement/Findings:

Transformation was carried out using a binary plasmid containing the kanamycin resistant selectable marker gene and the GUS reporter gene. The assembled binary vector was transformed into the electrocompetent Agrobacterium cells using electroporation. Explants from a promising BARI variety were co-cultivated with Agrobacterium cells and cultured on selection medium containing kanamycin and carbenicillin. DNA was extracted from the leaves of all the transformed seedlings to detect the presence of transgenes. Insertion of the transgenes has been confirmed in 12 transformed tomato plants by PCR using transgene-specific primers for nptII and gus, respectively. GUS testing was also performed for confirmation of the transgenic status. Seeds and DNA from the transgenic plants were extracted and preserved for further analyses

Knowledge Management: Report/Proceedings