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Transformation of Tomato for Broad Spectrum Resistance Against Leaf Curl Viruses (2019--2020)

M.A.Y. Akhond
Biotechnology Division, BARI, Joydebpur, Gazipur

K. Nahar
Biotechnology Division, BARI, Joydebpur, Gazipur

S. Ahmed
Biotechnology Division, BARI, Joydebpur, Gazipur

M. R. Kabir
Biotechnology Division, BARI, Joydebpur, Gazipur

Abstract/ Executive Summary:

Several experiments were conducted with a view to genetically transform tomato plants for broad-spectrum resistance against leaf curl viruses. Two binary vectors along with their four derivatives have been constructed for the optimization of transformation protocol. Based on the genome sequence of various ToLCV strains, DNA fragments from three diverse ToLCV species along with two tomato introns were amplified and cloned. Both the virus and intron sequences were assembled into sense/antisense configurations into Escherichia coli using standard protocols. In-house made vectors were tentatively named pBPA-BARI followed by a number. All the plasmid vectors were mobilized into electro-competent Agrobacterium cells prepared in-house and their presence in the Agrobacterium was confirmed by PCR analyses.

Keywords: Tomato, leaf curl virus, Reporter genes, PCR analyses

Location:

Start Date: 00-00-2019

End Date: 00-00-2020

Program Area: Pest Management

Commodity: Virus, Tomato

Objectives:

An attempt has been made to construct modular vectors in-house for the efficient transformation of tomato plants.

Methodology:

Two modular plasmid vectors having different promoter-terminator cassettes have been constructed and two reporter genes viz. gusA and Cycle 3 gfp (gfpC3) were inserted into each of the vectors using standard cloning techniques. Three introns – one from potato and two from tomato plants were amplified by PCR and cloned. Specific sequence fragments from the three most virulent species were amplified by PCR and also cloned. Both the virus and intron sequences were assembled into sense/antisense configurations into Escherichia coli using standard protocols. The integrity of newly constructed plasmid vectors was confirmed by sequencing. All the plasmid vectors were mobilized into electro-competent Agrobacterium cells prepared in-house.

Source of Information: Annual Research Report 2019-2020, Biotechnology Division, BARI, Joydebpur, Gazipur

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Two reporter genes viz. gusA and Cycle 3 gfp (gfpC3) were inserted into each of the two binary vector backbones having potato Ubi3 and CaMV35S promoter-terminator cassettes for optimization of tomato transformation protocol. Based on the genome sequence of various ToLCV strains, DNA fragments from three diverse ToLCV species and three tomato introns were amplified and cloned. The assembled fragments were further assembled and cloned into the optimized promoter-terminator cassettes. Vectors were tentatively named pBPA-BARI followed by a number. All the plasmid vectors were mobilized into electro-competent Agrobacterium cells prepared in-house and their presence in the Agrobacterium was confirmed by PCR analyses.

Knowledge Management: Report/Proceedings