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Genetic transformation of wheat using Agrobacterium tumifaciens - AR1002

Description

The UK public sector currently has no capacity to transform wheat using Agrobacterium tumifaciens, and the purpose of this research is to develop procedures for Agrobacterium co-cultivation of inflorescence and scutellum cultures, and to establish methods that are more facile and efficient than current direct-gene-transfer methods, and that could be applied to a range of wheat varieties.

Wheat, as a major UK crop, is a primary target for genetic manipulation, both for basic and applied research. Thus, improved transformation methods will have a profound impact on our understanding of the biology, on the productivity, and on the breeding of wheat.

Research funded by phase one of the CMG programme resulted in the development of a transient assay system for T-DNA delivery to immature wheat scutella tissue derived from model and commercial varieties. This allowed the assessment of a range of variables concerned with Agrobacterium-mediated transformation of wheat.

In this proposal we intend to examine the influence of two additional factors on T-DNA delivery and plant regeneration, and to to obtain transgenic plants by significantly scaling-up the numbers of plants regenerated.
Objective
The overall objective of this research to examine the influence of two additional factors on T-DNA delivery and plant regeneration, and to obtain transgenic plants by significantly scaling-up the numbers of plants regenerated.

1 Examine the influence of Silwet L-77 (a surfactant product) on T-DNA delivery and plant regeneration.

Silwet L-77 will be used to improve Agrobacterium introgression into epidermal and sub-epidermal tissues, and to test the effect of this on T-DNA delivery and regeneration. We have first hand evidence that Silwet has a marked positive effect on T-DNA delivery, however, at high concentrations, also inhibits the regeneration response. The concentration of Silwet will be optimised for a compromise between T-DNA delivery and regeneration.

2 Investigate the effect of selection pressure on regeneration after Agrobacterium co-cultivation.

The concentration and timing of PPT application will be studied to facilitate some selection, while allowing any transformants and some ‘escapes’ to regenerate.

3 Consolidation of protocols and scale-up of explant numbers for Agrobacterium co-cultivation and regeneration.

Combine existing knowledge regarding strain/vector combinations, media, inoculation/co-cultivation, pre-culture etc, with new data from objectives 1 and 2 above to establish a set of protocols that combines good T-DNA delivery with good level of regeneration, and to apply these to a large number (thousands) of individual explants. This will generate a porportionately large number (tens or hundreds) of plants, depending on the selection pressure etc. These plants will be screened by GUS assay, PCR and/or Southerns for the presence of transgenes.
Project Documents
• Final Report : Genetic transformation of wheat using Agrobacterium tumifaciens   (15k)
Time-Scale and Cost
From: 2000

To: 2001

Cost: £56,372
Contractor / Funded Organisations
Rothamsted Research (BBSRC)
Keywords
Arable Farming              
Biotechnology              
Crop Improvement              
Farming              
Genetically modified food and crops              
GM Food              
Wheat Production              
Fields of Study
Arable Crops