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BRACT - Biotechnology Resources for Arable Crop Transformation - AR1005

Description
An efficient transformation capability is of major importance as an analytical tool and for crop improvement. To facilitate the use of transformation technology within the UK there is a need for a centre of expertise to develop and distribute best practice among the scientific community. The main objective of this proposal is to provide a highly efficient, robust transformation capability for the main UK arable crops while also facilitating further improvements in transformation technology for the UK research community. This proposal, for the lead project within the DEFRA arable crop transformation technologies programme (CTD 0206) is a joint submission from the John Innes Centre and Rothamsted Research. The focus of the proposal is to draw together a complementary team that will use existing UK technology and expertise to best advantage. One of the main objectives will be to develop and apply clean gene technology, which is widely acknowledged as the way forward in the application of transformation in crop plants.

The key arable crops included in this proposal are wheat, barley and oilseed rape while others are included as secondary crops or addressed in separate proposals from other groups. For the three major crops, the programme will provide all the resources needed to facilitate transformation. These will include constructs requiring the minimum effort to insert genes or promoters of interest, through to efficient transformation methodology that builds in the capacity to produce transgenic plants free of selectable marker genes. To facilitate the production of marker free plants, Agrobacterium tumefaciens-mediated transformation will be the method of choice, although biolistic methodology will be available if required. The latest developments, from this and other projects within the programme, including improvements in transformation technology, strategies to increase the stability of transgene expression and new tissue specific promoters will be incorporated as they become available.

The principal route to inform users of the resources available will be via a web site. This will be based on the existing web site for the successful pGreen/pSoup vector system developed by Prof Phil Mullineaux at JIC and which will be utilised in this programme. The new web site will be updated and extended to include new vector sets and details of the transformation capability for the different crops. In developing and delivering a transformation capability for the UK research community, we propose to consult widely with potential users to assess future requirements. Through the involvement of a user group, resources will be tailored to meet demand as far as is practicable. It is envisaged that after three years it will be possible to maintain a transformation resource centre for the benefit of the research community by users building full costs for their transformation needs into grant proposals.
Objective
Main objective: To provide a highly efficient, robust transformation capability for the main UK arable crops while facilitating further improvements in transformation technology and delivery to the UK research community.

Technical and scientific objectives:

Objective 1 – Providing transformation resources to the UK research community. (JIC & R.Res)
01a Bring together the existing, most efficient and robust transformation protocols for wheat, barley and oilseed rape, including both Agrobacterium and biolistic-based methods, together with existing transformation vectors known to work well in these crops so that this technology can be offered as a transformation resource early in the project while improved methods and vectors are being developed.
01b Survey all potential users of transformation resources in the UK to determine demand for the facility.
01c Establish a user group to advise on the development of the facility and arrange a workshop for stakeholders to raise awareness of the facility.

Objective 2 – Vector construction. (JIC)
Construct a set of transformation vectors, based on the pGreen/pSoup vectors developed at JIC, that are Gateway compatible, allow clean gene technology to be applied if desired and can be easily modified to meet the transformation needs of the UK research community.

Objective 3 – Vector testing. (JIC & R.Res)
Test the new vectors in the appropriate transformation system for the main UK crops before making them available as a transformation resource.

Objective 4 – Develop an efficient Agrobacterium-mediated transformation system for wheat.
04a Re-establish basic protocol previously developed under MAFF funding (AR1002) and demonstrate stable transformations using pAL154/pAL156 in wheat varieties already shown to be amenable (Florida and Cadenza). Aim for at least 5% transformation efficiency. (R.Res)
4b Assess the highly regenerable Bobwhite lines with the aim of providing a higher efficiency Agrobacterium-mediated transformation system for wheat. Aim for greater than 5% transformation efficiency. (R.Res)
04c Apply protocols developed in 04a & 04b to testing efficacy of vectors generated in objective 2. (R.Res)
04d Transfer wheat transformation protocols to JIC. (JIC & R.Res)

Objective 5 – Agrobacterium-mediated transformation of barley. (JIC)
05a Extend the existing barley Agrobacterium-mediated transformation protocol to include a winter barley genotype. Aim for at least 5% transformation efficiency.
05b Transfer barley transformation protocols to RRes. (JIC & R.Res)

Objective 6 – Identify and test high throughput genotypes of oilseed rape. (JIC)
Improve Agrobacterium-mediated transformation of oilseed rape by selecting for high transformability using phenotypic markers developed and successfully used to select for this characteristic in Brassica oleracea with the aim of developing a high throughput transformation system for oilseed rape. Aim for at least 10% transformation efficiency.

Objective 7 – Production of marker free transgenic plants. (JIC & R.Res)
Demonstrate the production of marker-free transgenic cereals and oilseed rape using the Agrobacterium twin T-DNA approach.
07a Compare the frequency of unlinked T-DNA integrations between the dual vectors with individual T-DNAs (shown to be effective in rice at JIC) with the single vector with twin T-DNAs (shown to be effective in barley at CSIRO). Aim to analyse at least 10 segregating independent transformation events in both wheat and barley. (JIC & R.Res)
07b Develop marker free transgenic oilseed rape building on existing BBSRC funded research at JIC using the pGreen/pSoup vector system. Aim to analyse at least 10 segregating independent transformation events. (JIC)

Objective 8 – Stabilisation of transgene expression. (JIC)
Further test expression stabilised cassettes containing additional introns to confirm stabilisation of transgene expression using this approach.
08a Examine transgene expression in the existing wheat lines carrying expression stabilised cassettes in the T3 and T4 generations (extending work from project CEO171). (JIC)
08b Test the expression stabilised cassettes in barley. (JIC)

Objective 9 – Website development. (JIC & R.Res)
Update and extend the existing pGreen website so that this is the main initial source of information on crop transformation resources available to the UK research community.

Objective 10 – Planning future transformation resources. (JIC & R.Res)
10a Assess the future transformation requirements of the UK research community and establish a self-supporting transformation facility to meet these requirements.
10b Arrange a conference on crop transformation that brings together all CTD 0206 projects and provides a forum for discussion of the latest technology developments.
Project Documents
• Final Report : Biotechnology Resources for Arable Crop Transformation (BRACT)   (508k)
• Technical Summary : References for the approaches   (43k)
Time-Scale and Cost
From: 2003

To: 2007

Cost: £929,236
Contractor / Funded Organisations
Rothamsted Research (BBSRC), John Innes Centre (BBSRC)
Keywords
Arable              
Biotechnology              
Brassicas              
Cereals              
Genetically modified food and crops              
Genomics              
GM Food              
Oilseed Rape              
Sustainable Farming and Food              
Wheat              
Fields of Study
Arable Crops