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Scale-up of Agrobacterium mediated transformation of oats to detect low frequency stable integrations - AR1001

Description
The main objective of this project is to scale up Agrobacterium tumefaciens mediated transformation of oats in order to detect low frequency stable integrations and to regenerate and analyse transformed plants for transgene integration and expression. Experimental procedures will be based on our previous work with Agrobacterium mediated transformation of oats where the effectiveness of different tissue culture system, bacterial strains and vectors and plant tissue pretreatments designed to increase A. tumefaciens penetration and plasmid introduction into cells were optimised, and achieved high frequency T-DNA transfer to oat cells.
This work forms part of the MAFF Crop Molecular Genetics Programme and extends the work carried out in project CEO 161 with identical policy relevance.
The results of this work will be used to compare with the direct gene transfer system in order to define the most efficient and stable system for subsequent expression of agronomic genes in oats. For example some commercial interest has already been expressed in producing transgenic oat plants expressing biologically active proteins and enzymes, and academic interest in manipulating avenacin biosynthesis.
Objective

01 Scale-up of transformation to detect low frequencies of stable integration.

01.01 Mobilisation of Agrobacterium AGL1/pSoup/pAL157 plasmids into versions of AGL1 strains sensitive to cefotaxime.

01.02 Test large scale transformation of embryogenic callus of var Melys and leaf base sections of var Millennium with Agrobacterium vector AGL1/pSoup/pAL157 combining the optimum conditions for achieving T-DNA transfer as previously determined.

01.03 Determine if bar gene is expressed in gus expressing cultures. Assay cultures for bar gene expression and PAT activity and compare number and size of gus foci with and without PPT selection during 4-14 weeks culture.

01.04 Plant regeneration and molecular analysis of transformed plants for presence and expression of bar and gus genes.
Project Documents
• Final Report : Scale-up of Agrobacterium mediated transformation of oats to detect low frequency stable integrations   (3517k)
Time-Scale and Cost
From: 2000

To: 2001

Cost: £46,987
Contractor / Funded Organisations
Institute of Grassland and Environment Research (IGER)
Keywords
Arable Farming              
Biotechnology              
Cereal Production              
Crop Improvement              
Farming              
Genetically modified food and crops              
GM Food              
Fields of Study
Arable Crops