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Nuclear and plastid transformation of wheat and tritordeum using the streptomycin - selectable aadA marker gene - CE0173

Description
The overall objective is streptomycin-based nuclear and plastid transformation of wheat and tritordeum with the aadA selectable marker gene. This will involve the development of nuclear and plastid transformation vectors for wheat and tritordeum and their transformation into scutella nad immature inflorescence. The use of aadA will undoubtedly benefit wheat transformation by increasing the variety of selectable marker genes that are available. In addition, the proposal will advance our capacity to engineer cereal genomes by also targeting the plastic. The proposal is relevant to the agricultural requirements open competition CTD9802 Crop molecular genetics-improvement to cereal transformation.
Transformation of cereal plastids complements nuclear transformation and has considerable potential for increasing the levels of foreign gene expression and overcoming problems such as silencing. Differences between the nuclear/cytosolic and plastid expression machinery are potentially useful. For example, if the inherent structure (e.g. codon usage, internal mRNA destabilising sequences) of a gene prevents high levels of expression in the nucleus, expression in the plastic may overcome this problem. Once a gene has been integrated into plastid DNA its genetics and expression pattern can be predicted with a high degree of certainty. In addition, foreign genes located in plastics are not pollen transmitted which helps containment.
The technology developed has the capacity to facilitate UK breeding of crops by allowing the stable introduction and expression of foreign genes, with added value properties such as increased pathogen and stress resistance, into cereals. The results will be published in learned scientific journals and inventions will be scrutinised for patentability. The vecotrs and technologies we develop will be disseminated within the UK to promote their widespread application in the generation of UK based crops.
Objective
The overall objective is streptomycin-based nuclear and plastid transformation of wheat and Tritordeum with the aadA selectable marker gene. This can be broken down into a number of steps;

Manchester

1) Construction of vectors containing the aadA gene for nuclear transformation (6 months)
2) Analysis of plastid gene expression in wheat and Tritordeum during the regenration regime
used for transformation (8 months).
3) Mapping the 5’ and 3’ ends of transcripts from plastid genes that are constitutively expressed
or highly expressed in scutella and inflorescence (6 months).
4) Cloning and sequence analysis of plastid 5’ and 3’ regulatory regions (6 months).
5) Construction of cereal plastid expression cassettes (6 months).
6) Construction of targeting vectors for wheat and tritordeum plastid transformation (3 months).

Rothamsted

7) Particle bombardment of wheat scutella and tritordeum inflorescence with nuclear and plastid tranformation vectors (12 Months).
8) Analysis of transformed plants (6 months).
Project Documents
• Final Report : Nuclear and plastid transformation of wheat and tritordeum using the streptomycin - selectable aadA marker gene   (311k)
Time-Scale and Cost
From: 1998

To: 2001

Cost: £149,635
Contractor / Funded Organisations
University - Manchester
Keywords
Arable Farming              
Biotechnology              
Crop Improvement              
Farming              
Genetically modified food and crops              
GM Food              
Wheat Production              
Fields of Study
Arable Crops