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Transformation in Wheat



RRes


Contact: Huw D Jones - Web Page

Current status

Depending on the precise requirements, either particle bombardment or Agrobacterium tumefaciens is used as the DNA-delivery mechanism. Our optimisation experiments and protocols have been published widely (see link to relevant publications below). We have successfully transformed over 30 bread and durum wheat varieties but our variety of choice for biolistics is Cadenza (Spring), with Fielder (Spring), Florida (Winter) and the durum variety Ofanto preferred for Agrobacterium. Over the last few years we have been making 500-600 transgenic lines per year, mainly for collaborative gene-validation projects within Rothamsted but we also have many national and overseas collaborations and make a significant number of events as part of our efforts to further develop and improve our platform technologies for wheat transformation.

Costs and time scales

For wheat, the cost for producing 15 - 20 independent transgenic events with a single construct is £7,270 - £9937 depending on the method used, plant growth requirements and whether plants or seeds are required. Time scales are; 5 months to PCR positive, transportable, tissue-culture plantlets and 11 months to harvested T1 seed.

Current work and future directions at RRes

Reporter genes are used to study the effects of regulatory sequences including core and distal promoter elements, matrix attachment regions and introns on qualitative and quantitative patterns of gene expression. GUS and GFP have been used to validate 4 constitutive and 18 tissue specific, developmentally regulated or inducible promoters, and a catalogue of transformation vectors with defined spatial and temporal expression patterns is in preparation.
  • The relationship between patterns of gluten protein synthesis and deposition and grain processing quality: A transgenic approach
  • The role of gibberellin in the regulation of wheat grain development.
  • Transgenic approaches to study wheat grain development
  • Generation of new variation in bioactive compounds
  • Building cassettes of natural and modified R genes by genetic engineering
  • Over expression of genes encoding ion transport proteins to improve salt- and drought tolerance in wheat
  • AcDs tagging in wheat

Recent relevant publications

Book or Book Chapter

2009

2008

2007

2006

2005

Journal Article

2008

2007

2006

2005

NIAB


Contact: Professor Andy Greenland
    Centre for Plant Genetics,
    Breeding and Evaluation,
    NIAB,
    Cambridge. CB3 0LE
    +44(0)1223 342349
    andy.greenland@niab.com

Current status

The transgenic Initiative is a relatively new activity at NIAB. However, the key personnel have many years experience using the system developed by the Biogemma group in Cambridge. When the latter disbanded in 2006 the staff and transformation methodology transferred to NIAB, where the system is working very efficiently. T. aestivum NB1 is routinely transformed using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seed in planta as described in patent WO 00/63398 under licence from Biogemma. Transformation rates between 5-15% are routinely achieved. Some winter wheats and H.vulgare can also be transformed.

We would normally anticipate obtaining 30-40 independent transformed lines per construct. We can handle approximately 40 constructs per year, dependent on whether lines need to be grown in-house for seed production, or small plants passed on to third parties for seed production in their own facilities.

Regenerated plants are phenotypically normal, and carry the introduced genes at single insertion sites in 30 to 50% of transformation events. We routinely use the NptII selectable marker, though marker-free transformation can be achieved through co-transformation with two T-DNAs and subsequent segregation. Over-expression constructs can be made by classical cloning or Gateway technology (Invitrogen). RNAi constructs are made by Gateway technology.

Costs and time scales

We are happy to collaborate with scientists within the cereals community or to provide a contract transformation service, both under licensed agreement with Biogemma. Please contact us to discuss your specific requirements. Our charges for contract services depend on specification but are generally between £8,000-10,000 per construct. Time scales are; 5 months to PCR positive, transportable T0 plantlets and 9 months to harvested T1 seed.

We would be also be prepared to test the efficiency of other specific germplasm in transformation experiments, and are happy to develop new vectors in-house with third parties, or participate in the construct design process as necessary.

Current work and future directions

NIAB Research has interests in cereal disease resistance, novel traits and prebreeding targets. Our transgenic capability allows us to validate candidate genes at an early stage, in a time and cost effective manner prior to their incorporation into TILLING, ecoTILLING or traditional breeding programmes, in addition to commercial opportunities for transgenic crops.

We are constantly reviewing the varieties of wheat which can be efficiently transformed and expanding the available vector set, with particular emphasis on promoter identification and characterisation.


Recent relevant publications

Journal Article

2008

2006

2000





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