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MONOGRAM
UK focus on grain and grass research


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Transformation in Forage / amenity grasses



IBERS


Contact:Sue Dalton

Current status

Biolistic transformation of grasses has been in place since 1992, with the production of over 500 plants a year using genotypes selected for amenability. Recently Agrobacterium transformation has been achieved in embryogenic cell suspension cultures of two grass species, Festuca arundinacea and Lolium multiflorum. As most of the grass species transformed are out-breeders, transformed plants must be crossed with other genotypes to obtain progeny. The model species Brachypodium distaychon and Lolium temulentum are inbreeding.

Embryogenic cultures from shoot tips of the most amenable genotypes offer the most successful route for transformation. These elite embryogenic genotypes are multiplied and stored as in vitro plantlets, to ensure that intensely embryogenic material is always available. The amenability of these genotypes is heritable and so they can be used to cross in, and thereby improve, the transformability of particular genotypes of interest eg staygreen material. Plants can also be readily re-transformed by initiating new embryogenic cultures from primary transformants. The use of two different selectable marker genes, hph (hygromycin resistance) and nptII (paromomycin resistance), has allowed the addition of xylanase gene constructs to transgenic Festuca arundinacea plants already shown to have strong recombinant ferulic acid esterase activity. Festuca arundinacea, which is amenable to transformation by bombardment, is hexaploid and has relatively large cells. A tetraploid of an elite genotype of Lolium multiflorum showed an increase in transformation efficiency compared with the diploid, suggesting that cell size may be significant.

Agrobacterium -mediated transformation in Lolium multiflorum and Festuca arundinacea has been successful here, while most recent reports concern Lolium perenne, due to its economic importance. Other important species which have been transformed in this way are Poa pratensis, Cynodon dactylon, Dactylis glomerata, Zoysia japonica and Brachypodium distachyon.

Time scale and costs

Depending on species and time of year 10-20 independently transformed T0 plants can usually be produced within six months. Many species require vernalisation before flowers are produced so that a further six months at least may be needed to obtain T1 seed. Costs depend on the degree of collaboration.

Current work and future directions
  • Amenable genotypes of Festuca arundinacea, Lolium multiflorum or Brachypodium distaychon are the material of choice for investigating any new system, but Lolium perenne, Lolium temulentum, Festuca pratensis, Festuca rubra, Agrostis stolonifera, Poa pratensis and Dicanthium annulatum can be transformed.
  • Improvement of Agrobacterium-mediated transformation efficiency in our species of interest using new constructs being made available by BRACT and others
  • The development of Miscanthus transformation as a bioenergy crop
  • Important technology requirements for application to grass transformation are seen as
    1. development of large insert technology (eg for introduction of BAC clones),
    2. better Agrobacterium vectors and
    3. cell and tissue specific promoters.

Recent relevant publications

Journal Article

2008

2006

2003




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