The group conducts research in genetic engineering of various crops, primarily wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). High throughput, efficient and fine-tuned genetic transformation protocols have been developed by our team for these important cereals that are among the most transformation recalcitrant plants.
Our main objectives:
The protocol of Agrobacterium-mediated transformation of barley enables transformation efficiencies of up to 80% in model barley cv. Golden Promise. The protocol has also been successfully applied to the elite Australian barley varieties like Sloop, Clipper and Flagship and to the advanced breeding lines WI4330 and WI4259. Transformation frequencies are variable for these genotypes. Transgenic lines are predominantly produced with simple integration patterns and 40-60% typically have low copy inserts.
Current biolistic procedure uses co-transformation with the selectable marker gene for hygromycin resistance, which provides the option of segregating the marker gene away from the transgene in subsequent generations of the transgenic lines. A number of modifications have been made to the biolistic transformation protocol which have increased the transformation frequency and reduced the copy number of transgene insertions enhancing transgene stability. The improved procedure results in wheat transformation frequencies of up to 32% and produces transgenic lines in which 15% to 30% contain 1-2 copies of the transgene. We have successfully transformed all wheat cultivars and lines tested to date. These include model US cultivars Apogee, Bobwhite and modern Australian cultivars like Drysdale, Gladius, Rees, Westonia as well as durum line v.53380.
Agrobacterium-mediated transformation system for wheat (JT protocol) allows efficient production of transgenics in cultivars Fielder, Westonia, Gladius.
Transgene Copy Number and Expression
The group has developed techniques using quantitative-RT PCR (Q-PCR) to enable selection of transgenic plants which have low T-DNA copy number and have high expression of the transgene. This ensures transgenic plants ready for phenotypic analysis are identified rapidly.
Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method applicable to cereal breeding. CRISPR is an RNA-guided gene-editing platform that makes use of a bacterially derived protein (Cas9) and a synthetic guide RNA to introduce double strand breaks at specific locations within the genome. Editing is achieved by transiently introducing into plant cells the Cas9 protein along with specially designed guide RNAs (gRNA) that direct cutting through hybridization with its matching genomic sequence. ACPFGs transformation pipeline is developing a high-throughput methodology for both targeted knock-out and knock-in of gene sequences within the wheat and barley genomes. (Collaboration with Taj Arndell, Dr Ryan Whitford and Dr Nikolai Borisjuk).
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Recent selected Publications
Ismagul A, Iskakova G, Harris JC, Eliby S. (2014) Biolistic transformation of wheat with centrophenoxine as a synthetic auxin. Methods Mol Biol. 1145:191-202. http://link.springer.com/protocol/10.1007%2F978-1-4939-0446-4_15
Ismagul A, Mazonka I, Callegari C, Eliby S. (2014) Agrobacterium-mediated transformation of barley (Hordeum vulgare L.). Methods Mol Biol. 1145:203-11. http://link.springer.com/protocol/10.1007%2F978-1-4939-0446-4_16