3. Gene Transfer Techniques to Transform Wheat
The two prerequisites for the generation of transgenic cereals are the availability of in vitro culture techniques and techniques to transfer the gene of interest into the genome of the cell.
The gene of interest has to be integrated into cells that are totipotent. Totipotency describes the ability of a cell to regenerate a complete individual.
The gene transfer into the totipotent cell guarantees that each cell of the regenerated plant contains the genetic information encoded by the transgene.
Unlike many other crops, cereals, especially wheat, were thought to be recalcitrant to in vitro culture.
When the regeneration of whole wheat plants from embryos of immature seeds first succeeded in 1978 (Shimada, 1978), they became the explant of choice, and ongoing optimization of culture conditions has since enabled plants to be regenerated efficiently from many different wheat cultivars.
The formation of numerous somatic embryos and their regeneration is controlled by concentrations of phytohormones in the synthetic growth medium.
The medium also supplies the cells with carbohydrates, minerals and vitamins.
The major steps of an in vitro culture system for wheat are shown in Fig. 220.
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| Fig. 220 : In vitro culture steps to regenerate wheat plants from immature embryos |
A. Immature seeds are harvested from wheat spikes 10 - 15 days after anthesis, zygotic embryos are isolated and transferred to a synthetic growth medium.
B. Cells within the isolated immature embryo are induced to form callus. Callus induction is achieved by the phytohormone auxin in the medium. Each callus bears numerous somatic embryos with the potential to regenerate into individual plants. A change in auxin concentrations allows regeneration into plants.
The time required to regenerate a wheat plant by in vitro culture from immature embryos is about three months.
The efficiency of an in vitro culture system depends on the number of regenerated plants per isolated embryo.
For a culture system to be efficient enough for gene transfer, numerous somatic embryos per callus have to be formed, as only a small percentage of callus cells stably integrate the transferred gene.
When the in vitro culture system is used to generate transgenic plants, the gene transfer occurs immediately or 2 - 3 days after isolation of the immature embryos.
Together with the gene of interest, a selectable marker gene is co-transformed. The co-transformed selectable marker gene provides resistance to the selection agent in the medium component, usually an antibiotic or herbicide.
So only successfully transformed cells can survive, while cells that have not integrated the selectable marker gene cannot detoxify the antibiotic or the herbicide and die off. In this case the resistance to an antibiotic (e.g. kanamycin) or herbicide (e.g. BASTA®) serves the sole purpose of allowing the identification of the transformed cells and is actually an undesirable trait once the transgenic plant is ready for field testing.
Consumers are concerned that the use of antibiotics as selectable markers might increase the antibiotic resistance of human pathogens.
Although no conclusive evidence exists that these antibiotic resistance genes cause harm to humans, development of marker-free transgenic plants is a major field of research in genetic engineering in order to increase public acceptance of the technology.
The two most established techniques for wheat transformation are the biolistic method using the particle gun, also called microprojectile- mediated gene transfer, and transformation using Agrobacterium tumefaciens.
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