Graduate Thesis Or Dissertation
 

Identifying the Parameters for Wheat Pollen Electroporation Transformation

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  • Wheat (Triticum aestivum L.) is one of the most important crops in the world. It has been and continues to be one of the main sources of food for humans and animals. Although traditional wheat breeding has contributed greatly to the improvement of wheat both in productivity and biotic/abiotic stress tolerance/resistance, wheat breeding is restricted by the time it takes to produce new cultivars and species barriers that limit the sources of genes for wheat improvement. Genetic transformation, which allows for the rapid introduction of new genes without species barriers, has been used as a tool for crop improvement in many crops over the past twenty-five years. Wheat is one of the few major crops that has not benefited from genetic transformation. Biolistic- and Agrobacterium- mediated transformation are currently the two primary methods used for wheat genetic transformation though both have disadvantages related to genotype dependence on ability to produce plantlets in tissue culture and the involvement of selectable marker genes. Pollen electroporation transformation is a potential method that can overcome these disadvantages since there is no tissue culture requirement and no need for selectable markers to allow for growth and selection of transgenic plantlets. Therefore, the object of this research was to optimize the conditions needed for wheat pollen electroporation to utilize pollen electroporation as a method to produce transgenic wheat. To achieve this object experiments were conducted to determine the parameters for successful pollen electroporation. The first was to determine the optimum field strength for electroporation that allowed for the movement of macromolecules and DNA into the pollen grains while maintaining pollen viability. Based on the results of the Trypan blue test to determine movement of macromolecules into the pollen grain and the MTT (3-[4,5-Dimethyltiazo-2-yl]-2,5- diphenyltetrazolium bromide) test to determine pollen viability, the optimal field strength of wheat pollen electroporation transformation was 7.5 kV/cm. There was no interaction effect of pollen source and field strength indicating that the optimal field strength needed for transformation was independent of the genotype of the pollen source. The second experiment was to confirm that DNA would be taken up by the pollen grain after electroporation. Based on the results of the DNA uptake test, pollen could take up DNA from the surrounding liquid environment with and without an electric shock but uptake of DNA with an electric shock was greater. This demonstrated that electroporated pollen could act as a vector to transfer DNA into an egg cell and seed if fertilization occurs. To determine if transient expression of the gfp gene could be used in wheat to demonstrate successful introduction of foreign DNA into wheat via pollen electroporation, a plasmid carrying the gfp gene was shot into immature wheat embryo tissue using particle bombardment. Expression of the gfp gene was observed indicating that the plasmid DNA with the gfp gene could be used to visualize successful wheat transformation. For successful transformation with electroporated pollen, fertilization and seed development is needed. An experiment was conducted to pollinate several wheat genotypes with electroporated pollen to demonstrate the pollen was not only viable but could germinate allowing fertilization and subsequent seed development to occur. The use of four recipient cultivars (Brundage 96, Kaseberg, Bobtail and Rosalyn) was to determine if transformation with electroporated pollen could be a genotype independent method of wheat transformation. Seed set occurred on all four cultivars though it differed indicating the potential for an effect of the recipient female parent on the level of seed set. This study has proven that pollen electroporation transformation is a feasible method for wheat transformation and can provide a basis for the further refinement of pollen electroporation transformation of wheat.
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