Date of Award:

5-2003

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Plants, Soils, and Climate

Department name when degree awarded

Plants, Soils, and Biometerology

Committee Chair(s)

John G. Carman

Committee

John G. Carman

Committee

Anne J. Anderson

Committee

William F. Campbell

Committee

Jeanette M. Norton

Committee

John M. Stark

Abstract

Agrobacterium tumefaciens and A. rhizogenes are the causal agents of gall or hairy root disease, but normally the bacteria do not cause disease in wheat. However, both bacteria grew without inhibition when exposed to intact or wounded wheat roots or embryos, and they colonized wheat root surfaces to levels similar to dicotyledonous plants. A. tumefaciens and A. rhizogenes induced 23% cell death after a 1-h exposure to wheat embryo cells grown in 7.4 mM O2, while the extent of cell death at 2.1 mM O2 was 8%. Contact with A. tumefaciens or A. rhizogenes caused cultured wheat embryo and root cells to rapidly produce H2O2, which decreased when embryos and roots were cultured at 2.1 mM O2. Browning and autofluorescence, and an increase in ferulic acid in cell walls, were observed in wheat embryo and root epidermal cells exposed to Agrobacterium, but neither lignin nor callose was detected. Agrobacterium appeared to induce resistance-like responses in wheat that may limit transformation efficiency.

The inability to regenerate wheat plants using tissue culture has been a limitation to high efficiency transformation. Regeneration via somatic embryogenesis was improved significantly by simulating the in ovulo environment to which the immature wheat embryos are exposed. Triticum embryo culture medium (TEC) improved callus formation, somatic embryo formation, and regeneration from somatic embryos while reducing precocious germination when compared to growth on Murashige and Skoog medium. Regeneration frequencies were improved when embryos were cultured at the O2 concentration found in the wheat ovule (2.1 mM O2) rather than atmospheric O2 concentration (7.4 mM O2).

Agrobacterium-mediated transformation of wheat was limited by tissue necrosis following co-cultivation, and by poor plant regeneration. Reduction of necrosis and increased plant regeneration were accomplished by amending the culture medium with antioxidant compounds and by reducing the O2 tension in which the wheat embryos were cultured. Twelve days past anthesis (DPA), wheat embryos were co-cultivated with Agrobacterium tumefaciens strains WAg11 or EHA101, incubated on TEC medium containing antioxidant compounds (catalase, cysteine and ascorbic acid), and cultured at 2.1 mM O2 concentrations. Transformation was documented in 6.0% of regenerated A. tumefaciens WAg11 exposed wheat plants using the firefly luciferase (luc) reporter system.

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Comments

This work made publicly available electronically on September 25, 2012.

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