Doctoral Dissertations

Ru-Ju Chian

Date of Award

5-1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Peter M. Gresshoff

Committee Members

Fred Allen, Beth C. Mullin, Gary Stacey

Abstract

Nodulation is the consequence of the specific interaction between legume plants and the symbiotic bacteria Rhizobium (or its relatives Bradyrhizobium and Azorhizobium). Nodules provide a specific environment for bacterial growth and nitrogen-fixation. Although induced by bacteria or bacterial-derived compounds, (i.e.. Nod factors) organogenesis of nodules is directed by plant genes. Many cellular activities during nodule development are fundamental to plant growth, such as dedifferentiation of mature cells, induction of cell division, regulation of plant hormones, shoot-root communications, etc. Thus the study of nodulation is not only important to agricultural practice, but also provides an ideal system to address questions concerning plant-microbe interactions and plant developmental biology. To investigate gene regulation in nodule development using molecular biological tools, we established Agrobacterium rhizogenes-mediated transformation systems for soybean and Lotus japonicus, and regeneration protocols for L. japonicus. Plant genes important in nodule initiation and cell division were isolated. The ultimate goal of this project is to develop a system to study regulation of these genes in plants utilizing plant transformation. Part I of the thesis introduces general aspects of nodulation and Agrobacterium-mediated transformation. High frequency soybean root transformation based on A. rhizogenes strain K599 is reported in Part II. Meristematic nodules were harvested from hairy roots of different soybean cultivars and their N2-fixing activity was re-examined. The possible role of agrobacterial genes integrated into plant genome in interfering with programmed organ development is discussed. Part III introduces A. rhizogenes-mediated root transformation of model legume Lotus japonicus. This work reflects a logic transition of transformation from soybean to Lotus accompanied with advances in nodulation research. High frequency root transformation and simple and fast regeneration of transgenic L. japonicus plants were demonstrated. A brief comparison between the two systems was given. Isolation of a special early nodulin gene, the genomic clone of enod40 gene from L. japonicus is presented in Part IV. This gene is particularly interesting because its potential role in regulation of nodule induction and intracellular hormone balance. A division of all enod40 clones into two groups is proposed, based on sequence comparison. Because of the advanced L. japonicus root transformation and regeneration system, it has also become feasible to study gene functions in a homologous system with a model legume. Throughout the course of this research, the involvement of plant hormones, with relation to bacterial oncogenes (such as rol genes of A. rhizogenes) and plant regulatory genes (such as enod40), in morphogenesis of different organs were frequently surfaced. Part V integrates our knowledge from diverse research areas into relatively simple models which can help interpret some results observed in this study and stimulate thoughts for future studies.

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