Doctoral Dissertations

Date of Award

12-2000

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Plant, Soil and Environmental Sciences

Major Professor

B. V. Conger

Committee Members

Beth Mullin, von Arnim, Bob Trigiano

Abstract

Plant somatic cells have the ability to undergo sustained divisions and give rise to an entire organism. This remarkable feature, called plant cell totipotency, is dependent on a number of factors, including developmental state, genetic background and culture conditions. Totipotent morphogenesis has been observed to follow two main routes, organogenesis and embryogenesis. The initiation of embryogenic from nonembryogenic cells is usually induced by exogenous plant growth regulators, such as auxins. The precise nature of the triggering mechanisms for somatic embryogenesis are poorly understood. Dactylis glomerata L. (orchardgrass) possesses a high capacity for production of somatic embryos from leaf cultures. This presented an opportunity to study the molecular events taking place during somatic embryogenesis in a Poaceae species.

This dissertation describes the steps that were taken in pursuit of the identification of differentially expressed genes during initiation and early stages of somatic embryogenesis in orchardgrass. Basal leaf segments from the embryogenic genotype Embryogen-P were capable of producing somatic embryos after a four-day induction period on medium with the synthetic auxin 3,6-dichloro-2-methoxybenzoic acid (dicamba). Distal segments from the same genotype required 28 d of induction to acquire the same capacity for somatic embryo production. Leaf cultures from the nonembryogenic genotype Nonembryogen were induced by dicamba to form callus, but not embryos. The differential response of Embryogen-P and Nonembryogen to the embryo inductive medium was used to compare gene expression in these genotypes and to isolate cDNAs that appeared only in leaf cultures induced for somatic embryogenesis. RT-PCR differential display performed with total RNA from basal leaf segments cultured for 10 d and from distal leaf segments cultured for 28 d resulted in the isolation of 17 cDNA fragments that appeared to be characteristic for somatic embryogenesis from leaf cultures. Thirteen of them were cloned and sequenced. They represented between 186 and 362 bp from the 3' end of the transcripts, just before the poly(A) tail. Three of these cDNA fragments were used as probes for screening a cDNA library made from embryogenic leaf cultures.

Four clones were isolated from the cDNA library. One, designated as DGRPT6a was expressed in nonembryogenic and embryogenic leaf segments and suspension cultures. It was 81% identical to the 26S proteasome AAA-ATPase subunit RPT6a from Arabidopsis thaliana L. The other three clones were confirmed to have differential expression in embryogenic tissues. The DGMPP sequence showed high homology with a putative mitochondrial processing peptidase α-II chain precursor from rice. It was expressed only in embryogenic leaf cultures. The nucleotide sequence of DGE1 had a 49.7% identity with the DNA-binding protein WRKY3 mRNA from oat. The DGE2 full sequence showed no significant similarity with the GeneBank database. Both DGE1 and DGE2 encoded putative novel proteins. The putative DGEs appeared to be nuclear proteins controlled by phosphorylation and might be involved in transcription regulation. DGE1 was found to be expressed both in basal and distal leaf segments, while DGE2 was expressed only in distal leaf segments and in suspension cultures induced for embryogenesis. The isolation of three clones that were differentially expressed in embryogenic tissues of orchardgrass provides the opportunity for further more detailed studies of the molecular events that underlay the process of somatic embryogenesis in grass species.

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