Doctoral Dissertations

Date of Award

8-2001

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Gary Stacey

Committee Members

Fred L. Allen, Beth C. Mullin, Albrecht von ArniAl

Abstract

Experiments were carried out in the model legume L japonicus to determine the efficiency of promoter trapping and insertional mutagenesis using Agrobacterium tumefaciens (hypocotyl) and A. rhizogenes (hairy root) transformation systems. Three different binary vectors pAGUSBE^19, pGHMCS and pPCTVGUS, all with a promoterless gusA gene, were used for transformation. The frequency of promoter trapping was 1.9 % (6 out of 322 lines) in the hypocotyl system and 3.8 % (51 out of 1342) in the hairy root system. Some of the regenerated lines were sterile presumably due to somaclonal variation. Fourteen trapped lines showed root and nodule tissue-specific GUS expression. The trapped lines were divided into two classes based on the dependence of GUS expression upon rhizobium inoculation. GUS expression in lines CHEETAH, ARROWHEAD, MACHINE GUN, Lj335 and NEEL was not rhizobium inoculation dependent. Whereas, GUS expression in lines BAGEL, DONUT, FATA MORGANA, HYENA, TIMPA, Lj331-7, Lj331-2, and Lj343-3 was rhizobium inoculation dependent. CHEETAH expression was observed during both lateral root and nodule formation processes. FATA MORGANA, TIMPA, BAGEL, DONUT, HYENA, Lj33I-2, Lj331-7 and Lj343-3 expression was found only in nodules. MACHINE GUN and ARROWHEAD expression was detected only during root or lateral root formation process. Based on this result it was concluded that lateral root and nodule formation processes share at least some genetic control. A screening of the segregating generation (T2) of 209 transgenic lines resulted in the identification of 6 putative mutant phenotypes. These mutants were confirmed in the T3 generation for phenotypic reproducibility and segregation. However, none of the mutants were nodule or lateral root specific indicating that a larger number of independent insertion lines were needed to get observe such mutants.

Transgenic plants of the model legume Lotus japonicus were regenerated by hypocotyl transformation using a bar gene as a selectable marker. The bar gene encodes Phosphinothriein acetyl transferase that detoxifies phosphinothricin (PPT), the active ingredient of herbicides such as Ignite (AgrEvo) and Basta (Hoechst). Transgenie L. japonicus plants resistant to PPT were positive upon PGR using bar gene-specific primers. In 5 out of 7 independent lines tested, PPT resistance segregated as a single dominant allele indicating a single T-DNA locus into the plant genome. Typically, 15- 20% of transformed L. japonicus plants are lost to tissue culture-induced sterility when antibiotic selectable markers are used. All regenerated plants were fertile using PPT selection and void of visible abnormalities. The lack of tissue culture-induced variation, ease of PPT application and low cost of PPT make this protocol an attractive alternative for the regeneration of transgenic L. japonicus. Since several Lotus species are important forage crops, the production of PPT herbicide-resistant L. japonicus plants may have significant commercial applications in crop production.

Past studies of the effect of ethylene on nodulation have depended on the availability of ethylene insensitive mutants or application of precursors or inhibitors. Lotus japonicus plants expressing the dominant, negative etrl-1 allele from Arabidopsis thaliana were constructed and found to be ethylene insensitive by their lack of the triple response. Independent transgenic lines varied in their levels of insensitivity to ethylene. These lines were tested for their ability to be nodulated. The numbers of infection foci were enumerated using a Mesorhizobium loti strain constitutively expressing the lacZ gene. There was a direct correlation between the degree of nodulation and the level of ethylene insensitivity. Transgenic lines exhibiting a high level of insensitivity to ethylene were hypemodulated, compared to controls. In contrast, the level of nodulation in lines exhibiting mild insensitivity to ethylene did not differ significantly from controls. The position of infections was examined relative to root anatomy. Approximately 30% of the nodules or nodule primordia in transgenic plants were found between xylem poles, compared to only 5% in wild-type plants. The study indicated a role for ethylene in nodule initiation by influencing root cell infection by rhizobia and position of nodule initiation in relation to protoxylem pole. The hypemodulation of ethylene insensitive plants was nitrate sensitive. It is proposed that the hypemodulation due to ethylene insensitivity is due to defective 'local autoregulation' of nodulation. Since the hypernodulating plants grew similar to wild type, these findings may have agronomic importance.

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