Browsing by Author "Rongda Qu, Committee Chair"

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Agrobacterium-mediated Transformation of Tall Fescue (Festuca arundinacea Schreb.) for Fungal Disease Resistance.
(2006-05-08) Dong, Shujie; Rongda Qu, Committee Chair
Tall fescue (Festuca arundinacea Schreb.) is an open-pollinated, perennial, cool-season turf and forage grass species of great economic importance. It is a major turfgrass species for home lawns in North Carolina and the transition zone states in the US mostly because of its tolerance to the summer heat. Brown patch disease is the most serious and frequently occurring disease of tall fescue, caused by a basidiomycete fungus, Rhizoctonia solani (Kuhn). Gray leaf spot, induced by Pyricularia grisea (Cooke) Sacc., the asexual stage of Magnaporthe grisea (T.T. Hebert) Yaegashi & Udagawa, is becoming another important disease of tall fescue. The objectives of this research were to establish an efficient transformation system for tall fescue using Agrobacterium; to introduce disease resistance genes into tall fescue; and to study the effects of the transgenes in resistance to the two major fungal diseases. An efficient genetic transformation system, using Agrobacterium tumefaciens-mediated T-DNA delivery, was developed for tall fescue. Thirty four percent of the calli infected with A. tumefaciens were resistant to hygromycin B, the selection agent used to select/identify transformants, and the overall plant transformation frequency (the number of independently transformed plants over the number of calli infected) was about 8%. Southern analysis indicated the integration of transgenes into the plant nuclear genome and simple transgene copy patterns. The high efficiency observed was partly due to an elevated 2,4-D concentration (5 mg L-1) in the culture medium used during callus culture and co-cultivation. Inheritance studies revealed that the transgenes were transmitted to the progenies. Four genes with potential for fungal disease resistance, including: alfalfa β-1,3 glucanase AGLU1, T4 phage lysozyme, frog dermaseptin, and rice Pi9 genes, were introduced into two cultivars 'Coronado' and 'Matador' of tall fescue through Agrobacterium-mediated transformation. Of 29 T0 transgenic plants examined, six had a higher level of resistance to R. solani and 13 had enhanced resistance to P. grisea. The enhanced levels of resistance in most of these plants was highly significant. Five transgenic plants exhibited enhanced resistance to both fungal pathogens.
Gene Expression Regulation by the 5' Regulatory Sequences of the Rice rubi3 Gene in Transgenic Rice Plants
(2006-08-06) Lu, Jianli; Rongda Qu, Committee Chair
Plant biotechnology has found extensive applications in agricultural production, environmental protection, and molecular farming. A promoter of known expression pattern and regulation mode is of primary importance for these applications in that it helps tailor transgene expression in plants. The promoter of the rice polyubiquitin gene rubi3 was isolated in our laboratory. In transient assays, its expression was strongly enhanced by its 5' UTR intron and further elevated to a high level by sequences from the coding region of the rubi3 gene. In this thesis research, I analyzed the expression pattern of the rubi3 promoter, and studied the effects of its 5'UTR intron as well as the first 27 bp of the rubi3 coding sequence on its expression pattern and strength in transgenic rice plants. The expression pattern of the rubi3 promoter was studied by using both GUS and GFP as reporter genes to obtain more reliable information. Histochemical GUS localization and GFP visualization revealed that the rubi3 promoter, with its 5' UTR intron, was active in all the tissues and cell types examined, suggesting that it is a constitutive promoter although the expression levels among the tissues were not uniform. Its activity was especially strong in root apical meristems, leaf mesophyll cells, embryos, and parenchyma cells in the vascular system, including the vascular cylinders of roots and the vascular bundles of stems. The 5'UTR intron substantially enhanced the expression level of the rubi3 promoter without altering its expression pattern, but the magnitude of enhancement showed profound difference among various tissues. The increase in GUS enzyme activity was 3.1-fold in leaf, 23.6-fold in root, and 48-fold in callus tissue. Differential enhancement was also observed in other tissues of the GUS transformants and in the GFP transgenic plants. For example, enhancement was higher in embryos and aleurone layers, but obviously lower in stigma tissues. This report is the first one to demonstrate the tissue-dependency of intron-mediated enhancement of gene expression (IME) in stably transformed monocot plants. Moreover, differential enhancement was also found in different tissues at GUS mRNA level. The increase was 4.2-fold in leaf, 11.1-fold in root, and 12.1-fold in callus tissue. Further analysis broke down the enhancement effects of the rubi3 5' UTR intron into a pre-translational phase and a translational phase. The intron enhanced GUS gene expression mainly by elevating mature mRNA level in all the three tissues examined, suggesting that it stimulated GUS gene expression primarily at the pre-translational phase. However, the magnitude of enhancement in GUS activity was different from that at mRNA level, suggesting that the intron affected GUS gene expression at the translational phase as well. A fusion of the first 27 bp of the rubi3 coding sequence to the GUS gene further enhanced GUS activity over that conferred by the intron-containing rubi3 promoter without changing its expression pattern. The magnitude of the enhancement by this sequence also varied by tissue types. Analysis indicates that the enhancement effect took place completely at the translational phase without affecting mRNA accumulation. The results suggest that the 5' regulatory sequences of the rice rubi3 gene, including the promoter, the exon 1, the 5' UTR intron, and the exon 2 sequence, can be combined to direct strong and constitutive transgene expression in transgenic monocot plants. The observation also helps gain insight on the mechanisms of gene expression regulation, especially IME, in monocot plants.
St. Augustinegrass Improvement for Freezing Tolerance and Semi-dwarf Growth Habit
(2008-05-11) Li, Ruyu; Rongda Qu, Committee Chair; Art H. Bruneau, Committee Co-Chair; Melodee L. Fraser, Committee Member; Wayne W. Hanna, Committee Member; Grady L. Miller, Committee Member; David P. Livingston, Committee Member
A significant crop produced by the industry and sold in many states is St. Augustinegrass. However, it is sensitive to freezing temperature and has coarse plant architecture. The NC sod industry demands new St. Augustinegrass cultivars with further improved freezing tolerance and finer plant architecture (semi-dwarf growth habit). The objective of this project was to use various approaches, including germplasm collection, induction of somaclonal variations, and irradiation mutagenesis, to create variations and to screen for plant clones to meet the industry demands. Techniques were developed, which include establishment of a freezing tolerance test system under controlled conditions and improved tissue culture conditions. It was found that treating plants at 13°C for one week, followed by 3°C for another week was an effective condition for cold acclimation of St. Augustinegrass in a controlled environment for freezing tolerance test at or around -4°C for 3 hrs. An efficient tissue culture system was established for the somaclonal variation approach. In that effort, 11 explant tissues and four callus culture media were examined for tissue culture response. The best response came from immature embryo 7-14 days after pollination (DAP) on Murashige and Skoog (MS) medium containing 1 mg L-1 2, 4-D and 0.5 mg L-1 6-benzyladenine. The callus induction and regeneration rates were 97.7% and 47.6%, respectively. Thirty six germplasm accessions were collected. Nearly 8000 plants were regenerated through tissue culture. And 3300 plants recovered from mutagenized plant materials. They were screened for improved freezing tolerance and/or semi-dwarf growth habit. Using the freezing tolerance test system, it was revealed that Elm4, collected from downtown Raleigh, and SVC3, a somaclonal variant, had significantly improved freezing tolerance. Ray, a collection from Polk County, had finer plant architecture and comparable freezing tolerance to Raleigh. In addition, nineteen somaclonal variants from tissue culture and 12 mutants from mutagenesis showed semi-dwarf growth habit and grew vigorously. Most of them had shortened internodes and stolons. Field performance of 11 lines was evaluated for development of potential new cultivars.