Genetic engineering of sorghum and switchgrass for improved biofuel production

Dissertation supervisor: Dr. Zhanyuan J. Zhang.Includes vita.Biofuels, energy sources generated from biomass, have been seen as a potential route to meet energy demand and avoid political instability and environmental issues worldwide. Switchgrass has been considered as an excellent feedstock for biofuels due to the high cellulosic content, wide adaptation as well as the lower input energy for production. Sorghum is the fifth most important crop in the world for human staple food and also a versatile feedstock for grain, sugar, and biomass production. In current study, we demonstrated that expression of the Zea mays gibberellin 20-oxidase (ZmGA20ox) cDNA in switchgrass improved biomass production. Under greenhouse conditions, selected transgenic plants exhibited longer leaves, internodes and tillers, which resulted in 2-fold increased biomass. This is the first parallel report on the switchgrass biomass increase through genetic engineering approach. Our results suggest that the employment of ectopic ZmGA20ox, or selection for natural variants with high level expression of endogenous GA20ox are appropriate approaches to increase biomass production of switchgrass and possible other monocot biofuel crops. Additional contribution of this study is to optimize sorghum regeneration and transformation processes using standard binary vectors and bar gene as a plant selectable marker. The optimized transformation process enables reproducibly to achieve over 14% transformation frequency, the highest transformation efficiency through Agrobacterium-mediated transformation among the public laboratories. Of randomly analyzed independent transgenic events, 40-50% events carried a single copy of integrated T-DNA. The system developed here should be beneficial to sorghum biology study and genome exploration including genome editing.Includes bibliographical references

Morphology and DNA marker for distinguishing Paphiopedilum hangianum and Paphiopedilum emersonii from Vietnam

Genus Paphiopedilum has species having lovely flowers which are incredibly attractive to everyone. Their ornamental and commercial value caused over-collection and illegal poaching and trade. Due to these reasons, nowadays, the Venus slipper orchids are facing to deplete in nature. Therefore, it is important to consider these species conservation. Mainly, it is necessary to prioritize the identification and phylogenetic analysis methods of the genus Paphiopedilum which includes many species with similar morphological characteristics. Consequently, it isn't easy to distinguish the identical species of this genus when the plants are young or not yet fully flowering. Therefore, this study aimed to distinguish two Paphiopedilum species, i.e. P. hangianum and P. emersonii, which have similar morphological characteristics, through comparative morphological analysis and differences in DNA barcoding sequences. To solve the problem associated with species identifications, a morphological comparison table was created with the four DNA sequence markers matK, rbcL, rpoC1 and trnH-psbA. The results of the morphological analysis showed that P. hangianum and P. emersonii are significantly different from each other in the flower's characteristics. While the difference in leaf morphology of both selected species is found very little, it is also distinguishable upon careful comparison. Moreover, the DNA barcoding indicator gave accurate and rapid distinctions between the two species, even when     the plants are young or without flowers. Furthermore, this DNA barcoding can establish an evolutionary relationship between the two selected species and the other species of the genus Paphiopedilum. The results of this study also suggested that the indicator trnH-psbA is a suitable marker for distinguishing these two species and can be applied for the phylogenetic analysis of the genus Paphiopedilum in Vietnam

Agrobacterium-mediated transformation of Nang Thom Cho Dao, an indica rice variety

Agrobacterium-mediated transformation is the most efficient method to transfer gene of interest into the plant genome. However, the transformation efficiency of this method with indica rice varieties is still low and needs to be improved. In this study, the Agrobacterium-mediated method was used to transfer reporter genes into the Nang Thom Cho Dao, an indica rice variety. Different transformation parameters, such as selection to used hygromycin at concentrations of 0, 25, 30, 40, and 50 mg/L, bacterial density, and infected duration, were tested and optimized. The results showed that 30 mg/L of hygromycin was the most appropriate concentration for selecting transgenic callus in Nang Thom Cho Dao. In addition, the highest transformation efficiency of this Nang Thom Cho Dao rice cultivar was observed at the bacterial suspension density OD600nm of 0.1, the infected duration of 20 minutes, and the 3-day co-culture period. The presence and expression of transgenes were confirmed by gus staining and PCR with specific primers for hptII and gus genes. This transformation procedure should be used for further studies in genetic engineering of Nang Thom Cho Dao and other indica rice varieties

Simultaneously induced mutations in eIF4E genes by CRISPR/Cas9 enhance PVY resistance in tobacco

Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70% due to virus infections, especially by a potyvirus Potato virus Y (PVY). The replication of PVY relies on host factors, and eukaryotic translation initiation factor 4Es (eIF4Es) have already been identified as recessive resistance genes against potyviruses in many plant species. To investigate the molecular basis of PVY resistance in the widely cultivated allotetraploid tobacco variety K326, we developed a dual guide RNA CRISPR/Cas9 system for combinatorial gene editing of two clades, eIF4E1 (eIF4E1-S and eIF4E1-T) and eIF4E2 (eIF4E2-S and eIF4E2-T) in the eIF4E gene family comprising six members in tobacco. We screened for CRISPR/Cas9-induced mutations by heteroduplex analysis and Sanger sequencing, and monitored PVY(O) accumulation in virus challenged regenerated plants by DAS-ELISA both in T0 and T1 generations. We found that all T0 lines carrying targeted mutations in the eIF4E1-S gene displayed enhanced resistance to PVY(O) confirming previous reports. More importantly, our combinatorial approach revealed that eIF4E1-S is necessary but not sufficient for complete PVY resistance. Only the quadruple mutants harboring loss-of-function mutations in eIF4E1-S, eIF4E1-T, eIF4E2-S and eIF4E2-T showed heritable high-level resistance to PVY(O) in tobacco. Our work highlights the importance of understanding host factor redundancy in virus replication and provides a roadmap to generate virus resistance by combinatorial CRISPR/Cas9-mediated editing in non-model crop plants with complex genomes

Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds

Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits

Effects of Supplemental Light Spectra on the Composition, Production and Antimicrobial Activity of <i>Ocimum basilicum</i> L. Essential Oil

This study was performed to investigate the effects of different supplemental light spectra and doses (duration and illuminance) on the essential oil of basil (Ocimum basilicum L.) cultivated in the net-house in Vietnam during four months. Ten samples of basil aerial parts were hydrodistilled to obtain essential oils which had the average yields from 0.88 to 1.30% (v/w, dry). The oils analyzed using GC-FID and GC-MS showed that the main component was methyl chavicol (87.4–90.6%) with the highest values found in the oils of basil under lighting conditions of 6 h/day and 150–200 µmol·m−2·s−1. Additional lighting conditions caused the significant differences (p −2·s−1 in 6 h/day and (2) 43.5% Red: 43.5% Blue: 8.0% Green: 5.0% Far-Red at 100 μmol·m−2·s−1 in 6 h/day. The oils of basil in some formulas showed weak inhibitory effects on only the Bacillus subtilis strain. Different light spectra affect the biomass and essential oil production of basil, as well as the concentrations of the major components in the oil

Isolation and Cytotoxic Potency of Endophytic Fungi Associated with Dysosma difformis, a Study for the Novel Resources of Podophyllotoxin

AbstractEndophytic fungi are promising sources for the production of podophyllotoxin-an important anticancer compound, replacing depleted medical plants. In this study, the endophytes associated with Dysosma difformis-an ethnomedicinal plant species were isolated to explore novel sources of podophyllotoxin. Fifty-three endophytic fungi were isolated and identified by morphological observation and ITS-based rDNA sequencing, assigning them to 27 genera in 3 divisions. Fusarium was found the most prevalent genus with a colonization frequency of 11.11%, followed by Trametes (9.26%) and Penicillium (7.41%). Phylogenetic trees were constructed for the endophytic fungi community in two collection sites, Ha Giang and Lai Chau, revealing the adaptation of the species to the specific tissues and habitats. Cytotoxic activity of endophytic fungal extracts was investigated on cancer cell lines such as SK-LU-1, HL-60, and HepG2, demonstrating strong anti-cancer activity of six isolates belonging to Penicillium, Trametes, Purpureocillium, Aspergillus, and Ganoderma with IC50 value of lower than 10 µg/mL. The presence of podophyllotoxin was indicated in Penicillium, Trametes, Aspergillus and for the first time in Purpureocillium and Ganoderma via high-performance liquid chromatography, which implied them as a potential source of this anti-cancer compound

Flavonoids with hepatoprotective activity from the leaves of <i>Cleome viscosa</i> L.

<p>One new flavonol glycoside named visconoside C (<b>1</b>), together with seven known flavonol glycosides, quercetin 3-O-β-d-glucopyranoside 7-O-α-l-rhamnopyranoside (<b>2</b>), quercetin 7-O-α-l-rhamnopyranoside (<b>3</b>), astragalin (<b>4</b>), kaempferol 3-<i>O</i>-(4-<i>O</i>-acetyl)-<i>α</i>-l-rhamnopyranoside (<b>5</b>), kaempferol 7-<i>O</i>-<i>α</i>-l-rhamnopyranoside (<b>6</b>), kaempferitrin (<b>7</b>) and kaempferol 3-<i>O</i>-<i>β</i>-d-glucopyranoside 7-<i>O</i>-<i>α</i>-l-rhamnopyranoside (<b>8</b>) were isolated by various chromatography methods from the leaves of <i>Cleome viscosa</i> L. Their structures were elucidated by IR, UV, HR-ESI-MS and NMR (1D & 2D) experiments. The cytotoxicity and hepatoprotective activities using HepG2 human hepatoma cell line of <b>1</b> were measured by MTT assay. At the concentration of 25 μM and 50 μM, <b>1</b> showed cytotoxic activity against HepG2 cells (cell viability was decreased to 22.2 and 23.0%, respectively, compared with doxorubicin control), while at the concentration of 100 μM, <b>1</b> showed hepatoprotective activity against CCl₄-induced hepatotoxicity on HepG2 cells (34.3%, compared with quercetin control).</p