Zhang, Xing-Hai

Freshwater salinization and expanding desertification threaten global agriculture. Promise lies in salt resistance genes found in Salicornia europaea, a halophyte that thrives in high-salt conditions partly due to protein action. We focused one of its genes, SeNN24. It enhanced salt resistance in yeast and shows promise in improving crop resilience. Our research introduced SeNN24 into tobacco via agrobacterial transformation, testing the plants under salt and drought conditions. The transformed tobacco showed superior tolerance of up to 400mM NaCl and drought, maintaining health and even flowering under stress. This suggests that SeNN24 could potentially confer significant salt and drought resistance to vital crops, protecting them from environmental challenges and enhancing agricultural sustainability.

Lysine-rich KED was previously identified from wounded tobacco (Nicotiana tabacum) leaves before the alignment of protein sequences between NtKED (Nicotiana tabacum KED) and SlKED (Solanum lycopersicum KED) were discovered to display 55.1% identity. Using previously generated SlKED knockout plants by CRISPR/Cas9, we performed biological assays, to investigate the role of KED in wound response to biotic and abiotic stress. Previous studies implied that the KED gene functions as a role in the wound-induced mechanism, as well as suggested that it may also function in the plant defense system against biotic stress and insect herbivory. The results from bioassays using tobacco hornworm (Manduca sexta) have proven inconclusive thus far. Expression of KED is induced not only by mechanical wounding but also by touching such as brushing the leaves, indicating that this gene is sensitive to subtle environmental signal and may be involved in defense response against abiotic stress. To further investigate the KED gene’s role in the plant defense system, biological assays using both specialist and generalist herbivores, transcription analysis using various phytohormone mutant plants, and Evans blue cellular damage assays were performed. Our findings imply that the KED gene does not seem to have a long-term effect on insect herbivory but may have a shortterm anti-feeding effect against insect herbivores. Results from the Evans blue membrane damage assay indicate the KED gene may provide some benefit to mechanically damaged plants in a short-term period post-wounding of leaf tissues. Using the SlKED knockout as genetic tool, we conclude that this gene does not confer resistance to insect herbivores over a long-term but seems to provide a beneficial defense response in the short-term. Our membrane damage assay results also imply that this gene may be involved in membrane stabilization and repair of cellular damage after mechanical wounding.

DNA damage is one of the most harmful stress inducers in living organisms. Studies have shown that exposure to high doses of various types of radiation cause DNA sequence changes (mutation) and disturb protein synthesis, hormone balance, leaf gas exchange and enzyme activity. Recent discovery of a protein called Damage Suppressor Protein (Dsup), found in the tardigrade species Ramazzotius varieornatus, has shown to reduce the effects of radiation damage in human cell lines. We have generated multiple lines of tobacco plants expressing the Dsup gene and preformed numerous tests to show viability and response of these transgenic plants when exposed to mutagenic chemicals, UV radiation and ionizing radiation. We have also investigated Dsup function in association to DNA damage and repair in plants by analyzing the expression of related genes using RT-qPCR. We have also analyzed DNA damage from X-ray and UV treatments using an Alkaline Comet Assay. This project has the potential to help generate plants that are tolerant to more extreme stress environments, particularly DNA damage and mutation, unshielded by our atmosphere. The possibility of growing plants accompanying human space travel and extraterrestrial colonization inspires our imagination. Extremotolerant tardigrade genes such as Dsup may be a valuable avenue in helping to cultivate crops in these future endeavors.

Citrus greening, also known as Huanglongbing disease, is a phloem restrictive disease that affects orange as well as other citrus trees. The disease is caused by the gram negative bacteria Candidatus Liberibacter asiaticus. The bacteria is transmitted by the Asian psyllid, Diaphorina citri. The bacteria causes the tree to produce small and bitter oranges, the roots shrink and the leaves molt. There is currently no cure for this disease. The best way to manage citrus greening is by removing infected trees, implementing healthy planting material and controlling the psyllid population. Quantitative real time PCR (qPCR) was used to verify whether or not a given orange tree had citrus greening disease. DNA was extracted from leaves from eight trees. A qPCR analysis was performed using a primer with the bacteria DNA. Three trees were successfully diagnosed with citrus greening using this method.

Clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas) protein system, CRISPR/Cas9, uses single-guide RNA to guide Cas9 to the target site for genome editing. In this study, the CRISPR/Cas9 system was used to knockout KED in tomato (Solanum lycopersicum). KED was first identified while screening the wounded tobacco (Nicotiana tabacum) leaves. We found that alignment of the protein sequence of SlKED (Solanum lycopersicum KED) and NtKED (Nicotiana tabacum KED) showed 55.1% identity. To investigate, we generated SlKED knockout tomato plants with a single base pair deletion, a five base pair deletion and a three base pair deletion with a single base pair insertion. We performed wounding assays and analyzed gene expression and found that the wounded SlKED knockout plant showed no gene induction. Furthermore, the biological assay results revealed that the tobacco hornworm (Manduca sexta) gained more mass when fed on the SlKED knockout plant. Our studies show that the KED gene plays a role in wound-induced mechanism and suggested it may involve in the plant defense system against biological stress and insect feeding.

Advancements in biotechnology have allowed us to
study genetics and plant physiology by engineering
transgenic plants. For our research we transformed
Micro-Tom, a tomato variety developed for use in
genetic research, using Agrobacterium mediated
transformation. Within a time span of fourteen weeks,
we inserted two distinct plasmid constructs (pCAMBIA2301
and E1492). Plants have the unique ability to
regenerate their tissue and we took advantage of this
ability to regenerate the transgenic plants with antibiotic
selection. Approximately one third of the explants
endured the infection process and fourteen of these
survived in the presence of kanamycin. By the end of
the fourteenth week, eleven out of our fourteen plantlets
had fully developed roots but only four survived
to maturity. After verification with PCR and qPCR, we
found that we generated two transgenic plants. Here
we describe all the methods and techniques used to
achieve these compelling results.could be the potential
cause of this neurodegenerative disease, will help
elucidate the role of this amino acid in ALS.

The purpose of this research was to analyze the regulatory
pattern of the PUN promoter in the expression
of a marker gene, β-glucoronidase (GUS), within regenerated
tobacco plants. The genes for neomycin
phosphotransferase (NPT II) and GUS were included
in the coding region of the Ti plasmid construct. The
NPTII gene drove antibiotic resistance and was used
to select and identify homozygous lines through the segregation of the progeny. Analysis through histochemical
staining and genetic assays rendered putative
transgenic lines that were cultivated for further
assessment of progeny. First generation histochemical
analysis of 14-day tissue formation resulted in no
levels of expression for the GUS gene, which demonstrated
that the flower-specific PUN promoter was
not active in the leaf tissue. Further testing of gene
activity throughout all stages of tissue formation for
the first generation lines is required in order to assess
regulatory pattern of the PUN promoter.

The thesis consists of two chapters, and within each chapter is a different set
of methods and techniques that will be useful to me in future research endeavors. The
first is the transformation of tobacco chloroplast and the analysis of a resultant
chloroplast mutant, and the second is the establishment of a regeneration system for
the aquatic plant Typha domingensis, commonly known as cattail. The unifying
theme is plant transformation. The establishment of a regeneration system for a
potentially beneficial plant is useful for future transformations and the actual
transformation and analysis of mutants is useful for the characterization of
transformants. The chloroplast transformation was unsuccessful and analysis of the
mutation demonstrated it as a null mutation under normal growth conditions. Cattail
seeds were induced to form calli, then induced to regenerate into normal plants.

Many different ways to create mutants have been established. This research
demonstrates yet another variation of the promoter tagging technique that allows for a
single step selection of the putative transgenic plants that have a mutation in
constitutively expressed genes. While tomato transformants have not yet been
convincingly confirmed, tobacco transformation resulted in seven transgenic lines
showing resistance to high concentrations of kanamycin. Two transgenic lines were
further investigated and three putative promoters isolated. Transient expression analysis
of leaves transformed by particle bombardment with vectors carrying beta-glucuronidase
gene driven by these putative promoters suggests two of them to be functional. Further
investigation is needed to confirm the expression in the stably transformed plants as well
as cloning of the genes downstream of the functional promoters and research of their
functions.