2022 In Vitro Biology Meeting, June 4 – 7, San Diego, California

P-3048 Another Successful Target in the Suborder Sternorrhyncha (Hemiptera): Green Oligonucleotide Insecticides for Aphid Control Constitutive Gene adversi-ties. are small RNAs identified in Here, we investigated the role of miR169g, a conserved plant miRNA that targets CCAAT transcription factors in regulating plant development and stress response and the underlying physiological and molecular mechanisms using transgenic analysis in an important perennial grass species, creeping bentgrass (Agrostis Stononifera). Our data indicate that miR169 overexpression in transgenics alters plant development and significantly improves tolerance to drought and salt stresses associated with modified physiological and molecular characteristics. The results obtained demonstrate the importance of miR169 as a key coordina-tor in plant development and stress responses, providing information for the development of novel biotechnology approaches to genetically engineer crops for enhanced agricultural effects of soil salinity stress. The three-wheat genotypes responded differently to salt stress and MLE foliar spray. Plant cells are an attractive production platform for biotherapeutics and other recombinant proteins due to their ability to conduct posttranslational modifications (PTMs) without playing host to animal pathogens. The green microalgae Chlamydomonas reinhardtii shares these traits while also possessing a haploid genome, simple and inexpensive media requirements, easily scalable culturing conditions, and is classified by the US Food and Drug Administration as Generally Recognized as Safe. C. reinhardtii is also a model green algae with sequenced nuclear, mitochondrial, and plastid genomes and a large body of literature exists regarding the amplification of nuclear transgene expression. By investigating two PTMs in this species, including the glycophosphatidylinositol anchor (GPI anchor) common to eukaryotes and the hydroxyproline-O-glycosylation (Hyp-O-glycosylation) specific to higher plants and green algae, we intend to design and engineer new polypeptides with these two PTMs in C. reinhardtii for improved biomedical and industrial applications. Several GPI anchor signal peptides have been identified in silico in C. reinhardtii . Testing in vivo will determine whether these peptide sequences can be correctly processed, leading to the addition of the GPI glycolipid moiety to the target protein and displaying the protein on cell surface. Certain Hyp-O-glycosylation modules, such as the tandem repeats of the “Ser-Pro” motif, have been demonstrated to be functional in C. reinhardtii but their glycosylation patterns have not been closely examined. We expect their glycosylation patterns to be different from those of plants, which will be tested in this project. This abstract represents a doctoral research plan and is work-in-progress. Transformation in the U.S. and South America. The current study has optimized a protocol using callus induction from mature seeds with both Agrobacterium -mediated and biolistic transformation of the high-yielding U.S. tropical japonica cultivar Presidio. Gene editing efficiency was tested by evaluating knock-out mutations in the phytoene desaturase ( PDS ) and young seedling albino ( YSA ) genes, which provide a visible phenotype at the seedling stage for successful knockouts. Using the optimized protocol, transformation of 648 explants with particle bombardment and 532 explants with Agrobacterium led to a 33% regeneration efficiency. The YSA targets had ambiguous phenotypes, but 60% of regenerated plants for PDS showed an albino phenotype. Sanger sequencing of edited progeny showed a number of insertions, deletions, and substitutions at the gRNA target sites. These results pave the way for more efficient gene editing of tropical japonica rice varieties. Pre-harvest sprouting (PHS) is a representative moisture disaster in wheat, and is one of the factors that greatly damage the quantity and process ability of wheat. Transcriptome analysis was performed using samples obtained through experiments derived from PHS on 'Keumgang', 'Jeonju 377ho' and 'Woori'. The RNA-seq was constructed using the Truseq standard mRNA library prep kit (Illumina, CA, USA). In addition, the 100-base paired-end procedure was implemented by Illumina Novaseq 6000 sequencing system (Illumina, CA, USA). After clean-up was performed on raw data, the analysis of Eukaryotic Orthological Groups (KOG) and Kyoto Encyclopedia of Genomes (KEGG) analysis and Gene Ontology was conducted on established transcripts. SnRK1, and this study focused on the function of SnRK1C (LOC_Os05g45420) by evaluating the morphological characteristics of its mutants developed by CRISPR/Cas9 mediated targeted mutagenesis. Earlier, these mutants were found to contain homozygous -1 to -3 bp mutations leading to early stop codon in the SnRK1C reading frame. The T1 seeds harboring homozygous mutations were subjected to phenotypic assessment along with the wild type (WT) Oryza sativa var. japonica cv. Kitaake. Plants were grown in randomized block design in a controlled environment in the greenhouse. Our results show that the mutant lines had lower shoot biomass and a lower number of seeds per panicle when compared with the WT, while no difference in the weight of roots and weight of 100 seeds was observed. These results show the importance of SnRK1C in the vegetative growth and fertility of rice. In the ongoing study, these validated SnRK1C mutant lines will be used for transcriptomic analysis to understand the underlying signaling mechanisms. breeding. The ability to access and introgress these genes into a crop improvement regimen would require a robust and timely regeneration protocol for teosinte. This also requires the right in vitro culture system that aids successful regeneration. Here, we provide a detailed protocol for teosinte regeneration via tissue culture, with an outline from seed sterilization till T0 plants. Emphasis on media composition and its effect regeneration is well discussed. We also report the first protocol for genetic transformation of teosinte ( Zea parviglumis) using biolistic bombardment of seedling-derived callus tissues. We achieved a 4% transformation frequency, using a reporter plasmid pKL2155, which carries a mutant acetolactate synthase gene (HRA, for resistance of herbicide imazapyr) and a red fluorescent protein marker gene ( tdTo-mato ). This protocol provides not only a major enabling technology for studying domestication, gene function and crea-tion of an ideotype maize plant from its wild progenitor, but also opens the door to endless access to the teosinte genome, which may be key towards a soon hunger-free world. cells in Protoplast is a versatile in modern plant biology a platform for rapid analysis of diverse signaling pathways, studying functions of cellular machineries and functional genomics Protoplast the direct delivery of into the plant a reagents. protoplast protoplast leaf mesophyll protoplast Our results indicate, and may contribute to identification of for

Cannabis sativus L. is sensitive to plant growth regulators (PGR's) during micropropagation. Methods were developed to circumvent PGR use by developing in vitro systems that are akin to greenhouse stock plant systems. In sugar-free medium, increased light intensity directly promoted photoautotrophic growth of cannabis leaves, stems and roots. In photo-mixotrophic growth (micropropagation with sugar), apical shoot tips in vitro were removed multiple times (hedging) to enhance axillary bud divisions, and the micro-cuttings were rooted in ex vitro conditions. Increased light intensity in vitro improved performance of the photo-mixotrophic micro-cutting system during multiple harvests. When works was conducted on two different genotypes, increased light intensity in vitro subsequently increased the numbers of roots formed, the numbers of leaves formed, and the length of the leaves ex vitro. Light quality in vitro affected these responses differently in the two genotypes during the multiple harvests. The nutrient concentration in the plugs during ex vitro rooting after multiple harvests of shoot tips affected the size and quality of the ex vitro micro-cutting. The subsequent effects of mineral nutrition in the rooted plug affected the growth of vegetative stock plants in the greenhouse. Research continues to optimize the development of shoot tip cuttings without PGR's using a whole plant developmental model.

P-19
Cannabis Virology in 2022 Diagnostic and Remediation Options. JOSEPH RAMAHI. Ploidy LLC. Email: joseph. ramahi@gmail.com As Cannabis production expands across inter-state and international borders, many commercial cannabis production systems are negatively impacted by molecular pathogens such as Hop Latent Viroid, Lettuce Chlorosis Virus, Beet Curly Top Virus, and the list is growing. This presentation covers the last decade of Cannabis viral pathology, from early accounts of PCIA (Putative Cannabis Infectious Agents) in 2014, to the discovery & publicity of Hop Latent Viroid, which causes "dudding" loss of flower production systems totaling in the projected billions of dollars of crop loss. Common disease phenotypes, identification programs, and remediation solutions will be presented.
Abiotic stresses, including heat, cold, drought, and salinity, are significant causes of global crop loss. Plants are sessile organisms and hence cannot move away from environmental stresses. They must adapt to stress or succumb to these conditions. High throughput sequencing technologies, including NGS (Next Generation Sequencing) technologies, assist us in understanding the physiology of plant growth and development in stressful environments. The overall goal of our lab is to understand the molecular physiological mechanisms of stress tolerance in plants. We have studied various abiotic stresses in two plant species: (1) Paulownia elongata (salinity and heat stresses) and (2) turmeric (drought and salinity stresses). Plants were undergone through various abiotic stresses as described below, followed by RNAseq to compare the transcriptomes of stressed versus unstressed plants. A miseq instrument was used to sequence barcoded cDNAs from control and stressed plants. Paulownia plants were grown with 125 mM NaCl for ten days for the salt stress experiments. RNAseq analysis revealed differential expression of 645 genes due to salt stress, and some of the salt stress-related genes we found upregulated are given below: GDSL esterase/lipase, glucan-1,3-beta-glucosidase, and glyceraldehyde-3-phosphate dehydrogenase. For the heat stress, Paulownia and turmeric plants were grown at 40C for 24 hours. For Paulownia, we identified 4,435 genes that were differentially expressed when plants experienced heat stress, and we identified the following three genes, which were highly upregulated: tonoplast dicarboxylate transporter, heat shock protein, and triacylglycerol lipase. For drought stress, water was withheld from the turmeric plants grown for 14 days. We are in the process of conducting RNAseq with the turmeric plants undergoing heat and drought stress. Understanding how plants respond to abiotic stresses will help us develop stress-tolerant crops and enhance agricultural productivity in the future. The simplicity and specificity of CRISPR-Cas9 has become the primary method of editing asexually propagated polyploid plant species. One such plant, sweetpotato, endures genetic modification challenges due to multiple allelic polymorphisms or integrations. Though CRISPR-Cas9 and Cas13 have been utilized to improve sweetpotato using naked DNA or Agrobacterium tumefaciens delivery routes, removing foreign DNA through breeding is impossible. In addition, extended-expression of the constitutive promoter-driven Cas9 integration results in increased cleavage activities, often leading to continuous off-target editing. Therefore, this study seeks to develop the first DNA-free genome editing protocol in hexaploid sweetpotato utilizing the preassembled Cas9 ribonucleoprotein (RNP). Several single guide RNAs were designed to target the eukaryotic translation initiation factor 4E superfamily. Freshly isolated sweetpotato protoplasts were edited with CRISPR-CAS9-RNP complex via polyethylene glycol (PEG) mediated transfection under various parameters. The most efficient editing factors in sweetpotato were obtained with 25% PEG mixed with a 3:1 ratio of purified preassembled Cas9 protein and in-vitro transcribed guide RNAs under a 25-minute incubation period. Different allelic InDels were obtained with an editing efficiency of 10-20%, demonstrating that PEG-mediated RNP transfection can be a viable editing system toward developing DNA-free integration tools for polyploid and vegetatively propagated crops. Work Supported by USDA-NIFA Grants: 2017-38821-26414-GE, and Tuskegee University CAENS-GWCAES-NIFA-EVANS-ALLEN Grants.

P-1017
Constitutive Expression of a miR169 Gene Alters Plant Development and Enhances Drought and Salt Tolerance in Transgenic Creeping Bentgrass. XIAOTONG CHEN, Jason Yeung, Andrew Fiorentino, Qian Hu, Morgan Kuess, and Hong Luo. Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634. Email: xiaotoc@clemson.edu Abiotic stresses, such as salinity, drought and heat, are important limiting factors for plant growth and development, significantly impacting crop production and agriculture economy. Plants have evolved various protection mechanisms coping with different environmental adversities. Manipulation of genes involved in plant stress regulation to genetically engineer enhanced performance in transgenics plays an increasingly important role in sustainable modern agriculture. MicroRNAs (miRNAs) are endogenous small non-coding RNAs identified in plants that engage in post-transcriptional target gene regulation, crucial for plant development and environmental adaptation.
Here, we investigated the role of miR169g, a conserved plant miRNA that targets CCAAT transcription factors in regulating plant development and stress response and the underlying physiological and molecular mechanisms using transgenic analysis in an important perennial grass species, creeping bentgrass (Agrostis Stononifera). Our data indicate that miR169 overexpression in transgenics alters plant development and significantly improves tolerance to drought and salt stresses associated with modified physiological and molecular characteristics. The results obtained demonstrate the importance of miR169 as a key coordinator in plant development and stress responses, providing information for the development of novel biotechnology approaches to genetically engineer crops for enhanced agricultural production.

P-1018
T-DNA-Free Gene Editing through Transient Suppression of the POLQ Gene in Plants. HEQIANG  Plant cell culture has been established as a cost-effective alternative production platform for therapeutic proteins in industrial scale due to its intrinsic safety, low cost and the capability of post-translational modification. Due to its central role in bio-production and fundamental research, tobacco BY-2 cell has been referred to as the "CHO-cell in molecular farming" and the "HeLa cell in the biology of higher plants". However, major challenges exist for the BY-2 cell bioproduction system, such as low protein production and secretion, presence of large vacuoles, and difficulty in cryopreservation. These problems can largely be attributed to the distinctive plant cell wall structure that is composed of a complex matrix of interconnected polysaccharides. The complete cell wall structure may not be crucial for in vitro cultured plant cells because optimized culture conditions are provided to the cells to support their rapid propagation. This project aims to explore top-down cellular engineering approaches to create novel cell walldeficient or "animal cell-like" plant cell lines for enhanced therapeutic protein production. Major cellulose synthase A catalytic subunit (CESA) genes were targeted to inactivate using the CRISPR/Cas9 genome editing technology. The morphology, cell wall structural composition, cell growth, bioproduction properties and transcriptomics of the knockout BY-2 were characterized. A BY-2 cell line with the CESA3 gene knocked out showed significantly reduced cellulose content (40.2% reduction) and significantly changed cell wall sugar composition. This study establishes proof-of-concept for top-down plant cell wall engineering to create new plant cell lines for the production of recombinant proteins.

P-3002
Post . There was evidence of seasonal shifts in the alpha diversity of the bacterial communities, based on the ACE index. An unexpected result is that the plant species was not a significant factor in determining the alpha or beta diversity of the communities. These results emphasize the importance of protecting forests from repeated wildfire events.

P-3003
Advanced Reporter genes are useful tools to distinguish cells expressing proteins that are easily visualized, and/or quantitatively assayed. mNeonGreen (mNG) is a relatively new fluorescent protein that is several-fold brighter than green fluorescent protein (GFP), and does not require oxygen for the catalytic formation of its chromophore. mCherry is developed to avoid background autofluorescence in tissue cultured duckweed. Mushroom luciferase and nano-luciferase provide orders of magnitude greater signal-to-noise ratio based on fluorescence, with mushroom luciferin substrate being readily synthesized in plants rather than supplied as an exogenous substrate. A limitation with using reporter genes for transient expression, is that expression can occur in both the Agrobacterium vector, as well as the target plant cells. This work describes efforts to create introncontaining reporters to avoid Agrobacterium expression that includes different intron types, locations, while correcting for cryptic splice sites created by plant codon optimization. Three separate mNG CDS locations were chosen for insertion of the potato (PIV2) intron based structure predictions and previous placements in GFP -with very different performance. Begomovirus viral vector payloads were tested with different size introns to accommodate genome size constraints (Arabidopsis, AGO and Castor bean, PDK). Five different strong constitutive promoters displayed orders of magnitude different levels of expression of mushroom luciferase-intron, including testing of 35s promoters with different truncations, with and without TMV 5'UTR. These transient reporters were developed for the highly compact Agrobacterium binary vector pLSU1 (~4.5Kb), which has the characteristic of high copy number and stability in the absence of selection pressure to enhance transient expression. This extensive effort of improving these new reporter genes was undertaken to provide tools for the plant biotechnology community with a particular focus on optimizing Agrobacterium-mediated transient gene expression.

P-3004
Establishment of an An axenic co-culture of the phloem-feeding sweet potato whitefly (Bemisia tabaci) was established on tissue cultured plants. Establishment involved surface sterilization of eggs laid on photo-trophic meristem propagated sweet potato, followed by 'rescue' of the adults before contamination emerged from vascular feeding during egg pedicil attachment. The absence of microbial contamination in serially cultured cabbage for over a year suggests that the whitefly colony is free of culturable gut microbiome. Serial subculture on four cabbage seedlings in a GA7 tissue culture vessel can produce over 500 whiteflies from a dozen inoculated adults in roughly 6 weeks. Large differences in whitefly accumulation are observed for a dozen different plant species, but nearly all display full whitefly life-cycle development. This includes plant tissue culture initiated from surface-sterilized seed: Brassicaceae (cabbage, broccoli, radish), Solanacea (pepper, tomato), Cucurbitaceae (squash, cucumber), okra, greenbriar and others, as well as meristem-propagated sweet potato, and Asian yam (Dioscorea). Proliferation on two different Dioscorea species (cayenensis and rotundata) are dramatically different suggesting a native resistance. In addition to measurements of whitefly adult accumulation with time, ongoing studies include preference (in the same tissue culture vessel) as well quantitative viability assessments based on application of models of proliferation into planted screen cages. An acylsugar knockout line of Nicotiana benthamiana allows for healthy proliferation that does not occur on wild-type plants.
Since N. benthamiana is a host to a broad range of begomovirus plant pathogens, this work sets the stage for the study of in vitro virus transmission. An autoclavable membrane feeding device has been developed to provide feeding (as demonstrated by confocal microscopic uptake of fluorescent dye). The in vitro tissue culture environment provides for sufficient biocontainment for USDA-permitted study of virus transmission.

P-3005
Genome Starch breakdown involves a complex network of enzyme processes that are influenced by plant growth stages, hormones, and environmental factors. Alpha-amylase is one of the most important starch degrading enzymes in plants. The majority of information on gene evolution, copy number, conservation, and divergence in major cereal crops is based on protein isoforms, which is frequently erroneous. Genomewide analysis of the alpha-amylase gene family was done with the goal of performing extensive analysis of the structural copies of the gene, which found at least 10 gene copies in each of the diploid cereal ancestors. Physical mapping of the genes in wheat revealed gene copies on five of the seven chromosomes, including tandem duplications, implying that the alpha-amylase gene family in cereals has evolved in a complicated way. Wheat showed 36-fold amplification of the alpha-amylase gene copy. In rice, barley, maize and sorghum, the gene amplification ranged from 7 to 12 fold. Apart from group-specific patterns in gene structure, further study indicated that gene size exhibited 14-fold difference across all crops. The amino acid sequences of distinct alpha-amylase groups indicated substantial conservation. Furthermore, domains, motifs, and functional sites analysis showed that functional sites 6 and 13 contain group-specific signatures. Furthermore, promoter analysis revealed that the duplicated alpha-amylase gene family had different gains or losses of motifs, indicating likely functional divergence. Micropropagation is a powerful technology that has revolutionized horticulture due to rapid clonal propagation generating large amounts of disease-free planting material. While the goal is to produce genetically identical planting material, growers and researchers have noted an increased rate of mutations, referred to as somaclonal variation. In the late 20 th century, researchers developed numerous markers to investigate tiny fragments of DNA, such as Restriction Fragment Length Polymorphism (RFLP), Random Amplification Polymorphic DNA (RAPD), and Simple Sequence Repeats (SSR). Historically and even today, people have used these markers to evaluate genetic fidelity and justify true-to-type statements despite the abysmal whole genomic coverage. Such statements imply plants are genetically identical to the stock plants however, this is not valid to claim with only a few markers. Nevertheless, in the 21 st century, Genotyping-by-sequencing (GBS) was developed to further broaden Next-Generation Sequencing (NGS) uses with the sequencing and genotyping of thousands of markers compared to only hundreds with older genotyping platforms. GBS can sequence several million to billions of DNA bases throughout an individual genome or pooled population in a flexible, swift, and lowcost approach. In this study, we set out to compare the effectiveness of genotyping from GBS versus SSR using micropropagated cannabis. In the end, GBS discovered significant amounts of genetic diversity that SSR could not. As a result, this raises the question of using SSRs for genetic fidelity studies and previous research conclusions using them or similar markers. Thus, this study is to compare the power of GBS to SSR in evaluating mutation rates in micropropagated cannabis clones and encourages and supports NGS-based technologies as a standard for 21 st century genotyping research.

P-3007
Direct Avocado (Persea americana Mill.) is a tropical fruit crop highly appreciated for its nutritional value. Although the demand is growing, the availability of commercial clonal trees is limited. An alternative to producing high-quality plants while reducing the cost and time is micropropagation. Generally, this method has four stages; initiation, multiplication, rooting, and acclimation. However, woody plants like avocado are known to be recalcitrant to micropropagation. Additionally, material taken from adult trees requires an extra establishment stage before multiplication. This work studied the effect of the media on the initiation and establishment stages of in vitro cultures. Bud woods from adult trees of three avocado cultivars ('Mexicola', 'Pinkerton', and 'Lamb Hass') were collected and sterilized. Then, 64 buds per cultivar were subjected to four conditions (n=192); 1) initiation and establishment in Woody Plant medium (WPM), 2) initiation in WPM and establishment in WPM with coconut water (CW), 3) initiation in WPM and establishment in persea medium (P3G) with CW, and 4) initiation in P3G and establishment in P3G with CW. Every 6 weeks, the quality of the explants (leaves, stem and base) and the number of shoots were recorded before refreshing the medium. The initiation medium did not affect the establishment of the cultures for all 3 cultivars. Instead, the media and the presence of CW determined if the shoots establish in vitro after 24 weeks; the effect of the conditions was genotype-dependant. Cultures from cv 'Mexicola' established in both WPM and P3G with CW. Cultures from cv 'Lamb Hass' declined in all media. On the other side, shoots from cv 'Pinkerton' were established in three conditions, but quality and quantity were significantly better when P3G with CW was used. The in vitro establishment of avocado bud woods from adult avocado trees depends not only on the used medium but also on the cultivar's nutritional requirements.

P-3009
Isolation Velazquez-Marquez 2021, Senn 2022). Our study (Senn 2022) identified 6 isolates with high partial 16S sequence completion and low % similarity to the closest known species. Two of these isolates were WG14 and WG23 from the Datura rootzone, which were closely related to Chryseobacterium rhizospherae and C. phosphatilyticum, respectively. WGS data was then obtained from streak plates via Chelex DNA extraction. Cluster generation and DNBseq were performed by BGIA. For assembly and 16S ID we used TruBacID. The results gave definitive ID of each isolate as Chryseobacterium sp. nov. from 16S sequences. Subsystems analysis was performed using RAST. Microscopy of the two isolates revealed gram negative rods. Later attempts to subculture the isolates after cold storage were unsuccessful. The 2 isolates exhibited functions on the ascorbate pathway; ascorbate has antioxidant activity and was linked to gains in secondary metabolite production in common bean (Gaafar et al 2020). A high number of functions associated with biotin metabolism were observed, and with the degradation of lysine. Functions were associated with cyclic amino acid precursors. WG23 had sequence differences in 2 lipidrelated processing genes when compared with the genome of WG14. The first was lipid 3 flippase, and the second was Acetyl-coenzyme A synthetase, which is closely related to chalcone synthase. For functional differences, 15 TEs and a CRISPR protein appeared in the WG 23 genome but not in WG14. Further analysis will discuss evolutionary relationships between the new types and other species, structural differences in the genes, and the role that transposons may play in the evolution of enzymes whose products may support plant growth, or enhance the virulence of the bacteria. Moringa oleifera is well known as a miracle tree. It is considered as a vegetable in many countries. Moringa leaves are rich in iron, calcium, vitamin A and C, b-carotene, phenolic, and riboflavin. It has antioxidant properties and rich with many plant growth promoters like Zeatin and cytokinin. Zeatin stimulates cell division and elongation. Qatar's wheat production is very low. This low productivity is mainly due to the lack of well-adapted bread wheat varieties and suitable production technologies. High yielding varieties and production technologies should be developed considering the limited water resources and poor soil quality to pave the way for Qatar's sustainable wheat production. Several strategies have been applied to improve wheat growth under salt stress, including using exogenous phytoprotectants, seed priming, and application of plant growth promoters. The current study aimed to investigate the performance of three wheat genotypes under salt stress as a response to foliar application of Moringa leaf extract (MLE). Three wheat genotypes (Salt tolerant, moderate and sensitive) were sown in 48 pots; five seeds were sown in each pot filled with soil. Ten days after sowing, wheat seedlings were thinned, and three uniform seedlings per pot were left for future study. The salt stress treatment was applied in 15 days old plants with 150 mM NaCl in addition to control treatment (Distilled water). Each treatment was replicated four times. Salt stress treatments were applied to the plants at 10 days interval under shade-net house condition. Pots were divided into two groups. One group was foliar sprayed with Moringa leaf extract (MLE) and the other group was foliar sprayed with distilled water. Results revealed that salt stressed plants sprayed with MLE were significantly improved in growth characteristics such as flowering time, plant height, number of tillers, flag leaf area, spike length, number of grains per spike, and grain weight by alleviating the inhibitory effects of soil salinity stress. The three-wheat genotypes responded differently to salt stress and MLE foliar spray. Sorghum bicolor (L.) Moench, the fifth most important cereal worldwide, is a multi-use crop for feed, food, forage and fuel. Identifying genes associated with its notable abiotic stress tolerances requires a detailed molecular understanding of those traits. Our in-depth sorghum transcriptome study revealed over 40% of its transcriptome was not annotated. One approach to determine gene function is transformation; however, current sorghum transformation methods are time-consuming and genotype-dependent. Our efforts to develop efficient tools to engineer, edit, annotate and characterize sorghum's genes involved morphogeneassisted transformation (MAT). The morphogenes used were Zea mays Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is an AP2/ERF transcription factor that promotes cell proliferation during embryogenesis and Wus2 maintains stem cells in the shoot meristem. This led to accelerated times to achieve transformation, nearly half the time required for classical non-MAT approaches. MAT also resulted in more genotypes amenable to transformation, including several not previously transformed or historically recalcitrant, and improved transformation efficiencies reaching 52.8% with certain constructs. Morphogenes were also included in a CRISPR/Cas9 editing construct that led to a knock-out editing efficiency of 16.7% and the first report in sorghum of a biallelic edit in the T 0 generation. Another transformation approach, termed altruistic MAT, involved introducing a gene of interest in a separate Agrobacterium from the one with morphogenes, which leads to plants with the gene of interest but without the morphogenes. A high-throughput technique was used to identify single-copy plants, as well as a novel method to determine transgene-independent integration. Together, these advances provide a straightforward path to determine gene function in numerous genotypes of this multi-use crop.

P-3013
Inducible Expression for Acceleration of Design-Build-Test-Learn Cycles in the Metabolic Engineering of Oilcane. MONI QIANDE 1 , Dang Viet Cao 1,2 , Hui Liu 2,3 , John Shanklin 2,3 , and Fredy Altpeter 1,2 . 1 University of Florida -IFAS, Gainesville, FL; 2 DOE Center for Advanced Bioenergy and Bioproducts Innovation; and 3 Brookhaven National Laboratory, Upton, NY. Email: mqiande@ufl.edu, altpeter@ufl.edu Sugarcane is an ideal target crop to fuel the emerging bioeconomy. It combines superior biomass production and photosynthetic efficiency with hyperaccumulation of sucrose in its stem, which offers great prospects for diversion to alternative products. We recently reported the generation of oilcane, a sugarcane which has been metabolically engineered for hyperaccumulation of triacylglycerol (TAG) in its vegetative biomass (Zale et al. 2016;Parajuli et al. 2020). Inducible promoters may allow lipid production at will, at a time when tissue culture or critical stages of plant development are already completed. Since sugarcane tolerates elevated temperatures between 40°C and 45°C for an extended period of time, we explored different heat shock promoters (HSP) and the combination of heat-inducible and constitutively expressed lipogenic factors to accelerate Design-Build-Test-Learn cycles. Data describing transgene expression and resulting TAG accumulation before and after activation of different lipogenic factors and their combinations will be reported.

P-3014
Application Plant cells are an attractive production platform for biotherapeutics and other recombinant proteins due to their ability to conduct posttranslational modifications (PTMs) without playing host to animal pathogens. The green microalgae Chlamydomonas reinhardtii shares these traits while also possessing a haploid genome, simple and inexpensive media requirements, easily scalable culturing conditions, and is classified by the US Food and Drug Administration as Generally Recognized as Safe. C. reinhardtii is also a model green algae with sequenced nuclear, mitochondrial, and plastid genomes and a large body of literature exists regarding the amplification of nuclear transgene expression. By investigating two PTMs in this species, including the glycophosphatidylinositol anchor (GPI anchor) common to eukaryotes and the hydroxyproline-O-glycosylation (Hyp-O-glycosylation) specific to higher plants and green algae, we intend to design and engineer new polypeptides with these two PTMs in C. reinhardtii for improved biomedical and industrial applications. Several GPI anchor signal peptides have been identified in silico in C. reinhardtii. Testing in vivo will determine whether these peptide sequences can be correctly processed, leading to the addition of the GPI glycolipid moiety to the target protein and displaying the protein on cell surface. Certain Hyp-Oglycosylation modules, such as the tandem repeats of the "Ser-Pro" motif, have been demonstrated to be functional in C. reinhardtii but their glycosylation patterns have not been closely examined. We expect their glycosylation patterns to be different from those of plants, which will be tested in this project. This abstract represents a doctoral research plan and is work-in-progress.

P-3017
Development of an Hops are a high-value specialty crop grown in the US grown primarily for brewing beer. The rapid growth of the craft beer industry has led to increased demand and overall production has steadily expanded over the past decade. Hop growers face several challenges such as disease pressure from powdery mildew leading to increased input costs. Hop germplasm improvement by conventional breeding is a long process with an 8 to 15-year period between initial cross and commercial release of a new cultivar. With the recent development of genomic resources, including a high-quality chromosome-level genome assembly in hop, there is an opportunity for the application of modern biotechnological tools to enable investigation into gene function; utilization of these tools will inform breeding programs and ultimately decrease the timeline for generating varieties with enhanced agronomic traits. Our work has sought to establish a repeatable de novo shoot organogenesis and Agrobacterium-mediated transformation protocol in selected varieties of hop. We have screened several cultivars of economic importance in the US for their regeneration potential upon induction by various applications of exogenous hormones in tissue culture. Cultivars were also screened for their ability to uptake T-DNA using constructs carrying fluorescent markers. The effect of various media amendments, environmental conditions, and physical manipulations were then evaluated on explants of the cultivars that displayed the best performance in initial screens. The cultivar 'Cascade' gives a 40 to 50% regeneration frequency on media containing 10.0 mg/L BAP and 0.25 mg/L IAA, while 'Fuggle' achieves near the same frequency with 2.0 mg/L TDZ and 0.25 mg/L IAA. We will report on the results of several experiments aimed at increasing efficacy of T-DNA delivery, regeneration frequency, and the recovery of transgenic events. To date, we have found that different genotypes have very distinctive hormone requirements for regeneration. We thank the USDA-AFRI for grant support for our work (USDA grant #2021-67013-34739).

P-3018
Development of a Research was conducted to develop methods to isolate and store barley meristem explant tissues and establish a meristem-based transformation system for targeting an important but recalcitrant variety, Gemcraft. Mature barley embryonic axis and embryonic shoot tip explants were extracted from sanitized mature seeds and dried to 12% moisture. Prior to infection, the explants were rehydrated and treated with Agrobacterium strain AGL1 containing a a binary vector for delivery of T-DNA carrying a visual marker gene and NPTII, HPTII or bar as selection genes. After four days on coculture medium, treated explants were placed on selection medium for two weeks, following which green shoots were isolated and placed on rooting medium containing the selection agent. Rooted shoots were moved to soil and grown to maturity in the greenhouse. Transgenic T0 plants were obtained in 8-10 weeks from transformation to greenhouse. Marker gene expression was tracked in multiple leaves of T0 plants, and T1 seed from different spikes were separated to monitor chimerism and effects on transgene segregation. Germline transformation was confirmed via screenable marker and molecular assays of T1 progeny.

P-3019
Construction Agrobacterium-mediated transformation is a good way to integrate transgenes into the host genome of plant as a single copy event. However, there are still obstacles to achieve the Korean wheat transformation events using Agrobacterium in Korea, and even studies using particle bombardment had been low probability. Sequentially embryogenesis is one of most important factors in wheat transformation. Therefore, research to improve the Agrobacterium-mediated transformation efficiency of the Korean wheat varieties is essential.
In the previous study, we reported the successful possibility of immature embryogenesis culture method applied 'Speed Breeding' in Korea. In this experiment, wheat transformation was applied to immature embryos collected in 'Speed breeding' and the efficiency of wheat transformation was confirmed by measuring the transgene integration rate. In addition, the recently reported GRF-GIF chimera gene was cloned from cv. Keumgang, predominant variety in Korea, and constructed, and its ability to improve regeneration and shorten the tissue culture period was confirmed. As a result, the transgene integration was confirmed by PCR screening for calli and one regenerated callus that had been selected for 5 weeks by G418 antibiotics. Immunostrip test results using putative transformants, stably transgene-integration efficiency was 0.15%. Finally, PCR screenings using the putative transgenic plants were confirmed that was integrated transgene. This study can be useful information in the development of the wheat transformation system in Korea.
[Acknowledgements] This work was carried out with the support of " Similarly, mean transformation frequencies observed from 4 replications of IL3613 were 5.1%, 0%, 6.8%, and 7.8% for cytoplasm, ER, CPT, and dTP targeting vectors, respectively. Overall, the highest transformation frequencies were achieved with CPT and dTP mediated targeting of aadA1a, while the ER vector resulted in the lowest transformation frequencies across genotypes.
These results indicate that we have developed an improved genotype-flexible, meristem-based transformation system for soybean. This novel dTP-aadA1a selection marker now underpins the WCIC standard meristem-based soybean transformation system.

P-3021
Optimizing Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent the majority of rice production in the U.S. and South America. The current study has optimized a protocol using callus induction from mature seeds with both Agrobacterium-mediated and biolistic transformation of the high-yielding U.S. tropical japonica cultivar Presidio. Gene editing efficiency was tested by evaluating knock-out mutations in the phytoene desaturase (PDS) and young seedling albino (YSA) genes, which provide a visible phenotype at the seedling stage for successful knockouts. Using the optimized protocol, transformation of 648 explants with particle bombardment and 532 explants with Agrobacterium led to a 33% regeneration efficiency. The YSA targets had ambiguous phenotypes, but 60% of regenerated plants for PDS showed an albino phenotype. Sanger sequencing of edited progeny showed a number of insertions, deletions, and substitutions at the gRNA target sites. These results pave the way for more efficient gene editing of tropical japonica rice varieties. Biotechnology for crop improvement provides excellent opportunities to efficiently enhance agricultural production. However, transgenic technology application, especially in perennial grasses, raises the question of transgene escape into the nature and its unforeseen environmental consequence. We have developed an integrated strategy that combines a dual site-specific recombination system and total sterility induction mechanisms for transgene containment and removal in switchgrass and turfgrass. This should generate transgenic products self-contained for desirable transgene, but free of undesirable foreign DNAs, upon hybridization of two parental transgenic lines harboring different chimeric gene constructs. In the first line, a FLO/LFY RNAi expression cassette is separated from an upstream promoter by the Cre recombinase target site loxP-flanked phiC31 recombinase, hygromycin resistance (hyg) and Cas9 endonuclease genes. Another line contains an active herbicide resistance gene bar, recombinase gene Cre and FLO/LFY homolog gene guide RNA (sgRNA), and an inactive stressregulating gene, AVP1. When the two transgenic lines are cross-pollinated, the phiC31 recombinase in the hybrids would excise the phiC31 target site att-flanked bar, activating Cre target site loxP-flanked Cre and FLO/LFY sgRNA, and consequently removal of itself and the sgRNA. This will activate AVP1 leading to enhanced plant stress tolerance. Cre will also excise the loxP-flanked phiC31, hyg and Cas9, activating FLO/LFY RNAi, leading to total sterility. Additionally, Cas9/sgRNA-mediated gene editing will be active in the hybrids, ensuring FLO/LFY lockout for total sterility. Similar strategy can also be adapted to other crop species.

P-3023
Transcriptome Pre-harvest sprouting (PHS) is a representative moisture disaster in wheat, and is one of the factors that greatly damage the quantity and process ability of wheat. Transcriptome analysis was performed using samples obtained through experiments derived from PHS on 'Keumgang', 'Jeonju 377ho' and 'Woori'. The RNA-seq was constructed using the Truseq standard mRNA library prep kit (Illumina, CA, USA). In addition, the 100-base paired-end procedure was implemented by Illumina Novaseq 6000 sequencing system (Illumina, CA, USA). After clean-up was performed on raw data, the analysis of Eukaryotic Orthological Groups (KOG) and Kyoto Encyclopedia of Genomes (KEGG) analysis and Gene Ontology was conducted on established transcripts. Starch and sucrose metabolism, carbohydrate metabolism, energy supply in seeds, and germination mechanisms were identified. In GO analysis, related DEGs were mainly found. In addition, Ingenuity pathway analysis (IPA) experiment was conducted to confirm the expression in the protein.
Data from proteomics and RNA-seq experiments were compared and analyzed with bioinformatics data and confirmed through heatmap and clustering. For the expression test of germination and dormancy-related DEGs and pathway-related genes, additional validation was performed by the differential expression of genes through qRT PCR. It is necessary to identify genes and mechanisms related to seed germination and dormancy through continuous research and to identify previously unknown related mechanisms or other functions. This study to understand the mechanisms and genetic factors between seed germination and dormancy can increase the value of wheat in future agriculture. In addition, an understanding of genes and protein mechanisms is expected to help clear and precise wheat breeding program.
[Acknowledgements] This work was carried out with the support of "The RND program for forest science technology (Project No. 2021400C10-2225-CA02)" Korea Forest Service, Republic of Korea. These peptides are being tested singly as well as in combination with each other in order to reveal any antagonistic, additive or synergistic interactions. A combination of Cn-AMP1 and Skh-AMP1 peptides has been found to be particularly promising. Individually, these two HDPs did not inhibit the germination of F. sambucinum conidia by much: the highest percentages of inhibited conidia for Cn-AMP1 and Skh-AMP1 were 55% and 11%, respectively. However, when tested together, Cn-AMP1 and Skh-AMP1 inhibited over 95% of fungal conidia even at very low peptide concentration (0.5 μM each), indicating a strong synergistic interaction between these two peptides. The results of this research will pave the way for development of plants with potent disease resistance.

P-3025
Transforming Plant cell culture has proved to be a safe and cost-effective bioproduction platform for therapeutic proteins. A unique feature of the plant cells is that they could serve not only as the "bio-factory," but also the oral delivery vehicle for recombinant biologics. Recent advances have demonstrated that plant cell walls, made primarily of cellulose microfibrils, can act as an excellent natural capsule for oral delivery of biologic drugs. This project aims to leverage two unique posttranslational modifications -"glycosyl-phosphatidylinositol (GPI) anchor" and "plant-specific hydroxyproline (Hyp)-O-glycosylation" -to strategically design and engineer novel anti-TNFα biomolecules in plant cells to develop a new class of oral biologic drugs for the treatment of inflammatory bowel disease (IBD). The designer anti-TNFα biomolecules consist of three functional domains: a N-terminal single-chain fragment variable (scFv) of an anti-TNFα antibody, a proprietary Hyp-O-glycosylation module comprised of tandem repeats of the "Ser-Pro" motif, or (SP) n (n= 5 to 30), and a C-terminal GPI anchor. While the GPI anchor "displays" the expressed anti-TNFα biomolecules at the plant cell surface to presumably create a high local concentration of the biologics, the (SP) n glycomodule stabilizes the protein from degradation during both the bioproduction and oral delivery processes. Designer anti-TNFα biomolecules consisting of different sizes of the (SP) n glycomodule are investigated for their accumulations in tobacco BY-2 cells, biological activity, and stability in a simulated gastric fluid, which determines the (SP) 20 module as an optimal design for the biomolecules. The therapeutic effectiveness of the orally administrated designer anti-TNFα biologic (optimal design) in mitigating the IBD symptom is assessed in a dextran sulfate sodium (DSS)-induced colitis mouse model. The research may develop a new platform to produce effective oral biologic drugs for the treatment of UC and other inflammatory diseases of colon.

P-3027
Chromosomal  , 2004). Micronuclei may originate from acentric chromosome fragments (i.e. lacking a centromere), or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division. In the present work we examined the effect of Plant tissue culture and Somaclonal variation on mitotic abnormalities and the mitotic index (MI) of onion (Allium cepa. L), the preparations were made from the root tips and Callus of three onion varieties (Giza 20, Giza 6 and BeheriRed). The mitotic index for total dividing cell was ranged between 2.88±0.2 to 5.70±0.9, MI of donor plants of all tested cultivars was higher than derived Callus. Abnormalities of micronuclei percentage ranged from 0.05% (donor plants) to 0.76% (Callus), and abnormalities percentage of derived Callus of all cultivars were higher than donor plants and were significant in many cases. Dunaliella salina is a single celled eukaryotic algae that is capable of surviving high salt concentrations and high-intensity UV radiation. The ability to alter intracellular glycerol concentration and elastic cellular structure allow the algae to survive osmotic stress from very high or low salt concentrations. The cells are also capable of producing high intracellular concentrations of beta carotene to protect themselves from light and UV stress. Our lab has established protocols for the in vitro liquid culture of Dunaliella salina cells in a medium of filtered, sterilized, nutrient-fortified seawater. The growth of the cultures is being monitored with the use of a hemocytometer, and recordings have been made of live, motile cells. Cultures vary from green to reddish brown in coloration depending on beta carotene levels, and often contain floating, clumped colonies of cells. Cells from reddish brown cultures appear colorless on a hemocytometer, and are still alive and motile. An attempt was made to grow the cells in a medium with sharply increased salt concentration (3.5 M) compared to the baseline media in order to compare cell growth in the presence and absence of hypersaline stress. However, the cells were unable to survive the sharp increase in salinity, and no growth was observed. In the future, when testing the effects of increased salt concentration on growth, salinity will be increased in small increments to better acclimate the cells, and to produce a 'kill curve' across a range of salinities. Raman spectroscopy will be used to compare the proteomes of cells exposed to increasing levels of salt and UV stress. GC-MS will be used to determine other differences in chemical composition of cells in response to stress. The transcriptomes of cells exposed to increasing levels of stressors will be compared using RNA-Seq. The long-term goals of this study are to compare the transcriptomes, proteomes, metabolomes, chlorophyll content, and photosynthetic processes of cells exposed to varying levels of salinity, UV radiation, and possibly Mars-like conditions.

P-3029
Genetic to investigate physiological and molecular responses of lentil plants under flood stress or anoxia. In accomplishing this task flooding was induced for 4 days on 6-8 week old lentils. Molecular studies involved analyzing differential expression of the following genes by qPCR: acetyl-CoA carboxylase carboxyltransferase -subunit (accD), aldolase, chloroplast Cu/Zn superoxide dismutase (SD), PBA flash photosystem II D1 (PsbA), and petD-cytochrome b6/f complex subunit IV (PetD). The results of our qPCR experiments exhibited an upregulation of accD, aldolase, and petD genes; but the downregulation of PsbA and SD. Besides gene expression studies, physiological studies were also conducted. These tests incorporated catalase activity, chlorophyll content analysis, and sugar content estimation. Flooded lentils exhibited increased levels of production of soluble sugar. In addition, electron microscopy analysis revealed higher surface roughness and more wax deposition in flooded plants. However, waterlogged lentils exhibited lowered levels of catalase activity, chlorophyll content, and volatile emissions. Gas chromatography-mass spectrometry (GC-MS) analysis of the plant volatiles revealed that several wound compounds were emitted (2-hexenal, 3-hexenal, cis-3-hexenol, 3-pentanone, and cis-3-hexenyl acetate) and that the emissions were largely downregulated in the flood-stressed plants relative to the control plants. Our results will help us to understand molecular and physiological responses of lentils under anoxia and help us develop stress-tolerant lentil plants.

P-3030
Transcriptomic Turmeric (Curcuma longa) is primarily known for containing curcumin -a bioactive compound linked to anti-inflammatory, antioxidant and anti-cancer activity in humans, and a number of other potentially beneficial effects. Although turmeric has been used as a spice in India and Asia for centuries, its production has recently seen a significant increase to ≈1.7 million tons worldwide and is projected to increase further; this can be explained by a renewed interest in dietary supplement products, which market curcumin as a healthy supplement. With the global market for curcumin totaling US$21.9M (2019), turmeric is an economically important crop. However, while ample research is available on the bioactivity of curcumin and its related compounds -curcuminoids, there are not many reports on well-designed scientific investigations into the curcumin compounds production under various environmental stressful conditions. In this experiment we explore the effects of heat stress on gene expression and physiological responses in turmeric plants, potentially providing insight into the molecular physiology of the plant. Soil-grown turmeric specimen of the experimental group will be heat stressed at 42°C for 24 hours, then each plant will be tested for chlorophyll content, photosynthesis efficiency, catalase activity and curcumin content, and have samples collected for downstream RNA sequencing using Illumina MiSeq Next-Generation Sequencing procedure. The corresponding data from the control group, grown in normal conditions and sampled at the same time as the experimental group, will determine which properties are affected by the heat stress, including differential gene expression as identified by RNA sequencing. Correlating the physiological measurements with the gene expression data may additionally elucidate the pathways involved in heat stress mitigation. Furthermore, since curcumin is known to exhibit antioxidative properties, we speculate that its content might rise after a period of heat stress, to be detected in our assay.

P-3031
Expanding The scope of research questions that can be asked is limited by the tools available, and this is especially true in crop functional genomics research. For maize, a globally important crop, only a limited selection of promoters are available for modulating transgene expression. This is an issue, as process efficiency and genetic/epigenetic research is critically dependent on promoters behaving in an expected and specific manner. Currently, there are very few characterized promoters available which impart tissue/organ/timing-specific expression, which limits maize genetic research and enhancement efforts. To address the need for a wider array of characterized maize promoter choices, we developed protocols for the identification, cloning, and characterization of promoters in maize. We then used these methods to identify and characterize promoters which confer leaf, embryo, or root-specific gene expression across a variety of developmental points. Candidate promoters were identified through analysis of RNAseq data from the Maize Gene Expression Atlas with distance metrics to an idealized promoter profile. Additional analysis was performed to identify and remove genes with long-range activating regions with available ATAC-seq data in maize. Potential cis-regulatory regions within the promoters were also identified to be in synthetic promoters. All sequences were domesticated for use in Golden Gate cloning. Both unmodified and conjugate promoters sequences were used to drive expression of the GUS screenable marker gene. The constructs were transformed into maize inbred line LH244. MUG assays to quantify GUS protein levels are ongoing, but preliminary results indicated that 4 out of the 15 promoter sequences tested showed the desired tissue/organ-specific expression.

P-3032
The Plant Genetic Engineering Network. J. VAN  . Advanced analysis of their genome and genes linked for powdery mildew can be done through high-throughput genotyping. Synteny analysis helps in identification and isolation of homologous genes and its order for powdery mildew resistance and/or sensitivity in both the species. It may be helpful in future breeding program for disease resistance and wide hybridization. Hence, synteny analysis and its significance in pea and lathyrus will be discussed for its full exploitation in powdery mildew resistance. Transformation procedures (Agrobacterium tumefaciens and particle bombardment) to facilitate genome editing via CRISPR/Cas9 were developed in our labs. The lemonwhite 1 gene (lw1), first identified in Zea mays seeds and seedlings, was targeted. Due to the importance of designing gRNA sequences that could target homeologous genes, both miscanthus and sorghum orthologs of lw1 were aligned to identify conserved regions. Using various commercial software, three gRNA were designed and all were introduced into miscanthus embryogenic calli along with the neomycin phosphotransferase II (nptII) selectable marker gene. Selection was on media containing paromomycin (100-200 mg L -1 ). Transgenic regenerants displayed various leaf colors ranging from pale green to green/white stripes to pure white. Confirmed deletions, based on Sanger sequencing, ranged from 1-26 bases and one large deletion (433 bases) spanned two gRNA target sites. This is the first report of successful genome editing in miscanthus, and was confirmed in three species -M. sacchariflorus, M. sinensis and M. × giganteus. This also demonstrated the successful targeting of a new gene that can be used as a visual marker to indicate editing has taken place.

P-3035
Development of Tissue Culture-free Genetic Gene editing technologies, especially CRISPR/Cas have revolutionized crop engineering research. However, delivery of gene editing reagents is largely depending on genetic transformation and in vitro regeneration (tissue culture) of plants. Moreover, plant regeneration and genetic transformation are highly genotype-dependent. Therefore, the lack of efficient genotype-independent plant transformation methods in several recalcitrant crops including cotton, soybean, sorghum, common bean, etc. has been a major limitation in applying gene-editing technology in crop improvement. To overcome these challenges, we are creating a synthetic cascade to express developmental regulator genes involved in stem cell activity, rapid tissue differentiation, and the regeneration process. Importantly, these developmental regulators will be applied in vivo, and we envision the development of a robust regeneration and gene-editing methodology without a need for tissue culture. Currently, we are testing a series of developmental regulators in the model plant Nicotiana benthamiana (tobacco), and once successful, it will be applied to major recalcitrant crop species.

P-3036
Phenotypic For normal growth and development and to cope with stress, plants rely on several signaling mechanisms involving the sucrose non-fermenting related protein kinase 1 (SnRK1). This gene affects many aspects such as regulation of autophagy, gene expression, sugar signals and coordination of transcriptional networks. However, the role of SnRK1 in rice development and yield is not well understood. Rice contains three functional paralogs of SnRK1, and this study focused on the function of SnRK1C (LOC_Os05g45420) by evaluating the morphological characteristics of its mutants developed by CRISPR/Cas9 mediated targeted mutagenesis. Earlier, these mutants were found to contain homozygous -1 to -3 bp mutations leading to early stop codon in the SnRK1C reading frame. The T1 seeds harboring homozygous mutations were subjected to phenotypic assessment along with the wild type (WT) Oryza sativa var. japonica cv. Kitaake. Plants were grown in randomized block design in a controlled environment in the greenhouse. Our results show that the mutant lines had lower shoot biomass and a lower number of seeds per panicle when compared with the WT, while no difference in the weight of roots and weight of 100 seeds was observed. These results show the importance of SnRK1C in the vegetative growth and fertility of rice. In the ongoing study, these validated SnRK1C mutant lines will be used for transcriptomic analysis to understand the underlying signaling mechanisms.

P-3037
CRISPR RNA-guided Integrase System for Agrobacterium Genome Editing. EPHRAIM ALIU 1,2,3 , Keunsub Lee 1,2 , and Kan Wang 1,2 .  (Vo et al., 2021, Nature Biotech.). We optimized the expression of INTEGRATE system for Agrobacterium and generated single and simultaneous multi-gene insertional mutants with high efficiency. Moreover, Sanger sequencing analysis confirmed highly specific genome-wide DNA insertion across all unique target sites. Furthermore, in conjunction with the Cre-loxP recombination system, we show that the INTEGRATE system can be utilized to achieve precise large fragment DNA deletion over 20 kilobases. This work thus provides new genetic engineering tools for Agrobacterium species to accelerate gene functional analysis.

P-3038
Transformation Teosinte (Zea mays ssp. parviglumis) is considered as the progenitor of domesticated maize (Zea mays ssp. mays), with ample genetic diversity that can be useful for maize breeding. The ability to access and introgress these genes into a crop improvement regimen would require a robust and timely regeneration protocol for teosinte. This also requires the right in vitro culture system that aids successful regeneration. Here, we provide a detailed protocol for teosinte regeneration via tissue culture, with an outline from seed sterilization till T0 plants. Emphasis on media composition and its effect regeneration is well discussed. We also report the first protocol for genetic transformation of teosinte (Zea parviglumis) using biolistic bombardment of seedling-derived callus tissues. We achieved a 4% transformation frequency, using a reporter plasmid pKL2155, which carries a mutant acetolactate synthase gene (HRA, for resistance of herbicide imazapyr) and a red fluorescent protein marker gene (tdTomato). This protocol provides not only a major enabling technology for studying domestication, gene function and creation of an ideotype maize plant from its wild progenitor, but also opens the door to endless access to the teosinte genome, which may be key towards a soon hunger-free world.

P-3039
Physiological Turmeric (Curcuma longa) has been used as a medicinal plant for nearly 4000 years. In Southern Asia, it has been used not only as spices but also as a food preservative, beauty care product and a coloring material. In Indian culture, turmeric is used for religious ceremonies as well. However, it's concerning that, in many of the parts of Southern Asia, turmeric yield is getting affected due to different abiotic stresses. Drought is one of the most concerning abiotic stresses in areas around India. But there are not many reports about how drought stress can affect turmeric plants on a molecular level. In our study we are investigating physiological and biochemical changes of turmeric plants under drought stress. Understanding differential expression of genes in physiological stressful conditions will be crucial to comprehend the entire mechanism responsible for drought stress tolerance in turmeric plants. To mimic drought stress, turmeric plants were not watered for 20 days. Physiological and molecular studies were conducted following the drought stress. Physiological studies involved photosynthetic rate, chlorophyll content, moisture content and catalase activity estimation. While for molecular studies, RNA was extracted from drought stressed and controlled plants and then Illumina Truseq kit was used to prepare barcoded cDNA libraries. Illumina Miseq sequencing system will be used to sequence the barcoded cDNAs from controlled and drought-stressed plants. Omicsbox software will be used to quantify differential gene expression among control and drought-stressed turmeric plants. Differential expression of genes will be confirmed by qPCR. Furthermore, with identified upregulated and downregulated genes and physiological studies, we can hypothesize a distinct pathway which will be likely responsible for drought stress tolerance in turmeric plants.

P-3040
Towards Genetic Engineering of Sunflower Lineage to Produce Biodiesel. F. SHAURYA, B. Bharadwaj, and C. Basu. California State University, Department of Biology, Northridge, CA. Email: shaurya.lnu.768@my.csun.edu Sunflowers (,Helianthus annuus L) are widely grown crops all around the world. It's popularly known for its high oil content, which makes it an ideal crop for producing "Biodiesel". It yields well under various conditions which differentiates it from other oil crops as it's easy to grow regardless of farm sizes. Although, global warming is enormously becoming a major concern around the world. However, we developed effective ways to reduce the green house gas emission by using renewable sources of energy, such as biodiesel. Biodiesel is a domestically produced, clean-burning, renewable substitute for petroleum diesel. Therefore, the aim of our project is to develop genetically engineered sunflower plants for producing biodiesel. In this project, we plan to express germacrene synthase gene to increase production of the sesquiterpene compounds which happen to enhance over quality of biodiesel in sunflower. Germacrene synthase is hypothesized to increase the abundance of sesquiterpene in the crop. Sesquiterpene has similar chemical properties as biodiesel. Our hypothesis is to use the sesquiterpene biosynthesis pathway to overexpress germacrene synthase which would further lead to `overproduction of biodiesel in sunflower. For this purpose we will use agrobacterium-mediated transformation methods to make our transgenic sunflower plants which are expected to produce biodiesel precursors in excess. We successfully established a tissue culture-based sunflower regeneration system in our lab. We used sterile cotyledons to initiate callus formation. The tissue culture media was a Murashige and Skoog media supplemented with NAA and BAP. We are in the process of infecting the calli with the LBA4404 strain of Agrobacterium tumefaciens harboring the germacrene synthase gene. A successful production of transgenic sunflower plants could be used for production of enhanced quality of biodiesel.
The African continent is a secondary site of diversity for sweetpotato yet relatively few of the diverse and genetically unique farmer maintained sweetpotato landraces are securely conserved ex situ. The objective of this project was to collect, document, genotype, and ensure the secure long-term conservation of sweetpotato landraces from Ghana, Kenya, Mozambique, and Sierra Leone under the International Treaty for Plant Genetic Resources for food and Agriculture (ITPGRFA) to return virus-free plantlets to farmers in the contributing countries. Collected landraces from the different countries were established in vitro and shipped to the in trust sweetpotato collection at the CIPgenebank in Peru. The target was to ship 50 landraces per country in March 2020. However, shipment of the in vitro landraces was delayed by a year due to the COVID pandemic shut down of international shipments and sadly a significant amount of material was lost in vitro. Despite this, three shipments have been made resulting in 120 landraces undergoing phytosanitary cleaning in Peru, with a fourth and final shipment of 32 landraces scheduled for mid-2022. Genetic analysis (DArTseq) confirmed the unique nature of the material which clustered by country collected and as new pool when compared to the global sweetpotato collection in the CIP-Peru genebank. Although the project had letters of commitment from all countries to transfer the material under the ITPGRFA with the Standard Material Transfer Agreement (SMTA), to date, only Ghana and Sierra Leone have signed the SMTA due to within country challenges to obtain a signed SMTA. Lessons learned are that international collection projects of vegetatively-propagated materials should plan multiple shipments and contingency plans for maintaining material for a minimum of two years after collection. Additionally, a signed SMTA should be a prerequisite prior to shipping any materials. In the end, the project has been a success with novel sweetpotato genetic resources now securely conserved and available under the ITPGRFA for use in breeding, research, and training.

P-3042
Global Vault. These include vegetatively propagated crops and crops with recalcitrant or short-lived seed. There is a dire need to reliably protect these invaluable genetic resources, particularly in developing world, because collections are at risk of loss due to biotic and abiotic and many lack longterm resources for their secure maintenance. The study concluded: 1) cryopreservation is the best long-term conservation option for these collections; 2) cryopreservation has huge benefits for long-term safety back-up; 3) there is a critical need to accelerate the cryopreservation of clonal and recalcitrant seed collections; 4) the CGIAR is ideally situated to develop a proposal and seek donor sponsorship to meet these needs. To address this challenge, the CGIAR and Global Crop Diversity Trust, have proposed a collective effort, The Global Plant Cryopreservation Initiative (GPCI) to help countries worldwide securely safeguard vegetatively propagated crops long-term in cryopreservation. Phase One of the GPCI targets germplasm collections of five of humanity's ten most important crops (potato, cassava, sweetpotato, yam, banana) as well as five crops that are vital for the developing world (cacao, coffee, taro, coconut and ulluco). The initiative will build on the success of cryopreservation in potato and banana, where more than 80% of the global intrust collections have been safeguarded in cryobanks under strict quality guidelines. It will establish regional "Center of Cryopreservation Excellence" hubs, starting with one each in Latin America, Africa, and the EU. These hubs will work with partners globally to protect germplasm collections under the terms of the International Treaty for Plant Genetic Protoplasts are plant cells with degraded cell wall that behave like animal cells in vitro. Protoplast is a versatile system in modern plant biology that provides a platform for rapid analysis of diverse signaling pathways, studying functions of cellular machineries and functional genomics screening. Protoplast allows the direct delivery of DNA, RNA or protein into the plant cell and provides a highthroughput system to validate gene-editing reagents. However, this system is less exploited in several legumes crops (including soybean), and it is because of lower protoplast yields, transfection efficiencies and lack of working protocol for plant regeneration from protoplast Moreover, protoplast isolation in several plant is mainly focused on leaf mesophyll tissues. Although, root tissues provide several advantages, root protoplast isolation, transfection and gene-editing have not been established in soybean. To overcome these bottlenecks, we are developing a new robust method for high quality protoplast isolation and transfection from soybean roots (including transgenic hairy roots). With our newly developed the highest yield, 1.3 x 10 6 and 7.3 x 10 5 ) of protoplasts were obtained from soybean roots and hairy roots respectively. More importantly, we also describe a method for gene-editing in soybean protoplasts isolated from root tissues.

P-3050
Assessing x P. alba) can be transformed by particle bombardment at scale: Over a six-week period, three researchers bombarded 9000+ explants and generated a total of 125 independent, Geneticin resistant events with a transformation efficiency of ~1.5%. From stem-derived callus to putative transformed rooted plants, in soil it took on average 6 months. A systematic approach to expedite trait development from proof-ofconcept through production is described.

P-3011
In In vitro tissue culture methods provide an excellent alternative to reduce the overharvesting of medicinally important natural flora. Spilanthes (Asteraceae) is a group of medicinally important plants, recently declared threatened in the red data book due to its overexploitation in last few years. The plant is popularly known to possess anthelmintic, antimalarial, insecticidal, anti-inflammatory, anaesthetic and immunemodulating properties. In the current study, the large scale biomass and metabolites production are explored through in vitro adventitious root culture. The adventitious root culture of S. paniculata was established at shake flask level by using modified Murashige and Skoog (MS 1962) medium and further scaled-up in a stirred tank bioreactor (3 litre capacity), fitted with marine type impellers in the same medium composition. Aeration at 0.1 vvm (volume of air per volume of liquid per minute) was optimum for biomass growth. The growth kinetics studies showed that maximum biomass and metabolites accumulation occur on 15 th days of inoculation. After that, in vitro biomass was harvested from the bioreactor, dried and grounded to a fine powder, and the extracts were prepared using organic solvent. The extracts were analysed and characterized for the presence of N-alkylamides through High performance liquid chromatography (HPLC) and Mass spectrometry (MS). The presence of spilanthol was detected and quantified by using HPLC and further confirmed through MS analysis. Apart from spilanthol, extracts showed the presence of two other major metabolites, identified as Ethane; N-ethylcyclohexa-1,5-diene-1-carboxamide and N-Isobutyl-(2E,4Z,8Z,10E/Z)-dodecatetraenamide. The mass spectrometric analysis of the collected HPLC eluted peaks confirms their molecular weight as 211 and 247, respectively. The results of the study showed the presence of important N-alkylamides in in vitro grown adventitious roots. The study also provides an alternate technique for commercial production of bioactive metabolites of S. paniculata.

P-3043
Bioinspired Bacteria-associated infections and thrombus formation are two major complications plaguing the application of bloodcontacting medical devices. To prevent these complications, we developed ultra-low fouling coatings that simultaneously mimic multiple functions of antimicrobial agents and bioinspired superhydrophobic (SH) or liquid-infused surfaces (LIS) to prevent complications over the life span of a device. Firstly, SH paints consisting of perfluorosilane-coated hydrophobic zinc oxide (ZnO) and copper (Cu) nanoparticles (NPs), and polydimethylsiloxane in tetrahydrofuran were prepared and subsequently applied onto widely used medical-grade silicone rubber (SR) tubes. Then, the SH tubes were converted to the LIS tubes upon lubrication with low-viscosity silicone oil. The morphological features, chemical compositions, and wettability of the tubes were investigated by SEM, EDX, and contact goniometry. Furthermore, the antimicrobial, antifouling, and cytotoxic properties of the tubes were thoroughly examined against clinically relevant Escherichia coli (E. coli), platelets, and fibroblast cells. The introduction of hydrophobic ZnO and Cu NPs constructed micro/nanoscale structures on the SR surface, leading to superhydrophibicity (WCA = 159.16 ± 2.23°).
After the lubricant infusion, the hierarchical microscale structures and void gaps were occupied by silicone oil, leading to a flat liquid film on the LIS surface with a WCA of 105.29 ± 1.06°. The LIS tubes exhibited superior resistance to clot formation and platelet adhesion than uncoated and SH tubes. The SH and LIS tubes significantly reduced bacterial adhesion and biofilm formation of E. coli relative to control tubes ( > 5 log and > 3 log reduction, respectively). Also, 24 h cytotoxicity evaluation showed no cytotoxicity. This study demonstrates that novel LIS coating with antifouling and antimicrobial properties has huge potential to greatly increase the longevity of intravascular devices by reducing complications due to infections and thrombosis while improving patient care and quality of life.

P-3044
Influence Tinospora cordifolia, multipurpose woody liana has recently gained global recognition for its immense health benefits in immunizing the body against diabetes, cancer, SARS-CoV-2, dengue and other ailments. Along with, the plant participates in restoration and rejuvenation of the body as well. For fulfilment of requirement of the plant for food, drugs and as biomaterial, strategic methods for the germplasm conservation should also be employed. Plant cell culture is highly applicable approach which conserves the germplasm and provides superior plants for various applications, throughout the year. In the present study, in vitro cell culture was developed using leaf explants of T. cordifolia. Among tested media treatments, MS basal medium with growth regulators, BAP and NAA, gave highest callus proliferation rate in semi-solid and liquid culture medium. T. cordifolia of Menispermaceae family predominantly contains alkaloids, phenolics and terpenoid content. Likely, chemical analyses revealed higher alkaloid yield in in vitro cultures than the mother plant. Further, for exploring enhanced therapeutic and environmental benefits of in vitro cultures of T. cordifolia, they were transformed to nano-level. This report primarily proposed the methodology for biosynthesis of silver nanoparticles using in vitro cell cultures. The fabricated nanoparticles were characterized using UV-Vis spectroscopy, FTIR, SEM-EDX and FETEM. Results affirmed average particle size ranged from 10 nm to 24 nm at wavelength of ~450 nm, while the SAED pattern revealed the crystalline nature of the in vitro synthesized nanoparticle. This study is an initiative for tailoring cell culture mediated nanostructures and evaluating their future applications.

P-3045
Effect of BA The Musa sp. commonly known as banana, belongs to the family Musaceae, and is one of the most widely distributed and consumed fruit in tropical and subtropical countries. Nutritionally, it is one of the world's leading food crops with high content of minerals, vitamins, carbohydrates, flavonoids, and phenolic compounds. Banana leaves have a wide range of applications because of their large size, flexibility, and waterproof nature. They are used for cooking, wrapping and serving food and also as decorative elements in traditional ceremonies. However, apart from the traditional uses, the leaves of the banana are an undervalued commodity with a limited commercial value, and can be considered as an agricultural industry by-product and waste. Because banana leaves are widely available in large quantities, they can be used as a source of raw materials for the green technology industry. Rutin, a flavonoid, is naturally present in banana leaves and has several commercial properties. Rutin has several important pharmacological properties, which are beneficial to health and has powerful antioxidant properties. However, rutin cannot be used efficiently because of the high price. To reduce the cost of producing rutin, it is important to find a rich source, which is widely available and inexpensive. Therefore, the present study aims to report a methodology for extraction, and quantification of rutin from three different varieties of Musa sp., namely Malbhog (Musa paradisiaca, AAB), Chini champa (Musa paradisiaca, AAB) and Bhimkol (Musa balbisiana, BB), by using High Performance Liquid Chromatography (HPLC). Of the three varieties studied, Bhimkol variety showed the highest content of rutin with a crude methanolic extract of 387 mg, containing 11% of rutin, from 1 g of dried banana leaf powder. The results indicated that banana leaves, a food industry byproduct and agricultural waste, has the potential for use as an inexpensive and new source of rutin.

P-3052
Understanding the Dynamics of Soybean Root Nodule Development Using Single Cell Transcriptome Technology. L. D'AGOSTINO, L. Yong-Villalobos, L. Herrera-Estrella, and G. Patil. Texas Tech University, Institute of Genomics for Crop Abiotic Stress Tolerance, Lubbock, TX. Email: ldagosti@ttu.edu Beneficial microbes, especially rhizobium, offer a sustainable solution for improving nutrient uptake in legume crops and the research community understands a substantial amount about the processes involved in nitrogen fixation in legumes. Root nodules are exogenous organs formed through the symbiotic relationship between legume plants and the bacterial group rhizobium. Once formed, these nodules fix atmospheric nitrogen into usable ammonia for the plant, essentially replacing the need for nitrogen fertilizer application. However, the complexity of signal reception, metabolic-flux, nodule development, N-fixation, and nutrient uptake at the 'sub-cellular level' is elusive and not investigated in soybean or any other legume. In this project, we aim to capture the temporal and subcellular expression differences in relation to the nodule's growth stages (immature, mature, post-maturity) through a process known as single-cell transcriptomics. To do so, soybean genotype (Williams 82, reference genome) is inoculated with rhizobia to induce nodulation. Nodules are then collected at three stages and nuclei are extracted. These nuclei are processed through the 10X genomics single cell pipeline in order to generate a barcoded library made up of the individual cells. The transcriptional map generated from this data will help us to understand the dynamics of the gene-regulatory network and nitrogen fixation processes at a single cell level. On a larger scale, this project will provide in-depth understanding of symbiosis between rhizobium and soybean and will provide sustainable solutions to improve nutrient uptake in soybean and other legumes.

P-3053
Overexpression Blueberries (Vaccinium corymbosum) are rich in flavonoids, which are associated with positive effects on several aspects of human health. However, blueberry flesh, in contrast to bilberry flesh, is not pigmented and does not contain the phytochemicals associated with the skin. To understand the molecular basis of this difference, we stably transformed the highbush cultivar 'Draper X Legacy' with the anthocyanin transcription factor VcMYBA1 under control of a strong constitutive promoter, 35S CaMV. We genetically transformed leaf blade explants of 'Draper X Legacy", using GV3101, a disarmed strain of Agrobacterium tumefaciens, which contained a kanamycin selectable marker cassette in a binary vector, pART27. We produced numerous transformants and noted that only the transgenic lines, in contrast to the green regeneration control lines, produced deep red calluses in vitro. Deep red shoots were regenerated via adventitious organogenesis from some of these calluses. Molecular analyses confirmed the presence of nptII and VcMYBA transgenes in calli and shoots. These deep red shoots produced red roots in rooting medium and were exflasked into sphagnum moss for acclimation. They were then transferred to potting mix and grown to flowering. Anthocyanin content of the green leaves from wild type plants and deep red leaves from transgenic plants were compared by ultra-high performance liquid chromatography, followed by liquid chromatography, mass spectrometry to identify the differences in metabolites, including anthocyanins. Flowers will be manually pollinated and we will then examine the effects of overexpression of VcMYBA1 on the anthocyanin content in the flesh of the blueberry fruit. This research contributes to understanding the molecular regulation of anthocyanins and why the flesh in blueberries, does not contain anthocyanins. Such knowledge can inform breeding strategies to increase phytochemicals in fruit flesh, to improve nutritional dietary benefits and add consumer novelty.

P-3048
Another Successful Target  Aphids are serious pests of many field and forest crops. Among them, Macrosiphoniella sanborni Gillette, a common pest of chrysanthemums, causes significant damage to the world's floriculture. In addition to their direct negative effect on young shoots and buds, M. sanborni promote the spread of plant RNA viruses and provoke the development of sooty mold. These factors negatively affect the profitability of flower production. One of the issues facing floriculture is the urgent need to reduce the use of and dependence on contact chemical insecticides to lessen the ecotoxicological load on agrocenoses and reduce the danger to human health. Results from our earlier studies have demonstrated the high efficiency of green oligonucleotide insecticides (DNA insecticides) on armored scales, soft scales, mealybugs, and psyllids. This publication will describe for the first time an experiment on the aphid, M. sanborni. The DNA insecticide Macsan-11 5'-TGT GTT CGTTA-3' was developed based on the 5.8S rRNA sequence using the DNAinsector algorithm (dnain sector. com). The experiment was carried out on isolated Chrysanthemum morifolium plants under laboratory conditions at a temperature of 23°C. The density of the insects on the chrysanthemum plants at the beginning of the experiment was 3.44 individuals/cm 2 . Seven days after the plants received a single treatment with a solution of Macsan-11 at a concentration of 100 ng/μL (mg per m 2 of leaves), aphid mortality reached 67.28±2.54%; for the control insects, the mortality rate for the same period was 5.6±0.13% (p< 0.01). The nontarget oligonucleotide 5'-ACT GAC TGACT-3' had a moderate nonspecific effect of 17.1 ± 2.07% on the insects. Thus, we have created an effective DNA insecticide for M. sanborni control that allows us to reduce the number of aphids on the target plant quickly. We plan to increase the efficiency of the Macsan-11 insecticide in further experiments using a double treatment at a daily interval. The research was funded by the grant of the Russian Science Foundation (project no. 22-16-20052