ASHS 2024 Conference

In this interactive workshop, participants will be provided training on how to use Rosaceae (GDR, www.rosaceae.org), Vaccinium (GDV, www.vaccinoum.org) and Citrus (www.citrusgenomedb.org) specialty crop databases resources, as well as the Breeding Data Management tools, BIMS (www.breedwithbims.org), and the Field Book App for phenotype data collection. Using a case-study approach we will focus on how to use these integrated resources most efficiently for research and crop improvement efforts, how to apply FAIR data principles to sharing and submitting research data to these databases at the time of publication and facilitate a robust dialogue between researcher, breeders and the development team on needed improvements and long-term sustainability options for these resources.

This interactive workshop will bring together researchers and breeders to accelerate research and crop improvement in specialty crops. It will provide training and feedback on how to most effectively use the NIFA SCRI and National Research Support project 10 (NRSP10) funded Rosaceae, Citrus, and Vaccinium specialty crop databases and breeding data management resources (BIMS and Field Book), learn how to follow FAIR data principles, foster better two-way communication for increased feedback from researchers on further development of these resources, and continue to build a robust community around these research and crop improvement tools. The trainers include users and developers of these resources, ranging from early career to well-known and respected research enabling resource generators.

The Genome Database for Rosaceae (GDR), Genome Database for Vaccinium, and Citrus Genome Database (CGD) offer critical resources and tools to enable genomic, genetic, and breeding research for fruit, nut, and ornamental crops of great economic importance to the U.S. In addition, BIMS (BreedwithBIMS.org) and the Field Book APP provide widely used tools for managing plant breeding program data. While these resources are heavily used worldwide, many researchers are unaware of the full potential of using them and how they can contribute their own data for wider recognition and reuse. This interactive workshop aims to bridge this knowledge gap by providing hands-on training for specialty crop researchers on how to best use these resources and contribute their data. The workshop will also solicit ideas from participants on how to improve these databases and discuss the various options to make them sustainable in the long term. The workshop will bolster the utilization of integrated big data, promote future data sharing, and ensure that data is FAIR (Findable, Accessible, Interoperable, and Reusable).

The workshop aims to bring together researchers to accelerate research by more effective use of specialty crop databases and breeding data management resources, encouraging data submission at the time of publication, and gaining feedback from researchers. Through hands-on training, participants will become more familiar with the database resources and breeding data management tools, learn how to submit their data, and provide essential input for improving these databases and their long-term sustainability.

Coordinator(s)

• Dorrie Main, Washington State University, Pullman, WA, United States

Moderator(s)

• Cameron Peace, Washington State University, Horticulture, Pullman, WA, United States

Speaker/Participant(s)

• Dorrie Main, Washington State University, Pullman, WA, United States

Introduction to the Workshop and Specialty Crop Research Databases (15 mins)

• Jodi Humann, Washington State University, Horticulture, Pullman, WA, United States

How to efficiently use integrated genomics data and tools for research (20 mins)

• Sook Jung, Washington State University, Horticulture, Pullman, WA, United States
  How to efficiently use integrated genetics data and tools for research (20 mins)
  
  
• Nahla Bassil, USDA ARS, National Clonal Germplasm Repository, Corvallis, OR, United States
  How to efficiently use integrated germplasm and genotype data for research (15 mins)
  Summary:
  
  
• Trevor Rife, Clemson University, Plant and Environmental Sciences Department, Florence, SC, United States
  How to use the Field Book App for phenotypic data collection (15 mins)
• Sushan Ru, Auburn University, Auburn, United States
  How to use the Breeding Information Management System, BIMS, for Crop Improvement (20 mins)
• Cameron Peace, Washington State University, Horticulture, Pullman, WA, United States

Revolutionizing Grapevine Breeding: Overcoming Perennial Challenges with Genome Editing - Bridget Bolt
CRISPR/Cas9-Mediated Development of Low Mowing Frequency Perennial Ryegrass and Tall Fescue Varieties - Roshani Budhathoki
Precision Breeding Using CRISPR to Improve Production Traits in Blackberry - Pradeep Marri
Double CRISPR Knockout of Pectin Degrading Enzymes Improves Tomato Shelf-life While Ensuring Fruit Quality - Isabel Ortega Salazar
Functional Characterization of a Candidate Bacterial Wilt Resistance Gene in Tomato - James Duduit
Novel Short Synthetic Promoters for Constitutive Expression in Dicot Species - debao huang

Climate change poses a significant threat to perennial crops like grapevine in the USA, with projections indicating that 50-81% of acreage may become unproductive by 2040. To address this, breeding resilient varieties is imperative. However, traditional breeding for perennials is time-consuming (25-30 years) due to extended evaluation periods. Genome Editing, specifically CRISPR/Cas9, offers targeted modification potential but faces hurdles in perennial crops such as grapevine. These include somaclonal variation, inefficient transformation, transgene removal needs, and industry hesitance. We propose an efficient Genome Editing method for grapevine leveraging the systemic mobility of methylated dicistronic mRNA:tRNA molecules. This approach aims to: 1. Enable genome editing without transgene integration, negating the need for backcrossing and preventing unintended genetic changes. 2. Circumvent in-vitro culture, reducing somaclonal variation risk and preserving existing chimerism. Our method addresses key challenges in Genome Editing adoption for grapevine and could accelerate the development of climate-resilient varieties, crucial for sustainable agriculture in a changing climate.

Tall fescue (Lolium arundinacea Schreb.) and perennial ryegrass (Lolium perenne) are common cool-season turfgrass species. They are widely utilized in home lawns, athletic fields, golf courses, and roadsides. However, these grasses require frequent mowing to maintain lawn quality. Developing low-mowing frequency varieties is highly desirable because it can significantly reduce maintenance costs and fuel consumption. Gibberellin 20-oxidases (GA20ox) are key genes in the gibberellic acid (GA) biosynthesis pathway, mutations in which can induce a dwarf phenotype in plants and, therefore, reduce mowing frequency, as demonstrated by our dwarf turf varieties developed using conventional mutagenesis techniques. We have recently employed CRISPR/Cas9 technology to create knockout or knockdown mutations in the GA20oxidase1 gene to develop dwarf tall fescue and perennial ryegrass lines. The CRISPR/Cas9 construct used for editing LaGA20ox1 and LpGA20ox1 has ZmUbi and OsU6a promoters controlling the expression of Cas9 and sgRNA, respectively. We have delivered the CRISPR/Cas9 construct into tall fescue and perennial ryegrass via Agrobacterium-mediated transformation. We have observed a 35-50% reduction in plant height compared to the parental wild-type plants. Furthermore, the leaf widths in these mutants are reduced by 30-60%. Some mutants display a smoother leaf texture compared to the wild type. The T2 homozygous mutant progenies will undergo field evaluation for mowing frequency and fertilizer requirements. We anticipate that some of these gene-edited lines should exhibit a significant reduction in mowing frequency and fertilizer input.

Pairwise is a technology-focused food and agriculture company that makes tiny alterations to plant genetics to solve big problems. Powered by our best-in-class technology, Pairwise is harnessing new genomics technologies to create innovative new products. With our FulcrumTM platform, we accelerate innovation in plants and plant-based production systems, delivering value to people and our planet. Partnering with Plant Sciences, Inc. to access their elite germplasm, we created a platform to transform and edit tetraploid blackberry (Rubus sub. Rubus). We used the power of genetics and genomic tools to identify the candidate gene for thornless in blackberries and used CRISPR based gene editing to validate the trait. The identification of this gene will enable faster deployment of a thornless trait, an essential production trait, into blackberries. The gene may be broadly applicable to other Rubus species and horticultural crops such as roses.

Tomato fruit is an important and popular commodity worldwide. One of the main challenges for the fresh market tomato industry is postharvest deterioration, which is mainly determined by the rate of softening. This rate can affect tomato shelf-life, pathogen susceptibility, and fruit waste. The softening rate is regulated by multiple factors, but mainly by the pectin composition of the cell wall, which is remodeled, disassembled, and solubilized by enzymes during fruit ripening; a process that induces and determines the fruit softening in fleshy fruits. The main pectin-degrading enzymes that act on the pectin backbone are Polygalacturonases (PG) and pectate lyases (PL). In this study, we generated a double CRISPR knockout PGPL. We investigated the combined functions of SlPG2a and SlPL on fruit quality traits in postharvest, including shelf-life attributes like firmness and water loss, fruit marketability, and disease incidence. We also assessed additional attributes impacting consumer acceptance, such as taste and aroma. Our findings revealed that the tomato ripening enzymes SlPG2a and SlPL act additively, significantly affecting fruit firmness and shelf-life. Additionally, aspects of fruit quality, such as external color, sugar: acid ratio, and aroma volatiles, were improved or not affected in the double CRISPR knockout PGPL when compared to control. The discoveries of this research provide new insights into the influence of pectin backbone degradation on fruit physiology and postharvest quality, which can be used in crop improvement programs to make fruit more resilient in the supply chain without compromising consumer-based quality traits.

Bacterial wilt, caused by Ralstonia solanacearum (Rs), is an economically devastating plant pathogen that causes rapid death and has been widely distributed worldwide. Rs is a soil-borne bacterium that plugs plant xylem vessels, causing wilt and ultimately death in tomatoes (Solanum lycopersicum L.) and many other economically important crops. The most cost-effective and efficient means of managing Rs is planting resistant cultivars. However, acceptable Rs resistance in these genotypes is tightly linked to small fruit size, preventing development of BW-resistant large fruited tomato cultivars. Previous research has shown that a highly resistant cultivar's candidate resistance gene (Sl-BWR) is linked to qualitative resistance of Rs. Our preliminary data indicates that overexpression of the resistant allele in a susceptible cultivar background confers resistance comparable to the wild-type resistant allele. There is ongoing work to generate susceptible allele overexpression lines and knockout lines from the susceptible and resistant cultivars. We hypothesize the resistant allele knockouts will be susceptible to Rs, indicating that the candidate gene is the primary resistance factor. The gene could be functionally characterized to elucidate the Rs resistance mechanism in tomatoes to be deployed in a breeding program to develop resistant cultivars against bacterial wilt.

Determining the promoter motifs involved in regulating transcription, the first process of gene expression, is critical for synthetic promoter engineering. A gene’s promoter contains cis-regulatory elements, or motifs, which are binding sites for transcription factors (TFs) to initiate and drive transcription. Many bioinformatic tools have been developed for determining statistically overrepresented regions, representing sites of potential cis-regulatory elements, that are shared across groups of promoters. Combining the results produced by multiple bioinformatic tools can lead to improved detection accuracy of motifs conferring biological activity. In the present study, we compiled a set of 11 known soybean constitutive gene promoters under the assumption that some of the promoters are regulated by the same transcription factor(s). Seven bioinformatic tools capable of de novo motif discovery were used to determine potentially shared motifs within the promoters, which were then mapped back to the original promoter sequences. A total of 64 overlapping motif regions (OMRs) were commonly detected amongst the 11 constitutive promoters, and each OMR was cloned individually in front of the minimal CaMV 35S promoter driving GUSPlus reporter gene expression. Transient tobacco leaf agroinfiltration and subsequent quantitative GUS activity assays were used to determine each OMR’s ability to drive reporter gene expression. We found that 20 of the 64 bioinformatically-determined OMRs drove functional gene expression significantly higher than the basal levels conferred by the minimal 35S promoter. Of the 20 functional OMRs, 11 drove GUSPlus expression at levels from just twice that of the minimal 35S promoter up to nearly half of the full-length 35S promoter. We also transformed these 20 functional OMRs individually into Arabidopsis. While GUS staining is still ongoing, we identified a few OMRs which showed strong promoter strength in single-copied homozygous Arabidopsis seedlings. These functional OMRs are strong candidates for further characterization and can be used for crop improvement.

New insights into the Yellow2 locus and its role in beta-carotene accumulation in carrots (Daucus carota) - Michael Paulsmeyer
Overexpression of the Coding Sequence of Ma1 Leads to Enhanced Anthocyanin Biosynthesis in Apple via MYB73. - Mengxia Zhang
Rapid Detection and Coinfection Analysis of Aspergillus flavus in Peanut SeedsRapid Detection and Coinfection Analysis of Aspergillus flavus in Peanut Seeds - Emran Ali
RNA-seq Analysis Reveals Differentially Expressed Genes in Sweetpotato cv. Beauregard under Lead Stress - Mary Ann Munda
RNASeq Analysis Reveals Key Pathways Involved in Low Nitrogen Tolerance at the Onset of Storage Root Formation in Sweetpotato cv Bayou Belle - Lisa Arce
Introducing CHiDO – a No Code Genomic Prediction Software implementation for the Characterization & Integration of Driven Omics - Diego Jarquin

Carrots (Daucus carota) are a unique model for the accumulation of carotenoids. Beta-carotene accumulates in large amounts in the taproot if the proper alleles of the following three loci are present: OR, Y, and Y2. These three loci are not carotenoid biosynthetic genes but rather post-transcriptional regulation of carotenoid accumulation. The genes underlying the OR and Y loci have been characterized, but the gene underlying the Y2 locus is unknown. Through genomic and transcriptomic analyses, a single candidate that may interact with light signaling was found. To determine the function of this gene, the functional transcript from wild carrot was overexpressed in orange carrots and used in a transient infiltration assay with a GFP fusion tag in tobacco. The orange allele of this gene has a large transposon insertion that theoretically inactivates the gene. However, full length transcript can still be detected in orange carrots. This begs the question of whether the transposon is still active in certain accessions. In this study, the proportion properly assembled Y2 transcript was analyzed via qRT-PCR. A KASP marker was also developed to assist plant breeders in selection for the Y2 locus.

Anthocyanins, a group of secondary metabolites synthesized in the phenylpropanoid pathway, largely determine fruit peel color of fleshy fruits, but it is not known if its synthesis is linked to vacuolar malate accumulation that determines fruit acidity. Here, we show that when the coding sequence of Ma1, the major gene controlling apple fruit acidity, is overexpressed in ‘Royal Gala’ (RG), anthocyanin biosynthesis in the fruit peel is enhanced, corresponding to the downregulation of the expression of MYB73, an R2R3-MYB transcription factor. RNAi suppression of MYB73 expression via virus-induced gene silencing increases anthocyanin biosynthesis whereas its transient overexpression decreases anthocyanin biosynthesis in apple fruit peel. MYB73 binds to the promoter of the gene encoding UDP-glycose: flavonoid-3-O-glycosyltransferase (UFGT), the enzyme that catalyzes the last step in anthocyanin synthesis, to repress its expression. When MYB73 expression is suppressed by RNAi, UFGT expression is enhanced, leading to more anthocyanin synthesis, but this effect is blocked by RNAi suppression of UFGT expression. RNAi suppression of MYB73 enhances anthocyanin synthesis in wild-type RG apples whereas its overexpression decreases anthocyanin synthesis in Ma1-OE fruit. In the meantime, MYB73 competes with MYB1, one of the key activators of anthocyanin biosynthesis, binding to the promoter of UFGT and regulating its expression. These results indicate that MYB73 negatively regulates anthocyanin biosynthesis via repressing UFGT expression in apple peel. In Ma1-OE fruit, down-regulation of MYB73 releases UFGT from MYB73 repression and allows more MYB1 binding to UFGT promoter, leading to enhanced anthocyanin biosynthesis.

Aspergillus flavus is a widespread pathogen affecting crops like peanuts, contributing significantly to mycotoxin contamination and subsequent crop losses. Discriminating between toxigenic and non-toxigenic strains is crucial, yet conventional methods are often cumbersome and time-consuming. In this study, we developed rapid molecular tools to differentiate between these strains. Using morphological characteristics and species-specific PCR-sequencing, we identified isolates collected from peanut seeds in southern Georgia. Through primer optimization and qPCR targeting aflatoxin regulatory genes, we successfully distinguished aflatoxin-producing and non-producing isolates. Additional genes involved in aflatoxin biosynthesis were also analyzed, showing clear expression distinctions. Our findings demonstrate the specificity and efficiency of these primer sets, providing a valuable tool for managing A. flavus contamination in peanut seed lots. Additionally, research on the seed microbiome's impact on mycotoxin production remains limited. In this study, we assessed microbial communities in peanut seeds collected over various years using ITS gene sequencing. Our results revealed a diverse microbial population, including A. flavus and other fungal pathogens, highlighting the complexity of seed microbiota. This approach offers novel insights into peanut seed-associated microbiomes and aflatoxin contamination, shedding light on the correlation between microbial communities and aflatoxin pollution.

Lead (Pb) is a widespread toxic element in agricultural soils and Pb accumulation in plant roots represents a potential health risk for human beings. The sweetpotato (Ipomoea batatas L.) is a globally important root crop and one of the leading raw products for baby food processing. Limited information is available about the mechanism by which sweetpotato responds to Pb stress at the molecular level. Understanding the genetic mechanism of Pb uptake is essential for developing management approaches to mitigate Pb uptake in this crop. To address this knowledge gap, RNA-seq was used to characterize the transcriptome and identify differentially expressed genes from Pb-treated and untreated sweetpotato cv. Beauregard. Samples were taken from adventitious root tips at 5, 10, and 15 days after planting (DAP). Transcriptomic analysis revealed 4,077, 5,159, and 3,206 differentially expressed genes at 5, 10, and 15 DAP respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis shows that ABC transporters and sulfur metabolism pathways are upregulated at 5 DAP but are downregulated at 15 DAP, indicating that there may be a threshold in sweetpotato Pb tolerance. The results provide a deeper insight into the species-specific response of sweetpotato to Pb stress which can lead to the development of screening methods and evaluation of management strategies that reduce Pb uptake in this crop.

Nitrogen (N) is a key limiting macronutrient for crop growth and development and affects sweetpotato storage root formation and yield potential. In high-input production areas, excessive N application can suppress storage root formation and results in environmental pollution. The crop is also grown in low-input production systems with little or no N applications. In this study, sweetpotato cv Bayou Belle response to N deprivation during the establishment and storage root formation stages was investigated through a transcriptomic approach. RNA-seq data revealed a number of differentially expressed genes (DEGs) between N sufficient ( N) and N deficient (–N) conditions at 5, 10, and 15 days after planting (DAP). The number of significantly upregulated genes varied between timepoints. DEGs were further classified into functional categories and pathways to reveal putative functions. Gene Ontology annotation together with KEGG analysis revealed that majority of the DEGs are involved in sulfur compound metabolic process at 5 DAP and in ammonium transport for both 10 DAP and 15 DAP. These results provide valuable insights about the molecular mechanism of N regulation in sweetpotato adventitious roots undergoing storage root formation. These findings can lead to the development of tools and processes for improving N use efficiency and consistent storage root yields while reducing environmental impact in this globally important crop.

Climate change represents a significant challenge to global food security by altering environmental conditions critical to crop growth. Plant breeders can play a key role in mitigating these challenges by developing more resilient crop varieties; however, these efforts require significant investments in resources and time. In response, it is imperative to use current technologies that assimilate large biological and environmental datasets into predictive models to accelerate the research, development, and release of new improved varieties. Leveraging large and diverse data sets can improve the characterization of phenotypic responses due to environmental stimuli and genomic pulses. A better characterization of these signals holds the potential to enhance our ability to predict trait performance under changes in weather and/or soil conditions with high precision. This presentation introduces CHiDO, an easy-to-use, no-code platform designed to integrate diverse omics datasets and effectively model their interactions. With its flexibility to integrate and process data sets, CHiDO's intuitive interface allows users to explore historical data, formulate hypotheses, and optimize data collection strategies for future scenarios. The platform's mission emphasizes global accessibility, democratizing statistical solutions for situations where professional ability in data processing and data analysis is not available.

This year’s PB session will combine a keynote speaker session, business meeting and PB 2024 award ceremony together.
1. Keynote speaker: Hui Duan, USDA-ARS-REE (45 minutes) Biotechnology of Woody Ornamental Plants
2. PB business meeting (30 minutes) Chair annual report, elect new chair, chair elected and secretory. Plan next year’s work.
3. 2024 Plant Biotechnology Interest Group annual award ceremony (45 minutes)

Leaf Culture and Regeneration in Two Cultivated Strawberries (Fragaria x ananassa) - Kedong Da
Leaf Culture and Regeneration in Hydrangea Macrophylla - Kedong Da
Immature Embryo Germination of Camellia sinensis (L) O. Kuntze. - Sarita Paudel
Development of an In Vitro Transformation System for Gene Editing Powdery Mildew Resistance in Gerbera jamesonni - Heather Gladfelter
Declining Phosphorus Availability Increases Sucrose Synthase Activity and Storage Root Formation in Sweetpotato cv Beauregard Adventitious Roots - Marissa Barbosa

Strawberry (Fragaria × ananassa) holds significant commercial importance as a fruit crop. Tissue culture regeneration techniques offer a promising avenue for rapid propagation, genomic study, and biotech breeding, including genome editing and genetic transformation. However, literature reports on cultivated variety-based strawberry regeneration and transformation/genome editing is limited. This study aims to investigate the influence of various tissue culture conditions on the regeneration efficiency of two major cultivated strawberry varieties, 'Chandler' and 'Festival', by assessing the impact of different culture conditions and hormonal treatments on the regeneration of selected varieties' in vitro leaf cultures. The first three open leaves from 4-week-old in vitro cultures were collected and cultured on Murashige and Skoog (MS) medium supplemented with varying concentrations of Thidiazuron (TDZ) (0, 0.5, 1, 2, 4 mg/L) combined with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.2 mg/L). The cultures were maintained in a growth chamber with varying light (dark/light) and photoperiod conditions. Regeneration parameters, including regeneration rate, regeneration number, and root development, were recorded over an 8-week period. Preliminary results indicate a significant influence of TDZ concentrations and light conditions on shoot regeneration rate and number. The combination of TDZ at 1 mg/L and 2,4-D at 0.2 mg/L under a 20-day dark pretreatment followed by a light (16/8-hour) condition resulted in the highest regeneration efficiency. Rooting was observed when the cultures were transferred to a hormone-free MS medium.

Hydrangea is a genus of flowering plants that includes a diverse range of species and cultivars. These plants are known for their large, showy flower heads and are commonly grown as ornamental shrubs in gardens and landscapes. Hydrangea macrophylla, commonly known as bigleaf hydrangea, is a popular ornamental shrub in the hydrangeaceae family, well-known for its large, showy flower heads and broad leaves. Tissue culture regeneration techniques offer a promising avenue for rapid propagation, genomic study, and biotech breeding, including genome editing and genetic transformation. However, literature on hydrangea macrophylla regeneration and transformation/genome editing report is limited. This study aims to investigate the influence of various tissue culture conditions on the regeneration efficiency of Hydrangea macrophylla 'Blaumeise,' 'H2020-59,' and 'H15298' by assessing the impact of different culture conditions and hormonal treatments on the regeneration of selected varieties in vitro leaf cultures. The first three open leaves from 4-week-old in vitro cultures were collected and cultured on Murashige and Skoog (MS) medium, supplemented with varying concentrations of 6-Benzylaminopurine (BAP) (0, 0.5, 1, 2 mg/L) combined with 1-Naphthaleneacetic Acid (NAA) (0.01 mg/L). The cultures were maintained in a growth chamber with varying light (dark or light) and photoperiod conditions. Regeneration parameters, including regeneration rate, regeneration number, and root development, were recorded over a 4-week period. Preliminary results indicate a significant influence of BA concentrations and light conditions on shoot regeneration rate and number. The combination of BA at 2 mg/L and NAA at 0.01 mg/L under dark conditions resulted in the highest regeneration efficiency. Rooting was observed when the cultures were transferred to a hormone-free MS medium.

Camellia sinensis (L.) O. Kuntze is a woody perennial, evergreen shrub or small tree native to Southwest China, whose leaves are processed into various types of teas for consumption. Harvesting mature tea seeds typically requires 12 to 14 months, with an additional 2 to 3 months needed for germination using traditional methods. In an effort to expedite this breeding cycle, we conducted aseptic germination experiments on immature embryos of Camellia sinensis fruits collected from different months, spanning from May to September. Visible embryos were observed in immature fruits harvested in late July, exhibiting an average size of 2.17 ± 0.33 cm, and demonstrated germination capabilities only after mid-August. These embryos were excised and cultured on Murashige and Skoog (MS) medium supplemented with 3% sucrose and 0.65% agar. Cultures were maintained in a growth chamber set at 24°C under a 16-hour photoperiod. To enhance the germination process, cultures were kept in darkness for the initial two weeks. Immature embryos initiated germination approximately one to two weeks after the initiation of culture. After two months, plantlets were transplanted into a substrate consisting of peat moss and perlite (1:1 v/v) and acclimatized in a mist system within a greenhouse. Five month post-culture initiation, the average shoot length, root length, and leaf number of transplanted plantlets were recorded as 2.24 ± 0.89 cm, 5.61 ± 4.58 cm, and 8.36 ± 2.84, respectively. Preliminary findings suggest promising outcomes, demonstrating the feasibility of in-vitro embryo germination in tea plants. This methodology holds the potential to mitigate late-term abortion of hybrid embryos and significantly shorten the breeding cycle, critical aspects in breeding triploid tea varieties through crossbreeding diploid and tetraploid tea plants.

Gerbera is among the significant players in the global cut flower market, valued at 30 billion USD in 2023. Gerbera daisies, along with chrysanthemums are the largest segment of the cut flower market with a share of 40%. The quality and quantity of gerbera cut flower production is negatively impacted by the fungal disease powdery mildew causing a significant loss of revenue. Effects from powdery mildew infection include reduced flower quality, stunted growth, decreased flower longevity, and decreased marketability. Powdery mildew is currently controlled using fungicides which increases the cost of cut flower production and has the potential for the fungus to develop resistance to the fungicides. One strategy is to use gene editing and CRISPR technology to produce gerbera plants resistant to powdery mildew. The gene “mlo” which stands for “Mildew Locus O” plays a crucial role in the plant’s defense against powdery mildew infections. Plants with mutations in the “mlo” gene exhibit a type of broad-spectrum resistance providing protection against various strains of powdery mildew. We developed a tissue culture regeneration and transformation system for the Gerbera jamesonii cultivar ‘Flori Line Maxi Yellow’ which is sensitive to powdery mildew. The Agrobacterium tumefaciens strain GV3101 containing a binary plasmid encoding for an enhanced GFP marker gene and hygromycin selection gene was used to develop the transformation system. Young, 10mm sized in vitro leaves with part of the petiole were used as explants. A sensitivity test with the explants showed hygromycin at 7.5 mg/L was optimal for selection of callus with positive GFP expression. The next step is to regenerate GFP plants to confirm successful transformation and design CRISPR constructs to inactivate the “mlo” gene in the powdery mildew sensitive gerbera cultivar via gene editing.

Sucrose Synthase (SuSy) plays a crucial role in sugar metabolism mainly in the sink tissues of plants. In sweetpotato, increased SuSy activity has been associated with increased storage root development and correlated with sink strength. However, little is known about the specific variables associated with increased SuSy activity. Evidence from model systems supports the hypothesis that phosphorus (P) starvation is associated with increased accumulation of carbohydrates in roots. In the first study, we measured SuSy gene expression in ‘Beauregard’ sweetpotato grown in a split root system and subjected to the following P treatments: positive control ( / ), negative control (0/0), declining P (-/-), and split P ( /-). The declining P treatment corresponded to 25, 50, 75 and 0% progressive reduction in P and was imposed on days 6, 9, 12, and 15, respectively. A second study was conducted to measure storage root development at 50 days. The (-/-) treatment was associated with increased SuSy activity in developing adventitious starting at 11 days after planting. Moreover, plants grown with the declining P treatment produced storage roots with larger diameter (>2cm) significantly higher than the positive control. Decreased SuSy activity was associated with reduction in storage root number among P-deficient (0/0) plants. Taken together, these results support the hypothesis that P availability in the root zone is associated with sink strength and storage root formation signaling in adventitious roots. These findings can be used to develop tools and management practices to increase P fertilizer efficiency for consistent storage root yields in sweetpotato.

A forum for discussion of potential collaborations with regards to technology in horticulture – i.e. biotechnology, UAVs, cameras, sensors, artificial intelligence, etc.

PoDRM2: Unveiling a Key Regulator of Biomass Production and Starch Accumulation in Arabidopsis thaliana - JianHuang
Efficient high molecular weight DNA isolation and whole genome sequencing of papaya for molecular applications - Jon Suzuki
Tissue Culture Regeneration of Miscanthus Sinensis ’Gracillimus’ - Kedong Da
In Vitro Shoot Regeneration Protocol For Southern Highbush Blueberry (SHB) Cultivars - Anandi Karn

Starch, a vital dietary component and crucial for bio-ethanol generation, is synthesized by plants during photosynthesis. Augmenting starch output holds promise for human and animal nutrition, as well as bioenergy. Our previous work involved cloning the homolog gene DRM2 from Purslane and subsequently overexpressing PoDRM2 in Arabidopsis. Comparative analysis between wild-type Columbia and homozygous PoDRM2 transgenic lines revealed a substantial increase in plant size and nearly a 90% rise in fresh biomass per plant in PoDRM2 lines, indicating a potentially heightened efficiency in photosynthesis. We conducted further investigations into starch synthesis and accumulation in leaves. Iodine staining revealed that PoDRM2 transgenic Arabidopsis lines accumulated significantly more starch than the control under both dark and light conditions. Additionally, total carbohydrates in the leaves of transgenic lines more than doubled that of the wild type. Furthermore, PoDRM2 lines exhibited higher chlorophyll content compared to the control. These findings strongly indicate that PoDRM2 serves as a crucial regulator of starch accumulation. PoDRM2, encoding a methyltransferase, was implicated in altering the methylation status of over 2,500 genes through genome-wide bisulfite sequencing. Notably, 55 out of 61 genes involved in the photosynthesis pathway were affected, underscoring the significant role of DNA methylation in regulating starch accumulation and photosynthesis in plants.

Plant whole genome sequencing provides detailed information on gene content, genome organization, and evolutionary relationships as well as supports biotechnological applications such as gene editing. The first 3X draft genome sequence of papaya based on whole genome shotgun reads from the transgenic ‘SunUp’ papaya cultivar was published in 2008. Since then, advancements in sequencing and whole genome assembly enabled a near complete sequence of ‘SunUp’ and a detailed picture of events resulting from particle gun-mediated transformation. With current technology, the 372 Mb genome size of papaya makes it tractable for routine whole genome sequencing to characterize different cultivars and molecular events. In this study, we improved ease and speed of preparation, efficiency of recovery, and DNA quality through a combination of classical and contemporary plant nuclei or high molecular weight DNA isolation methods. Leveraging Hi-Fi sequencing and Hi-C technology, we achieved rapid chromosome-level sequence assembly of two local Hawaiian cultivars, Kapoho and Waimanalo. The assembled genomes of Kapoho and Waimanalo spanned 341.6 Mb and 337.4 Mb, respectively, with a total of 20,343 and 20,165 annotated protein-coding genes.

The genus Miscanthus is considered an ideal choice for both ornamental and biofuel purposes, owing to its appealing aesthetics and significant potential for high-energy biomass production. Traditional breeding efforts in Miscanthus have predominantly focused on enhancing nutrient efficiency and tolerance to both biotic and abiotic stresses. However, these endeavors are often time-consuming. The emergence of plant genome editing technologies has opened up a new and efficient avenue for Miscanthus breeding. These innovative techniques hold promise for accelerating the breeding process, allowing for more rapid and targeted improvements in desired traits. The development of an efficient plant regeneration system is crucial for the application of modern genome editing technologies in Miscanthus breeding and for achieving large-scale biomass production. Among the Miscanthus species, Miscanthus sinensis poses a particular challenge in tissue culture regeneration. In this report, we present an effective system for callus induction and regeneration in Miscanthus sinensis. Callus was induced from the stems of in vitro-cultured Miscanthus sinensis 'Gracillimus' using a modified MS media supplemented with varying levels of 2,4-D. Regeneration-competent callus was achieved through continuous selection on the callus maintenance/selection medium over a period of 6 months. Remarkably, 100% of the callus successfully regenerated new shoots on a modified MS medium containing Benzylaminopurine (6-BA) and α-Naphthaleneacetic acid (NAA). This marks the first efficient 'Gracillimus' regeneration system using in vitro culture as the starting material. The established system demonstrates a high potential for the micropropagation of Miscanthus sinensis 'Gracillimus' with a propagation rate of 3.5. Currently, efforts are underway for genome editing of Miscanthus sinensis utilizing this established system.

Blueberry (Vaccinium sp. L.) is one of the most important fruit crops from the Ericaceae family and the highbush blueberry (Vaccinium corymbosum) is the most widely grown species. It's popularity is increasing day-by-day because of their unique flavor and rich nutritional content. Consequently, significant efforts have been made to develop superior cultivars with high yield, biotic and abiotic stress resistance using conventional breeding. However, due to high heterozygosity, polyploidy and long juvenile period, traditional breeding approaches can often be tedious and time consuming. Therefore, there is need to integrate modern precision breeding tools with traditional ones, to accelerate blueberry crop improvement. However, the success of novel biotechnological tools like gene editing and conventional transformation relies on successful shoot regeneration system. Many studies in blueberry show a lack of reliable regeneration protocols and their genotype-dependency. Furthermore, most of the reported regeneration studies have been conducted on northern highbush blueberry (NHB) cultivars. Therefore, the current study aims to develop shoot regeneration protocol for seven important southern highbush blueberry (SHB) cultivars (Colossus, Optimus, Albus, Arcadia, Keecrsip, FL 14-242