DOMESTICATING VANILLA THROUGH PLANT BREEDING AND FUNCTIONAL GENOMICS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1027898
• Grant No.: 2022-67013-36248
• Cumulative Award Amt.: $299,745.00
• Proposal No.: 2021-07610
• Project Start Date: Dec 1, 2021
• Project End Date: Nov 30, 2024
• Grant Year: 2022
• Program Code: [A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
(N/A)

Non Technical Summary
Vanilla has potential to be among the highest grossing agricultural commodities, and could revitalize some of our distressed agricultural industry (Fig. 1). Spices like vanilla comprise a small portion of our diet, but have major impacts on the sensory quality of our food. Vanilla extract comes from the cured beans of either Vanilla planifolia or V. x tahitensis as legally defined by the FDA. V. planifolia is native to North and Central America, but Madagascar is today's leading vanilla grower. Domestic vanilla production is becoming increasingly attractive as international supplies are perennially strained and demand for vanilla extract increases as companies like MARS, Unilever, and Nestle pledge to remove artificial ingredients from their products. Most vanillin, the primary flavor component of vanilla extract, is chemically synthesized, but vanilla extract has the potential to support growers in Florida, Hawaii, and Puerto Rico striving to meet an evolving consumer base favoring local, organic, and natural products. Vanilla is somewhat unique in that the species not been domesticated through plant improvement, and today's industry relies cultivated, wild clones. Therefore, domesticating vanilla could have major impacts on productivity and profitability. The short-term goals of this project are 1) identify superior vanilla accessions for domestic commercialization, and 2) develop breeding tools for vanilla. The long-term goals and outcomes of this program are to 1) create a self-sustaining domestic vanilla industry, and 2) train field-based plant breeders.Domestic vanilla production would increase vanilla supply, enhance quality, and improve raw material traceability. Domestic production could create new specialty crop opportunities for growers leveraging V. planifolia or V. x tahitensis. The high value, low supply, and consistent demand for vanilla could help to increase income stability and profitability for domestic growers especially with the release of new cultivars. There is also great potential for optimized production systems to reduce production costs and increase sustainability through local production. ?

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1099	1081	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1099 - Tropical/subtropical fruit, general/other;

Field Of Science
1081 - Breeding;

Keywords

genomics

plant breeding

vanilla

Goals / Objectives
Vanilla has potential to be among the highest grossing agricultural commodities, and could revitalize some of our distressed agricultural industry (Fig. 1). Spices like vanilla comprise a small portion of our diet, but have major impacts on the sensory quality of our food. Vanilla extract comes from the cured beans of either Vanilla planifolia or V. x tahitensis as legally defined by the FDA. V. planifolia is native to North and Central America, but Madagascar is today's leading vanilla grower. Domestic vanilla production is becoming increasingly attractive as international supplies are perennially strained and demand for vanilla extract increases as companies like MARS, Unilever, and Nestle pledge to remove artificial ingredients from their products. Most vanillin, the primary flavor component of vanilla extract, is chemically synthesized, but vanilla extract has the potential to support growers in Florida, Hawaii, and Puerto Rico striving to meet an evolving consumer base favoring local, organic, and natural products. Vanilla is somewhat unique in that the species not been domesticated through plant improvement, and today's industry relies cultivated, wild clones. Therefore, domesticating vanilla could have major impacts on productivity and profitability. The short-term goals of this project are 1) identify superior vanilla accessions for domestic commercialization, and 2) develop breeding tools for vanilla. The long-term goals and outcomes of this program are to 1) create a self-sustaining domestic vanilla industry, and 2) train field-based plant breeders.

Project Methods
Objective 1.1 Characterize accessions in the vanilla collection, and 1.2 Establish new vanilla populations. Techniques. Bean length, bean weight, and days to harvest will be measured for each accession each year. Beans will be harvested when the flower end of the bean begins to yellow indicating peak maturity. Beans will be conventionally cured and extracted as previously described. Filtered extracts are analyzed by HPLC using authentic standards for vanillin and glucovanillin. A basic HPLC method for quantifying vanillin has already been published. We are budgeting for 500-600 HPLC samples each year. For Objective 1.2, breeding populations will be established as we have previously reported . Breeding parents will be selected using preliminary data on bean length, vanillin content, and adaptability to Florida's growing conditions. Interspecific hybrids will include crosses with V. planifolia, the commercial species, and each other species possible. Our current shade house has room for 4,600 individual vanilla trellises.Objective 2.1 includes optimizing gene delivery with multiple gold particle sizes (0.6, 1.0, and 1.6 µm), bombardment pressures (450 to 1,800 psi), and explant spacing within the PDS-1000 gene gun tested in duplicate. Treated vanilla protocorms will be maintained on O139 orchid replate medium amended with 2 mg/ml BA (6-benzylaminopurine). Hygromycin (50 mg/L) will be used to select for transformed cell masses 2 weeks after bombardment. For Objective 2.2, we propose to test CRISPR-based gene editing in vanilla in combination with rapid cycle breeding cycle seed-to- seed generations in vitro as has been done for other orchid species. The construct includes three sites for gRNA target cloning, the GFP reporter, hygromycin resistance, and V. planifolia FT (flowering time) overexpression. Candidate CRISPR targets include phytoene desaturase (PDS, albino phenotype) and CPLC (vanillin biosynthesis). Vanilla PDS and CPLP have 174 and 139 high specificity NGG PAM motifs, respectively, using CRISPOR. This construct will be delivered using the results from Objective 2.1. CRISPR edits will be verified by extracting DNA from GFP-positive calli, PCR amplification of gene targets using gene-specific primers, cloning of amplicons, and sequencing 8 clones per transformant.Objective 3. KASP assays will be designed using primer design tools available through LGC genomics and will be designed to target six CPLP SNPs and three PAL SNPs that distinguish alleles. The KASP assay includes a DNA template (25 ng), two target-specific primers with adapters (12 µM), and the KASP mastermix that includes fluorophore-labeled adapters and reaction enzymes. The assays are run on our Roche LightCycler. All accessions in the collection will be screened for correlation between SNPs and vanillin content.Objective 4. We propose setting camera traps to monitor both V. planifolia and V. phaeantha through the flowering season (~6 weeks each year). Vanilla flowers must be pollinated during a 6-hour window. Any potential pollinator visits will be quantified by the number of flowers visited per pollinator, pollinator time spent working a single flower, the number of pollinators visiting flower, and other metrics. A few pollinators will be captured for imaging and identification.Objective 5. Extension activities will be enhanced through the results of this study. An extension agent network for vanilla has already been established and includes representation from the six southernmost Florida counties. Tropical fruit in-service trainings are offered every other year for extension agents. Symposia for growers and the general public will be offered once per year. Pollination and harvesting/curing demonstrations will be offered once per year.

Progress 12/01/23 to 11/30/24

Outputs
Target Audience:The target audience for the last performing period of this project includes commercial growers, industry collaborators, academics, and students. Vanilla is a high-value tropical crop particularly well-suited for small-scale growers in U.S. tropical regions, where agriculture faces significant challenges from urbanization and development pressures. Industry partners interested in modern horticultural practices, elite agronomic traits, and improved supply chains have actively engaged in the project's collaborations. Ethnic minority and socially disadvantaged growers have shown interest in leveraging vanilla production to establish small agricultural businesses, integrating farming with tourism, recreational, and educational programs. The economic potential and unique characteristics of vanilla have positioned it as a top choice for these growers. As the project progressed, the scope of its target audience has expanded from national to international stakeholders, driven by research advancements in germplasm conservation and diversity evaluation. Outreach efforts have included invited presentations, regular tours of laboratory and field vanilla collections, internships, professional workshops, extension/outreach training programs, and scientific publications. In 2024, nine presentations were delivered as part of this project. Four were presented at professional academic meetings, including the 2024 National Association of Plant Breeding Conference. The remaining five were directed at growers, county extension agents, and plant breeding professionals to disseminate key research findings. These presentations were conducted by the Principal Investigator (PI) and a graduate student supported by the project. A new extension factsheet was developed to educate growers interested in cultivating vanilla. Furthermore, the PI has maintained active communication with brokers and growers abroad through email and phone calls to monitor market trends, customer demands, and other critical crop information. These combined efforts have significantly enhanced knowledge-sharing and fostered broader engagement within the vanilla industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A PhD student specializing in plant breeding has been extensively trained in molecular biology, bioinformatics, plant breeding, and horticulture. This student successfully defended his dissertation and is set to graduate in December 2024. To ensure continuity in research activities, a master's student was recruited to carry the project forward. Additionally, a high school student trained under this project has since enrolled in college to pursue a bachelor's degree in horticulture. The project also provided research training experiences to volunteers interested in learning about vanilla cultivation, equipping them with knowledge and skills for future vanilla commercial production. How have the results been disseminated to communities of interest?In 2024, we disseminated research findings through an In-Service Training program designed for county extension faculty and professionals specializing in commercial horticulture production and natural resources. A total of 28 attendees participated, gaining a deeper understanding of vanilla cultivation and its potential. We developed a new extension publication to replace the outdated version, incorporating updated, scientifically validated information. Additionally, two more extension publications focusing on postharvest and curing processes are currently under review to address grower interests. Our research outputs were shared at four professional conferences through oral presentations, along with one poster presentation. Furthermore, one manuscript has been published, and another is in the final stages of the review process. What do you plan to do during the next reporting period to accomplish the goals?Although this marks the final reporting period for this project, the support provided by this funding has enabled us to secure additional internal and external resources to address critical challenges, including horticultural production and disease and pest management. Although not encouraged by the grant reviewers, we feel it is very important to develop biotechnology tools for this crop given some of the challenges this crop is facing. To this end, we developed two protocols to achieve both gene editing and genetic engineering in vanilla as proof of concept. This work led to two peer reviewed research articles. also developed These efforts have laid a strong foundation for continued advancements in vanilla cultivation in the tropical regions of the U.S. We remain committed to expanding our germplasm evaluation and breeding programs, with a focus on identifying and developing distinct genetic materials. Particular emphasis will be placed on unique flavor profiles and enhanced disease resistance to support the long-term sustainability and profitability of vanilla production.

Impacts
What was accomplished under these goals? Objective 1) Phenotype a vanilla diversity collection and establish plant breeding populations Objective 1.1 Characterize accessions in the vanilla collection for key quality metrics Vanilla beans take nine months from pollination to maturity. In 2024, the harvest of vanilla accessions was limited to 25 due to the impact of a prevalent disease and extreme heat reported in 2023. Despite this challenge, we successfully collected data on bean weight, length, and vanillin content, revealing significant differences among the accessions in our collection. Although the number of harvested accessions was limited, we evaluated 35 commercial vanilla beans representing diverse flavor profiles from around the world through international collaboration. Flavor profiling of the evaluated accessions highlighted a few selections with superior flavor quality and exceptional potential for commercial production. These identified accessions represent valuable genetic resources for breeding programs aimed at improving vanilla's agronomic and commercial traits. Objective 1.2 Establish new vanilla populations through plant breeding In 2024, we expanded four breeding populations initially developed in 2023 and created four additional populations with a specific focus on improving two key commercial traits: vanillin content and bean dehiscence. The parental selections for the expanded populations were based on vanillin content data from 2022 and 2023. The newly created populations were developed using one accession known for its non-splitting beans and three accessions with high vanillin content. These parental accessions included both Vanilla planifolia and Vanilla pompona. Some of the expanded and newly developed hybrids are currently in tissue culture and will be hardened to adapt to field conditions in 2025. The majority of seeds are still in the germination stage due to the unique requirements of propagating vanilla orchids. These efforts are critical steps toward achieving significant genetic improvements in this economically valuable crop. We developed the first vanilla haploid induction protocol and few haploid breeding lines under this objective. The results were published as peer-reviewed articles. Objective 1.3 Expand the genetic diversity of the vanilla collection In 2024, we introduced 14 additional accessions of commercial vanilla species from diverse sources. These newly acquired accessions, known for producing commercial-grade beans, come from varied genetic backgrounds. Using genotyping-by-sequencing techniques, we genetically characterized these accessions and compared them with our existing collection. The addition of these accessions significantly enhances the genetic diversity within our collection of commercial vanilla species. This expanded genetic variation is a critical resource for developing unique and improved vanilla varieties with desirable traits. Objective 2) Develop KASP markers for high-value traits Objective 2.1 Test phenylalanine ammonia-lyase (PAL) variants for increased vanillin The work conducted in the previous year did not reveal a significant correlation between the PAL-derived KASP marker and vanillin content. Due to the limited bean yield in 2024, we were unable to advance this sub-objective by incorporating additional phenotypic data from the collection. As a result, we opted to adopt an omics-based approach to investigate broader genomic regions and genes, including PAL, associated with vanillin biosynthesis. This approach aligns with the goals outlined in Objective 2.3 and provided more comprehensive insights into the genetic basis of vanillin production. Objective 2.2 Test cysteine protease-like protein (CPLP) variants for increased vanillin In the previous year, our research identified two CPLP genes potentially associated with vanillin content accumulation. However, no significant correlation was found between the CPLP-derived KASP markers and vanillin content. Due to the limited bean yield in 2024, we were unable to further this sub-objective by integrating additional phenotypic data from the collection. To address this challenge, we decided to adopt an omics-based approach to explore broader genomic regions and genes involved in vanillin biosynthesis, including PAL. This strategy, detailed under Objective 2.3, aims to provide a more comprehensive understanding of the genetic factors influencing vanillin content. Objective 2.3 Vanillin pathway interrogation using NextGen and targeted resequencing We conducted two experiments to investigate the vanillin biosynthesis pathway using transcriptome approach. First, a comparative transcriptome study was performed on two vanilla accessions from our collection with distinct vanillin content, identified through HPLC and LC-MS/MS analyses. Two bean tissue types were sampled at three late developmental stages (months 7, 8, and 9) to identify differentially expressed genes related to vanillin biosynthesis. This study revealed 70 significantly expressed genes associated with the vanillin biosynthesis pathway, of which 50 genes with relevant protein functions were used to reconstruct the vanillin biosynthesis pathway. The findings suggest the existence of multiple vanillin biosynthesis pathways within vanilla beans. Building on these results, a time-series transcriptome study was conducted to track gene expression throughout the nine-month bean development process with PacBio sequencing. This study aimed to identify gene clusters regulating vanillin accumulation during bean development, complementing the comparative transcriptome findings. The time-series analysis identified three broad gene expression clusters corresponding to bean development stages, including a critical transition point from physiological development to chemical compound biosynthesis. The integration of these two studies provided critical insights into the genetic mechanisms underlying vanillin biosynthesis. Notably, we identified six genes closely associated with vanillin synthesis, including two PAL gene copies and one CPLP gene copy, corroborating our research directions outlined in Objectives 2.1 and 2.2. Additionally, three regulatory genes with significant roles in vanillin biosynthesis were identified, offering new targets for marker development. These findings represent a significant advancement for the vanilla breeding program. The identified genes will serve as focal points for uncovering key genetic variations and developing marker-assisted selection strategies, enabling the creation of superior vanilla cultivars with improved vanillin content. Objective 3) Enhance extension, communication, and outreach We developed a new factsheet as an extension publication to provide growers with valuable information on vanilla cultivation. Additionally, we organized a training program attended by 28 county extension faculty members and professionals, focusing on the history and cultivation of vanilla. This year, we conducted four office visits with potential growers and facilitated three in-depth field visits to nurseries. Further efforts were made to strengthen connections between growers and nurseries, aiming to improve plant availability and support the expansion of vanilla cultivation.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: First Gynogenesis of Vanilla planifolia for Haploid Production and Ploidy Verification Protocol
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Unraveling the Genetic Makeup of Vanilla: Transcriptomic Insights into Enhancing Vanillin Biosynthesis for Local Production in the USA. 2024 NAPB conference
• Type: Theses/Dissertations Status: Submitted Year Published: 2024 Citation: MODERN TOOLS FOR HIGH VANILLIN CONTENT VANILLA BREEDING
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: CRISPR/Cas9-mediated editing of the phytoene desaturase gene in Vanilla planifolia enabling targeted domestication.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Agrobacterium-mediated transformation, selection and regeneration of Vanilla pompona

Progress 12/01/21 to 11/23/24

Outputs
Target Audience:The target audience for this project includes professionals, academics, industry representatives, growers, community members, and students, with a particular emphasis on first-generation growers and economically or educationally disadvantaged growers. Demographic categories encompass Asian, Black/African American, Hispanic/Latino, Caribbean Islander, and White communities. Vanilla, a high-value tropical crop, presents significant opportunities for small-scale growers in U.S. tropical regions, where agriculture is increasingly challenged by urbanization and development pressures. The crop's unique characteristics and substantial economic potential make it an attractive option for commercial growers seeking alternative high-value crops and for industry partners aiming to advance domestic production with improved horticultural practices, elite agronomic traits, and more efficient supply chain strategies. For ethnic minority and socially disadvantaged growers, vanilla serves as a gateway to establishing small agricultural businesses that integrate farming with tourism, recreation, and educational programs. The program's efforts to reach these diverse groups have expanded both nationally and internationally through formal classroom instruction, regular laboratory sessions, internships, grower and community workshops, professional conferences, and extension/outreach initiatives. Over 40 public and private seminars were delivered to community members throughout southern Florida, potential commercial growers, extension agents, government officials, and private companies interested in supporting and continuing this work. These presentations were conducted by the principal investigator (PI), graduate students, staff, and undergraduate interns funded by the project. To further support growers, a new extension factsheet was developed to educate those interested in vanilla cultivation. Additionally, the PI has maintained active communication with brokers and growers abroad through email and phone calls, ensuring regular updates on market trends, customer demands, and other critical crop information. These combined efforts have significantly enhanced knowledge-sharing and fostered broader engagement within the vanilla industry, while strengthening collaborations with industry partners and expanding the reach of this high-value crop. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One PhD student specializing in plant breeding graduated with support from this project The student received extensive training in molecular biology, bioinformatics, plant breeding, and horticulture. Additionally, two undergraduate student interns gained hands-on experience and skills in horticulture and botany. A high school student trained under the project has since enrolled in college to pursue a bachelor's degree in horticulture. The project also provided research training opportunities to volunteers interested in vanilla cultivation, equipping them with valuable knowledge and practical skills to support potential future commercial production efforts. How have the results been disseminated to communities of interest?Extension activities such as In-service training for extension agents, field days, workshops and publications (both extension and research) were performed to reach out to over 300 attendees. Presentations were also made to private companies to establish collaborations with industry partners. Posters and scientific talks were also presented to scientific communities in academic conferences such as Florida Society of Horticulture Sciences, American Society of Horticulture Sciences and National Association Plant Breeding. Three research publications were accepted to advance the knowledge of vanilla. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1) Phenotype a vanilla diversity collection and establish plant breeding populations. Objective 1.1 Characterize accessions in the vanilla collection for key quality metrics.A total of 59 vanilla accessions growing in Florida was pollinated and harvested for vanilla bean evaluation over the three years. Bean weight, length, dehiscence and vanilla content were recorded and assessed as key quality metrics. In addition, flavor profiling was performed in few accessions to compare with 35 commercial vanilla beans representing diverse flavor profiles from around the world. Significant differences were identified for the bean quality metrics. Flavor profiling of the evaluated accessions highlighted a few selections with superior flavor quality and exceptional potential for commercial production in Florida.Objective 1.2 Establish new vanilla populations through plant breeding.More than 20 breeding populations were initiated as part of this project. The resulting hybrids exhibited diverse phenotypic responses during germination and the seedling stage. From these, five breeding populations were selected for further development, each aligned with specific breeding objectives. These objectives included enhancing vanillin content, improving adaptation to Florida's environment, reducing bean splitting, and developing unique flavor profiles. Parental selections for these breeding populations were based on phenotypic evaluations outlined in Objective 1.1. To broaden the genetic pool, enhance plant vigor, and improve disease resistance, the selected parental accessions included both Vanilla planifolia and Vanilla pompona. Additionally, three haploid breeding lines were successfully developed, accompanied by the establishment of the first vanilla haploid induction protocol. This groundbreaking work is documented in a peer-reviewed manuscript, marking a significant milestone in vanilla breeding research.Objective 1.3 Expand the genetic diversity of the vanilla collection.We have acquired 35 additional accessions of Vanilla planifolia, V. pompona, V. x tahitensis, V. odorata, and other wild species from around the world to enhance the genetic diversity of our vanilla collection. Germplasm conservation efforts are actively underway to preserve these accessions in vitro. Concurrently, horticultural practices are being employed to maintain the new accessions in the shadehouse environment. To evaluate the genetic diversity of these accessions in comparison to our established collection, we utilized genotyping-by-sequencing techniques. The goal of this genetic diversity assessment is to establish a core collection that represents the broader genetic variability within our collection, serving as a foundation for future breeding initiatives.Objective 2) Develop KASP markers for high-value traits.Molecular markers were developed as fast and efficient KASP markers to differentiate between three commercial vanilla species. Sequence variants of the PAL and CPLP genes were identified and utilized for validating these KASP assays. However, the presence of multiple gene copies introduced complexity to the sequence variation analysis. To address this, transcriptome studies were conducted with a focus on identifying key genes associated with the vanillin biosynthesis pathway that influences vanillin content across different genetic materials. These studies led to the identification of six genes linked to vanillin synthesis, including two copies of the PAL gene and one copy of the CPLP gene. Additionally, three regulatory genes with critical roles in vanillin biosynthesis were discovered, providing valuable new targets for further marker development and genetic improvement efforts.Objective 3) Enhance extension, communication, and outreach.A total of 35 extension talks on vanilla cultivation were delivered to an audience of over 150 participants, significantly enhancing outreach, communication, and extension efforts. These presentations were conducted through various platforms, including in-service training sessions, grower communications, workshops, field days, and interest group meetings. In addition to the extension talks, detailed consultations were provided to six commercial growers to assist in establishing trial sites in South Florida. A new factsheet was developed as an extension publication, offering growers valuable insights into vanilla cultivation. Further efforts focused on strengthening connections between growers and nurseries to enhance plant availability and support the broader adoption and expansion of vanilla cultivation in the region.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2022 Citation: Diagnostic KASP markers differentiate Vanilla planifolia, V. odorata, V. pompona, and their hybrids using leaf or cured pod tissues
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: First Gynogenesis of Vanilla planifolia for Haploid Production and Ploidy Verification Protocol
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: CRISPR/Cas9-mediated editing of the phytoene desaturase gene in Vanilla planifolia enabling targeted domestication
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Agrobacterium-mediated transformation, selection and regeneration of Vanilla pompona
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Comparative transcriptomics in vanilla identifies key genes involved in vanillin biosynthesis
• Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: MODERN TOOLS FOR HIGH VANILLIN CONTENT VANILLA BREEDING
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Vanilla, an Emerging Specialty Crop for Tropical Agriculture
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Chambers A. Vanilla breeding and genomics for domestic production. American Society for Horticultural Sciences Webinar.

Progress 12/01/22 to 11/30/23

Outputs
Target Audience:Target audience includes potential commercial growers, industry collaborators, academics, and students. Potential commercial growers who are seeking alternative high value crops are specifically attracted by the potential of this high value crop. Industry partners who are interested in domestic production of this crop with improved horticulture practices, elite agronomic traits and better supply chain have been reaching out for collaboration. We have expanded the demographic of the audience from both nationally and internationally. Efforts include invited presentations, laboratory instruction on a regular basis, internships, professional workshops, and extension/outreach. Thirteen public and private seminars were delivered this year related to this funding project. Five of these presentations were to potential commercial growers, industry collaborators, government officials who have great interest in this crop. Additional presentations were also made to community members and academic professionals to disseminate the information for scientific exchange. Additionally, we presented five research findings in professional conferences and to private industry partners interested in collaborations. These presentations were made by PD, postdoc, graduate student, and staff who were supported by this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One PhD graduate student majoring in plant breeding is being trained in molecular biology, bioinformatics, plant breeding, and horticulture. We also provided research experience to one PhD student intern and two undergraduate student interns. How have the results been disseminated to communities of interest?In 2023, we have disseminated research findings through workshop (vanilla symposium) with ~50 total attendees. We also updated the EDIS publications based on scientific-validated information. Four research talks and one poster were presented at professional conferences such as American Society of Horticulture Sciences and National Association of Plant Breeders (NAPB). Three presentations were delivered to national and international industry partners which have results long-term research collaborations. What do you plan to do during the next reporting period to accomplish the goals?Due to personal changes and unforeseeable climate challenges, we have requested a one-year extension to this project. We will continue to evaluate the phenotype of the germplasm collection in the next year. We recognized the limitation of cloning genes in small group of accessions; thus, we will utilize a sequencing-based approach to identify the genetic variations of the CPLP and PAL genes and associated with larger group of accessions for associate analysis. We plan to publish the first manuscript of comparative transcriptome analysis in the last year of this project. We will present the research information to growers, researchers, and industry.

Impacts
What was accomplished under these goals? Objective 1) Phenotype a vanilla diversity collection and establish plant breeding populations Objective 1.1 Characterize accessions in the vanilla collection for key quality metrics Fifty-nine vanilla accessions were harvested from our collection in 2022. In 2023, we harvested 57 accessions which contributed to the second-year bean evaluation of our collection. We collected data on bean weight, length, and vanillin content. Significant differences were found for each trait among the accessions as well as between the two years. We have identified accessions that have a significantly higher amount of vanillin content as compared to the commercial standards. Objective 1.2 Establish new vanilla populations through plant breeding In 2023, we generated four populations with specifical goal to increase vanillin content, the most important valuable commercial trait of this crop. The parents of these four populations were selected based on the vanillin content generated from 2022. All the hybrids were from commercial species and meet the FDA regulations. These populations are created in April 2023 and currently maintained in tissue culture, the hybrids will be hardened in January 2024 and transferred to shade house for phenotypic evaluation. Objective 1.3 Expand the genetic diversity of the vanilla collection We have solicited an additional seven accessions that are commercial species from various sources. The seven accessions have elite traits such disease resistance, non-split beans and high vanillin content. The new accessions will be useful to understand the genetic variations of important traits and can be used to narrow down our breeding goals. Objective 2) Develop KASP markers for high-value traits Objective 2.1 Test phenylalanine ammonia-lyase (PAL) variants for increased vanillin We have identified four PAL genes in Vanilla planifolia, the full length CDS sequences were cloned and sequenced using sanger sequencing. Genetic variants were discovered between different copies of PAL gene. KASP markers were designed and tested among seven vanilla accessions showing high and low vanillin content. Genomic DNAs from leaf, root, stem and pod tissue of the seven accessions were extracted for KASP analysis. However, we did not identify any correlation between the KASP marker and vanillin content. Objective 2.2 Test cysteine protease-like protein (CPLP) variants for increased vanillin We have identified three CPLP genes in Vanilla planifolia, the full length CDS sequences were cloned and sequenced using sanger sequencing. Genetic variants were discovered between different copies of CPLP gene. KASP markers were designed and tested among seven vanilla accessions showing high and low vanillin content. Genomic DNAs from leaf, root, stem, and pod tissue of the seven accessions were extracted for KASP analysis. We have identified that one copy of CPLP is only expressed in leaf sample. Thus, we focused on the remaining two copies for the analyses. However, we did not identify any correlation between the KASP marker and vanillin content. Objective 2.3 Vanillin pathway interrogation using NextGen and targeted resequencing In 2022, we quantified the vanillin content of seven accessions and selected the accessions with the highest and lowest vanillin content for comparative analyses. We collected the beans at six, seven and eight months after the pollination. The bean samples were also dissected into the green(outer) and white(inner) tissue. A total of 36 samples were subjected for the RNAseq analysis. No differentially expressed gene was identified between the sample tissue of individual accession across different time points. A total of 500 genes were identified as DEGs between the same tissue of the two accessions at the same time point. A total of 21 genes were identified to be tightly associated with potential vanillin biosynthesis pathways. The co-expression network is ongoing to explore the dominant biosynthesis pathway and the major genes that regulate vanillin content in different accessions. A manuscript is being developed to present the findings of this objective. Objective 3) Enhance extension, communication, and outreach Thirteen talks related to vanilla have been given in 2023. Other activities also include one grower communication through workshops with 50 attendees and two updated extension publications as public resources. Detailed consultations have been delivered to six commercial growers in establishing their trial sites in south Florida. In addition, efforts have been made to connect the growers with the nurseries to enhance plant availability.

Publications

Progress 12/01/21 to 11/30/22

Outputs
Target Audience:Target audience incudesprofessionals, academics, industry representatives, growers, community members, andstudents. These groups include first generation growers and economically/educationally disadvantaged growers. Demographic categories includeAsian, Black/African American, Hispanic/Latino, Caribbean Islander, and White. Efforts include formal classroom instruction as invited, laboratory instruction on a regular basis, internships, grower/community workshops, andextension/outreach. We had 22 public and private seminars this year related to this fundign project. Most of these were to community members throughout southern Florida. Additionally, we presented our research findings of professional conferences and to private companies that are interested in the continuation of this work. Presentations were made by the PD as well as the graduate student and undergraduate interns funded by this project. Changes/Problems:None. The project is proceeding according to plan. What opportunities for training and professional development has the project provided?We have provided training to one graduate student relating to molecular biology, bioinformatics, plant breeding, and horticulture. We have provided research experience to two undergraduate student interns. How have the results been disseminated to communities of interest?We have disseminated research findings at six grower/community events with ~150 total attendees. We have presented at professional conferences reaching ~200 total attendees. We have also presented to three private companies. What do you plan to do during the next reporting period to accomplish the goals?Finish KASP development, validation, and testing. Conduct RNAseq analysis of vanilla beans. Further develop genetic resources through plant breeding. Continue communication to growers, researchers, and industry.

Impacts
What was accomplished under these goals? Objective 1) Phenotype a vanilla diversity collection and establish plant breeding populations Objective 1.1 Characterize accessions in the vanilla collection for key quality metrics We have assayed beans from 59 vanilla accessions growing in Florida for vanillin content. We also collected data on bean weight and length. Significant differences were found for each trait. Objective 1.2 Establish new vanilla populations through plant breeding Our first round of populations have been hardened and are growing in the shade house. Plants will be transferred to final growing locations in Q4 2022. 20 additional populations were created in 2022 and will be growing in the shade house by mid 2023. Objective 1.3 Expand the genetic diversity of the vanilla collection Additional accessions of V. x tahitensis, V. odorata, and others have been collection and are being maintained in the shade house. Objective 2) Develop KASP markers for high-value traits Our first KASP manuscript conducted as part of this work was published in 2022. Objective 2.1 Test phenylalanine ammonia-lyase (PAL) variants for increased vanillin Sequence variants have been bioinformatically identified andKASP assays have been developed. We are currently cloning full length CDS sequences for KASP controls and analysis. Objective 2.2 Test cysteine protease-like protein (CPLP) variants for increased vanillin KASP assays have been designed and are being validated. We are currently cloning full length CDS sequences for KASP controls and analysis. Objective 2.3 Vanillin pathway interrogation using NextGen and targeted resequencing Beans are being collected in Q3 and Q4 of 2022 for this analysis. Objective 3) Enhance extension, communication, and outreach 22 talks on vanilla have been given in 2022 since the start of the project. This includes and in-service training and grower communication through workshops.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Diagnostic KASP markers differentiate Vanilla planifolia, V. odorata, V. pompona, and their hybrids using leaf or cured pod tissues