Recipient Organization
CHI BOTANIC INC.
1962 MAIN ST STE 200 # 234
SARASOTA,FL 342369519
Performing Department
(N/A)
Non Technical Summary
Vanilla is a widely used ingredient found in thousands of products. Most commercial vanilla production has been offshored. The U.S. manufacturers that utilize natural vanilla must import this crop but this material often has issues with high prices, inconsistent supply chains, variation in quality, and imparts a heavy toll on local people and ecosystems where it is produced.We aim to solve the current issues with vanilla by the development of a commercial Vanilla planifolia plant cell culture. The liquid culture of plants is an established technology in which plants are grown as a fine suspension of small clumps or single cells. Unlike V. planifolia plants, an V. planifolia cell culture can be domestically-produced in a safe and high quality way without pesticides or adulterants.?Our high-throughput approaches will explore more V. planifolia genotype-phenotype and environment-phenotype space than ever before. This research will establish V. planifolia cell culture technology as an innovative and domestic way to produce vanilla.
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
(N/A)
Goals / Objectives
The overall objective of the proposal is to generate a vanilla cell culture for the use in fragrances, foods,drinks, or other products that contain vanilla. While the generation of plant cell cultures is guided bystandard tissue culture practices, the specific tissues, genotypes, hormone concentrations, and optimalmedium are plant species specific. Our Technical Objectives are to further expand the application of plantcell culture to cell suspension culture of vanilla and the production of valuable glycosides, specificallyvanillin. The specific objectives of the program can be enumerated as follows:1. Generate V. planifolia plant cell culture lines that contain a fluorescent reporter that correlates tothe expression of the phenylpropanoid biosynthetic pathway2. Identify environmental variables, elicitors, and chemicals that increase vanillin production.3. Isolate V. planifolia plant cell lines that have increased vanillin production.4. Quantitative determination of vanillin levels from best lines to confirm phenotype.
Project Methods
Technical Objective 1. Transformation of V. planifolia cultures.Task 1.1 Synthesis of Vanilla Planifolia Phenylpropanoid Pathway Reporter (pVaP3R) vectors.We propose to leverage the 5' upstream promoter regions of genes encoding three key enzymatic steps inthe biosynthesis of vanillin (Fig. 2) for the creation of transgenic fluorescent reporter plasmids.In order to generate the pVaP3R family of reporter plasmids, synthetic reporter fragments including: p35s::GFP::t35s (pVaP3R-1, + control), pPAL::GFP::t35s (pVaP3R-2), pVAN::GFP:t35s (pVaP3R-3) or pHBS::GFP::t35s (pVAP3R-4) will be cloned into the pCambria-1300-Pac1 multiple cloning site (MCS).Task 1.2 Transformation of V. planifolia cell suspension culturesWe propose to introduce the pVaP3R constructs outlined above (Fig. 2) into V. planifolia suspensioncultures using particle bombardment.Bombardment methods will be performed as described previously for maize(Vain et al, 1993). In brief, cultures will be grown to early log phase prior to gene introduction. Cellclumps and small cell clusters will be aseptically filtered through sterile glass fiber filters and placed onsemi solid media containing mannitol and sorbitol for 4 hours pre-bombardment to plasmolyze the targetcells. After bombardment, filters containing cells will be returned to media containing mannitol andsorbitol for recovery and then transferred to media without selective agent (hygromycin) for 4-7 days.After this additional recovery, bombarded cells will be transferred to hygromycin containing media forselection of transgenic cell lines.Task 1.3 Confirmation of positive controls and baseline GFP fluorescenceGFP expression in cell lines will be tracked and quantified using an automated image collection andanalysis system as described previously (Finer et al., 2006, Chiera et al., 2007). In brief, transgenic V.planifolia lines will be plated on various media in Petri dishes, and those dishes placed on the2-dimensional robotics platform for monitoring of GFP expression. Images will be collected from eachpiece of tissue, every hour for 100 hours for quantification of gfp expression and generation of time lapseanimations of tissue displaying gfp expression.Technical Objective 2. Identify Conditions and Elicitors that Increase VanillinTask 2.1 - Environmental and chemical elicitation of diverse phenotypes.Elicitors are chemicals that can trigger specific phenotypic responses in plants, oftenassociated with biotic or abiotic stresses. Some common eliciters include flagellin, LPS, salicylic acid,methyl salicylate, benzothiadiazole, benzoic acid, chitosan, and jasmonic acid. In addition, a chemicalgenomics approach will be taken to identify small molecules in the "Library of AcTive Compounds onArabidopsis" (LATCA) that stimulate vanillin production.Technical Objective 3. FACS to Isolate High Vanillin LinesTask 3.1 Fluorescence cell sorting for characteristic native fluorescence of Vanilla glycosides-filledphenylplast-type plastids.To select for Vanilla lines thatproduce increased levels of Vanilla glycosides, a mixed population of Vanilla cells will be analyzed andthe top 1% of cells with green fluorescences (GFP channel) will be sorted and isolated. This sortingprocedure will be repeated multiple times to enrich for cells that have increased green fluorescences andthus Vanilla glycosides. These cells will then be subjected to secondary screening with a fluorescent platereader and the subsequent top lines will have vanillin content measured via HPLC (Task 4).Task 3.2 Fluorescence cell sorting of pVaP3R Reporter LinesV. planifolia cell suspension cultures that have been transformed with pVaP3R constructs outlined in Task1 that produce GFP in response to upregulation of the phenylpropanoid pathway will be screened viaFACS.Task 3.3 Secondary screening via fluorescence plate reader.To assess vanillin content in lines isolated in Task 3.1, a Bio-tek Cytation 1 Cell Imaging Multi-ModeReader (Cyt1V) will be used to assay fluorescence from 96-well plate libraries.Technical Objective 4. Quantitation of Vanilla Glycosides (Vanillin) from Best LinesTask 4.1 Extraction and HPLC analysis of VanillinVanillin content of V. planifolia cell suspension cultures identified in Tasks 1-3 will be quantified. V.planifolia biomass will be isolated, hydrolyzed, and extracted. Extracts will be separated on an HPLCwith a C18 column using an isocratic mobile phase. Vanillin will be detected by measuring absorbance at271 nm (Adawiya 2020).Task 4.2 ConditioningIsolation of cells shown to accumulate VAN and correlated glucovanillin will be followed bysimulation of taditional vanilla bean curing process as well as treatment with commercial β-glucosidasesto liberate Vanilla glycosides like transformation of glucovanillin to vanillin.