Source: UNIVERSITY OF FLORIDA submitted to
DISEASE-RESISTANT SEEDLESS MUSCADINE GRAPE CULTIVARS UTILIZING ECO- AND CONSUMER-FRIENDLY CISGENIC MODIFICATION TECHNOLOGIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0225619
Grant No.
2011-51181-30668
Project No.
FLA-APO-005098
Proposal No.
2011-02115
Multistate No.
(N/A)
Program Code
SCRI
Project Start Date
Sep 1, 2011
Project End Date
Aug 31, 2016
Grant Year
2011
Project Director
Gray, D. J.
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Mid-Florida Research and Education Center, Apopka
Non Technical Summary
The project seeks to essentially develop a new type of grape that can be grown in the Southeast. The grape will be a seedless, rot resistant version of traditional muscadine grape. Development will be accompanied by studies of shelf life. Significantly, economic analyses and extension activities will be conducted to educate the public and smooth the way for deployment of the new grape product.
Animal Health Component
(N/A)
Research Effort Categories
Basic
30%
Applied
30%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2031130104015%
2031130105015%
2031130108015%
2041130104010%
2041130105015%
2041130108010%
6041130105020%
Goals / Objectives
The purpose of the proposed Standard Research and Extension Grant (SREP) is to modify quality attributes of muscadine grape (Vitis rotundifolia Michx. and hybrids), thus overcoming existing obstacles to consumer acceptance, resulting in increased production and market penetration. The objectives are to 1) add fungal fruit rot resistance and seedlessness to target cultivars via a technique of molecular biology termed "cisgenics", that promises to be consumer friendly, 2) modify onset of post-harvest rot by pre- and post-harvest handling, 3) develop best management practices and 4) facilitate grower/wholesaler/retailer/consumer education to increase acceptance of rot resistant seedless muscadine grape. Muscadine is an enduring fruit crop grown in the southeastern United States. Cultivars have been greatly improved by breeding so that berry size can exceed 20 g (i.e., a small plum), with excellent flesh and skin texture. However, poor shelf life and the lack of seedlessness in fruit of all cultivars are limiting factors to significantly increased production and distribution (see appendix for letters of support from growers). The project uses newly available muscadine genetic engineering technology to confer fungal disease resistance and seedlessness into elite germplasm. Uniquely, only genetic constructs obtained from grape will be used in an approach termed "cisgenics". Further, incorporation of female cultivars, which do not produce viable pollen, eliminates concerns regarding foreign gene flow. This "green approach" to genetic modification is ecologically and consumer friendly inasmuch as it perturbs the grape genome much less than conventional breeding, and should cause fewer GMO-related concerns to the public.
Project Methods
The obstacle to using conventional breeding to develop seedless Muscadine cultivars has led us to lay the groundwork for a genetic engineering approach. We have developed the requisite core technology with which to develop seedless Muscadine cultivars and prototype plants are in greenhouse testing phases. Technology advancements now available for the project include the following: 1) a genetic engineering system for Muscadines, 2) a grape-derived promoter that functions only in seeds and 3) two grape-derived antifungal genes that, when re-engineered back into grape, already are shown to result in disease-resistant plants, 4) grape-derived T-DNA border sequences, enhancers and terminators. Thus, using only grape-derived genetic elements, a "cisgenic" or "green approach", all of which are in hand, we propose to accomplish the following: 1) construct a genetic vector to attach various seed-disrupting genes to the seed-specific promoter, genetically engineer the vector into Muscadine cultivars and test for seedlessness; 2) insert both antifungal genes into Muscadine cultivars and test for rot resistance; 3) simultaneously develop plants containing both seedlessness and antifungal genetic elements; 4) study post-harvest pathology and physiology of the cisgenic plants compared to non-cisgenic controls; 5) develop horticultural practices to facilitate optimum production in the field; . 6) conduct economic analyses, including grower/consumer surveys to delineate preferences and raise awareness of the crop; 7) develop best management practices (BMPs) for growers, wholesalers and retailers to promote the resulting fruit product; and 8) develop and maintain a website as a portal for education, support and information gathering for the project (http://mrec.ifas.ufl.edu/muscadine).

Progress 09/01/11 to 08/31/16

Outputs
Target Audience:The target audiences reached are other scientists and, importantly, consumers, as we have begun to successfully deploy "Precision Breeding" technology,which became refined during this project. Numerous successful presentations of the PB conceptvia local extension presentations as well as presentation to the ubiquitous tours that pass through the MREC. An easily understandable brochure was produced, of which hundreds have been distributed to date. Changes/Problems:The project suffered from a severe accounting problem. Despite recieving over $1M from the grant in IDC and with the project ending on September 31, 2016, Gray was repeatedly informed by UF/IFAS accountingat the end of 2015 and early 2016 that all grant funds were exhausted, causing staff layoffs andending the project prematurely.However, Gray determined on his own early in 2016 that $200k remained unused in a subaccount for Co-PI Dr. Sims in the UF/IFAS Department of Food Science and Human Nutrition.This was highly damaging to completion of the project because it sabotaged a carefully planned conclusion to the project and planning for future funded projects. Although UF/IFAS accounting, once informed that the $200k existed,eventually made it available for use in the project, the damage was done. The project could not terminate with the level of success planned and most of these lostfunds went unused. Oddly, there was no admission from accounting that they had made such a large error and the faulty accounting system remains in place today. Concurrently Gray's 33 year-old grape biotechnology research program began to recieve a highly significant lack of support from a new IFAS administration, with the problems stemming from such currently in dispute. Because of the way that these funds for the project were handled and there appears to be an ongoing problem with UF accounting practices,it is suggested that the accounts related to this project be audited. What opportunities for training and professional development has the project provided?Six Master's and one Ph.D. degree-level students were trained during the project and received their degrees. Similarly, a Postdoctoral Research Associate was trained, including attending multi-day grant-writing workshop. How have the results been disseminated to communities of interest?A total of 10 refereed journal articles are in print, with three more submitted. There were 15 presentations at international, national and regional scientific meetings. An explanatory brochure was created and distributed widely. Six theses and a dissertation were produced. Ten presentations to extension groups, visiting scientific organizations, citizenand grape grower meetings occurred. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? VITIS GENETICS and PRECISION BREEDING (Gray and Li): Prior to the present project, we had spent many years learning how to identify promising genetic elements and to create proprietary tools needed for their insertion into grapevine improvement. A number of Vitis-derived genes were tested. In many cases, large gene families composed of orthologous sequences were discovered. We systematically cloned these and tested each ortholog for activity, resulting in the following active versions of listed genes: Putative seedlessness inducers: Anti-Vv Sucrose synthase gene (VvSU-SY), VvWOX9 gene; Selectable markers: Myb2-A (anthocyanin), Acetolactate synthase; Disease resistance: Pathogenesis-related protein group 1 (PR-1), Pathogenesis-related protein group 5 = VvThatumatin-like protein (VvTL1), Vv2S Albumin (Vvalb), VvAMP/defensing, VvSnakin. As such, many putative genes for fruit rot resistance and seedlessness were made available for the current project.The results obtained with these Vitis models developed by us over many years provided a fast track to apply genes with pretested traits for muscadines, which, as mentioned above, were being inserted into muscadines. These "precision-bred" in the strict sense and had no commercial value. Field testing to evaluate precision bred grape lines Fungal disease resistance in the field. Prior to the first deployment of modified muscadine plants under the current permit, we had already begun to test other genes into model cultivars. Florida (Gray & Li).:Prototype PB muscadine 'Delicious' plants containing the anti-VvSuSy gene first fruited in 2015. Compared to control 'Delicious', the modified version possessed the same berry size and number of seeds, however more seeds from the modified lines did not contain embryos or endosperm, i.e., were non-viable. The non-viable seeds retained a hard seed coat, but these results pave the way for specific targeting of seed coat abortion, which would result in true seedless cultivars. Mississippi (Stringer): Fourty-five muscadine grape plants obtained from UFL. They were established in the field in June and July 2013 in the test site located at the USDAS-ARS research farm near Perkinston, MS. In Aug., 2015, plants supposed to have been a transcript of the commercial cultivar 'Delicious', "Delicious HS-9", were determined by Gray during a site visit to in fact be the UFL cultivar 'Southern Jewel', leaving only 'Alachua' and "ALA-214DGFP" as valid strains for the study.Observations on fruits showed that both muscadine grape strains produced similar berries possessing similar qualities, but the slightly lower value for soluble solids content as well as slightly higher pH value suggested that berries of the ALA-214DGFP transcript may ripen slightly later than unmodified 'Alachua'. US Virgin Islands (Zimmerman): In February, 2013, muscadine grapes were obtained by IFAS-Mid-Florida Research and Education Center. In 2015, plants started to flush out by February 17. One 'Delicious' had a flower cluster but no fruit were set. Plants were weak and chlorotic. Application of fertilizer and Fe EDDHA had no effect on control of chlorosis. It is speculated that the root system had penetrated the six inches of top soil and was into the high pH calcareous subsoil. Plant survival rate continued to decline. In early November, the vines dropped their leaves. On November 13, prior to the termination of the release permit, the plants were devitalized by autoclaving the plant material. PATHOLOGY (Trigiano): Two species of fungi, Colletotrichium gleosporides and Pestalotia macrospora were identified as the initial contaminants responsible for fruit rot in muscadines and were isolated from muscadine berries as pure cultures. During this reporting period, muscadine grapevines were inoculated with a mixture of 5 Colletotrichium gleosporides and 4 Pestalotia macrospora isolates. The question of which pathogen is the primary attacker, leading the way for others was investigated. FOOD SCIENCE (Sims): Two M.S. students were supported by this project and finished their degrees. Research was to investigate the range in acceptability of muscadine grape cultivars and to correlate overall liking to other quality measurements to determine the main drivers of liking was conducted. Evaluating the perception of muscadine grapes by consumers showed significantly different ratings by the two groups for appearance, overall liking, textures of skin and pulp, flavor and seeds. ECONOMICS (Fonsah): We developed and investigated a stochastic farm enterprise budget framework for muscadine grapes production and marketing that accounts for structural dependency between yields and prices as well as uncertainty in estimates of profits and investment decisions. EXTENSION AND SURVEYS (Olmsted and House): Focus groups: A focus group with 10 participants was conducted in 2012 to explore questionnaire wording and development. Although participants were able to understand the difference between transgenic and cisgenic processes, they saw them both as genetic modification and not as acceptable as "natural breeding". Consumer surveys: Consumer surveys to determine attitudes towards the consumption of genetically engineered foods and the acceptance of the general concepts offered by cisgenics, in addition to the willingness to pay (WTP) for genetically engineered seedless grapes. A second consumer survey was conducted in 2015 to further understand consumer preferences for genetically engineered foods. The final dataset in this survey included 6,000 participants. Grape preference taste panels were conducted with C. Sims and in cooperation with two other universities (Washington State University and Purdue University), in areas of the United States not familiar with muscadine consumption. The extension team worked with C. Sims (Co-PD) to conduct consumer sensory panels for determination of variety preference. Evaluation of extension activity impact: Evaluation of participants in workshops held in conjunction with the annual meeting of the American Society for Horticultural Science was used to direct activities of the extension, which included the revision of the grape production manual for Florida and authoring of an extension document on consumer attitudes towards using biotechnology for seedless muscadine grapes (House, Olmstead, Ligon). Extension and outreach efforts were concentrated on collecting data and writing documents for description of breeding methods and a muscadine/bunch grape best management practices manual. In the final year of the proposal we will identify current marketing outlets and strategies for muscadine products and delineate gaps for precision bred grape products.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Gray D. J., Z. T. Li, T. N. L. Grant, D. A. Dean, R. N. Trigiano and S. A. Dhekney. 2016. The Application of Precision Breeding (PB) for Crop Improvement is Fully Consistent with the Plant Life Cycle: The Utility of PB for Grapevine. ACTA Hort. 1115:46-56.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Dhekney, S.A., Kandel, R., Bergey, D., Asanakunov, B., Sitther, V., Li, Z.T. and Gray, D.J. (2016). Development of novel plant phenotypes using plant pigment-associated genes. Acta Hort. 1140, 169-174.
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Brown, K. Acceptability of muscadine grapes and those characteristics related to consumer acceptance. MS thesis, University of Florida, 2015.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Awondo, S. N., G.E. Fonsah and D.J. Gray. Incorporating Structure and Stochasticity in Muscadine grapes Enterprise Budget and Investment Analysis. HortTech. (Accepted).
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Fonsah, E. G., S.N. Awondo. Cost Estimates and Investment Analysis for Muscadine Grapes Production in Georgia. Journal of Food Distribution Research, (47(1):1-5.
  • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Edenbrandt, A., L. House, Z. Gao, M. Olmstead, and D. Gray. 2015. Consumer acceptance of cisgenic food and the impact of information and reference points. Agricultural Economics. (submitted).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Awondo, S.N, and E.G. Fonsah (2015). Cost Estimates and Investment Analysis for Muscadine Grapes Production in Georgia, Poster Presented at the Southeast Regional Fruits and Vegetable Conference, Savannah, January 08-11
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: b. Fonsah, E. G., S.N. Awondo (2015). Cost Estimates and Investment Analysis for Muscadine Grapes Production in Georgia. Food Distribution Research Society Annual Conference, Philadelphia, October 9-12
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Brown, K., C. Sims, P. Conner, A. Odabasi, E. Dreyer, and D. Gray. 2014. Quality attributes related to the consumer acceptability of muscadine grape cultivars. Institute of Food Technologists Annual Meeting, New Orleans, LA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Olmstead et al., Understanding Breeding, Cisgenic and GMO Terminology, Southeast Fruit Professional Workers Conference, Athens GA, 9-19-13
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: House, L., M. Olmstead, D. Gray, and J. Xie. Consumer Reactions to Biotechnology in Muscadine Grapes, Southeast Fruit and Vegetable Conference, Savannah, GA, 1-10-2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Edenbrandt, A., L. House, M. Olmstead, Z. Gao, and D. Gray. Consumer attitudes towards genetic engineering in Muscadine grapes, Southern Region of the American Society for Horticultural Science.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Edenbrandt, A., L. House, M. Olmstead, Z. Gao, and D. Gray. Consumer attitudes towards genetic engineering in Muscadine grapes, Grapes of Arkansas Workshop, Clarksville, AR, 9-15-16.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dhekney S. A., Li, Z .T., Gray, D. J. 2016. An update on the development of commercial crop cultivars using precision breeding. P-21. In Vitro Cell. Dev.Biol.  Animal 32: P-21.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Gray, D.J., Li, Z.T., Grant, T.N.L., Dean, D.A., and Dhekney, S.A. 2016. The new biology of precision breeding. P-20. In Vitro Cell. Dev. Biol.  Animal. 52: P-20.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Gray, D.J., Li, Z.T., Cordts, J., Grant, T.N.L., Dean, D.A., and Dhekney, S.A. 2016. The biology of precision breeding. Annual conference of the American Society of Horticultural Sciences, Atlanta GA, August 8-11.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dhekney S.A., Li, Z.T., Grant, T.N.L. and Gray, D.J. 2016. New cultivars released or in development using precision breeding. Annual conference of the American Society of Horticultural Sciences, Atlanta GA, August 8-11.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Li, Z.T., Dhekney S.A., Grant, T.N.L. and Gray, D.J. 2016. Identification and testing of endogenous genetic elements for precision breeding. Annual conference of the American Society of Horticultural Sciences, Atlanta GA, August 8-11.


Progress 09/01/14 to 08/31/15

Outputs
Target Audience:The target audiences reached are other scientists and, importantly, consumers, as we have began to successfully deploy "Precision Breeding" technology,which became refined during this project. Numerous successful presentations of the PB conceptvia local extension presentations as well as presentation to the ubiquitous tours that pass through the MREC. An easily understandable brochure was produced, of which hundreds have been distributed to date. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Postdoctoral Research Associate, Dr. Deborah Dean attended the Plant and Animal Genomics meeting in San Diego January 2014 where she made two presentations. Dr. Dean also attendeda two-day grant writing workshop at the Gainesville campus in January 2015. How have the results been disseminated to communities of interest?Results were disseminated via refereed publications and at a number of international, national and regional meetings, via invited platform presentations and published abstracts. The meetings included the annual meetings of the American Society for Horticultural Sciences (2014 & 2015), International Horticultural Congress (2014), Society for In Vitro Biology (2014 & 2015), Southeast Fruit and Vegetable Conference (2015) in addition to numerous presentations to local growers and tour groups. What do you plan to do during the next reporting period to accomplish the goals?The above described research projects will continue and/or be finalized in 2016. This reportingperiod marks the end of this project, which will be sumnmarized in a final report after August 30, 2015.

Impacts
What was accomplished under these goals? VITIS GENETICS (Gray and Li): Via a multi-decade project, a number of Vitis-derived genes are currently in field test using model Vitis cultivars and are now being deployed into Vitis rotundifolia. In many cases, large gene families composed of orthologous sequences exist. We systematically cloned these and tested each ortholog for activity, resulting in the following active versions of listed genes: Putative seedlessness inducers: Anti-Vv Sucrose synthase gene, VvWOX9 gene; Selectable markers: Myb2-A, Acetolactate synthase; Disease resistance: VPR-1, VvPR-5, Vv2S Albumin, VvAMP/defensing, VvSnakin-1. Plasmids and Vectors: A proprietary plasmid was created to have an open site for insertion of PB vectors. The plasmid includes proprietary Vitis-derived elements so that, upon insertion into the Vitis genome, the vector inserts will contain only Vitis DNA with zero exceptions. The genes already are being field-evaluated in non-muscadine cultivars of Vitis vinifera and Vitis hybrid grapevines. For example, Vitis hybrid 'Seyval Blanc' plants containing the PR-1 gene show remarkable fungal disease resistance in the field for over three years to date. Beyond excellent results with PR-1, we inserted a gene complex containing both VvTL-1 and Vv2S Albumen, both of which have been shown by us to provide fungal disease resistance separately. We have VvTL-1/Alb genetic constructs functioning in Thomson Seedless in the field. It's too early to tell, but these may exceed the PR-1 results, based on early observations in the field. The results obtained with these Vitis models developed by us over many years provides a fast track to pretested traits for muscadines, which, as mentioned above, are being inserted into muscadines now. FIELD TESTING: Evaluating grapevines in the field for improved traits consumes a number of years because of the long grape lifecycle and the strict requirement that desired traits hold-up for the vine's productive lifespan, which can be decades. Typically, evaluation for seven or more years is required to make a putative commercial selection, which then can be provided to growers for final evaluations. For the current project, the first two modified PB prototypes were deployed nearly four years ago into to three USDA/AHIS/BRS-approved field sites in Florida, Mississippi and the US Virgin Islands. Florida (Gray & Li). Prototype PB muscadine 'Delicious' plants containing the anti-VvSuSy gene first fruited in 2015. Compared to control 'Delicious', the modified version possessed the same berry size and number of seeds, however more seeds from the modified lines did not contain embryos or endosperm, i.e., were non-viable. The non-viable seeds retained a hard seed coat, but these results pave the way for specific targeting of seed coat abortion, which will result in true seedless cultivars. Due to a relatively dry year, we were unable to evaluate 'Alachua' plants containing the VvTL-1 because they showed no difference from controls in disease resistance. Mississippi (Stringer). Data were collected in April on Alachua and ALA-214DGFP on vine length and leaf bud emergence. Observations on leaf and berry diseases were made on 7/29/15, and from mid-July until late Aug, vines were harvested weekly and berry samples were taken to the laboratory for fruit quality analysis. US Virgin Islands (Zimmerman). Grape plants dropped their leaves from late November to early December and had a short dormancy. In late January, the grapes sprouted from the basal ½ meter of the plants. Only three Delicious plants flowered and set fruit in February. The fruit matured during early April. Plant growth stopped by the end of April. The plants had grown to 1.5m for 'Delicious', 1.1m for 'Alachua', the precision bred prototype lines were similar at 0.7 m. PATHOLOGY (Trigiano): Two species of fungi, Colletotrichium gleosporides and Pestalotia macrospora were identified as the initial contaminants responsible for fruit rot in muscadines and were isolated from muscadine berries as pure cultures. During this reporting period, muscadine grapevines were inoculated with a mixture of 5 Colletotrichium gleosporides and 4 Pestalotia macrospora isolates. This study is still underway. FOOD SCIENCE (Sims): An MS project was finalized entitled "Acceptability of muscadine grapes and those characteristics related to consumer acceptance". Another project on the consumer acceptability of muscadine grapes is underway. Two different groups (n=100 each) of consumers were recruited to evaluate 2 varieties of muscadine grapes: consumers who consume muscadine grapes each year and consumers who have never eaten a muscadine grape. These two groups rated the acceptability of muscadine grapes using the hedonic general labeled magnitude scale and then rated their hedonic responses to a series of questions related to their liking of a variety of foods and other experiences. The data is being analyzed to determine differences between the 2 groups and the characteristics of each of these groups. This will hopefully lead to suggestions on how best to reach out to new consumers who may like muscadine grapes if given a chance to taste them. ECONOMICS (Fonsah): We developed and investigated a stochastic farm enterprise budget framework for muscadine grapes production and marketing that accounts for structural dependency between yields and prices as well as uncertainty in estimates of profits and investment decisions. We travelled/visited and work with major muscadine grapes producers in Georgia to gather data and develop a traditional enterprise budget based on theoretical and accounting methods recommended by the American Agricultural Economics Association (AAEA) Task Force on Commodity Costs and Returns. In the end, we compared estimates and investment decisions derived from the traditional and stochastic approaches. Results from this study have been presented at the Southeast Regional Fruits & Vegetable Conference in Savannah, GA, in January 2015, and the Food Distribution Research Society Annual Conference in Philadelphia, October, 2015. A paper based on the study is under consideration for publication in the Journal of Food Distribution Research, and another is about to be submitted to HortScience. A survey and experiment is currently being developed to elicit consumers' beliefs, acceptability and willingness to pay for genetically-improved precision-bred, disease-resistant, seedless muscadine grape cultivars, as ongoing research. EXTENSION AND SURVEYS (Olmsted and House): Extension and outreach efforts have been concentrated on collecting data and writing documents for description of breeding methods and a muscadine/bunch grape best management practices manual. In the final year of the proposal we will identify current marketing outlets and strategies for muscadine products and delineate gaps for precision bred grape products. A final consumer survey will be conducted on determining information source and the effect of consumer attitudes towards precision breeding

Publications

  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2016 Citation: Gray D. J., Z. T. Li, T. N. L. Grant, D. A. Dean, R. N. Trigiano and S. A. Dhekney. 2016. The Application of Precision Breeding (PB) for Crop Improvement is Fully Consistent with the Plant Life Cycle: The Utility of PB for Grapevine. Text of an invited keynote presentation at the Int. Hort. Congress, Brisbane. ACTA Hort. (in press).
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Gray D. J., Z. T. Li and S. A. Dhekney. 2014. Precision breeding of grapevine (Vitis vinifera) for improved traits. Plant Sci., 228: 3-10.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Gray D. J., Z. T. Li, T. N. L. Grant, D. A. Dean, R. N. Trigiano, D. R. Bergey, S. A. Dhekney. 2015. La s�lection v�g�tale de precision pour la mise au point de nouveaux c�pages: Une nouvelle technologie g�n�tique dam�lioration des plantes, enti�rement en accord avec le cycle de vie de la vigne. Revue des Oenologues No 157 sp�cial. November 2015. (Translation of The application of precision breeding (PB) for crop improvement is fully consistent with the plant life cycle: the utility of PB for grapevine).
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Li, Z. T., D. L. Hopkins and D. J. Gray. 2015. Overexpression of antimicrobial lytic peptides protects grapevine from Pierces disease under greenhouse but not field conditions. Trans. Res. 24: 821-836.
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Brown, K., C. Sims, A. Odabasi, L. Bartoshuk, P. Conner, D. Gray. 2015. Acceptability of muscadine grapes and those characteristics related to consumer acceptance. J. Food Science (submitted)
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Brown, K. Acceptability of muscadine grapes and those characteristics related to consumer acceptance. MS thesis, University of Florida, 2015.
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Fonsah, E. G., S.N. Awondo (2015). Cost Estimates and Investment Analysis for Muscadine Grapes Production in Georgia. Journal of Food Distribution Research, (submitted).
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Awondo, S. N., G.E. Fonsah and D. J. Gray. Incorporating Structure and Stochasticity in Muscadine grapes Enterprise Budget and Investment Analysis. Hortscience (submitted).
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Edenbrandt, A., L. House, Z. Gao, M. Olmstead, and D. Gray. 2015. Consumer acceptance of cisgenic food and the impact of information and reference points. Agricultural Economics. (submitted).
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Awondo, S. N., E. G. Fonsah, D. J. Gray. 2015. Incorporating Structure and Stochasticity in Farm Enterprise Budget and Investment Analysis: The case of Muscadine grapes production in Georgia. HortScience (submitted).


Progress 09/01/13 to 08/31/14

Outputs
Target Audience: Nothing Reported Changes/Problems:It has become important to more clearly define the Precision Breeding technology. Therefore, we are changing certain key terms. To meet the evolving genetic comparisons and to clarify terminology, the following terms: "Cisgenic" and "Intragenic" are replaced with "Precision Breeding" and its derivatives. This change reflects that this developing technology has become comparable to "Conventional Breeding". Former comparisons to "Transgenic" that were enabled by the previous "cisgenic" terminology are no longer valid to describe the evolving applications of Precision Breeding (PB). What opportunities for training and professional development has the project provided?Postdoctoral Research Associate, Sunny Kim, attended a grant writing workshop at UF/IFAS in 2012. How have the results been disseminated to communities of interest?The current target audiences are growers of muscadine grapes and interested consumers, who are with whom we interact through regional presentations about the project and potential consumers who will be contacted via surveys. What do you plan to do during the next reporting period to accomplish the goals?Precision-bred prototype plants continue to be constructed and placed into permitted field trials at three locations. As they grow to maturity, we will begin to see the results of out attempts to create fruit rot-resistant, seedless muscadine grape varieties.

Impacts
What was accomplished under these goals? During this second reporting period, we continued to train a newly hired Postdoctoral Research Associate and a Ph.D. candidate in the Gray program and a M.S. candidate in the House/Olmstead program. Following the Precision Breeding approach of the project, grape-derived genes and genetic elements continued to be isolated from the grape genome, as described in refereed publications, and prepared for testing, including the following: 1) 31 promoter sequences were recovered and tested for driving gene expression (see publication), 2) A functional terminator sequence was recovered and tested, 3) Putative antimicrobial genes for fruit rot resistance were recovered, including Vitis vinifera thaumatin-like protein gene (VVTL-1), Pathogenesis-related protein 1 gene (PR-1), Snakin/Defensin gene, Antimicrobial peptide genes 1 & 2 (AMP-1, AMP-2). 4) Putative genes to induce seedlessness were recovered including an Anti-sucrose synthase gene (SuSy) and a WOX gene. Partially grape-derived vectors were created and inserted into grapevine. Each vector currently contains a non-grape-derived NPTII gene for antibiotic selection. The vectors are being inserted into our high-performance (non-muscadine) model germplasm for early testing of disease resistance and seedlessness. Prototype muscadine plants were produced and propagated for field trials. These plants contain the VVTL-1 gene (Alachua) and the Anti-SuSy gene (Delicious). An application to USDA/APHIS for a field trail permit was submitted in June and appears to be technically approved. In anticipation of a permit, field sites have been prepared in Florida, Mississippi and the US Virgin Islands. This includes installation of trellises, irrigation and soil amendment. In order to conduct future evaluation of fruit rot resistance and post-harvest storage, An economic analysis is being conducted to compare net return of conventional vs.Precision-Bred muscadine. This net return analysis will be essential in determining grower's acceptability to adopt Precision-Bred muscadines. A focus group will be conducted to assist in development of a survey designed to evaluate consumer acceptance of Precision-Bred muscadine fruit compared to fruit developed by conventional breeding or a transgenic approach. Information about the project was disseminated at one regional and one international meeting, PARTICIPANTS: Principal Investigators/Project Directors: Dennis J. Gray, Professor Grape Genetics and Developmental Biology, Director - Grape Biotechnology Core Laboratory, University of Florida/IFAS, Apopka, FL 3. Directed the project; conducted research to develop Precision Bred muscadine plants; prepared a field site; developed a field testing permit application for USDA/APHIS to enable testing in Florida, Mississippi and the US Virgin Islands. Dr. Esendugue Greg Fonsah, Associate Professor and Extension Economist - Fruit and Vegetables, University of Georgia, Tifton, GA. Began development of an economic analysis for cost effectiveness of Precision-Bred muscadines. Dr. Lisa House, Professor and Director of the Florida Agricultural market Research Center, University of Florida/IFAS, Gainesville, FL. Began development of a focus group will be conducted to assist in development of a survey designed to evaluate consumer acceptance of Precision-Bred muscadine fruit compared to fruit developed by conventional breeding or a transgenic approach. Dr. Mercy Olmstead, Assistant Professor Horticultural Science and Extension. University of Florida/IFAS, Gainesville, FL. Began development of a focus group will be conducted to assist in development of a survey designed to evaluate consumer acceptance of Precision-Bred muscadine fruit compared to fruit developed by conventional breeding or a transgenic approach; was invited to discuss the project at the Southeast Fruit and Vegetabole Conference. Dr. Charles A. Sims, Professor Food Science and Extension, University of Florida/IFAS, Gainesville, FL. Recruiting a graduate student to work on the project. Dr. Stephen J. Stringer, Research Horticulturalist, USDA ARS, Poplarville, MS. Prepared a field site for testing of Precision-Bred muscadines. Dr. Robert N. Trigiano, Professor, Plant Pathologist, University of Tennessee, Knoxville, TN. Isolated strains of the pathogen that causes fruit rot and is evaluating their epidemiology. Thomas W. Zimmerman, Research Associate Professor, University of the US Virgin Islands, Kingshill, St. Croix, VI. Developed a permitted field site for testing of Precision-Bred muscadines. Key Personnel: Dr. Zhijian T. Li, Senior Biological Scientist, University of Florida/IFAS, Apopka, FL. Conducted research to develop the genetic constructs for the project. Developed prototype muscadine plants for field testing. Postdoctoral Research Associate: Dr. Kyung H. Kim, Postdoctoral Research Associate, University of Florida/IFAS, Apopka, FL. Developing biolistic methods for gene insertion. Graduate Student: Mr. Jonathan Jasinski, Ph.D. Candidate, University of Florida/IFAS, Apopka, FL. Learned culture systems and currently involved in course work. Training or Professional Development: Postdoc Kim was terminated for non-performance. Mr. Jasinski obtained training in molecular genetics through course work. TARGET AUDIENCES: The current target audiences are growers of muscadine grapes and interested consumers, who are with whom we interact through regional presentations about the project and potential consumers who will be contacted via surveys. Report Date 07/21/2014

Publications

  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Z. T. Li, K-H Kim, J. R. Jasinski, M. R. Creech and D. J. Gray. 2012. Large-scale characterization of promoters from grapevine (Vitis spp.) using quantitative anthocyanin and GUS assay systems. Plant Sci. 196: 132-142.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Li, Z.T., K-H Kim, J.R. Jasinski, M.R. Creech and D.J. Gray. 2012. Large-scale characterization of promoters from grapevine (Vitis spp.) using quantitative anthocyanin and GUS assay systems. Plant Sci. 196: 132-142.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Dreyer, E., C. Sims, R. Rouseff, D. Gray and M. Sipowicz. 2013. Sensory and compositional characteristics of Blanc du Bois wine. Amer. J. Enol. Vitic. 64: 118-125.
  • Type: Other Status: Published Year Published: 2012 Citation: Gray, D. J., Z. T. Li, and S. A. Dhekney. 2012. Progress in the development of intragenic grapevine. World Congress on In Vitro Biology, In Vitro Cell. Dev. Biol. 48:S18.
  • Type: Other Status: Published Year Published: 2013 Citation: Gray, D. J., Z. T. Li, and S. A. Dhekney. 2013. Transcriptional activity and practical use of ubiquitin and PR-1 gene promoters from grapevine (Vitis spp.) in transgenic plant recovery. In Vitro Cell. Dev. Biol. Animal 49: S60.
  • Type: Other Status: Published Year Published: 2013 Citation: Gray, D. J, Z. T. Li, K.-H. Kim, D. D. Silva. S. A. Dhekney. 2013. Development of cisgenic grapevine. HortScience 48(9):S172.
  • Type: Other Status: Published Year Published: 2013 Citation: Kim, K.-H., Z. T. Li, J. R. Jasinski, D. J. Gray. 2013. An improved and efficient micropropagation system for grapevine (Vitis sp.). Southern Reg. Amer. Soc. Hort. Sci. annual meeting. HortScience 48(9):S34.
  • Type: Other Status: Published Year Published: 2013 Citation: Kim, K.-H., Z. T. Li, J. R. Jasinski, D. J. Gray. 2013. Optimizing grapevine transient transformation via biolistic bombardment of somatic embryos. In Vitro Cell. Dev. Biol. Animal 49:S38.


Progress 09/01/12 to 08/31/13

Outputs
Target Audience: The current target audiences are growers of muscadine grapes, who will be informed through regional presentations about the project and potential consumers who will be contacted via surveys. We expect that the target audiences will expand as the project continues past its first year. Efforts during this second year included laboratory training of the postdoc and graduate student in the genetic modification protocol. Outreach was accomplished through three invited presentations at a regional and international meeting as well aspublished refereed scientific article and published abstract. Changes/Problems: The postdoctoral research associate in the Gray lab was not renewed after the second year due to non-performance. This created a significant slow-down in planned progress to create modified grape lines for testing. What opportunities for training and professional development has the project provided? Dr. Kyung H. Kim, Postdoctoral Research Associate, University of Florida/IFAS, Apopka, attended a grant writing workshop, sponsored by UF. Mr. Jonathan Jasinski, Ph.D. Candidate, University of Florida/IFAS, Apopka,learned culture systems and is currently involved in course work. Training or Professional How have the results been disseminated to communities of interest? Dr. Gray gave three invited presentations discussing the project to scientists. Dr. Olmstead gave a presentation on genetic engineering terminology to a major group of extension agents, specialists and other fruit professional workers in September, 2013 as part of the Southeastern Fruit Professional Workers Conference, in Athens, GA. What do you plan to do during the next reporting period to accomplish the goals? Emphasis is on testing the usefullness of grape-derived genes and promoting elements and in creating genetically-modified muscadine lines for field evaluation.

Impacts
What was accomplished under these goals? D.J. Gray & Z.T. Li: Successful utilization of cisgenes to develop resistant engineered plants against various pathogens requires extraordinary efforts to determine which gene(s) among numerous candidate genes isolated from the host genome are responsible for the desired resistance characteristics. Following our efforts to characterize the functional capacity of various promoters and genetic elements from grapevine, major emphasis was give to gene characterization in 2013. Detailed operations and significant findings are given in the following outlines. To test the functional fidelity of gape-derived genes, a total of 227 plants including non-transformed and marker-containing only and resistance gene-expressing plants were transplanted to the USDA-permitted field site in May of 2013. Resistance genes being evaluated include Vitis vinifera thaumatin-like protein gene (VVTL-1), pathogenesis-related protein 1 gene (PR-1), Snakin/Defensin gene, 2S albumin gene (ALB) and antimicrobial peptide genes 1 & 2 (AMP-1, AMP-2). During the first growing season in 2013, all newly transplanted plants were subject to heavy fungal and bacterial infestation due to abundant supply and ready movement of pathogens from the adjacent field plot that was designed and had been maintained for 7 years to evaluate Pierce's disease resistance in transgenic grapevine plants. Severe disease symptoms were detected mostly from control plants along with some transgenic plants. Consequently, all non-transformed control plants and marker-controlling transgenic plants died. On the other hand, survival percentages of transgenic plants for PR-1 (101 plants), VVTL-1 (11 plants), Snakin (3 plants), AMP (6 plants) and ALB/VVTL-1 combination genes (79 plants) ranged 84.1, 27.3, 100, 83.3, R.N. Trigiano: Although Colletotrichum gleosporides is the recognized casual agent of muscadine grape rot, we frequently isolate a species of Pestalotia from rotting grapes. Isolates of C. gleosporides and Pestalotia sp. were tested for the ability to produce cellulases and pectinases, both enzymes are involved in fruit rots. Both species were prolific producers of both exo- and endo- cellulases and pectinases. It is very possible that Pestalotia may contribute to muscadine postharvest rot of muscadine grapes and should be considered when evaluating cisgenic grapes for disease resistance and enhanced shelf life. We also developed a pressure/puncture protocol for measuring “ripeness or rottiness” of grapes. C.A. Sims: A study looking at the sensory acceptability of about 25 muscadine varieties/lines and how that relates to texture of muscadine pulp and skin (with Patrick Conner). A study looking at a comparison of the standard scale used to measure how much people like things vs. a new scale of measuring that.We plan to use the new scale to measure how much people like muscadines in the sensory studies we will eventually do withgenetically altered lines. Twenty-two different cultivars grown in southern Georgia at the Tifton University of Georgia campus were tested over the span of the approximately two month muscadine growing season. Each week, between 4 and 6 cultivars were harvested at optimum maturity and tested at the University of Florida. At the last session, four commercial grape varieties (red, green, black, (vitis vinifera) and Concord) were tested as well for comparison purposes. A sensory panel of approximately 85 people was recruited with the screening criteria of them having previously tasted muscadine grapes. The scale used for this sensory panel is the hedonic general labeled magnitude scale or Hg-LMS. Questions the panelists were asked to rate include: overall liking, appearance liking, flavor liking, pulp texture liking and skin texture liking. Textural analysis was completed on the muscadine grapes at the same time as the ongoing sensory panel. Texture Profile Analysis (TPA) and puncture testing were done using a TA.XT plus texture analyzer with a 50kg load. These tests were performed on 20 grapes of each cultivar. Other analyses that were completed include Brix, pH, and titratable acidity. All the collected data was subjected to analysis of variance (using Statistical Analysis Software or SAS) to determine differences between cultivars. Correlation and regression analysis and principal component analysis were used to identify relationships between sensory scores and the quality data. From the preliminary analysis of variance data collected, there were significant differences detected between the cultivars by the panelists. The average soluble solids were 14.9 degrees Brix, the mean pH recorded was 3.76, and the average ratio between soluble solids and acidity was 48.79%. The three cultivars with the highest overall liking scores were GA 2-8-21, GA 6-2-46, and GA 5-1-34. Conversely, the three cultivars that scored the lowest overall liking scores were Triumph, GA 1-1-48, and Carlos. In relation to scores received by the table grape varieties, only the GA 2-8-21 cultivar scored higher with an average rating of 32.13. Correlation data revealed that the factors that are highly correlated with overall liking scores were muscadine flavor liking/disliking (R=0.94), and muscadine pulp liking/disliking (R=0.88). S.A. Stringer: Field study,Stone County, MS. Fourty-five muscadine grape plants consisting of 18 plants of ‘Delicious’ HS9, 18 plants of ‘Delicious’, 9 plants of ‘Alachua’ and 10 plants of ‘Alachua’ 214D GFP+ were established in the field in June and July 2013 in the test site located at the USDAS-ARS research farm near Wiggins, MS. Plants care included irrigation, fertilization, and use of wire mesh around plants to prevent deer feeding injury. The site was inspected by USDA – APHIS in August 2013, and was found to be in compliance. M.A. Olmstead & L.A. House: Helped to direct and collect data on consumer acceptance of cisgenic muscadine fruit compared to fruit developed by conventional breeding or transgenic approaches. This student was successful in finishing her M.S. Thesis. Publications on the students’ results are being developed and will be published in 2014. Dr. Olmstead gave a presentation on genetic engineering terminology to a major group of extension agents, specialists and other fruit professional workers in September, 2013 as part of the Southeastern Fruit Professional Workers Conference, in Athens, GA.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2012 Citation: Li, Z.T., K-H Kim, J.R. Jasinski, M.R. Creech and D.J. Gray. 2012. Large-scale characterization of promoters from grapevine (Vitis spp.) using quantitative anthocyanin and GUS assay systems. Plant Sci. 196: 132-142.


Progress 09/01/11 to 08/31/12

Outputs
OUTPUTS: During this first reporting period, certain staffing needs were met, including hiring a Postdoctoral Research Associate and a Ph.D. candidate in the Gray program and a M.S. candidate in the House/Olmstead program. Following the cisgenic approach of the project, grape-derived genes and genetic elements were isolated from the grape genome and prepared for testing, including the following: 1) 31 promoter sequences were recovered and tested for driving gene expression (see publication), 2) A functional terminator sequence was recovered and tested, 3) Putative antimicrobial genes for fruit rot resistance were recovered, including Vitis vinifera thaumatin-like protein gene (VVTL-1), Pathogenesis-related protein 1 gene (PR-1), Snakin/Defensin gene, Antimicrobial peptide genes 1 & 2 (AMP-1, AMP-2). 4) Putative genes to induce seedlessness were recovered including an Anti-sucrose synthase gene (SuSy) and a WOX gene. Partially cisgenic vectors were constructed containing each of these genes driven by the best constitutive promoter discovered (see publication) and terminator. Each vector currently contains a non-cisgenic NPTII gene for antibiotic selection. The vectors are being inserted into our high-performance (non-muscadine) model germplasm for early testing of disease resistance and seedlessness. Prototype muscadine plants were produced and propagated for field trials. These plants contain the VVTL-1 gene (Alachua) and the Anti-SuSy gene (Delicious). An application to USDA/APHIS for a field trail permit was submitted in June and appears to be technically approved. In anticipation of a permit, field sites have been prepared in Florida, Mississippi and the US Virgin Islands. This includes installation of trellises, irrigation and soil amendment. In order to conduct future evaluation of fruit rot resistance and post harvest storage, 10 isolates of Colletotrichum were recovered from muscadine fruit. An economic analysis is being conducted to compare net return of conventional vs. cisgenic muscadine. This net return analysis will be essential in determining grower's acceptability to adopt cisgenic muscadine. A focus group will be conducted to assist in development of a survey designed to evaluate consumer acceptance of cisgenic muscadine fruit compared to fruit developed by conventional breeding or a transgenic approach. Information about the project was disseminated at one regional and one international meeting, which include the following: 1) Olmstead et. al., "Seedlessness and disease resistance in muscadine: bringing it to reality", Southeast Fruit and Vegetable Conference, Savannah GA, 1-7-12, and 2) Gray et al., "Progress in the development of intragenic grapevine", World Congress on In Vitro Biology, Belleview WA, 6-3-12. PARTICIPANTS: Principal Investigators/Project Directors: Dennis J. Gray, Professor Grape Genetics and Developmental Biology, Director - Grape Biotechnology Core Laboratory, University of Florida/IFAS, Apopka, FL3. Directed the project; conducted research to develop cisgenic muscadine plants; prepared a field site; developed a field testing permit application for USDA/APHIS to enable testing in Florida, Mississippi and the US Virgin Islands. Dr. Esendugue Greg Fonsah, Associate Professor and Extension Economist - Fruit and Vegetables, University of Georgia, Tifton, GA. Began development of an economic analysis for cost effectiveness of cisgenic muscadines. Dr. Lisa House, Professor and Director of the Florida Agricultural market Research Center, University of Florida/IFAS, Gainesville, FL. Began development of a focus group will be conducted to assist in development of a survey designed to evaluate consumer acceptance of cisgenic muscadine fruit compared to fruit developed by conventional breeding or a transgenic approach. Dr. Mercy Olmstead, Assistant Professor Horticultural Science and Extension. University of Florida/IFAS, Gainesville, FL. Began development of a focus group will be conducted to assist in development of a survey designed to evaluate consumer acceptance of cisgenic muscadine fruit compared to fruit developed by conventional breeding or a transgenic approach; was invited to discuss the project at the Southeast Fruit and Vegetabole Conference. Dr. Charles A. Sims, Professor Food Science and Extension, University of Florida/IFAS, Gainesville, FL. Recruiting a graduate student to work on the project. Dr. Stephen J. Stringer, Research Horticulturalist, USDA ARS, Poplarville, MS. Prepared a field site for testing of cisgenic muscadines. Dr. Robert N. Trigiano, Professor, Plant Pathologist, University of Tennessee, Knoxville, TN. Isolated strains of the pathogen that causes fruit rot. Dr. Thomas W. Zimmerman, Research Associate Professor, University of the US Virgin Islands, Kingshill, St. Croix, VI. Preparing a field site for testing of cisgenic muscadines. Key Personnel: Dr. Zhijian T. Li, Senior Biological Scientist, University of Florida/IFAS, Apopka, FL. Conducted research to develop the genetic constructs for the project. Developed prototype muscadine plants for field testing. Postdoctoral Research Associate: Dr. Kyung H. Kim, Postdoctoral Research Associate, University of Florida/IFAS, Apopka, FL. Developing biolistic methods for gene insertion. Graduate Student: Mr. Jonathan Jasinski, Ph.D. Candidate, University of Florida/IFAS, Apopka, FL. Learned culture systems and currently involved in course work. Training or Professional Development: Dr. Kim attended a short course on grant writing. Mr. Jasinski is obtaining training in molecular genetics through course work. TARGET AUDIENCES: The current target audiences are growers of muscadine grapes, who will be informed through regional presentations about the project and potential consumers who will be contacted via surveys. We expect that the target audiences will expand as the project continues past its first year. Efforts during this first year included laboratory training of the postdoc and graduate student in the genetic modification protocol. Outreach was accomplished through two invited presentations at a regional and international meeting as well as a published refereed scientific article and a published abstract. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Muscadine grape is a highly nutritious fruit, with higher fiber and antioxidant levels than other types of grape. However, muscadine grape is an underutilized fruit crop due to poor shelf life and lack of seedless varieties. This project utilizes a consumer-friendly genetic modification approach, termed "cisgenesis" or "precision breeding", to produce advanced varieties with fruit rot resistance and seedlessness. Fruit of rot resistant varieties will be able to be marketed to a much broader geographic region due to increased shelf life. Seedless varieties will have increased desirability to consumers. The impact of this project is that demand for muscadine grape will increase significantly. This will result in greatly increased acreage devoted to production in the southeastern United States along with more jobs in production, distribution and marketing.

Publications

  • D. J. Gray, Z. T. Li and S. A. Dhekney. 2012. Progress in the development of intragenic grapevine, World Congress on In Vitro Biology, In Vitro Cell. Dev. Biol. 48:S18 (abstract).
  • Z. T. Li, K-H Kim, J. R. Jasinski, M. R. Creech and D. J. Gray. 2012. Large-scale characterization of promoters from grapevine (Vitis spp.) using quantitative anthocyanin and GUS assay systems. Plant Sci. 196: 132-142.