Progress 01/01/12 to 12/31/15
Outputs
Target Audience:Results were presented at the Annual meeting of project directors for NIFA food science investigators. Handa AK, Campanella OH Mitcham EJ, de Freitas ST (2015). Industrial pectin of genetically modified fruit pectin and reduction in calcium deficiency disorders. USDA/NIFA - NRI/AFRI Function and Efficacy of Nutrients, Bioactive Components of Foods, Improved Processing Technologies, Improving Food Quality and Value, and Reducing Food Allergies by Improving Food Quality Project Director's Meeting. McCormick Place - Chicago July 10, 2015 Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Project has allowed training of undergraduate and graduate students and postdocs to do relevant agricultural research and learn to use emerging technologies including biotechnology and instrumentation. How have the results been disseminated to communities of interest?Results were presented at the Annual meeting of project directors for NIFA food science investigators Handa AK, Campanella OH Mitcham EJ, de Freitas ST (2015). Industrial pectin of genetically modified fruit pectin and reduction in calcium deficiency disorders. USDA/NIFA - NRI/AFRI Function and Efficacy of Nutrients, Bioactive Components of Foods, Improved Processing Technologies, Improving Food Quality and Value, and Reducing Food Allergies by Improving Food Quality Project Director's Meeting. McCormick Place - Chicago July 10, 2015 What do you plan to do during the next reporting period to accomplish the goals? Develop proposal to test new hypothesis emerged from these investigation and pursue them if funded.
Impacts
What was accomplished under these goals?
All stated objectives of the proposed research were accomplishes, however, further experimentation is required to test some of the hypothesis emerged from these studies. . The following paragraph summarizes some of these accomplishments. Characterization of genetically engineered reduced PME activity on cell wall chemistry and pectin recovery from fruit: Characterization of six independent transgenic line with introduced PME antisense gene showed that the introduced PME antisense gene impaired expression of PME both at transcript accumulation and total enzymatic activity levels that was stably maintained in sexually propagated progenies for at least up to 6 generations. The evaluation of cell wall composition revealed presence of significantly higher levels of galacturonic acid and rhamnose in transgenic fruits with reduced PME activity compared to parental lines, suggesting impaired PME activity reduced cell wall hydrolysis and maintaining the higher the size of pectin compared to wild type parental fruits. Cell wall sugar linkage analyses confirmed these results as there was significant increase increases in 2-Rha, 4-GalA, 3,4 GalA and 5-Ara(f) linkages in the cell walls of low PME compared to parental non-engineered ripening fruits. Collectively, results showed genetic engineering of PME activity lead to higher retention of pectin in the transgenic fruits and providing a strong evidence for improving both pectin quantity and quality by genetically engineering of tomato. Transcriptome characterization of ripened fruits showed that reduction in PME activity was associated with a massive changes in gene expression. Reduction in PME activity led to up and down regulation of over 1000 genes in ripening tomato fruit. These genes represented a range of enzymatic activity with a potential to further enhance fruit pectin quality. These results suggest that pectin generated poly/oligo-uronides plays significant role in expression of genes, however, more work/experiments are needed to further this hypothesis. Evaluation of the roles of these genes during fruit ripening, using emerging genome editing technology, would likely yield a wealth of information to improve fruit quality. Our results showed that a significant improvement can be achieved in the pectin degree of methoxylation by regulating in vivo PME activity to an optimum level and would likely increase yield of pectin and improve processing attributes of tomato juice. Characterization of genetically engineered tomato pectin including rheological attributes: Pectin from genetically modified tomato fruits with dramatic reduction in PME activity was extracted and evaluated using a battery of tests to determine its suitability for processed product requiring high methoxyl pectin. The steady-state shear ramps performed to discern differences in rheology between pectin prepared from tomato fruits with varying PME activity and ripeness stage showed that reduction in PME activity greatly increased the final viscosity of prepared tomato juice even from advanced stages of fruit ripening that generally lowers juice quality due to cell wall degrading enzymes. The difference in viscosity for juice was more distinct than that for serum, indicating the importance of PME activity on the fruit cell wall and water-insoluble pectins. Nuclear Magnetic Resonance (NMR) spectra and Gas Chromatography (GC) both indicated the presence of galacturonic acid residues in tomato serum, and NMR showed that these galacturonic acid residues are indeed from pectin. NMR spectra established the effectiveness in down-regulating PME on the degree of methylation of serum pectins, confirming with rheological measurements that increased DM of pectin yields higher viscosity products. Practical application of this information will prove valuable in understanding the effect of genetically regulating enzyme activity in vivo and determining the optimal level of PME for creating tomato products with desirable quality attributes. This work has been published as a master thesis and is being written for publication for general dissemination. Effects of modified pectin chemistry on levels of free and bound calcium in fruits and its effects on calcium related physiological disorders: Our preliminary results showed that fruits from a pectin methylesterase (PME)-silenced line containing 10% of wild type PME activity had lower fruit susceptibility to BER during growth and development. These observations led to hypothesis that (PME) expression and activity increases Ca2+ binding to the cell wall, reducing Ca2+ content in other cellular pools of Ca2+ and increasing fruit tissue susceptibility to blossom-end rot (BER) development. A series of experiments analyzing the changes in BER incidence, plasma membrane leakage, cell turgor pressure, PME activity, fruit elasticity and firmness did not corroborated this hypothesis as a direct correlation between PME activity and PME was not observed. The presence of BER in tomato fruit enhanced PME activity in the affected tissue but this relationship is not reciprocal as genetically silencing PME expression and activity did not avoid the development of BER. Exogenous Ca2+ treatment did not significantly affect development of BER in wild type (WT) and 8 PME-silenced genotypes but the lack of Ca2+ fertilization significantly increased the % of BER in all genotypes. Results showed that the transportation of calcium to the bottom of the fruit is low as the percentage of total calcium was lower in bottom tissues than the top tissue in WT and low PME fruits suggesting initiation of BER related biological processes. Within the same genetic line, tomatoes with BER displayed lower apoplastic calcium than tomatoes without BER; however, a tomato line with low apoplastic calcium does not necessarily show BER development. Preliminary results from the RNAseq experiments revealed significant changes expression of genes related heat stress/desiccation between top and bottom cores of tomatoes with BER. Further evaluation of these changes would help understand the molecular signals leading to development of BER. Finally our results have identified several growth regulators that caused reduction in BER presence.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Handa A.K., Anwar R. and Mattoo A. K. (2014) Biotechnology of Fruit Quality. In: Fruit Ripening: Physiology, Signalling and Genomics (eds P. Nath et al.). CAB International 2014. Pp 259-290.
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Handa A.K., Anwar R. and Mattoo A. K. (2014) Genetic Regulation of Phytonutrients in Fresh Produce for Health Benefits International Symposium on Quality Management of Fruits and Vegetables for Human Health. FVHH2013, Bangkok, Thailand, August 5-8, 2013
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Anwar, R., Mattoo, A.K. and Handa, A.K. (2014) Polyamine Interactions with Plant Hormones: Crosstalk at Several Levels. In T. Kusano, H. Suzuki (eds.), Polyamines universal molecular nexus for growth, survival and specialized metabolism, Springer, Japan, pp.267-302; DOI 10.1007/978-4-431-55212-3_22
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Handa AK (2014) Biotechnology and enhanced pro-health nutrient metabolome of fruit Annual meeting pf ASHS 2014, Orlando, FL
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Handa A.K. (2016) Regulation of fruit quality, shelf life and architecture of tomato fruit by genetically enhanced spermidine levels Utsunomiya University, Utsunomiya, Japan, March 15 2016
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Handa AK, Datsenka T, Carpita NC, McCann M (2014) Characterization of pectin and rhamnogalacturonan-I network in the cell walls of transgenic fruits with altered expression of a pectin methylesterase and a putative rhamnogalacturonan lyase. Annual meeting of ASPB, Portland, OR
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Progress 01/01/14 to 12/31/14
Outputs
Target Audience: Results were presented at the Annual meeting of project directors for NIFA food science investigators held on April 26, 2014 as a part of Experimental Biology 2014 meeting In San Diego Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Project has allowed training of graduate student and technician to do relevant agricultural research and learn to use emerging technologies including biotechnology and instrumentation. How have the results been disseminated to communities of interest? Results were presented at the Annual meeting of project directors for NIFA food science investigators Handa AK, Campanella OH, Mitcham EJ (2014) Industrial Properties of Genetically Modified Fruit Pectin and Related Reductions In Calcium Deficiency Disorders. Annual meeting of project directors for NIFA food science investigators Convention Center, San Diego, CA, April 24, 2014 What do you plan to do during the next reporting period to accomplish the goals? Objective 1: We are presently writing manuscript on the results that we have obtain. These results will be published in a refereed journal Objective 2: As stated above research is still in progress but we hope to complete it by the end of December 2015. Objective 3: To determine the effect of degree of pectin methoxylation on processing efficiency: Most of this objective has been completed. We will be writing this data for publication to disseminate information. Objective 4: To determine the effects of modified pectin chemistry on calcium related physiological disorders. Experiments are in progress at UC Davis in Prof Mitcham laboratory to determine how low PME activity alters fruit quality by regulating effects of endogenous calcium. We hope to finish this work by the end of 2015 and publish results in a refereed journal.
Impacts
What was accomplished under these goals?
Objective 1: To determine the effects of altered PME activity on cell wall chemistry The effects of decreasing PME activity on pectin chemistry were characterized. Our results show that reduced PME activity modifies the carbohydrate composition of fruit pectin and significantly altered sugar linkages of cell wall polymers. Pectin from the low PME fruits retains significant higher levels of galacturonic acid likely by reducing accessibility of sugar-linkages to the cell wall hydrolases leading to impaired hydrolysis of fruit pectin. Objective 2: To determine the effects of altered degree of methyl esterification on pectin molecular size and the resulting rheological properties. We have begun to characterize the effects of reduced PME activity on overall gene expression and fruit quality. This work is in progress Objective 3: Physical and chemical attributes of a genetically modified fruit pectin Significant progress was made in accomplishing this objective in Prof. Campanella laboratory. Carl Litterell has completed his master project to accomplish objectives of the proposed research and completed his M.S under Prof. Campanella and submitted his thesis to Purdue University. Summary of his findings included in his thesis are as follows: The steady-state shear ramps were performed to discern differences in rheology between prepared tomato samples of varying PME activity and ripeness stage, for whole tomato juice, separated juice serum, and tomato juice solids reconstituted with both water and tomato serum. Our results show that a reduction in PME activity greatly increases the final viscosity of prepared tomato samples, and the maturation process decreases overall viscosity through the degradation and demethylation pf plant cell walls and pectin. The difference in viscosity for juice was more distinct than that for serum, indicating the importance of PME activity on the fruit cell wall and water-insoluble pectin. Nuclear Magnetic Resonance (NMR) spectra and Gas Chromatography (GC) both indicated the presence of galacturonic acid residues in tomato serum, and NMR showed that these galacturonic acid residues are indeed from pectin. Furthermore, NMR spectra confirmed the effectiveness in down-regulating PME and its impact on the degree of methylation of serum pectin, confirming with rheological measurements that increased DM of pectin yields higher viscosity products. Practical application of this information will prove valuable in understanding the effect of genetically regulating enzyme activity in vivo and determining the optimal level of PME for creating tomato products with desirable quality attributes. Furthermore, the present work seeks to use tomato pectin as a model system for elucidating information regarding the relationship between genetic alterations of pectin and the effect on its chemical structure which affect the physical and functional attributes. Objective 4: To determine the effects of modified pectin chemistry on calcium related physiological disorders. Professor Mitcham's laboratory conducted a large number of experiments using nine tomato genotypes, impaired in expression of PME, developed at Purdue. To delineate the role of PME in calcium related disorders, various parameters were evaluated at 15, 30 and 45 days after pollination: quantification of BER incidence, fruit firmness and cell turgor pressure, and correlated with fruit PME activity in different tomato tissues and preliminary studies of expression of the PME gene LOC544090. Results from these investigations revealed that 1) PME enzymatic activity and initial BER development are located in different tomato tissues, 2) BER developed in tomato fruit independently from PME activity and calcium in nutrient solution, 3) BER altered firmness and turgor pressure, and 4) BER activated the expression of the PME gene at early stages of development. However, this relationship was not reciprocal; genetically silencing PME expression and activity did not avoid the development of BER. To confirm these results, further studies will include color, firmness, membrane leakage, total soluble calcium, apoplastic calcium, total calcium, PME activity (in four different sections of tomato) and PME gene expression (specific sections of tomato and four different PME genes) at 15, 30 and 45 DAP.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Handa AK, Campanella OH, Mitcham EJ (2014) Industrial Properties of Genetically Modified Fruit Pectin and Related Reductions In Calcium Deficiency Disorders. Annual meeting of project directors for NIFA food science investigators Convention Center, San Diego, CA, April 24, 2014
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Handa A.K., Anwar R. and Mattoo A. K. (2014) Biotechnology of Fruit Quality. In: Fruit Ripening: Physiology, Signalling and Genomics (eds P. Nath et al.). CAB International 2014. Pp 259-290.
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2015
Citation:
Escribano, S. and Mitcham, E. J. (2015) Effects of modified pectin chemistry on tomato fruit susceptibility to the calcium deficiency disorder blossom-end rot. In: Proceedings of the Fifth International Conference Postharvest Unlimited. Eds. G.A. Manganaris, P.M. Toivonen and P. Kalaitzis. ISHS. Acta Horticulturae 1079. (In Press)
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2014
Citation:
Carl Patrick Littrell (2014)PHYSICAL AND CHEMICAL ATTRIBUTES OF A GENETICALLY MODIFIED FRUIT PECTIN. MS Thesis, Purdue University
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Progress 01/01/13 to 12/31/13
Outputs
Target Audience: food processors Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Project has allowed training of both undergraduate and graduate student to do relevant agricultural research and learn to use emerging technologies including biotechnology and instrumentation. How have the results been disseminated to communities of interest? Results were presented at the Annual meeting of project directors for NIFA food science investigators Handa AK, Campanella OH, Mitcham EJ (2013) Industrial Properties Of Genetically Modified Fruit Pectin And Related Reductions In Calcium Deficiency Disorders USDA/NIFA grant 12-67017-30159. National Meeting of Project Directors. IFT2013, Chicago August 16, 2013. What do you plan to do during the next reporting period to accomplish the goals? Objective 1: We have determined the effect of altered PME on cell wall chemistry in WT and one of the transgenic fruit. To establish the role of PME in the cell wall sugar composition and linkage analyses, we will be analyzing additional genotypes with altered PME activity. Other experiments will include (1) Sequential extraction of wall polymer fractions, (2) Fourier transform infrared (FTRI) micro-spectroscope to determine the effect of pectin engineering on cell wall composition. Objective 2: A standard procedure for sample preparation was established and will be used on all samples moving forward to ensure consistent results that provide better insight into the role of PME on functional attributes of pectin. The analytical techniques required for accurately characterizing the chemistry of the pectins of the transgenic lines at different maturation levels were decided and will be implemented in congruence with the rheological and particle size data gathered to provide a precise view into the structure-function relationship of pectin. Objective 3: To determine the effect of degree of pectin methoxylation on processing efficiency: Most of this objective has been completed. We will be writing this data for publication to disseminate information. Objective 4: To extend and validate our preliminary results, many new PME-silenced tomatoes are growing in the UC Davis greenhouse. Fruit of wild-type and selected PME-silenced homozygous lines will be analyzed at 15, 30 and 45 days after pollination for BER incidence. Fruit pericarp tissue will be analyzed for plasma membrane leakage, cellular turgor pressure, PMEs activity and expression, degree of extracted water soluble and insoluble pectin methoxylation, total tissue Ca2+ concentration , total tissue water soluble and insoluble Ca2+ concentration, cell wall Ca2+ concentration, water soluble and insoluble pectin Ca2+ concentrations. Pericarp tissue will also be analyzed for in situ immunolocalization of high and low methylated pectins to characterize the cell wall distribution of these pectins during fruit growth and development and increasing fruit susceptibility to BER incidence.
Impacts
What was accomplished under these goals?
1. Genotype of each transgenic line used in the research was reconfirmed both by PCR and segregation analyses. Fruits from each genotype were also evaluated for the PME activity to confirm the stable gene silencing of PME and to use transgenic events resulting in different levels of endogenous PME activity in ripening tomato fruits. 2. Since older seeds from some of the PME lines exhibited reduced germination on soil, they were rescued using agar plate and planted in the greenhouse and fresh seeds recollected. Seeds of many of these lines were sent to UC, Davis for determining BER and other parameters. 3. Effects of decreasing PME activity on pectin chemistry and rheological properties are being characterized. . Our initial results show that reduced fruit PME results in higher retention of pectin in fruits. Reduction in the PME activity significantly altered sugar linkages of cell wall polymers as fruit pectin from low PME fruits retains significant higher levels of galacturonic acid. Our results suggest that the high degree of methoxylation reduces accessibility of several linkages to cell wall hydrolases and impair hydrolysis of fruit pectin. 4. We have been evaluating the rheological properties, including sugar content, particle size, and tomato juice serum composition of genetically modified pectin. Low PME transgenic fruit juice and serum exhibited markedly higher viscosity than high PME juice. Juice displays non-Newtonian (pseudoplastic) behavior, while serum showed purely Newtonian behavior. Measurements of the rheological steady-shear rates for whole juice and serum from transgenic fruits exhibited 11% and 100% of that of the WT parental fruit. These differences are indicative of the higher degree of methoxylation in pectin juice pulp due to reduced PME activity, yielding positive attributes for processing. 5. Scanning Electron Microscopic (SEM) images displayed a noticeable difference in the plant cell structure and water carrying capability between the wild type and transgenic fruits. 6. Fruits from Ohio 8245 lines having 4%, 7% and 67% (transgenic lines) and 100% (parental line) PME activities were evaluated for BER both at Purdue and UC Davis laboratories. Trails in the Purdue green house indicated that PME activity of about 10% of the WT fruits reduced development of BEP in tomato fruits. UC Davis research suggested no correlation between PME activity and BER development. BER development was also independent of Ca2+ levels in the nutrient solution in this investigation. PME silencing did not affect solute leakage from fruit membranes and ripening associated fruit firmness. However, UC Davis research indicated that PME activity dramatically affected the turgor pressure of the cells both in the presence and absence of Ca2+ .presence or absence of calcium and in the absence of calcium modified elasticity of the fruit around the equatorial plate.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Handa AK (2013) Genetic Regulation of Phytonutrients in Fresh Produce for Health Benefits. International Symposium on
Quality Management of Fruits and Vegetables for Human Health. FVHH2013, Bangkok, Thailand, August 5-8, 2013
|
Progress 01/01/12 to 12/31/12
Outputs
Target Audience: food processors Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Project has allowed training of both undergraduate and graduate student to do relevant agricultural research and learn to use emerging technologies including biotechnology and instrumentation How have the results been disseminated to communities of interest? Results were presented at the Annual meeting of project directors for NIFA food science investigators Handa AK, Campanella OH, Mitcham EJ (2013) Industrial Properties Of Genetically Modified Fruit Pectin And Related Reductions In Calcium Deficiency Disorders USDA/NIFA grant 12-67017-30159. National Meeting of Project Directors. IFT2013, Chicago August 16, 2013. What do you plan to do during the next reporting period to accomplish the goals? Objective 1: We have determined the effect of altered PME on cell wall chemistry in WT and one of the transgenic fruit. To establish the role of PME in the cell wall sugar composition and linkage analyses, we will be analyzing additional genotypes with altered PME activity. Other experiments will include (1) Sequential extraction of wall polymer fractions, (2) Fourier transform infrared (FTRI) micro-spectroscope to determine the effect of pectin engineering on cell wall composition. Objective 2: A standard procedure for sample preparation was established and will be used on all samples moving forward to ensure consistent results that provide better insight into the role of PME on functional attributes of pectin. The analytical techniques required for accurately characterizing the chemistry of the pectins of the transgenic lines at different maturation levels were decided and will be implemented in congruence with the rheological and particle size data gathered to provide a precise view into the structure-function relationship of pectin. Objective 3: To determine the effect of degree of pectin methoxylation on processing efficiency: Most of this objective has been completed. We will be writing this data for publication to disseminate information. Objective 4: To extend and validate our preliminary results, many new PME-silenced tomatoes are growing in the UC Davis greenhouse. Fruit of wild-type and selected PME-silenced homozygous lines will be analyzed at 15, 30 and 45 days after pollination for BER incidence. Fruit pericarp tissue will be analyzed for plasma membrane leakage, cellular turgor pressure, PMEs activity and expression, degree of extracted water soluble and insoluble pectin methoxylation, total tissue Ca2+ concentration , total tissue water soluble and insoluble Ca2+ concentration, cell wall Ca2+ concentration, water soluble and insoluble pectin Ca2+ concentrations. Pericarp tissue will also be analyzed for in situ immunolocalization of high and low methylated pectins to characterize the cell wall distribution of these pectins during fruit growth and development and increasing fruit susceptibility to BER incidence.
Impacts
What was accomplished under these goals?
Genotype of each transgenic line used in the research was reconfirmed both by PCR and segregation analyses. Fruits from each genotype were also evaluated for the PME activity to confirm the stable gene silencing of PME and to use transgenic events resulting in different levels of endogenous PME activity in ripening tomato fruits. Since older seeds from some of the PME lines exhibited reduced germination on soil, they were rescued using agar plate and planted in the greenhouse and fresh seeds recollected. Seeds of many of these lines were sent to UC, Davis for determining BER and other parameters. Effects of decreasing PME activity on pectin chemistry and rheological properties are being characterized. . Our initial results show that reduced fruit PME results in higher retention of pectin in fruits. Reduction in the PME activity significantly altered sugar linkages of cell wall polymers as fruit pectin from low PME fruits retains significant higher levels of galacturonic acid. Our results suggest that the high degree of methoxylation reduces accessibility of several linkages to cell wall hydrolases and impair hydrolysis of fruit pectin. We have been evaluating the rheological properties, including sugar content, particle size, and tomato juice serum composition of genetically modified pectin. Low PME transgenic fruit juice and serum exhibited markedly higher viscosity than high PME juice. Juice displays non-Newtonian (pseudoplastic) behavior, while serum showed purely Newtonian behavior. Measurements of the rheological steady-shear rates for whole juice and serum from transgenic fruits exhibited 11% and 100% of that of the WT parental fruit. These differences are indicative of the higher degree of methoxylation in pectin juice pulp due to reduced PME activity, yielding positive attributes for processing. Scanning Electron Microscopic (SEM) images displayed a noticeable difference in the plant cell structure and water carrying capability between the wild type and transgenic fruits. Fruits from Ohio 8245 lines having 4%, 7% and 67% (transgenic lines) and 100% (parental line) PME activities were evaluated for BER both at Purdue and UC Davis laboratories. Trails in the Purdue green house indicated that PME activity of about 10% of the WT fruits reduced development of BEP in tomato fruits. UC Davis research suggested no correlation between PME activity and BER development. BER development was also independent of Ca2+ levels in the nutrient solution in this investigation. PME silencing did not affect solute leakage from fruit membranes and ripening associated fruit firmness. However, UC Davis research indicated that PME activity dramatically affected the turgor pressure of the cells both in the presence and absence of Ca2+ .presence or absence of calcium and in the absence of calcium modified elasticity of the fruit around the equatorial plate.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Handa AK (2013) Genetic Regulation of Phytonutrients in Fresh Produce for Health Benefits. International Symposium on Quality Management of Fruits and Vegetables for Human Health. FVHH2013, Bangkok, Thailand, August 5-8, 2013
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