Progress 12/15/03 to 04/14/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Environmental stresses, such as temperature extremes, are major determinants of the economic viability of fruit crop production and postharvest quality. Frost and freeze damage, as well as heat stress, can significantly reduce yields and, in the case of freeze damage, sometimes cause the loss of entire orchards. Both fall and spring frosts, as well as midwinter freezes, represent distinct sources of injury to fruit trees and result in economic losses to growers and the fruit industry that can reach millions of dollars in years when weather is severe. Deficiencies in micronutrients, especially zinc and copper, in apple trees result in poor fruit quality and poor growth. Foliar or soil applications of micronutrients to compensate for deficiencies can lead to excessive build- up of metals in
the soil. High levels of metals, such as zinc and copper, in soils is of environmental concern and can lead to the restricted use of these compounds. The project assembles a team of plant physiologists, molecular biologists, and tissue culture specialists to address critical environmental stress and nutrient problems in the fruit industry with an emphasis on the development and use of apple biotechnology. Genetic approaches will be utilized to identify and evaluate the role of specific genes in stress tolerance of fruit trees, and infrared thermography will be used to gain a better understanding of ice nucleation and propagation. Additional studies will determine the ability of overexpression of Zn- transporters to alleviate problems of Zn-deficiency in apple trees. Genetic studies will focus on the identification and characterization of transcription factors, newly identified dehydrin genes, and the role of two seasonally regulated proteins in cold tolerance. The ability of
antioxidant genes to enhance resistance to environmental stress will be evaluated. Concomitantly, attempts will be made to improve the safety and efficacy of apple transformation methodology by constructing tissuespecific promoters. This project is part of National Program 302, Plant Biological and Molecular Processes and addresses ARS Strategic Plan Goal 1 (Enhance Economic Opportunities for Agricultural Producers), Objective 1.2 (Contribute to the Efficiency of Agricultural Production Systems). Accomplishing the objectives will benefit fruit tree breeding programs, growers, consumers, agricultural extension agents, the commercial fruit industry, scientists, and educators by providing new products and information. Increased resistance to environmental stress will provide growers with germplasm that is adaptable to adverse climate conditions. More effective methods of managing Zn nutrition will help to avoid environmental contamination and may eliminate the need for applying Zn
fertilizers. Specific products that can be anticipated are: a) data on gene identification and function that can be used in mapping projects and in markerassisted breeding programs, b) genetically-enhanced apple cultivars with increased resistance to environmental stress and increased capacity to absorb micronutrients, such as zinc, and; c) genes, promoters, and methodology that enhance the acceptance and utility of apple biotechnology. 2. List by year the currently approved milestones (indicators of research progress) The milestones for this Project Plan were revised and approved in FY2005. FY 2003 Objective 1: Genetic Regulation of Biotic and Abiotic Stress 1. Overexpression of Antioxidant Genes in Apple - Construction of vectors containing both SOD and APX genes and transformation of apple with constructs. 2. Cold Binding Transcription Factor (CBF) Studies - Cloning of CBF gene from apple. 3. Isolation, Cloning, and Characterization of Seasonally Regulated Proteins- Obtain
full-length clones of 16 and 19 kDa proteins. 4. Inducible and Tissue-specific Promoters - Construct a customized vector for promoter strength analysis and construct an artificial flower-specific promoter. Objective 2: Genetic Regulation of Micronutrient Uptake and Transport 5. Overexpression of Zn-Transporter Genes in Apple - Construction of expression cassettes, transformation of apple, initial selection of transgenic lines on antibiotic culture medium. FY 2004 Objective 1: Genetic Regulation of Biotic and Abiotic Stress 1. Overexpression of Antioxidant Genes in Apple - Substantially complete lab and greenhouse evaluation of apple lines. 2. CBF Studies - Begin construction of overexpressing and silencing CBF constructs. 3. Isolation, Cloning, and Characterization of Seasonally Regulated Proteins- Initiate characterization of antifreeze and cryoprotective activity of 16 and 19 kDa proteins. 4. Inducible and Tissue-specific Promoters - Initiate testing of flower- specific promoter
constructs in Arabidopsis. Objective 2: Genetic Regulation of Micronutrient Uptake and Transport 5. Overexpression of Zn-Transporter Genes in Apple - Verify transgenic lines by PCR. FY 2005 Objective 1: Genetic Regulation of Biotic and Abiotic Stress 1. Overexpression of Antioxidant Genes in Apple - Complete lab and greenhouse evaluation of transgenic lines overexpressing APX gene. 2. CBF Studies - Complete construction of overexpressing CBF constructs. 3. Isolation, Cloning, and Characterization of Seasonally-Regulated Proteins This study was completed in 2004, and it was determined that neither the 16 nor 19 kDa protein had antifreeze or cryoprotective properties. As a result, this component was terminated in FY 2005. Functional genomic/proteomic approaches will be used to identify additional genes/proteins associated with cold hardiness in tree fruit crops and this will be a key component in the new, approved Project Plan 1931-21000-016-00D. 4. Inducible and Tissue-Specific
Promoters - Continue to test flower- specific promoter constructs in Arabidopsis. Objective 2: Genetic Regulation of Micronutrient Uptake and Transport 5. Overexpression of Zn-Transporter Genes in Apple - Complete verification of transgenic lines by PCR, Northerns, and Southerns; initiate screening of lines for efficiency of Zn absorption and transport. FY 2006 Objective 1: Genetic Regulation of Biotic and Abiotic Stress 1. Overexpression of Antioxidant Genes in Apple - Complete all studies on transgenic plants overexpressing APX and SOD and make transgenic lines available to other scientists. 2. CBF Studies - Make CBF RNAi constructs and transform apple with both overexpressing and silencing constructs. 3. Inducible and Tissue-specific Promoters - Substantially complete evaluation of inducible promoters and flower specific promoters in Arabidopsis. Objective 2: Genetic Regulation of Micronutrient Uptake and Transport 4. Overexpression of Zn-Transporter Genes in Apple - Substantially
complete greenhouse evaluation of transgenic lines overexpressing Zn transporter genes. 4a List the single most significant research accomplishment during FY 2006. A new peach dehydrin has been identified and characterized: Very little information on proteins associated with dehydration and drought responses in fruit trees is available, including a lack of genetic information for selecting dehydration resistant/tolerant varieties. In collaboration with scientists from Penn State University and Gabriel Lippmann (Luxembourg), we identified and isolated a gene encoding a second peach dehydrin (PpDhn2). We conclude from the promoter sequence analysis and characterization of expression that this gene is more responsive to dehydration than to cold per se. This dehydrin gene could be used in transgenic tree fruit to provide additional protection from dehydration, or used to identify Quantitative Trait Loci associated with drought tolerance. This accomplishment addresses NP302, Component 2,
"Biological processes that improve crop productivity and quality" and relates to Problem Statement 2B, "Understanding plant interactions with their environment". Research on dehydrin genes is continuing in a new, approved project (1931- 21000-016-00D), "Using Functional and Applied Genomics to Improve Stress and Disease Resistance in Fruit Crops." 4b List other significant research accomplishment(s), if any. A peach cab gene promoter can be used to control tissue-specificity of fused genes: Most constructs to express genes of interest in plants use the Cauliflower Mosaic Virus promoter (35S) which does not have tissue- specificity. Our studies show that a reporter gene under control of a peach photosynthetic promoter is expressed in a tissue-specific manner in transformed tomato plants. The constructs show increased expression in leaves and young fruit but significantly reduced levels in ripe (edible) fruit and substantially reduced levels roots. Industry has requested the vector for
evaluation in transgenic peaches and other fruit. This accomplishment addresses NP302 Component 3, "Plant biotechnology risk assessment", Problem Statement 3A, "Improving and assessing genetic engineering technology". 4d Progress report. As indicated in Question 3A, the majority of milestones have either been completed or have been designated for continuation in the new Project Plan (1931-21000-016-00D). 5. Describe the major accomplishments to date and their predicted or actual impact. Genomic response of peach to low temperature and short photoperiod: Most information regarding the identity and expression of genes associated with cold hardiness has come from studies of herbaceous plants where cold- responsive mechanisms may be different from those used by woody plants. Little or no information on the global regulation of cold-responsive genes is available for woody fruit trees. Using suppression subtractive hybridization (SSH), several novel genes were identified in peach that may
play a significant role in the regulation of stress tolerance. In collaboration with Penn State University, several genes were identified that may serve as useful genetic markers or candidate genes for genetic engineering of stress tolerance. Some of these genes are novel with respect to cold-responsive studies, as they have not been associated with cold hardiness in the literature. This knowledge will facilitate the development of new tree fruit cultivars with improved resistance to drought and cold stress and will contribute to the development of more accurate models of cold-induced gene regulation in fruit trees. The need for functional genomic knowledge in fruit crops has been identified by the industry as a high priority in order to provide the knowledge to address complex problems facing growers, such as cold and drought stress, improved tree architecture, and fruit quality. This accomplishment relates to NP302, Component 1, "Functional utilization of plant genomes: translating
plant genomics into crop improvement" , Problem Statement 1B, "Applying genomics to crop improvement" and also addresses Component 2, "Biological processes that improve crop productivity and quality", Problem Statement 2B, "Understanding plant interactions with their environment". Transgenic plants resistant to oxidative stress: In collaboration with researchers from Oregon State University, we have produced transgenic apple plants (and tomato plants) that are resistant to oxidative stress. Oxidative stress is responsible for sunscald or sunburn of apples which results in a browning of the apple skin which in turn dramatically lowers the value of the fruit and affects overall fruit quality. Transgenic apple plants (and tomato plants) were produced that overexpress the antioxidant enzyme gene, ascorbate peroxidase (APX). The resulting transgenic lines of apple show significantly greater resistance to injury induced by high temperatures, prolonged exposure to UV-B, as well as freezing
temperatures. This research has been published and demonstrates that genetic enhancement of fruit crops is a feasible approach to improving resistance to environmental stress. If adopted by tree fruit breeders, this technology has the potential to generate new apple varieties with improved resistance to cold, UV radiation, and heat stress. This accomplishment addresses the NP302, Component 1, "Functional utilization of plant genomes: translating plant genomics into crop improvement" , Problem Statement 1B, "Applying genomics to crop improvement" and addresses Component 2, "Biological processes that improve crop productivity and quality" , Problem Statement 2B, "Understanding plant interactions with their environment". Trait modification of apple using genetic engineering: Traditional breeding is a very long process for fruit trees and often desirable traits are 'mixed' with undesirable traits. Furthermore, some traits are lacking in available germplasm (e.g. resistance to certain
pests/diseases) and other methods have to be used for transferring the desired trait. Over 35 transgenic apple lines were created to address designated milestones. They include overexpressing SOD lines and Zntransporter lines, as well as lines involved in Carbohydrate/ Fire Blight studies, Rootstock Trait Modification, and Promoter Analysis. All these new lines carry the potential of improving resistance to drought, UV radiation, cold, and disease, as well as, improving nutrient uptake from the soil. They are also essential for evaluating methods of precisely regulating gene expression in fruit crops. This accomplishment addresses the NP302, Component 1, "Functional utilization of plant genomes: translating plant genomics into crop improvement" , Problem Statement 1B, "Applying genomics to crop improvement" and Component 3, "Plant biotechnology risk assessment", Problem Statement 3A, "Improving and assessing genetic engineering technology". A new "promoterless" vector that allows
direct comparison of promoter strength to the 35S promoter: There is a need to develop new promoters (small pieces of genetic code that regulate when a gene is expressed and when it is turned off) that can be used to regulate gene expression in transgenic plants. Thus far, however, it has been difficult to compare the strength and specificity of new promoters with promoters that are currently available, including the most widely-used promoter, 35S. A gene construct was made that allows one to compare the strength and specificity of a new promoter with 35S. The vector relies on the use of two different reporter genes (genes that allow one to easily evaluate patterns of gene expression) in the same construct. One reporter gene is regulated by 35S and the other by the new promoter that is being tested. Using this construct, researchers can determine how new promoters compare to an existing standard promoter, 35S. This accomplishment relates to NP302, Component 3, "Plant biotechnology
risk assessment", Problem Statement 3A, "Improving and assessing genetic engineering technology". New genomic information is made publicly available: Genomic information for tree fruits has been lacking compared to model plants and herbaceous crops. There is a critical need to develop genomic information on fruit trees in order to realize functional utilization of the genomes of these important crops for improvement. Genetic sequences from peach and apple were deposited in GenBank at the public National Center for Biological Information (National Medical Library), making them available to the global research community free of charge. Supplying gene sequences from fruit crops to a public database will make this information available to researchers worldwide addressing critical problems in fruit production. Information generated from these sequences will be used in breeding programs to facilitate selection of desirable stress-resistant traits and could also be used in genetic
engineering programs to enhance the properties of existing, popular fruit tree varieties. This accomplishment addresses NP302, Component 1, "Functional utilization of plant genomes: translating plant genomics into crop improvement", Problem Statement 1B, "Applying genomics to crop improvement" and Component 2, "Biological processes that improve crop productivity and quality", Problem Statement 2B, "Understanding plant interactions with their environment". 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? 1. Peach photosynthetic gene promoter constructs have been requested by a biotechnology company and sent to them for testing and should be available in approximately ten years. 2. Peach photosynthetic gene promoter constructs have been evaluated by
other scientists for RNAi-mediated plum pox virus resistance in tobacco, tomato and plum and should be available in approximately five to eight years. Constraints to adoption? Issues associated with public acceptance of genetically-modified- organisms (GMO's). 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). "Overexpression of a Cytosolic, Ascorbate Peroxidase Gene in Apple (Malus domestica Borkh) Improves Resistance to Environmental Stress", presented at the International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species (Daytona Beach, FL, October, 2005). "Inducible DNA Promoters for Use in Apple", presented at the International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species, Orlando, FL, October 9-15, 2005. "Comparative Expression of Peach Genes Regulated by Low Temperature and Photoperiod",
presented at the Annual Plant and Animal Genome conference, San Diego, CA, January 2006. "Trait modification through genetically engineered rootstocks", presented at the Washington Tree Fruit Research Commission, Apple Horticulture /Pathology Review, Pasco, WA, January 19, 2006.
Impacts
(N/A)
Publications
- Wisniewski, M.E., Bassett, C.L., Renaut, J., Farrell, Jr., R., Tworkoski, T., Artlip, T.S. 2006. Differential regulation of two dehydrin genes from peach (prunus persica) by photoperiod, low temperature and water deficit. Tree Physiology. 26:575-584.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Abiotic stresses are major determinants of the economic viability of fruit crop production and postharvest quality. Unfavorable environmental conditions such as frost and freeze damage and heat stress result in a reduction of annual yields and, in the case of freeze damage, sometimes the loss of entire orchards. Both fall and spring frosts, as well as midwinter freezes, represent distinct sources of injury to fruit trees and economic losses to growers. Deficiencies in micronutrients, especially zinc and copper, in apple trees result in poor fruit quality and poor growth. Foliar or soil applications of micronutrients to compensate for deficiencies can lead to excessive build-up of metals in the soil. High levels of metals, such as zinc and copper, in soils is of environmental concern and can lead to
the restricted use of these compounds. The project assembles a team of plant physiologists, molecular biologists, and tissue culture specialists to address critical environmental stress and nutrient problems in the fruit industry with an emphasis on the development and use of apple biotechnology. Genetic approaches will be utilized to identify and evaluate the role of specific genes in stress tolerance of fruit trees, and infrared thermography will be used to gain a better understanding of ice nucleation and propagation. Additional studies will determine the ability of overexpression of Zn- transporters to allevieate problems of Zn-defieciency in apple trees. Genetic studies will focus on the identification and characterization of transcription factors, newly identified dehydrin genes, and the role of two seasonally regulated proteins in cold tolerance. The ability of antioxidant genes to enhance resistance to environmental stress will be evaluated. Concomitantly, attempts will be
made to improve the safety and efficacy of apple transformation methodology by constructing tissue- specific promoters. This project is part of National Program 302, Plant Biolological and Molecular Processes and addresses ARS Strategic Plan Goal 1 (Enhance Economic Opportunities for Agricultural Producers), Objective 1.2 (Contribute to the Efficiency of Agricultural Production Systems). Accomplishing the objectives will benefit fruit tree breeding programs, growers, consumers, agricultural extension agents, the commercial fruit industry, scientists, and educators by providing new products and information. Increased resistance to environmental stress will provide growers with germplasm that is adaptable to adverse climate conditions. More effective methods of managing Zn nutrition will help to avoid environmental contamination and may eliminate the need for applying Zn fertilizers. Specific products that can be anticipated are: a) data on gene identification and function that can be
used in mapping projects and in marker-assisted breeding programs, b) genetically-enhanced apple cultivars with increased resistance to environmental stress and increased capacity to absorb micronutrients, such as zinc, and; c) genes, promoters, and methodology that enhance the acceptance and utility of apple biotechnology. 2. List the milestones (indicators of progress) from your Project Plan. FY2003 Overexpression of Antioxidant Genes in Apple. 1. Construction of vectors containing antioxidant genes (APX or SOD) and transformation of apple. CBF (Cold Binding Transcription Factor) Studies. 2. Cloning of CBF gene from apple. Isolation, Cloning, and Characterization of Seasonally-Regulated Proteins. 3. Obtain full-length clones of 16 and 19 kDa proteins. Inducible and Tissue-Specific Promoters. 4. Construct a customized vector for promoter strength analysis and a construct containing a flower-specific promoter. Overexpression of Zn-Transporter Genes in Apple. 5. Construction of
expression cassettes, transformation of apple, initial selection of transgenic lines on antibiotic culture medium. FY2004 Overexpression of Antioxidant Genes in Apple. 1. Substantially complete of lab and greenhouse evaluation of transgenic lines. CBF (Cold Binding Transcription Factor) Studies. 2. Begin construction of overexpressing and silencing CBF constructs. Isolation, Cloning, and Characterization of Seasonally-Regulated Proteins. 3. Initiate characterization of antifreeze and cryoprtoective activity of 16 and 19 kDa proteins. Inducible and Tissue-Specific Promoters. 4. Initiate testing of flower-specific promoter constructs in Arabidopsis. Overexpression of Zn-Transporter Genes in Apple. 5. Verify transgenic lines by PCR. FY2005 Overexpression of Antioxidant Genes in Apple. 1. Complete of lab and greenhouse evaluation of transgenic lines overexpressing APX gene. CBF (Cold Binding Transcription Factor) Studies. 2. Complete construction of overexpressing CBF constructs.
Isolation, Cloning, and Characterization of Seasonally-Regulated Proteins. 3. Complete characterization of antifreeze and cryoprtoective activity of 16 and 19 kDa proteins. Inducible and Tissue-Specific Promoters. 4. Continue to test flower-specific promoters constructs in Arabidopsis. Overexpression of Zn-Transporter Genes in Apple. 5. Complete verification of transgenic lines by PCR, Northerns, and Southerns. Initiate screening of lines for efficiency of Zn absorption and transport. FY2006 Overexpression of Antioxidant Genes in Apple. 1. Complete all studies on transgenic plants overexpressing APX and SOD and make transgenic lines available to other scientists CBF (Cold Binding Transcription Factor) Studies. 2 Make CBF RNAi constructs and transform apple with both overexpressing and silencing constructs. Inducible and Tissue-Specific Promoters. 3. Substantially complete evaluation of inducible promoters and flower specific promoters in Arabidopsis. Overexpression of
Zn-Transporter Genes in Apple. 4. Substantially complete greenhouse evaluation of transgenic lines overexpressing zinc transporter genes. FY2007 CBF (Cold Binding Transcription Factor) Studies. 1. Complete transformation of apple with constructs to either overexpress or silence the CBF gene and verify transgenic lines. Inducible and Tissue-Specific Promoters. 2. Complete evaluation of flower specific promoters in Arabidopsis. Overexpression of Zn-Transporter Genes in Apple. 3. Complete greenhouse evaluation of transgenic lines overexpressing zinc transporter genes. FY2008 CBF (Cold Binding Transcription Factor) Studies. 1. Evaluate overexpressing and silenced CBF transgenic lines of apple for levels of cold hardiness. Inducible and Tissue-Specific Promoters. 2. Make a genetic construct of a dehydrin gene under the control of a flower-specific promoter for transformation of apple. Overexpression of Zn-Transporter Genes in Apple. 3. Determination of success of the hypothesis and
identification of lines suitable for further study. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Completion of lab and greenhouse evaluation of transgenic lines overexpressing APX gene. Milestone Fully Met 2. Complete construction of overexpressing CBF (cold binding transcription factor)constructs. Milestone Fully Met 3. Complete characterization of antifreeze and cryoprotective activity of 16 and 19 kDa proteins. Milestone Fully Met 4. Continue to test flower-specific promoter constructs in Arabidopsis. Milestone Substantially Met 5. Complete verification of transgenic lines by PCR, Northerns, and Southerns. Initiate screening of lines for efficiency of Zn absorption and transport. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over
the next 3 years under each milestone? FY 2006 Milestone: Complete all studies on transgenic plants overexpressing APX and SOD and make transgenic lines available to other scientists Activity: We will determine if the hypothesis that higher levels of antioxidant enzymes improve resistance to environmental stress is correct. A manuscript will be prepared to disseminate this information and lines will be made available to other researchers. Further field research in this area will not be conducted due to insufficient resources and a change in research direction as outlined in a new project plan that is being prepared for review. Milestone: Make CBF (Cold Binding Transcription Factor) RNAi constructs and transform apple with both overexpressing and silencing constructs. Activity: Constructs designed to overexpress or silence the transcription factor (CBF) will be used to transform apple in order to understand the role of CBF in cold acclimation of fruit crops. This research will
continue in a new CRIS project plan. Milestone: Substantially complete evaluation of inducible promoters and flower specific promoters in Arabidopsis. Activity: Flower-specific promoter evaluation: Transgenic lines of Arabidopsis carrying the constructs designed to evaluate the specificity of the promoters will continue in order to determine if the promoters are truly flower specific. Transgenic lines of Arabidopsis will be created that should express a stress tolerance gene only in flower tissues and improve the freezing tolerance of the flowers.. If these constructs work well in Arabidopsis, we will begin transforming apple for subsequent testing. These experiments will continue under a new CRIS project plan. Research on a homone-inducible promoter (XVE) will be completed. Extramural funding for this project has terminated and no further research in this area is planned. Milestone: Substantially complete greenhouse evaluation of transgenic lines overexpressing zinc transporter
genes. Activity: We will continue to confirm and characterize expression of zinc transporter genes in transgenic apple and will begin to determine the efficiency of zinc absorption and transport in these transgenic lines. This research will allow us to determine if our hypothesis that overexpression of zinc transporter genes can be used to solve problems of zinc deficiency in fruit crops is correct. FY 2007 Milestone: Complete transformation of apple with constructs to either overexpress or silence the CBF (Cold Binding Transcription Factor) gene and verify transgenic lines. Activity: Confirmation of transgenic lines of apple overexpressing CBF (Cold Binding Transcription Factor) will be completed as will the identification of lines that have reduced or silenced levels of CBF. Milestone: Complete evaluation of inducible promoters and flower specific promoters in Arabidopsis. Activity: Transgenic Arabidopsis lines carrying dehydrin constructs that should be expressed specifically in
flower tissues will be tested for frost resistance. Make constructs that use other dehydrin genes that we have cloned from peach so that they will be expressed specifically in floral tissues in order to maximize this approach for frost protection. It is proposed that these experiments will continue under a new CRIS project plan after review by OSQR. Specificity of expression and evaluation of cold tolerance in Arabidopsis transformants will precede transformation in apple, thus allowing us to select the best possible combinations for expression in apple. Milestone: Complete greenhouse evaluation of transgenic lines overexpressing zinc transporter genes. Activity: We will complete the characterization of the expression of zinc transporter genes in transgenic apple and also complete the assessment of the efficiency of zinc absorption and transport in these transgenic lines. FY 2008 Milestone: Evaluate overexpressing and silenced CBF (Cold Binding Transcription Factor) transgenic lines
of apple for levels of cold hardiness. The phenotypes of transgenic lines of apple will be characterized where the transcription factor CBF (Cold Binding Transcription Factor) has been either silenced or overexpressed. This will determine the role of this transcription factor in regulating cold hardiness in fruit crops. Milestone: Make a genetic construct of a dehydrin gene under the control of a flower-specific promoter for transformation of apple. Evaluation: Transformation of apple will begin with select constructs. Confirmed transgenic plants will be moved to the greenhouse. Flowering in apple will take 3-4 years and so we will have to wait this long before we can test any apple transgenic lines for frost tolerance for improved flower hardiness. Milestone: Determination of success of the hypothesis and identification of lines suitable for further study. Activity: The utility and success of overexpressing zinc transporter genes in apple to improve zinc absorption and
translocation will be determined. If successful, this research will demonstrate an approach that can reduce or eliminate zinc sprays in apple and other fruit crops. Reduction or elimination of zinc sprays will greatly benefit the natural environment by reducing the potential risk of heavy metal soil contamination. It will also identify lines suitable for independent evaluation. If approved, this research will continue in a new project plan that is currently being prepared for review by OSQR. 4a What was the single most significant accomplishment this past year? Genomic Response of Peach to Low Temperature and Short Photoperiod. Using subtractive/suppressive hybridization (SSH), several novel genes were identified in peach that may play a significant role in the regulation of stress tolerance. In collaboration with Penn State University several genes were identified that may serve as useful genetic markers or candidate genes for genetic engineering of stress tolerance. This knowledge
will facilitate the development of new tree fruit cultivars with improved resistance to drought and cold stress. The need for functional genomic knowledge in fruit crops has been identified by the industry as a high priority in order to provide the knowledge to address complex problems facing growers, such as cold and drought stress, improved tree architecture and fruit quality. 4b List other significant accomplishments, if any. Regulating Gene Expression in Transgenic Apple: Currently, there is no technology available that allows one to precisely control gene expression in apple. In cooperative research between the USDA-ARS, Kearneysville, WV; Cornell University, Geneva, NY; and ARO, Volcani Center, Bet Dagan, Israel, such apple-compatible technology was demonstrated, allowing gene expression to be chemically regulated. This technology may provide a precise method of studying the effect of a given gene on a plant but also may mitigate some of the consumer and environmental
concerns facing the commercialization of genetically-engineered apple varieties and rootstocks. The potential of this approach is still being assessed. Trait Modification of Apple Using Genetic Engineering: Over 35 transgenic apple lines were created to address designated milestones. They include overexpressing SOD lines and Zn-transporter lines, as well as lines involved in Carbohydrate/ Fire Blight studies, Rootstock Trait Modification, and Promoter Analysis. All those new lines carry the potential of improving resistance to drought, UV radiation, cold, and disease, as well as improving nutrient uptake from the soil. They are also essential for evaluating methods of precisely regulating gene expression in fruit crops. 4d Progress report. Research was completed on inducible DNA promoters and their ability to regulate gene expression in transgenic apple. The research was conducted under a reimbursable agreement between ARS, Israel (ARO), Cornell University and the US-Israel
Binational Agricultural Development Fund (BARD). Additional details can be found in the final report for the subordinate project, 1931-21220-014-01R, Regulated expression of site- specific DNA recombination for precision genetic engineering in apple, and the present annual report. Transformation vectors constructed to investigate graft-transmissible gene silencing were placed in apple. The research is being conducted under a trust agreement between USDA-ARS and the Washington Tree Fruit Research Commission (WTFRC). USDA-ARS is providing the expertise for constructing the vectors and evaluating the resulting transformants while WTFRC is providing general expertise and will be involved in the evaluation of the resulting transgenic lines. Further details can be found in the annual report of the subordinate project 1931-21220-014-03T, Trait modification of conventional apple scions mediated by genetically engineered apple rootstocks. A new project was initiated to identify ESTs
associated with susceptible and resistance responses of apple to infection by fire blight. The research is being conducted under a reimbursable agreement between USDA- ARS, Cornell University, Pennsylvania State University, and CSREES (NRI). Details can be found under the annual report for the subordinate project 1931-21220-014-04R, Functional genomic response of apple to fire blight. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. In collaboration with researchers from Oregon State University, we have produced transgenic apple plants (and tomato plants) that are resistant to oxidative stress. Oxidative stress is responsible for sunscald or sunburn of apples which results in a browning of the apple skin which in turn dramatically lowers the value of the fruit and affects overall fruit quality. Transgenic apple plants (and tomato plants) were produced that overexpress the antioxidant enzyme gene, ascorbate peroxidase
(APX). The resulting transgenic lines of apple show significantly greater resistance to injury induced by high temperatures, prolonged exposure to UV-B, as well as freezing temperatures. This research has been published and demonstrates that genetic enhancement of fruit crops is a feasible approach to improving resistance to environmental stress. If adopted by tree fruit breeders, this technology has the potential to generate new apple varieties with improved resistance to cold, UV radiation, and heat stress. This accomplishment addresses the research component dealing with overexpression of APX and SOD in apple and partially completes the 48 Month Milestone. Construction and analysis of a suppression subtractive hybridization (SSH) library has led to the identification of genes globally expressed in peach bark under different photoperiods and after exposure to low temperature. This accomplishment is crucial to our understanding of how trees adapt to cold stress as it has led to the
identification of several novel genes that respond to low, cold-acclimating temperature (5DGC, 41 DGF) . Detailed analysis of these novel genes will allow us to evaluate their contribution to the development of cold acclimation in trees. Through manipulation of these genes or their use in traditional breeding programs, we will be able to obtain fruit tree varieties with enhanced ability to withstand cold temperature extremes with little or no damage to the plant. A gene construct was constructed that allows one to restrict foreign gene expression to vegetative portions of fruit trees while greatly reducing or eliminating expression in fruit tissues. This research addresses the need to develop methods to more precisely regulate gene expression in order to enhance safety. The construct, along with a reporter gene that allows one to determine in what tissues genes are being expressed, was transformed into a model plant system (tomato) and shown to produce high levels of expression in
leaves and significantly lower and decreasing expression in ripe fruit, flowers and roots. These results indicate that this technology can be used to provide high levels of expression of a foreign gene in leaves without causing significant expression of the gene in fruits or roots, thus potentially more consumer-acceptable transgenic plant. This accomplishment addresses the research component dealing with inducible and tissue-specific promoters and partially completes the 48 Month Milestone. There is a need to develop new promoters (small pieces of genetic code that regulate when a gene is expressed and when it is turned off) that can be used to regulate gene expression in transgenic plants. Thus far, however, it has been difficult to compare the strength and specificity of new promoters with promoters that are currently available, including the most widely-used promoter, 35S. A gene construct was made that allows one to compare the strength and specificity of a new promoter with
35S. The vector relies on the use of two different reporter genes (genes that allow one to easily evaluate patterns of gene expression) in the same construct. One reporter gene is regulated by 35S and the other by the new promoter that is being tested. Using this construct, researchers can determine how new promoters compare to an existing standard promoter, 35S. This accomplishment addresses the research component dealing with inducible and tissue-specific promoters and completes the 14 Month Milestone. Genetic sequences from peach and apple were deposited in GenBank, making them available to the global research community free of charge. There is a critical need to develop genomic information on fruit crops. Supplying gene sequences from fruit crops to a public database will make this information available to researchers worldwide addressing critical problems in fruit production. This accomplishment relates to the research component on isolation, cloning, and characterization of
seasonally- regulated proteins in fruit trees and is in addition to the stated Milestones. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A patent entitled, 'Method for reducing freeze and chill damage in plants' was filed. This technology was developed through a CRADA with industry. This technology provides a novel method of low temperature protection for chilling-sensitive and frost-sensitive herbaceous plants based on the use of particle films. Constraints to adoption include the liability of manufacturer and inconsistent control due to environmental variables and variability in the freezing response of different species of horticultural crops. 7. List your most important publications in the popular press and presentations to organizations and
articles written about your work. (NOTE: List your peer reviewed publications below). Presentations: Michael Wisniewski: Proteomic responses of peach to low temperature and short photoperiod' Gordon Conference on Temperature Stress in Plants, Harbor Towne, California, January, 2005. Carole Bassett: Cold and short day photoperiod responsive genes from peach bark. Presented at the Plant and Animal Genomics Conference, San Diego, CA, Jan 2005. Carole Bassett and Michael Wisniewski: Proteomics of cold acclimation and photoperiod in woody plants: Differential in gel electrophoresis (2- DIGE) of soluble proteins extracted from peach bark. Presented at the Plant and Animal Genomics Conference, San Diego, CA, Jan 2005. Carole Bassett: Characterization of sequences up-regulated in peach bark in response to low temperature. Presented at the American Society for Horticultural Science Annual Conference, Las Vegas, NV, July 2005. Carole Bassett: From the Field to the Lab and Back Again: The
peach chlorophyll a/b-binding protein promoter regulates tissue-specific expression of target genes, Department of Horticultural Sciences, NYSAES, Cornell University, June 2005. Carole Bassett: Identification and characterization of dehydrins in peach, University of Illinois, Biotechnology Center, Champagne-Urbana, IL, Aug 2005. Michael Wisniewski: Progress Report,'Overexpression of Antioxidant Genes in Apple for Improved Stress Resistance', Washington Tree Fruit Research Commission, January 6, 2005. Michael Wisniewski and Dariusz Swietlik: Progress Report, 'The Use of Transporter Genes to Increase Zinc Absorption and Transport in Apple', Washington Tree Fruit Research Commission, January 6, 2005. Michael Wisniewski: Poster, Differential patterns of expression and regulation of two dehydrin genes in peach bark tissues. The American Society for Horticultural Science Annual Conference, Las Vegas, NV, July 2005.
Impacts
(N/A)
Publications
- Griffith, M., Lumb, C., Wiseman, S., Wisniewski, M.E. 2005. Antifreeze proteins modify the freezing process in planta. Plant Physiology. 138: 330- 340.
- Wang, Y., Wisniewski, M.E., Meilan, R., Webb, R., Fuchigami, L., Boyer, C. 2005. Overexpression of cytosolic ascorbate peroxidase in tomato (lycopersicon esculentum) confers tolerance to chilling and salt stress. Journal of the American Society for Horticultural Science 130(2):167-173. 2005.
- Gusta, L.V., Wisniewski, M.E., Nesbitt, N.T., Gusta, M.L. 2004. The effect of water, sugars, and proteins on the pattern of ice nucleation and propagation in acclimated and non-acclimated canola (brassica napus) leaves. Plant Physiology. July 2004, Vol. 135, pp. 1642-1653.
- Bassett, C.L., Nickerson, M.L., Farrell, R.E., Artlip, T.S., El Ghaouth, A. , Wilson, C.L., Wisniewski, M.E. 2005. Characterization of an s-locus receptor protein kinase-like gene from peach. Tree Physiology. 25: 403-411.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Abiotic stresses are major determinants of the economic viability of fruit crop production and postharvest quality. Unfavorable environmental conditions such as frost and freeze damage and heat stress result in a reduction of annual yields and, in the case of freeze damage, sometimes the loss of entire orchards. Both fall and spring frosts, as well as midwinter freezes, represent distinct sources of injury to fruit trees and economic losses to growers. Deficiencies in micronutrients, especially zinc and copper, in apple trees result in poor fruit quality and poor growth. Foliar or soil applications of micronutrients to compensate for deficiencies can lead to excessive build-up of metals in the soil. High levels of metals, such as zinc and copper, in soils is of environmental concern and can lead to
the restricted use of these compounds. The project assembles a team of plant physiologists, molecular biologists, and tissue culture specialists to address critical environmental stress and nutrient problems in the fruit industry with an emphasis on the development and use of apple biotechnology. Genetic approaches will be utilized to identify and evaluate the role of specific genes in stress tolerance of fruit trees, and infrared thermography will be used to gain a better understanding of ice nucleation and propagation. Additional studies will determine the ability of overexpression of Zn- transporters to alleviate problems of Zn-deficiency in apple trees. Genetic studies will focus on the identification and characterization of transcription factors, newly identified dehydrin genes, and the role of two seasonally regulated proteins in cold tolerance. The ability of antioxidant genes to enhance resistance to environmental stress will be evaluated. Concomitantly, attempts will be made
to improve the safety and efficacy of apple transformation methodology by constructing tissue- specific promoters. This project is part of National Program 302, Plant Biolological and Molecular Processes and addresses ARS Strategic Plan Goal 1 (Enhance Economic Opportunities for Agricultural Producers), Objective 1.2 (Contribute to the Efficiency of Agricultural Production Systems). Accomplishing the objectives will benefit fruit tree breeding programs, growers, consumers, agricultural extension agents, the commercial fruit industry, scientists, and educators by providing new products and information. Increased resistance to environmental stress will provide growers with germplasm that is adaptable to adverse climate conditions. More effective methods of managing Zn nutrition will help to avoid environmental contamination and may eliminate the need for applying Zn fertilizers. Specific products that can be anticipated are: a) data on gene identification and function that can be used
in mapping projects and in marker-assisted breeding programs, b) genetically-enhanced apple cultivars with increased resistance to environmental stress and increased capacity to absorb micronutrients, such as zinc, and; c) genes, promoters, and methodology that enhance the acceptance and utility of apple biotechnology. 2. List the milestones (indicators of progress) from your Project Plan. Although the current project was initiated in December, 2003, Milestones covering 14 months were addressed in bridge CRIS 1931-21220-012-00D, Management of Biotic and Abiotic Stress in Fruit Crops, and reported on in FY 2003 Annual Report. a. Overexpression of Antioxidant Genes in Apple. FY2003 Construction of vectors containing antioxidant genes (APX or SOD) and transformation of apple. FY2004/05 Completion of lab and greenhouse evaluation of transgenic lines. FY2006/07 Field establishment of selected lines. FY2008 Identification of transgenic lines suitable for independent evaluation. b. CBF
and Dehydrin Studies. FY2003 Cloning of CBF gene from apple. FY2004/05 Prepare two-hybrid library from cold-stressed bark tissues and CBF gene. FY2006/07 Characterize sequences obtained from yeast two hybrid system. FY2008 Develop a model of cold-induced gene regulation in fruit trees. c. Isolation, Cloning, and Characterization of Seasonally-Regulated Proteins. FY2003 Obtain full-length clones of 16 and 19 kDa proteins. FY2004/05 Complete characterization of antifreeze and cryoprtoective activity of 16 and 19 kDa proteins. FY2006/07 Produce transgenic apple plants overexpressing 16 and 19 kDa proteins. FY2008 Characterize phenotype of transgenic lines overexpressing 16 and 19 kDa proteins. d. Inducible and Tissue-Specific Promoters. FY2003 Construct a customized vector for promoter strength analysis and a construct containing a flower-specific promoter. FY2004/05 Test flower-specific promoters in transient assays: test Inducible promoters in apple. FY2006/07 Complete
evaluation of inducible promoters and flower specific promoters. FY2008 Make a genetic construct of a dehydrin gene under the control of a flower-specific promoter. e. Overexpression of Zn-Transporter Genes in Apple. FY2003 Construction of expression cassettes, transformation of apple, initial selection of transgenic lines on antibiotic culture medium. FY2004/05 Verification of transgenic lines by PCR, Northerns, and Southerns. Initial screening of lines for efficiency of ZN absorption and transport. FY2006/07 Selection of lines for field evaluation. FY2008 Determination of success of the hypothesis and identification of lines suitable for further study. 3. Milestones: Although the current project was initiated in December 2003, Milestones covering 14 months were addressed in a bridge CRIS 1931-21220-012-00D, Management of Biotic and Abiotic Stress in Fruit Crops, and reported on in FY 2003 Annual Report. A. Milestones to be addressed in FY 2004: Substantial progress has been
made on all milestones and we are on target to complete all milestones listed in the project plan. Some modifications have been made as noted below: Completion of lab and greenhouse evaluation of transgenic lines of apple overexpressing APX or SOD antioxidant genes. Evaluation of transgenic tomato lines overexpressing SOD have not shown increased stress resistance over transgenic tomato lines overexpressing APX. Therefore, overexpressing SOD in apple will not be pursued. Evaluation of transgenic apple lines overexpressing APX is on target for completion of the FY04/05 milestone. CBF gene has been cloned from apple and construction of yeast two hybrid system is on target for completion of the FY04/05milestone. Characterization of cryoprotective and antifreeze properties of 16 and 19 kDa seasonally-regulated proteins in peach is on target for completion of the FY04/05 milestone. The evaluation of inducible gene promoters in apple is on track for completion of the the FY04/05
milestone. Construction of tissue-specific promoters and specialized vectors is on target for completion of the FY04/05 milestone. A customized vector for the evaluation of promoter strength has been constructed (pCAMBIAgfp94). A stigma/style-specific promoter (ZPT2-10) driving expression of GUS has been placed in the plant transformation vectors, pBINplusARS and pCAMBIA2301. Another promoter (ZPT3-3) specifying expression of GUS in petal limbs and carpels has been placed in pCAMBIA2301. Verification of transgenic lines of apple overexpressing Zn-transporter genes is on target for completion of the FY04/05 milestone. B. Milestones to be addressed over the next 3 years (2005, 2006, 2007) FY 2005 Laboratory and greenhouse evaluation of transgenic lines of apple overexpressing the antioxidant gene, ascorbate peroxidase (APX) will be completed and we will determine if the hypothesis that higher levels of antioxidant enzymes improve resistance to environmental stress is correct. If the
hypothesis is correct we will also have identified which transgenic lines are suitable for further evaluation under field conditions. Construction of a yeast two-hybrid system using CBF or another candidate protein as a bait will be completed and proteins interacting with the bait will be in the process of being identified. The results from this research will greatly improve our knowledge of the genetic regulation of cold tolerance in fruit crops. We will determine if two, specific, seasonally-regulated proteins in peach have either antifreeze or cryoprotective properties. If they do we will have evidence that these proteins play an important role in cold tolerance. Such information will be especially important to gene mapping projects and to marker-assisted breeding programs. The ability to use light or specific chemicals to induce transgene expression in apple will be evaluated. Plant transformation vectors constructed in FY2004 to evaluate the activity of the light-inducible CAB
promoter and the chemically-inducible XVE promoter are currently being transferred to 'Galaxy' apple by Agrobacterium-mediated transformation. Transgenic plants containing the vectors will be selected and transformation will be confirmed by laboratory tests. The activity of the promoters in apple will then be evaluated in the laboratory by monitoring promoter-driven marker gene expresssion following exposure to various amounts of the inducers. Completion of construction of pZPT3-3GUS in pBINplusARS is anticipated in FY2005. In addition we plan to place both promoters alone, separately into another plant transformation vector (pCGN1578) to improve the efficiency of obtaining apple transformants carrying genes that confer frost protection. We will test specificity of promoters in Arabidopsis plants transformed with pBINzpt2GUS and pBINzpt3GUS. Contruction of the peach Ppdhn1 gene under control of the promoters in pBINplusARS and pCGN1578 will be completed by the end of the reporting
period. We will confirm and characterize expression of zinc transporter genes in apple and will begin to determine the efficiency of zinc absorption and transport in these transgenic lines. This research will allow us to determine if our hypothesis that overexpression of zinc transporter genes can be used to solve problems of zinc deficiency in fruit crops is correct. FY 2006 Select lines of transgenic apple overexpressing a cytosolic, APX gene will be established in field plantings and evaluation of the phenotypes will commence. If increased stress resistance is observed and no adverse effects are observed in the transgenic lines then our hypothesis that higher levels of antioxidant enzymes confers increased resistance to environmental stress will be considered confirmed. This information will be useful to breeders involved in improving stress resistance in fruit crops and provide unique germplasm within the guidelines of current regulatory laws and interest by the industry.
Identification and characterization of proteins identified in the yeast two-hybrid system will be completed. This information will increase our basic understanding of the regulation of cold tolerance in fruit crops. Genes encoding the peach 16 kDa and 19 kDa proteins will be overexpressed in apple and evaluation of the effect of these genes on stress tolerance will be commenced. Additionally, genomic or proteomic analyses will be employed to identify other putative 'cold hardiness' genes. Evaluation of the light- and chemically-inducible promoters in apple will be completed after testing their activity in greenhouse-grown plant material. Arabidopsis and apple will be transformed with the two promoter-Ppdhn1 constructs in pBINplusARS and pCGN1578. Specificity of expression and evaluation of cold tolerance in Arabidopsis transformants will precede transformation in apple, thus allowing us to select the best possible combinations for expression in apple. Evaluation of the efficiency
of zinc absorption and transport in transgenic apple lines overexpressing a zinc transporter gene from either Arabidopsis or Thlaspia will be completed and the most promising lines will be selected for field evaluation. FY 2007 Field evaluation of transgenic lines of apple overexpressing an APX gene will identify lines that are potentially suitable for independent evaluation in other university, state, and federal fruit crop programs. In addition to the transgenic genotypes, the information from this line of research will be useful to classical breeding programs as it will provide a biochemical marker for resistance to environmental stress. Based on the information obtained from the yeast two-hybrid system a model of cold-induced gene regulation in fruit crops will be developed. This information will greatly increase our fundamental knowledge of cold tolerance in fruit crops and will be especially useful for marker- assisted breeding programs. Characterization of transgenic apple
lines overexpressing genes encoding the peach 16 and 19 kDa proteins will be near completion. The effect of these proteins on stress tolerance will be assessed. If the impact on stress tolerance is positive without any adverse effects this information will greatly increase our knowledge of cold hardiness in fruit crops. This information will be of great value to marker-assisted breeding programs and will also be used to develop new strategies to improve cold tolerance in fruit crops. Evaluation of inducible promoters will be completed. Evaluation of Arabidopsis promoter-Ppdhn1 constructs will be completed. We will complete transformation of apple with flower specific gene constructs and will move confirmed transgenic plants to greenhouse. The utility and success of overexpressing zinc transporter genes in apple to improve zinc absorption and translocation will be determined. If successful, this research will demonstrate an approach that can reduce or eliminate zinc sprays in apple
and other fruit crops. Reduction or elimination of zinc sprays will greatly benefit the natural environment by reducing the potential risk of heavy metal soil contamination. It will also identify lines suitable for independent evaluation. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment: A subtractive hybridization-PCR suppression technique identified genes expressed in peach bark in response to either a short photoperiod or cold temperature. This accomplishment, which represents a collaborative effort with the Penn State University, is crucial to our understanding of how trees adapt to cold stress and has led to the identification of several novel genes that appear to be associated with short-day photoperiods and/or response to cold temperature. Analysis of these sets of genes will allow us to develop a more accurate understanding of how trees adapt to cold temperatures and through manipulation of select genes or their
use in marker-assisted breeding programs, we will be able to obtain fruit tree varieties with enhanced ability to withstand cold temperature extremes. B. Other significant accomplishment: A patent was filed to cover technology developed to protect plants from chilling and frost injury. C. Signficant activities that support special target populations: None D. Progress Report: Work was completed on a specific cooperative agreement between USDA-ARS and the Washington Tree Fruit Research Commission (WTFC). The project was terminated in July, 2004. USDA-ARS provided expertise to produce transgenic apple plants (cv Gala) overexpressing a cytosolic ascorbate peroxidase (APX) gene. Resulting lines exhibited increased resistance to oxidative stress induced by freezing, high temperature, and UV-B. Industry supplied general expertise and partial support. Details can be found in the final report for the parent project 1931-21220-012-00D, Management of Biotic and Abiotic Stress in Fruit
Crops, and also in the final report for the subordinate project, 1931-21220-014-02T, Genetic engineering of multiple stress resistance in apple through enhancement of antioxidant enzymes, and in the present annual report. Inducible DNA promoters were evaluated for their ability to regulate gene expression in transgenic apple. The research was conducted under a reimbursable agreement between ARS, Israel (ARO), Cornell University and the US-Israel Binational Agricultural Development Fund (BARD). Additional details can be found in the final report of the previous parent project 1931-21220-012, Management of Biotic and Abiotic Stress in Fruit Crops, the annual report for the subordinate project, 1931-21220- 014-01R, Regulated expression of site-specific DNA recombination for precision genetic engineering in apple, and the present annual report. Plant transformation vectors were constructed to investigate graft- transmissible gene silencing in apple. The research is being conducted under
a trust agreement between USDA-ARS and the Washington Tree Fruit Research Commission (WTFC). USDA-ARS is providing the expertise for constructing the vectors and evaluating the resulting transformants while WSTFC is providing general expertise and will be involved in the evaluation of the resulting transgenic lines. Further details can be found in the annual report of the subordinate project 1931-21220-014-03T, Trait modification of conventional apple scions mediated by genetically engineered apple rootstocks. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. In collaboration with researchers from Oregon State University, we have produced transgenic apple plants (and tomato plants) that are resistant to oxidative stress. Oxidative stress is responsible for sunscald or sunburn of apples which results in a browning of the apple skin which in turn dramatically lowers the value of the fruit and affects overall fruit quality.
Transgenic apple plants (and tomato plants) were produced that overexpress the antioxidant enzyme gene, ascorbate peroxidase (APX).The resulting transgenic lines of apple show significantly greater resistance to injury induced by high temperatures, prolonged exposure to UV-B, as well as freezing temperatures. This research demonstrates that genetic enhancement of fruit crops is a feasible approach to improving resistance to environmental stress. This accomplishment addresses the research component dealing with overexpression of APX and SOD in apple and partially completes the 48 Month Milestone of the project plan and relates to Objective 1.2 of the National Program Action Plan. A plasmid construct was developed containing the regulatory (promoter) region of a photosynthetic gene (cab) and a reporter gene (GUS). This research addresses the need to develop safer plant transformation constructs which do not express foreign genes in edible plant parts such as fruit. The construct was
transformed into a model plant system (tomato) and shown to produce high levels of GUS expression in leaves and significantly lower and decreasing expression in ripe fruit, flowers and roots, respectively. These results indicate that the cab promoter can be used to provide high levels of expression of a foreign gene in leaves without causing significant expression of the gene in fruits or roots, thus potentially yielding a safer, more acceptable transgenic plant. This accomplishment addresses the research component dealing with inducible and tissue-specific promoters and partially completes the 48 Month Milestone of the project and relates to Objective 1.2 of the National Program Action Plan. A single vector was constructed which carries both a control gene (GUS) under the regulation of a commonly used promoter (35S), and a promoter- less gene encoding a version of the green fluorescent protein (gfp) optimally modified for plants. This accomplishment will assist researchers in
assessing ectopic expression of genes in transformed plants by providing an internal control. Using this construct, we can determine promoter expression more accurately without the complication of position effects arising as a consequence of independent insertion into the genome. Improved testing of tissue-specific promoters will result in better promoter selection for targeted genetic engineering. This accomplishment addresses the research component dealing with inducible and tissue-specific promoters and completes the 14 Month Milestone of the project plan and relates to Objective 1.2 of the National Program Action Plan. Genetic sequences from peach and apple were deposited in GenBank, making them available to the global research community free of charge. There is a critical need to develop genomic information on fruit crops. Supplying gene sequences from fruit crops to a public database will make this information available to researchers worldwide addressing critical problems in
fruit production. This accomplishment relates to the research component on isolation, cloning, and characterization of seasonally- regulated proteins in fruit trees and is in addition to the stated Milestones in the project plan and relates to Objective 1.2 of the National Program Action Plan. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A patent entitled,'Method for reducing freeze and chill damage in plants' was filed. This technology was developed through a CRADA with industry. This technology provides a novel method of low temperature protection for chilling-sensitive and frost-sensitive herbaceous plants based on the use of particle films. Constraints to adoption include the liability of manufacturer and inconsistent control due to
environmental variables and variability in the freezing response of different species of horticultural crops. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Presentations: 'From Genes to Whole Plants and the Mysteries That Lie In Between: An Overview of Cold Hardiness in Woody Plants.' Invited Lecture, University of Luxembourg, Luxembourg. May 3, 2004. Progress Report,'Overexpression of Antioxidant Genes in Apple for Improved Stress Resistance', Washington Tree Fruit Research Commission, January 6, 2004. Progress Report, 'The Use of Transporter Genes to Increase Zinc Absorption and Transport in Apple', Washington Tree Fruit Research Commission, January 6, 2004. Poster, 'Effect of photoperiod, temperatue and drought on the expression of two dehydrin genes in peach', at Keystone Symposium (Plant Responses to Abiotic Stress), Santa Fe, NM Feb. 19-24, 2004. 'Identification of genes expressed in peach
bark in response to cold treatment and different photoperiods'. Presented at the Rosaceae Genetics Conference, Clemson, SC. May, 2004.
Impacts
(N/A)
Publications
- FULLER, M., HAMED, F., WISNIEWSKI, M.E., GLENN, D.M. PROTECTION OF CROPS FROM FROST USING A HYDROPHOBIC PARTICLE FILM AND AN ACRYLIC POLYMER. ANNALS OF APPLIED BIOLOGY. 2003 Vol. 143, pgs. 93-97.
- WISNIEWSKI, M.E., GLENN, D.M., GUSTA, L., FULLER, M., DUMAN, J., GRIFFITH, M. USING INFRARED THERMOGRAPHY TO STUDY ICE NUCLEATION AND PROPAGATION IN PLANTS. ACTA HORTICULTURAE. 2003. 618:485-492
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