Source: AGRICULTURAL RESEARCH SERVICE submitted to
GENETIC ENGINEERING OF FLORAL BULB CROPS FOR VIRUS AND NEMATODE RESISTANCE
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0410400
Grant No.
(N/A)
Project No.
1230-21000-047-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
May 8, 2006
Project End Date
Sep 30, 2010
Grant Year
(N/A)
Project Director
KAMO K K
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
(N/A)
WASHINGTON,DC 20250
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2042120100050%
2122121104050%
Goals / Objectives
Utilize genetic engineering to improve resistance of bulb crops to fungal diseases and plant parasitic nematodes. (a) Develop transformation technology for Easter lily; (b) test candidate genes to confer resistance to Fusarium in Gladiolus; and (c) determine whether dsRNA expressed in plants can target RNAi to control plant parasitic nematodes.
Project Methods
Transform a commercially important Gladiolus cultivar,Easter lilies, and Ornithogalum with genes for resistance to either cucumber mosaic virus or ornithogalum mosaic virus. Determine if the genetically engineered plants are resistant to these viruses. Complete experiments involving cucumber mosaic virus resistance in transgenic Gladiolus plants that contain either the CMV coat protein subgroup 1 or subgroup 2, CMV replicase, or single chain antibodies to the CMV coat protein. Optimize the transformation system for Easter lilies and Ornithogalum using either the gene gun or Agrobacterium. Develop a transformation system for roots of Easter lilies using Agrobacterium rhizogenes. Transgenic Gladiolus plants with D4E1 and cpo have been developed using the gene gun and are being grown for challenging with Fusarium in the greenhouse. Gladiolus will also be transformed various chitinase genes for possible resistance to Fusarium. Evaluate effectiveness of genetically engineered resistance to Fusarium oxysporum in Gladiolus by comparison to biological control technologies. Develop Easter lilies that are resistant to the nematode Pratylenchus penetrans, the root lesion nematode. Genes involved in nematode development will be isolated and tested for their ability to kill Pratylenchus penetrans or affect its reproduction using RNAi.

Progress 05/08/06 to 09/30/10

Outputs
Progress Report Objectives (from AD-416) Utilize genetic engineering to improve resistance of bulb crops to fungal diseases and plant parasitic nematodes. (a) Develop transformation technology for Easter lily; (b) test candidate genes to confer resistance to Fusarium in Gladiolus; and (c) determine whether dsRNA expressed in plants can target RNAi to control plant parasitic nematodes. Approach (from AD-416) Transform a commercially important Gladiolus cultivar, Easter lilies, and Ornithogalum with genes for resistance to either cucumber mosaic virus (CMV) or ornithogalum mosaic virus. Determine if the genetically engineered plants are resistant to these viruses. Complete experiments involving cucumber mosaic virus resistance in transgenic Gladiolus plants that contain either the CMV coat protein subgroup 1 or subgroup 2, CMV replicase, or single chain antibodies to the CMV coat protein. Optimize the transformation system for Easter lilies and Ornithogalum using either the gene gun or Agrobacterium. Develop a transformation system for roots of Easter lilies using Agrobacterium rhizogenes. Transgenic Gladiolus plants with D4E1 and cpo have been developed using the gene gun and are being grown for challenging with Fusarium in the greenhouse. Gladiolus will also be transformed various chitinase genes for possible resistance to Fusarium. Evaluate effectiveness of genetically engineered resistance to Fusarium oxysporum in Gladiolus by comparison to biological control technologies. Develop Easter lilies that are resistant to the nematode Pratylenchus penetrans, the root lesion nematode. Genes involved in nematode development will be isolated and tested for their ability to kill Pratylenchus penetrans or affect its reproduction using RNAi. Progress was made on both objectives. Under Objective 1, Gladiolus plants were developed with either the cucumber mosaic virus coat protein serotype I or serotype II, replicase, or antibody to coat protein serotype I or serotype II genes. Plants containing the replicase gene were found most resistant to cucumber mosaic virus (CMV), and transgene expression levels correlated with resistance. For Objective 2, the gene gun technology was applied to lilies and a reproducible transformation system was established although the efficiency of transformation remains very low. Conditions have been optimized for transient transformation of lilies using Agrobacterium tumefaciens making it feasible to attempt stable transformation in the future using Agrobacterium. Because of the low level of transformation with lilies, two cry genes have been transformed into hairy roots of tomato allowing us to test the ability of these two cry genes to confer resistance to the root lesion nematode in the future. Gladiolus plants have been developed with antifungal genes (the synthetic peptide D4E1, chloroperoxidase, exo and endochitinase). The antifungal genes affect the phenotype of these plants making it difficult to multiply them for challenge. Thus, as an alternative, Fusarium has been transformed with fluorescence genes that will allow us to study the interaction of the fungus with the transgenic plant lines. This is the final report for this project. National Programs 301 and 302 are being merged, and a bridge project was approved by the Office of National Programs in July, 2010 (new bridge 1230-21000-048-00D) and will start 10/1/10. Accomplishments 01 Development of Gladiolus with virus resistance. Cucumber mosaic virus (CMV) is one of the most important plant viruses because it infects approximately 1000 plant species of both food and ornamental crops. Flower bulb crops are particularly susceptible to viruses because they a propagated vegetatively by their bulbs, and the virus remains within the bulb. Flowers infected with CMV have streaking on the petals making the unmarketable, and infected plants have decreased vigor resulting in a lo yield of bulbs. Gladiolus, representing a flower bulb crop, was genetically enhanced with several antiviral genes, CMV coat protein serotype I, coat protein serotype II, replicase, antibody to coat protei serotype I, and antibody to coat protein serotype II. The replicase gen was most effective at conferring short-term virus resistance. This information is useful to scientists who are engineering flower bulb crop for virus resistance. Gladiolus plants are currently being multiplied f a field challenge, and if found to be resistant in the field, Gladiolus growers will have a source of CMV-resistant germplasm as resistance is n present in commercially important cultivars.

Impacts
(N/A)

Publications

  • Kamo, K., Jordan, R., Guaragna, M.A., Hsu, H.T., Ueng, P. 2010. Resistance to cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase or coat protein subgroup II gene from Cucumber mosaic virus. Plant Cell Reports. 29:695-704.
  • Kamo, K. and Joung, H.Y. 2009. Long-term gus expression from Gladiolus callus lines containing either a bar-uidA fusion gene or bar and uidA delivered on separate plasmids. Plant Cell Tissue Organ Culture. 98:263- 272.
  • Kamo, K., Joung, Y.H., Green, K. 2009. GUS expression in Gladiolus plants controlled by two Gladiolus ubiquitin promoters. Floriculture and Ornamental Biotechnology. 3:10-14.


Progress 10/01/08 to 09/30/09

Outputs
Progress Report Objectives (from AD-416) Utilize genetic engineering to improve resistance of bulb crops to fungal diseases and plant parasitic nematodes. (a) Develop transformation technology for Easter lily; (b) test candidate genes to confer resistance to Fusarium in Gladiolus; and (c) determine whether dsRNA expressed in plants can target RNAi to control plant parasitic nematodes. Approach (from AD-416) Transform a commercially important Gladiolus cultivar,Easter lilies, and Ornithogalum with genes for resistance to either cucumber mosaic virus or ornithogalum mosaic virus. Determine if the genetically engineered plants are resistant to these viruses. Complete experiments involving cucumber mosaic virus resistance in transgenic Gladiolus plants that contain either the CMV coat protein subgroup 1 or subgroup 2, CMV replicase, or single chain antibodies to the CMV coat protein. Optimize the transformation system for Easter lilies and Ornithogalum using either the gene gun or Agrobacterium. Develop a transformation system for roots of Easter lilies using Agrobacterium rhizogenes. Transgenic Gladiolus plants with D4E1 and cpo have been developed using the gene gun and are being grown for challenging with Fusarium in the greenhouse. Gladiolus will also be transformed various chitinase genes for possible resistance to Fusarium. Evaluate effectiveness of genetically engineered resistance to Fusarium oxysporum in Gladiolus by comparison to biological control technologies. Develop Easter lilies that are resistant to the nematode Pratylenchus penetrans, the root lesion nematode. Genes involved in nematode development will be isolated and tested for their ability to kill Pratylenchus penetrans or affect its reproduction using RNAi. Significant Activities that Support Special Target Populations Gladiolus plants transformed with either an exo or endochitinase gene isolated from Fusarium or a chloroperoxidase gene have been developed. Leaf extracts were used in an in vitro assay to screen for those transgenic lines that showed inhibition of Fusarium oxysporum spore germination. The three transgenic plant lines of Gladiolus found resistant in vitro to Cucumber mosaic virus strain S (subgroup II) and three lines resistant to CMV serotype I were verified by Southern hybridization to contain the CMV coat protein subgroup II and CMV defective replicase genes, respectively. These plants have been multiplied in vitro this season for future challenge outdoors. Lily callus has been used for gene gun-mediated transformation with the gus reporter gene to test factors that might optimize the very low transformation efficiency. In 7 experiments, the cystatin gene has been used because it has been shown to be effective in control of nematodes. Experiments have shown that Agrobacterium-mediated transformation of Easter lilies occurs at a low frequency.

Impacts
(N/A)

Publications

  • Kamo, K. 2009. Transgene expression for Gladiolus plants grown outdoors and in the greenhouse. Scientia Horticulturae. 11:275-280.
  • Kamo, K. and Han, B.H. 2008. Biolistic-mediated transformation of Lilium longiflorum dv. Nellie White. HortScience. 43:1864-1869.


Progress 10/01/07 to 09/30/08

Outputs
Progress Report Objectives (from AD-416) Utilize genetic engineering to improve resistance of bulb crops to fungal diseases and plant parasitic nematodes. (a) Develop transformation technology for Easter lily; (b) test candidate genes to confer resistance to Fusarium in Gladiolus; and (c) determine whether dsRNA expressed in plants can target RNAi to control plant parasitic nematodes. Approach (from AD-416) Transform a commercially important Gladiolus cultivar,Easter lilies, and Ornithogalum with genes for resistance to either cucumber mosaic virus or ornithogalum mosaic virus. Determine if the genetically engineered plants are resistant to these viruses. Complete experiments involving cucumber mosaic virus resistance in transgenic Gladiolus plants that contain either the CMV coat protein subgroup 1 or subgroup 2, CMV replicase, or single chain antibodies to the CMV coat protein. Optimize the transformation system for Easter lilies and Ornithogalum using either the gene gun or Agrobacterium. Develop a transformation system for roots of Easter lilies using Agrobacterium rhizogenes. Transgenic Gladiolus plants with D4E1 and cpo have been developed using the gene gun and are being grown for challenging with Fusarium in the greenhouse. Gladiolus will also be transformed various chitinase genes for possible resistance to Fusarium. Evaluate effectiveness of genetically engineered resistance to Fusarium oxysporum in Gladiolus by comparison to biological control technologies. Develop Easter lilies that are resistant to the nematode Pratylenchus penetrans, the root lesion nematode. Genes involved in nematode development will be isolated and tested for their ability to kill Pratylenchus penetrans or affect its reproduction using RNAi. Significant Activities that Support Special Target Populations Genetic engineering of Gladiolus for Fusarium resistance Leaf extracts were used in an in vitro assay to screen for those transgenic lines with the D4E1 antimicrobial peptide gene that showed the most inhibition of Fusarium oxysporum spore germination. Five lines were selected, and these will be grown in the greenhouse in Fusarium-infected soil to determine their level of resistance. Putatively transformed plants with either an exo or endochitinase gene isolated from Fusarium, a chitinase gene from Trichoderma, or an antibody gene to Fusarium combined with an antifungal gene have been developed. This research is covered under National Program 302, Component 1 (Analysis and Modification of Plant Genomes), Problem Area 1b (Plant Transformation Systems and Influence of Transgenes on Genome Structure and Function), ARS Strategic Plan Goal 2. Genetic engineering of Gladiolus and Ornithogalum for virus resistance Four transgenic plants lines of Gladiolus have been found resistant in vitro to CMV strain S (serotype 2), and four lines were resistant to CMV serotype 1. These plants have been multiplied in vitro and this season were planted in soil in the greenhouse for future challenge outdoors. This research is covered under National Program 302, Component 1 (Analysis and Modification of Plant Genomes), Problem Area 1b (Plant Transformation Systems and Influence of Transgenes on Genome Structure and Function), ARS Strategic Plan Goal 2. Genetic engineering of Easter lilies Stable integration of the reporter gene, uidA, has been verified by showing inheritance of this gene following cross pollination of transgenic lilies with non-transgenic lilies. We were unable to verify integration of the transgene by Southern hybridization so this information was needed before proceeding on transformation of lilies for nematode and virus resistance genes. This research is covered under National Program 302, Component 1 (Analysis and Modification of Plant Genomes), Problem Area 1b (Plant Transformation Systems and Influence of Transgenes on Genome Structure and Function), ARS Strategic Plan Goal 2.

Impacts
(N/A)

Publications

  • Joung, H.J., Cantor, M., Ellis, D.D., Kamo, K.K. 2007. Cryopreservation of Gladiolus shoot tips derived from cormels. CryoLetters. 48:251-255.
  • Kamo, K.K. and Joung, Y.H. 2007. Gladiolus. Biotechnology in Agriculture and Forestry. 61:289-298.


Progress 10/01/06 to 09/30/07

Outputs
Progress Report Objectives (from AD-416) Utilize genetic engineering to improve resistance of bulb crops to fungal diseases and plant parasitic nematodes. (a) Develop transformation technology for Easter lily; (b) test candidate genes to confer resistance to Fusarium in Gladiolus; and (c) determine whether dsRNA expressed in plants can target RNAi to control plant parasitic nematodes. Approach (from AD-416) Transform a commercially important Gladiolus cultivar,Easter lilies, and Ornithogalum with genes for resistance to either cucumber mosaic virus or ornithogalum mosaic virus. Determine if the genetically engineered plants are resistant to these viruses. Complete experiments involving cucumber mosaic virus resistance in transgenic Gladiolus plants that contain either the CMV coat protein subgroup 1 or subgroup 2, CMV replicase, or single chain antibodies to the CMV coat protein. Optimize the transformation system for Easter lilies and Ornithogalum using either the gene gun or Agrobacterium. Develop a transformation system for roots of Easter lilies using Agrobacterium rhizogenes. Transgenic Gladiolus plants with D4E1 and cpo have been developed using the gene gun and are being grown for challenging with Fusarium in the greenhouse. Gladiolus will also be transformed various chitinase genes for possible resistance to Fusarium. Evaluate effectiveness of genetically engineered resistance to Fusarium oxysporum in Gladiolus by comparison to biological control technologies. Develop Easter lilies that are resistant to the nematode Pratylenchus penetrans, the root lesion nematode. Genes involved in nematode development will be isolated and tested for their ability to kill Pratylenchus penetrans or affect its reproduction using RNAi. Significant Activities that Support Special Target Populations Genetic engineering of Easter lilies: Easter lilies have been transformed with a marker gene in order to develop a tranformation protocol. Expression of the marker gene has been verified, but it has not been possible to verify integration of the marker gene into the genome of lily. AFLP markers using primers that recognized the gene of interest and a restriction site that the genomic DNA had been cut with was tested as a method for showing integration of a transgene into genomic DNA, but it was unsuccessful. Alternatively seeds from cross-pollinated transgenic lily lines are being collected and placed on medium for embryo rescue. The progeny will be analyzed by PCR for the presence of the marker gene. A molecular analysis of the transgenic plants is necessary to verify stable integration of the transgene and to identify which plants are independent transformants rather than genetic duplicates. This research is needed prior to the accomplishment of one of the FY 2007 milestones (Analyze transgenic lilies by PCR and Southern hybridization to confirm presence of selectable marker gene), and it is covered under National Program 302, Component 1 (Analysis and Modification of Plant Genomes), Problem Area 1b (Plant Transformation Systems and Influence of Transgenes on Genome Structure and Function), ARS Strategic Plan Goal 2. Accomplishments Genetic engineering for virus resistance in Gladiolus Viruses are a major problem in floral bulb crops because their symptoms make the plants unmarketable. There are no commercially available cultivars of Gladiolus that are resistant to viruses available for breeding. We have engineered eight lines of Gladiolus that show resistance to cucumber mosaic virus 2-3 months after the plants growing in tissue culture have been inoculated with the virus. One line of the cultivar Peter Pears stands out as being quite resistant. These plant lines are being multiplied for growth in the greenhouse and another round of inoculation with the virus. The research is covered under National Program 302, Component 1 (Analysis and Modification of Plant Genomes), Problem Area 1b (Plant Transformation Systems and Influence of Transgenes, . Genome Structure and Function), ARS Strategic Plan Goal 2. Genetic engineering for Fusarium resistance: The gene gun has been used to transform Gladiolus cells with several antifungal genes (chitinase from Trichoderma; exochitinase and endochitinase from Fusarium; an antibody gene to Fusarium; and synthetic antimicrobial peptide �D4E1� and chloroperoxidase (CPO). Gladiolus plants containing the antifungal D4E1 and CPO transgenes are growing in the greenhouse, and the soil was inoculated with Fusarium to determine their possible resistance to Fusarium. This study is ongoing and it is anticipated that results on antifungal resistance will be available at the end of the growing season when there will be data on the number of plants that survived Fusarium. For the other anti-fungal genes, tissue cultured cells are under selection to isolate putative transformants. Fusarium is the main fungal pathogen affecting Gladiolus and many other flower bulb crops. This research is two of the FY 2007 milestones (Grow D4E1 and CPO-containing Gladiolus plants in the greenhouse and multiply chitinase plants in vitro), and it is covered under National Program 302, Component 1 (Analysis and Modification of Plant Genomes), Problem Area 1b (Plant Transformation Systems and Influence of Transgenes on Genome Structure and Function), ARS Strategic Plan Goal 2. Genetic engineering for virus resistance: Viruses continually affect the flower bulb crops as the viruses are present in the flower bulbs that are planted each season. Viral symptoms include streaking on leaves and flowers, stunting of the plant, and deformed leaves making the plants unmarketable. Flower bulbs have been genetically engineered for virus resistance. Transgenic Gladiolus plants containing either the cucumber mosaic virus (CMV) coat protein or replicase genes that have shown short-term resistance to 2 �g CMV are being challenged with higher concentrations (5 and 10 �g) of CMV. The challenged plants are growing in tissue culture and are analyzed for the presence of virus as soon as there is enough plant tissue available for the ELISA assay, and this is typically 2-3 months after the plant has been inoculated with the virus. This research is part of a FY 2007 milestone (Grow Gladiolus plants containing replicase and coat protein genes in the greenhouse) because it determines which plants show the most resistance to virus and only the most promising plants will be grown in the greenhouse. The research is covered under National Program 302, Component 1 (Analysis and Modification of Plant Genomes), Problem Area 1b (Plant Transformation Systems and Influence of Transgenes on Genome Structure and Function), ARS Strategic Plan Goal 2. Construct chimeric OrMV::OrV3 dsRNA constructs: Thev bulbous crops Ornithogalum and Lachenalia are commonly infected with potyviruses that affect crop quality and productivity. Phylogenetic analysis of multiple virus isolates from Ornithogalum and Lachenalia revealed that isolates of Ornithogalum mosaic virus (OrMV) are differentiated into several subclades, and that one OrMV isolate from Lachenalia is quite distinct from other OrMV isolates. A representative OrMV isolate and an OrV3 isolate were selected to create a hybrid, partial inversely duplicated OrMV::OrV3 construct for plant transformation. Genetic engineering of Ornithogalum to induce resistance to OrMV and OrV3 should allow sustainable production of superior interspecific hybrid cultivars, without the losses due to viral infection. This research is part of one of the FY 2007 milestone, and is covered under National program 302, Component 1 (Analysis and Modification of Plant Genomes), Problem Area 1b (Plant Transformation Systems and Influence of Transgenes on Genome Structure and Function), ARS Strategic Plan Goal 2. Technology Transfer Number of Web Sites managed: 1

Impacts
(N/A)

Publications

  • Joung, Y.H. and Kamo, K. 2006. Expression of a polyubiquitin promoter isolated from Gladiolus. Plant Cell Reports. 25:1081-1088.


Progress 10/01/05 to 09/30/06

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? The environmental horticulture industry is one of the fastest growing segments of the nation's agricultural economy and is comprised of a variety of businesses involved in production, distribution and services associated with ornamental plants, landscape and garden supplies and equipment. The National Gardening Association reported that 85 million U. S. households spent $39.6 billion at lawn and garden retail outlets in 2002. The USDA National Agricultural Statistics Service reported that in 2005 the wholesale value of floral and nursery crops was $15.22 billion with 12,258 growers employing 187,563 workers. Over one quarter of these growers (28%) had sales less than $100,000. The quality of many floral crops is adversely affected by diseases, and many crops lack natural resistance to disease. Even if disease resistance genes are known in related species, transferring resistance into horticulturally acceptable ornamentals by traditional methods may take many cycles of breeding to achieve. Currently growers utilize significant amounts of pesticides to produce high quality crops free of disease, but the pesticides have an economic and environmental cost. This project is centered around the transformation of floral monocotyledonous bulb crops to introduce resistance to important diseases that affect crop quality. Genetic engineering of floral bulb crops for disease resistance offers an alternative to pesticide usage and lowers production costs in an increasingly competitive market. Disease resistance genes can be introduced into commercially important cultivars that lack the necessary genes for disease resistance. The wholesale value in the U.S. of flower stems grown domestically was $397 million for 2005. Gladiolus ranks fifth for the number of cut-flowers sold worldwide in 2004, and their value in the U.S. was $24 million. The highest value for cut-flowers was for lilies that had a wholesale value of $77 million in 2005. Most of the cut-flower lilies are grown in California. The wholesale value of potted lilies was $38 million in 2005. Gladiolus will be transformed with antiviral genes to cucumber mosaic virus (CMV) and antifungal genes against Fusarium oxysporum. Ornithogalum will be transformed with antiviral genes to Ornithogalum mosaic virus (OrMV). The transformation technology will be developed for lilies that are susceptible to viruses and nematodes. These transformation systems will allow both basic and applied research on gene expression. This research is under National Program 302, Component I (Analysis and Modification of Plant Genomes), Problem Area 1b (Plant Transformation Systems and Influence of Transgenes on Genome Structure and Function). 2. List by year the currently approved milestones (indicators of research progress) Year 1 (2006) CMV resistance in Gladiolus and lilies: - Sequence the Gladiolus isolate of CMV and use it to make the CMV dsRNA construct. - Test a lily isolate of CMV in Easter lily for pathogenicity and subclone as a CMV dsRNA construct for transformation. - Multiply in vitro the transgenic Gladiolus plants with CMV coat protein and replicase genes that have been found to confer resistance to CMV. OrMV resistance in Ornithogalum: - Clone and sequence additional portions of OrV2 and OrV3 to select gene regions for use. - Continue analysis of Ornithogalum promoters and transient expression of various constructs in Ornithogalum. - Transform callus and suspension cell cultures of Ornithogalum with various selectable markers. Nematode resistance in lilies: - Transform lilies with various selectable marker genes. - Develop an Agrobacterium rhizogenes mediated transformation system for lily. - Sequence and subclone clones from an EST library of the root lesion nematode into vectors for RNAi feeding experiments. Fusarium resistance in Gladiolus: - Multiply Gladiolus plants in vitro that contain antifungal genes, D4E1 and cpo. - Develop transgenic Gladiolus plants to contain exo and endochitinases. Year 2 (2007) CMV resistance in Gladiolus and lilies: - Make transgenic Gladiolus and lilies containing CMV dsRNA. - Grow transgenic Gladiolus plants containing CMV replicase and coat protein genes in the greenhouse. OrMV resistance in Ornithogalum: - Construct chimeric OrMV::OrV3 dsRNA constructs. Nematode resistance in lilies: - Analyze transgenic lilies by PCR and Southern hybridization to confirm the presence of selectable marker genes. - Feed RNAi constructs to the root lesion nematode. Fusarium resistance in Gladiolus: - Grow D4E1 and cpo-containing Gladiolus plants in the greenhouse. Molecular analysis of these lines. - Multiply chitinase-containing Gladiolus plants in vitro. Year 3 (2008) CMV resistance in Gladiolus and lilies: - Multiple in vitro the Gladiolus and lilies containing CMV dsRNA. - First year field study of virus resistance in transgenic Gladiolus plants that contain the CMV coat protein and replicase genes. OrMV resistance in Ornithogalum: - Analyze transgenic Ornithogalum with marker genes and transform with antiviral constructs. Nematode resistance in lilies: - Transform lilies with various promoters. - Subclone nematode EST clones of interest into plant vectors. Fusarium resistance in Gladiolus: - Greenhouse challenge of Gladiolus plants containing D4E1 and cpo with Fusarium oxysporum. - Grow chitinase transgenic Gladiolus plants in the greenhouse. Molecular analysis of these plants. Year 4 (2009) CMV resistance in Gladiolus and lilies: - CMV challenge in vitro of the transgenic Gladiolus plants containing CMV dsRNA. - CMV challenge in the greenhouse of the lilies transformed with CMV dsRNA. - Second year field study of CMV resistance in Gladiolus plants with the replicase and coat protein genes. OrMV resistance in Ornithogalum: - Develop transformants of Ornithogalum for resistance to OrMV. Nematode resistance in lilies: - Analyze transgenic lilies containing various promoters for levels of GUS expression in the roots and shoots. - Transform lilies with nematode resistance genes. Fusarium resistance in Gladiolus: - Greenhouse challenge of the Gladiolus plants with the chitinase genes. Year 5 (2010) CMV resistance in Gladiolus and lilies: - Molecular analysis of Gladiolus and lilies containing CMV dsRNA. OrMV resistance in Ornithogalum: - Evaluate Ornithogalum for expression of antiviral constructs. Nematode resistance in lilies: - Multiply lilies with nematode resistance genes for establishing plants in the greenhouse. Plants will be used for future molecular analysis of the transgenes and challenge by nematodes. 4a List the single most significant research accomplishment during FY 2006. Transformation of Gladiolus for virus resistance Several antiviral genes are being tested in Gladiolus to determine if they confer resistance to Cucumber mosaic virus (CMV). Viruses are a major problem affecting plants that are propagated vegetatively because they are carried through the plants to the next crop. In collaboration with Projects 1230-22000-012-00D and 1230-21000-040-00D, several transgenic gladioli lines have been produced which express either CMV replicase and/or coat protein. We have identified several lines of transgenic Gladiolus plants containing the CMV replicase and coat protein serotype II genes that show short term resistance to CMV. These lines will be multiplied and grown in the field to determine if they have long term resistance to CMV. This work will facilitate the evaluation of virus resistance in transgenic gladiolus plants to yield improved floral quality and productivity. This research is under National Program 302, Component I, Problem Area Ib and under ARS Strategic Plan Goal 1, Performance measure 1.2.8 and Goal 3, Performance Measure 3.2.5. 4b List other significant research accomplishment(s), if any. Transformation of Gladiolus for Fusarium resistance Several antifungal genes are being tested in Gladiolus to determine if they confer resistance to Fusarium. Fusarium is the main problem affecting Gladiolus and other bulb crops. Methyl bromide has been used traditionally to control Fusarium in the field, but this option is no longer available so development of plants resistant to Fusarium will be useful. We have identified several lines of transgenic Gladiolus expressing the antifungal genes, D4E1 and cpo. These lines will be multiplied for future challenge with Fusarium. This research is under National Program 302, Component I, Problem Area Ib and under ARS Strategic Plan Goal 1, Performance measure 1.2.8 and Goal 3, Performance Measure 3.2.5. 4d Progress report. Cloning and sequencing of the 3 region of Ornithogalum-infecting potyviruses Infection of Ornithogalum by Ornithogalum mosaic virus (OrMV) and related potyviruses is a major factor affecting plant quality and yield; whereas species Ornithogalum can be propagated via seed to recover virus-free plants, horticulturally superior interspecific Ornithogalum hybrid cultivars cannot be propagated by seed as the desirable characters would segregate in the progeny. Genetic engineering of viral resistance into the interspecific hybrids should allow sustainable vegetative production of these superior cultivars. We have cloned and sequenced approximately 1. 8kb of the 3-terminal region of the genome of four distinct potyviruses naturally infecting Ornithogalum and Lachenalia. Sequence comparisons of the cloned portions have been made in order to identify appropriate regions of high homology for construction of inverted repeat constructs to obtain virus resistance by induction of virus-specific gene silencing (RNA interference). This research was a milestone for Year 1 (2006). This research is under National Program 302, Component I, Problem Area Ib and under ARS Strategic Plan Goal 1, Performance measure 1.2.8 and Goal 3, Performance Measure 3.2.5. 5. Describe the major accomplishments to date and their predicted or actual impact. Developed transformation system for Easter lilies. Nematodes are the major problem when growing lilies in the field, and efforts to control them using chemicals or cultural practices have been unsuccessful. A procedure was developed to transform Easter lilies. Future research will be involved in using this system to create transgenic nematodes-resistance lilies. This research is a milestone for Year 4 (2009). This research is under National Program 302, Component I, Problem Area Ib and under ARS Strategic Plan Goal 1, Performance measure 1.2.8 and Goal 3, Performance Measure 3.2.5. Developed Gladiolus plants expressing antiviral and antifungal genes. Cucumber mosaic virus and Fusarium are major problems with floral bulb crops. Several different antiviral and antifungal genes have been introduced into Gladiolus. Future research will involve screening the transgenic plants for resistance/tolerance. All commercial cultivars of Gladiolus are susceptible to viruses and there does not appear to be any natural resistance, so genetic engineering is the only alternative. The Fusarium fungus is the biggest problem when growing Gladiolus and other bulb crops in the field. Since the use of methyl bromide for its control will no longer be available and there are limited effective chemicals available for treatment, transgenic resistance will be an important control strategy. This research was a milestone for Year 1 (2006). This research is under National Program 302, Component I, Problem Area Ib and under ARS Strategic Plan Goal 1, Performance measure 1.2.8 and Goal 3, Performance Measure 3.2.5. 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 display describing current research was presented at the Mid-Atlantic Nursery Trade Show in Baltimore, MD, January, 2006. Participated in the Floral and Nursery Crops Research Unit Open House for the Green Industry, May 9, 2006 in Washington DC, and presented Overview of ornamentals research at the U.S. National Arboretum. The technology to transform Easter lilies has been shared with scientists at the Rural Development Administration in Korea. 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). Attended the Second Floral and Nursery Crops Researchers Workshop held June 12-15 2006 in Portland, OR, and presented Transgenic plants of Gladiolus containing the coat protein and replicase genes of cucumber mosaic virus. Attended the Society for In Vitro Biology Meeting held June 3-6, 2006 in Minneapolis, MN and presented a poster by Kamo, K., Ueng, P., Aebig, J., Hsu, H.T., Guaragna, M.A., and Jordan, R., entitled Transgenic plants of Gladiolus containing the coat protein and replicase genes of cucumber mosaic virus.

Impacts
(N/A)

Publications

  • Kamo, K.K., Ueng, P.P., Aebig, J.A., Hsu, H., Guaragna, M.A., Jordan, R.L. 2006. Transgenic plants of gladiolus containing the coat protein and replicase genes of cucumber mosaic virus. [abstract]. Society for In Vitro Biology Proceedings. 42:44-A.