Progress 09/15/04 to 09/14/07
Outputs
We have previously obtained seeds from 6 transgenic lines of Mexican lime that are putatively CTV-resistant from Dr. Leandro Pena at the Instituto Valenciano de Investigaciones Agrarias in Valencia, Spain. Micro-satellite analysis was used to distinguish nucellar (hemizygous) and zygotic (homozygous) seedlings in the transgenic lines and a wild-type control. A nucellar seedling from each of the six transgenic lines and the wild-type control was propagated onto sixteen C-35 citrange rootstocks and grown in the greenhouse. These grafted plants have now attained a suitable size for field evaluation. We have also received the required permits from the Hawaii Department of Agriculture and USDA-APHIS allowing the field trial to commence. We have sequenced over 100 coat protein genes from Hawaiian CTV. This, as well as coat protein gene sequence data of CTV strains from around the world obtained from GenBank, were used to generate a synthetic transgene that was ~95% identical
to all of these genes. We have generated multiple lines of transgenic Mexican lime plants containing this synthetic gene in sense, anti-sense, and inverted-repeat configurations. PCR and genomic Southern analysis have confirmed the transgenic nature of these plants, and we are continuing to generate additional lines for each of the transgene configurations. Several of the transgenic lines have been propagated by bud-grafting for virus-resistance bioassays.
Impacts
Citrus tristeza virus (CTV) and its most effective vector, the brown citrus aphid (BrCA), have been present in Hawaii for over 50 years. Their relationship may be responsible for the high incidence and genetic diversity of CTV that makes Hawaii an ideal location to test citrus for CTV resistance. We have recently established a field site dedicated to test for broad CTV resistance in citrus and are conducting studies at this site using plants of susceptible citrus varieties to work out a useful testing protocol. Our first objective is to use this field site and protocol to challenge putatively resistant transgenic Mexican lime plants (Citrus aurantifolia) developed and in transit from the Instituto Valenciano de Investigaciones Agrarias in Spain. These plants represent the first transgenic citrus that offer resistance to CTV in laboratory and greenhouse experiments, but have thus far been untested under field conditions. Our second objective is to use a synthetic
resistance transgene based on the coat protein gene sequence of over 100 Hawaiian and 14 other CTV strains from around the world to transform Mexican lime and evaluate its ability to induce broad resistance against CTV via post-transcriptional gene silencing. Overall, we believe that research utilizing Hawaii's diverse CTV population can better prepare citrus-producing regions where the BrCA and severe strains of CTV are, or will soon become permanently established.
Publications
- Melzer, M. .J., Borth, W.B., Zee, F., Hilf, M.E., Garnsey, S.M., and Hu, J.S. 2006. Incidence, distribution, and diversity of Citrus tristeza virus in the Hawaiian islands. In: Proc. 16th Conf. IOCV pages 179-186.
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Progress 10/01/05 to 09/30/06
Outputs
Objective 1. Evaluate putatively resistant transgenic Mexican lime plants from Spain at our field site dedicated to testing for CTV resistance. We have previously obtained seeds from 6 transgenic lines of Mexican lime that are putatively CTV-resistant from Dr. Leandro Pena at the IVIA in Valencia, Spain. Budwood from nucellar seedlings of each line (and a wild-type control) were propagated onto C-35 citrange rootstock. Sixteen grafted plants were created for each line and have now had 13-15 months of growth under greenhouse conditions, are have attained a size suitable for field evaluation. In October 2006 we received the required permits from the Hawaii Department of Agriculture and USDA-APHIS allowing the field trial to commence. Objective 2. Develop CTV-resistant citrus using the synthetic gene approach. Previously we had sequenced over 100 coat protein genes from Hawaiian CTV. This, as well as coat protein gene sequence data of CTV strains from around the world
obtained from GenBank, was used to generate a synthetic transgene that was 95% identical to all of these genes. We have generated multiple lines of transgenic Mexican lime plants containing this synthetic gene in sense, anti-sense, and inverted-repeat configurations. PCR and genomic Southern analysis have confirmed the transgenic nature of these plants, and we are continuing to generate additional lines for each of the transgene configurations. Several of the transgenic lines are growing well in the greenhouse, and will be propagated by bud-grafting for virus-resistance bioassays by the end of 2006.
Impacts
Citrus tristeza virus (CTV) and its most effective vector, the brown citrus aphid (BrCA) have been present in Hawaii for at least 50 years. As no serious control strategies have ever been employed to control CTV or the BrCA, resulting in high incidence and diversity of the virus. As such, Hawaii is the ideal location to test plants putatively resistant to CTV that have been developed in Hawaii, the US mainland, or elsewhere. The development of citrus resistant to Hawaiian CTV strains would be beneficial to not only Hawaii, which has not been self-sufficient in citrus for decades, but also other citrus-producing regions in the US and abroad. Many of the strains that are found in these regions are also present in Hawaii, making plants resistant in Hawaii potentially resistant elsewhere.
Publications
- Melzer, M. .J., Borth, W.B., Zee, F., Hilf, M.E., Garnsey, S.M., and Hu, J.S. 2006. Incidence, distribution, and diversity of Citrus tristeza virus in the Hawaiian islands. In: Proc. 16th Conf. IOCV pages 179-186.
- Melzer, M. .J., Borth, W.B., Zee, F., Hilf, M.E., Garnsey, S.M., and Hu, J.S. 2006. Incidence and diversity of Citrus tristeza virus in the Hawaii. Phytopathology 96: 77.
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Progress 10/01/04 to 09/30/05
Outputs
Objective 1. Evaluate putatively resistant transgenic Mexican lime plants from Spain at our field site dedicated to testing for CTV resistance. We have obtained seeds of 6 transgenic lines of Mexican lime from Dr. Leandro Pena at the IVIA in Valencia, Spain. These and seeds from non-transgenic controls were germinated in vitro and transferred to our greenhouse facilities. Visual evaluation and microsatellite analyses were used to discriminate between nucellar and zygotic plants. Budwood from a nucellar plant of each line (and control) was propagated onto C-35 citrange rootstock. Sixteen grafted plants were created for each line and should be ready for transplant to the field in early 2006. A set of Performance Standards requested by the Hawaii Department of Agriculture for field release of transgenic citrus were generated and accepted by the HDOA and USDA-APHIS, allowing the field trial to commence. Objective 2. Develop CTV-resistant citrus using the synthetic gene
approach. Previously we had sequenced over 100 coat protein genes from Hawaiian CTV. This, as well as coat protein gene sequence data of CTV strains from around the world obtained from GenBank, was used to generate a synthetic transgene that was 95% identical to all of these genes. We have recently created transformation constructs that incorporate this synthetic transgene in various orientations: sense, antisense, and an inverted repeat that is linked together with the p23 gene from CTV. Based on information from other laboratories, we are also in the process of creating an inverted repeat of the p23 gene linked with the synthetic coat protein gene. We are currently using these constructs to transform Mexican lime.
Impacts
Citrus tristeza virus (CTV) and its most effective vector, the brown citrus aphid (BrCA) have been present in Hawaii for at least 50 years. As no serious control strategies have ever been employed to control CTV or the BrCA, resulting in high incidence and diversity of the virus. As such, Hawaii is the ideal location to test plants putatively resistant to CTV that have been developed in Hawaii, the US mainland, or elsewhere. The development of citrus resistant to Hawaiian CTV strains would be beneficial to not only Hawaii, which has not been self-sufficient in citrus for decades, but also other citrus-producing regions in the US and abroad. Many of the strains that are found in these regions are also present in Hawaii, making plants resistant in Hawaii potentially resistant elsewhere.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
Objective 1: Collect virus dissemination data at a field site dedicated to testing CTV-resistant citrus plants and develop a standard testing protocol. We have imported 7 Mexican lime lines from Dr. Leandro Pena (IVIA, Spain) that have shown various degrees of CTV resistance in greenhouse assays. These were brought in as seed in March of 2004 and have been grown to produce budwood for propagation. We estimate these plants will be ready for field testing in October 2005, although we also plan to put some seedlings into the field site in early 2005 for preliminary results. We are also working to import another, much more promising line of resistant Mexican lime from Dr. Pena, however for unknown reasons, the permitting process for this application has gone much slower at the State level, and we are still waiting for approval. Objective 2: Transform Mexican lime with a synthetic transgene designed for broad CTV resistance. Previously we had been having difficulties
creating fully transgenic Mexican limes. Most of the regenerated plantlets were chimeric, with one sector transgenic, and the remainder non-transgenic. Recently, Dr. Pena visited our laboratory and gave us very helpful suggestions for creating transgenic Mexican lime and other citrus. As a result, after exposure to Agrobacterium containing a plasmid with uidA-intron, we now routinely produce explants that show heavy GUS staining, as opposed to minor spots as we had obtained previously. We are much more confident we can produce fully transformed plants with our current protocol.
Impacts
Our first objective is to exploit Hawaii diverse CTV population by creating a field site dedicated to testing for broad CTV resistance in citrus. We have established such a site at a former citrus arboretum using existing trees as well as newly planted trees that are being inoculated with CTV isolates collected from across Hawaii. Before this site becomes operational, we need to conduct preliminary studies using susceptible citrus varieties to work out a standard testing protocol. This will greatly facilitate our ability to reliably test putatively CTV-resistant citrus from Hawaii and abroad. Our second objective is to use a synthetic resistance transgene based on the coat protein gene sequence of over 100 Hawaiian and 14 other CTV strains from around the world to transform Mexican lime and evaluate its ability to induce resistance against CTV via post-transcriptional gene silencing. Overall, we feel that research utilizing Hawaii diverse CTV population can better
prepare citrus-producing regions around the world where the BrCA and severe strains of CTV are, or will soon become, permanently established.
Publications
- No publications reported this period
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