Progress 09/01/04 to 08/31/07
Outputs
Somatic Hybridization: Emphasis throughout the grant period was on conducting somatic hybridization experiments combining mandarin with pummelo in efforts to re-synthesize a superior CTV-resistant sour orange rootstock, with the most experiments using selected seedling pummelo parents that showed resistance to CTV-induced quick decline after two years in the field. This work was highly successful as somatic hybrid plants were recovered from 19 combinations. All of these hybrids were propagated via rooted cuttings for CTV testing and horticultural evaluation. Seed trees of these hybrids were planted in the field along with previously produced mandarin/pummelo somatic hybrids, and a few of these trees fruited for the first time this year. Fruit and seed were collected to determine amenability of these hybrids to traditional seed propagation. Hybrids from this program have potential to combine the attributes of sour orange including wide soil adaptation and good fruit
quality, with resistance to CTV-induced quick decline and tree size control. Tetraploid Roostock Breeding: hundreds of seed from crosses (diploid and tetraploid) were germinated directly in the greenhouse screen for soil adaptation and Phytophthora resistance, and more than 250 superior hybrid seedlings were selected for further study. All of these hybrids were challenged by grafting with CTV-infected sweet orange, with very few showing any CTV-induced symptoms in the greenhouse. Seed trees of each hybrid were also produced and planted in the field. Transformation: The protoplast CTV-challenge assay was completed on more than 150 transgenic grapefruit plants containing CTV-derived constructs (392 sequence and the hairpin p232 sequence) for potential CTV resistance. Four White Marsh grapefruit plants repeatedly showed altered banding patterns in northern analysis, suggesting some resistance. All of these plants were inoculated by blind-bud grafting with C. macrophylla infected with the
T-36 quick-decline isolate of CTV in the greenhouse. ELISA analysis will be conducted on these plants this winter in efforts to validate the protoplast assay results. Field Assay to assess CTV-resistance in new hybrids: A top-working field assay was established to test new material for resistance/tolerance to CTV. Material tested includes new mandarin/pummelo somatic hybrids, selected tetrazygs, and selected pummelo parents used in somatic fusion experiments. This test has shown that its possible to bypass the confounding affects of seedling yellows that interferes with greenhouse screens, and several promising mandarin/pummelo hybrids were identified. These hybrids show little or no CTV replication and good growth as compared to sour orange that shows high CTV replication and virtually no growth. Some hybrids showing high CTV replication are also showing good growth with no quick-decline symptoms. Summary: this research has moved us much closer to having a suitable CTV tolerant or
resistant replacement for sour orange rootstock, and possibly CTV resistant transgenic White Marsh grapefruit.
Impacts
Significant progress was made regarding the development of a replacemente rootstock for sour orange. Several new hybrids with strong parentage were produced, and a new field CTV-challenge assay has identified hybrids with resistance/tolerance to CTV-induced diseases. Transgenic 'White Marsh' grapefruit plants with potential resistance to CTV were also identified using our protoplast challenge assay, with validation underway.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
Somatic Hybridization: Emphasis continued on conducting somatic hybridization experiments combining mandarin with pummelo in efforts to re-synthesize a superior CTV-resistant sour orange rootstock, with the most recent experiments using selected seedling pummelo parents that showed resistance to CTV-induced quick decline after two years in the field. Somatic hybrid plants were recovered from the following 10 combinations: Amblycarpa mandarin + pummelo HBJL-12, Amblycarpa + pummelo 5-1-99-2; Nova mandarin hybrid + pummelo 7-3-99-1; Nova + 7-2-99-2; Nova + 8-2-99-1; Nova 8-1-99-4B;W. Murcott tangor + pummelo HBJL-12; W. Murcott + pummelo 5-1-99-2; W. Murcott + pummelo 7-2-99-2; and W. Murcott + 4-3-99-2. All of these hybrids are being propagated for CTV testing and horticultural evaluation. Hybrids from this program have potential to combine the attributes of sour orange including wide soil adaptation and good fruit quality, with resistance to CTV-induced quick decline
and tree size control. Tetraploid Roostock Breeding: Seed from 2005 crosses (diploid and tetraploid) was germinated directly in the greenhouse screen for soil adaptation and Phytophthora resistance, and more than 150 superior hybrid seedlings were selected for further study. All of these hybrids were challenged by grafting with CTV-infected sweet orange. Seed trees of each hybrid were also produced. Additional crosses were conducted during 2006, and recovered seed planted in the soil/Phytophthora screen. Transformation: The protoplast CTV-challenge assay was completed on more than 150 transgenic grapefruit plants containing CTV-derived constructs (392 sequence and the hairpin p232 sequence) for potential CTV resistance. Four White Marsh grapefruit plants repeatedly showed altered banding patterns in northern analysis, suggesting some resistance. These plants are being inoculated by traditional greenhouse methods to validate this result. Field Assay to assess CTV-resistance in new
hybrids: A top-working field assay was established to test new material for resistance/tolerance to CTV. Material being tested includes new mandarin/pummelo somatic hybrids, selected tetrazygs, and selected pummelo parents being used in somatic fusion experiments. This test is showing promise to bypass the confounding affects of seedling yellows that interferes with greenhouse screens.
Impacts
Significant progress was made regarding the development of a replacemente rootstock for sour orange. Several new hybrids with strong parentage were produced, and a new field assay is being developed to expedite identification of hybrids with appropriate resistance/tolerance to CTV-induced diseases. Transgenic 'White Marsh' grapefruit plants with potential resistance to CTV were also identified using our protoplast challenge assay.
Publications
- Orbovic, V, and J.W. Grosser. (2006). Citrus: Sweet orange (Citrus sinensis L. Osbeck Valencia) and Carrizo citrange [Citrus sinensis (L.) Osbeck x Poncirus trifoliata (L.) Raf.]. In: K. Wang (Ed.), Agrobacterium protocols-methods in molecular biology (pp. 177-189), Humana Press Inc., USA.
- Ananthakrishnan, G., V. Orbovic, G.,Pasquali., and J.W. Grosser. 2007. Transfer of CTV-derived resistance candidate sequences to four grapefruit cultivars through Agrobacterium-mediated genetic transformation. In Vitro Cell. Dev. Biol.--Plant (in press).
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Progress 10/01/04 to 09/30/05
Outputs
Emphasis has been on conducting somatic hybridization experiments combining mandarin with pummelo in efforts to re-synthesize a superior CTV-resistant sour orange rootstock, with the most recent experiments using selected seedling pummelo parents that showed resistance to CTV-induced quick decline after two years in the field. Somatic hybrid plants were recovered from the following 20 combinations: Amblycarpa mandarin + pummelo 4-3-99-2; Amblycarpa + pummelo 4-4-99-6; Amblycarpa + pummelo 7-3-99-1; Amblycarpa + pummelo MG1; Amblycarpa + pummelo MG10; Amblycarpa + pummelo HBJL3; Amblycarpa + pummelo HBJL5; Amblycarpa + pummelo HBJL7; Amblycarpa + pummelo Chandler A1-11; Page tangor + pummelo HBJL3, Page + pummelo HBJL4; Page + pummelo HBJL5, Page + pummelo HBJL12, Page + pummelo 7-3-99-1; and Murcott tangor + pummelo HBJL5; Changsha mandarin + HBJL3; Changsha mandarin + HBJL5; Changsha mandarin + HBJL7; W. Murcott tangor + HBJL3; and W. Murcott + HBJL7 (these are the
first mandarin + pummelo somatic hybrids made with Changsha or W. Murcott). All of these hybrids are being propagated for CTV testing and horticultural evaluation. Hybrids from this program have potential to combine the attributes of sour orange including wide soil adaptation and good fruit quality, with resistance to CTV-induced quick decline and tree size control. Seed from 2004 crosses (diploid and tetraploid) was germinated directly in the greenhouse screen for soil adaptation and Phytophthora resistance, and more than 150 superior hybrid seedlings were selected for further study. These include more than 50 diploid hybrids of Cleopatra mandarin with pummelo. All of these hybrids were challenged by grafting with CTV-infected sweet orange. Seed trees of each hybrid were also produced. Southern analyses was conducted on transgenic grapefruit plants containing CTV-derived constructs (392 sequence and the hairpin p232 sequence) for potential CTV resistance, as necessary to validate
transformation and to determine copy number. Most transgenic plants contained one to two copies of the respective transgene. Assay of these plants for CTV resistance is underway.
Impacts
This research is expected to develop a replacement rootstock for sour orange. Currently, the industry does not have an adequate replacement rootstock for use on challenging soils. New hybrid rootstocks produced in this project should exhibit the attributes of sour orange, but also resistance to CTV-induced quick decline and potential for tree size control to facilitate harvesting. Approximately 15 million trees on sour orange remain in Florida, but are being lost at a rate of about 1 million per year. A replacement rootstock would have significant economic impact on the industry. Ours is the only program in the USA using protoplast fusion technology to solve this problem.
Publications
- Grosser, J. W., V. Medina-Urrutia, G. Ananthakrishnan, and P. Serrano. 2004. Building a Replacement Sour Orange Rootstock: Somatic Hybridization of Selected Mandarin + Pummelo Combinations. J. Amer. Soc. Hort. Sci. 129:530-534.
- Grosser, J.W. and Gmitter, F.G. Jr. 2005. 2004 SIVB Congress Symposium Proceedings, Thinking Outside the Cell; Applications of somatic hybridization and cybridization in crop improvement, with citrus as a model. In Vitro Cell. Dev. - Plant 41:220-225.
- Ananthakrishnan, G., Gowda, S. , Orbovic, V., Dawson, W.O. and J.W. Grosser. 2006. Regeneration of Transgenic Sweet Orange and Carrizo citrange Plants Containing a 742bp Citrus Tristeza Virus-Derived Sequence 392. Proc. Int. Soc. Citriculture (2004) Vol. 1:135-138.
- Orbovic, V., G. Ananthakrishnan, S. Gowda, W. O. Dawson, and J. W. Grosser (2004). Genetic Engineering of Resistance to CTV in Grapefruit through Agrobacterium-mediated Genetic Transformation. World Congress on In Vitro Biology, San Francisco, California, May 22-27, 2004. pp. 68-Abstract.
- Ananthakrishnan, G., Calovic, M., Serrano, P., and J.W. Grosser. 2006. Production of additional allotetraploid somatic hybrids combining mandarins and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock. In Vitro Cell Dev.Biol. - Plant 42:367-371.
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Progress 09/01/04 to 09/30/04
Outputs
This report covers progress made during the first 3 months of the project. Emphasis has been on conducting somatic hybridization experiments in efforts to re-synthesize a superior CTV-resistant sour orange rootstock. Numerous fusion experiments were conducted combining protoplasts of selected mandarin embryogenic suspension cultures including Amblycarpa, Murcott, Page, and Changsha, with leaf protoplasts of selected pummelo seedlings. Starting with more than 250 pummelo seedlings pre-selected for adaptation to high ph soil and Phytophthora resistance, we have now narrowed the list of parents down to the top 30 that are showing resistance to CTV-induced quick decline in the field after 2 years in a challenging field site with high ph, calcareous soil. Somatic hybrid plantlets were recovered from the following combinations: Amblycarpa + pummelo 4-3-99-2; Amblycarpa + pummelo 7-3-99-1; and Amblycarpa + pummelo MG10. Somatic embryos were recovered from Amblycarpa +
pummelos HBJL3, HBJL5, HBJL7; Page + pummelos HBJL3, HBJL5, HBJL12, and 7-3-99-1; and Murcott + pummelos HBJL 7, HBJL12, 8-1-99-2B, 7-3-99-1, 4-3-99-2, and 7-2-99-2. The first embryogenic calli has been recovered from a few fusions using Changsha mandarin for the first time. Hybrids from this program have potential to combine the attributes of sour orange including wide soil adaptation and good fruit quality, with resistance to CTV-induced quick decline and tree size control. Additional trees of the top ten pummelo seedlings were propagated to provide additional leaves for ongoing fusion experiments.
Impacts
This research is expected to develop a replacement rootstock for sour orange. Currently, the industry does not have an adequate replacement rootstock for use on challenging soils. New hybrid rootstocks produced in this project should exhibit the attributes of sour orange, but also resistance to CTV-induced quick decline and potential for tree size control to facilitate harvesting. Approximately 15 million trees on sour orange remain in Florida, but are being lost at a rate of about 1 million per year. A replacement rootstock would have significant economic impact on the industry. Ours is the only program in the USA using protoplast fusion technology to solve this problem.
Publications
- No publications reported this period
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