Progress 08/17/10 to 08/16/15
Outputs
Target Audience:Other scientists in academia and industry, students as well as agricultural producers. Changes/Problems:No major problems have been encountered in carrying out the project. What opportunities for training and professional development has the project provided?One undergraduate student, one graduate student and four postdoctoral trainees are being trained while working on the projects described. How have the results been disseminated to communities of interest?Some of the results from the research projects have been published or are in the process of writing up. What do you plan to do during the next reporting period to accomplish the goals?Will continue to work on these projects on cotton and any other projects that may come up.
Impacts
What was accomplished under these goals?
To improve cottonseed quality, substantial reductions in seed-gossypol levels have been obtained by RNAi-mediated silencing of delta-cadinene synthase gene. From the new set of experiments with the silencing construct LCT66, an additional line (#66-317) was selected for field trial in 2015. In addition, the ultra-low gossypol cottonseed (ULGCS) lines (#66-49B and #66-274) were also grown in the field to obtain tissue samples for NPTII protein analysis in various tissues. As seen in 2009 - 2014, the non-seed tissues from all three lines continue to show terpenoids at levels similar to those found in the wild-type plants under field conditions in the year 2015. Importantly, the RNAi seeds continued to show the ULGCS phenotype. These results are very encouraging as they show the inheritance and stability of the ULGCS trait under field conditions in a different set of transformants. In 2015, we have conducted five additional, multi-location field trials with the two selected ULGCS lines (#66-49B and #66-274) for their eventual deregulation. We have conducted most of the molecular characterization of the transgene integration in these two lines. We have completed the assembly of an 'All-cotton Construct' where "All-cotton cassette" and the "selectable marker cassette" will be on two separate T-DNAs. Several transformation experiments have been conducted with this new construct. In the project involving RNAi-mediated resistance to nematodes in the cotton plant, we have obtained some encouraging results against reniform nematodes with one of the RNAi constructs. We are still investigating several other constructs for their ability to confer resistance to root-knot nematodes and/or reniform nematodes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
URRIOLA, J. & RATHORE K. S. (2015) Overexpression of a glutamine synthetase gene affects growth and development in sorghum. Transgenic Res (2015) 24:397407
- Type:
Book Chapters
Status:
Published
Year Published:
2015
Citation:
RATHORE, K. S., CAMPBELL, L. M., SHERWOOD, S. & NUNES, E. (2015) Cotton (Gossypium hirsutum L.) In: Methods in Molecular Biology, Vol. 1224: Agrobacterium Protocols, 3rd Ed.,Vol 2, ed. K. Wang (Springer, New York), pp. 11-23.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Other scientists in academia and industry, students as well as agricultural producers. Changes/Problems: No major changes or problems to report. What opportunities for training and professional development has the project provided? One undergraduate student and three postdoctoral trainees were trained while working on the projects described. How have the results been disseminated to communities of interest? Most of the results of research have been published or are in the process of writing up. What do you plan to do during the next reporting period to accomplish the goals? Will continue to work on the projects on cotton and any other projects that may come up.
Impacts
What was accomplished under these goals?
To improve cottonseed quality, substantial reductions in seed-gossypol levels have been obtained by RNAi-mediated silencing of delta-cadinene synthase gene. From the new set of experiments with the silencing construct LCT66, three additional lines (#66-239, #66-H-49, and #66-S-49) were selected for field trial in 2014. As seen in 2009 - 2013, the non-seed tissues from all three, new lines continue to show terpenoids at levels similar to those found in the wild-type plants under field conditions in the year 2014. Importantly, the RNAi seeds continued to show the ultra-low gossypol cottonseed (ULGCS) phenotype. These results are very encouraging as they show the inheritance and stability of the ULGCS trait under field conditions in a different set of transformants. We have further screened for and identified additional, single transgene copy ULGCS lines from second round of transformation with LCT66. These will be evaluated for trait stability over several generations. We have three additional, multi-location field trials with two selected ULGCS lines ((#66-49B and #66-274) for their eventual deregulation. We have begun a molecular characterization of the transgene integration in these two lines. We have begun to assemble an 'All-cotton Construct' where "All-cotton cassette" and the "selectable marker cassette" will be on two separate T-DNAs. Once assembled, this vector will be used for transforming cotton with the expectation that the selectable marker gene can be segregated out in the progeny. In the project involving AtNPR1-mediated disease resistance in transgenic cotton lines, we found that AtNPR1 can provide tolerance to Fusarium oxysporum f. sp. Vasinfectum, but only when the isolate produces little or no FA. In an extension of our earlier work on tobacco osmotin gene, we have shown that this gene also confers drought tolerance to carrot.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
URRIOLA, J. & RATHORE K. S. (2014)
Temporal and spatial activities of a rice glutelin promoter in transgenic sorghum. Plant Cell Tiss Organ Cult. 116: 227-234.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
ANNON, A., RATHORE K. S. & CROSBY, K. (2014)
Overexpression of tobacco osmotin gene in carrot (Daucus carota L.) enhances drought tolerance. In Vitro Cell. and Develop. Biology Plant, 50: 299-306.
|
Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Other scientists in academia and industry, students as well as agricultural producers. Changes/Problems: No major changes or problems to report. What opportunities for training and professional development has the project provided? One undergraduate student, a graduate student and three postdoctoral trainees were trained while working on the projects described. How have the results been disseminated to communities of interest? Most of the results of research have been published. What do you plan to do during the next reporting period to accomplish the goals? Will continue to work on the projects on cotton and any other projects that may come up.
Impacts
What was accomplished under these goals?
To improve cottonseed quality, substantial reductions in seed-gossypol levels have been obtained by RNAi-mediated silencing of delta-cadinene synthase gene. From the new set of experiments with the silencing construct LCT66, three additional lines (#66-103, #66-163C, and #66-193B) at T3 generation were selected for field trial in 2013. As seen in 2009 - 2012, the non-seed tissues from all three, new lines continue to show terpenoids at levels similar to those found in the wild-type plants under field conditions in the year 2013. Importantly, the RNAi seeds continued to show the ultra-low gossypol cottonseed (ULGCS) phenotype. These results are very encouraging as they show the inheritance and stability of the ULGCS trait under field conditions in a different set of transformants. We have further screened for and identified additional, single transgene copy ULGCS lines from second round of transformation with LCT66. These will be evaluated for trait stability over several generations. We have also initiated multi-location field trials with two selected ULGCS lines ((#66-49B and #66-274) for eventual deregulation of one of these. We have begun to regenerate plants from a new series of transformation experiments using an ‘All-cotton Construct’ to obtain a different set of ULGCS lines. All the components of the silencing cassette in the All-cotton construct are from cotton. Thus far >100 lines have been transferred to soil from these experiments and several more are in various stages of regeneration. We have obtained seeds from majority of these lines. These have been tested for gossypol levels, but we have not yet found one with ULGCS trait. We will continue the screening process as and when additional material becomes available. In an extension of our earlier work on AtNPR1-mediated disease resistance in transgenic cotton lines, we transformed Fusarium oxysporum f. sp. Vasinfectum (isolate Fov11) with a GFP gene and obtained transgenic fungal lines expressing this gene. One of these Fov11 lines was used to understand the mechanism of resistance in the AtNPR1-expressing cotton lines. This transgenic strain showed that NPR1-expression significantly slowed the progression of the disease in the transgenic plants. We had transformed sorghum to overexpress glutamine synthetase to assess its effect on the growth and development of the plant. When grown under optimal nitrogen conditions, these Gln1 transgenic lines showed greater tillering and up to 2.1-fold increase in shoot vegetative biomass. Even under greenhouse conditions, we observed a seasonal component to both these parameters and the grain yield. Our results, showing that the growth and development of sorghum Gln1 transformants are also affected by N availability and other environmental factors, suggest complexity of the relationship between GS activity and plant growth and development.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
KUMAR, V., JOSHI, S. G., BELL, A. A. & RATHORE K. S. (2013) Enhanced resistance against Thielaviopsis basicola in transgenic cotton plants expressing Arabidopsis NPR1 gene. Transgenic Res. 22: 359-368.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
PALLE, S. R., CAMPBELL, L. M., PANDEYA, D., PUCKHABER, L., TOLLACK, L. K., MARCELL, S., SUNDARAM, S., STIPANOVIC, R. D., HINZE, L., WEDEGAERTNER, T.C. & RATHORE, K. S. (2013) RNAi-mediated Ultra-low Gossypol Cottonseed Trait: Performance of Transgenic Lines under Field Conditions. Plant Biotech J. 11: 296-304.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
REN, Y., BANG, H., GOULD, J., RATHORE K. S., PATIL, B. S. & CROSBY, K. M. (2013) Shoot regeneration and ploidy variation in tissue culture of honeydew melon (Cucumis melo L. inodorus). In Vitro Cell Dev. Biol. Plant 49: 223-229.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
REN, Y., BANG, H., LEE, E. J., GOULD, J., RATHORE K. S., PATIL, B. S. & CROSBY, K. M. (2013) Levels of phytoene and b-carotene in transgenic honeydew melon (Cucumis melo L. inodorus). Plant Cell Tiss. Organ Cult. 113: 291-301.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
GAO, S-J., DAMAJ, M. B., PARK, J. W., BUENROSTRO-NAVA, M. T., MOLINA, J., WANG, X., CIOMPERLIK, J. J., MANABAYEVA, S. A., ALVARARADO, V. Y., RATHORE K. S., SCHOLTHOF, H. B. & MIRKOV, T. E. (2013) Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors. PLoS One. 8: 1-13.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2013
Citation:
BIOTECHNOLOGICAL APPROACHES FOR GENETIC IMPROVEMENT OF SORGHUM - JAZMINA ITZEL URRIOLA SIMONS
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: To improve cottonseed quality, substantial reductions in seed-gossypol levels have been obtained by RNAi-mediated silencing of delta-cadinene synthase gene. From the new set of experiments with the silencing construct LCT66, three lines (#66-274, #66-316, and #66-317) at T3 generation were selected for field trial in 2012. As seen in 2009 - 2011, the non-seed tissues from all three, new lines continue to show terpenoids at levels similar to those found in the wild-type plants under field conditions in the year 2012. Importantly, the RNAi seeds continued to show the ultra-low gossypol cottonseed (ULGCS) phenotype. These results are very encouraging as they show the inheritance and stability of the ULGCS trait under field conditions in a different set of transformants. We have further screened for and identified additional, single transgene copy ULGCS lines from second round of transformation with LCT66. These will be evaluated for trait stability over several generations. A new series of transformation experiments is being conducted using an All-cotton construct to obtain a different set of ULGCS lines. All the components of the silencing cassette in the All-cotton construct are from cotton. Thus far 56 lines have been transferred to soil from these experiments and several more are in various stages of regeneration. We have obtained seeds from 21 lines thus far. These have been tested for gossypol levels, but we have not yet found one with ULGCS trait. We will continue the screening process as and when the material becomes available. In collaboration with Dr. Tzachi Samocha, we had conducted a small, shrimp-feeding study using two of our ULGCS lines. The results suggest that ULGCS lines were as good as the glandless lines in supporting the growth of shrimp in aquaculture. In an extension of our earlier work on AtNPR1-mediated disease resistance in transgenic cotton lines, we transformed Fusarium oxysporum f. sp. Vasinfectum (isolate Fov11) with a GFP gene and obtained transgenic fungal lines expressing this gene. One of these Fov11 lines will be used to understand the mechanism of resistance in the AtNPR1-expressing cotton lines. In the third year of field trial, the NPR1 lines survived the usual biotic and abiotic pressures of field conditions and grew to maturity showing that transgenic lines are robust enough to withstand rigors of field conditions. We had transformed sorghum expressing a rice glutelin promoter-driven reporter gene and have confirmed its endosperm-specific activity in sorghum. The transgenic lines are being characterized further in terms of spatial and temporal activity of the promoter. The results suggest that it will be a useful promoter for transgenic improvement of grain quality in sorghum. PARTICIPANTS: One undergraduate student, a graduate student and three postdoctoral trainees were trained while working on the projects described. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Gossypol elimination research will have a significant impact on the value of cottonseed because after oil extraction, the meal can then be utilized directly as food or as a feed for animals other than cattle, especially for poultry and aquaculture. Gossypol-free cottonseed will also reduce the cost of processing and production of cottonseed oil. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases and nematodes in cotton production, thus positively impacting the disease-resistance aspect of our cotton improvement program. Sorghum is an important crop not just for food and feed, but is also being considered as a source to generate biofuels. Thus, the rice glutelin promoter, that has proven to be highly endosperm-specific in its activity in sorghum, will serve as an important tool in future projects to improve the quality of grain sorghum.
Publications
- RATHORE, K. S., SUNDARAM, S., SUNILKUMAR, G., CAMPBELL, L. M., PUCKHABER, L., MARCELL, S., PALLE, S. R., STIPANOVIC, R. D. & WEDEGAERTNER, T.C. (2012) Ultra-low gossypol cottonseed: generational stability of the seed-specific, RNAi-mediated phenotype and resumption of terpenoid profile following seed germination. Plant Biotech J. 10: 174-183.
- KUMAR, V., PARKHI, V., JOSHI, S., CHRISTENSEN, S., KOLOMIETS, M. & RATHORE K. S. (2012) A novel, conditional, lesion mimic phenotype in cotton cotyledons due to the expression of an endochitinase gene from Trichoderma virens. Plant Science 183: 86-95.
- Rathore, K. S. (2012) Use of RNAi to obtain Ultra-low gossip[ol cottonseed to enhance global food and feed security. Plant and Animal Genome XX Conference (abstract), San Diego, CA. January, 2012
- Palle, S.R., Rathore, K.S., Sundaram, S., Ganesan, S., Campbell, L. Puckhaber, L., Marcel, S. Stipanovic, R. and Wedegaertner, T. (2012) Ultra-low gossypol cottonseed: generational stability of the seed-specific phenotype and resumption of terpenoid profile following seed germination. Plant Biology (abstract), Austin, TX. July, 2012
- Urriola, J. and Rathore K.S. (2012) Activity of a rice glutelin promoter in transgenic sorghum. Plant Biology (abstract), Austin, TX. July, 2012
- Joshi, S. Kumar, V., Christensen, S., Jayaprakasha, G., Patil, B., Kolomiets, M. and Rathore, K. S. (2012) A novel, conditional, lesion mimic phenotype in cotton cotyledons due to the expression of an endochitinase gene from Trichoderma virens. Plant Biology (abstract), Austin, TX. July, 2012
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: To improve cottonseed quality, substantial reductions in seed-gossypol levels have been obtained by RNAi-mediated silencing of delta-cadinene synthase gene. From the original and new set of experiments with the silencing construct LCT66, three lines (#66-49B, #66-81 and #66-250) at T7 generation were selected for field trial. As seen in 2009 and 2010, the non-seed tissues from both lines continues to show terpenoids at levels similar to those found in the wild-type plants under both, field and greenhouse conditions in the year 2011. Importantly, the RNAi seeds continued to show the ultra-low gossypol cottonseed (ULGCS) phenotype. These results are very encouraging as they show the inheritance and stability of the ULGCS trait under field conditions over three years. We have screened for and identified several new, ULGCS, single transgene copy lines, including #66-250 from the second round of transformation with LCT66. These will be evaluated for trait stability over several generations. Hundreds of new lines recovered following transformation with artificial miRNA were tested for ULGCS trait, however, the desired phenotype was not obtained using this approach. A new series of transformation is being conducted using an All-cotton construct to obtain a different set of ULGCS lines. All the components of the silencing cassette in the All-cotton construct are from cotton. Thus far ten lines have been transferred to soil from these experiments and several more are in various stages of regeneration. In an extension of our earlier work on AtNPR1-mediated disease resistance, we further tested lines #68L-19 and #68L-20 against Thialaviopsis basicola and two additional, previously untested strains of Fusarium oxysporum f. sp. vasinfectum. Both lines showed significant resistance to these two serious pathogens, including the two new strains tested. These results confirm and extend the broad-spectrum nature of the resistance conferred by NPR1 expression. In the second year of field trial, the NPR1 lines survived the usual biotic and abiotic pressures of field conditions and grew to maturity. We established a reliable and rapid, Agrobacterium-mediated transformation system for sorghum, an important food/feed crop and a potential biofuel plant and published the results in 2011. We have transformed sorghum expressing a rice glutelin promoter-driven reporter gene and have confirmed its endosperm-specific activity in sorghum. The results suggest that it will be a useful promoter for transgenic improvement of grain quality in sorghum. PARTICIPANTS: One undergraduate student, a graduate student and three postdoctoral trainees were trained while working on the projects described. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Gossypol elimination research will have a significant impact on the value of cottonseed because after oil extraction, the meal can then be utilized directly as food or as a feed for animals other than cattle, especially for poultry and aquaculture. Gossypol-free cottonseed will also reduce the cost of processing and production of cottonseed oil. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases and nematodes in cotton production, thus positively impacting the disease-resistance aspect of our cotton improvement program. A rapid and efficient transformation system to introduce transgenes into sorghum is of great value in improving this crop that is not only an important food and feed crop, but is also being considered as a source to generate biofuels. The rice glutelin promoter has proven to be highly endosperm-specific in its activity in sorghum, thus it will serve as an important tool in future projects to improve the quality of grain sorghum.
Publications
- KUMAR, V., CAMPBELL, L. M. & RATHORE K. S. (2011) Rapid recovery- and characterization of transformants following Agrobacterium-mediated T-DNA transfer to sorghum. Plant Cell Tiss. Organ Cult. 104: 137-146.
- MURTHY, K.N.C., JAYAPRAKASHA, G. K., KUMAR, V., RATHORE, K. S. & PATIL, B.S. (2011) Citrus Limonin and its Glucoside Inhibit Colon Adenocarcinoma Cell Proliferation through Apoptosis. J. Agricultural & Food Chem. 59:2314-2323.
- K. S. RATHORE, G. SUNILKUMAR, L. M. CAMPBELL. Cotton plant with seed-specific reduction in gossypol (2011). Patent No.: US 7,999,148.
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: To improve cottonseed quality, substantial reductions in seed-gossypol levels have been obtained by RNAi-mediated silencing of delta-cadinene synthase gene. From the original set of experiments with the silencing construct LCT66, two lines (#66-49B, and #66-81) at T6 generation were selected for field trial. As seen in 2009, the non-seed tissues from both lines continues to show terpenoids at levels similar to those found in the wild-type plants under both, field and greenhouse conditions in the year 2010. Importantly, the RNAi seeds continued to show the ultra-low gossypol cottonseed (ULGCS) phenotype. These results are very encouraging as they show the inheritance and stability of the ULGCS trait even under field conditions. We have also identified several new lines from the second round of transformation with LCT66 that show ULGCS phenoyupe. In addition, we are beginning to recover lines following transformation with artificial miRNA constructs that are yet to be tested for ULGCS trait. A new series of transformation has been initiated using an ALL-COTTON construct to obtain a different set of ULGCS lines. All the components of the silencing cassette in the ALL-COTTON construct are from cotton. The selectable marker gene cassette is on a separate binary vector, thus it can be segregated out in subsequent generations. In a further extension of our earlier work on disease resistance, we exposed AtNPR1-expressing cotton lines #68L-19 and #68L-20 to Thialaviopsis basicola. Both lines showed significant resistance to this serious pathogen. Molecular analysis revealed that the roots of AtNPR1 line exhibit stronger and faster induction of several defense-related genes, including PR1, thaumatin, glucanase, lipoxygenase and chitinase in response to a challenge from T. basicola. Higher-level activities of some of the defense-related genes were further confirmed in the roots of NPR1 lines growing under field conditions. The NPR1 lines survived the usual biotic and abiotic pressures of field conditions and grew to maturity. Results from T. basicola infection assays confirm and extend the broad-spectrum resistance conferred by NPR1 expression. We have established a reliable, Agrobacterium-mediated transformation system for sorghum, an important food, feed crop, and a potential biofuel plant. A manuscript based on this work has been submitted for publication. We are now using this method to obtain transgenic sorghum to characterize glutelin promoter from rice for its endosperm-specific activity in sorghum. Preliminary results are encouraging in that we do observe some seed-specific activity. These results need to be further confirmed by more extensive analysis. If it shows the expected tissue specificity, it will prove to be highly useful promoter for transgenic improvement of grain quality in sorghum. PARTICIPANTS: One undergraduate student, a graduate student and three postdoctoral trainees were trained while working on the projects described. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Gossypol elimination research will have a significant impact on the value of cottonseed because the meal can then be utilized directly as food or as a feed for animals other than cattle, especially for poultry and aquaculture. Gossypol-free cottonseed will also reduce the cost of processing and production of cottonseed oil. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases and nematodes in cotton production, thus positively impacting the disease-resistance aspect of our cotton improvement program. A rapid and efficient transformation system to introduce transgenes into sorghum is of great value in improving this crop that is not only an important food and feed crop, but is also being considered as a source to generate biofuels.
Publications
- PARKHI, V., KUMAR, V., CAMPBELL, L. M., BELL, A. A. & RATHORE K. S. (2010) Expression of Arabidopsis NPR1 in transgenic cotton confers resistance to non-defoliating isolates of Verticillium dahliae but not the defoliating isolates. Journal of Phytopathology 158: 822-825.
- PARKHI, V., KUMAR, V., CAMPBELL, L. M., BELL, A. A., SHAH, J. & RATHORE K. S. (2010) Resistance against various fungal pathogens and reniform nematode in transgenic cotton plants expressing Arabidopsis NPR1. Transgenic Research 19: 959-975.
- DAMAJ, M. B., KUMPATLA, S. P., EMANI, C., BEREMAND, P, D., REDDY, A. S., RATHORE, K. S., BUENROSTRO-NAVA, M. T., CURTIS, I. S., THOMAS, T. L., MIRKOV, T. E. (2010) Sugarcane DIRIGENT and O-METHYLTRANSFERASE promoters confer stem-regulated gene expression in diverse monocots. Planta 231: 1439-1458.
|
Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: To improve cottonseed quality, substantial reductions in seed-gossypol levels have been obtained by RNAi-mediated silencing of delta-cadinene synthase gene. From the original set of transformation experiments with the silencing construct LCT66, two lines (#66-49B, and #66-81) at T5 generation were selected for field trial. Non-seed tissues from both lines continues to show terpenoids, at levels similar to those found in the wild-type plants under both, field and greenhouse conditions. Importantly, the RNAi seeds continued to show the ultra-low gossypol cottonseed (ULGC) phenotype. These results are very encouraging as they show the inheritance and stability of the ULGC trait even under field conditions. Fiber quality, fiber and seed yields, and seed-oil content were higher in the field-grown RNAi plants compared to the wild-type counterparts. Biochemical/molecular analyses were conducted on various parts of a young seedling growing under normal conditions and in seedlings exposed to some biotic challenges to examine the state of terpenoid (gossypol)-based defense response. In response to biotic challenges, the roots of RNAi seedlings are able to launch terpenoid-dependent defense response. Developing embryos from selected RNAi lines were subjected to molecular analysis to detect the presence of dCS siRNA. These lines did show the presence of siRNA in embryos confirming that the low seed-gossypol trait in mature seeds was a result of RNAi against the target gene. siRNA was detected in embryos, mature seeds and the cotyledon portion of a young seedling. Their presence does not seem to interfere with the ability of the root to produce defensive terpenoids. In a further extension of our earlier work on disease resistance, we subjected NPR1-expressing cotton line #68L-19 to Fusarium X reniform nematode complex. The transgenic line showed significant resistance in terms of both plant growth and the number/weight of bolls. The results suggest that NPR1 expression not only confers resistance to Fusarium and reniform nematodes individually, but also to the disease complex. The roots of NPR1-transformants show higher activities of some of the defense-related genes prior to or following the exposure to reniform nematodes. Similar analysis following a challenge with Rhizoctonia solani showed that defense-related transcripts were induced earlier and at higher levels in the roots of AtNPR1-expressing transgenic cotton lines compared to the WT counterparts. These factors may contribute towards their resistance to various pathogens. We continue to work on a project to establish an Agrobacterium-mediated transformation system for sorghum, an important food, feed, and a potential biofuel crop plant. We investigated the parameters related to cocultivation, culture, and regeneration and have been able to obtain transgenic sorghum plants in as little as 2.5 months. Also, our results suggest that CaMV 35S promoter activity in T0 generation is very low during the early stages of development of a transgenic sorghum plant, and is not indicative of the expression level during the later stages of development or in the next generation. PARTICIPANTS: One undergraduate student, a graduate student and four postdoctoral trainees were trained while working on the projects described. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Gossypol elimination research will have a significant impact on the value of cottonseed because the meal can then be utilized directly as food or as a feed for animals other than cattle. Gossypol-free cottonseed will also cut down the cost of processing and production of cottonseed oil. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases and nematodes in cotton production, thus positively impacting the disease-resistance aspect of our cotton improvement program. A rapid and efficient transformation system to introduce transgenes into sorghum will be of great value in improving this crop that is not only an important food and feed crop, but is also being considered as a source to generate biofuels.
Publications
- SUNILKUMAR, G., WAGHELA, S. D., CAMPBELL, L. M. & RATHORE K. S. (2009) Expresion of anti-K99 scFv in transgenic rice tissues and its functional characterization. Transgenic Research 18: 347-360.
- PARKHI, V., KUMAR, V., SUNILKUMAR, G., CAMPBELL, L. M., SINGH, N. K. & RATHORE K. S. (2009) Expression of apoplastically secreted tobacco osmotin in cotton confers drought tolerance. Molecular Breeding 23: 625-639.
- KUMAR, V., PARKHI, V., KENERLEY, C. & RATHORE K. S. (2009) Defense-related gene expression and enzyme activities in transgenic cotton plants expressing an endochitinase gene from Trichoderma virens in response to interaction with Rhizoctonia solani. Planta 230: 277-291.
|
Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: To improve cottonseed quality, substantial reductions in seed-gossypol levels were obtained by RNAi-mediated silencing of δ-cadinene synthase gene. From about 80 independent RNAi lines, 13 lines were selected for initial screening. Seeds from nine of these transgenic cotton lines have now been analyzed for gossypol levels up to T5 generation. Two lines in particular, #66-49B and #66-81, have continued to show low seed-gossypol trait (>98% reduction) for five generations. These results show the inheritance and stability of the low seed-gossypol trait. Levels of gossypol and related terpenoids were also examined in various parts of a young seedling growing under normal conditions and in seedlings exposed to some biotic challenges. The results show that the cotyledon and hypocotyl (two of the largest parts of a dry cottonseed) do not synthesize new terpenoids as the seed germinates, however, the growing root and the new leaves of RNAi seedling are capable of producing normal levels of defensive terpenoids. Moreover, in response to biotic challenges, the roots of RNAi seedlings are able to launch normal, terpenoid-dependent defense response. Importantly, the RNAi lines were no more susceptible to Rhizoctonia solani infection compared to the wild-type plants. In a project to confer disease resistance to cotton, 3 additional NPR1 lines were screened for resistance against F. oxysporum (Fov-11), and Line #68L-16 showed significant resistance to the disease. Two of the NPR1 lines #68L-19 and #68L-20 that had shown significant resistance to F. oxysporum (Fov-11) in earlier studies were further tested for their resistance to various strains of V. dahliae. Both these lines showed significant resistance to a non-defoliating strain (TS-2) of V. dahliae. These two lines were also found to show significant resistance to a different non-defoliating strain (4A-EZ). In a preliminary experiment, we examined NPR1 lines #68L-19 and #68L-20 for their tolerance to reniform nematodes. Both the lines showed significant tolerance, in terms of nematode count at 10-week post-inoculation time point, boll retention per plant, and average boll weight. This is a very exciting result will need to be confirmed. In a project to explore the use of plants to manufacture phytopharmaceuticals, we transformed rice plants for the expression of scFv antibodies against a certain adhesion protein (K99) on the surface of enterotoxigenic K99+ E. coli (ETEC). This ETEC causes severe diarrhea and eventually death in newborn calves. The scFvs are produced in leaves and in the embryo portion of the seed. Additional experiments revealed that endosperm tissue does not produce this antibody and also contains storage proteins of similar weight that interfere with scFv detection by Western blot analysis. ELISA method, however, does not suffer from this interference. A number of physiological and biochemical parameters that are involved in tolerance to drought were examined in two cotton lines expressing the tobacco osmotin. In greenhouse experiments, the transgenic lines were found to be tolerant to drought conditions and suffered lesser yield penalty compared to controls. PARTICIPANTS: One undergraduate student and four postdoctoral trainees were trained while working on the projects described. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Gossypol elimination research will have a significant impact on the value of cottonseed because the meal can then be utilized directly as food or as a feed for animals other than cattle. Gossypol-free cottonseed will also cut down the cost of processing and production of cottonseed oil. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases of cotton thus positively impacting the disease-resistance aspect of our cotton improvement program. The results obtained with osmotin-transformed cotton plants show that this gene may be useful in conferring drought-tolerance trait to cotton plants. If recombinant antibodies against K99 protein can be mass-produced at low cost, these can be used to prevent the development of severe and often deadly infections in neonatal dairy calves.
Publications
- Rathore, K. S., Sunilkumar, G., Cantrell, R. G., Hague, S. & Reding, H.K. (2008) Cotton. In: Compendium of Transgenic Crop Plants, Vol. 7: Transgenic Sugar, Tuber and Fiber Crops, eds. C. Kole and T. C. Hall (Wiley-Blackwell. Chichester, West Sussex, UK), pp. 199- 238.
- Sunilkumar, G., Waghela, S. D., Campbell, L. M. & Rathore, K. S. (2008) Expresion of anti-K99 scFv in transgenic rice tissues and its functional characterization. Transgenic Reseach DOI 10.1007/s11248-008-9223-2.
- Ren, S, Mandadi, K. K., Boedeker, A. L., Rathore, K. S. & McKnight, T. D. (2007) Regulation of telomerase in Arabidopsis by BT2, an apparent target of TELOMERASE ACTIVATOR1. Plant Cell 19: 23-31.
|
Progress 01/01/07 to 12/31/07
Outputs
In the project to improve quality of cottonseed by reducing gossypol from the seed, substantial reductions in seed-gossypol levels were obtained when we utilized a powerful gene silencing technology, RNAi. T1, T2, and T3 generation seeds were obtained from several RNAi lines. Results from terpenoids analyses had shown that seeds from some lines showed as much as 98% reduction in gossypol values. The low seed-gossypol trait was maintained in T3 seeds from several lines. The results show stability of the trait. Molecular analysis was performed on selected lines and their progeny to ascertain transgene integration and copy numbers. These results also show stability and inheritance of the transgenic trait. It was shown earlier that the foliage, other floral organs, and roots in these lines show normal, wild-type levels of gossypol and related terpenoids. On the basis of gossypol values in T3 seeds and the results from the molecular analysis, T3 progeny from nine promising
lines were selected and are being grown in the greenhouse to obtain next generation seeds. The generational studies are intended to ascertain the stability of the trait and to select the best possible lines. The original research publication of 2006 has been followed by publication of two additional review articles in 2007 that describe the impact of this research. In a project to confer disease resistance to cotton, we made crosses between osmotin-expressing and chitinase-expressing (obtained in a previous project) cotton lines in order to stack the two disease-resistance genes. These plants showed some resistance to Fusarium wilt. In a related project, Arabidopsis NPR1 gene was used to transform cotton. NPR1 controls the expression of various PR genes that are involved in plant defense against pathogens and because it acts upstream of PR genes, it is thought to confer a stronger and a more broad-spectrum resistance to diseases. NPR1-expressing cotton plants and their progeny has
been identified and show significant resistance to Fusarium and Verticillium wilts. These plants will be evaluated further for their resistance to other cotton diseases. In a project to explore the use of plants to manufacture phytopharmaceuticals, we transformed rice plants for the expression of scFv antibodies against a certain adhesion protein (K99) on the surface of enterotoxigenic K99+ E. coli (ETEC). This ETEC causes severe diarrhea and eventually death in newborn calves. The antibody produced in the rice leaves was purified and used to examine functionality in in vitro assays. The results show that the rice-produced scFv was functional. Early indications were that the leaves of the rice plants expressed the recombinant scFv while the seeds did not accumulate this antibody. A thorough investigation revealed that the scFvs are produced in the embryo portion of the seed, however, certain factor(s) present in the endosperm portion interfere with the detection. Attempts are underway
to resolve this issue.
Impacts
Gossypol elimination research will have a significant impact on the value of cottonseed because the meal can then be utilized directly as food or as a feed for animals other than cattle. Gossypol-free cottonseed will also cut down the cost of processing and production of cottonseed oil. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases of cotton thus positively impacting the disease-resistance aspect of our cotton improvement program. If recombinant antibodies against K99 protein can be mass-produced at low cost, these can be used to prevent the development of severe and often deadly infections in neonatal dairy calves.
Publications
- RATHORE K. S. (2007) Cotton. In: Biotechnology in Agriculture and Forestry, Vol. 61: Transgenic Crops VI, eds. E. C. Pua and M. R. Davey (Springer-Verlag, Berlin Heidelberg, Germany), pp. 107- 127.
|
Progress 01/01/06 to 12/31/06
Outputs
In the project to improve quality of cottonseed by reducing gossypol from the seed, use of antisense technology provided 75% reduction in gossypol levels in the seed. More substantial reductions in seed-gossypol levels were obtained in another project that utilized a more powerful gene silencing technology, RNAi. T1 and T2 generation seeds were obtained from several RNAi lines. Results from terpenoids analyses show that seeds from some lines show as much as 98% reduction in gossypol values. However, the foliage, other floral organs, and roots in these lines show normal, wild-type levels of gossypol and related terpenoids. The results also show stability and inheritance of the transgenic trait. Several promising lines will be evaluated for 2-3 additional generations under greenhouse conditions to identify lines with the lowest seed-gossypol levels that are stable. The first phase of this project is complete and has been published in 2006. In a project to confer disease
resistance to cotton, we produced over one hundred transgenic plants containing the tobacco osmotin gene. The transgenic plants were evaluated for the transgene expression to identify lines that express the osmotin gene at high levels. The progeny from selected, high transgene expressing lines were examined for the osmotin expression and T2 seeds from homozygous lines were obtained. This generation is being tested for resistance to various fungal diseases. We are also making crosses between osmotin-expressing and chitinase-expressing (obtained in a previous project) cotton lines in order to stack the two disease-resistance genes. In a project to explore the use of plants to manufacture phytopharmaceuticals, we transformed rice plants for the expression of scFv antibodies against a certain adhesion protein (K99) on the surface of enterotoxigenic K99+ E. coli. Although the leaves of the rice plants expressed the recombinant scFv, the seeds produced from these lines did not accumulate
the antibodies. The antibodies produced in the rice leaves will be purified and used to examine functionality in in vitro assays.
Impacts
Gossypol elimination research will have a significant impact on the value of cottonseed because the meal can then be utilized as food or to feed animals other than cattle. Gossypol-free cottonseed will also cut down the cost of processing and production of cottonseed oil. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases of cotton thus positively impacting the disease-resistance aspect of our cotton improvement program. If recombinant antibodies against K99 protein can be mass-produced at low cost, these can be used to prevent the development of severe and often deadly infections in dairy calves.
Publications
- RATHORE, K. S., SUNILKUMAR, G. & CAMPBELL, L. M. (2006) Cotton (Gossypium hirsutum L.) in Methods in Molecular Biology, Vol. 343: Agrobacterium Protocols, 2nd Ed., Vol 1, ed. K. Wang (Humana Press Inc. Totowa, NJ), pp. 267- 279.
- SUNILKUMAR, G., CAMPBELL, L. M., PUCKHABER, L., STIPANOVIC, R. D. & RATHORE K. S. (2006) Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. PNAS 103:18054-18059.
|
Progress 01/01/05 to 12/31/05
Outputs
In the project to improve quality of cottonseed by reducing gossypol from the seed, use of antisense technology provided 75% reduction in gossypol levels in the seed. Another project, utilizing RNAi-based silencing, was initiated in order to obtain a more substantial reduction in seed-gossypol levels. Seeds from the primary RNAi transformants are being analyzed. Results obtained thus far seem to indicate that T1 seeds, on average, show 95% reduction in gossypol values while the foliage of T1 plants show normal, wild-type levels of gossypol and other terpenoids. These results will be confirmed in subsequent generations. Another project to improve the nutritional aspects of cottonseed, using antisense technology to down-regulate delta-12 desaturase to obtain low linoleic/high oleic acid seeds is now complete and has been published. In a disease resistance project in cotton, we produced over one hundred transgenic plants. The transgenic plants were evaluated for the
transgene expression to identify lines that express tobacco osmotin at high levels. The progeny from 10 selected, high transgene expressing lines will be examined for the osmotin expression and will then be tested for resistance to fungal diseases. In a project to explore the use of plants to manufacture phytopharmaceuticals, we transformed rice plants for the expression of scFv antibodies against a certain adhesion protein (K99) on the surface of enterotoxigenic K99+ E. coli. The leaves of the rice plants were analyzed and were found to be expressing the recombinant scFv. Seeds produced from these lines will be examined for the production and accumulation of these antibodies.
Impacts
Gossypol elimination research will have a significant impact on the value of cottonseed because the meal can then be utilized as food or to feed animals other than cattle. Gossypol-free cottonseed will also cut down the cost of processing and production of cottonseed oil. Improvements in the fatty acid profile in cottonseed oil will provide a healthier oil and this will positively impact its value and enhance its competitive position against other oils. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases of cotton thus positively impacting the disease-resistance aspect of our cotton improvement program. If recombinant antibodies against K99 protein can be mass-produced at low cost, these can be used to prevent the development of severe and often deadly infections in dairy calves.
Publications
- SUNILKUMAR, G., CAMPBELL, L. M., MONJUR, H. M., CONNELL J. P., HERNANDEZ, E., REDDY A. S., SMITH C. W. and RATHORE K. S. (2005) A comprehensive study of the use of a homologous promoter in antisense cotton lines exhibiting high seed-oleic acid phenotype. Plant Biotechnology Journal 3: 319-330.
|
Progress 01/01/04 to 12/31/04
Outputs
In a project to improve quality of cottonseed by reducing gossypol from the seed, T3 and T4 seeds from one of the low-gossypol antisense lines continued to show low-gossypol trait (75% reduction). Importantly, the leaves from T3 and T4 seedlings had gossypol levels equivalent to the leaves from non-transgenic controls. In another project to improve the nutritional aspects of cottonseed, we selected four antisense delta-12 desaturase lines that exhibit low linoleic/high oleic acid trait in their seeds. In one of these lines, the low linoleic acid/high oleic acid trait was found to be stable up to T3 generation of seeds. In a disease resistance project in cotton, we produced several hundred transgenic callus lines. Several lines became embryogenic and have begun to regenerate plants. The transgenic plants will be evaluated for the transgene expression to identify lines that express tobacco osmotin at high levels. The progeny from these lines will then be tested for
resistance to fungal diseases. In a project to explore the use of plants to manufacture phytopharmaceuticals, we initiated research to evaluate whether certain adhesion proteins on the surface of enterotoxigenic K99+ E. coli, when produced recombinantly, have the ability to block the attachment of bacteria to calf intestinal cells. The results show that the recombinant K99, does indeed inhibit attachment of the infectious bacteria to host cell receptors.
Impacts
Gossypol elimination research will have a significant impact on the value of cottonseed because the meal can then be utilized as food or to feed animals other than cattle. Gossypol-free cottonseed will also cut down the cost of processing and production of cottonseed oil. Improvements in the fatty acid profile in cottonseed oil will positively impact its value and enhance its competitive position against other oils. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases of cotton thus positively impacting the disease-resistance aspect of our cotton improvement program. If recombinant K99 proteins can be mass produced at low cost, it can be used to prevent the development of severe and often deadly infections in dairy calves.
Publications
- JAY, C. M., BHASKARAN, S., RATHORE, K. S. and WAGHELA, S.D. (2004) Enterotoxigenic K99+ Escherichia coli attachment to host cell receptors inhibited by recombinant pili protein. Veterinary Microbiology 101: 153-160.
|
Progress 01/01/03 to 12/31/03
Outputs
In a project to improve quality of cottonseed by reducing gossypol from the seed, T2 seeds from one of the low-gossypol antisense lines continued to show low-gossypol trait (60% reduction). Importantly, the leaves from T2 seedlings had gossypol levels equivalent to the leaves from non-transgenic controls. In another project to improve the nutritional aspects of cottonseed, we identified six antisense delta-12 desaturase lines that exhibit low linoleic/high oleic acid trait in their seeds. This low linoleic acid/high oleic acid trait has been found to be stable up to T3 generation of seeds. We had identified three transgenic cotton lines expressing chitinase gene from Trichoderma virens at a very high level. T2 progeny from these lines were significantly resistant to Rhizoctonia solani and Alternaria alternata. In a related disease resistance project in cotton we have produced several hundred transgenic callus lines. These will be taken through various stages of tissue
culture before they become embryogenic. A new project has been initiated to explore the use of plants to manufacture phytopharmaceuticals. Two constructs have been made for the expression of scFv-antibodies in plants. These scFvs target E. coli surface proteins, F5 that are involved in the development of severe and often deadly infections in dairy calves.
Impacts
Gossypol elimination research will have a significant impact on the value of cottonseed because the meal can then be utilized as food or to feed animals other than cattle. Gossypol-free cottonseed will also cut down the cost of processing and production of cottonseed oil. Improvements in the fatty acid profile in cottonseed oil will positively impact its value and enhance its competitive position against other oils. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases of cotton thus positively impacting the disease-resistance aspect of our cotton improvement program. Success in producing functional antibodies in plants will allow us to produce important proteins of pharmaceutical importance in more economical ways.
Publications
- EMANI, C., GARCIA, J. M., LOPATA-FINCH, E., POZO, M., URIBE, P., KIM, D-J., SUNILKUMAR, G., COOK, D. R., KENERLEY, C. M. and RATHORE, K. S. (2003) Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens. Plant Biotechnology Journal 1: 321-336.
- RATHORE, K. S., SUNILKUMAR, G. AND MOHR, L. (2003) CaMV 35S promoter-regulated expression of green fluorescent protein in cotton: from transient activity to transgenic plants. Cotton Improvement Conference. 2003 Beltwide Cotton Research Conferences, Nashville, TN.
|
Progress 01/01/02 to 12/31/02
Outputs
In the project to engineer cotton for resistance to phloem feeding insects, three lines were identified that express snow drop lectin at high level. T2 plants from the highest expressing line have been evaluated for resistance against cotton aphids. These plants were found to be resistant to cotton aphid. In a project to improve quality of cottonseed by reducing gossypol from the seed, 62 transgenic cotton lines, with antisense delta-cadinene synthase gene from Gossypium hirsutum driven by two different seed specific promoters, were obtained. T2 seeds from one of the low-gossypol lines continued to show low-gossypol trait. In another project to improve the nutritional aspect of cottonseed, we identified six antisense delta-12 desaturase lines that exhibit low linoleic/high oleic acid trait in their seeds. This low linoleic/high oleic acid trait was also confirmed in T2 generation seeds. We also obtained 82 lines of transgenic cotton plants expressing chitinase gene
from Trichoderma virens. T2 progeny from three of the high chitinase-expressing lines were evaluated for disease resistance against Rhizoctonia solani and Alternaria alternata. Transgenic cotton plants were found to be significantly resistant to these two fungal pathogens of cotton. In an effort to understand expression profile of the CaMV 35S promoter, cotton was transformed with green fluorescent protein gene under the control of this promoter. Results show that during early embryogenesis, this promoter was inactive. Following mid-embryonic stage, the expression of the promoter was constitutive in the remaining stages of growth and development of the plant with varying levels of expression in various tissues. Promoter activity was very low in the endosperm and in the pollen. Developing fiber did exhibit promoter activity although at low level. In the remaining tissues of the cotton plant, the promoter was active at moderate to high levels.
Impacts
Positive results from insect resistance research will have an immediate impact on the host plant resistance aspect of cotton breeding program at TAMU. Gossypol elimination research will have a significant impact on the value of cottonseed because the meal can then be utilized to feed animals other than cattle. Gossypol-free cottonseed will also cut down the cost of processing and production of cottonseed oil. Improvements in the fatty acid profile in cottonseed oil will positively impact its value and enhance its competitive position against other oils. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases of cotton thus positively impacting the disease-resistance aspect of our cotton improvement program. The CaMV 35S promoter work has significance in relation to Bt cotton because the Bt gene in Bollgard Cotton is under the control of the same promoter. Our results have confirmed that the activity of the promoter is very
low in pollen and supports the studies that Bt toxin levels are low in the Bollgard cotton.
Publications
- EMANI, C., SUNILKUMAR, G. and RATHORE, K. S. (2002) Transgene silencing and reactivation in sorghum. Plant Science: 162:181-192.
- SUNILKUMAR, G., MOHR, L., LOPATA-FINCH, E., EMANI, C. and RATHORE, K. S. (2002) Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP. Plant Molecular Biology 50: (3) 463-474.
- SUNILKUMAR, G., CONNELL J. P., SMITH C. W., REDDY A. S., and RATHORE K. S. (2002) Isolation and functional characterization of alpha-globulin promoter from cotton in transgenic cotton, Arabidopsis and tobacco. Transgenic Research 11: 347-359.
|
Progress 01/01/01 to 12/31/01
Outputs
On the project to engineer cotton for resistance to phloem feeding insects, three lines have been identified that express snow drop lectin at high level. T2 plants from the highest expressing line are being evaluated for resistance against whiteflies and aphids. On another project to improve quality of cottonseed by reducing gossypol from the seed, sixty two transgenic cotton lines, with antisense delta-cadinene synthase gene from G. hirsutum driven by two different seed specific promoters, were obtained. T1 seeds from these lines were analyzed and some lines with low seed-gossypol have been identified. T2 seeds from these selected lines will be analyzed to test for further reduction in gossypol levels. We have also obtained and transferred to soil over forty lines of transgenic cotton plants expressing chitinase gene from Trichoderma virens. Of these, more than twenty lines have transgenic chitinase activity higher than twenty fold from basal control activity. T1
progeny from high chitinase expressing lines is being evaluated for disease resistance against various fungal pathogens of cotton. Transgene expression assays on transgenic sorghum plants obtained last year showed that the reporter gene (gusA), but not selectable marker gene (bar), was silenced in T0, T1 and T2 generations. Further analyses revealed that silencing was largely due to transgene methylation.
Impacts
Positive results from insect resistance research will have an immediate impact on the host plant resistance aspect of cotton breeding program at TAMU. Gossypol elimination research will have a significant impact on the value of cottonseed because the meal can then be utilized to feed animals other than cattle. Gossypol-free cottonseed will also cut down the cost of processing and production of cottonseed oil. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases of cotton thus positively impacting the disease-resistance aspect of our cotton improvement program. Our results with transgenic sorghum suggest that transgene methylation can be a serious problem with consistent expression of the introduced genes in sorghum. Means to avoid transgene methylation will significantly improve transgene expression in sorghum.
Publications
- SUNILKUMAR, G. and RATHORE, K. S. 2001. Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration. Molecular Breeding 8:37-52.
- EMANI, C., SUNILKUMAR, G. and RATHORE, K. S. 2002. Transgene silencing and reactivation in sorghum. Plant Science 162:181-192.
- SUNILKUMAR, G., CONNELL J. P., SMITH C. W., REDDY A. S., and RATHORE K. S. 2002. Cotton alpha-globulin promoter: Isolation and functional characterization in transgenic cotton, Arabidopsis and tobacco, Transgenic Research (in press).
|
Progress 01/01/00 to 12/31/00
Outputs
On a project to engineer cotton for resistance to phloem feeding insects, several transgenic plants have tested positive for expression the of snowdrop lectin gene by Western analysis. Seeds have been obtained from some of the plants and the progeny will be evaluated for resistance against whiteflies and aphids. On another project to improve quality of cottonseed by reducing gossypol from the seed, transformation experiments were carried out utilizing antisense delta-cadinene synthase gene from G. hirsutum driven by two different seed specific promoters. Sixty two transgenic plant lines have been moved to soil in the greenhouse and seeds have been obtained from 22 lines. We will analyze seeds from these plants for gossypol levels. We have also characterized one of the seed-specific promoter, alpha-globulin promoter from G. hirsutum, that was used for some of these transformations. The results show that this promoter can drive expression of transgenes in cotton as well
as two other heterologous dicot species during seed development. We have also obtained and transferred to soil over forty lines of transgenic cotton plants expressing chitinase gene from Trichoderma virens. Of these, more than twenty lines have transgenic chitinase activity higher than twenty fold from basal control activity. T1 progeny from these will be tested for disease resistance against various fungal pathogens of cotton. We have been successful in obtaining ten transgenic sorghum plants from two lines. Progeny from one of the lines is being analyzed for transgene expression.
Impacts
Positive results from insect resistance research will have an immediate impact on the host plant resistance aspect of cotton breeding program at TAMU. Gossypol elimination research will have a significant impact on the value of cottonseed because the meal can then be utilized to feed animals other than cattle. Gossypol-free cottonseed will also cut down the cost of processing and production of cottonseed oil. Alpha-globulin promoter from cotton will be very useful in any project to improve seed quality in dicots and also to drive other genes coding for proteins of pharmaceutical value in seed tissue. Disease resistant cotton will significantly cut down the losses suffered from various fungal diseases of cotton thus positively impacting the disease-resistance aspect of our cotton improvement program.
Publications
- GANESAN, S. K. and K. S. RATHORE. 2000. Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration. Submitted to Molecular Breeding.
- GANESAN, S. K., CONNELL J. P., SMITH C. W., REDDY A. S., AND RATHORE K. S. 2000. Cotton alpha-globulin promoter: a seed-specific promoter for dicots. ASPP, 2000 annual meeting, San Diego,CA Plant Biology. Abst. pp 194.
|
Progress 01/01/99 to 12/31/99
Outputs
On a project to engineer cotton for resistance to phloem feeding insects, several transformation experiments were carried out and transgenic plants obtained from these experiments are now being transferred to soil. These transformants carry either the CpTI or the snowdrop lectin (GNA) gene driven by phloem specific promoter. After obtaining seeds from these plants, the progeny will be evaluated for resistance against whiteflies and aphids. On another project to improve quality of cottonseed by reducing gossypol from the seed, transformation experiments were carried out utilizing antisense delta-cadinene synthase gene from G. hirsutum driven by two different seed specific promoters. Putative transgenic plants have been recovered from twenty lines from these experiments. Several have been transferred to soil in greenhouse to obtain seeds. Seeds from these plants will then be analyzed for gossypol levels. We are also characterizing one of the seed-specific promoter,
alpha-globulin promoter from G. hirsutum, that was used for these transformations. The results show that this promoter can drive expression of transgenes in tobacco, Arabidopsis and cotton during seed development. We are also working to develop an efficient transformation system utilizing the gene gun to transform sorghum with the ultimate aim of introducing genes for antifungal proteins to address the problem of grain mold. Work on establishing an efficient transformation procedure for soybean is also underway so that this crop can be utilized for producing recombinant antibodies.
Impacts
Positive results from insect resistance research will have an immediate impact on the host plant resistance aspect of cotton breeding program at TAMU. Gossypol elimination research will have a significant impact on the value of cottonseed because the meal can then be utilized to feed animals other than cattle. Gossypol-free seed will also cut down the cost of processing and production of cottonseed oil.
Publications
- Rathore, K.S. and Stipanovic, R.D. 1999. Analysis of gossypol and related terpenoids in antisense transgenic cotton plants. Proceedings Beltwide Cotton Conferences. Pp. 445.
|
Progress 01/01/98 to 12/31/98
Outputs
On a project to engineer cotton for resistance to phloem feeding insects, we made several binary vector constructs utilizing phloem-specific and constitutive promoters to drive snowdrop lectin (GNA) and cowpea trypsin inhibitor (CptI) genes and have begun to transform cotton with these. On another project to improve quality of cottonseed by reducing gossypol from the seed, we have begun to evaluate transgenic cotton plants that were transformed with antisense constructs of delta-cadinene synthase (a key enzyme involved in the biosynthesis of gossypol) genes from G. arboreum. We have also carried out a new set of transformation experiments utilizing delta-cadinene synthase gene from G. hirsutum. It will take another six months before transgenic plants are recovered from the transformed culture lines. We are also working to develop an efficient transformation system for sorghum with the ultimate aim of introducing genes for antifungal proteins to address the problem of
grain mold. Work on establishing an efficient transformation procedure for soybean is also underway so that this crop can be utilized for producing recombinant antibodies.
Impacts
(N/A)
Publications
- Rathore, K.S. and Stipanovic, R.D. 1998. Evaluation of transgenic approach to reduce gossypol in cottonseed. Plant Physiol. (Supp.) 117:85.
|
|