Progress 09/01/10 to 08/31/13
Outputs
Target Audience: (1) The target audience is plant biotechnologists and plant breeders, particularly those interested in forage crops and transgene containment. (2) Regulatory agencies, who should be aware of the genetic diversity available in Medicago for the mode of plastid inheritance. Changes/Problems: We have found that the A17 x Borung and A17 x Paraggio F2 segregates ~ 25% pigment deficient progeny complicating further genetic analyses. Genetic analyses of plastid inheritance call for crossing different accessions and analyzing a segregating population. Because the A17 and tissue culture responsive 2HA lines are genetically related, we shall focus future efforts on the R108 line, which is also tissue culture responsive and hopefully does not segregate pigment deficient progeny in inter-line crosses. What opportunities for training and professional development has the project provided? Dr. Tarinee Tungsuchat-Huang, a Research Associate, developed the 2HA tissue culture system and conducted plastid transformation in Medicago truncatula. She also supervised the undergraduate students who participated in the project. She is currently employed by Genewiz, Inc., Piscataway, NJ. Mr. Csanad Gurdon, a Ph.D. student in the Plant Biology Graduate Program developed PCR-based Medicago truncatula plastid DNA markers and assembled and annotated plastid genomes from Illumina and SOLiD reads of total genomic DNA. He conducted the plastid inheritance survey in Medicago truncatula and set up the A17 male sterile line with Borung plastids to test plastid inheritance in the F2 of A17 x Paraggio cross. He is expected to graduate in 2014. Ms. Kristina Slivinski, a former undergraduate genetics major who prepared an honors thesis in the laboratory, was employed as a technician during the first year of the project. She constructed Medicago-specific plastid vectors with aurea spectinomycin resistance marker genes. She is currently employed by the Sloan-Kettering Cancer Center in New York running a genomics lab as a technician. Undergraduate students during the semester work for credits and full time during the tree summer month supported by a fellowship. Three undergraduate students were trained through the project. Megan Radler and Kanak Verma developed new dicistronic plastid marker genes for plastid transformation. Ms. Radler is now employed as a technician in a research laboratory at the Sloan-Kettering Cancer Center in New York, with support to take courses towards a PhD degree. Kanak Verma earned Highest Honors in Genetics for her thesis research, and the Department of Genetics Howard C. Passmore Award for Academic Achievement. She is currently enrolled in medical school. Mr. Simone Lovano transformed Medicago truncatula with a nuclear gentamycin resistance gene. He is currently a Plant Biology graduate student at Rutgers. How have the results been disseminated to communities of interest? Dissemination of results to the communities of interest was though publications and presentations at meetings. The publications are listed under products. During the life of the project PD disseminated the results at the following meetings: (1) Plant and Animal Genome Conference XIX, San Diego, CA, January 14-19, 2011. Somatic Cell Genetics Workshop, Invited Speaker. (2) International Conferenc on Plant Transformation Technologies II, Vienna, Austria, February 19-22, 2011. Invited Speaker. (3) Plant-Based Vaccines & Antibodies, 8-10 June 2011, Porto, Portugal, Invited Speaker. (4) Plant and Animal Genome Conference XX, San Diego, CA, January 14-18, 2012, Plant Organellar Genetics Workshop organizer and speaker. (5) 7th Annual Tripartite Meeting of the Americas between the University of Sao Paulo, Rutgers University, and The Ohio State University, Columbus, Ohio, May 31 - June 2, 2012; invited speaker. (6) Annual Project Director’s Meeting for Biotechnology Risk Assessment Grants (BRAG) Program, Riverdale, MD, June 5-6, 2012; speaker (7) 3rd International Symposium on Chloroplast Genomics and Genetic Engineering, New Brunswick, NJ, May 8-12, 2013; Conference Chair and invited speaker. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
(1) Screening the 30 deeply sequenced Medicago lines for mode of plastid inheritance. We have screened a total of ten Medicago truncatula accessions for the mode of plastid inheritance using A17 line as the maternal parent. We have chosen the A17 line as maternal parent because in this background a nuclear male sterility mutation is available avoiding the problem of manually removing the anthers to prevent self-pollination. We have found that plastids are biparentally transmitted in each cross, except one line (Paraggio), in which predominantly maternal plastid inheritance was found. Thus, there is potential to restrict pollen transmission of plastid transgenes by choosing the appropriate genetic background. As part of collaboration, we surveyed eighteen commercial Medicago sativa (alfalfa) cultivars for their mode of plastid inheritance. We confirmed predominantly paternal or biparental plastid inheritance in most of the crosses. However, we have found two exceptional lines transmitting plastids inefficiently both as paternal and maternal parents. This we attribute to the incompatibility of plastids with the hybrid nuclear background that results in the preferential maintenance of the recurrent (RegenSY) chloroplasts. Our finding suggests that containment of transgenic plastids can be obtained by accumulating appropriate nuclear genes required for plastid maintenance. (2) Testing mode of plastid inheritance in the Borung x Paraggio F1 hybrids and in the F2 generation. We developed the genetic tools for the project, a male sterile A17 line with Borung plastids and an F2 population of A17 x Paraggio cross. Development of the alloplasmic A17 line is a new addition to the project causing delay, therefore his part of the project will be completed at a later date. (3) Marking the plastids of the Jemalong 2HA line with a transgenic aurea spectinomycin resistance gene. Most effort was invested in project 3. We developed the tissue culture system to recover transgenic clones by selection for spectinomycin resistance, constructed two sets of plastid transformation vectors (targeting insertions in the trnV/rps12 and trnK/trnH intergenic regions) carrying the spectinomycin resistance (aadA) gene and two variants, an aurea version that yields yellow leaves and one co-expressing a green fluorescence protein (GFP) that is readily detectable under blue light. Experiments of the size that yield hundreds of transplastomic tobacco clones failed to yield any transplastomic Medicago truncatula lines. The biological reason for the recalcitrance of Medicago truncatula to plastid transformation in not known. Because the marker genes first have been tested in tobacco where plastid transformation is routine, we are planning to publish the positive results. (4) Testing the mode of plastid inheritance in reciprocal crosses within the Jemalong 2HA line using the aurea spectinomycin resistance gene as marker. Objective (4) is dependent on achieving plastid transformation in Medicago truncatula. Because we did not obtain transplastomic lines, we could not execute this part of the project. (5) Testing plastid genome recombination in Medicago truncatula containing mixed plastids. Because of the large number of samples, we test only one marker to genotype the plastid DNA (ptDNA). This approach is valid only if no ptDNA recombination occurs when two different plastids are present in a Medicago truncatula cell. Therefore, we developed markers for the entire plastid genome based on whole genome sequencing and excluded plastid genome recombination in the A17 x R108 cross by testing multiple markers.
Publications
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2014
Citation:
Maliga, P. and Tungsuchat-Huang, T. (2014) Plastid transformation in Nicotiana tabacum and Nicotiana sylvestris by biolistic DNA delivery to leaves. In Chloroplast Biotechnology: Methods and Protocols (Maliga, P. ed.) New York: Springer Science+Business Media, pp. in press.
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2014
Citation:
Tungsuchat-Huang, T. and Maliga, P. (2014) Plastid marker gene excision in greenhouse-grown tobacco by Agrobacterium-delivered Cre recombinase In Chloroplast Biotechnology (Maliga, P. ed. New York: Springer Science+Business Media, pp. in press.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Csanad Gurdon and Pal Maliga (2014) Two Distinct Plastid Genome Configurations and Unprecedented Intraspecies Length Variation in the accD Coding Region in Medicago truncatula. DNA Research, submitted.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Csanad Gurdon and Pal Maliga (2013) Hypervariable plastid genomes in Medicago truncatula. Poster Abstract P106, presented at the 3rd International Symposium on Chloroplast Genomics and Genetic Engineering, New Brunswick, NJ, May 12-13, 2013.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Tarinee Tungsuchat-Huang, Kanak Verma, Megan R. Radler and Pal Maliga (2013) Rapid identification of transplastomic clones by GFP expression from dicistronic operons. Poster Abstract P209, presented at the 3rd International Symposium on Chloroplast Genomics and Genetic Engineering, New Brunswick, NJ, May 12-13, 2013.
- Type:
Journal Articles
Status:
Other
Year Published:
2013
Citation:
Brigitta Dudas, Csanad Gurdon, Reka Pesti, Gyorgy Botond Kiss, Barnabas Jenes and Pal Maliga (2014) Survey reveals potential for plastid transgene containment by accumulating incompatibility genes in Medicago sativa L. in preparation
- Type:
Journal Articles
Status:
Other
Year Published:
2014
Citation:
Csanad Gurdon and Pal Maliga (2014) Biparental plastid inheritance in Medicago truncatula yields cells with mixed plastids but no genetic recombination between plastid genomes. In preparation
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Gurdon, Cs. and Maliga, P. Poster P0055. Next generation sequencing of Medicago truncatula plastid genomes. Plant & Animal Genome XX, San Diego, CA, January 14-18, 2012
- Type:
Book Chapters
Status:
Published
Year Published:
2012
Citation:
Maliga, P. (2012) Plastid transformation in flowering plants. In Genomics of Chloroplasts and Mitochondria (Bock, R. and Knoop, V. eds). Springer, pp. 393-414.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Dudas, B., Kiss, G.B., Jenes, B. and Maliga, P. (2012) Spectinomycin resistance mutations in the rrn16 gene are new plastid markers in Medicago sativa. Theor Appl Genet 125, 1517-1523.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: ACTIVITIES: (1) The target for the genetic analyses of plastid inheritance is Paraggio, a cultivar, which showed low levels of plastid transmission as pollen parent. We are now constructing suitable F2 populations and tester lines to test segregation for the maternal and biparental modes of plastid inheritance. (2) The objective of plastid transformation in Medicago truncatula is to provide genetic and phenotypic markers in crosses. The plastid transformation vectors carry the aurea-aadA gene and highly-expressed GFP genes. The transforming DNA is introduced into alfalfa leaves by the biolistic protocol and putative transplastomic clones are selected by spectinomycin resistance. Plastid transformation is in progress. EVENTS: PI in 2012 gave lectures and disseminated information at the following events and locations: (1) Plant and Animal Genome Conference XX, San Diego, CA, January 14-18, 2012, Plant Organellar Genetics Workshop organizer and speaker (2) 7th Annual Tripartite Meeting of the Americas between the University of Sao Paulo, Rutgers University, and The Ohio State University, Columbus, Ohio, May 31 - June 2, 2012; speaker (3) Annual Project Director's Meeting for Biotechnology Risk Assessment Grants (BRAG) Program, Riverdale, MD, June 5-6, 2012; speaker PARTICIPANTS: Mr. Csanad Gurdon, a Ph.D. student in the Plant Biology Graduate Program developed PCR-based plastid DNA markers and assembled and annotated plastid genomes from Illumina and SOLiD reads of total genomic DNA. He now conducts the plastid inheritance study in crosses with cultivar Paraggio, and will screen additional Medicago truncatula lines for plastid inheritance. Dr. Tarinee Tungsuchat-Huang, a Research Associate, took on the supervision of the Medicago truncatula plastid transformation project. Two undergraduate genetics major students contribute to plastid transformation in Medicago truncatula. Ms. Megan Radler successfully tested novel GFP-expressing spectinomycin resistance markers in tobacco. These genes are already used for plastid transformation in Medicago. Ms. Kanak Verma, another undergraduate student, is testing variant kanamycin resistance genes in tobacco, to be used in future plastid transformation vectors. Collaborations PI is collaborating with Dr. Brigitta Dudas at the Agricultural Biotechnology Center, Godollo, Hungary, on a parallel study of plastid inheritance in Medicago sativa. The Maliga laboratory shared information that facilitated the development of plastid markers in Medicago sativa and provided plastid transformation vectors to obtain genetically marked lines. In Godollo, plastid-encoded spectinomycin resistance mutations were isolated in Medicago sativa, which modify restriction endonuclease cleavage sites. These AFLP markers are now utilized as universal DNA markers in a survey on the mode of plastid inheritance. The collaborative effort yielded one joint publication. TARGET AUDIENCES: (1) The target audience is plant biotechnologists and plant breeders, particularly those interested in forage crops and transgene containment. (2) Regulatory agencies, who should be aware of the genetic diversity available in Medicago for the mode of plastid inheritance. PROJECT MODIFICATIONS: Originally, we were planning to screen the 30 deeply sequenced Medicago truncatula lines for their mode of plastid inheritance. We now decided to screen Medicago truncatula ssp. tricycla lines instead, in the hope of capturing more diversity. The seed was obtained from Dr. Stephanie L. Greene, USDA, ARS National Temperate Forage Legume Germplasm Resources Unit, Prosser, WA.
Impacts
The complete plastid genome sequence of Medicago truncatula cultivars Borung, Jemalong 2HA and Paraggio has been deposited in GenBank.
Publications
- Maliga, P. (2012) Plastid transformation in flowering plants. In Genomics of Chloroplasts and Mitchondria (Bock, R. and Knoop, V. eds). Springer, pp. 393-414.
Dudas, B., Kiss, G.B., Jenes, B. and Maliga, P. (2012) Spectinomycin resistance mutations in the rrn16 gene are new plastid markers in Medicago sativa. Theor. Appl. Genet. 125: 1517-1523.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: (1) Excluding plastid genome recombination in Medicago truncatula. Because of the large number of samples, we test only one marker to genotype the ptDNA. This approach is valid only if there is no ptDNA recombination in the mixed plastid population. We therefore developed markers for the entire plastid genome based on whole genome sequencing and excluded plastid genome recombination in the F1/RF1 progeny of the A17 x R108 cross by testing multiple markers around the plastid genome. (2) Study of plastid inheritance in the Borung x Paraggio cross has been initiated. (3) We developed a protocol for spectinomycin selection in cultured Medicago truncatula cells and developed Medicago-specific vectors with the aurea spectinomycin resistance gene. Plastid transformation is in progress. EVENTS: PI gave lectures and disseminated information at the following events and locations: (1) Plant and Animal Genome Conference XIX, San Diego, CA, January 14-19, 2011. Somatic Cell Genetics Workshop, Invited Speaker; (2) International Conferenc on Plant Transformation Technologies II, Vienna, Austria, February 19-22, 2011. Invited Speaker; (3) Plant-Based Vaccines & Antibodies, 8-10 June 2011, Porto, Portugal, Invited Speaker. PARTICIPANTS: Participants Mr. Csanad Gurdon, a Ph.D. student in the Plant Biology Graduate Program developed PCR-based plastid DNA markers and assembled and annotated plastid genomes from Illumina and SOLiD reads of total genomic DNA. He now conducts the plastid inheritance study in the Boring x Paraggio cross, and will screen additional Medicago truncatula lines for plastid inheritance. Ms. Kristina Slivinski is a former undergraduate genetics major who conducted her thesis research in the laboratory testing the aurea spectinomycin resistance marker genes. Kristina is currently employed as a technician. She constructed Medicago-specific plastid vectors, and is in the process of introducing the aurea spectinomycin resistance marker genes into the Medicago truncatula plastid genome. There are two undergraduate genetics major students in the laboratory. Ms. Megan Radler is testing new marker genes for plastid transformation in tobacco. Mr. Simone Lovano transformed M. truncatula with a nuclear gentamycin resistance gene. Collaborations PI is collaborating with Brigitta Dudas at the Agricultural Biotechnology Center, Godollo, Hungary, on a parallel study of plastid inheritance in Medicago sativa. The Maliga laboratory shared information that facilitated the development of plastid markers in Medicago sativa and provided plastid transformation vectors to obtain genetically marked lines. TARGET AUDIENCES: The target audience is plant biotechnologists and plant breeders, particularly those interested in forage crops and transgene containment. PROJECT MODIFICATIONS: (1) Biparental plastid inheritance in Medicago truncatula is very common. We realized that plastid genome recombination in a mixed plastid population can distort the plastid inheritance study, if it is based on testing a single marker. Therefore, we gave priority to a new study that excluded ptDNA recombination in Medicago truncatula crosses.
Impacts
(1) Excluding plastid genome recombination in Medicago truncatula plants with two different plastid types validated testing a single marker for plastid genotyping.
Publications
- No publications reported this period
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