Progress 05/01/15 to 04/30/17
Outputs
Target Audience:The target audience for this research includes scientists and seed companies with research interests in apomixis and/or haploidembryo development.The PI was a speaker in the Sporogenesis, Meiosis and Apomixis session of The 24th International Congress on Sexual Plant Reproduction. Data from this research was also presented by the Co-PI at the 11th International Congress of Plant Molecular Biology and at the Plant & Animal Genome Conference XXIV and XXV. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?The results of this research have been published in a scientific journal and multiple talks at various plant conferences have been given. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Through the analysis of transgenic rice and maize lines carrying various forms of the PsASGR-BBML transgene, we were able todemonstrate that PsASGR-BBML transgenescanpromote various levels of parthenogenesis in monocot crop species. This discoverysupports thatgenetically engineering apomixis, a naturally occurring mode of asexual reproduction in flowering plants that results in seed formation without the involvement of meiosis or fertilization of the egg, in crop speciesmay be possible using apomictic derived genes. Apomixis in crop species is ahighly desirable trait as seed formed on an apomictic plant contain embryos genetically identical to the mother. Apomixis has the potentialto preserve hybrid vigor in highly productive crop plant genotypes from one generation to the next.Thisresearch also shows the potential of using PsASGR-BBML transgenes as an alternative method for haploid induction in maize, rice and potentially other monocot crop species. The production of double-haploid lines are increasingly being used in commercial breeding programs to quickly produce homozygous imbred lines that can be used to generate high-yeilding, high-quality hybrids. The major goal of determining if the PsASGR-BBML transgene could induce parthenogenesis/haploid seed development in rice and maize was accomplished in the following way. Three different constructs containing various forms of the PsASGR-BBML transgene were created. The gPsASGR-BBMLconstruct contained the PsASGR-BBML promoter, the genomic PsASGR-BBML coding region and native 3' UTR. The cPsASGR-BBML construct contained the PsASGR-BBML promoter, the PsASGR-BBML open reading frame and native 3' UTR. The DD45-gPsASGR-BBML construct contained egg cell-specific promoter DD45/EC1.2 (At2g21740), PsASGR-BBML genomic coding region and native 3' UTR. The three constructs were transformed into rice and17 to 24 independent lines for each contrust was assayed for the ability to induce parthenogenesis/haploid seed productionbased on bulkseed flow cytometry of mature seed. All three constructs had the abilityto induce parthenogenesis, although penetrance of the trait for each constructs varied. Eighty-seven and 89% of the transgenic rice lines containingthe gPsASGR-BBML and DD45-gPsASGR-BBML constructs showed some level of parthenogenesis in mature seed. Only 25% of the transgenic rice lines containingthecPsASGR-BBML construct showed some level ofparthenogenesis in mature seed.While not conclusive, the differencein penetrance between the genomic and cDNA codingconstructs suggestsa potentialgene regulatory regionwithin the introns ofPsASGR-BBML that will need further study. Functional haploid seed production was confirmed inten rice lines.The most sucessful lineproduced four haploid seedlings with the majority of lines producingone to threehaploidseedlings from 25 germinated seed. Non-reduced T1 diploidplants inheriting the transgene continued to produce haploid T2 seed via bulk flow cytometry of mature seed, showing that the trait can be inherited through generations. The gPsASGR-BBML and DD45-gPsASGR-BBML constructs were transformed into maize. Twenty-five independent lines for each construct were assayed for the ability to induce parthenogenesis/haploid seed productionbased on bulk seed flow cytometry of mature seed.Both constructs had the abilityto induce parthenogenesis and create haploid seed in maize, although the penetrance of the constructs varied. Forty-seven and 80% of the transgenicmaize lines containingthe gPsASGR-BBML and DD45-gPsASGR-BBML constructs showed some level ofparthenogenesis viabulk seed flow cytometry of mature seed. Whilemore transgenic plants would need to be produced, the results suggest that the DD45-gPsASGR-BBMLconstructmay work better in multiple monocot species and that promoters could play a critical role in increasing the efficiency of the gPsASGR-BBMLtransgene.Haploid seed production in maize was confirmed forboth constructs. With a limited number of seed germinated, one DD45-gPsASGR-BBML line produced all haploid seedlings. The results of this study conclusively show that the PsASGR-BBML transgenein various forms canpromote parthenogenesis in the monocot crop speciesrice and maize with varying degrees of penetrance.The most successful lines indentified in this study as of yet could not yet be used in agricultural development of engineered apomixis due to seed set.Total seed setfor the variousrice and maize lines showing parthenogenesis/haploid seed production wasstatistically less than seed set fromwild-type or transgene negativelines. Additional research will be needed for fine-tuning the use of this transgene in crop plants for 100% penetrance which can be found in natural apomictic plants. The most successful lines indentified in this studywould behigh enough for efficient double-haploid recovery in agriculture breeding. However,a different expression system for the PsASGR-BBML transgenethe would need to be engineered to inactivate expression of the PsASGR-BBML transgeneonce the desired double-haploid was recovered.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Conner, Joann A., Maricel Podio, and Peggy Ozias-Akins. "Haploid embryo production in rice and maize induced by PsASGR-BBML transgenes." Plant reproduction 30.1 (2017): 41-52.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Conner, J. A., Podio, M, & Ozias-Akins, P. A Conserved Apomict-Derived Babyboom Gene Promotes Parthenogenesis in Multiple Cereals. Plant & Animal Genomes XXII Conference, San Diego, CA January 14-18, 2017. Workshop: Components of Apomixis
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Conner, J. A., Podio, M, & Ozias-Akins, P. Induced Parthenogenesis with an Apomict-Derived Gene. Plant & Animal Genomes XXII Conference, San Diego, CA January 9-13, 2016. Workshop: Components of Apomixis
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Conner, J. A., Induced parthenogenesis and haploid seed production in millet, maize and rice with PsASGR-BBML transgenes. 24th International Congress on Sexual Plant Reproduction, Tucson, AZ March 18-23, 2016
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Ozias-Akins, P., Conner, J. A., & Podio, M. A gene for parthenogenesis from a natural apomict is sufficient for haploid parthenogenesis in sexual grasses. 11th International Congress of Plant Molecular Biology. Iguazu Falls, Brazil Oct 25-30, 2015
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Progress 05/01/15 to 04/30/16
Outputs
Target Audience:The target audience for this research includes scientists and companieswith research interests inapomixis and/or haploid embryo development. The PI was a speaker in the Sporogenesis, Meiosis and Apomixis session of The 24th International Congress on Sexual Plant Reproduction and the preliminary data from this research was also presented at the 11th International Congress of Plant Molecular Biology . Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Preliminary data from this research has been presented to the scientific communityas presentations at the 24th International Congress on Sexual Plant Reproduction and the 11th International Congress of Plant Molecular Biology. What do you plan to do during the next reporting period to accomplish the goals?We willfinish assaying theremaining maize lines by seed flow cytometry to complete the goals of this project and determine the best transgenic lines to continuethe characterization of thePsASGR-BBML transgene .
Impacts
What was accomplished under these goals?
Apomixis is a naturally occurring mode of asexual reproduction in flowering plants that results in seed formation without the involvement of meiosis or fertilization of the egg. Seeds formed on an apomictic plant contain genetically identical embryos, thus the trait has significant potential for preserving hybrid vigor from one generation to the next in highly productive crop plant genotypes. To createapomictic seed,two processes arerequired; apomeiosisto create unreduced embryo sacs that are genetically identical to the mother and parthenogenesisto promote embryoformation without fertilization. The PsASGR-BBML transgene, whichpromotes embryo formation without fertilization in pearl millet, can also induceembryo formation without fertilizationin transgenic maize and rice lines. This resultbrings us closer to engineering apomixis in crop species. In addition, the PsASGR-BBML transgenic lines were sucessful in creating viable haploidplants in both rice and maize lines and therefore could be usedan alternative method for haploid inductionof maize and rice. Doubled-haploid lines are currently being used in many commercial maize breeding programs in North America and Europe. Rice lines weregenerated containing 1) the PsASGR-BBML transgene originally used in millet which contains the native promoter and genomic coding region of the gene, 2) the PsASGR-BBML transgene expressed under the control of the egg-cell specific promoter DD45 from Arabidopsis, and 3) the PsASGR-BBML transgene in cDNA form (without introns) expressed under the native promoter. Lines were assayed for the complete open reading frame of the transgene and 17,24, and 23 independent linesfor each construct were obtained. 76%, 71% and 25% of the independent linesshowed some level of haploid seed production for each transgene construct based on bulk flow cytometric seed screening of ~15 seed per line.These results show that the promoter used to express the transgene did not significantly effect the phenotype. However,as thegenomic constructlines created a more penetrent phenotype of haploid seed production than the cDNA form, a level of RNA regulation for the transgene is suggested. Maize lines were generated containing 1) the PsASGR-BBML transgene originally used in millet which contains the native promoter and genomic coding region of the gene, 2) the PsASGR-BBML transgene expressed under the control of the egg-cell specific promoter DD45 from Arabidopsis. Lines were assayed for the complete open reading frame of the transgene and26 independent linesfor each construct were obtained. We have not yet finished assaying all the lines for haploid seed production; however in the lines we have analyzed, haploid seed production has occured from both constructs. The research to date shows that the PsASGR-BBML transgene can beexpressed in other monocot crop species and retain the same activity found in the natural apomict plant. The PsASGR-BBML transgene promotes parthenogenesis, embryo formation without fertilization.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Induced Parthenogenesis and Haploid Seed Production in Millet, Maize and Rice with PsASGR-�BBML Transgenes
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
A gene for parthenogenesis from a natural apomict is sufficient for haploid parthenogenesis in sexual grasses
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