Progress 12/01/23 to 11/30/24
Outputs
Target Audience: 1. Our department greenhouse, where we grow our sorghum plants, participated in the Door's Open Milwaukee Event. Over 1080 guests visited the greenhouse during the 2-day event. We showcased our ongoing sorghum research to the visitors. 2. I presented our research on utilizing Nuclear Male Sterility (NMS) for crop hybrid breeding in my Molecular Genetics class, attended by both graduate and undergraduate students. 3. In collaboration with the UWM research foundation, our patent [Zhao, D. Xin, Z. (2024) Sterile mutant and two-line breeding system, US Patent 12,065,658] has been issued. Several seed companies have begun reaching out to explore potential collaborations. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We provided training in sorghum research to my postdoc Xia Wan, Ph.D. student Uyiosasere Dennis Aigbe, and undergraduate student Hammad Khan. How have the results been disseminated to communities of interest?1. We showcased our ongoing sorghum research to the visitors in the Door's Open Milwaukee Event. Our department greenhouse, where we grow our sorghum plants, participated in the Door's Open Milwaukee Event. Over 1080 guests visited the greenhouse during the 2-day event. 2. Worked with the UWM research foundation, we keep publicizing our Nuclear Male Sterility (NMS)-based two-line system for sorghum hybrid breeding to seed companies and sorghum breeders. 3. Worked with the UWM research foundation, our patent [Zhao, D. Xin, Z. (2024) Sterile mutant and two-line breeding system, US Patent 12,065,658] has been issued. Several seed companies have begun reaching out to explore potential collaborations. What do you plan to do during the next reporting period to accomplish the goals?1. In the next reporting period, we aim at completing experiments in objective 2 and hopefully obtain the bridge plants. 2. Continuously train the postdoc, Ph.D. students, and undergraduate students. 3. Submit a manuscript about characterizing a male sterile mutant in sorghum. 4. In case that we are unable to generate the bridge plants, we will repeat the transformation process, although this will take considerable time to achieve new results. We remain optimistic about the ongoing work.
Impacts
What was accomplished under these goals?
Progress in 2024: Objective 1: Optimize the 3-Component (3C) transgene construct for developing a new nuclear male sterility system in sorghum. In 2024, we continued examining two independent sorghum transgenic lines derived from our first-generation 3C construct. While the pollen killer gene effectively eliminated 50% of the pollen grains in both lines, the seed sorting marker gene did not function as expected. Furthermore, despite extensive troubleshooting, we were unable to detect the transgenes via PCR. As a result, we have decided to discontinue further analysis of these two transgenic lines. To address these issues, we redesigned four new 3C constructs and successfully produced all of them. We have obtained 7 transgenic lines derived from two constructs from Wisconsin Crop Innovation Center (WCIC) at University of Wisconsin-Madison are currently conducting sorghum transformation. We have analyzed these plants and found that three lines carry transgenes and exhibited 50% of non-viable pollen grains. We have also recruited the postdoc Xia Wan, and the Ph.D. student Uyiosasere Dennis Aigbe joined the lab in early 2024. I referenced last year's note to highlight the challenges we previously faced regarding personnel. "We are frustrated with hiring a postdoc research associate. We began our search for a postdoc research associate in 2022 and extended several offers. One candidate who accepted our offer in early April 2023 faced visa difficulties, leading to a prolonged waiting period. In October 2023, we identified another candidate. Additionally, we faced setbacks with Ph.D. students. The Ph.D. student who joined the PD's lab in fall 2022 had to discontinue our Ph.D. program in early 2023 due to health-related issues. A new Ph.D. student expected to join the project in the fall of 2023 was unable to do so due to the failure of obtaining his visa." Objective 2: Generate the bridge plant that produces pure male sterile plants for two-line hybrid sorghum breeding. To generate the bridge plants (3C/3c ms8/ms8) using the transgenic lines derived from our two new 3C constructs, we crossed MS8/ms8 plants with 3C/3c plants. The MS8/ms8 plants were identified by the derived Cleaved Amplified Polymorphic Sequence (dCAPS) analysis, and the presence of transgenes were verified by PCR. The F1 plants with four genotypes (3C/3c MS8/MS8, 3C/3c MS8/ms8, 3c/3c MS8/MS8, and 3c/3c MS8/ms8) are currently growing in the greenhouse. We will conduct dCAPS and PCR genotyping to identify the 3C/3c MS8/ms8 plants. Objective 3: Test the bridge plant in genetically diverse backgrounds. We will initiate experiments in this objective, as soon as we generate the bridge plant.
Publications
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Progress 12/01/22 to 11/30/23
Outputs
Target Audience:1. The PD had a Zoom meeting with S&W Seed Company. Participants in the meeting included a sorghum breeding lead scientist and several sorghum breeders from the S&W Seed Company, IP staff at UWM, and the co-PD. During the meeting, the PD presented our approaches for developing the two-line hybrid sorghum breeding system and updated our progress. 2. I presented our research that employs Nuclear Male Sterility (NMS) for crop hybrid breeding in my Cell Biology and Molecular Genetics classes. These courses were attended by both graduate and undergraduate students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We provided training in sorghum research to my senior Ph.D. student Benjamin Gentile, although his Ph.D. project is unrelated to this project. Benjamin volunteered to perform certain experiments for this project. How have the results been disseminated to communities of interest?1. The PD had a Zoom meeting with S&W Seed Company. Participants in the meeting included a sorghum breeding lead scientist and several sorghum breeders from the S&W Seed Company, IP staff at UWM, and the co-PD. During the meeting, the PD presented our approaches for developing the two-line hybrid sorghum breeding system and updated our progress. 2. The PD attended the National Association of Plant Breeders 2023 Annual Meeting in Greenville, South Carolina. 3. The PD presented our research that employs Nuclear Male Sterility (NMS) for crop hybrid breeding in my Cell Biology and Molecular Genetics classes. These courses were attended by both graduate and undergraduate students. 4. Worked with the UWM research foundation, we publicized our Nuclear Male Sterility (NMS)-based two-line system for sorghum hybrid breeding to seed companies and sorghum breeders. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will continuously optimize the 3-Component (3C) transgene construct. We will generate two to four 3C constructs and generate 3C sorghum transgenic plants accordingly. We plan to introduce about five independent transgenic lines to the ms8 mutant background. We will recruit a postdoc in 2024.
Impacts
What was accomplished under these goals?
Sorghum is the fifth most produced grain crop, and the U.S. is the largest producer in the world. Due to its resilience to abiotic stresses and marginal soils, sorghum is emerging as a promising food, feed, and biofuel crop adapted to low-input conditions. At present, hybrid sorghum breeding relies entirely on the Cytoplasmic Male Sterility (CMS)-based three-line system. Not only is the CMS system complicated and expensive in producing hybrids, but also it limits the efficient exploration of diverse germplasm resources for developing all types of commercially desired hybrids. Nuclear Male Sterility (NMS)-based two-line systems in rice and maize have been shown to be equal or more effective at capturing heterosis as conventional systems; however, no such system has been developed or tested in sorghum. In the two-line breeding system, hybrid seeds are produced between a male-sterile seed parent and a male-fertile pollen parent. In hybrid maize breeding, maize plants produce separate male and female inflorescences; therefore, the complete male sterile lines can be achieved by physically removing tassels. In rice, conditional male sterile lines are used to generate male sterile plants for hybrid seed production under the restrictive environmental condition, and to maintain themselves under the permissive environmental condition. Sorghum plants produce bisexual flowers and sorghum conditional male sterile lines are not found. Thus, the overall goal of this project is to develop the bridge plant which can be used for two-line hybrid breeding in sorghum. If successful, the outcome of our project system will accelerate breeding efficiency in sorghum by simplifying and expediting sorghum breeding process, and by broadening the spectrum of hybrid combinations that can be rapidly evaluated. Such a system would help sorghum breeders achieve better genetic gains and could potentially protect the U.S. sorghum crop from devastating diseases. Therefore, our project will address critically needed agricultural issues. We welcome sorghum breeders to test and improve this novel system in the field. The proposed project will also provide valuable opportunities to incorporate research into educational activities in plant biology via integrating the proposed research with training of undergraduate students and promoting science education for high school students. To achieve our overall goal, we proposed three Objectives: Objective 1: Optimize the 3-Component (3C) transgene construct for developing a new nuclear male sterility system in sorghum. We expect to develop a seed sorting marker in sorghum. Based on this result, we will complete generation of the 3C construct that can rescue the ms8 mutant male fertility, specifically devitalize pollen grains that harbor transgenes, and sort non-transgenic and transgenic sorghum seeds. Objective 2: Generate the bridge plant that produces pure male sterile plants for two-line hybrid sorghum breeding. First, we expect to obtain 10 independent lines of candidate transgenic plants which are hemizygous for the 3C transgenes (3C/3c). These candidate transgenic plants will produce nearly 50% of dead pollen grains and seeds with strong RFP signals. Second, we will generate 5 independent lines of bridge plants (3C/3c ms8/ms8) that are homozygous for the ms8 mutation but hemizygous for the 3C transgenes. The bridge plants will be capable of not only producing 100% pure transgene-free male sterile plants but also maintaining themselves. Objective 3: Test the bridge plant in genetically diverse backgrounds. We expect to generate bridge plants in the backgrounds of five publicly available elite sorghum lines. These bridge plants with different genetic backgrounds produce 100% pure transgene-free male sterile plants, which can be pollinated with any elite sorghum lines to efficiently generate diverse hybrids for selecting desirable hybrid vigor. Even though a small number of seeds may contain the transgenes, they can be culled out by the seed sorting marker. In addition, those bridge plants can maintain themselves from self-pollination. This system can also achieve ideal hybrid vigor for breeding all desired sorghum hybrids, including grain, forage, sweet, feed, bioenergy sorghum, and those with demanding abiotic and biotic stress resilience. Progress in 2023: Objective 1: Optimize the 3-Component (3C) transgene construct for developing a new nuclear male sterility system in sorghum. In 2023, we continued our examination of two independent sorghum transgenic lines derived from our first generation of 3C construct. Although the pollen killer gene can effectively eliminate 50% of pollen grains in these two lines, the seed sorting marker gene did not perform as anticipated. Unexpectedly, we encountered difficulties in detecting transgenes through PCR. We are currently troubleshooting the PCR-based transgene detection process. To address these issues, we have redesigned four new 3C constructs and successfully produced all of them. Wisconsin Crop Innovation Center (WCIC) at University of Wisconsin-Madison are currently conducting sorghum transformation using two of these 3C constructs. We hope that WCIC can solve the transformation problems that we previously encountered. We are frustrated with hiring a postdoc research associate. We began our search for a postdoc research associate in 2022 and extended several offers. One candidate who accepted our offer in early April 2023 faced visa difficulties, leading to a prolonged waiting period. In October 2023, we identified another candidate. Additionally, we faced setbacks with Ph.D. students. The Ph.D. student who joined the PD's lab in fall 2022 had to discontinue our Ph.D. program in early 2023 due to health-related issues. A new Ph.D. student expected to join the project in the fall of 2023 was unable to do so due to the failure of obtaining his visa. Objective 2: Generate the bridge plant that produces pure male sterile plants for two-line hybrid sorghum breeding. We are making attempts to generate the bridge plants (3C/3c ms8/ms8) using the transgenic lines derived from our first generation of 3C construct. Without establishing a reliable PCR method for detecting transgenes, the process will be significantly slowed down or we may not be able to generate the bridge plants. We are optimistic that the transgenic lines from our new 3C constructs will lead to the successful generation of the bridge plants. Objective 3: Test the bridge plant in genetically diverse backgrounds. We have not initiated any experiments for this objective yet, as we first need to generate the bridge plant.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Smith, A., Gentile, B.R., Xin, Z. and Zhao, D. (2023) The effects of heat stress on male reproduction and tillering in Sorghum bicolor. Food and Energy Security 00, e510. https://doi.org/10.1002/fes3.510.
Submitted additional materials for the patent application: Sterile Mutant and Two-line Breeding System (pending, Application Number: PCT/US19/12217)
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Progress 12/01/21 to 11/30/22
Outputs
Target Audience:1. Worked with the UWM research foundation, we publicized our Nuclear Male Sterility (NMS)-based two-line system for sorghum hybrid breeding to seed companies and sorghum breeders. 2. I talked about our molecular genetic approaches that use Nuclear Male Sterility (NMS) for crop hybrid breeding in my Cell Biology and Molecular Genetics classes. Both graduate and undergraduate students took these classes. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We provided trainings for the new Ph.D. student Brandon Sherman and the undergraduate student Breckyn Hockers. Breckyn received the UWM Support for Undergraduate Research Fellows (SURF) twice in 2022. How have the results been disseminated to communities of interest?1. Worked with the UWM research foundation, we publicized our Nuclear Male Sterility (NMS)-based two-line system for sorghum hybrid breeding to seed companies and sorghum breeders. 2. I talked about our molecular genetic approaches that use Nuclear Male Sterility (NMS) for crop hybrid breeding in my Cell Biology and Molecular Genetics classes. Both graduate students undergraduate students took these classes. 3. Planned to attend 2022 National Association of Plant Breeders (NAPB) Annual Meeting. The trip was cancelled due to Covid. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will continuously optimize the 3-Component (3C) transgene construct. We will generate two to four 3C constructs and generate 3C sorghum transgenic plants accordingly. We plan to introduce about five independent transgenic lines to the ms8 mutant background. We will recruit a postdoc in 2023.
Impacts
What was accomplished under these goals?
Sorghum is the fifth most produced grain crop, and the U.S. is the largest producer in the world. Due to its resilience to abiotic stresses and marginal soils, sorghum is emerging as a promising food, feed, and biofuel crop adapted to low-input conditions. At present, hybrid sorghum breeding relies entirely on the Cytoplasmic Male Sterility (CMS)-based three-line system. Not only is the CMS system complicated and expensive in producing hybrids, but also it limits the efficient exploration of diverse germplasm resources for developing all types of commercially desired hybrids. Nuclear Male Sterility (NMS)-based two-line systems in rice and maize have been shown to be equal or more effective at capturing heterosis as conventional systems; however, no such system has been developed or tested in sorghum. In the two-line breeding system, hybrid seeds are produced between a male-sterile seed parent and a male-fertile pollen parent. In hybrid maize breeding, maize plants produce separate male and female inflorescences; therefore, the complete male sterile lines can be achieved by physically removing tassels. In rice, conditional male sterile lines are used to generate male sterile plants for hybrid seed production under the restrictive environmental condition, and to maintain themselves under the permissive environmental condition. Sorghum plants produce bisexual flowers and sorghum conditional male sterile lines are not found. Thus, the overall goal of this project is to develop the bridge plant which can be used for two-line hybrid breeding in sorghum. If successful, the outcome of our project system will accelerate breeding efficiency in sorghum by simplifying and expediting sorghum breeding process, and by broadening the spectrum of hybrid combinations that can be rapidly evaluated. Such a system would help sorghum breeders achieve better genetic gains and could potentially protect the U.S. sorghum crop from devastating diseases. Therefore, our project will address critically needed agricultural issues. We welcome sorghum breeders to test and improve this novel system in the field. The proposed project will also provide valuable opportunities to incorporate research into educational activities in plant biology via integrating the proposed research with training of undergraduate students and promoting science education for high school students. To achieve our overall goal, we proposed three Objectives: Objective 1: Optimize the 3-Component (3C) transgene construct for developing a new nuclear male sterility system in sorghum. We expect to develop a seed sorting marker in sorghum. Based on this result, we will complete generation of the 3C construct that can rescue the ms8 mutant male fertility, specifically devitalize pollen grains that harbor transgenes, and sort non-transgenic and transgenic sorghum seeds. Objective 2: Generate the bridge plant that produces pure male sterile plants for two-line hybrid sorghum breeding. First, we expect to obtain 10 independent lines of candidate transgenic plants which are hemizygous for the 3C transgenes (3C/3c). These candidate transgenic plants will produce nearly 50% of dead pollen grains and seeds with strong RFP signals. Second, we will generate 5 independent lines of bridge plants (3C/3c ms8/ms8) that are homozygous for the ms8 mutation but hemizygous for the 3C transgenes. The bridge plants will be capable of not only producing 100% pure transgene-free male sterile plants but also maintaining themselves. Objective 3: Test the bridge plant in genetically diverse backgrounds. We expect to generate bridge plants in the backgrounds of five publicly available elite sorghum lines. Thesebridge plants with different genetic backgrounds produce 100% pure transgene-free male sterile plants, which can be pollinated with any elite sorghum lines to efficiently generate diverse hybrids for selecting desirable hybrid vigor. Even though a small number of seeds may contain the transgenes, they can be culled out by the seed sorting marker. In addition, those bridge plants can maintain themselves from self-pollination. In 2022, we examined our two independent sorghum transgenic lines resulting from our first generation of 3C construct. In this construct, the seed sorting marker gene does not work as expected; however, the pollen killer gene can kill 50% of pollen grains. We have narrowed down three sublines and are testing its ability in rescuing the ms8 mutant male fertility. In 2022, we made slow progresses for multiple reasons. First, the Wisconsin Crop Innovation Center (WCIC) at University of Wisconsin-Madison failed to generate sorghum transgenic plants from two new 3C constructs. Second, we have a hard time in identifying a qualified postdoc. Third, a Ph.D. student left the PD's lab unexpectedly for a health-related reason. We have redesigned two new 3C constructs and we will work with the WCIC to solve the sorghum transformation issues. A new Ph.D. student joined the PD's lab in fall, 2022. He has been involved in this project. We plan to recruit a postdoc in 2023.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Huang, J., Zhao, L., Malik, S., Gentile, B.R., Xiong, V., Arazi, T., Owen, H.A., Friml, J. and Zhao, D. (2022) Specification of female germline by microRNA orchestrated auxin signaling in Arabidopsis. Nature Communications 13 (1): 6960. doi: 10.1038/s41467-022-34723-6.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Malik, S. and Zhao, D. (2022) Epigenetic regulation of heat stress in plant male reproduction. Frontiers in Plant Science 13: 826473. doi: 10.3389/fpls.2022.826473.
- Type:
Books
Status:
Published
Year Published:
2022
Citation:
Xu, M., Pedmale, U., Zhao, D., Song, J., Wu, G., and Yamaguchi, N. (Eds). E-book: Epigenetics in Plant Development. Frontiers in Plant Science (2022). ISBN 978-2-88974-727-6, doi: 10.3389/978-2-88974-727-6.
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