Progress 06/01/14 to 01/31/15
Outputs
Target Audience:A Sorghum Doubled Haploid Breeding system will revolutionize sorghum crop improvement world-wide. Sorghum is known for its drought and heat tolerance and is grown in many arid and semi-arid regions of the world. This includes countries in North and South America, Africa, Asia, and Europe. Any public and/or private breeding program in these regions will benefit from SDH technology. Climate change seems likely to increase the planting of sorghum in many areas of the world. Global sorghum production currently averages 60 million metric tons annually. It is a well-known fact that production of this versatile crop has not reached its full potential. There is much room for the sorghum industry to grow. It is likely that SDH technology will be deployed in the United States first before it is implemented internationally. Sorghum is the 3rd largest cereal crop in the United States, with an annual value recently estimated at $1.6 billion. In the United States today, there are 20 public and private breeding entities engaging in sorghum crop improvement. Breeders, geneticists, and business leaders within these programs are target audiences. We also plan to make policymakers, farmer organizations and other public sector stakeholders aware of the success of the SDH effort, so that sorghum improvement efforts can be seen with a new appreciation of the progress that it will enable. Our target audiences during Phase I were public and private breeding programs critical to the success of our initial research. These included breeders and geneticists at Kansas State Univesity and also a few key people in private sorghum breeding companies. Toward the end of Phase I, we extended this reach to a few private seed companies as well as a few key food companies who have a strategic interest in advancing sorghum. After our SDH breeding system is developed in the USA, we will tap additional potential beyond the U.S. borders. There are approximately 15 public and private entities that are currently engaged in international sorghum breeding and genetics. Furthermore, international NGOs in the food and agriculture sector with a stake in improving the lives of small holder farmers in developing nations would have interest in this technology. Finally, anyone with an interest in the future of food and agriculture, a safe and affordable food supply, and environmentally friendly biofuels, biomaterials, and plant fiber should be considered a target audience. This includes policy makers and anyone participating in the supply chain from producers to consumers. Anyone who eats food has a stake in the success of this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?HPI's Phase I project has provided training opportunitiesfor all team members, including technicians at HPI and KSU. These opportunities included: Developing a protocol for regenerating new roots on young and mature field grown plants. Harvesting and preparing regenerated roots for root tip analysis. Squashing prepared roots for chromosome counts. Evaluation of field plants for signs of haploidy Field and Greenhouse breeding/crossing methods None of these methods were previously practiced in HPI, and this training has strengthened the company's core capacities significantly. How have the results been disseminated to communities of interest?With the discovery of HIP1, we have been cautious about disseminating our preliminary results in detail. However, HPI has made key industry partners aware ofour progress. These communications have been extended primarily through conference calls with individual companies, including private seed companies in the USA and Europe. We have received feedback that these companies are interested in becoming customers and/or joining HPI on some aspects of development and commercialization after Phase II is complete. By protecting the identity of HIP1 and other HIPs as we discover them, we aim to preserve patentability, and once the haploid inducing properties are fully confirmed, we plan to protect this invention. It is becoming known throughout the sorghum breeding community that HPI has had some success with discovering a Haploid Inducing Pollinator and building a haploid breeding system around it. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
On December 4, 2014, HPI discovered the first haploid sorghum progeny, designated DH2831-1. This haploid arose from a cross between a Haploid Inducing Pollinator ("HIP") now designated HIP1 and an A-line (male sterile line), P9511. More than 12 individual root tip cells from DH2831-1 were observed and photographed showing only 10 chromosomes. No cells were observed with more than 10 chromosomes. These haploid cells came from multiple roots that were regenerated on field grown plants. HIP1 is a photoperiod-sensitive, white seeded durra with long awns. Careful observation by KSU collaborators, Drs. Tesso and Perumal confirmed that haploids were present at a rate of 9-12 percent among the progeny of crosses between HIP1 and 3 different A-lines (P9511, KS116 and KS118). A second year of field trials was planned and implemented during Phase I to expand the hunt for useful pollinators. 150 new, diverse, Sorghum bicolor lines were strategically identified for the 2nd year of HIP-hunting crosses. These new males were planted on June 9-10th of 2014 for crossing with two of the same male sterile females as before. In late August 2014, these crosses were successfully performed. Progeny seeds have been harvested, cleaned and prepared for non-destructive physical characterization using high-throughput sorting. These seeds will be planted in early summer of 2015 and evaluated for the presence of haploids through the fall growing season. From the onset of this SDH project, HPI was open to testing and evaluating non-sorghum types as potential haploid inducers, including corn and other grasses. This approach would be labor intensive and require a different scientific plan. For example, embryo rescue would likely be necessary to obtain a viable haploid plant, and cytogenetic analysis and microscopic observation of pollinated flowers would be required to determine the fate of alien pollen chromosomes (Objectives 2.1 and 2.2 from Phase I). Each would require substantially more labor and resources. Since it has been shown that viable haploid seeds are formed in crosses using some S. bicolor pollinators, it is no longer necessary to pursue this alternative approach. Objective 2.3. Produce doubled haploid plants. An adequate supply of haploid seedlings is required to test and develop an efficient method for producing doubled haploid plants. This was not possible in Phase I due to the timing of the discovery of HIP1. In future rounds of identifying and characterizing haploid plants, HPI plans to propagate haploids vegetatively via tillering to obtain small plants that could be used to test conditions for chromosome doubling. If sufficient haploid seedlings are obtained from the second round of HIP1 crosses, some of them will be retained for testing chromosome-doubling methods. This objective will be addressed Post Phase I. HPI successfully completed the USDA/SBIR CAP1 program to establish a comprehensive commercialization plan for our SDH technology. Currently, three different business models are being carefully considered for generating revenue from the SDH breeding system. Intellectual property strengthens HPI's competitive position in all three of these models. They are: Model 1: Produce SDHs on a Fee for Service Basis; No IP Claims on Customers' Materials Model 2: License Haploid Inducing Pollinators (HIPs) and other protected components of the SDH system; Licensee Produces SDHs in Accordance with License Terms. License could be non-exclusive or exclusive within fields of use. Model 3:Offer SDH Technology and Production System for Sale. Furthermore, HPI submitted a SBIR Phase II proposal in February 2015. Phase II support will allow HPI to build on Phase I success by developing a robust system for producing doubled haploids and using them to breed sorghum. To be useful, the SDH breeding system must embody several key features. 1) The system must allow a wide range of sorghum germplasm (ideally, all sorghum germplasm) to be pollinated using an easily propagated haploid-inducing pollinator (HIP), resulting in a reliably high frequency of viable sorghum haploid seeds. Ideally, this would be accomplished using a single HIP, but a small set of HIPs that are each compatible with a well-defined subset of sorghum germplasm would be acceptable. 2) For easy use in a commercial setting, the SDH breeding system must include an efficient method for segregating haploid seeds away from their diploid siblings. Ideally, the feature that permits rapid identification and segregation of haploids should be inherently a reflection of haploidy, and independent of the genotype of the embryo donor line. 3) The resulting haploid seeds must germinate efficiently to yield viable haploid seedlings that can be converted at high efficiency to doubled haploids. 4) The doubled haploid plants must produce seeds at a high rate, so that HPI can deliver a sufficient number of seeds to enable customers to realize the full benefits of using the DH method. 5) The logistics of the system need to be simple, with minimal requirements of time, labor and specialized facilities. HPI's SBIR Phase II program will be aimed at delivering the knowledge and tools necessary to build such a robust SDH breeding system.
Publications
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Progress 06/01/14 to 01/31/15
Outputs
Target Audience:
Nothing Reported
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?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? HPI will continue to screen for Haploid Inducing Pollinators (HIPs) and will begin to genetically characterize the newly discovered HIP1. Seeds from the second round of crosses will be prepared for high throughput seed sorting for Summer 2015 planting. Furthermore, we will continue to plan for future experiments to screen for additional HIPs.
Impacts
What was accomplished under these goals?
Executive Summary Sorghum is a drought and heat tolerant crop that can help meet global needs for food, feed, and fuel. However, sorghum lags behind other crops in making progress in breeding, yield and trait improvement. Heartland Plant Innovations is putting sorghum improvement on a new trajectory by developing a robust system for producing and utilizing Sorghum Doubled Haploids (SDHs). Developing a SDH production system will offer a quicker route to new, improved hybrids with increased yields for farmers. HPI's goal is to discover and use pollinators that induce formation of haploid embryos and viable haploid seeds in diverse lines of sorghum. This approach has been successfully applied in most species where it has been attempted, including maize, wheat, and barley. Working with collaborators, HPI has been conducting research under Phase I to: 1) Confirm haploid-inducing pollinators (HIPs) identified in an initial experiment and determine the genetic diversity of these lines; 2) Plan and initiate a 2nd year of field trials to expand screening for useful pollinators. Research conducted Since June 2014 and the beginning of Phase I funding, HPI has been observing and evaluating progeny plants that are located on 5 acres of KSU's north Agronomy farm. These progeny were derived from crosses between approximately 140 diverse males and four different male-sterile lines. This experiment is described in more detail below. This 5 acre plot of land represents approximately 130,000 progeny that were observed 2-3 times throughout the growing season. Plants that expressed sterile inflorescences as well as various recessive traits, including awnless, juicy mid-rib, and tan plant type, as well as reduced plant stature and vigor were tagged for further evaluation. These physical plant characteristics are all possible indicators of haploidy. Candidate haploid plants were then subjected to root tip squashing and chromosome counting, which revealed if any HIPs) are present in this first round of screening. Diploid plants will have 20 chromosomes and haploid plants will have 10. Research results and potential commercial application Preliminary research results are that HPI has found 1 HIP. This male parent produced haploid progeny at an estimated rate of 9-12 percent across multiple females. Further testing will be required to confirm these preliminary findings. Once this HIP is confirmed, it will become the target of future studies. This HIP and additional HIPs found in the coming season will provide the foundation for developing a commercial SDH breeding system. Sorghum breeders who are working to deliver new varieties to farmers will greatly benefit from incorporating SDH lines into their programs. SDH lines could be used to accelerate traditional breeding, or to rapidly introgress high-value traits for resistance to diseases, insects or abiotic stress. Success with this sorghum DH research will permit expansion of HPI's business to include as customers sorghum breeders around the world. HPI expects to market production of SDHs on demand, or to partner with others in making DH technology available for customers to use. Research conducted prior to Phase I funding Year 1 Experiment (Manhattan and Hays, Kansas; June 2013 - May 31, 2014) Four male-sterile A-lines of sorghum that carry recessive traits such as tan plant type, white-seeded, awned, liguleless and brown mid-rib (BMR) were pollinated using a group of publicly-available males that carry dominant wild-type alleles for these same traits. These crosses were conducted in the field. The males were chosen from a genetically diverse panel that has been assembled by public sorghum breeders. Progeny seeds from these crosses were harvested in late 2013. Not all of the possible crosses were successful, however a sufficient number of crosses succeeded for the start of Phase I experiments. Also, a subset of crosses from the Year 1 Experiment was re-planted in winter nurseries in Mexico during the fall of 2013. These progeny seeds were harvested and prepared for planting. Progeny from field and winter nursery crossing was replanted and examined for evidence of haploidy in three ways: 1) non-destructive physical characterization of seeds prior to germination; 2) visual inspection of young seedlings in the field at early stage of development; 3) visual inspection of mature plants in the field. Automated high-throughput methods of seed screening were used to identify a sub-population of progeny seeds that exhibit likely indicators of haploidy. Information about seeds will be recorded, and after germination, the characteristics of seedlings or plants will be reverse-correlated with seed properties. Seed characteristics that serve as reliable indicators of haploidy would be highly valuable as HPI goes about developing a useful system for producing and using SDH lines. Research conducted under Phase I funding Goal 1: Confirm haploid-inducing pollinators identified in an initial experiment and determine the genetic diversity of these lines Directly following the non-destructive physical characterization of seeds, HPI worked with cooperators at Kansas State University to plant all the progeny seeds from field and winter nursery crosses. Specifically, HPI worked with KSU sorghum breeders, to plant these seeds on 5 acres of land on the KSU North Farm in Manhattan, KS. Planting occurred on June 18-19th, 2014. Three different sets of DH lines were planted across the 5-acre field. Beginning in late July 2014, HPI consultants observed field plants weekly evaluating each plant at different stages of development. This evaluation identified approximately 90 progeny plants that were uprooted from the field. These progeny plants represented 50 different males that could be candidate Haploid Inducing Pollinators. All 50 candidate haploid inducing pollinators were planted in 2 winter nursery locations for further evaluation. These uprooted plants were processed for root tip evaluation and chromosome counts. HPI conducted this root analysis after intense training with a KSU scientist in the Wheat Genetics and Genomics Resource Center who specializes karyotype analysis. The methods and protocols for sorghum were tested in the months leading up to field evaluations. Random sorghum lines grown in pots the greenhouse were used for these tests. The ideal root tip squash reveals one or more cells with chromosomes visible for counting. Diploids have 20 chromosomes, while haploids will have 10. On December 4, 2014, HPI discovered its first haploid progeny seed. The Haploid Inducing Pollinator (now known as HIP1) that fathered these seeds will become the target of future studies. Goal 2: Plan and initiate a 2nd year of field trials to expand screening for useful pollinators A second round of new, diverse, males with dominant traits were strategically identified for the 2nd year of field trials. These new males were planted on June 9-10th and will undergo screening for useful pollinators. The same male-sterile A-lines with recessive traits from the 1st experiment were planted alongside these new males. In late August, these male and female plants were successfully crossed. This trial produced a second set of progeny seeds that have been harvested, cleaned and prepared for non-destructive physical characterization with high-throughput sorting. These seeds will be planted in early summer of 2015 and evaluated for haploidy through the fall growing season. Both diploid and haploid progeny from HIP1 will be of interest for further study. Diploids will be evaluated for transmission of the HIP characteristic. Haploids will be counted and documented via chromosome counting. Some haploids will become the subjects for developing methods for chromosome doubling in sorghum.
Publications
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