SOYBEAN DOUBLED HAPLOID TECHNOLOGY BY ANDROGENESIS

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 1012879
• Project Start Date: Jul 1, 2017
• Project End Date: Feb 25, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
ASU-College of Agriculture

Non Technical Summary
This project is aimed at the development of an enabling technology to advance the pace of soybean breeding and genetics: androgenesis, the development of a plant directly from the male gamete. This is typically accomplished in plants by anther culture or isolated microspore culture. In soybean, only a few genotypes have been observed to generate plants from anther culture, at very low frequencies (less than 2%). We have been screening soybean anther cultures from three genotypes for putative gametic calli, and testing the hypothesis that technical advances made in androgenesis from other plant species may be useful for promoting soybean androgenesis. This strategy identified a number of factors that promote the formation of putative gametic calli, such as nitrogen starvation, use of high levels of 2,4-D, 10°C/8°C (day/night) for 3 days pretreatment of donor plants, and 11°C initial incubation temperature. We have obtained one of the "responsive" genotypes, IAS-5, reported to produce embryos from anther cultures at up to 2% frequency. We propose to test IAS-5 using the best reported protocol vs. our best protocol. If our protocol has any merit, we should obtain significantly more than 2% embryos from culture. Assuming IAS-5 does respond as previously reported, or better, we will continue to test androgenic pyramidal triggers to optimize the system. We will continue to evaluate isolated microspore culture for sustained cell divisions (proliferation of haploid calli) or embryo production. We will assess the ploidy and/or origin of proliferating cultures to validate haploid production. A reliable system for producing haploid or doubled haploid plants of soybean will have a great impact on the soybean industry and academia.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1820	1050	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1820 - Soybean;

Field Of Science
1050 - Developmental biology;

Keywords

soybean

androgenesis

haploids

anther culture

microspore culture

Goals / Objectives
The overall research goal of this project is to increase soybean production by reducing the time and cost of soybean improvement, in order to address global food security and regional climate variability. This will be accomplished through the development and deployment of an androgenetic DH platform for soybean in order to identify new traits quickly and generate new soybean crop improvement tools.The specific research objectives needed to accomplish this goal include the following:Objective 1:Screen additional soybean genotypes for responsiveness to the protocol.Only certain genotypes are responsive to androgenesis, even in "responsive" plant species. We plan to test IAS-5, reported to produce up to 2% embryogenic anther cultures (Moraes et al. 2004), which we recently obtained through USDA-GRIN.Objective 2:Continue to develop a protocol that promotes soybean PGC formation. There are additional androgenetic stressors remaining to be tested in soybean.Objective 3:Establish isolated microspore culture for soybean using the anther culture protocol.This greatly improves the opportunity for gametic - rather than somatic - tissue response.Objective 4:Test the transient expression of embryo-identity genes in soybean microspore cultures as a means to stimulate plant regeneration.It is challenging to recover plants from legume androgenetic systems, even when embryos are initiated. Transient expression of embryo-identity genes should stimulate the embryo formation and plant development from soybean androgenetic cultures.Objective 5:Confirm the recovery of haploid tissues and/or plants.We can verify that the androgenesis protocol is actually successful or efficacious only by proliferating haploid tissues or recovering haploid plants that can be tested for ploidy level.

Project Methods
From the previous work performed at Arkansas State University (Garda et al. 2015, 2016), we have identified several potential factors for optimization of soybean androgenesis: Floral bud stage-We have been using buds 2.5 - 3.5 mm in length (early uninucleate to early binucleate microspores). Pretreatment temperatures-We found the use of 10°C/8°C (day/night) for 3 days shock prior to harvesting the buds was superior to 4°C overnight shock for promoting PGCs. Incubation temperature-We found that incubation of anthers at 11°C for 30 days in the dark to be superior to 4, 18, 25 or 33°C incubation for PGC formation. High Auxin-We found culture media containing 40 mg/L 2,4-D to be superior to 0, 10 or 20 mg/L 2,4-D for promoting PGCs. Nitrogen Starvation-We found culture media containing Nitsch and Nitsch (1969) major salts to be superior to full-strength BABI major salts.Objective 1: Our previous results and the literature suggested that the ability to produce plants from anther cultures of soybean may be genotype-dependent (Garda et al. 2015, 2016). We used Williams82, Thorne and Jack soybeans in our previous experiments, all three of which gave putative gametic responses but clearly defined embryos were not formed. One genotype that has been reported to produce androgenetic embryos from soybean anther cultures is 'IAS-5' (Kaltchuk-Santos et al. 1997, Moraes et al. 2004). Recently the USDA Germplasm Resources Information Network (GRIN) added a deposit of 'IAS-5,' which we have obtained. Use of IAS-5 should permit us to obtain embryos from anther cultures and thus prove the concept of our technology.? IAS-5 Experiment: We will compare our protocol (above) against the protocol of Moraes et al. 2004. They found no effect of pretreatment temperatures, so we will compare no pretreatment to our 10°C/8°C (day/night) for 3 days shock. They used 25°C continuous, which we will compare to our 11°C initial incubation temperature. They used B5 Long basal medium with Yeung's amino acid supplements, which we will compare to our modified BABI (also a B5 derivative). They used 2 mg/L 2,4-D + 0.5 mg/L BA as the hormone treatment, which we will compare to 0 (control) and 40 mg/L 2,4-D. They used 9% sucrose, which we will compare to our 2% sucrose + 2% sorbitol treatment. They did not use nitrogen starvation, which we will compare to our nitrogen starvation treatment. If the protocol we have developed has any value, then we should obtain significantly more than the 2% embryogenic anther cultures reported by Moraes et al. 2004, and we should have comparable embryo-like structures available to determine ploidy and/or origin (see objective 5).Objective 2: Assuming the IAS-5 experiment described above is successful, a continuing priority is to further investigate leads for protocol optimization generated during our initial experiments.? Auxin Experiment: Our previous results indicated that high auxin in the form of 40 mg/L 2,4-D promoted putative gametic response (Garda et al. 2015); lower concentrations of 2,4-D did not appear beneficial. However, higher concentrations of 2,4-D need to be tested. Also, because the Cicer anther culture system responded to Picloram as the auxin source in combination with BA (Panchangam et al. 2014), we will test the following 8 treatments: 40 mg/L 2,4-D (control); 70 mg/L 2,4-D; 100 mg/L 2,4-D; 40 mg/l 2,4-D + 0.1 mg/L BA; 0.5 mg/L Picloram; 5 mg/L Picloram; 50 mg/L Picloram; 5 mg/L Picloram + 0.1 mg/L BA.? Inorganic Nitrogen Experiment: In our previous experiments, putative gametic responses were promoted by the substitution of Nitsch and Nitsch Major salts for the BABI Major salts formula (Garda et al. 2015; see Table 1, Figs. 1 and 2). This substitution represented a reduction in nitrate from 29 mM (BABI) to 18 mM (NN), with little change in ammonium concentration (8-9 mM). It is not clear whether soybean gametic tissues are responding to nitrate reduction, total nitrogen starvation, or a specific ratio of nitrate to ammonium. We make the assumption that 50 mM nitrogen is the maximum needed, and that less than 10 mM nitrogen is probably too little. Accordingly, we will test the following 11 treatments: BABI (original control); NN (new control); 1.5x NN; 0.5x NN; 40:10 nitrate:ammonium (FN medium, Finer and Nagasawa 1988); 30:10; 20:10; 10:10; 24:6; 15:5; 10:5.? pH Experiment: Santarem et al. (1997) demonstrated that pH has a strong impact on soybean somatic embryogenesis. We will compare pH 5.8 (control) to pH 7.0.Objective 3: A logical extension of the anther culture system is to adapt it to the culture of isolated microspores. One drawback with anther cultures of soybean is the generation of somatic calli as well as gametic calli or plants (Kaltchuk-Santos et al. 1997). Microspores, if sufficiently clean and pure, greatly reduce the occurrence of somatic calli and plants in culture (Rodrigues et al. 2006). We will compare cultures of anthers with isolated microspores using the protocol of Rodrigues et al. (2006) as our initial control, and adapting portions of our anther culture protocol to date to that system. If clean microspore cultures can be established, and if sustained cell divisions occur, then we will be able to proliferate tissues to test for ploidy level (with or without plant regeneration).Objective 4: Transient expression of embryo-identity genes such as BABY BOOM has been tested in cacao to stimulate embryogenesis in cultured tissues (Florez et al. 2015); transient expression provided moderate stimulation of somatic embryogenesis which resulted in plant recovery, whereas stable transformation with cacao BABY BOOM, leading to ectopic expression of this transcription factor, inhibited plant development from the embryos. In a further effort to recover plants from our culture systems, we will use transient expression of embryo-identity genes in our microspore cultures. Both BABY BOOM (BBM; Gordon-Kamm et al. 2010) and EMBRYOMAKER (EMK; Tsuwamoto et al. 2010) are examples of AP-2 type transcription factors associated with transition from vegetative to embryogenic development. EMK is of special interest to us because it was identified initially through screening of gene expression during androgenesis of isolated microspores of Brassica napus. We propose to use both BBM and EMK in the forms present in Arabidopsis (AtEMK and AtBBM), in the forms of the Arabidopsis counterparts but with both synthesized for soybean preferred codon usage (At/GmBBM and At/GmEMK), and their native soybean forms (GmBBM and GmEMK), all driven by the soybean polyubiquitin constitutive promoter (Gmubi; Hernandez-Garcia et al. 2009). Transformation will be accomplished using particle bombardment with pGmubi as the expression vector. Peak transient expression occurs within 24-72 h post-cocultivation or -bombardment.If embryos are induced, the frequency of embryo formation will be used to optimize the bombardmentconditions. If no or very few embryos are induced, we will use a green fluorescent protein (gfp) gene in the expression vector (e.g., as per Lindbo 2007) in order to visualize where and when transient expression is occurring, and we will use that strategy to optimize bombardment conditions.Objective 5: A high priority is to recover plants from our culture system(s) as a proof of concept for this technology.We will treat the microspore-derived embryos as somatic embryo cultures following the protocols outlined by Samoylov et al. (1998) to recover plants. We will utilize SSR markers described by Rodrigues et al. (2004) and flow cytometry to determine ploidy level and zygosity of recovered plants.

Progress 07/01/17 to 02/25/21

Outputs
Target Audience:This project is aimed at development and validation of an enabling technology: Soybean doubled haploids via androgenesis. Our target audiences include soybean geneticists and breeders, and legume biotechnologists. Earlier in the project a dissertation was completed. This year a thesis was completed, two technical progress reports were completed, and two peer reviewed articles were accepted forpublication in appropriatejournals. Changes/Problems:The objective related to transient expression of embryo-identity transcription factors was postponed. Our collaborators at a major American agricultural chemical and seed company are experts and the patent holders on these embryo-identity transcription factors, while they have been able to optimize the system for maize somatic embryogenesis they have not been able to achieve the same reliability for soybean or other dicots. We reproducibly obtain microspore embryos in culture, so induction is not the biggest issue. Instead, we shiftedfocus on embryo conversion to plants. What opportunities for training and professional development has the project provided?A total of sixundergraduate students have performedresearch internships with training in microscopy, media preparation, sterile technique, dissection of floral bud parts, data collection and analysis, and soybean reproductive biology. The undergraduate students are in animal sciences or health sciences. One graduate student completed a doctoral dissertation in Molecular Biosciences. One graduate student completed a master's degree in Plant Sciences and a secondmaster's student completed a degree in Molecular Biosciences. The graduate students presented research posters at the annual meetings of the Society for In Vitro Biology and at state and local venues. One master'sstudent completed a summer internship at Corteva Agriscience. How have the results been disseminated to communities of interest?Results have been disseminated through poster presentations at local and national conferences and by presentations to two major seed companies, Bayer Crop Science and Corteva Agriscience. Two technical reports were completed. One doctoral dissertation, a master's thesis and two journal articles have been completed or accepted for publication. At least one more publication is under preparationfrom the completed thesis. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Obj. 1: We have tested IAS-5, Embrapa-1, Williams82, Maverick in isloated microspore cultures, all genotypes achieve sustained cell divisions and embryo formation. Obj. 2: We have not been able to improve the protocol beyond last year, no other stressors added to the protocol have been beneficial. Induction of donor plants at 10ºC/8ºC for 3 days followed by 4ºC overnight when floral buds are at 3.5 mm in length. Initial incubation of isolated microspores at 11ºC in dark for 4 days using NLN basal medium plus 10% coconut water plus 10 mg/L 2,4-D plus 0.1 mg/L BA. Cultures moved to low light at 18ºC for 3 days and supplemented with phytohormone-free medium as needed to maintain volume. Cultures moved to 25ºC low light for the remainder. Anaverage of 10 embryogenic masses per mL culture are recovered. At 2 weeks cultures are spun at 10K RPM for 2 minutes and resuspended in phytohormone-free medium. Cultures are supplemented with BABI basal medium plus 0.006 mg/L picloram plus 0.35 mg/L BA. Obj. 3: The isolated microspore culture protocol achieves sustained cell divisions and embryo formation in at least 90% of the replicates. Microspore embryogenesis in soybean resembles the Brassica model system. Obj. 4: We have discussed this approach with collaborators at Corteva Agrisciences. It may be too challenging to accomplish this objective within the timeframe specified. Focus has shifted to promote the advancingdevelopment of embryogenic masses as a means to recover haploid or doubled haploid plants. Use of silver nitrate and activated charcoal are being explored as agents that may promote embryo development from these cultures. Obj. 5: Flow cytometric analyses have demonstrated that freshly isolated microspores are haploid using our isolation protocol, and that spontaneous doubling of the chromosomes sometimes occurs within the first days of culture. Faster isolation procedures result in contamination with trichomes from the gynoecium, which resemblemicrospore embryos but which are somatic,diploid cells. We recommend our slower but more stringent isolation protocol of isolating the anthers prior to microspore isolation to avoid confusion with trichome contaminants.

Publications

• Type: Other Status: Published Year Published: 2019 Citation: Hale, B., C. Phipps, C. Kelley, and G.C. Phillips. 2019. Evaluation of soybean androgenesis by isolated microspore culture. Arkansas Soybean Research Studies 2019:31-37.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Garda, M., B. Hale, N. Rao, M. Lowe, M. Bright, S. Goodling, and G.C. Phillips. 2020. Soybean androgenesis I: Identification of pyramidal stressors in anther culture that sustain cell divisions and putative embryo formation in isolated microspore cultures. In Vitro Cell. Dev. Biol. Plant. 56(4):415429.
• Type: Other Status: Published Year Published: 2020 Citation: Hale, B., C. Phipps, N. Rao, and G.C. Phillips. 2020. Advances in soybean microspore culture. Arkansas Soybean Research Studies 2020:74-80.
• Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: Hale, B. 2020. Induction and Characterization of Microspore Embryogenesis in Soybean (Glycine Max [L.] Merrill). Thesis, Arkansas State University, Jonesboro, Fall 2020.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Hale, B., C. Phipps, N. Rao, A. Wijeratne, and G.C. Phillips. 2020. Differential expression profiling reveals stress-induced cell fate divergence in soybean microspores. Plants 9:15101525.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2021 Citation: Hale, B., C. Phipps, N. Rao, C. Kelley, and G.C. Phillips. 2021. Soybean androgenesis II: characterization of early embryogenesis in isolated microspore cultures. In Vitro Cellular and Developmental Biology - Plant 57: (In press). 2021 Spring

Progress 10/01/18 to 09/30/19

Outputs
Target Audience: This project is aimed at development and validation of an enabling technology: Soybean doubled haploids via androgenesis. Our target audiences include soybean geneticists and breeders, and legume biotechnologists. This year a thesis was completed which was presented at the Society for In Vitro Biology annual meeting and will be published in peer reviewed journals. Changes/Problems:The objective related to transient expression of embryo-identity transcription factors may be delayed. Our collaborators at a major American agricultural chemical and seed company are experts and the patent holders on these embryo-identity transcription factors, while they have been able to optimize the system for maize somatic embryogenesis they have not been able to achieve the same reliability for soybean or other dicots. We reproducibly obtain microspore embryos in culture, so induction is not the biggest issue. Instead, we should focus on embryo conversion to plants. What opportunities for training and professional development has the project provided?Two undergraduate students have been performing research internships with training in microscopy, media preparation, sterile technique, dissection of floral bud parts, data collection and analysis, and soybean reproductive biology. The undergraduate students are in health sciences, one is accepted to medical school and the other is pursuing clinical lab sciences. One graduate student completed a master's degree. A second master's student presented a research poster at the annual meeting of the Society for In Vitro Biology. How have the results been disseminated to communities of interest? Results have been disseminated through poster presentations at local and national conferences.Two journal articles are being prepared from the completed dissertation and thesis. What do you plan to do during the next reporting period to accomplish the goals?We are on track to accomplish the goals of the project. One objective may be delayed because of its complexity to execute. The focus for the coming year is to attempt to convert microspore embryos into plantlets.

Impacts
What was accomplished under these goals? Obj. 1: We have tested IAS-5, Embrapa-1, Williams82, Maverick in isloated microspore cultures, all genotypes achieve sustained cell divisions and embryo formation. Obj. 2: We have not been able to improve the protocol beyond last year, no other stressors added to the protocol have been beneficial. Obj. 3: The isolated microspore culture protocol achieves sustained cell divisions and embryo formation in at least 90% of the replicates. Microspore embryogenesis in soybean resembles the Brassica model system. Obj. 4: We have discussed this approach with collaborators at Corteva Agrisciences. It may be too challenging to accomplish this objective within the timeframe specified. Obj. 5: Flow cytometric analyses have demonstrated that freshly isolated microspores are haploid using our isolation protocol, and that spontaneous doubling of the chromosomes occurs within the first days of culture. Faster isolation procedures result in contamination with trichomes which respond similarly as microspores but which are somatic and diploid cells. We recommend our slower but more stringent isolation protocol to avoid confusion.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Hale, B., M. Garda, C. Phipps, C. Kelley, and G. Phillips. 2019. Soybean Isolated Microspore Culture: Sustained Cell Divisions and Embryo Formation. Annual Meeting of the Society for In Vitro Biology, June 8-12. P-2033 (Abstr.). Tampa, FL.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:This project is aimed at development and validation of an enabling technology: Soybean doubled haploids via androgenesis. Our target audiences include soybean geneticists and breeders, and legume biotechnologists. This year a dissertation was completed which will be presented at the Society for In Vitro Biology annual meeting and published in peer-reviewed journals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three undergraduate students have been performing research internships with training in microscopy, media preparation, sterile technique, dissection of floral bud parts, data collection and analysis, and soybean reproductive biology. One undergraduate student is a plant science major and will be presenting her results at the university research forum Create@State. Two other undergraduate students are in health sciences, one is accepted to medical school, the other is pursuing clinical lab sciences. One graduate student received her doctorate based on this project and attended the annual meeting of the Society for In Vitro Biology. One other graduate student presented a research poster at the statewide Arkansas Biosciences Institute Conference. How have the results been disseminated to communities of interest?Results have been disseminated through poster presentations at local or national conferences. Two journal articles are being prepared from the completed dissertation. What do you plan to do during the next reporting period to accomplish the goals?The project is going well, we are on track to complete the project on schedule.

Impacts
What was accomplished under these goals? Objective 1: IAS-5 responds similarly as do Jack, Williams 82, and Thorne. These 4 genotypes produce PGCs at an average rate of 25%. An F1 hybrid involving IAS-5 (Embrapa-1) responds at 47% PGC. This protocol is not genotype limited. Objective 2: We have tested additional androgenetic stressors including pH, osmoticum, pretreatment conditions. Androgenic induction appears to be optimized using: Donor plant pretreatment at 10C day/8C night for 3 days, 4C overnight, initial incubation at 11C dark, then 18C in light and 25C in light, nitrogen starvation medium for initial incubation, pH 5.8, 2% sucrose + 2% sorbitol. Objective 3: The androgenic induction stressors identified above were tested with isolated microspore cultures. Addition of 2,4-D and BA is necessary to support sustained cell divisions. 100% of the cultures exhibit sustained cell divisions aand formation of embryos. Objective 4:Transient expression of embryo-identity genes is planned for 2019. Objective 5:Flow cytometric analyses demonstrate the haploid status of freshly isolated microspores. Under certain culture conditions, evidence of spontaneous chromosome doubling was observed.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Garda, M. 2018. Soybean Androgenesis Advances. Dissertation, Arkansas State University, Molecular BioSciences Program, Jonesboro, AR, 118 pp.

Progress 07/01/17 to 09/30/17

Outputs
Target Audience:This project is aimed at development and validation of an enabling technology: Soybean doubled haploids via androgenesis. Our target audiences includesoybean geneticists and breeders, andlegume biotechnologists. This year we presented results to Bayer Crop Sciences and the Society for In Vitro Biology, respectively, to reach our target audiences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four undergraduate students gained research training and experience on this project. One of those undergraduate students presented his research results at the university's research forum (Create@State),co-presented results at the annual meeting of the Society for In Vitro Biology, did a summer internship at Danforth Plant Science Center, and received offers of full-rides at two prestigious graduate schools (Cornell, UGeorgia). The doctoral student presented her results at the Society for In Vitro Biology, at Bayer Crop Sciences, and received specialized training in microscopy image analysis at the Danforth Plant Science Center. How have the results been disseminated to communities of interest?Results have been communicated by research poster at the Society for In Vitro Biology, and by taped seminar presentation at Bayer Crop Sciences. What do you plan to do during the next reporting period to accomplish the goals?So far, everything is on track. No change in strategy or immediate plans are warranted.

Impacts
What was accomplished under these goals? Objective 1: We tested IAS-5. IAS-5 responds similarly to Jack, Williams82 and Thorne, but perhaps more consistently. IAS-5 is more reliable as a donor than the other genotypes tested. Objective 2: We have tested additional androgenic stressors, such as pH, pH stabilization using biological buffers, different donor plant pre-treatments. Results this year have resulted in an optimized anther culture protocol. Objective 3: Preliminary experiments have shown sustained cell divisions and embryo formation in multiple replicates. The anther culture protocol was adapted to isolated microspore culture for induction, but both auxin (2,4-D) and pH had to be optimized separately. Experiments are ongoing to optimize the isolated microspore culturesystem. Objective 4: Not yet underway, dependent upon Obj. 3 completion. Objective 5: Confirmation of haploid status for anther culture derived putative gametic calli is underway. Purity of isolated microspores in our culture system assures that any embryos obtained are haploid in origin.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Garda, M., B. Hale, M. Lowe, N. Rao, M. Bright, S. Goodling, K. Bade, and G.C. Phillips. 2017. Further studies of putative gametic calli from soybean anther cultures. Annual Meeting of the Society for In Vitro Biology, June 10-14. P-3028 (Abstr.) Raleigh, NC.