Recipient Organization
ARKANSAS STATE UNIVERSITY
(N/A)
STATE UNIVERSITY,AR 72467
Performing Department
Agriculture Studies
Non Technical Summary
This is a New, Regular, Research proposal addressing USDA Research Need Area 2) Applied Studies in the Food and Agricultural Sciences (P, B). This proposal seeks to develop an enabling technology to advance soybean breeding and genetics: androgenesis, the development of a plant directly from the male gamete. This is accomplished in plants typically by anther culture or isolated microspore culture. In soybean, anther cultures have generated embryos at very low frequencies (< 2%), and only rarely a plantlet. We screened soybean anther cultures from multiple genotypes for putative gametic callus/embryo formation. This strategy identified factors that promote the formation of putative gametic calli, such as nitrogen starvation, 10°C/8°C (day/night) for 3 days plus 4°C overnight pretreatment of donor plants, and 11°C initial incubation temperature. Our anther culture protocol now yields putative gametic callus/embryo structures at 12-20% frequencies. However, it is reported that both haploid (gamete-derived) and anther wall (somatic-derived) tissues give rise to these structures in soybean. Isolated microspore culture would avoid this problem. We have adapted our protocol to the culture of isolated microspores of soybean, and for the first time (to our knowledge) we have obtained sustained cell divisions and embryo formation. Our objectives are to optimize the isolated microspore culture system for sustained cell divisions; explore treatments to induce chromosome doubling in culture; optimize the system for embryo formation; and convert these embryos into plants. A reliable system for producing doubled haploid plants of soybean will have a great impact on the soybean industry.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
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, by isolated microspore culture technology, 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: Optimize the isolated microspore culture system for sustained cell division. Adaptation of the anther culture protocol to isolated microspore culture may require additional optimizations, e.g., the transition from semi-solid anther cultures to liquid microspore cultures may benefit from adjustments to osmoticum and pH (Ferrie and Caswell 2011). Also, growth regulator signals may need adjustment to stimulate more cell division.Objective 2: Evaluate treatments to induce chromosome doubling. While most DH systems first regenerate haploid plantlets and then induce chromosome doubling among those plantlets to produce the DH (Ferrie and Caswell 2011), there have been attempts to induce chromosome doubling in microspore cultures prior to regenerating plantlets. If there is a developmental block in soybean haploid tissues preventing them from developing into plantlets, then induction of chromosome doubling in microspore culture should result in DH tissues with the normal somatic number, which should develop similarly to somatic embryos. Objective 3: Optimize the frequency of embryo formation. This step is related to Objective 1, especially in relation to growth regulator signals, but osmoticum and pH may also play a role in embryo induction (Ferrie and Caswell 2011). Also, if Objective 2 is successful, then we can screen typical somatic embryogenesis induction protocols for soybean.Objective 4: Evaluate treatments to convert embryos into plants. It is challenging to recover plants from legume androgenetic systems, even when embryos are initiated. However, if Objective 2 is successful, then we should have DH tissues with the normal somatic number. If those tissues respond to typical somatic embryogenesis induction protocols during Objective 3, then they should also respond to the typical somatic embryo differentiation and development protocols.
Project Methods
Methods:We initiated our studies into soybean androgenesis with the Soybean Doubled Haploids Workshop held at Arkansas State University on February 23, 2015. Seven experts were invited to present their work in relation to soybean genetics, breeding, and doubled haploids. Presenters included David Grant, Alison Ferrie, Eliane Kaltchuk de Santos, John Finer, Wayne Parrott, Tom Clemente, and Kan Wang (could not attend but sent her presentation). Attendees included Elizabeth E. Hood, Greg Phillips, Pengyin Chen, Martina Garda, Ken Korth, Vibha Srivastava, and Argelia Lorence. This workshop provided stakeholder input into our efforts.From the previous work performed at Arkansas State University (Garda et al. 2015, 2016, 2017), we have identified several factors for optimization of soybean androgenesis:- Floral bud stage: Immature buds 2.5 - 3.5 mm in length (early uninucleate to early binucleate microspores).- Pretreatment of Donor Plants: Three days shock at 10°C/8°C (day/night) plus overnight at 4°C prior to harvesting the buds.- Incubation temperature: Incubation at 11°C in the dark for 1 week, move to 18°C in diffuse light for 1 week, then move to 25°C in diffuse light.- Nitrogen Starvation: Nitsch and Nitsch (1969) major salts and vitamins plus BABI minor salts during the induction phase.- Genotype: Multiple genotypes appear to respond, but we are using IAS-5 because it produces more floral buds in the growth chambers than do the other genotypes tested.- Auxin: No auxin is needed in anther cultures, but the addition of 10-40 mg/l 2,4-D enhances anther culture PGC responses. Isolated microspore cultures likely require hormone stimulation in order to initiate cell division; we will use 10 mg/l 2,4-D as an initial control for isolated microspore cultures.Objective 1: Optimize for sustained cell division. We started with the isolated microspore protocol of Rodrigues et al. (2006) for genotype IAS-5, which had not resulted in sustained cell divisions for them or for us. We adapted the androgenic triggers we had identified in our previous anther culture work (described immediately above) to this protocol: donor plant pretreatment, nitrogen starvation, initial incubation temperature, 2,4-D as auxin - which has resulted in sustained cell divisions. Guided by the recommendations of Ferrie and Caswell (2011) and Lulsdorf et al. (2011), additional experiments will be conducted to optimize key variables to stimulate sustained cell divisions. We are using the R program for statistical analyses. We design experiments with a minimum of 10 replicates per treatment. When all equipment works properly, we can initiate one new experiment (replicate) each week. Our goal is for each replicate to respond with sustained cell divisions. These variables include:? Cell culture density. In general, cell density must meet a minimum and not exceed a maximum cell density in order to proliferate, but the optimal range of cell densities has yet to be determined for soybean. Currently we use the anthers from 60-80 floral buds for one prep (one prep generates four replicates, or one replicate of each of four treatments), and 60 buds appears to be the minimum for this set-up. We will compare 60, 80, 100, and 120 buds per prep in order to optimize cell density for sustained cell division.? pH. While anther cultures preferred pH 5.8 with no biological buffer, pH is a critical variable that may change as we transition from anther culture (semi-solid media, microspores encased in tissue) to microspore culture (liquid media, free microspores). We will test pH 5.8 (no buffer), pH 4.0 (Na-citrate), pH 5.8 (MES), pH 7.0 (MOPS), pH 9.0 (CHES) (see Figure 6 above).? Osmoticum. Osmoticum is another variable that may need adjustment as we transition from anther culture to microspore culture. We will test 9% sucrose (Moraes et al. 2004), 12% sucrose (Rodrigues et al. 2006), 2% sucrose + 2% or 4% sorbitol (see Figure 4 above).? Growth regulator treatments. Anther cultures benefitted from the addition of 10-40 mg/l 2,4-D during our previous experiments though they did not require it. The recent report of chickpea haploid plant recovery from anther cultures used a combination of 10 mg/l 2,4-D + 15 mg/l AgNO3 (Abdollahi and Rashidi 2018). We will compare 10 mg/l 2,4-D plus and minus 15 mg/l AgNO3. Picloram at 0.5 mg/l has been used for induction of androgenesis of chickpea (Panchangam et al. 2014), and it should be compared to 2,4-D as auxin. Addition of benzyladenine as cytokinin (0.1-2 mg/l) should stimulate more cell division. Concentrations of auxin and cytokinin will be optimized to stimulate frequency of sustained cell divisions, and to balance this response with optimal embryo formation during Objective 3.Objective 2: Spontaneous doubling of chromosomes often occurs in isolated microspore culture, and increases as the cultures age (Ferrie and Caswell 2011; Lulsdorf et al. 2011). Additional treatments to promote chromosome doubling include addition of 0.1-0.2% colchicine to the culture medium at an early stage of culture (Wurschum et al. 2012); we will test this treatment at 0.1% and optimize from there if it appears promising. Another report utilized a 21% mannitol treatment of microspores prior to culture in combination with cold pretreatment (Li and Devaux 2003). Recent reports of using AgNO3 in the culture medium for anther cultures of different species have suggested that it may promote spontaneous chromosome doubling (Keles et al. 2015; Abdollahi and Rashidi 2018). We will conduct replicated experiments to evaluate each of these three potential strategies for soybean, separately and in combinations.Objective 3: Embryo induction is dependent upon androgenic stimuli and growth regulators used (Ferrie and Caswell 2011; Lulsdorf et al. 2011). We will optimize these factors to promote higher frequencies of embryo formation. Because we are already observing embryo formation in preliminary cultures, this step should be relatively easy to optimize once we have optimized for sustained cell divisions during Objective 1. We are primarily concerned that the growth regulator treatments used for sustained cell divisions do not detract from embryo formation, and vice versa.Objective 4: Assuming at least partial success during Objective 2, the isolated microspore cultures may already be induced for chromosome doubling. That means that the microspore-derived embryos should be at the normal somatic number. Microspore-derived embryos with the normal somatic chromosome number should behave like somatic embryos in culture. If so, we will use the protocols of Samoylov et al. (1998) to recover plants from microspore-derived embryos and optimize as needed. Alternatively, we will test the protocols of Abdollahi and Rashidi (2018), who used 10 mg/l 2,4-D + 15 mg/l AgNO3 to regenerate haploid and dihaploid plants of chickpea from anther cultures, the first report of multiple plant recovery from a legume anther culture system.