IMPROVEMENT OF THE GENETICS OF PANICUM VIRGATUM (SWITCHGRASS)

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0226451
• Project No.: ARZT-1367580-H25-213
• Project Start Date: Jul 1, 2011
• Project End Date: Sep 30, 2015

Project Director
Feldmann, KE.

Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824

Performing Department
Plant Science

Non Technical Summary
Switchgrass (Panicium virgatum) is a tetraploid to octoploid and phenotypically very heterogeneous. This is in part due to the vast polymorphism in the species and the fact that it is an obligate outcrosser. While plants can be identified that are higher yielding or more stress tolerant it will take many years of breeding to dramatically improve the germplasm. If a superior plant was also apomictic, or even-self-fertilizing, yields could be doubled, especially during the establishment phase, in just a few years. Switchgrass does possess a low level of self-fertility, and likely apomixis, and this will be increased using a mutagenesis and selection approach. In addition, to ensure success, that is doubling yield, an efficient clonal propagation system will be established so that tens of millions of clones can be derived from a single superior plant and transplanted to the field. In this way, although more expensive, yields can also be doubled in one or two years.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
202	1629	1080	60%
205	1629	1080	20%
206	1629	1080	10%
201	1629	1080	10%

Knowledge Area
206 - Basic Plant Biology; 205 - Plant Management Systems; 202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1629 - Perennial grasses, other;

Field Of Science
1080 - Genetics;

Keywords

panicum virgatum

switchgrass

apomixis

clonal propagation

obligate outcrossing

Goals / Objectives
1) Generate mutagenized populations of switchgrass plants. We will screen the M1s for phenotypes and collect M2 seeds individually from 3,500 M1 plants 2) Identify self-compatible lines. We should identify several lines that generate seeds and these will need to be tested to distinguish between self-compatability and apomixis. 3) Identify apomictic lines. We should identify several lines that generate seeds and these will need to be tested to distinguish between self-compatability and apomixis. 4) Improve clonal propagation for superior switchgrass plants. We have 10 genotypes growing in the field that will be tested. Testing of these lines will result in two publications.

Project Methods
Objective 1) A chemically mutagenized seed population has been generated and transferred to the field. To identify a concentration of EMS that would induce mutations but allow seedling survivability and good seed production, we established a dose response curve using 0.05 to 0.8% EMS at both 10 and 24 hrs. We settled on 0.1% EMS for 10 hrs. Assuming 4 Genetically Effective Cells (GECs)and a 4n chromosome complement, 2,000 M1s should be enough to generate multiple mutations at each locus. Objective 2) It has been reported that, while switchgrass is an obligate outcrosser, 0.5% of the seed on average result from self-fertility. To further characterize this and to identify plants that have higher rates of self-compatibility, assuming a GEC Number of 4, four positionally-selected panicles on each M1 plant and one panicle on an M2 plant will be bagged prior to flowering. The labeled bags will be collected after 5-6 weeks and evaluated for the number of progeny. We will bag 3,500 plants in YR1. Objective 3) Switchgrass populations are extremely polymorphic and thus phenotypically heterogeneous. The phenotype of plants within a released variety can range from 4 to 10 ft tall and possess from 80 to 300 panicles on irrigated land in AZ. Some plants have an upright appearance while others are drooped over with a very ornamental appearance. Plants that are 10 ft tall with 200-300 larger panicles, but with low seed output, so that they are less susceptible to lodging, are the most desirable plants. However, there is currently no way to generate a pure breeding population. If we could identify an apomictic gene and transfer it to a superior plant, the yield (tons/acre) could be doubled in one year. To identify plants that are apomictic we will screen any selfed progeny. Once self-fertile plants are identified we will need to ascertain whether it is a result of a loss of self-incompatibility or apomixis. Progeny resulting from apomixis will be identical to the parent plant while plants that result from self-fertility will have variable phenotypes, depending on the sets of alleles contained in the parent. Objective 4) It is highly advantageous in an obligate outcrossing species to be able to generate thousands and even tens millions of clones of a single superior plant. We have been working on nodal propagation in switchgrass and we are able to get essentially 100% of the buds in cut nodes from some genotypes to form a shoot. We can dip these in a root stimulant and establish plants directly in soil. We do see genotypic differences in the ability to generate plants from the nodes. This is a very simple procedure that allows us to generate 1,000s of clones from a single plant. We aim to characterize some of the factors related to the success of clonal propagation over a number of genotypes. In addition, we have shown that we can use meristem culture to generate millions of clones from a single superior plant. Using this approach and contracting with a high throughput nursery and a company that specializes in transplanting seedlings it would be possible to plant thousands of acres with a single high yielding clone.

Progress 07/01/11 to 09/30/15

Outputs
Target Audience:Graduate student, Josh Weaver, presented a talk at the In Society of In Vitro Biology Annual Conference in Tucson in June, 2015. 50-70 participants attended the talk. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has been the primary responsibility of a Native American graduate student, Josh Weaver. In addition, a number of undergraduates have been trained on various aspect ofthis project over the past 5 years. How have the results been disseminated to communities of interest? Weaver, J.M., Laura Sofia Montes Sujo, L.S.M. and Feldmann, K.A. 2014. A simplified technique for the propagation of shoots from nodes of select switchgrass (Panicum virgatum L.) genotypes. BioEnergy Research, DOI 10.1007/x12155-014-9470-4. Mr. Weaver has also presented the results at Purdue University and the In Vitro Cell meetings. A publication describing the epicuticular waxes on wildtypes two types of variants will be submitted soon. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Mutagenized population was generated and screened thoroughly for visible phenotypes. Glossy and glaucous epicuticular was mutants were identified and characterized and the results will be submitted for publication soon. 2 and 3) 1,800 lines were tested for self-compatibility and apomixis and while we identified a number of candidates, none of them showed a similar phenotype in the next generation. 4) Clonal propagation was improved and a paper was published describing these results..

Publications

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Our target audience includes anyone working with switchgrass who is interested in cloning variants of this species. In addition, our target audience would include researchers interested in epicuticular wax or drought tolerance in grasses. Changes/Problems: We knew from the start that it might be difficult to identify apomictic and self-compatible mutants in an M1 population, especially a tetraploid but we thought it was worth a try. While we identified some tillers on some plants that gave rise to more seeds than expected in the first attempt, new tillers arising from this same region in YR2 did not show more seed set. In phenotyping our M1 population we identified many leaf and epicuticular was variants and decided to characterize the EW in hopes of identifying plants that were more drought tolerant. The waxes have been characterized and we have made clones for testing drought tolerance and for genetic crosses. What opportunities for training and professional development has the project provided? The graduate student has presented his results at a scientific meeting on switchgrass and he will be presenting additional results at the upcoming In Vitro Cell Biology meetings in Tucson. How have the results been disseminated to communities of interest? Via publication and scientific meetings What do you plan to do during the next reporting period to accomplish the goals? I will edit the epicuticular wax paper and share it with collaborators before submission.

Impacts
What was accomplished under these goals? 1) and 4) above were completed. While we found candidates for 2) and 3) they did not breed true. As such, we decided to characterize other variants that we identified in the population. We have characterized, both chemically and structurally,the epicuticular waxes on a number of glossy and glaucous mutants and a paper describing these results will be submitted soon. In addition, various mutants are being crossed to help ascertain the number of loci involved in was production in our variants.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Weaver, Joshua M.; Sujo, Laura Sofia Montes; Feldmann, Kenneth A. A Simplified Technique for the Propagation of Shoots from Nodes of Switchgrass (Panicum virgatum L.) Genotypes BIOENERGY RESEARCH Volume: 7 Issue: 4 Pages: 1351-1357 Published: DEC 2014

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: We are studying naturally occurring epicuticular wax variants in switchgrass (Alamo). These phenotypic variants may be important in drought tolerance or in biomass production. One obstacle to studying these variants was to generate enough genetically identical clones of each variant to conduct replicated experiments. While there was a published cloning procedure it was more tedious than it needed to be so we developed a much more efficient vegetative propagation system that does not require any special equipment. We have published this procedure in BioEnergy. We identified both glossy and glaucous variants and extracted the waxes for analysis by GC-MS. We have also examined the wax morphology using SEM. Using the results from these two methods, the wax variants were placed into one of three classes. We are now crossing the lines within each variant class to test for allelism and preparing a manuscript that describes the waxes in these variants and wildtype. Changes/Problems: We bagged 1,800 plants and collected and counted the seeds that resulted. We had a half dozen candidates from this analysis but rebagging them the next year showed that it was spurious seed production. We have observed a number of different interesting phenotypes in the field while we were doing this work and the student chose to work on epicuticular waxes. What opportunities for training and professional development has the project provided? The native American PhD student on this project has published his first paper, done his first SEMs, and for the first time has extracted waxes and learned how to analyze them. How have the results been disseminated to communities of interest? The vegetative propagation result has been emailed to interested colleagues and published in BioEnergy Research. What do you plan to do during the next reporting period to accomplish the goals? Work closely with the student to identify the resources needed to complete the analysis of the epicuticular wax variants including drought studies. We also aim to propagate a number of different morphological types from the field this year so that we can do replicated trials with these variants in the field next year.

Impacts
What was accomplished under these goals? We have completed goals 1) and 4) but we were unable to identify either apomictic or self-fertile lines. Instead, we decided to study other variants in the population including epicuticular waxes and there possible involvement in drought tolerance/susceptibility. We have published a first paper, and as corresponding author, and a second will soon be ready for submission. We will acknowledge NIFA support in the second paper.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Weaver, J.M., Laura Sofia Montes Sujo, L.S.M. and Feldmann, K.A. 2014. A simplified technique for the propagation of shoots from nodes of select switchgrass (Panicum virgatum L.) genotypes. BioEnergy Research, DOI 10.1007/x12155-014-9470-4.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Mapped the entire mutagenized field for mutant phenotypes Repeated the vegetative propagation study on select genotypes Cleaned mutagenized seeds collected in 2011 Rebagged interesting putative mutants as well as plants that were missed in 2011 PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Identified 6 plants that made more than the expected number of seeds. These were good candidates for apomixis or improved self-fertility. Vegetatively propagated an interesting sectored plant that may have commmercial value Collected waxes from a number of putative epicuticular wax mutants as well as wildtype and analyzed these in collaboration with Matt Jenks at the USDA

Publications

• No publications reported this period

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: 1. Transplanted 3,500 mutagenized switchgrass plants to the field. 2. Bagged 2,000 of these plants and collected the resulting progeny. 3. Screened these plants for mutant phenotypes. 4. Identified both glaucous and glossy phenotypes and extracted waxes for analysis. 5. Completed nodal propagation studies on ten distinct switchgrass genotypes. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. Identified genotypes that are promising for nodal propagation. 2. Identified nodal positions that are the best for nodal propagation. 3. Improved the ease of propagating the plants with nodal culture; a manuscript is in preparation which describes these improvements.

Publications

• No publications reported this period