Biochemical Characterization of Cuticular Wax Variants in Switchgrass (Panicum Virgatum L.)
Item Type text; Electronic Dissertation
Authors Weaver, Joshua Micheal
Publisher The University of Arizona.
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Link to Item http://hdl.handle.net/10150/628411
BIOCHEMICAL CHARACTERIZATION OF CUTICULAR WAX VARIANTS IN SWITCHGRASS (PANICUM VIRGATUM L.)
by
Joshua M. Weaver
______Copyright © Joshua M. Weaver 2018
A Dissertation Submitted to the Faculty of the
SCHOOL OF PLANT SCIENCES
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2018
2
THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Joshua M. Weaver, titled Biochemical Characterization of Cuticular Wax Variants in Switchgrass (Panicum virgatum L), and recommend that it be accepted as fulfilling the ti on requirement for the Degree of Doctor of Phil oso � �::, July 17,2018 Kenneth A. Feldmann
_M_at_ t_h_ew_A_.__e_J n_ks______Date; ulyJ 9, 2018 Matthew A. Jenks
�-- /':ub�/Q/------Dale: July 9, 2018 Chieri Kubota
--;/� �� Date: Mike Ottman
Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copies of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfillingthe dissertation requirement.
______Date: July 17, 2018 431ssertation� Director: Kenneth A. Feldm-ann 3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of the requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that an accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
SIGNED: Joshua M. Weaver 4
ACKNOWLEDGMENTS
I would first like to thank the outstanding mentors (Drs. Russell Weigel, Kenneth A. Feldmann and Matthew A. Jenks) for helping to shape my outlook on the natural world and instilling in me the tools for success. I am deeply indebted to my committee members (Drs. Mike Ottman and Cheri Kubota) for their critical advice, pursuit of the highest standards and unfailing patience. I would also like to thank Drs. Edzard van Santan, David Bransby, Mike Casler, Christian Tobias and Kenneth Vogel for their work as pioneers in the field and for laying the ground work that all current and future switchgrass research is based. I would be amiss not to mention my collaborators at the USDA-ARS, in Maricopa, including Dr. John Dyer, Pernell Tomasi and Greg Lohrey. I would also like to give a special thanks to my lab members K. Milward, L. S. Montes-Sujo, and M. J. Feldmann. I thank the University of Arizona faculty member Drs. Edward J. Bedrick, of the Bio-5 Institute, for statistical consultation and Brooke Beam Massani, staff scientist, W.M. Keck Center for Nano-Scale Imaging in the Department of Chemistry and Biochemistry. I would like to acknowledge the funding support by the Alfred P. Sloan Foundation’s Indigenous Graduate Partnership, University of Arizona Graduate College and Mississippi Band of Choctaw Indians Tribal Scholarship Program. Sponsorship was provided by the Office of Diversity and Inclusion, Helen Roberti Charitable Trust and TA-ship by School of Plant Sciences. I would like to give many thanks to members of the Office of Diversity and Inclusion Dr. Frans Tax and Donna Treloar, Sloan Foundation faculty members Drs. Kevin Gibson, Ronald Trosper and the late Maria Teresa Velez, and Mississippi Band of Choctaw Indians Tribal Scholarship Program’s scholarship officer Melanie Carson.
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DEDICATION
This dissertation work is dedicated to Michelle J. Pace, Zachary D. Weaver, parents (Douglas P. and Sandra K. Weaver) and grandparents (Irvin D. and Katrine R. Weaver) for their continuous and loving support.
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TABLE OF CONTENTS
LIST OF FIGURES AND TABLES 9
DISSERTAION FORMAT 11
ABSTRACT 13
INTRODUCTION 16
Chapter 1: Switchgrass (Panicum virgatum L.) 16 1. Introduction 16 2. Mircopropagation 18 3. Switchgrass Molecular Breeding 20 3.1 Molecular Markers (RFLP, RAPD, SSR, EST-SSR, EST-STS, AFLP and SNP) 20 3.2 Linkage Maps (Molecular Mapping) 22 4. Development as a Biofuel Crop 23 4.1 Conservation, Feedstock Development and Agronomic Practice 23 4.2 Conversion of Lignocellulosic Biomass to Ethanol (Acid Hydrolysis, Enzymatic Hydrolysis and Simultaneous Saccharification and Fermentation (SSF)) 26 4.3 Early Breeding Efforts in Switchgrass 27 5. Future Use as a Biofuel 29 5.1 Governmental Influences 29 5.2 Crop Improvements (Artificial Seeds, S-Z- Incompatibility and Reduced Lignin) 32
PRESENT STUDY 35
Present Study (Chapter 2, Appendix A and Appendix B) 35 1. Chapter 2: Mutagenesis and Genetic Improvements of Panicum virgatum 35 2. APPENDIX A: A Simplified Technique for the Propagation of Shoots from Nodes of Switchgrass (Panicum virgatum L.) Genotypes 36 3. APPENDIX B: Cuticular Wax Variants in a Population of Switchgrass (Panicum virgatum L.) 36
Chapter 2: Mutagenesis and Genetic Improvements of Panicum virgatum 38 1. Introduction 38 1.1 Diploid Switchgrass 38 1.2 Self-Fertility 39 1.3 Apomixis 40 1.4 Ethyl Methanesulfonate (EMS) Induced Variation 41 7
TABLE OF CONTENTS – Continued
2. Materials and Methods 43 2.1 Plant Material and Growth Conditions 43 2.2 Screen for Self-Fertility and Apomixis 45 2.3 Cleaning Seed 46 2.4 Scoring Plant Morphology 47 3. Results 48 4. Discussion 50 5. Conclusions 51
Chapter 3: Research Findings 52 1. Summary 52 1.1 EMS Population 52 1.2 Screen for Self-Fertility 53 1.3 Nodal Propagation 53 1.4 Characterization of Wax Variants 54 1.5 Crossing Block 56 2. Future Research 57
FIGURES AND TABLES 59
LEGENDS 62
REFERENCES 63
APPENDIX A - A Simplified Technique for the Propagation of Shoots from Nodes of Switchgrass (Panicum virgatum L.) Genotypes 72 1. Abstract 75 2. Introduction 76 3. Materials and Methods 78 3.1 Plant Material 78 3.2 Harvesting Nodal Segements 79 3.3 Shoot Induction from Axillary Buds 80 3.4 Root Induction from Shoots 81 3.5 Statistical Analysis 82 4. Results and Discussion 83 4.1 Shoot Production from Harvested Nodal Explants 83 4.2 Root Formation from Axillary Shoots 84 5. Legends 88 6. Figures 89 7. References 91
APPENDIX B - Cuticular Wax Variants in a Population of Switchgrass (Panicum virgatum L.) 93 1. Abstract 96 8
TABLE OF CONTENTS – Continued
2. Introduction 97 3. Materials and Methods 99 3.1 Plant Material and Growth Conditions 99 3.2 Leaf Cuticle Wax Extraction 100 3.3 Chemical Analyses of Waxes 101 3.4 Scanning Electron Microscopy 103 4. Results 103 5. Discussion 106 6. Legends 109 7. Figures and Table 111 8. Supplemental Figures 114 9. References 116
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LIST OF FIGURES AND TABLES
Chapter 2: Mutagenesis and Genetic Improvements of Panicum virgatum 38
Figure 1 59 EMS Mutagenesis Procedure in Switchgrass Figure 2 59 EMS Dose Response Curve Figure 3 60 Chlorophyll Mutants in Switchgrass Figure 4 60 Field Bagging in Fall 2011 Table 1 61 Seed Germination in Switchgrass (EMS and Control) Table 2 61 Scoring of Morphological Traits
APPENDIX A - A Simplified Technique for the Propagation of Shoots from Nodes of Switchgrass (Panicum virgatum L.) Genotypes 72
Figure 1 89 Procedure for Nodal Propagation of Switchgrass Figure 2 89 DFT (Deep Flow Technique) Raft-Type Hydroponics System Figure 3 90 Shoot Formation by Node Position Figure 4 90 Root Formation in Two Rooting Conditions
APPENDIX B - Cuticular Wax Variants in a Population of Switchgrass (Panicum virgatum L.) 93
Figure 1 111 Photographic Images of Switchgrass Variants Figure 2 111 Wax Chemical Profiles by Chemical Class Figure 3 112 Comparison of C33 β-diketones Figure 4 112 Ultrastructural Characterization of Leaf Epicuticular Wax
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LIST OF FIGURES AND TABLES – Continued
Table 1 113 Mean Comparison of Leaf Cuticular Waxes Supplemental Figure 1 114 GC-MS Chromatogram in ST Switchgrass Supplemental Figure 2 115 Mass Spectrum of C33 β-diketone (tritiacontane-12,14-dione) Supplemental Figure 3 115 Mass Spectrum of C33 hydroxy-β-diketone (5-hydroxytritriacontane- 12,14-dione)
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DISSERTATION FORMAT
The research goals of this dissertation are to 1) create a population of 1,849 Ethyl
Methanesulfonate (EMS) treated switchgrass (Panicum virgatum L., cv. Alamo) 2) develop a means for nodal propagation using commonly available greenhouse materials and 3) characterize three unique cuticular wax variant types (Non-Glaucous (NG),
Reduced Glaucous (RG) Highly Glaucous (HG)) and Standard-type (ST). The dissertation is organized as five chapters that are each presented in three topic areas
(literature review, present study and future work) and two appendices (A and B).
Chapter one, entitled “Switchgrass (Panicum virgatum L.)”, is a literature review that is composed of five sections with additional sub-sections. This chapter highlights the current understanding and advances in switchgrass research from its’ earliest accounts as a prairie grass, adaptation for restoration, identification as a “model” bioenergy crop, breeding, propagation, conversion into ethanol and advanced molecular genetic techniques. Chapter two, entitled “Mutagenesis and Genetic Improvements of Panicum virgatum”, is the first of the three chapters, including the two published articles found as
Appendices (A and B), that comprise the present study. The outline of this chapter is similar to a published manuscript (including an introduction, materials and methods, results, discussion and conclusions) but aside from the dissertation this chapter will not be submitted for publication. Chapter two outlines our initial interest in the creation, characterization, and screening, via bagging, of an EMS mutagenized population of switchgrass for dominant mutations leading to self-fertility and apomixis. The third and final chapter, entitled “Research Findings”, is the final topic area of this dissertation and 12 is a continuation of our present study. This chapter contains two sections, including summary and future research.
The two published studies, provided in Appendices A and B, were conducted as part of the present study. In Appendix A, my coauthors and I developed an improved propagation system for switchgrass. In ten genotypes of switchgrass cultivar (cv.) Alamo, we examine shoot formation from axillary buds at three node positions (Low, Mid, and
High), during a six-week incubation period, in a Deep Flow Technique (DFT) type hydroponics system. Root formation was also tested in two rooting conditions, over four weeks. This article was published in BioEnergy Research in 2014.
In Appendix B, my coauthors and I describe three uniquely identified cuticular wax variant types (NG, RG and HG) in a population of 1,849 plants of switchgrass cv. Alamo.
Gas chromatography with mass spectrometry and field emission scanning electron microscopy are used to compare visually identified cuticular wax variants to the most abundant Standard Type (ST) switchgrass. This manuscript was published in Industrial
Crops and Products in 2018.
13
ABSTRACT
Taxonomic distinction of switchgrass (Panicum virgatum L.) as Lowland (L-) or
Upland (U-) ecotypes is made in reference of their distinct morphology, regions of adaptation and ploidy level. Widely adapted to southern latitudes, below 45°N, of the
United States, L- ecotypes possess a number of favorable agronomic traits, including resistance to flooding, high yield, tall stems, thick stems, delayed anthesis and pathogen resistance, which are generally considered more useful as an herbaceous bioenergy crop.
The overall objective of this research was the identification, propagation and characterization of natural variants in L- switchgrass cv. Alamo plants possessing valuable agronomically important traits. Our experiments focused on the identification and characterization of traits including self-fertility/apomixis and cuticle wax formation.
In the present study, we attempted to introduce variation by creating a population of
1,849 M1 plants. We observed sectored leaves on a number of M1 plants indicating that the mutagenesis treatment was successful. While several plants were identified in the first generation that set more seeds after bagging individual plants, they were not confirmed in the second year and the screen for self-fertility/apomixis was deemed unsuccessful. We turned our attention to the natural variation that existed in this population of plants. In scoring these plants in the field during development, we identified plants that had 1) more or fewer tillers, 2) narrow or wide leaves, 3) shorter or taller tillers, 4) thicker tillers, 5) variegation and 6) plants with altered amounts of visual glaucousness. Plants having changes in visible glaucousness (Non-Glaucous (NG), Reduced Glaucous (RG) and
Highly Glaucous (HG)) that differed from Standard Type (ST) became the focus of this study. 14
To propagate a larger number of each variant plant for subsequent testing, we developed a custom Deep Flow Technique “DFT” style raft-type hydroponics system for the nodal propagation of identical plants by direct organogenesis from axillary meristems.
In this study, ten plants, which varied in height and tiller number, were clonally propagated in the greenhouse by splitting the root ball. Eight clones of each plant were then transplanted back into the field for use in this study. Comparisons of shoot formation at three node positions (Low, Med and High) on the tiller indicated that shoot formation was greatest at the Low position. Three of the ten genotypes that were tested have increased shoot formation at Mid and High nodes. Comparison of two rooting conditions indicated that rooting in five genotypes were significantly different. Four of the five genotypes favor the rooting conditions of soil and commercial rooting powder, containing indole-3-butyric acid (IBA). Applicable for even novice propagators with limited tissue culture experience, this procedure utilizes common greenhouse material and is ideal for a teaching lab on bioenergy crops. It can also be scaled-up, for use in a breeding program, to replicate thousands of identical clones by including a six-week propagation followed by a two-month vegetative period.
In the first comprehensive analyses of wax ultrastructure and chemical composition of L- switchgrass, we described three unique cuticular wax variant types (NG, RG and
HG) from a population of 1,849 switchgrass plants. Gas chromatography with mass spectrometry and field emission scanning electron microscopy were used to compare visually identified cuticular wax variants to the most abundant ST switchgrass. ST had rod- and plate-shaped cuticle wax structures on the ab- and ad-axial surfaces, respectively. NG types have reduced amounts of total wax, reduced amounts of β- 15 diketones, and absence of rod-shaped wax structures, suggesting similarity to cer mutants in wheat (Triticum aestivum) and barley (Hordeum vulgare). This study demonstrates the potential genetic diversity for the glaucous trait present in switchgrass germplasm.
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INTRODUCTION
Chapter 1: Switchgrass (Panicum virgatum L.) 1. Introduction Switchgrass (Panicum virgatum L.) is a member of the Paniceae tribe in the grass family Poaceae. Native to North America, wild populations of switchgrass were found in areas below 55˚N latitude and east of 100˚W longitude, i.e. east of the Rocky Mountain
Range (1). One of the three dominant warm season C4 perennial prairie grasses, along with indiangrass (Sorghastrum nutans L.) and big bluestem (Andropogon gerardii
Vitman), switchgrass once inhabited the vast tall grass prairies, savanna, open woods, brackish marshes, and pinewood ecosystems of North America (2, 3, 4). Wild populations of switchgrass have a bunch type growth and reproduce by seed, tillers and rhizomes (1).
The taxonomic division of switchgrass, as distinct Upland (U-) and Lowland (L-) ecotypes, is based on plant morphology, chloroplast DNA (cpDNA) sequence and ploidy
(5, 6). Widely distributed above 34˚ N latitude in North America, U- ecotypes were described as being short plants having a semi-decumbent stature, narrow leaves and slow growth rate (4, 7). L- ecotypes, present below 45˚ N latitude in North America, were tall plants with broad leaves, thick stems and bluish color (1, 4). Most reliably distinguished by cpDNA sequence, L- ecotypes possess a single restriction site that is not present in U- ecotypes (6, 8).
Using light microscopy and flow cytometry, switchgrass is identified as having a basic chromosome number of x = 9 (9). In a major distinction, L- ecotypes were generally found to be tetraploid (2n = 4x = 36) and U- ecotypes were octoploid (2n = 8x
= 72). However, ploidy is not limited to these ecotype designations. Several wild 17 populations were found having varied chromosome counts, including U- tetraploids, L- octoploids and very rarely as infertile dihaploid plants (2n = 2x = 18) (10, 11, 12). The presence of aneuploidy, as deviations of the expected somatic chromosome number, was found in populations with naturally higher ploidy levels (10). Martinez-Reyna and Vogel
(13) described switchgrass as an allogamous, wind pollinated, obligate outcrossing species with an incompatibility system, present at both pre- and post-fertilization, and very low self-fertilization rates in both tetraploids (0.35%) and octoploids (1.39%).
However, a recent report of L- ecotype, cultivar (cv.) NL94, having 61.2% self- compatibility, under growth chamber conditions, indicates that seed formation is amendable (14).
Vogel (1) showed that switchgrass has susceptibility to biotic factors, including insect herbivores such as grasshoppers (Acrididae family), and to fungal pathogens including rust (caused by Puccinia species), smut (caused by Tilletia maclaganii), anthracnose
(caused by Colletotrichum graminicola), Elsione spot blot (caused by Elsinoe panici), spot blotch (caused by Bipolaris sorokiniana), Phoma leaf spot (caused by Phoma species) and Fusarium root rot (caused by Fusarium species). U- ecotypes of switchgrass have unique adaptations to the abiotic conditions found in northern regions, including drought resistance, cold tolerance and earlier senescence (15). Adapted to areas of flooding, L- ecotypes have greater biomass yields attributed to delayed anthesis and rust resistance (16, 1, 4). As a species, switchgrass is thought to retain resistance to a wide range of biotic and abiotic conditions in regional gene pools established on a latitudinal gradient, adapted to photoperiod and temperature, and along a longitudinal gradient, with drought and disease resistance (15, 4). 18
2. Micropropagation The Conger group, at the University of Tennessee, established protocols, in switchgrass, for the in vitro propagation of cells or tissues. Embryogenic calli (EC), masses of dividing pluripotent cells, were formed in mature caryopses and young leaf explants of cv. Alamo. The explants were placed on Murashige and Skoog (MS) agar media containing 30 g L-1 maltose and a combination of plant growth regulators (PGRs):
22.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and either 5 µM 6-benzylaminopurine
(BAP) or 45 µM BAP (17). Somatic embryogenesis (SE) was observed from EC re- cultured on MS media, without PGRs, and in the presence of light. Denchev and Conger
(18) optimized the procedure for SE, from mature caryopses, by combining PGRs, 11.3
µM or 22.5 µM 2,4-D and 45 µM BAP, in a four-week germination treatment followed by re-culturing EC on MS media, every 30 days. Gupta and Conger (19) developed a means for direct organogenesis from caryopses, of U- ecotype cvs. Trailblazer and
Blackwell and L- ecotype cv. Alamo, cultured on MS media with 4.5 µM 2,4-D and 18.2
µM 1-phenyl-3-(1,2,3-thiadiazol-5YL)-urea (TDZ).
Alexandrova et al. (20) achieved SE from flowering immature inflorescences on MS media containing 5 µM BAP. Axenic spikelets were re-cultured on MS media, with 22.5
µM 2,4-D and 5 µM BAP, for 4 weeks in the dark, prior to their transfer to MS media, devoid of PGRs. This technique is valuable for both in vitro fertilization, haploid anther culture and as a source of explants for gene transfer, using either micro-projectile bombardment or Agrobacterium-mediated transformation. In parallel, the in vitro propagation by direct organogenesis from the axillary buds of culm node segments was established on MS media containing 12.5 µM BAP. Using this technique, approximately
500 plantlets could be established from the culm nodes of a single donor plant in as little 19 as 12 weeks (21). This had the advantage of limiting the exposure to potentially deleterious growth regulators, reduced the regeneration time from agar-based EC techniques and allowed for selection of unique genotypes prior to cultivation.
In the first transgenic protocol developed for switchgrass, Richards et al. (22) used particle bombardment of ECs, established using the Alexandrova et al. protocol (20), to introduce a dual marker plasmid containing the visual marker green fluorescent protein
(GFP) reporter (sgfp) gene and selectable phosphinothricin acetyltransferase (bar) gene conferring herbicide resistance. In the first successful transformation of a forage grass using Agrobacterium tumefaciens strain AGL1, Somleva et al. (23) introduced a binary vector pDM805, carrying a selectable bar and β-glucuronidase (gus) genes, into EC of
Alamo. Somleva et al. (24) introduced an expression cassette pMBXS155 containing a functional multigene pathway capable of forming polyhydroxybutyrate (PHB), a renewable and biodegradable bio-based plastic. PHB accumulated in leaf tissue and in whole tillers at 3.72% and 1.23% dry weight, respectively. Xi et al. (25) used A. tumefaciens strain EHA105, containing the hygromycin phosphotransferase (hph) gene, to introduce a selectable marker with antibiotic resistance into EC, which enabled the early identification of transgenic plants.
In an initial report of type 2 calli in switchgrass, Burris et al. (26) extended the transformation lifetime of type 1 callus, by more than six months, by replacing MS media with LP9 media, containing 100 mg proline, 5 mg L-1 2,4-D, and no BAP or myo-inositol.
The type 2 callus is described as rapidly growing, friable, with higher viability, and is better suited for transformation by A. tumefaciens. Li and Qu (27) increased the transformation efficiency, up to 90% in switchgrass cv. Performer and 50% in cv. Alamo 20 and Colony, by initiating type 2 calli in MB media containing 5 µM 2,4-D, 1 µM BAP, 3 g L-1 Phytagel, 30 g L-1 maltose and L-proline. The enhancements to transformation efficiency and low transgene copy number, i.e. less chance for transgene silencing, associated with A. tumefaciens have made it the preferred transformation method in switchgrass.
3. Switchgrass Molecular Breeding 3.1 Molecular Markers (RFLP, RAPD, SSR, EST-SSR, EST-STS, AFLP and SNP) Restriction Fragment Length Polymorphism markers (RFLPs) rely on restriction enzymes to digest genomic DNA. Restriction fragments were visualized when separated by agarose gel electrophoresis, transferred via Southern Blot and hybridized with a probe.
Comparison of individuals and progeny allowed for the genotyping of a population, as homozygotes or heterozygotes, based on the RFLP allele that were present (28). Using
RFLPs, Hultquist et al. (6) distinguished L- and U- cytotypes by a change in a restriction site in chloroplast DNA (cpDNA). Martinez-Reyna et al. (29) observed cpDNA to be maternally inherited in reciprocal crosses of tetraploid L- and U- ecotypes. In the first low-density linkage map of switchgrass, constructed from 102 RFLPs, a single marker, created from a 49 bp deletion in trnL-UAA intron, was used to distinguish between U- and L- ecotypes (30, 31).
A number of molecular markers (RAPD, SSR, EST-SSR, EST- STS, AFLP and SNP) use polymerase chain reaction (PCR) to associate molecular variation with traits of interest. Random Amplified Polymorphic DNA markers (RAPDs) use short oligonucleotide primers and low annealing temperatures in PCR (28). PCR products were used to genotype several loci by comparison of the segregation analyses in parental lines and progeny. Using RAPDs, Gunter et al. (32) analyzed 91 polymorphic loci to verify the cytotype designations of cpDNA, made by Hultquist et al. (6). RAPDs require no prior 21 knowledge of DNA sequence, but also have a much lower resolving power than site- specific markers.
Microsatellite markers (SSR, EST-SSR and EST-STS) rely on primer pairs, in repetitive oligonucleotide sequences, to identify polymorphisms and distinguish homozygous and heterozygous alleles (28). In SSRs (Single Sequence Repeats), short tandem repeats, of 2-6 bp, were widely distributed throughout the genome and present in noncoding or regulatory regions (33). Expressed Sequence Tag - SSR (EST-SSRs) and
EST - Sequence Tagged Sites (EST-STS) markers were designed by using cDNA libraries to identify a gene (200-800 bp) or a predicted intron, respectively. Development of expressed SRRs was less costly to establish than genomic SSRs and were limited to gene-rich regions (34).
In the first described EST-SSRs in switchgrass, Tobias et al. (35, 36) created 32 primer pairs from cDNA sequences, in callus, crown and seed tissues, of switchgrass cv.
Kanlow. Tobias et al. (37) developed an additional 830 EST-SSRs in genes that play a putative role in cell-wall biogenesis. Narasimhamoorthy et al. (38) used 24 grass conserved and 39 switchgrass EST-SRRs to identify genetic variability and confirm ploidy designations of 31 populations of U- and L- ecotypes of switchgrass. Okada et al.
(39) described establishing primer pairs, as SSRs, EST-SSRs and EST-STS, in the construction of high density linkage maps of L- by L- ecotypes. Wang et al. (40) sequenced clones enriched in single sequence repeats and developed 1,030 SSRs and detected 802 polymorphic alleles in a cross of L- ecotypes. Zalapa et al. (41) used 55
SSRs and 6 cpDNA sequences to classify 18 cvs. by ecotype, ploidy and geographical origin. In 67 switchgrass accessions, Zhang et al. (8, 12) used 19 SSRs and 5 cpDNA 22 sequences to distinguish a high frequency of natural hybridization, propose that divergence, in ploidy and ecotype, began 1.5 – 1M years before present (ybp) and identified the central and eastern Gulf Coast glacial refuge as the primary origin in all but two switchgrass accessions. Liu et al. (33) used 499 loci to create a more saturated linkage map in a selfed generation (S1) of northern L- genotype, to gain a better understanding of heterosis breeding for the future use of inbred lines. Finally, Serba et al.
(7) used SSRs, EST-SSRs and diversity array technology (DArT) markers, for microarray-based chip analysis that used amplified PCR products of interest as probes, in the first high density linkage map of an L- by U- ecotype.
In another PCR-based technique, amplified fragment length polymorphisms (AFLPs) require DNA restriction with two enzymes and a restriction site-specific adaptor that is ligated to genomic DNA (45, 28). Todd et al. (42) used these dominant markers to amplify 452 polymorphic bands and distinguish the genetic diversity of U- and L- ecotypes. Although a powerful tool for the identification of polymorphisms, this labor- intensive method requires more skill and equipment than other molecular markers.
Alternatively, Single Nucleotide Polymorphisms (SNPs) employ a PCR-based method that relies on forward and reverse primers to detect variation at the nucleotide level (23).
Ersoz et al. (43) created 149,000 SNPs and 13 short-read sequences generated from
Illumina short read sequences and EST libraries. Although more rigorous data processing is required than in other molecular markers, the decreasing cost of sequencing will increase the usage of SNPs in linkage mapping.
3.2 Linkage Maps (Molecular Mapping) Linkage mapping of a polyploidy, such as switchgrass, relies on genotyping with molecular markers that segregate 1:1 in a full-sib pseudo-test backcross mapping 23 population (44, 45). The progeny of three crosses were used as mapping populations in four published linkage maps of switchgrass. Missaoui et al. (30) indicate switchgrass was an autotetraploid with a high degree of preferential pairing between homologous chromosomes, suggesting disomic inheritance. Okada et al. (39) found that L- switchgrass has 18 linkage groups, distributed into two highly homologous sub-genomes, with a 1.5 Gb (haploid) genome that confirms complete or near-complete disomic inheritance. Okada et al. (39) also suggest that switchgrass is an allopolyploid, created by the hybridization of two species, having disomic inheritance, i.e. two alleles at each locus, rather than behaving as a true tetraploid with tetrasomic inheritance, i.e. four alleles per locus. Liu et al. (33) found 18 linkage groups, with a cumulative length of
2085.2 cM, and homology between the nine linkage groups of NL94. Serba et al. (7) used comparative analysis to show highly collinear maps with similar recombination rates, suggesting chromosome exchange can occur between different ecotypes. The increased use of molecular markers will provide the basis for genetic research including quantitative trait locus (QTL) mapping, marker assisted breeding and enhance the understanding of heterosis breeding (46).
4. Development as a Biofuel Crop 4.1 Conservation, Feedstock Development and Agronomic Practice Since the 1940’s, interest in switchgrass for pasture, rangeland, and prairie soil restoration has stemmed from its broad adaptability on marginal land (1, 4). As part of the Soil Conservation Service (SCS), an agency of the United States Department of
Agriculture (USDA), seed collections from wild populations were used to establish switchgrass accessions at 15 Plant Materials Centers (PMC) (1, 4). Improvements to wild populations, identified in various regions of the US including Nebraska, North Carolina,
Oklahoma, and Wisconsin, capitalized on there between population genetic variability for 24 a number of traits, including establishment, yield quality, plant height, seed yield, disease resistance, and maturity (1, 15). Regionally adapted accessions of switchgrass were used to develop registered seed for release as a cultivar named after their region of origin, including cvs. Grenville (released 1940) and Blackwell (released 1944). In 1949, the first certified seed, or progeny of registered seed, U- ecotype cv. Nebraska 28 was released by the USDA at the University of Nebraska (ARS, Lincoln and Nursery Division; 47).
In 1978, the US Department of Energy (DOE) established the Bioenergy Feedstock
Development Program (BFDP), at Oak Ridge National Laboratory (ORNL), to evaluate herbaceous and short rotation forest systems on marginal lands for use in bioenergy (48).
The DOE transferred technical and administrative management of the Short Rotation
Woody Crops Program (SRWCP) to ORNL, and initiated the Herbaceous Crops
Research Program (HECP) in 1984 (49). As part of the 1985 Food Security Act, the
Conservation Reserve Program (CRP) was established to reduce soil loss on highly erodible agricultural land. The CRP, extended in the 1990 Farm bill (Food, Agriculture,
Conservation and Trade Act), enrolled 18 million hectares of land and dedicated 12 million hectares to forage and native grasses (50). During this time the agronomic practices for switchgrass were improved which aided its development as a bioenergy crop.
Screening trials, in 34 species, were conducted at six universities, including Auburn,
Cornell, Purdue, Virginia Tech, Iowa State University and North Dakota State, and an
Ohio based company, Geophyta (49). At Auburn University, stands of L- ecotype cv.
Alamo, planted in 1988, were reported as having high yields (up to 34.6 Mg (Metric Ton) ha-1) in unirrigated research plots and a consistently high 13-year average (23 Mg ha-1 yr- 25
1) was evidence of the lasting quality in yield (48, 49). At Purdue, U- ecotype switchgrass, cv. Cave-in-Rock, observed the highest biomass yield (17 Mg ha-1) of a perennial grass and average yields (9 - 12 Mg ha-1 yr-1) were more consistently observed.
The major findings of each region were that sorghum (Sorghum bicolor L.), switchgrass and reed canarygrass (Phalaris arundinacea L.) have a higher yield potential than other candidate species including pearl millet (Pennisetum americanum L.), bermudagrass
(Cynodon dactylon L.), sericea lespedeza (Lespedeza cuneate) Johnsongrass (Sorghum halenpsnse L.), tall fescue (Fesctuca arundinacea) and energy cane (Saccharin hybrid)
(49).
As congress imposed funding limitations on the DOE and HECP, concerns for potential erosion in annual grasses lead to the recommendation of a single candidate species. In 1991, the DOE designated switchgrass as a model bioenergy crop for the many attributes that it possesses, including established cultivation practices, high biomass yield, propagation through seed dispersion, perennial nature, and adaptability to poor soils (49). In 1992, the BFDP initiated seven switchgrass research projects, at five universities and two government laboratories, and the Natural Resources Conservation
Service (NRCS) expanded their field testing sites to include six USDA-National
Materials Testing Centers (51). Harvest yield performance comparisons of nine switchgrass cvs. indicate average yields of 16 Mg ha-1 (7.14 ton acre-1) in unirrigated plots (48). The maximum yields (> 20 Mg ha-1) were observed in the best switchgrass cvs. including Alamo in the southern latitudes, Alamo and Kanlow in mid latitudes and
Cave-in-Rock, Trailblazer, and Sunburst for northern latitudes of the US (51, 48). The ideal use of supplemental nitrogen was determined in establishment year (< 50 kg ha-1) 26 and was increased (80 - 120 kg ha-1) in the following years (52, 48). Comparisons of 1- and 2- cut systems indicated that in eastern regions of the US, a higher total biomass was observed when harvested twice annually, but was not observed in regions of Texas subject to drought stress (53). Over five years, the 1- and 2- cut systems averaged roughly the same biomass (27 Mg ha-1 yr-1), ultimately indicating superiority in a properly timed
1- cut system. The additional energy input in eastern regions and reduced crop yields in drought stressed regions discouraged the use of a two cut system (48).
4.2 Conversion of Lignocellulosic Biomass to Ethanol (Acid Hydrolysis, Enzymatic Hydrolysis and Simultaneous Saccharification and Fermentation (SSF)) Lynd et al. (54) described the fermentation to ethanol from various types of lignocellulosic biomass including agricultural residues, short rotation crops, herbaceous energy crops and municipal solid waste. Lignocellulosic biomass was found to be composed of cellulose (35-50%), hemicellulose (20-35%), and lignin (15-25%), with the remaining components known as extractives and ash (55). The isolation of fermentable sugars from cellulose, glucans composed of cellobiose, and hemicellulose, glycans composed of various sugars mostly xylose, were inherently more difficult than the well- established fermentation of starch and sugar (56). A number of processes, including Acid
Hydrolysis (AH), Enzymatic Hydrolysis (EH) and Simultaneous Saccharification and
Fermentation (SSF), were used to break down the fermentable polysaccharides and separate lignin, a non-fermentable phenylpropene polymer (57).
In an industrial scale bio-refinery, lignocellulosic biomass from woody and herbaceous crops, including switchgrass, were first mechanically milled to a small particle size. In separate processes, a short pretreatment of AH, a combination of moderate heat (140 - 160˚C) and low acid (1.5 % sulfuric acid), was followed by EH, sequential exposure to cellulase enzymes including endoglucanase, exoglucanase and 27 glucosidase, to break down the cellulose and hemicellulose into fermentable sugars (55).
The use of high heat, 200 – 240˚C, or high acid concentration were discouraged for their apparent drawbacks, including degradation byproducts (hydroxymethylfurfural, furfural and tar) and the absence of low cost recovery technology (57). In the final step, glucose- rich broth is transferred into a separate fermenter where yeast, Saccharomyces cerevisiae, converts the glucose into ethanol. In an alternative process, simultaneous saccharification and fermentation (SSF) utilized a combination of cellulase via fed-batch enzyme production, fungus (Trichoderma reesei) and yeast to produce higher yields of ethanol by immediate conversion of the available glucose and limiting sugar losses by out competing other microorganisms (54, 57, 58, 59, 60, 61, 62).
4.3 Early Breeding Efforts in Switchgrass In the 1950’s, breeding efforts at the University of Nebraska focused on the genetic improvements in forage quality for use in livestock production (4). Using In Vitro Dry
Matter Disappearance (IVDMD), a two stage acidification assay for laboratory simulation of rumen digestion, an increase in 40 g kg-1 of IVDMD was observed to increase cattle production by 67 kg ha-1 (1). Rapid cycling of high IVDMD switchgrass led to the development of switchgrass cv. Shawnee, a cross of cv. Trailblazer and high yielding cv. Cave-in-Rock (63). In 1988, Twidwell et al. (64) showed that a decrease in forage quality, as a reduction in N concentration and IVDMD, was observed in the month after anthesis. Inversely related to IVDMD, lignin content was found to be highest in the stem and increased with maturation. The variation in cell types of the leaf blade, composed mostly of parenchyma cells, and stem, having heavily lignified cortex, xylem, and bundle sheath, were found to influence digestibility (65). Sanderson and Wolf (53) observed reduced ash content by delaying harvest until after first frost, after nutrients (N, 28
P, and K) and carbohydrates were able to translocate from leaf tissue to the crown and roots. An increase in ash and K, were attributed to plants with a higher stem to leaf ratio and can lead to the accumulation of partial fused deposits on the walls exposed to heat, i.e. boiler slagging (51). This suggests that the forage quality of switchgrass is amendable and highly dependent on time of harvest. The attributes, of increased IVDMD and decreased ash and K, selected for in leafy plants were inadvertently also beneficial in the conversion to ethanol, via a reduction of lignin (51, 4).
In the 1990’s, the BFDP conducted switchgrass research in areas of best management practices, breeding, physiological characteristics, genetics and propagation. Research at
Oklahoma State University (OSU) focused on annual cycles of Recurrent Restriction
Phenotypic Selection (RRPS) aimed to increase the desirable genes by crossing the best yielding U- and L- ecotypes (51). After genetic gains were evaluated for three RRPS cycles, major constraints, including inadequate seed set, low correlations of biomass yield in the establishment year and strong environmental influences of plant development, limited the effectiveness of phenotypic selection, in the first year, as an indicator of biomass yield (51). In a modified recurrent genotypic selection (RGS), which extended the selection cycle to four years, it was determined that genotypic comparisons of half-sib progeny were a better indicator of breeding value as broad genetic populations were observed to correlate with an increase in yield, i.e. hybrid vigor (51, 48)
In 1996, switchgrass research at the University of Georgia was initiated to address environmental challenges faced in the southeastern US. Breeding selections based on the honeycomb plot design, that allowed for site variability interactions to be considered in parallel with genetic variability, led to the development of four synthetic lines that were 29 observed to have increases of 7.4% yr-1 and 30% gains over parental lines (66). This success stimulated the United States Department of Agriculture - Agricultural Research
Service (USDA-ARS) to establish smaller breeding projects, in Lincoln, Nebraska,
University of Tennessee and, Stillwater, Oklahoma, where gains in yield varied in L- ecotypes, 1-2% yr-1, and U- ecotypes, 3-5% yr-1, similar to heterotic breeding of corn
(Zea mays L.) during the 1930’s (51, 48).
5. Future Use as a Biofuel 5.1 Governmental Influences In the US, the 1990 Clean Air Act Amendments (CAAA) mandated the reduction of benzene and aromatic hydrocarbon additives in a phase-in of clean fuel, including gasoline containing 2.7% oxygen (1992) and 2% oxygen year-round (1995), for the nine worst cities to exceeded the National Ambient Air Quality Standards (NAAQS) for ozone
(O3) and carbon monoxide (CO) (54). Having a low volatility and toxicity, ethanol is inherently better for the environment than the combustion of fossil fuels that increase greenhouse gas carbon dioxide (CO2) emission, thereby trapping heat in the atmosphere i.e. climate change (57). In 1991, ethanol sold for $0.32 to $0.37/L ($1.20 to $1.40/gal) and 3.8 GL (1 billion gallons) per year were produced from corn grain (57, 67). Wyman
(67) estimated that the cost of ethanol from lignocellulosic biomass of $0.34/L
($1.27/gallon) was similar to that of producing ethanol from corn. Lynd et al. (54) estimated that the net energy return ratio (R = output:input) for energy production, accounting for all exported electricity, associated inputs and energy gained by combustion of lignin, of ethanol from lignocellulosic biomass (R = 5) was greater than that derived from corn (R = 1.2). McLaughlin and Walsh (68) estimated the net energy ratio, dependent on handling and harvesting, of switchgrass (R = 12.4), yielding 13.5 Mg ha-1 yr-1, to corn (R = 8), including stover and fiber as energy products. The available 30 technology in 1998, indicates that the energy required in fermentation of corn is 4.5 times higher than the simultaneous saccharification and fermentation (SSF) process, described above in Ch. 4.2, used in switchgrass. Taking this into consideration, McLaughlin and
Walsh (68) found the total energy ratio of switchgrass (R=4.34), including the SSF process, to be higher than that for corn (R=1.21), similar to the finding of Lynd et al.
(54). These attributes stimulated a decade of research by the BFDP to reduce the cost of ethanol from lignocellulosic biomass making it a more competitive gasoline replacement.
In the first large field scale trials of switchgrass, Schmer et al. (69) determined the net energy of cellulosic biomass using material inputs and agronomic practices available in
2001. On 10 farms, in North Dakota, South Dakota, and Nebraska, switchgrass field plots managed for bioenergy observed annual biomass yields of 5.2 - 11.1 Mg ha-1 (2.32 - 4.95 tons acre-1). Using a conservative conversion rate (0.38 L kg-1) for cellulosic biomass to ethanol, the harvested biomass translates to roughly 1976 - 4218 L ha-1 using approximately 91 Gal ton-1. Schmer et al. (69) estimate that switchgrass has a net energy yield (60 GJ ha-1 yr-1) that is 540% more renewable fuel produced than was consumed over five years and indicates that ethanol production results in 94% lower greenhouse gas emissions than gasoline (69). Advances in molecular breeding techniques, including the use of molecular markers and inexpensive genotyping, will accelerate yield gains, and enhancements to the conversion process will further encourage higher net energy yields.
The Biomass Research and Development Act of 2000 created the Biomass R&D
Technical Advisory committee to advise the Secretaries of Agriculture and Energy on the nation’s reliance on foreign oil. Upon suggestion, Congress envisioned a 30% replacement of current petroleum consumption with roughly 227 billion liters (60 billion 31 gallons) of ethanol by 2030 (70). In 2002, the DOE’s crop production research was temporarily de-emphasized, effectively removing the BFDP, until renewed interest began after the publication of the ‘Billon Ton Study (BTS)’ in 2005 (71). The BTS outlines that meeting the goal of 30% fuel replacement, is achievable but requires more than 1 billion dry tons of biomass annually from agricultural lands, allocated as crop residues, perennial crop, grains, forestry bi-products and animal manures, while also meeting food and feed demands. The fermentation of sugar and starch from first generation crops, including sugarcane (Saccharum officinarum L.) and corn, lost favor due to the limited production outside of the subtropical regions of the US and low net energy gained, respectively (49).
The Renewable Fuel Standard (RFS2), as part of the Energy Independence and
Security Act of 2007 (EISA), mandates an increase in advanced biofuels to 21 billion gallons by 2022 and a cap of 15 billion gallons from corn (72). A number of non-food crops, that can be grown on marginal lands, have been considered for second-generation lignocellulosic bioenergy crops, including switchgrass, Miscanthus giganteus, energy cane, elephant grass (Pennistum purpureum), Napier grass (Pennisetum purpureum),
Agave species, and poplar (Populus trichocarpa) (72). Recent updates to the BTS, known as the 2011 U.S. Billion Ton Update (2011 BT2) and the 2016 Billion Ton Report
(BT 2016), have enhanced the original BTS by providing an economic model of feedstock availability that includes the projections for a large range of potential sources of bioenergy, including algae species Chlorella sorokiniana (freshwater) and
Nannochloropsis salina (saline water), waste and a wide array of individual bioenergy crops. In addition to cost predictions of feedstock supplies delivered to potential bio- refineries, the BT 2016 report assesses the current and projected use of advanced biofuels 32 targeted by RFS2. This indicates that cellulosic ethanol and advanced biofuels were well behind the expected target values as of 2015 and that biodiesel was found to have surpassed its expected usage. This is not entirely unexpected as ethanol is subject to the
E10 wall, limiting usage, and biodiesel is not subject to any such blending restrictions
(73). By 2022, the largest projected gains were still expected to come from cellulosic by the advancement in molecular breeding techniques and fermentation processes.
5.2 Crop Improvements (Artificial Seeds, S-Z- Incompatibility and Reduced Lignin) As a dedicated energy crop, the focus of switchgrass breeding is to increase total yield and enhance the plant composition for the conversion of biomass to ethanol.
Current yield increases of 20-30% were observed by heterosis breeding but seed viability is around 1%. Synthetic (artificial) seeds, using Sodium Alginate and CaCl2, have been used in several species including papaya (Carica papaya L.) and alfalfa (Medicago sativa
L.; 74, 75). Propagation of a single or few high yielding elite genotypes, via synthetic seeds, would provide an immediate increase in yields, by reducing genotypic variability, with little associated costs. In the first report of switchgrass cultured on liquid media,
Gupta and Conger (76) developed fine embryogenic suspensions by culturing EC on MS liquid media. Odjakova and Conger (77) found that in 10 day old EC, an osmotic pretreatment of sorbitol and mannitol increased SE initiation. Mazarei et al. (78) observed type 2 calli, similar to Burris et al. (25), after 8 weeks of replacing EC suspensions with liquid MS media containing 9 µM 2,4-D, 4.4 BAP and 30 g L-1 maltose. Type 2 calli would make an ideal meristematic tissue for use as synthetic seeds.
In an outcrossing species, thought to contain a S-Z- incompatibility mechanism, switchgrass is inherently heterozygous at many loci (13). Heterosis is observed at the tetraploid level and genetic maps suggest that chromosomal exchanges, between U- and 33
L- ecotypes, allow for the introgression of favorable traits (79, 7). Using linkage maps,
Okada et al. (38) observed segregation distortion in two linkage groups (I-a and VII-b) thought to carry orthologs of the S-Z- self-incompatibility loci. Serba et al. (7) found the linkage group VII-b to contain an ortholog to the Z- loci in rye (Secale cereal) and tentatively localized S- loci to homolog group III. In comparison, Liu and Wu (14) provided a linkage map of the self-compatible genotype (NL94).
In addition to increased yields, crop improvements can be made in the amount of available lignin present in vivo. Lignin is present in both the middle lamella and secondary cell wall, where it forms complex hydrophobic polymers that play a role in plant defense and structural rigidity. Common in grasses, the type 2 cell wall structure is composed of fermentable polysaccharides (cellulose, hemi-cellulose and pectin) and phenolic polymer lignin. The lignin monomers p-coumaryl alcohol, conifer alcohol, and sinapyl alcohol, synthesized as part of the shikimic acid and phenylpropanoid pathways, were polymerized, via oxidative coupling, to p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) residues, respectively (79). The biosynthetic genes for lignin production have been identified in the model organism Arabidopsis thaliana. In alfalfa (81), downregulating early precursors, C3H and HCT, in the lignin bio-synthetic pathway, were found to reduce biomass by 40%, and increase sugar production by 166%. This suggests a correlation in lignin and fermentable glucose production, thus reducing the cost of AH in ethanol production. In sorghum, Sattler et al (82) indicate that, in novel bmr loci, lignin reduction by measure of Klason lignin, ash corrected residue after two stage AH, did not correlate with increased glucose yield after EH. Although the potential benefit for enhanced ethanol yields by a reduction of lignin has not currently been 34 decided, both studies suggest that a conformational change in lignin residues, as lower
S/G ratio due to a reduction in S-residues, is possible and may be enhanced in double mutants.
35
PRESENT STUDY
The following includes one chapter and a brief description of the major finding of two manuscripts, found in Appendices A and B. Please reference the appendices for the materials and methods, results, discussion and conclusions.
Present Study (Chapter 2, Appendix A and Appendix B) 1. Chapter 2: Mutagenesis and Genetic Improvements of Panicum virgatum This chapter describes the creation of an Ethyl Methanesulfonate (EMS) mutagenized population of switchgrass cv. Alamo. Our initial interests were in screening, via bagging and seed count, for dominant mutations leading to self-fertility and apomixis. Similar to the expected rate of dominant mutations (1%) observed in Arabidopsis, we identified variegation in 2.97% of the M1 plants as sectors of yellow-green or white tissues in the leaves and/or stems. Arabidopsis has two Genetically Effective Cells (GECs) and
Panicum likely has four GECs, so it is not unexpected to find double the number of induced sectors. In addition, it was not the goal of this mutagenic treatment to collect seeds from the M1s and it is likely that the mutagenic treatment used in this study was greater than that typically used for Arabidopsis. From 2011 – 2012, 1,482 bagged plant tillers were collected and ranked (1 - 5) for seed formation. A mean seed formation (1.28
+ 0.78) indicates seed counts of < 30 were most common. In the second year, we retested the 19 plants that had the highest seed formation in our first year’s baggings but the higher number of selfed seeds were not repeated. At that point we determined the experiment to be unsuccessful. However, while scoring the field, we identified three variant types of plants having altered leaf surface wax (Non-Glaucous (NG), Reduced
Glaucous (RG) and High Glaucous (HG)). The propagation and biochemical 36 characterization of these cuticular wax variants are the primary focus of the remaining sections in the dissertation.
2. APPENDIX A: A Simplified Technique for the Propagation of Shoots from Nodes of Switchgrass (Panicum virgatum L.) Genotypes In this manuscript, my coauthors and I describe a propagation technique using common greenhouse materials and a simplified procedure for novice propagators. In this procedure, three node positions (Low, Mid and High) were dissected from plant tillers harvested from eight replicates of ten randomly selected genotypes of L- ecotype switchgrass cv. Alamo. Shoot formation was highest from low culm nodes incubated in a simple DFT style hydroponic system for six weeks. Excised shoots from the low nodes rooted best in soil and commercial rooting powder (Rootone) conditions after four weeks.
The two genotypes with the highest percentage shoot formation at Mid (90%) and High
(60%) nodes, were also identified to have 90% root formation in RC1. In each of these gentoypes, a single plant containing an average of 150 tillers is estimated to produce 648 identical rooted plants in as little as 10 weeks.
3. APPENDIX B: Cuticular Wax Variants in a Population of Switchgrass (Panicum virgatum L.) This manuscript is the first comprehensive cuticle analysis, including ultrastructural and chemical composition, of a L- ecotype of switchgrass. In a population of 1,849 outcrossing plants, 92 wax variants were identified based on visible glaucousness. My coauthors and I describe a standard type (ST) switchgrass cv. Alamo as having moderate amounts of visible glaucousness (bluish-white color). Analyses of leaf wax content and composition using GC/MS confirm the presence of C33 β-diketone and C33 hydroxyl-β- diketone. In ST, we describe the presence of rod- and plate-shaped structures on the ab- and adaxial surfaces, respectively. We define three variant types of switchgrass (NG, RG 37 and HG). The NG types were observed to have lower visual glaucousness, devoid of rod shaped wax structures with reduced amounts of β-diketones. These NG types were the first visual glaucous variants described in switchgrass and were similar to cer mutants in wheat and barley. This study demonstrates the potential genetic diversity for the glaucous trait in switchgrass germplasm.
38
Chapter 2: Mutagenesis and Genetic Improvements of Panicum virgatum 1. Introduction Switchgrass (Panicum virgatum L.) behaves as an allotetraploid and contains two copies of two subgenomes (AABB) that are inherited as a haploid (38). As an obligate outcrossing species, the preference for heterozygosity is impeding progress toward making switchgrass amenable to genetic studies. The ability to conduct future genetics experiments on switchgrass will rely on the identification of a fertile diploid plant, an increase in self-fertility, a plant that exhibits a high level of apomixis or the ability to do vegetative propagation, via asexual tissue culture. The original interest in creating an
EMS-mutagenized population in switchgrass was to screen, via bagging and increased seed set, for dominant mutations leading to increased self-fertility and apomictic plants.
For a mutation to be transferred to a subsequent generation it must be present in one of the Genetically Effective Cells (GECs) of the embryo or in their progenitor cells. In angiosperms, seed formation can occur through a sexual pathway, leading to the fusion of gametes, and by asexual reproduction of sporophytic or gametophytic origin (83). We originally aimed to identify increased self-fertility and/or apomixis, by screening a population of 1,849 M1 plants. Similar to the expected rate of dominant mutations (1%) observed in Arabidopsis, we identified how many M1 plants (2.97%) with sectors of yellow-green or white tissues in the leaves and/or stems.
1.1 Diploid Switchgrass Native switchgrass stands are observed to have a wide range in ploidy levels that are found primarily in L- and U- ecotypes as tetraploids (84) and octoploids (9). Naturally occurring diploid switchgrass plants, containing two sets of chromosomes, were isolated from Chippewa Falls, Wisconsin (84). Conger (85) describes an inefficient means for producing diploid switchgrass, via anther culture, but the tissue origin for shoot 39 formation, was not determined. Christian Tobias, at the USDA in Albany, identified two dihaploid switchgrass plants (2n = 2x = 18), via fusion of two reduced gametes, in a sexual cross of L- ecotypes, cvs. Alamo and Kanlow (11; 86). These plants were small in stature, functionally sterile with reduced vigor, limiting their ability for asexual propagation through cuttings (11). Using florescence in-situ hybridization (FISH) analysis, Young et al. (86) identified a plasmid probe CentC, developed from the maize centromere, which was able to identify a dihaploid plant (ALB280), described in Young et al. (11). Tobias suspects that a low level of apomixis is present in some populations of switchgrass and the research using CentC is ongoing (Tobias per. comm., 2017).
1.2 Self-Fertility An S-Z- incompatibility system is present in switchgrass, as two independently segregating genes with a series of alleles that control self-fertility similar to that found in other perennial grass species (87; 13; 7). The complementary interaction of S- and Z- genes, of female gametophyte and pollen grain, initiates fertilization and double fertilization leads to the formation of normal size brown seeds. Martinez-Reyna and
Vogel (13) observed a very low self-fertilization rate that was present in both tetraploids
(0.35%) and octoploids (1.39%). The presence of 61.2% self-compatibility, in L- ecotype switchgrass cv. NL94, provides evidence that seed production is amendable under growth chamber conditions (14).
In pre-fertilization incompatibility, Martinez-Reyna and Vogel (13) indicate fertilization is inhibited when both S- and Z- genes match between the pollen grain and stigma. Pre-fertilization incompatibility can be identified by small seed with either small or shriveled endosperm. Small seed is indicative of a failure in embryo development and could be the result of either incompatible pollen or post-fertilization incompatibility, via 40 chromosome imbalance preventing intermating between ploidy levels. In accordance with the endosperm balance number (EBN) hypothesis, switchgrass requires a 2:1 ratio, maternal to paternal genetic contribution, for proper embryo and endosperm development
(88). For example, a tetraploid by octoploid cross has unique seed characteristics determined by ploidy of the female parent. When the female is of lower ploidy, a small shriveled seed will form and at higher ploidy, a floury endosperm will form.
1.3 Apomixis Apomixis refers to the asexual reproduction of seed containing embryos that are genetically identical to the maternal genotype (89). The embryo will develop without fertilization of the egg cell, referred to as parthenogenesis (83). In theory, apomictic seeds could be a valuable means for crop improvement useful in maintaining a maternal variant or genotype exhibiting heterosis. As most apomictic species are facultative, the presence of epigenetic barriers and effects of gene dosage have limited the introgression of apomictic traits into crop plants (90).
In apomixis, the origin of tissue leading to the formation of an embryo are either an unreduced egg cell (gametophytic) or maternal sporophytic tissue (sporophytic) (89).
Gametophytic apomixis are further distinguished by a reduction of the megaspore mother cell (MMC) in meiosis or as an unreduced MMC directly entering into mitosis, referred to as apospory or diplospory, respectively. In apospory, mitosis occurs in a diploid somatic cell positioned next to the MMC, referred to as the aposporous initial cell, rather than in a haploid megaspore. While in diplospory the MMC may immediately begin mitosis or enter meiosis before aborting the process (83). In addition to apomeiosis, a bypass of meiosis in embryo sac formation, and parthenogenesis, the female gametophyte in gametophytic apomixis may vary in the number of cells and their ploidy leading to an 41 imbalance in the male to female ratio of endosperm. The fertilization of the endosperm is required for pseudogamous apomicts but an unfertilized central cell is also possible, in the less common autonomous apomicts (90). This is unlike that of sporophytic apomixis, where development of the embryo sac, following the sexual pathway, still occurs and a sexually derived embryo may also be present (89).
1.4 Ethyl Methanesulfonate (EMS) Induced Variation Naturally occurring variation is essential to crop improvement but often does not occur frequently enough to introduce traits of interest into elite lines. Exposure of seeds to mutagenic agents including radiation (X- and ᵧ- rays) or chemical agents, such as Ethyl
Methanesulfonate (EMS), are commonly used to introduce variation for a desired trait
(91; 92; 93). Genetic studies using EMS mutagenesis are described in plants including, A. thaliana (94), barley (95; 96), corn (97), rice (Oryza sativa) (98), Sorghum bicolor (99), wheat (100), pearl millet (Pennisetum glaucum) (101) among many other plant species.
Considerable research in the model plant Arabidopsis has led to the characterization of many EMS induced mutants.
In Arabidopsis, there are three cell layers in the “tunica-corpus” type Shoot Apical
Meristem (SAM). The outer “tunica” layer, is composed of two cell layers including the outer most layer (L1) that forms the epidermis and the second layer (L2) containing the sub-epidermal layer of the palisade mesophyll and abaxial spongy mesophyll tissues. The inner “corpus” layer contains the third layer (L3) that forms the deep mesophyll and vascular tissue (102). Mutations that arise in distinct cell lineages, as periclinal (complete cell layer) and mericlinal (partial cell layer) chimeras, affect photosynthesis and differ in genotype from there adjacent tissues. Sectorial chimeras also result from a mutation in 42 the Genetically Effective Cells (GECs), or a progenitor cell, and give rise to mutated tissues that traverse each of the SAM layers.
Typically, Arabidopsis seeds are treated with a 20 - 100 mM EMS solution for 8 - 20 hours and rinsed with water to remove residual EMS, before planting onto moist soil
(103). In low doses, EMS provides an abundant source of point mutations, having a strong bias for G/C to A/T transitions, and a low level of chromosome breaks, leading to reduced fertility (104; 105). In the protein-coding region the mispairing of nucleotides, forming nonsense, missense and silent mutations, results in the conversion to a stop codon, codon substitution (conservative and non-conservative) or one that does not alter the amino acid sequence, respectively (106).
Only mutations in the GECs, giving rise to the sporogenous tissue, are passed to the next generation. Induced recessive mutations are not possible to visualize by phenotypic changes in the mutagenized (M1) seeds/plants, and identification of recessive mutants require screening of the M2 generation in a self-fertilizing species. Dominant mutations, in the M1 generation, have been identified in Arabidopsis, rice and rye, among other species (107; 108).
Variegation refers to any plant having somatic tissues containing altered pigmentation
(yellow or white). The green leaf sectors contain cells that have morphologically normal chloroplasts. The pigmentation sectors contain plastids that are deficient in chlorophyll and/or carotenoid pigments. Variegated plants occur by mutations, in the nuclear, chloroplast and mitochondrial genes, leading to neighboring cells with genetically distinct cell lineages, referred to as chimeras (109). In addition to chimerism, variegated plants can also occur by a number of mutations including transposable elements, RNA 43 silencing, plastome mutations, mitochondrial genome mutations and plastid-nucleus incompatibility (109). In a dominant mutation, the transmission of the variegation trait often does not have nuclear genetic basis and does not obey Mendel’s law of segregation i.e. maternal inheritance. In the ideal mutagenesis treatment, leaf sectors, occur in 1% of the M1 plants, often as part of a leaf but it can be as much as half of the plant in a species with two GECs (110; Feldmann per. comm., 2017.).
A good indicator of how well the mutagenesis worked in the M1 generation is percent germination and fecundity, ability to produce offspring, of the M1 plants. Typically, a dose response curve is used to establish the ideal EMS treatment regime. The number of
GECs in a self-pollinating species, such as Arabidopsis, is calculated by the segregation ratio of seeds in the M2 generation (111, 112). A mutation in one of the GECs is transmitted to the M2 generation, 1/8th of the progeny for a recessive mutation will be homozygous (92; 111).
2. Materials and Methods 2.1 Plant Material and Growth Conditions P. virgatum (switchgrass) seeds of L- cv. Alamo were kindly donated by Dr. David
Bransby, formerly of Auburn University. In spring 2011, a dose response curve was created by exposure of switchgrass seeds to EMS (ethyl methanesulfonate, Sigma-
Aldrich, LLC, St. Louis MO) in five increasing EMS concentrations (0, 40, 80, 160 and
320 mM) each at two exposure times (8 and 24 hours). The dose response curve indicated that 80 mM EMS, for 8 hours, was the best treatment and larger batches of seeds were treated with interest for transplantation to the field. In total 4,000 seeds were used for each EMS concentration and exposure time. The weight of 2000 seeds (1.95 g) were allocated, in two replications, into each of 18 conical tubes (15 ml) containing 10 ml of
100 mM potassium phosphate, EMS and buffered to pH 7.5. The tubes were shaken 44 vigorously by hand for several minutes and then set horizontally in plastic holding containers on top a shaker table, set to 140 rpm, and placed inside an incubator, set at
26˚C (Ch. 2 Fig. 1A). After the appropriate amount of time, the EMS was dumped off and the seeds were rinsed three times with distilled water and air dried on filter paper for
30 minutes, prior to planting (Ch. 2 Fig. 1B).
Dried seed from each tube, containing 2,000 seeds, was divided in half and approximately 1,000 seeds (from the replicate tubes) were planted into each of four half flats (STI-201, T.O. Plastics, Clearwater MN), with a total of 4,000 seeds planted for each concertation and exposure time. The flats contained a soilless mix of 3:2 ratio of
Sunshine mix #1 (LC1, Sun Gro Horticulture Canada Ltd, Seba Beach AB, Canada) to
Vermiculite (Medium grade, Thermo-O-Rock, Chandler AZ) amended with 3 tbsp. time release fertilizer (Osmocote®, Scotts-Sierra Horticultural Products Company, Marysville
OH). The seeds were topped with 1/8th inch layer of soilless mix, watered, and covered with a humidity dome (2010 Tray, Landmark Plastic Corporation, Akron OH). The flats were transferred into a cold room for three days at 4˚C. The flats were transferred to the greenhouse and germinated beneath two layers of 30% shade cloth. A layer of shade cloth was removed after week two and again at week three, at which time the humidity domes were removed completely (Ch. 2 Fig. 1C). In Table 1, the germination data, collected at three weeks, were compared by χ2-test using JMP software, version 12 (SAS
Inc. Cary, NC). The statistical significance was assessed at the 5% probability level unless otherwise indicated.
Three weeks after germination a total of 3,476 plants, M1 and Control (0 mM EMS), including both rounds of mutagenesis, were transplanted into Ray Leach “cone-tainers” 45
(SC - 10 Super- White Low Density, Hummert International, Springfield MO) containing a soilless mix. Prior to their transfer into the field, all plants were cut back to 30 cm and hardened off by exposure to ambient temperature and humidity inside a shaded head house. Transfers took place in two batches, on March 30, 2011 and May 10, 2011, and individual plants were spaced in a grid containing 47 rows each with between 47 to 102 equally spaced plants (61 cm between plants and 91 cm between rows). The plants were hand watered, with a hose, immediately after planting and twice weekly for the first month.
All field experiments were conducted at the University of Arizona’s West Campus
Agricultural Center (CAC) (32˚ 13’48” N, 110˚ 57’11” W; elevation 776 m). The West
CAC soils are a Vinton Soil Series and greater than 1 m deep (113). Flood irrigation, provided up to six times per year, was slightly alkaline and high in calcium deposits. The semi-arid climate (low relative humidity RH < 20%) has annual precipitation concentrated in a bimodal fashion during the summer monsoons, July - September, and during winter storm months, December - January. From 2011 - 2013, the temperatures were slightly higher than previous 30 - year average (69.4˚F) and June 2013 was the hottest on record with a monthly average high temperature of 105.4˚F. Tucson observed annual temperatures of 69.9, 71.3 and 70.8˚F, respectively. A near average annual precipitation observed in 2011 (31.06 cm) decreased in both 2012 (20.09 cm) and 2013
(21.67 cm), respectively. Weather data were collected from the National Oceanic and
Atmospheric Administration (NOAA) website (114).
2.2 Screen for Self-Fertility and Apomixis In August, bagging was conducted during the R1 stage of reproductive floral development (115). Three tillers, of similar height, from separate sides of a single plant 46 were covered with a Lawson pollination bag (no. 404 Tassel Bag, Lawson Bags Co. Inc.,
Illinois, USA). The tillers were forced into a side perforation, with the bag folded onto itself around the tillers, leaving room for the panicles to expand. The bags were stapled
(P22 plier type stapler, Arrow Fastener Co., LLC, New Jersey, USA) shut to secure the tillers and reinforced across the top seam (Ch. 2 Fig. 3). The bags were labeled, using wax pencils, to identify the row and plant number. In September, approximately 40 bags were reapplied on newly flowering tillers, as previously described, after the first bags were removed by adverse weather conditions. In December, bags were collected using gardening shears to sever the tillers and any exposed panicles were discarded. Pollination bags were stored in black garbage bags, transferred to the UA 6th street greenhouse and seeds collected over several months. In 2012, bagging was conducted to capture any elusive plants missed in the first year’s screening and to replicate some of the findings. In the second year, 19 plants, with increased seed formation in the first year, were re-bagged with up to three bags, depending on their tiller number.
2.3 Cleaning Seed In December 2011, seeds were collected, from each pollination bag, by threshing the panicles between a rubber mat, with slotted grooves, and a hand pad. Large leaves were removed prior to threshing and stem debris removed prior to screening. Seeds were cleaned using a set of three screens (4.5/64, 3.55/18 and 2.5/64, Seedburo Equipment
Company, Chicago, USA), placed in a stainless steel pan and self-blown to remove excess chaff. Count data for normal size brown colored seed formation was scored on a five-point scale with 1 indicating < 30 seeds formed and 5 indicating > 120 seeds formed.
The seed were stored in coin bags (no. seven coin, Colombian, Minneapolis, USA). The 47 seeds were placed in a light-tight metal box and stored in a climate-controlled room. For seed formation, we calculated the mean, standard deviation, median, mode and range.
2.4 Scoring Plant Morphology In May 2012, scoring of ten morphological traits, including plant color, plant health, plant height, tiller angle, tiller number, stem width, leaf carriage, leaf width, leaf curling and variegation, was conducted on field-grown plants during the second year. Plant color/reflectance was scored on a 4 - point scale with 1 having bright green (glossy) appearance, 2 being light green, 3 being green/blue with blueish white (glaucous) and 4 having leaves with an abundance of glaucousness. Morphological traits, including plant health, plant height, tiller angle, tiller number, leaf width, variegation, were scored on a 4
- point scale based on severity. Plant health was scored a 1 indicating healthy tissue, no wilting or necrosis (biotic or abiotic stress), and 4 indicating wilting or necrosis present in
> 75% of the plant. Plant height was scored with 1 being less than 1 ft. and 4 being greater than 4 ft. As indication of plant stature, tiller angle was scored with a 1 representing procumbent (parallel to soil surface) and 4 representing erect (90˚ with the soil surface). Tiller number was scored with 1 indicating < 25 tillers and 4 indicating the presence of > 125. Leaf width was scored with 1 being < .4 in. and 4 being > 1 in.
Variegation was scored with 1 indicating no variegation and 4 indicating variegation on >
75% of the plant.
Morphological traits of leaf curling, stem width and leaf carriage were scored on a 3 - point scale. Leaf curling was scored with 1 being no curl and 3 being tightly curled. For stem width 1 indicates small stems < 6 mm and 3 indicate a thick stem > 12 mm. Leaf carriage was scored from 1 erect to 3 droopy. For the entire field (n = 1849), we calculated the mean, standard deviation, median, mode and range for all traits (Table 2). 48
3. Results Seed germination was observed to be highest in the control (0 mM EMS) with significantly lower (P < 0.001) seed germination found at each increasing EMS concentrations (40, 80, 160, and 320 mM) and exposure time (8 and 24 hrs.; Ch. 2 Table
1). As a percentage, the seed germination of the 0 EMS control and increasing (40, 80
160 and 320 mM) EMS concentrations for 8 hrs., were 23.6, 13.7, 7.3, 2.3 and 0.3%, respectively. The germination in similarly increasing (40, 80, 160 and 320 mM) EMS concentration for 24 hrs. were 3.8, 0.7, 0.05 and 0.0%, respectively. An EMS dose response curve is presented (Ch. 2 Fig. 2) as a percentage of seed viability, or number of seeds able to germinate under appropriate conditions, calculated from Ch. 2 Table 1. All surviving M1 plants were transplanted to cone-tainers and to the field. All further experiments focus on the population of 1,849 M1 plants (80 mM EMS), in rows 1 - 29.
From the M1 population, Standard Type (ST) plants with green/blue leaf color were most abundant and possessed a moderate amount of glaucousness (bluish white) on both ab- and ad-axial leaf surfaces. Plants possessing color that varied from the ST, including light green (n = 136) and purple (n = 26), were not observed to vary in the amounts of visible glaucousness. However, an altered leaf surface reflectance, and total wax amount, was most apparent in the three variant types (Non-Glaucous (NG; n = 3), Reduced
Glaucous (RG; n = 14) and Highly Glaucous (HG; n = 75)) found throughout the field.
The NG types have a bright green abaxial leaf surface which appears glossy, with a glaucous adaxial leaf surface, similar to ST. On the abaxial surface, the RG types appeared less glaucous than ST and the reduction in glaucousness, on NG and RG, is indication of a general reduction in leaf wax. Detailed comparisons of the variant types and ST are discussed further in Ch. 4. 49
Plant height is the greatest indicator of biomass yield (Casler per. comm., 2013).
Morphological variants were identified in the population, including 44 tall plants (> 4 ft.), and 110 short plants (< 2 ft.). Six of the short plants were also found to contain an abundance of leaf glaucousness, similar to HG types. Fifty-five variegated plants with pigmentation sectors on leaves and tillers were observed. Two plants with sectored tillers, i.e. albino, were identified during transplanting and maintained their phenotype in the field during the first year (Ch. 2 Fig. 1A - B). Eight plants contained sectored leaves with light pin striping on one leaf (Ch. 2 Fig. 1C). Forty-three plants were observed to have yellow-green sectors on one leaf and a single plant was observed with a glossy sector (not shown). Leaf sectors on just one or a few tillers, is probable indication of an induced mutation in a single region of the SAM. One variegated plant was observed having a pronounced leaf dis-coloration with green mid-veins on the entire plant, likely a naturally occurring variation (Ch. 2 Fig. 1D).
In 2011, a total of 1,482 bagged plant tillers were collected and the mean seed formation (1.28 + 0.78) indicates that seed counts of < 30 are most common. Seed formation was observed to have a median, mode, and range of (1, 1, 4), respectively.
Bags collected with seed formation > 120 were observed 37 times. To confirm the seed formation in plants from the first 15 rows, a subset of 19 plants, with elevated seed formation, were re-bagged in the following year and remaining 18 plants were not considered. From these 19 plants, one had a similar seed formation, 17 were significantly lower, and one was not collected (data not shown). Over winter two variegated plants, having albino leaf sectors, did not retain the trait past the first season.
50
4. Discussion We observed an inverse correlation in seed viability with increased concentration (40
- 320 mM) and duration (8 and 24 hrs.) of exposure to the chemical mutagen EMS. These findings are similar to that described in A. thaliana, at similar concentrations (11 - 100 mM) and duration (0.5 - 54 hrs.; 91; 92). In addition to embryo lethality, variegations identified in 2.97% of the M1 plants, as sectors of yellow-green or white tissues in the leaves and/or stems, are indicative of dominant mutations in GECs or their derived cells lineages. As indication of abiotic stressors, leaves with light green color could be indication of a lack of available soil nitrogen and a purple coloration is generally associated with anthocyanins. However, variegated plants are caused by mutations in the nuclear, chloroplast and mitochondrial genes that lead to pigmentation sectors and contain plastids that are deficient in chlorophyll and/or carotenoids. In an obligate out- crossing species, the inherent heterozygosity is apparent when considering the range in observed plant morphologies. As it is not possible to visualize any phenotypic changes due to recessive mutations in the M1 generation, the cuticular wax variants are likely due to the natural variation present in switchgrass cv. Alamo.
Self-fertility and apomixis were pursued as a means to improve switchgrass for genetic studies. As such, we attempted to induce dominant mutations that might result in increased seed set indicating that we had affected self-fertility or apomixis. In the first year of bagging, we did find 19 plants that produced more seeds than normal. However, in repeating the bagging on these plants in the 2nd year, these results were not repeated.
This indicates that either the S-Z- allele combination of tillers bagged in the second year were similar, however it is unlikely with multiple bags, or possibly that seeds in the first 51 year were formed by pollen contamination due to bagging after inflorescences were already fertile.
5. Conclusions In the first reported chemical mutagenesis experiment on switchgrass, we successfully established a population of 1,849 M1 switchgrass plants. We were not successful in identifying plants having more self-fertility or apomixis. As seed formation in an obligate outcrossing species is very unlikely, we maintain that this strict selection criterion makes it the ideal situation for the identification of dominant mutations affecting seed production. In our phenotypic screening we identified a large amount of heterogeneity in the population. While scoring the field, we identified three variant types of plants having altered leaf surface wax (Non-Glaucous (NG), Reduced Glaucous (RG) and High Glaucous (HG)). The propagation and biochemical characterization of these cuticular wax variants are the primary focus of the remaining chapters in the dissertation.
52
Chapter 3: Research Findings 1. Summary 1.1 EMS Population In the Control, seed germination (23.6%) in L- switchgrass (Panicum virgatum L., cv.
Alamo) is observed to be similar to the expected diploid segregation ratio (9:3:3:1) for two genes (S-Z- genes), where ¼ of the seed are heterozygous and produce viable progeny. At increasing EMS concentrations (40, 80, 160, 320 mM) and exposure time (8 and 24 hrs.), seed germination rates were observed to be significantly lower (P < 0.001) than Control. In the first reported chemical mutagenesis experiment on switchgrass, we successfully created a field-established population of 1,849 M1 plants
In scoring ten naturally occurring morphological traits, including plant color, plant health, plant height, tiller angle, tiller number, stem width, leaf carriage, leaf width, leaf curling and variegation, we identified a large amount of heterogeneity in the 1,849 plants.
The visible glaucousness of three variant plant types (Non-Glaucous (NG), Reduced
Glaucous (RG) and Highly glaucous (HG)) was observed in 4.98% of the population and differed from Standard type (ST). As it is not possible to visualize any phenotypic changes due to recessive mutations in the M1 generation, the cuticular wax variants and other variants scored in the M1 population are likely due to the natural variation present in this switchgrass seed.
Fifty-two variegated plants, 43 yellow-green sectors, 8 pin-striped and 1 glossy, were observed with pigmentation sectors on just a few tillers and are probable indication of a mericlinal mutation (partial cell layer) of the L2 layer. The pigmentation sectors that arise as mutations in the SAM, and maintained in their derived cells lineages, are thought to be the result of dominant mutations induced by the mutagenesis treatment. Two variegated plants, with sectored albino tillers, are likely sectorial chimeras with a mutation present in 53 all three cell-layers. One variegated plant observed with prominent leaf dis-coloration and green mid-veins resembles that of a periclinal mutation (complete cell layer) of the L2 layer (Ch. 2 Fig. 1D). As the periclinal mutation is uniform in phenotype throughout the entire plant it is likely a heritable mutation found in the germplasm. Any further characterization, as to the origin of these variegations, will require further study.
1.2 Screen for Self-Fertility A total of 1,482 bagged plant tillers (~4,000 bags total) were collected and the mean number of seeds was observed to be low (< 30 seeds). In the first year, most bags contained < 10 seeds, similar to the expected 1% seed formation of a L- ecotype, but 37 plants possessed elevated seed formation (> 120 seeds). In the second year, we did not replicate our findings in 18 of the 19 plants tested for elevated seed formation. This indicates that pollen contamination (late bagging) in the first year was likely the cause for seed formation.
1.3 Nodal Propagation As a means to capture variants for genetic study, we developed a custom DFT style propagation method that utilizes nodal segments to induce shoot formation from axillary buds. We multiplied 10 genotypes of cv. Alamo, each varied for height and morphology, in the greenhouse, by halving, before field planting. In total, 70 tillers per genotype were collected and 99 nodal segments were harvested at each of three positions (Low, Mid and
High). The shoots were incubated in the growth chamber for 6 weeks before planting in two rooting conditions for 4 weeks. After six weeks, a two-way ANOVA test strongly indicated genotype by node position interactions and means comparisons, using Tukey
HSD multiple comparison, of shoot formation at three node positions (Low, Med and
High) were made for genotype and node position. The statistical significance was assessed at 5% probability. 54
Shoot formation was greatest at the low node position. Three of the ten genotypes tested had increased shoot formation at Mid and High nodes. Comparison of two rooting conditions, by χ2-test, indicated that rooting in five genotypes was significantly different.
Four out of the five genotypes favor the rooting conditions of soil and commercial rooting powder. Propagation of variant lines allowed us to maintain parental lines in the greenhouse and to establish field copies for use in complementation testing into allelic groups, see below.
1.4 Characterization of Wax Variants In the first comprehensive analysis, providing ultrastructural and chemical composition, we compare three variant types (NG, RG and HG) to the ST switchgrass.
The most abundant ST plants were observed to have a moderate amount of glaucousness
(bluish white) on both ab- and ad-axial leaf surfaces. The NG types have a bright green abaxial leaf surface that appears glossy, with a glaucous adaxial leaf surface similar to
ST. On the abaxial surface, the RG types appeared to have a glaucousness intermediate to
NG and ST. The HG exhibited a more intense bluish-white color than ST.
Using gas chromatography with mass spectrometry and field emission scanning electron microscopy, we compared the visually identified cuticular wax variants (NG, RG and HG) to the STs. In each variant type, four leaf wax samples were collected in three representative genotypes. Using GC-FID, 53 wax constituents were identified in six chemical classes and a representative chromatography of ST is presented (Appendix B
Supplemental Fig. 1). The presence of C33 and C33OH β-diketone constituents were verified, in ST, using GC-MS (Appendix B Supplemental Fig. 2 and 3). Means comparisons, by t-test, of variants to ST were made for six chemical classes and total wax. A low standard error (SE) was found, indicating a similarity in type designation, 55 and the significance assessed (P < 0.05). Significantly different amounts of total wax were found for each variant type. The comparison of chemical classes indicates that NG types were similar to ST in 1-alcohol, RG types were similar to ST in β-diketones and
HG types were significantly lower than ST in 1-alcohol and significantly higher in β- diketones. In a percentage of total wax for each type, the chemical class of β-diketones was HG (46%), RG (23%), ST (18%) and NG (1%). The chemical wax profiles for each variant and ST indicate the relative amounts of wax constituents in each of the six chemical classes. In acids, the C32 constituents are highest in HG types and ST. A similar pattern of alkanes, 1-alcohols, and aldehydes were found among each of the variants and
ST. In the chemical class of β-diketones, C33 constituents were the largest in each variant except for NG types which had reduced amounts of C33 constituent and absence of
C33OH β-diketones. In amount of C33 β-diketones, a means comparison, by t-test, indicate that NG types were significantly lower that ST, RG types were similar to ST and HG types were significantly higher than ST.
Using field emission scanning electron microscopy to compare the abaxial (facing away from the stem) and adaxial (facing the stem) leaf surfaces, we see the abaxial side of each variant type and ST have plate-shaped epicuticular wax structures, commonly associated with alcohols. Both ST and NG types were observed to have similar amounts of alcohol. In the abaxial side, we observed that ST, RG and HG types all have rod- shaped epicuticular wax structures, commonly associated with β-diketones, and NG types are devoid of cuticular wax structures. In NG types the reduced visual glauouness, reduced β-diketones and absence of rod-shaped structures suggest similarity to cer mutants descried in wheat and barley. This is the first comprehensive analyses, including 56 wax ultrastructure and chemical composition, of L- ecotype switchgrass. We are the first to identify rod- and plate-shaped structures on the ab- and adaxial surfaces of ST. We define three variant types (NG, RG, and HG) based on visible glaucousness and this study demonstrates the potential genetic diversity for glaucousness trait present in switchgrass germplasm
1.5 Crossing Block In summer 2015, variant plants were propagated, from a field-established population, using the propagation methods described by Weaver et al. (113). In August 2015, we established crossing blocks with six genotypes having reduced amounts of visual glaucousness (NG and RG types). In four replications, blocks containing a single copy of each genotype were planted with 3 ft. spacing. In a test for allelism, reciprocal crosses were made, in three replications, by crossing tillers from neighboring plants. As a control, selfed crosses made using the techniques described in Ch. 2.2. To insure pollination, it is recommended to remove the remaining inflorescence meristems from unbagged tillers, as a means to encourage growth within the bags.
Bags were labeled, using wax pencils, with plant identification numbers referencing the cross and tillers were individually labeled, using tape and a sharpie marker, for plant number. In December 2015, the bags were harvested, the tillers were then separated into individually labeled bags and stored in the UA 6th street greenhouse. Over several months seed were cleaned using the techniques described above in Ch. 2.3. We identified a single
NG type plant that was consistently able to produce seed in reciprocal crosses (53 - 411 seeds) and when selfed (98 seeds). A similar finding, for self-fertility and apomixis, was previously described in a L- ecotype of switchgrass (14). The ability to make reciprocal crosses suggests unique alleles of both S-Z- genes that differ from that found in the 57 remaining genotypes tested or possibly the absence of S-Z- alleles. As the selfed seed was found in a single replication these findings warrant further experiments be conducted to validate these findings.
2. Future Research In a population of 1,849 plants (Panicum virgatum L., cv. Alamo) established in
2011, we identified 92 variant plant types having visible glaucousness and total wax
(three Non-Glaucous (NG), 14 Reduced Glaucous (RG), 75 Highly glaucous (HG)) that differ from Standard type (ST). The glaucous trait has been linked with drought and heat tolerance in crop plants (116). My research interests are focused on the identification of allelic relationships among these wax variants. Assignment of variants into allelic groups is important to establish the number of wax-associated loci in this population, and subsequently for development of associated gene-specific molecular markers for breeding purposes. With interests for yield enhancement in dedicated bioenergy crops, the identification of genes underlying agronomically important traits is currently at the forefront of switchgrass research (117; 118; 119).
The introduction of enhanced self-fertility in variant lines is proposed as a means to conduct genetic studies in switchgrass. In switchgrass, an obligate outcrosser, seed formation is specified gametophytically by a complementary interaction of its S- and Z- genes and selfed seed formation is very low in both U- (1.39%) and L- (0.35%) ecotypes
(18; 41). Okada et al. (38) suggest that switchgrass has disomic inheritance, i.e. two alleles at each locus, rather than behaving as a true tetraploid. In 2011, seed germination
(23.6%) was observed in an allogamous population of switchgrass and is similar to the diploid segregation of two genes, S and Z, present in a 9:3:3:1 ratio, where ¼ of the progeny are heterozygous, i.e. viable. In a 2015 field-established crossing block, we 58 identified an NG type plant that was able to produce seed (53 - 411 seeds) in reciprocal crosses with six genotypes and when selfed (98 seeds). A similar finding, for self-fertility and apomixis, was previously described in a L- ecotype of switchgrass (14; 32).
I intend to replicate our findings, for self-fertility, by reciprocal and self-crosses, in 12 variant plants with reduced (NG and RG types) or increased (HG types) amounts of glaucousness. Then I intend to produce self-fertile variant lines, by introgression of self- fertility, in a series of backcrosses with the identified plants. Finally, I propose a genetic study, intended to determine the number of genes, allelic complementation, and dominance, by reciprocal crosses with variant lines in two groups with altered amounts of glaucousness.
Allelic complementation will result in the presence of ST progeny and dominance determined by crossing variant lines and ST. In genomic DNA of parental and variant lines, simple sequence repeat (SSR) marker segregation analysis, using PCR amplified primer pairs, will identify polymorphisms and confirm the inheritance of homozygous and heterozygous alleles. The proposed research will require a greenhouse and a quarter acre of field space. As part of this research, the scoring of morphological traits and characterization of leaf wax by GC-MS, will be conducted at multiple time points to examine cuticle wax development. It is my aim to develop molecular markers associated the glaucous trait in switchgrass for use as QTLs in a breeding program.
59
FIGURES AND TABLES
Figure 1:
Figure 2: 60
Figure 3:
Figure 4:
61
Table 1
Control 8 Hrs 24 Hrs EMS (mM) 0 40 80 160 320 40 80 160 320 Germination (4,000 seeds) 944 548 *** 292 *** 91 *** 13 *** 151 *** 27 *** 2 *** 0 ***
Table 2
Trait Mean Std Dev Median Mode Range Plant Color 2.95 0.40 3 3 3 Plant Health 1.06 0.25 1 1 2 Plant Height 3.08 0.45 3 3 3 Tiller Angle 3.05 0.45 3 3 3 Tiller Number 2.89 0.49 3 3 3 Stem Width 2.03 0.21 2 2 2 Leaf Carriage 1.74 0.82 1 1 2 Leaf Width 2.97 0.45 3 3 3 Leaf Curling 1.37 0.68 1 1 3 Variegation 1.03 0.18 1 1 3
62
LEGENDS
Figure 1:
Photographic images of switchgrass EMS mutagenesis procedure. A) Seeds are exposed to chemical mutagen (EMS) at increasing concentrations (0, 40, 80, 160, and 320 mM) and at two durations (8 and 24 hrs.). B) After treatment, seeds are rinsed three times with DI water and air dried on filter paper. C) The number of germinating seeds in each tray was scored after three weeks in greenhouse conditions.
Figure 2:
EMS dose response curve is presented as a percent of seed viability of Control (0 mM) and increasing concentrations (40, 80, 160, and 320 mM) at two durations (8 and 24 hrs.). Each data point represents a single replication of 4,000 seeds germinated per exposure concentration and time.
Figure 3:
Photographic images of the chlorophyll mutants of switchgrass. A) Sector identified, by white arrow, in “cone-tainers” prior to field planting. B) The field planting retained the albino sector at time of scoring. C) A plant identified with variegation, in leaf sectors, in field conditions D) A plant identified with prominent variegation in field conditions
Figure 4:
Photographic image of field bagging during Fall 2011.
Table 1:
The germination of 4,000 switchgrass seeds per EMS concentration and Control. Seeds of cv. Alamo (Control) compared to those exposed to chemical mutagen (EMS) in four increasing concentrations (40, 80, 160, and 320 mM) and at two durations (8 and 24 hrs.). Significance values (χ2-test) are indicated by the asterisks; *** P < 0.001.
Table 2:
Plant scoring data, provided in mean, standard deviation, median, mode and range, for ten morphological traits in switchgrass cv. Alamo.
63
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72
APPENDIX A
A Simplified Technique for the Propagation of Shoots from Nodes of
Switchgrass (Panicum virgatum L.) Genotypes
Copyright 2014 Joshua M. Weaver, Laura Sofia Montes Sujo and Kenneth A. Feldmann
Published in BioEnergy Research 7:4
doi:10.1007/s12155-014-9470-4
73
SPRINGER NATURE LICENSE TERMS AND CONDITIONS Jul 16, 2018
This Agreement between Mr. Joshua Weaver ("You") and Springer Nature ("Springer Nature") consists of your license details and the terms and conditions provided by Springer Nature and Copyright Clearance Center. License Number 4390891424979 License date Jul 16, 2018 Licensed Content Publisher Springer Nature Licensed Content Publication BioEnergy Research A Simplified Technique for the Propagation of Shoots from Licensed Content Title Nodes of Switchgrass (Panicum virgatum L.) Genotypes Joshua M. Weaver, Laura Sofia Montes Sujo, Kenneth A. Licensed Content Author Feldmann Licensed Content Date Jan 1, 2014 Licensed Content Volume 7 Licensed Content Issue 4 Type of Use Thesis/Dissertation Requestor type academic/university or research institute Format electronic Portion full article/chapter Will you be translating? no Circulation/distribution <501 Author of this Springer yes Nature content BIOCHEMICAL CHARACTERIZATION OF Title CUTICULAR WAX VARIANTS IN SWITCHGRASS (PANICUM VIRGATUM L.) Instructor name Joshua M. Weaver Institution name University of Arizona Expected presentation date Aug 2018
74
Keywords
Nodal culture, Micropropagation, Panicum virgatum L., Indole-3 butyric acid (IBA)
75
1. Abstract Nodal propagation of switchgrass (Panicum virgatum L.) is an important technique for multiplying a variant plant, with a potentially valuable agronomic trait, for replicated field trials. To develop an improved propagation system, shoot induction from three node positions (Low, Mid, and High) was tested in ten genotypes of switchgrass cv. Alamo.
Nodal segments were incubated in a custom hydroponics system with 1/4 strength
Murashige and Skoog liquid medium for six weeks. In all genotypes, shoot formation was highest at the Low node position, the largest nodes, ranging from 60 – 98%. From the ten genotypes tested, three genotypes (4, 5, and 6) had a significantly higher shoot formation at Mid and High nodes with a shoot formation greater than 80% and 50%, respectively. Shoot formation for all other genotypes at the same two positions was less than 40% and 10%, respectively. In a single replication, 30 shoots from the lowest node position of each genotype were rooted for four weeks in each of two rooting conditions
(RC1 - Roottone dip and soilless media and RC2 – IBA in a half strength liquid MS media). Four genotypes showed greater root formation in RC1 while one genotype showed greater root formation in RC2. In this paper, we describe a procedure for generating a population of genetically identical plants from switchgrass, in as little as six- weeks, that is relatively inexpensive, efficient and does not require specialized equipment needed for tissue culture.
76
2. Introduction Panicum virgatum L. (switchgrass) is a warm season C4 perennial prairie grass species native to North America. A dominate species of the tall grass prairies, stands of wild populations are found in areas of North America below 55˚N latitude and east of the
Rocky Mountain Range, i.e. 100˚W longitude (1). Switchgrass has traditionally been used both as a forage crop and to restore grasslands (1). Members of the Poaceae family
(true grasses), including proso millet, maize, sorghum, rice and switchgrass, are the most efficient producers of biomass and have the most potential for use as cellulosic biofuels
(2). Switchgrass has many attributes that make it a nearly ideal energy crop including high biomass yield, perennial nature, high water use efficiency and ability to recycle nutrients back to the root system during senescence (3). Since 1990, the DOE’s
Bioenergy Feedstock Development Program at Oak Ridge National Laboratory and others have touted switchgrass as a model biomass energy crop (4).
Using light microscopy and flow cytometry, it was shown that switchgrass has a basic chromosome number of x = 9 and that it is found primarily as either a tetraploid (2n = 4x
= 36), lowland ecotypes, or octoploid (2n = 8x = 72), upland ecotypes (5). Stands of lowland ecotypes, commonly found in flood plains, are thought to have more economic value as they occur in a bunch formation, are more disease resistant and have greater biomass. Upland ecotypes, mostly octoploids but some tetraploids, are regionally adapted to northern longitudes and have better cold hardiness. Regional adaptability is largely determined by photosensitivity and gains in biomass in lowland ecotypes are attributed to delayed anthesis (6). Both upland and lowland switchgrass ecotypes can be found within a single stand but are generally regionally isolated. 77
As an obligate out-crossing and an allopolyploid species, switchgrass is inherently heterogeneous and polymorphic. In fact, it has been estimated that the selfing rate in switchgrass is 0.35% in tetraploids and 1.39% in octoploids (7; 8). A large number of phenotypic variants can be observed in a switchgrass population including those for plant height, stature, leaf width, leaf curling, number of tillers, color, time of flowering, senescence and others, some of which could be useful traits to be used in a breeding program. However, because of its outcrossing nature, seed collected from a single plant possessing a variant trait, such as epicuticular wax, height, or biomass, do not breed true, making it impossible to do replicated trials of phenotypic variants of interest.
Vogel 2008 describes the need for asexual propagation of parents of F1 hybrids as a means of capturing non-additive genetic variance (9). In addition, the development of a sexually self-compatible or apomictic system is needed so that variation or improvement can be maintained. Until this is possible, the best solution for generating a population of genetically identical plants from a specific variant is to develop an efficient vegetative propagation system. An in-vitro method for the vegetative propagation of switchgrass has been described (10) but it is laborious, requires specialized equipment and has not been widely utilized for generating clones of switchgrass variants. In this paper, we describe a vegetative (nodal) propagation system that is inexpensive, efficient, fast and can be modified for use in various situations.
78
3. Materials and Methods 3.1 Plant Material In 2009, a large population of cv. Alamo, lowland switchgrass ecotype, was grown under well-watered field conditions at the Maricopa Agricultural Center (MAC) (33˚
04’37” N, 111˚58’26” W) in Maricopa, AZ. In the summer of 2009, ten genotypes were selected for differences in plant height and unique architecture, e.g. upright vs. decumbent and narrow vs. wide leaves. Clones from these plants were produced by using nodal cuttings from each of these ten genotypes, with a technique similar to that described below. In 2010, these clones were grown to maturity in the University of
Arizona, 6th Street Parking Garage greenhouses, on the main campus in Tucson, AZ. In
October 2010, mature plants were transplanted to the field as described below.
Field experiments were conducted at the University of Arizona West Campus
Agricultural Center (CAC) (32˚ 13’48” N, 110˚57’11” W; elevation 776 m). The 3-ha
West CAC has a Vinton Soil Series (0-3% slope, fine sandy loam, Typic Torrifluvents) with the source of sediment being the bordering Santa Cruz River (SWES Dept. U of AZ, unpublished data). The field was flood-irrigated four times during the growing season with slightly alkaline, irrigation water high in calcium deposits. Tucson is semi-arid, has low relative humidity RH (< 20%), and an annual rainfall of 30 cm. Precipitation distribution is bimodal with winter storms from the Pacific and summer monsoons.
Average monthly high and low summer temperatures, from 1918 - 2010 for the months of June to September, are 100.3, 99.7, 97.4, 94.5˚ F and 69.3, 74.4, 73.3, 68.6 ˚ F, respectively. Average monthly high and low winter temperatures, from 1918 - 2010 for the months of December to February, are 64.8, 65.5, 68.5 ˚ F and 39.1, 39.8, 42.2 ˚ F, respectively. Weather data were collected from the National Oceanic and Atmospheric
Administration website (11). 79
In the summer of 2009, eight plants from each of the ten genotypes were established in the greenhouse in 2L plastic pots. In October of 2010 these potted plants were trimmed to 5 - 7 cm above the soil surface, leaving the lowest culm node intact. Four plants for each genotype were transferred to the West CAC field (61 cm between plants and 91 cm between rows) to test winter hardiness. In April of 2011, the other four pots of greenhouse established plants were trimmed and split vertically. Half of each plant was transplanted to the field, thus completing ten rows of eight plants. The remaining half of the trimmed plants were repotted and kept as a back-up in the greenhouse.
3.2 Harvesting Nodal Segments In the fall of 2011, the eight field-grown plants representing each genotype were indistinguishable in size and architecture except for one clone of genotype 9, and it was culled. To test for propagation efficiency, ~60 - 70 tillers each containing 6 - 7 nodes (~2 m tall) were harvested from each of the ten genotypes of one-year-old field-established clones of switchgrass cv. Alamo (Fig. 1A) The tillers were harvested just above the lowest visible node and prior to anthesis or the R4 stage of reproductive-floral development (12). The bases of the bound tillers were placed in water and transported to the lab to harvest the nodes.
Nodal segments from the ten genotypes were dissected 5 cm above and 2 cm below each culm node. Each excised nodal segment now contained one culm node, an axillary bud (AB) and a short length of internode above and below. Nodal segments from each genotype were separated by position (Low, Mid, and High) on the tiller, and placed in 2L beakers filled with 500 ml reverse osmosis (RO) water to remove any soil and debris remaining from the field. Starting from the base of the tiller, the Low position includes culm nodes 2 - 3, while Mid and High positions include culm nodes 4 - 5 and 6 - 7, 80 respectively. The top or inflorescence nodes were not used, and if a harvested tiller did not have the requisite six nodes, additional size-appropriate culm nodes were harvested from other tillers.
3.3 Shoot Induction from Axillary Buds The nodal segments were placed, basal (or proximal) end submerged, in a DFT (Deep
Flow Technique) raft-type hydroponics system for up to six weeks to induce shoot formation from the ABs (Fig. 1B). The DFT system ensured uniformity of the experimental conditions for shoots formed in the absence of any exogenously supplied plant growth regulators. The DFT system included 10 cm diameter plastic pots (Fig. 2B) lined with a black fiberglass screen (Fig. 2A) to prevent the nodal segments from falling through the holes in the pots. The DFT raft (Fig. 2C), in which ten pots were placed, was constructed with a 1” thick expanded polystyrene sheet. The raft was cut to fit inside of a black plastic planting tray (1020 Tray, Landmark Plastic Corporation, Akron OH), which served as the reservoir (Fig. 2D). Pot-size holes were cut into the rafts in an alternating array so that ten pots would fit into one raft and tray. Nine pots containing a total of 297 nodal segments, representing one genotype, were transferred to the DFT system. One pot from each genotype was transferred into each of nine trays, such that each tray contained a single cup from each genotype. The pots were positioned so genotypes (1 - 10) were increased by one position in each of the nine trays. In this way no single genotype was in the same tray position, between any two trays. Nine trays fit inside a single shelved E-15
Conviron® growth chamber. The growth chamber was equipped with a combination of fluorescent and incandescent light sources (16:8 hr light/dark cycle; 23/18˚ C day/night temp regime; 220 PPF). Ambient air is circulated within the incubator by in- and out- vents. The reservoirs were filled with 4L of nutrient solution containing 1/4 strength 81
(1.075 g/L) Murashige and Skoog (13) liquid medium (M5524, SIGMA-ALDRICH, St.
Louis MO). Pot and tray positions were rotated, in a clockwise direction, every three days, and liquid nutrient levels were adjusted to 4L. Shoots that formed from the axillary buds were excised at the base with a scalpel and collected after weeks 4, 5 and 6. To ensure uniformity, the 42-day duration allowed each pot and tray to move into every position inside the growth chamber over the course of the experiment and the DFT system provided abundant nutrient availability.
3.4 Root Induction from Shoots To induce rooting, two rooting conditions (RC1 and RC2) were tested in a growth chamber (16:8 hr light/dark cycle; 23/18˚ C day/night temp regime; 90 PPF). Sixty excised shoots from the Low node position for each of the ten genotypes were divided equally between the two rooting conditions. Sixty shoots were used because this is how many shoots Genotype 1, the poorest shooting genotype, produced (Fig. 3). For RC1 newly excised shoots were dipped into commercially available rooting hormone (0.1%
IBA Rhizopon® AA #1, Hortus USA Corp, NY NY) creating a thin layer of powder over the cut surface. These shoots were transferred to individual wells of 72 well square plug propagation trays (P-72 SQ, Landmark Plastic Corporation, Ohio) containing a 3:2
(gallons) ratio of Sunshine mix #1 (LC1, Sun Gro Horticulture Canada Ltd, Seba Beach,
AB Canada) to Vermiculite (medium grade, Thermo-O-Rock, Chandler AZ) amended with 3 tbsp. time release fertilizer (Osmocote®, Scotts-Sierra Horticultural products
Company, Marysville OH). The 72 well propagation trays were placed inside black plastic planting trays so that the plants could be sub-irrigated with distilled water as needed. In RC2, newly excised shoots were placed directly into a DFT system. The ten genotypes were transferred to a single reservoir with ½ strength (2.15 g/L) MS liquid 82 medium supplemented with 10-6 IBA (I5386, SIGMA-ALDRICH, St. Louis MO). Every three days, the reservoir level was adjusted to 4L with the MS IBA solution. In each of the two experimental conditions, rooted clones were harvested after weeks 2, 3, and 4.
3.5 Statistical Analysis In the shooting experiment 2,970 nodal segments were placed into nine DFT systems.
Each DFT raft contained ten pots, and each pot contained 33 nodal segments. This allowed for three replications in three node positions of ten genotypes to be maintained in a single growth chamber. The average of these three replications was converted to a percentage and presented in Fig 3 with standard deviation error bars. A comparison of the treatment means was conducted using the Tukey HSD multiple comparison procedure.
The effects of genotype and node position on shoot formation were analyzed using a two way factorial ANOVA design. Each model was conducted using JMP software version
10 (SAS Inc. Cary, NC) and the statistical significance was assessed at the 5% probability level unless otherwise stated.
In the rooting experiment, shoots collected from the Low node position in each of the ten genotypes were equally divided among two experimental rooting conditions (RC1 and RC2). For each treatment, a 30 shoot sample per genotype is present in a single replication. The genotypic comparisons of response to rooting conditions are presented in
Fig. 4. Here the total number of shoots forming roots for each genotype over the four week period is converted into a percentage. A comparison of root formation between
RC1 and RC2 for each genotype is also made by chi-square test, and statistical significance assessed at the 5% probability level.
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4. Results and Discussion 4.1 Shoot Production from Harvested Nodal Explants Meristems contain undifferentiated cells that give rise to donor cells that differentiate to form organs in response to environmental, hormonal and genetic factors. In grasses, tillers initiate from the shoot apical meristems to form leaves, stems and roots. Aerial portions of each tiller develop as repeating units, phytomers, consisting of a leaf, culm node, internode, intercalary meristem, and axillary meristem (14). The intercalary meristem develops at the base of nodes and leaf blades and allows for rapid leaf growth.
Axillary meristems form at the intersection of leaf and stem tissues and subsequently give rise to axillary buds. On the tiller, the AB’s are dormant due to the basipetal movement of auxin. While high levels of auxin promote apical dominance, a diminished supply of auxin and the acropetal movement of cytokinin promote meristem size and AB emergence, in the form of a new shoot. In nature the release of AB dormancy is important in determining plant architecture and maximizing productivity (15).
In these experiments, a break in AB dormancy was observed as shoot formation in all ten genotypes by the end of week 3. The shoots that formed either protruded through the outer protective leaf sheath at a 45˚ angle to the stem (Fig. 1C) or grew behind it and emerged at the distal end of the nodal segment (Fig. 1D). In Figs 1C and 1D, the leaf sheaths were removed to help visualize the shoot orientation relative to the nodal segment. Interestingly, the 45˚ orientation was observed after just one week in genotype 4 and was more prevalent in genotypes 4, 7 and 9. Each node formed a single shoot with the exception of genotype 4 which occasionally formed two. It is not clear whether genotype 4 made two ABs or whether an AB gave rise to two shoots. Nodal segments of genotype 4 also gave rise to roots from the basal end of the new shoots and the exposed surface of the culm node (not shown). Genotypes 2 and 4 produced a near maximum 84 number of shoots by the end of week three and by week four were finished producing shoots (data not shown). Each of the remaining eight genotypes continued to form shoots and at six weeks a maximum number of shoots were formed in all genotypes. After six weeks, the nodal segments that failed to form shoots were accumulating algae and fungal contamination and were discarded.
A genotype x node position interaction was strongly indicated by two-way ANOVA.
Significant differences were found when comparing shoot formation among genotypes and node positions. Mean comparison of genotypes, grouped genotypes by comparable means, indicate that group A produced the most shoots (genotypes 6, 5, and 4), group B fewer (genotypes 3, 9, and 7), group BC even less (genotypes 2, 10, and 12) and group D the least (genotype 1). Mean comparison of node position across all genotypes indicate that the Low nodes produced the most shoots (27.4) out of 33, the Mid nodes intermediate (14.1), and the High nodes the least (8.1). An inverse correlation of node position and percent shoots generated were observed for all genotypes. Genotypes 4, 5, and 6 produced more shoots than the other genotypes at the Mid and High node positions
(Fig. 3).
4.2 Root Formation from Axillary Shoots In total, 60 shoots from each genotype were collected from the Low culm nodes and equally allocated into two experimental rooting conditions (RC1 and RC2). In RC1 a single treatment of IBA (10-3) was applied directly to the cut surface of shoots, which were then placed in a soilless media. In contrast, a lower concentration of IBA (10-6) was maintained in RC2 for the four week duration. Root formation was achieved in both rooting conditions, and recorded at the end of four weeks (Fig. 4). The rooted shoots were 85 successfully transplanted into the greenhouse and grown in containers (SC10 cell, S+S,
SC) with soilless media similar to that previously described.
Auxin supplied exogenously initiates root production by direct organogenesis. In
RC2, where root elongation could be visualized, roots formed in as little as two weeks and rooted shoots were transplanted after weeks 2, 3, and 4. Adventitious roots grew directly into the media layer, either into a storm screen or media plug (Figs. 1E and 1F).
The presence of lateral roots in both rooting conditions is consistent with the extended exposure of roots to exogenous IBA. IBA is structurally similar to the endogenous indole-3-acetic acid (IAA), and is widely used commercially to induce adventitious roots from stem cuttings (16). A combination of biotic and abiotic environmental factors limited the success of rooting in RC2 over time due to contamination of the media with algae.
A genotypic comparison, by chi-square test, reveals five genotypes (2, 3, 5, 6 and 10) that have a significant difference in root formation between RC1 and RC2; four (2,3,5,6) of which favor the RC1treatment. Two genotypes (1 and 4) have p-values close to but not within the 5% probability level. Four genotypes (2, 4, 6 and 7) produced > 90% root formation in RC1. Because RC1 produces more rooted shoots for most of the genotypes and as it is the simpler of the two procedures, it is the recommended rooting procedure.
The combination of high shoot and root formation in genotypes (4 and 6) mean that they produced the greatest number of viable clones. From the results described here, it is estimated that a single plant of genotypes 4, 5 and 6, yielding approximately 600 nodal segments, will generate as many as 500 rooted clones. After transplanting these 500 plants to the field, in the next year, using this same technique, it should be possible to 86 produce more than 250,000 rooted clones for transplant to the field. The recommended method for multiplying a variant would be to harvest the lower nodes, 2, 3 and 4, in combination with dipping any resulting shoots in IBA (RC1) before transplant to a soilless mix followed by transplantation to the field.
The regeneration of elite genotypes in switchgrass has focused on the establishment of embryonic callus in-vitro by utilizing leaf, mature caryopses, inflorescence and axillary nodal segments (6; 10; 17; 18). Each of these successes is based on cv. Alamo and each of these techniques requires both specialized knowledge and equipment necessary for in-vitro tissue culture. A similar technique utilized mature caryopses, to compare both upland and lowland cultivars of switchgrass, for the regeneration of multiple shoot clumps; all cultivars responded in a similar manner but at different frequencies (19). From the similarity in shoot formation of upland and lowland cultivars, we might expect a similar result of varied frequency in genotype and cultivar response in the formation of shoots from axillary nodal segments when using the procedure outlined in this experiment.
In this paper, we describe a vegetative (nodal) propagation system that is an inexpensive procedure for generating hundreds or even millions of identical cv. Alamo clones of naturally occurring or ethyl methanesulfonate (EMS) induced plant variants.
This procedure has advantages over similar tissue culture procedures in that phenotypic selection can be made prior to propagation, and it limits exposure to plant growth hormones that may cause somaclonal variation. Minor amendments to this procedure can be expected for plant selections used in a breeding program. For example, using this method, replicated trials can be set up to test the importance of various phenotypes (wide 87 leaves, thick tillers, tall plants) for biomass or to impose various environmental stresses, such as drought on variants with more or less epicuticular wax, etc.. This procedure has been adapted to a greenhouse setting and conducted with only readily available products such as Styrofoam cups or similar containers. However, the cups need to be filled regularly to prevent desiccation of the nodal segments. The DFT system ensures that the nodal segments do not dry out. This procedure would also be ideal for educational purposes in any school setting.
Acknowledgements: We would like to thank Zachary Weaver, Ms. Kathryn
Millward, and Mitchell Feldmann for technical assistance. We thank Dr. David Bransby for the Alamo seeds. Finally, we are indebted to the advice provided by Drs. Mike
Ottman and Edzard van Santen.
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5. Legends
Figure 1:
A procedure for the nodal propagation of switchgrass. A) Field-grown switchgrass cv. Alamo was used for nodal propagation. B) Shoot induction from nodal segments in the DFT hydroponics system with 1/4 strength MS liquid medium. C, D) Removal of the leaf sheath from the node exposed the primary orientation of new shoot formation at a 45˚ angle to vertical (C) or as parallel to the nodal segments (D). E) Nodal segments rooted in soilless media in growth chamber conditions after four weeks. F) Nodal segments from a single genotype rooted in 1/2 strength MS liquid medium containing 10-6 IBA in a custom DFT hydroponics system, and in growth chamber conditions for four weeks. G) A 72 well tray containing multiple genotypes, of rooted clones, after a total of eight to ten weeks; clones are ready for transfer to the greenhouse for hardening before transplanting to the field.
Figure 2:
Model DFT (Deep Flow Technique) raft-type hydroponics system ensured uniformity of the experimental conditions for the ten genotypes for six weeks. A) Black fiberglass screen prevents the nodal segments from falling through the holes in the pots. B) The 10 cm diameter plastic pots hold nodal segments. C) Raft, in which ten pots were placed, was constructed from an expanded polystyrene sheet cut to fit inside of the tray. D) Black plastic planting tray served as the reservoir.
Figure 3:
Shoot formation by position on the tiller for the ten genotypes. Nodes were harvested from ten genotypes at three node positions (Low, Mid, and High) on the tiller and scored for shoot production after 4, 5 and 6 weeks (the graph represents total shoot formation over 6 weeks). For each genotype (1 - 10) there are a maximum of 33 nodes per replication at each of the three node positions. The graph represents a total of three replications. The y-axis represents the mean number of shoots formed per replication including standard deviation error bars. For every genotype, the highest number of shoots formed from the lowest node position. Genotypes 4, 5, and 6 have a significantly greater shoot formation at Mid and High node positions than the other genotypes.
Figure 4:
Root formation on shoots from the Low node position for ten genotypes. Two experimental rooting conditions were tested (RC1 and RC2) for each genotype, each consisting of a single 30 shoot sample. For RC1, shoots were dipped in Rootone® rooting powder (0.1% IBA Rhizopon®) and placed in soilless media. The treated shoots were transferred to a growth chamber and watered as needed. For RC2 nodal segments were rooted in ½ strength MS liquid medium supplemented with 10-6 IBA, with cut tip submerged in a custom DFT hydroponics system in growth chamber for four weeks. Root formation in 4/10 genotypes (2,3,5,6) is higher in the RC1 treatment, while genotype 10 rooted better in the RC2 treatment.
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6. Figures
Figure 1
A) Screen
B) Cup
C) Raft
D) Tray
Figure 2 90
Node Position Effect on Shoot Formation 100
90 Genotype 1 80 Genotype 2 70 Genotype 3 60 Genotype 4 50 Genotype 5 40 Genotype 6 Genotype 7 30
PrecentShoot Formation Genotype 8 20 Genotype 9 10 Genotype 10 0 Low Nodes (2-3) Mid Nodes (4-5) High Nodes (6-7)
Figure 3
Figure 4
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7. References
1. Vogel, K.P. 2004. Switchgrass. In: L.E. Moser, L. Sollenberger, and B. Burson, editors, Warm-season (C4)
grasses. ASA-CSSA-SSA Monograph, Madison, WI. 561-588.
2. Jakob, K., F. Zhou, and A. H. Paterson. 2009. Genetic improvement of C4 grasses as cellulosic biofuel
feedstocks. In vitro Cell. Dev. Biol.- Plant 45, 291-305
3. Schwartz, C., and R. Amasino. 2013. Nitrogen recycling and flowering time in perennial bioenergy crops.
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feedstock in the United States. Biomass and Bioenergy 28, 515-535.
5. Hopkins, A.S., C.M. Taliaferro, C.D. Murphy, and C. D’Ann. 1996. Chromosome number and nuclear DNA
content variation among switchgrass populations. Crop Sci. 36, 1192-1195.
6. Denchev, P.D., and B.V. Conger. 1995. In vitro culture of switchgrass: Influence of 2,4-D and picloram in
combination with benzyladenine on callus initiation and regeneration. Plant Cell Tissue Organ Cult. 40, 43-
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7. Martinez-Reyna, J.M. and K.P Vogel, 2002. Incompatibility systems in switchgrass, Crop Sci. 42, 1800-
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8. Lundqvist, A. 1956a. Self-incompatibility in rye. I. Gentetic control in the diploid. Hereditas 42, 293-348
9. Martinez-Reyna, J.M. and K.P Vogel, 2008. Heterosis in switchgrass: spaced plants, Crop Sci. 48, 1312-
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10. Alexandrova, K.S., P.D. Denchev, and B.V. Conger. 1996. Micropropagation of switchgrass by node culture.
Crop Sci. 36, 1709-1711.
11. National Oceanic and Atmospheric Administration (NOAA). 2013. Monthly and Daily Normals (1981-2010)
plus Daily Extremes (1895-2013) for Tucson, AZ. Department of Commerce, Washington, DC.
http://www.wrh.noaa.gov/twc/climate/tus.php (accessed 1 July. 2013).
12. Moore, K.J., L.E. Moser, K.P. Vogel, S.S. Walker, B.E. Johnson, and J.F. Pedersen. 1991. Describing and
quantifying growth stages of perennial forage grasses. Agron. J. 83, 1073-1077
13. Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue
cultures. Physiol. Plant. 15, 473-497.
14. Pautler, M., W. Tanaka, H. Hirano, and D. Jackson. 2013. Grass Meristem I: Shoot apical meristem
maintenance, axillary meristem determinacy and the floral transition, Plant Cell Physiol. 54(3), 302-312 92
15. Whipple, C.J., T.H. Kebrom, A.L. Weber, F. Yang, D. Hall, R. Meeley, R. Schmidt, J. Doebley, T. P.
Brutnell, and D. P Jackson. 2011. Grassy tillers1 promotes apical dominance in maize and responds to shade
signals in the grasses. Proc. Natl Acad. Sci. 108, E506-E512.
16. Wei, K., W. Liyuan, H. Cheng, C. Zhang, C. Ma, L. Zhang, W. Gong, L. Wu. 2013. Identification of genes
involved in indole-3- butyric acid-induced adventitious formation in nodal cuttings. Gene 514, 91-98
17. Denchev, P.D., and B.V. Conger. 1994. Plant regeneration from callus cultures of switchgrass. Cell Biology
& Molecular Genetics 34, 1623-1627.
18. Alexandrova, K.S., P.D. Denchev, and B.V. Conger. 1996. In vitro development of inflorescences from
swithcgrass nodal segments. Crop Sci. 36, 175-178.
19. Dutta Gupta S. and B.V. Conger. 1998. In vitro differentiation of multiple shoot clumps from intact seedlings
of switchgrass. In Vitro Cell. Dev. Biol. - Plant 34, 196-202.
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APPENDIX B
Cuticular Wax Variants in a Population of Switchgrass
(Panicum virgatum L.)
Copyright 2018 Joshua M. Weaver, Greg Lohrey, Pernell Tomasi, John M. Dyer, Matthew A. Jenks and Kenneth A. Feldmann
Published in Industrial Crops and Products 117
doi:10.1016/j.indcrop.2018.02.081
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95
Keywords
Panicum virgatum; Poaceae; switchgrass; leaf; cuticular wax variants; β-diketones
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1. Abstract Leaf cuticular waxes are known to influence both biotic and abiotic stress tolerances of plants. The objective of this work was to characterize the wax phenotypic diversity present in a population of 1,849 switchgrass plants. We identified 92 visually distinct variant plants that possessed altered leaf glaucousness relative to the common standard type (ST), which exhibited a bluish-white (glaucous) leaf color. The variants could be grouped into three classes: 1) non-glaucous types (NG) that possessed a shiny green leaf surface, 2) reduced glaucous types (RG) that appeared less glaucous than ST, and 3) highly glaucous types (HG) that exhibited more intense bluish-white color than ST.
Analyses of total cuticular wax content averaged over each of three NG (mean 304.79 +
15.16 µg/dm2), RG (mean 533.33 + 21.62 µg/dm2) and HG types (mean 1228.23 + 45.74
µg/dm2) showed significant differences (P < 0.001) from three selected STs (mean
810.92 + 30.57 µg/dm2). Analysis of wax composition among these selected types revealed that the C33 β-diketones were the most abundant wax compounds in all but NG types. Field emission scanning electron microscopy showed that abaxial leaf surfaces exhibited predominantly rod-shaped crystals, and adaxial surfaces exhibited predominantly plate-shaped wax crystals on all lines, except for NG that lacked wax crystals on the abaxial leaf surface. As a target for crop improvement, this study reveals that a large amount of variation for cuticle waxes exists within this switchgrass germplasm.
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2. Introduction The plant cuticle is a hydrophobic barrier that coats most aerial plant surfaces, and is composed of aliphatic compounds deposited both within and above the structural cutin matrix of the cuticle membrane (Kosma and Jenks, 2007). The cuticle plays a role in limiting plant transpiration and improving plant water conservation, which is especially important during climatological drought (Riederer and Schreiber, 2001). It also helps to protect the plant from insect herbivory, fungal pathogens and reduces potential heat stress by increasing reflection of solar radiation (Reicosky and Hanover, 1978; Jenks et al.,
1995). The cuticular waxes on most plants are dominated by very long chain fatty acids
(VLCFAs; ~C20 to C36) and their derivatives including aldehydes, primary alcohols, alkanes, and the longer-chain length wax esters that can reach roughly 70 carbons long
(Buschhaus et al., 2007). Other common wax classes include secondary alcohols, ketones and, in some species, β-diketones (von Wettstein-Knowles, 1972; Kunst and Samuels,
2003). When visualized by scanning electron microscopy, the epicuticular waxes are deposited as smooth coatings, or crystallize into a diverse array of amorphous, rod-like, or dendritic structures, depending on species and organ surface (Jeffree, 2007).
Tulloch and Hoffman (1980) identified the primary leaf surface wax components of switchgrass (Panicum virgatum L.) as C33 β-diketone (tritiacontane-12,14-dione) and a
C33 hydroxy-β-diketone (5-hydroxytritriacontane-12,14-dione), accounting for 69% and
6% of the total wax load, respectively. β-diketones have been identified in the wax of several members of the Poaceae including wheat (Triticum aestivum L.) and barley
(Hordeum vulgare L.) (Hen-Avivi et al., 2016), and other non-Poaceae such as
Rhododendron (Evans et al., 1975), and Hosta (Jenks et al., 2002). The current 98 understanding of the impact that β-diketones have on glaucousness is previously described (Jeffree, 2007, Zhang et al. 2015, Hen-Avivi et al., 2016).
The glaucous trait, created by a visible deposition of epicuticular wax crystals, has been a target for research in several crop species (Zhang et al., 2015). Casler et al. (2012) described the waxy bloom as a blue-colored (glaucous) coating on the stems and leaves of lowland ecotypes of switchgrass. In wheat, a similar bluish-gray leaf coating was linked to the presence of β-diketones and increased grain yields. Work to elucidate genetic control over the synthesis of β-diketones in wheat and barley revealed the involvement of the Cer-cqu gene cluster, which contains three independent genes in the
β-diketone synthase polyketide pathway (Schneider et al., 2016). In barley, β-diketones are dramatically reduced in cer-c and cer-q mutants, but in cer-u a compensatory increase in β-diketones was observed in the absence of hydroxy-β-diketones (von Wettstein-
Knowles, 1972). Switchgrass is a model herbaceous species for bioenergy production, and significant emphasis is now being placed on elucidating agronomically important traits and the underlying genes that determine biomass yield, including stress tolerance
(Wright and Turhollow, 2010; Lowry et al, 2015). Characterization of existing variability in leaf wax structure and composition in switchgrass germplasm is an important first step toward understanding the underlying potential for this trait in future crop improvement strategies. In this report, a survey of the ultrastructure and chemical composition of leaf surface waxes existing on standard and glaucousness variants of the lowland ecotype
(Alamo) switchgrass is presented.
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3. Material and Methods 3.1 Plant Material and Growth Conditions Seeds of lowland P. virgatum cv. Alamo were kindly provided by Dr. David Bransby, formerly of Auburn University. Seeds were treated with 80 mM EMS (ethyl methanesulfonate, Sigma-Aldrich, LLC, St. Louis MO) for four hours and rinsed several times. The initial interest in creating an EMS population was to screen, via bagging, for dominant mutations leading to self-fertility and apomixis. The EMS concentration and time for treatment were chosen from a dose response curve that was conducted. As no dominant mutants were observed, and there was no possibility of identifying recessive mutants in an obligate outcrossing population, the EMS treatment is not considered further in the report. After treatment, approximately 1,000 seeds were planted in each of eight flats that were stratified for three days at 4˚C then transferred to a greenhouse. After three weeks, seedlings were transplanted into Ray Leach “cone-tainers” (SC-10 Super-
White Low Density, Hummert International, Springfield MO). Before transplanting to the field, plants were trimmed to a height of 30 cm and hardened off by exposure to ambient temperature and humidity inside a screened headhouse. In March of 2011, plants were transferred to the field in an equally spaced (61 cm between plants and 91 cm between rows) grid containing 29 rows with between 47 to 81 plants per row. Field experiments were conducted at the University of Arizona’s West Campus Agricultural Center (CAC)
(32˚ 13’48” N, 110˚ 57’11” W; elevation 776 m). The West CAC field has a Vinton Soil
Series (0-3% slope, fine sandy loam, Typic Torrifluvents) with irrigation water that is slightly alkaline and high in calcium (Weaver et al., 2014). This semi-arid climate (low relative humidity RH < 20%) has a bimodal annual precipitation that is concentrated during the summer monsoon months, July through September, and during the winter 100 storm months of December through January. In 2011, Tucson observed an average yearly temperature of 69.9 ˚F and annual precipitation of 31.06 cm, which was similar to the previous 30-year averages of 69.4˚F and 29.34 cm, respectively (NOAA, 2014). In
September 2011, variants were scored for plant height, stature, number of tillers, leaf width and curling and plant color/reflectance. Among the 1,849 transplants, most of the plants displayed a moderate amount of leaf glaucousness and are referred to as standard type (ST). 92 plants exhibited distinct visible differences in surface glaucousness when compared to the ST. The 92 variants were grouped into three distinct categories including three non-glaucous (NG), 14 reduced glaucous (RG), and 75 highly glaucous (HG) types.
In addition, six plants were identified with extreme glaucousness on both leaf surfaces along with a very short stature and few tillers. These six plants were not considered further. From these primary visual screens, we selected three representatives from the ST,
NG, RG, and HG types for a more detailed analysis of the glaucousness trait. For five successive growing seasons these individuals expressed their respective glaucous phenotypes. None of the glaucous variants, or any of the other phenotypes that were identified, was chimeric as would be expected if the mutagenesis treatment contributed to any of the observed phenotypes in the M1 population. As such, the variation observed is due to natural variation in the population.
3.2 Leaf Cuticle Wax Extraction To best represent a typical wax chemical composition for the ST and each type of variant, leaf samples were collected during the first year anthesis or the R4 stage of reproductive-floral development (Moore et al., 1991). From tillers having fully expanded second leaves below the flag leaf, leaf samples were collected in each of the three selected plants within a class. Leaf samples were excised and placed individually in 101 pollination bags (No. 404, Lawson, Northfield IL) for transport to the lab and wax extraction (in less than 1 hour). Mean wax amounts are presented in this report as the average of three selected plants representative of the ST and each of the variant types. In mid-leaf sections (22.5 x 1.3 cm (L x W)) including the mid-vein, leaf area was determined using a flatbed scanner and image analysis software (LIA32ver.0.377e,
Nagoya University, Nagoya, Japan). Leaf sections were cut into three segments, to fit inside 20 ml scintillation vials (Teflon-lined caps, VWR International, LLC, Brisbane
CA), and submerged in 15 ml of hexane (99% GC hexane, Sigma-Aldrich LLC, St.
Louis, MO) for 45 seconds before the wax extracts were dispensed into clean scintillation vials, as described by Jenks et al. (1995). The leaf segments were briefly (2 sec) rinsed once more with new hexane (5 ml) and hexane fractions combined for each leaf sample.
The samples appeared clear with a slight yellow tint (absent in green coloration typical of chlorophylls) before being evaporated to dryness.
3.3 Chemical Analyses of Waxes Gas chromatography (GC) with flame ionization detection was performed using an
Agilent 7890A GC-FID. The internal standard hexadecane (5 µg) was added to evaporated samples and waxes were derivatized by heating in (75 µl) N,O- bis(trimethylsilyl) trifluoroacetamide (BSTFA, Sigma-Aldrich LLC, St. Louis, MO) at
100°C for 15 min. The BSTFA was evaporated under nitrogen and the samples were suspended in hexane (25 µl) for analyses. Compounds were injected in the GC and then separated using a 12 m, 0.2 µm HP-1 capillary column with helium as the carrier gas. The
GC was programmed with an initial temperature of 80°C and increased at 15°C/min. to
260°C where the temperature was held for 10 min., then increased at 5°C/min. to 320°C, where the temperature was held for 24 min. for a total run time of 58 min. The 102 composition of waxes were analyzed by a GC-Mass Spectrometer (MS) using an Agilent
7890A GC and 5975C Triple-Axis detector MS with 12 m, 0.2 µm HP-Ultra 1 capillary column with helium gas as a carrier, using a similar temperature profile as used for GC-
FID. The molecular identities of individual wax compounds were determined by quadrupole electron impact GC-MS, using relative retention time and mass fragment spectra of each molecular species, in addition to comparisons to NIST MS Search 2.0 database and bona fide standards run on the same instrument. However, those missing from the library were compared to previously published spectra or elucidated from their ion fragmentation patterns (Tulloch and Hoffman, 1980; Wen and Jetter, 2009).
Quantification was based on FID peak areas, using Enhanced data analysis G1701EA software (Agilent Technologies, Santa Clara, CA), relative to the internal standard.
Specific correction factors were developed from external standards and applied to the peak areas of the free fatty acids (C20, C21, C24, C26, and C28), primary alcohols (C20, C22,
C24, C25, C26, C28, C29, and C30) and alkanes (C23, C24, C25, C26, C27, C28, C29, C30, C31, C32 and C33). For all other peaks, a correction factor of 1 was assigned. A representative chromatogram of the 53 wax compounds identified and characterized using GC-MS, which included acids, aldehydes, 1- alcohols, alkanes, β-diketones and esters, is shown in
Supplemental Fig. 1. Further experiments are needed for definitive structural identification of five putative compounds: C19 acid, C20 acid, C21 acid, C33 ketol and C33
OH ketol. Additionally, three tentatively identified compounds, C31 acid, C32 alcohol and
C33 β-diketone (tritriacontane-14,16-dione), were found to be co-eluting with C33 β- diketone (Supplemental Fig. 1B). Since these molecular identities were not confirmed, they were summed in the total unknowns and not discussed further. Total wax amounts 103
(summed across all quantified compounds) were averaged across the three selected individuals of each phenotypic class and expressed as a function of leaf surface area
(µg/dm2) (Table 1). Wax composition was analyzed using JMP software, version 12
(SAS Inc. Cary, NC), to calculate the Pearson product-moment correlation coefficient and for means comparisons by Student’s t-test. The statistical significance was assessed at the 5% probability level unless otherwise indicated.
3.4 Scanning Electron Microscopy Ultrastructural characterization of abaxial (facing away from the stem) and adaxial
(facing the stem) leaf surface waxes was performed on three leaves of a representative plant of the standard type (ST) and a representative plant for each of the three variant types (NG, RG, and HG). Leaf samples were air-dried ca. 32 hours and then small pieces mounted on pin stubs using double-sided carbon tape. Leaf samples were sputter coated twice at 0 and 40 with ca. 7 nm total of platinum (Hummer 6.1 sputtering Device,
Anatech USA, Union City, USA). Micrographs were produced using a field emission scanning electron microscope (4800 FE-SEM; Hitachi, Tokyo, Japan).
4. Results Of the 1,849 plants screened for visible glaucousness, 1,751 plants had a bluish-white color and moderate amounts of visible glaucousness on both the abaxial and adaxial leaf surfaces. This phenotype is referred to as the standard type (ST) for Alamo. 92 plants were identified as having overall normal growth architecture and a leaf surface reflectance that differed dramatically from ST and these variants were classified into three unique types (Fig. 1A-D). The three non-glaucous types (NG) were characterized by a shiny green abaxial leaf surface and an adaxial surface that retained the glaucous
(bluish-white) trait (Fig. 1B). The 14 reduced glaucous types (RG) were distinguished by 104 having a visual glaucousness intermediate to NG and ST, i.e. reduced blue coloration, on the abaxial surface only (Fig. 1C). Although not performed in the current study of switchgrass waxes, Jetter and Schaffer (2001) describe the use of adhesive gum arabic for the extraction of one side of the leaf surface in P. laurocerasus leaves, a technique that could provide a means to extracting abaxial and adaxial waxes separately for the future detailed analysis of this trait in switchgrass. Seventy-five highly glaucous types (HG) had visible glaucousness greater than observed on ST and this occurred on both abaxial and adaxial leaf surfaces (Fig. 1D).
Analysis of leaf wax content and composition using GC/FID/MS confirmed the presence of prominent isomers C33 β-diketone (tritiacontane-12,14-dione) and C33 hydroxyl-β-diketone (5-hydroxytritriacontane-12,14-dione), which were supported by mass spectra that contained diagnostic ions at m/z 100, 183, 122 and m/z 159, respectively (Supplemental Figs. 2 and 3). Mean total wax amounts were determined for each of three ST (mean 810.92 + 30.57 µg/dm2), NG (mean 304.79 + 15.16 µg/dm2), RG
(mean 533.33 + 21.62 µg/dm2) and HG (mean 1228.23 + 45.74 µg/dm2) types as representatives of all of the plants in each class (Table 1). The relatively small standard errors indicate that the visible glaucousness trait for these selections is closely associated with total wax loads.
Comparisons of chemical classes revealed that NG types were similar to STs in amounts of 1-alcohols, but significantly lower (P < 0.01) than STs in the mean amounts of acids, aldehydes, alkanes, β-diketones, esters and unknowns. The RG types were significantly lower (P < 0.01) than STs in all chemical classes except for the β-diketones, which were similar to ST’s. The HG types were similar to STs in the amounts of acids, 105 aldehydes, alkanes, and unknowns. The HG types had significantly lower (P < 0.01) amounts of 1-alcohols and esters than STs, but were significantly higher (P < 0.001) in amounts of β-diketones. In terms of the percentage of total wax (calculated from Table
1), β-diketones in the HG (46.14%) and RG (22.88%) types were higher than the STs
(18.25%) and NG type (1.69%).
Comparisons between wax profiles showed differences in the carbon length distributions of several chemical classes that are worth noting (Fig. 2). For fatty acids, the
2 2 C32 acids were the highest in STs (81.11 + 9.53 µg/dm ) and HG (75.31 + 4.68 µg/dm ) types, but were not significantly different from each other. The C32 acids in RG (55.12 +
4.11 µg/dm2) and NG types (6.90 + 0.78 µg/dm2) were significantly lower (P < 0.05) than in the other types, with the C30 acids in NG types (12.01 + 1.18) being the most abundant
(Fig. 2 B).
The C33 β-diketones were the most abundant compounds on the leaf surface of ST,
RG and HG types, whereas β-diketones were extremely low in NG types (Fig. 2). The
2 C33 β-diketones of NG (5.17 + 1.12 µg/dm ) were significantly lower (P < 0.001) than
STs (144.48 + 21.10µg/dm2), while HG (553.79 + 25.58µg/dm2) types were significantly higher (P < 0.001) than STs (Fig. 3). The mean amounts of C33 β-diketones for RG
(119.30 + 11.57µg/dm2) were similar to STs.
FE-SEM imaging revealed that the adaxial leaf surfaces of all types (ST, NG, RG and
HG) exhibited plate-shaped structures (Fig. 4A-D) whereas the abaxial surfaces of all types except NG exhibited rod-shaped wax crystals (Fig. 4 E, G, H). The NG abaxial surfaces completely lacked wax crystals and displayed a flattened and smoothed-over waxy surface (Fig. 4F). 106
5. Discussion We report here for the first time an ultrastructural analysis of epicuticular waxes on
ST lowland Panicum virgatum, revealing the presence of densely distributed diverse plate- and rod-shaped epicuticular wax crystals over the switchgrass leaf surface. We also report that the cuticular wax constituents of the ST switchgrass cultivar Alamo were comparable to those previously reported in upland P. virgatum cv. Nebraska 28 (Tulloch and Hoffman, 1980). Besides presenting this more detailed analysis of waxes on ST switchgrass, we report a comprehensive analysis of the wax ultrastructure and chemical composition on three newly defined types of switchgrass: non-glaucous (NG), reduced glaucous (RG), and highly glaucous (HG). These variant types were identified based on the level of visible glaucousness. Three representatives of each type were sampled and an average wax ultrastructural and chemical compositional profile across each type was determined as a way to describe the representative wax profiles. The waxes present on the three individuals within each type were similar, as evidenced (in part) by the very low standard errors we report for the average wax amounts determined using GC-MS.
Whether the variants within a type might arise by mutations in the same wax-associated gene, or different genes, is uncertain. Moreover, allelism tests within these phenotypic groups are still needed to ascertain whether the selected individuals represent the same or different complementation (allelic) groups. The results presented here clearly demonstrate the potential genetic diversity for the glaucousness trait present in the switchgrass germplasm, as well as providing a useful description of the microstructural and chemical profiles of the surface waxes on ST and new variant types for glaucousness.
The NG and HG types produced significantly lower and higher amounts of C33 β- diketones in comparison to STs, respectively. NG types also lacked the abaxial wax 107 structures that were present in RG, HG and ST types, which is consistent with NG’s glossy leaf phenotype. Alcohols were found to be equally abundant in NG and STs, suggesting the presence of alcohols is associated with the plate-shaped wax structures on the adaxial side. In contrast to the lower amounts of β-diketones and absence of abaxial wax structures on NG, we can glean from this that the presence of rod-shaped abaxial wax structures on ST indicates the presence of β-diketones. The biochemical and morphological differences in wax content and structure suggest that the β-diketones play a role in determining the glaucous trait in switchgrass. Notably however, the RG switchgrass exhibited reduced glaucousness but little change in β-diketones, indicating that other wax compounds also affect the appearance of the switchgrass leaf glaucousness trait.
Similar to the Hordeum mutants (Cer-c, -q and -u) and the Triticum wl locus mutants, which have reduced amounts of β-diketones or the absence of hydroxyl-β-diketone, respectively, our findings suggest that NG types similarly possess a major inhibition of the β-diketone biosynthetic pathway (Schneider et al. 2016). Relative to ST, we observed an increase in production of all wax constituents on HG types, and a general reduction in all wax constituents on RG types, but no significant reduction in total β-diketones.
Whether the chemical differences observed in these types are due to modifications in metabolic, regulatory, and/or other types of gene expression awaits further studies.
This is the first comprehensive description of glaucous variants in switchgrass. Our results show that the visible wax phenotypes (i.e. high or low glaucousness) on variants were closely correlated to the total amount of waxes as calculated using GC-MS, and thus the amount of visual glaucousness was an indicator of total wax load. Notwithstanding, 108 visible wax amount (intensity of glaucousness) is not always associated with total wax weight, and this is best explained by the fact that glaucousness is positively correlated with the size and density of reflective surface wax crystal facets (Jenks et al., 1994; Grant et al., 2003). In some plants, waxes do not form crystals (and so do not reflect light) even though the wax load may be relatively high. Switchgrass devoid of rod-shaped wax structures, i.e. NG types, has the potential for pathogen resistance, as observed in the irg1/palm1 mutant in Medicago truncatula, and resistance to phytophagous insects, as observed in the bm mutants in Sorghum bicolor (Stark and Weibel, 1981; Uppalapati et al., 2012). The genetic resources described here provide valuable new tools for future studies to elucidate the eco-physiological functions of cuticular waxes, and the potential to apply genetic approaches in switchgrass to improve its biomass yield in more arid and similarly challenging environments. Further studies are needed to examine the genetic, biochemical, and eco-physiological implications for surface waxes on leaves using the three unique wax types identified by this screen.
Acknowledgments JMW would like to acknowledge the funding supported by the Alfred P. Sloan
Foundation’s Indigenous Graduate Partnership, University of Arizona Graduate College, and the Mississippi Band of Choctaw Indians Tribal Scholarship Program. We thank K.
Milward, L. S. Montes-Sujo and M. J. Feldmann, for planting, collection of leaf material and wax extraction. We also thank Ed Bedrick, of the Bio-5 Institute at the University of
Arizona, for statistical consultation. All SEM images and data were collected in the W.M.
Keck Center for Nano-Scale Imaging in the Department of Chemistry and Biochemistry at the University of Arizona with funding from the W.M. Keck Foundation Grant.
109
6. Legends
Figure 1:
Phenotypic characterization of ST and three variant types: NG, RG and HG. A-D) Photographic images of whole plants reveal abaxial leaf surfaces that vary in glaucousness from green (NG type) to whitish blue (HG type). The insets indicate changes in glaucousness based on leaf sidedness with the abaxial surfaces identified by white arrows and the adaxial identified by black arrows.
Figure 2:
Wax chemical profiles, presented as chemical classes, on the leaf segments of ST (n=3), and three variant types NG (n=3), RG (n=3), and HG (n=3). A-D) Carbon chain lengths of individual constituents are shown for each wax chemical class in µg/dm2 (mean ± SE). Numbers beside bars indicate actual amounts where the total is above the Y-axis scale. Esters are a sum of five identified ester compounds.
Figure 3:
Amounts of C33 β-diketones on leaf segments of switchgrass for ST (n=3), and three variant types NG (n=3), RG (n=3), and HG (n=3). Data represent the average and standard error (presented in µg/dm2). Significance values (t-test) are indicated by the asterisks; *** P < 0.001.
Figure 4:
Ultrastructural characterization of leaf epicuticular wax on switchgrass of ST, NG, RG and HG types. A-D) Micrographic images of the adaxial surfaces of leaves reveal plate-shaped wax crystal morphology of ST, NG, RG and HG. E-H) Rod-shaped wax crystal morphology on the abaxial surface of ST (similar to RG and HG) and absence of wax crystals on NG switchgrass.
Table 1:
Amounts of total leaf cuticular waxes, and totals for six chemical classes, on leaves in ST (n=3), and three variant types NG (n=3), RG (n=3), and HG (n=3). Data is presented in µg/dm2 (mean ± SE). Acid is free fatty acid; 1-alcohol is primary alcohol. Significance values (t-test) are indicated by the asterisks; * P < 0.05, ** P < 0.01, *** P < 0.001.
Supplemental Figure 1:
GC-MS chromatogram of hexane soluble cuticular wax components obtained after BSTA derivatization from the ST. A) Retention times 5.6-24 min., numbered constituents are as listed below. B) Retention times 24-42 min., symbols within the peaks at baseline indicated minor coelution compounds as listed below. ITSD: internal standard hexadecane (5 µg). (1) C23 alkane, (2) C19 acid, (3) C20 alcohol, (4) C24 alkane, (5) C20 acid, (6) C25 alkane, (7) C21 acid, (8) C22 alcohol, (9) C26 alkane, (10) C24 aldehyde, (11) C22 acid, (12) C27 alkane, (13) C25 aldehyde, (14) C23 acid, (15) C24 alcohol, (16) C28 alkane, (17) C26 aldehyde, (18) C24 acid, (19) C29 alkane, (20) C27 aldehyde, (21) C25 acid, (22) C26 alcohol, (23) C30 alkane, (24) C28 aldehyde, (25) C26 acid, (26) C27 alcohol, (27) C31 alkane, (28) C29 aldehyde, (29) C27 acid, (30) C28 alcohol, (31) C25 alcohol, (32) C32 alkane, (33) C30 aldehyde, (34) C28 acid, (35) C29 alcohol, (36) C33 alkane, (37) C29 acid, (38) C30 alcohol, (39) C32 aldehyde, (40) C30 acid, (41) C31 alcohol, ( ) C31 acid, ( ) C32 alcohol, (42) C33 β- diketone (tritriacontane-12,14-dione), ( ) C33 β-diketone (tritriacontane-14,16-dione), (43) C33 ketol, (44) C32 acid, (45) C34 alcohol, (46) C33 hydroxy-β-diketone (5-hydroxytritriacontane-12,14-dione), (47) C33 OH ketol, (48) C34 acid, (49) ester 1, (50) ester 2, (51) ester 3, (52) ester 4, (53) ester 5.
110
Supplemental Figure 2:
Mass spectrum of C33 β-diketone (tritiacontane-12,14-dione) with notable diagnostic ions at m/z 100, 183 and 122.
Supplemental Figure 3:
The mass spectrum of C33 hydroxy-β-diketone (5-hydroxytritriacontane-12,14-dione) with notable diagnostic ion m/z 159.
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7. Figures and Table
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8. Supplemental Figures
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