Biochemical Characterization of Cuticular Wax Variants in Switchgrass (Panicum Virgatum L.)

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. 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-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.