Characterization and Improvement of Novel Bioenergy Grasses (Tripidium spp.)
Nathan Maren, Ph.D. Final Seminar
Under the direction of Dr. Tom Ranney and Dr. Hudson Ashrafi
Thursday, September 17, 2020, at 1:00 pm
Join Zoom Meeting: https://ncsu.zoom.us/j/97092441009?pwd=aTZxYWVpUjNuS1g0dXkxdUlPVkl2Zz09

Abstract
Growing economies, limited fossil fuel reserves, and environmental concerns have justified expanded research on renewable energy sources, including bioenergy crops.  Taxa in the Poaceae Subtribe Saccharinae have gained attention as bioenergy crops based on their broad adaptability, pest resistance, and high biomass yields.  Recently reclassified, Tripidium (syn. Erianthus, syn. Saccharum) is of particular interest due to perenniality, cold-hardiness, and high biomass yields. Tripidium ravennae is a cold-hardy, diploid species (2n = 2x = 20).  Tripidium arundinaceum is a sub-tropical polyploid species (2n = 3x, 4x, 6x = 30, 40, 60) with high biomass yields.

Conventional breeding efforts focused on developing Tripidium as a competitive bioenergy feedstock for temperate climates.  Advanced interspecific hybrids between T. arundinaceum and T. ravennae were evaluated in field plots relative to Miscanthus ×giganteus  over three years.  Collected data evaluated biomass yield, plant fertility, cytogenetics, and compositional analyses for lignocellulosic ethanol and forage utility.  Cytology and cytometry confirmed hybrids were tetraploid with 2n = 4x = 40 (2C genome size = 5.06 pg).  Dry biomass yields varied as a function of year and accession and increased each year, ranging 3.4 – 10.6, 8.6 – 37.3, and 23.7 – 60.6 Mg/ha for Tripidium hybrids compared to 2.3, 16.2, and 27.9 Mg/ha for M. ×giganteus in 2016, 2017, and 2018, respectively. Variations in yield and compositional analyses contributed to variations in theoretical ethanol yields ranging from 10,181 to 27,546 L/ha for Tripidium accessions compared to 13,095 L/ha for M. ×giganteus.  These initial findings for Tripidium hybrids are promising and warrant further development of Tripidium as a temperate bioenergy feedstock.

A robust understanding of the molecular mechanism of flowering in Tripidium will enable future biotechnology applications of floral and seed development. Therefore, a differential gene expression (DEG) analysis was conducted to identify the DEGs associated with flower and seed development in T. ravennae.  In the early phases of inflorescence development, the type II subfamily of MADS-box transcription factors were over-represented in both GO enrichment and differential expression analyses.  As developing inflorescences matured, there was increased expression of inflorescence determinacy regulators, meiotic, and multicellular organism developmental process transcripts.  In seed developmental samples, multiple unigenes with oxidative-reductive processes were identified.  These results provide insights into the molecular regulation of reproductive development and provide a foundational database for future investigations and analyses, including genome annotation, functional genomics characterization, gene family evolutionary studies, comparative genomics, and precision breeding.

The potential to improve Tripidium hybrids via biotechnology applications for value-added traits would further enhance crop improvement opportunities.  The objective of this portion of the research was to develop an efficient regeneration and transformation procedure with utility for genetic modification of Tripidium hybrids.  Multiple studies elucidated the effects of various hormones, tissue culture media adjuncts, culture duration, Agrobacterium density, and hygromycin concentration on callus induction, maintenance, regeneration, and transformation efficiency.  Callus induction media containing 10 – 40 µM 2,4-D with 12.5 mM L-proline generated callus that maximized shoot number (mean of 37 to 45 shoots٠explant-1) following regeneration.  Callus maintenance media containing four µM 2,4-D and 12.5 mM L-proline for durations less than 12 weeks resulted in callus that maximized shoot number (mean of 13 to 18 shoots٠explant-1) following regeneration. Final experiments evaluating hygromycin concentration and bacterial density on transformation efficiency are in progress.

Collectively, these research projects served to 1) evaluate and characterize new Tripidium hybrids as potential bioenergy crops, 2) establish foundational transcriptomic resources on flowering and reproductive development of Tripidium, and 3) develop a regeneration and transformation system to enable future biotechnology applications in these crops.  These efforts provide strategic advances in the development of Tripidium as a new bioenergy crop.