Computational tool-assisted primer design for real-time reverse transcription (RT) PCR (qPCR) analysis largely ignores the sequence similarities between sequences of homologous genes in a plant genome. It can lead to false confidence in the quality of the designed primers, which sometimes results in skipping the optimization steps for qPCR. However, the optimization of qPCR parameters plays an essential role in the efficiency, specificity, and sensitivity of each gene’s primers. Here, we proposed an optimized approach to sequentially optimizing primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference (and target) gene. Our approach started with a sequence-specific primer design that should be based on the single-nucleotide polymorphisms (SNPs) present in all the homologous sequences for each of the reference (and target) genes under study. By combining the efficiency calibrated and standard curve methods with the 2−ΔΔCt method, the standard cDNA concentration curve with a logarithmic scale was obtained for each primer pair for each gene. As a result, an R2 ≥ 0.9999 and the efficiency (E) = 100 ± 5% should be achieved for the best primer pair of each gene, which serve as the prerequisite for using the 2−ΔΔCt method for data analysis. We applied our newly developed approach to identify the best reference genes in different tissues and at various inflorescence developmental stages of Tripidium ravennae, an ornamental and biomass grass, and validated their utility under varying abiotic stress conditions. We also applied this approach to test the expression stability of six reference genes in soybean under biotic stress treatment with Xanthomonas axonopodis pv. glycines (Xag). Thus, these case studies demonstrated the effectiveness of our optimized protocol for qPCR analysis.

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High biomass yields have been documented for Tripidium spp. (Erianthus spp., Saccharum spp.), but targeted breeding for bioenergy applications has been limited. Advanced, interspecific hybrids between Tripidium ravennae and T. arundinaceum were planted in replicated field plots in 2016. Comparative feedstock evaluations examined biomass yields, cytogenetics, plant fertility, and compositional analyses relative to Miscanthus × giganteus. Dry biomass yields varied as a function of year and accession and increased each year ranging from 3.4 to 10.6, 8.6 to 37.3, and 23.7 to 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. Cytology and cytometry confirmed that Tripidium hybrids were tetraploid with 2n = 4x = 40 (2C genome size = 5.06 pg) and intermediate between T. ravennae with 2n = 2x = 20 (2C genome size = 2.55 pg) and T. arundinaceum with 2n = 6x = 60 (2C genome size = 7.61 pg). Plant fertility characteristics varied considerably with some accessions producing no viable seeds or fewer than that observed for M. × giganteus. Accessions varied significantly for flowering culm number and height and dates of peak anthesis ranging from 14 September to 2 October. 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. Relative feed value (RFV) indices for winter-harvested Tripidium accessions varied from 52.8 to 60.0 compared to M. × giganteus with 45.4. RFV for summer-harvested Tripidium accessions varied from 71.6 to 80.5 compared to M. × giganteus with 61.0. These initial findings for Tripidium hybrids, including high biomass yields, cold hardiness, and desirable traits for multiple markets (e.g., forage, bioenergy, bioproducts), are promising and warrant further development of Tripidium as a temperate bioenergy feedstock. K E Y W O R D S bioenergy, bioprocessing, Erianthus, fertility, forage, lignocellulose, Miscanthus, plant breeding, polyploidy, reproductive biology, Saccharum, sterility, Tripidium [Correction added on 13 April 2020 after first online publication: the genome values have been updated in the Abstract and Results sections in this version.] 362 | MAREN Et Al.Note: Superscript letters indicate statistically significant value p ≤ .05. 1 2C DNA values represent the mean value of three to six samples conducted for each taxon.2 Taxa followed by an asterisk were confirmed with cytology. 3 Values represent the mean ± SEM. Mean separation within columns by Fisher's least significant difference at p ≤ .05. 4 Calculated as (seed set × seed germination)/100. 5 Flowering was not observed within the growing season at the Fletcher, NC evaluation site.

Plant transformation and regeneration remain highly species- and genotype-dependent. Conventional hormone-based plant regeneration via somatic embryogenesis or organogenesis is tedious, time-consuming, and requires specialized skills and experience. Over the last 40 years, significant advances have been made to elucidate the molecular mechanisms underlying embryogenesis and organogenesis. These pioneering studies have led to a better understanding of the key steps and factors involved in plant regeneration, resulting in the identification of crucial growth and developmental regulatory genes that can dramatically improve regeneration efficiency, shorten transformation time, and make transformation of recalcitrant genotypes possible. Co-opting these regulatory genes offers great potential to develop innovative genotype-independent genetic transformation methods for various plant species, including specialty crops. Further developing these approaches has the potential to result in plant transformation without the use of hormones, antibiotics, selectable marker genes, or tissue culture. As an enabling technology, the use of these regulatory genes has great potential to enable the application of advanced breeding technologies such as genetic engineering and gene editing for crop improvement in transformation-recalcitrant crops and cultivars. This review will discuss the recent advances in the use of regulatory genes in plant transformation and regeneration, and their potential to facilitate genotype-independent plant transformation and regeneration.

Background Tripidium ravennae is a cold-hardy, diploid species in the sugarcane complex (Poaceae subtribe Saccharinae) with considerable potential as a genetic resource for developing improved bioenergy and ornamental grasses. An improved understanding of the genetic regulation of reproductive processes (e.g., floral induction, inflorescence development, and seed development) will enable future applications of precision breeding and gene editing of floral and seed development. In particular, the ability to silence reproductive processes would allow for developing seedless forms of valuable but potentially invasive plants. The objective of this research was to characterize the gene expression environment of reproductive development in T. ravennae. Results During the early phases of inflorescence development, multiple key canonical floral integrators and pathways were identified. Annotations of type II subfamily of MADS-box transcription factors, in particular, were over-represented in the GO enrichment analyses and tests for differential expression (FDR p-value < 0.05). The differential expression of floral integrators observed in the early phases of inflorescence development diminished prior to inflorescence determinacy regulation. Differential expression analysis did not identify many unique genes at mid-inflorescence development stages, though typical biological processes involved in plant growth and development expressed abundantly. The increase in inflorescence determinacy regulatory elements and putative homeotic floral development unigenes at mid-inflorescence development coincided with the expression of multiple meiosis annotations and multicellular organism developmental processes. Analysis of seed development identified multiple unigenes involved in oxidative-reductive processes. Conclusion Reproduction in grasses is a dynamic system involving the sequential coordination of complex gene regulatory networks and developmental processes. This research identified differentially expressed transcripts associated with floral induction, inflorescence development, and seed development in T. ravennae. These results provide insights into the molecular regulation of reproductive development and provide a foundation for future investigations and analyses, including genome annotation, functional genomics characterization, gene family evolutionary studies, comparative genomics, and precision breeding.

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