Plant Breeding for Intercropping in Temperate Field Crop Systems: A Review.

Monoculture cropping systems currently dominate temperate agroecosystems. However, intercropping can provide valuable benefits, including greater yield stability, increased total productivity, and resilience in the face of pest and disease outbreaks. Plant breeding efforts in temperate field crops are largely focused on monoculture production, but as intercropping becomes more widespread, there is a need for cultivars adapted to these cropping systems. Cultivar development for intercropping systems requires a systems approach, from the decision to breed for intercropping systems through the final stages of variety testing and release. Design of a breeding scheme should include information about species variation for performance in intercropping, presence of genotype × management interaction, observation of key traits conferring success in intercropping systems, and the specificity of intercropping performance. Together this information can help to identify an optimal selection scheme. Agronomic and ecological knowledge are critical in the design of selection schemes in cropping systems with greater complexity, and interaction with other researchers and key stakeholders inform breeding decisions throughout the process. This review explores the above considerations through three case studies: (1) forage mixtures, (2) perennial groundcover systems (PGC), and (3) soybean-pennycress intercropping. We provide an overview of each cropping system, identify relevant considerations for plant breeding efforts, describe previous breeding focused on the cropping system, examine the extent to which proposed theoretical approaches have been implemented in breeding programs, and identify areas for future development.

Perennial groundcovers: an emerging technology for soil conservation and the sustainable intensification of agriculture.

Integrating perennial groundcovers (PGC) - sometimes referred to as living mulches or perennial cover crops - into annual cash-crop systems could address root causes of bare-soil practices that lead to negative impacts on soil and water quality. Perennial groundcovers bring otherwise absent functional traits - namely perenniality - into cash-crop systems to preserve soil and regenerate water, carbon, and nutrient cycles. However, if not optimized, they can also cause competitive interactions and yield loss. When designing PGC systems, the goal is to maximize complementarity - spatial and temporal separation of growth and resource acquisition - between PGC and cash crops through both breeding and management. Traits of interest include complementary root and shoot systems, reduced shade avoidance response in the cash-crop, and PGC summer dormancy. Successful deployment of PGC systems could increase both productivity and profitability by improving water- and nutrient-use-efficiency, improving weed and pest control, and creating additional value-added opportunities like stover harvest. Many scientific questions about the inherent interactions at the cell, plant, and ecosystem levels in PGC systems are waiting to be explored. Their answers could enable innovation and refinement of PGC system design for multiple geographies, crops, and food systems, creating a practical and scalable pathway towards resiliency, crop diversification, and sustainable intensification in agriculture.

Genetic Transformation of Recalcitrant Upland Switchgrass Using Morphogenic Genes.

Switchgrass (Panicum virgatum) is an excellent feedstock for biofuel production. While genetic transformation is routinely done in lowland switchgrass, upland cultivars remain recalcitrant to genetic transformation. Here we report the establishment of an efficient and reproducible transformation protocol for two upland cultivars, 'Summer' and 'Blackwell', by ectopic overexpression of morphogenic genes, Baby boom (Bbm) and Wuschel2 (Wus2). Two auxotrophic Agrobacterium strains, LBA4404Thy- and EHA105Thy-, each harboring the same construct containing ZmBbm, ZmWus2, and a green fluorescence protein (GFP) gene, ZsGreen1, were used to infect immature leaf segments derived from in vitro grown seedlings. The Agrobacterium strains also contain a transformation helper plasmid that carry additional copies of Agrobacterium virulence genes. GFP-expressing calli were identified and selected for regeneration. The highest transformation efficiency of 6% was obtained for the tetraploid cultivar Summer when LBA4404Thy- was used for infection, which is twice of that for the octoploid cultivar Blackwell. LBA4404Thy- consistently outperformed EHA105Thy- on transformation frequency across the two cultivars. Fifteen randomly selected putative transgenic plants of Summer and Blackwell, representing independent callus events, were confirmed as transgenic by the presence of the transgene, ZmAls, and the absence of AtuFtsZ, a chromosomal gene specific to the Agrobacterium strain LBA4404 using polymerase chain reaction. Transgene integration and expression was further confirmed by the detection of GFP in roots, and the resistance to herbicide injury to leaves of selected putative transgenic plants. The ZmBbm and ZmWus2 genes were successfully removed from 40 to 33.3% of the transgenic plants of Summer and Blackwell, respectively, via the Cre-Lox recombination system upon heat treatment of GFP-expressing embryogenic calli. Our successful transformation of recalcitrant upland switchgrass provides a method for gene function analysis and germplasm enhancement via biotechnology.

Functional Analysis of the teosinte branched 1 Gene in the Tetraploid Switchgrass (Panicum virgatum L.) by CRISPR/Cas9-Directed Mutagenesis.

Tillering is an important biomass yield component trait in switchgrass (Panicum virgatum L.). Teosinte branched 1 (tb1)/Branched 1 (BRC1) gene is a known regulator for tillering/branching in several plant species; however, its role on tillering in switchgrass remains unknown. Here, we report physiological and molecular characterization of mutants created by CRISPR/Cas9. We successfully obtained nonchimeric Pvtb1a and Pvtb1b mutants from chimeric T0 mutants using nodal culture. The biallelic Pvtb1a-Pvtb1b mutant plants produced significantly more tillers and higher fresh weight biomass than the wild-type plants. The increased tiller number in the mutant plants resulted primarily from hastened outgrowth of lower axillary buds. Increased tillers were also observed in transgene-free BC1 monoallelic mutants for either Pvtb1a-Pvtb1b or Pvtb1b gene alone, suggesting Pvtb1 genes act in a dosage-dependent manner. Transcriptome analysis showed 831 genes were differentially expressed in the Pvtb1a-Pvtb1b double knockdown mutant. Gene Ontology analysis revealed downregulation of Pvtb1 genes affected multiple biological processes, including transcription, flower development, cell differentiation, and stress/defense responses in edited plants. This study demonstrates that Pvtb1 genes play a pivotal role in tiller production as a negative regulator in switchgrass and provides opportunities for further research aiming to elucidate the molecular pathway regulating tillering in switchgrass.

Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9.

The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self-infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar 'Alamo' switchgrass. We first developed a transient assay by which a non-functional green-fluorescent protein gene containing a 1-bp frameshift insertion in its 5' coding region was successfully mutated by a Cas9/sgRNA complex resulting in its restored function. Agrobacterium-mediated stable transformation of embryogenic calli derived from mature caryopses averaged a 3.0% transformation efficiency targeting the genes of teosinte branched 1(tb1)a and b and phosphoglycerate mutase (PGM). With a single construct containing two sgRNAs targeting different regions of tb1a and tb1b genes, primary transformants (T0) containing CRISPR/Cas9-induced mutations were obtained at frequencies of 95.5% (tb1a) and 11% (tb1b), respectively, with T0 mutants exhibiting increased tiller production. Meanwhile, a mutation frequency of 13.7% was obtained for the PGM gene with a CRISPR/Cas9 construct containing a single sgRNA. Among the PGM T0 mutants, six are heterozygous and one is homozygous for a 1-bp deletion in the target region with no apparent phenotypical alterations. We show that CRISPR/Cas9 system can generate targeted mutagenesis effectively and obtain targeted homozygous mutants in T0 generation in switchgrass, circumventing the need of inbreeding.

BdVRN1 Expression Confers Flowering Competency and Is Negatively Correlated with Freezing Tolerance in Brachypodium distachyon.

Vernalization is an essential process by which many temperate plant species acquire competence for flowering. Brachypodium distachyon is a model plant for temperate grasses including many cool-season forage and turfgrasses and cereals. Grasses with spring growth habit do not require vernalization for flowering and are typically less winter hardy. Yet the connection between vernalization and freezing tolerance remain unclear. The diverse requirement of vernalization for flowering makes it an ideal choice for studying the relationship between vernalization and freezing tolerance. Here, we isolated and analyzed the spatial and temporal expression patterns of two vernalization related homologous genes, BdVRN1 and BdVRN3 in Bd21, a non-vernalization-requiring accession, and Bd29-1, an accession shown to be vernalization-requiring. We showed that expression of BdVRN1 and BdVRN3 is independent of vernalization in Bd21, but is vernalization dependent in Bd29-1. Moreover, vernalization-induced expression of BdVRN1 appears to precede that of BdVRN3 in Bd29-1. Bd21 RNAi knockdown mutants for BdVRN1 conferred vernalization requirement for flowering, and reduced the expression of BdVRN3. Both Bd29-1 and the BdVRN1 RNAi mutants of Bd21 exhibited reduced freezing tolerance upon vernalization treatment. Cold-responsive genes BdCBF2, BdCBF3, BdCBF5, BdCBF6, and BdDREB2A were all constitutively expressed at a high level in the BdVRN1 RNAi mutants of Bd21. Taken together, our results suggest that expression of BdVRN1 promotes flowering by upregulating BdVRN3, and gaining the competency for flowering reduces freezing tolerance in Brachypodium.

Characterization of BdCBF genes and genome-wide transcriptome profiling of BdCBF3-dependent and -independent cold stress responses in Brachypodium distachyon.

Freezing stress substantially reduces crop yields and limits plant distribution. The identification of genes critical for cold acclimation is thus of great importance. C-repeat binding factors (CBFs) are transcription factors that play key regulatory roles in the cold acclimation process, which dramatically increases freezing tolerance in plants. We report here that B. distachyon can successfully cold acclimate and we identified a CBF gene family consisting of eight genes in a tandem array and are designated as BdCBF1-8. Expression analysis indicated that all the eight BdCBF genes are induced by cold. Freezing tolerance experiments showed that the knockdown of BdCBF3 gene in RNAi cbf3 mutant plants results in a significant reduction in survival after an exposure to freezing temperatures. RNA-seq transcriptomic analysis was conducted using the wild type and cbf3 mutant plants under both normal and cold conditions. We identified 460, 3213, 2839 and 1871 differentially expressed genes exhibiting different expression levels by pairwise comparisons of cbf3 (23°C) vs. WT (23°C), WT (23°C) vs. WT (4°C), cbf3 (23°C) vs. cbf3 (4°C), and cbf3 (4°C) vs. WT (4°C), respectively. These differentially expressed genes were enriched in several biological pathways. Combined analyses of differentially expressed genes in some of the enriched pathways provide insights into mechanisms of plant response to cold in the BdCBF3-dependent, -independent or -compensatory categories.

Down-regulation of BdBRI1, a putative brassinosteroid receptor gene produces a dwarf phenotype with enhanced drought tolerance in Brachypodium distachyon.

Brassinosteroids (BRs) play important roles in plant growth, development and responses to a range of environmental cues. Although the mechanism of how BRs regulate growth and development is well-understood in Arabidopsis, the effect of BRs on stress tolerance, particularly drought tolerance remains unknown. We isolated a BRI1 (BRASSINOSTEROID INSENSITIVE 1) homologous gene, BdBRI1 from Brachypodium distachyon, a model for temperate grasses and cereals, created and characterized RNA interference (RNAi) knockdown mutants for BdBRI1 in Brachypodium. The loss-of-function BdBRI1-RNAi mutants exhibited reduced plant height, shortened internodes, narrow and short leaf, and reduced expression of BR signaling genes, BdBES1, BdBZR1, BdBLE2, and enhanced expression of BR biosynthesis genes BdD2, BdCPD and BdDWF4. More importantly, BdBRI1 RNAi mutants exhibited enhanced drought tolerance, accompanied by highly elevated expression of drought-responsive genes, BdP5CS, BdCOR47/BdRD17, together with BdERD1 and BdRD26, two putative targets of the transcription factors BES1 and BZR1 that are key components of the BR signaling pathway. Our results suggest that BR signaling and biosynthesis are largely conserved among Arabidopsis, rice and Brachypodium, and that BR signaling plays an important role in drought tolerance by directly regulating expression of key drought-responsive genes. The effect of BR biosynthesis or crosstalks between BR and other hormones or components of stress signaling pathways on drought tolerance is discussed.

Brassinosteroid signaling network: implications on yield and stress tolerance.

The steroidal hormone brassinosteroids (BRs) play important roles in plant growth and development. Genetic, genomic and proteomic studies in Arabidopsis have identified major BR signaling components and elucidated the signal transduction pathway from the cell surface receptor kinase BRI1 to the BES1/BZR1 family of transcription factors. BRs interact with other plant hormones in coordinating gene expression and plant growth and development. In this review, we provide an update on the latest progress in characterizing the BR signaling network and discuss its interactions with other hormone pathways in determining yield component traits and in regulating stress responses.

Ice recrystallization inhibition proteins of perennial ryegrass enhance freezing tolerance.

Ice recrystallization inhibition (IRI) proteins are thought to play an important role in conferring freezing tolerance in plants. Two genes encoding IRI proteins, LpIRI-a and LpIRI-b, were isolated from a relatively cold-tolerant perennial ryegrass cv. Caddyshack. Amino acid alignments among the IRI proteins revealed the presence of conserved repetitive IRI-domain motifs (NxVxxG/NxVxG) in both proteins. Quantitative reverse transcriptase PCR (qRT-PCR) analysis indicated that LpIRI-a was up-regulated approximately 40-fold while LpIRI-b was up-regulated sevenfold after just 1 h of cold acclimation, and by 7 days of cold acclimation the transcripts had increased 8,000-fold for LpIRI-a and 1,000-fold for LpIRI-b. Overexpression of either LpIRI-a or LpIRI-b gene in Arabidopsis increased survival rates of the seedlings following a freezing test under both cold-acclimated and nonacclimated conditions. For example, without cold acclimation a -4 degrees C treatment reduced the wild type's survival rate to an average of 73%, but resulted in survival rates of 85-100% for four transgenic lines. With cold acclimation, a -12 degrees C treatment reduced the wild type's survival rate to an average of 38.7%, while it resulted in a survival rate of 51-78.5% for transgenic lines. After cold acclimation, transgenic Arabidopsis plants overexpressing either LpIRI-a or LpIRI-b gene exhibited a consistent reduction in freezing-induced ion leakage at -8, -9, and -10 degrees C. Furthermore, the induced expression of the LpIRI-a and LpIRI-b proteins in transgenic E. coli enhanced the freezing tolerance in host cells. Our results suggest that IRI proteins play an important role in freezing tolerance in plants.

Visualization by atomic force microscopy and FISH of the 45S rDNA gaps in mitotic chromosomes of Lolium perenne.

The mitotic chromosome structure of 45S rDNA site gaps in Lolium perenne was studied by atomic force microscope (AFM) combining with fluorescence in situ hybridization (FISH) analysis in the present study. FISH on the mitotic chromosomes showed that 45S rDNA gaps were completely broken or local despiralizations of the chromatid which had the appearance of one or a few thin DNA fiber threads. Topography imaging using AFM confirmed these observations. In addition, AFM imaging showed that the broken end of the chromosome fragment lacking the 45S rDNA was sharper, suggesting high condensation. In contrast, the broken ends containing the 45S rDNA or thin 45S rDNA fibers exhibited lower density and were uncompacted. Higher magnification visualization by AFM of the terminals of decondensed 45S rDNA chromatin indicated that both ends containing the 45S rDNA also exhibited lower density zones. The measured height of a decondensed 45S rDNA chromatin as obtained from the AFM image was about 55-65 nm, composed of just two 30-nm single fibers of chromatin. FISH in flow-sorted G2 interphase nuclei showed that 45S rDNA was highly decondensed in more than 90% of the G2/M nuclei. Our results suggested that a failure of the complex folding of the chromatin fibers occurred at 45S rDNA sites, resulting in gap formation or break.

Identification of genes associated with cold acclimation in perennial ryegrass.

Cold acclimation dramatically increases freezing tolerance in many temperate plant species. An understanding of cold acclimation is important for extending adaptation areas of perennial ryegrass. Freezing tolerance is greatly increased in perennial ryegrass cv. 'Caddyshack' after cold acclimation. Genes differentially regulated during cold acclimation were identified by analyzing the abundance of expressed sequence tags (ESTs) randomly sampled from two cDNA libraries, one constructed from 14-d cold-acclimated (CA; LT(50)=-12.2 degrees C) and the other from nonacclimated (NA; LT(50)=-7.6 degrees C) leaves of 'Caddyshack'. More than 1400 quality ESTs were generated for each library. Over 60 EST groups were either increased or decreased three times or more in the CA library than in the NA library. Functional classification showed that for nine gene ontology (GO) subcategories, the ratio of CA ESTs to NA ESTs increased twice or more, whereas the ratio decreased by half or more for seven other GO subcategories. Expression analysis of 23 selected genes confirmed that 19 of them exhibited expression patterns consistent with the EST abundance analysis. Our results suggest that up-regulation of cold-regulated (COR), dehydration-responsive, and ice recrystallization inhibition (IRI) genes, and down-regulation of photosynthesis and respiration-related genes are important to increasing freezing tolerance in perennial ryegrass.

45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp.

BACKGROUND: In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported. METHODS AND RESULTS: During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions) at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region. CONCLUSIONS: The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.

Functional and phylogenetic analysis of a DREB/CBF-like gene in perennial ryegrass (Lolium perenne L.).

The dehydration-responsive element binding proteins (DREB1)/C-repeat (CRT) binding factors (CBF) function as transcription factors and bind to the DRE/CRT cis-acting element (core motif: G/ACCGAC) commonly present in cold-regulated (COR) genes and subsequently upregulate the expression of such genes in Arabidopsis. We identified a DREB1A/CBF3-like gene, designated LpCBF3, from perennial ryegrass (Lolium perenne L.) by using RT-PCR and RACE (rapid amplification of cDNA end). The LpCBF3 gene contains all the conserved domains known to exist in other CBF genes. A comprehensive phylogenetic analysis using known and computationally identified CBF homologs in this study revealed that all monocot CBF genes are separately clustered from eudicot CBF genes and the LpCBF3 is the ortholog of rice OsDREB1A/CBF3 gene. Similar to other DREB1A/CBF3 homologs, expression of the LpCBF3 is induced by cold stress, but not by abscisic acid (ABA), drought, or salinity. Overexpression of the LpCBF3 cDNA in Arabidopsis induced expression of the Arabidopsis DREB1A/CBF3 target COR genes, COR15a and RD29A, without cold acclimation. Ion leakage in leaves of the overexpression transgenic plants was significantly reduced, an indication of enhanced freezing tolerance. Our data demonstrated that LpCBF3 not only resembles DREB/CBF genes of Arabidopsis, but is also capable of functioning as a transcriptional regulator in Arabidopsis, a species distant to the grass family.