Genetic Variation in Tolerance to Iron Deficiency among Species of <i>Oryza</i>

Transplanted rice cultivation has caused groundwater depletion in several regions globally. Direct-seeded rice under aerobic conditions is a water-saving alternative. However, under aerobic conditions, iron in the soil is oxidized from the ferrous to ferric forms, which are not easily available to rice crops, resulting in iron-deficiency-induced chlorosis (IDIC) and causing significant reductions in yield. Cultivated rice accessions have limited variations in IDIC tolerance, while the wild Oryza germplasm could be a potential source of IDIC tolerance. In this study, 313 Oryza accessions were evaluated for IDIC tolerance at the tillering stage under aerobic conditions and 20 IDIC-tolerant lines were identified. The twenty lines showed no signs of chlorosis and had high levels of iron content and SPAD values, while the eight cultivated controls exhibited varying degrees of chlorosis symptoms and low levels of SPAD and iron content. To confirm their tolerance, the selected lines were evaluated again in a subsequent year, and they showed comparable levels of tolerance, indicating that these lines were efficient in iron uptake and utilization, resulting in maintained high chlorophyll and leaf area index. These accessions may be useful for developing IDIC-tolerant cultivars for aerobic rice cultivation and future studies of the molecular basis of IDIC tolerance

Agrobacterium- and a single Cas9-sgRNA transcript system-mediated high efficiency gene editing in perennial ryegrass.

Genome editing technologies provide a powerful tool for genetic improvement of perennial ryegrass, an important forage and turfgrass species worldwide. The sole publication for gene editing in perennial ryegrass used gene-gun for plant transformation and a dual promoter based CRISPR/Cas9 system for editing. However, their editing efficiency was low (5.9% or only one gene-edited plant produced). To test the suitability of the maize Ubiquitin 1 (ZmUbi1) promoter in gene editing of perennial ryegrass, we produced ZmUbi1 promoter:RUBY transgenic plants. We observed that ZmUbi1 promoter was active in callus tissue prior to shoot regeneration, suggesting that the promoter is suitable for Cas9 and sgRNA expression in perennial ryegrass for high-efficiency production of bi-allelic mutant plants. We then used the ZmUbi1 promoter for controlling Cas9 and sgRNA expression in perennial ryegrass. A ribozyme cleavage target site between the Cas9 and sgRNA sequences allowed production of functional Cas9 mRNA and sgRNA after transcription. Using Agrobacterium for genetic transformation, we observed a 29% efficiency for editing the PHYTOENE DESATURASE gene in perennial ryegrass. DNA sequencing analyses revealed that most pds plants contained bi-allelic mutations. These results demonstrate that the expression of a single Cas9 and sgRNA transcript unit controlled by the ZmUbi1 promoter provides a highly efficient system for production of bi-allelic mutants of perennial ryegrass and should also be applicable in other related grass species

An Improved Procedure for Agrobacterium-Mediated Transformation of &lsquo;Carrizo&rsquo; Citrange

Although several protocols for genetic transformation of citrus have been published, it is highly desirable to further improve its efficiency. Here we report treatments of Agrobacterium cells and citrus explants prior to and during co-cultivation process to enhance transformation efficiency using a commercially used rootstock &lsquo;Carrizo&rsquo; citrange [Citrus sinensis (L.) Osb. &times; Poncirius trifoliata (L.) Raf.] as a model plant. We found explants from light-grown seedlings exhibited higher transformation efficiency than those from etiolated seedlings. We pre-cultured Agrobacterium cells in a 1/10 MS, 0.5 g/L 2-(N-morpholino) ethanesulfonic acid (MES) and 100 &micro;M acetosyringone liquid medium for 6 h at 25 &deg;C before used to infect citrus explants. We incubated epicotyl segments in an MS liquid medium containing 13.2 &micro;M 6-BA, 4.5 &micro;M 2,4-D, 0.5 &micro;M NAA for 3 h at 25 &deg;C prior to Agrobacterium infection. In the co-cultivation medium, we added 30 &micro;M paclobutrazol and 10 &micro;M lipoic acid. Each of these treatments significantly increased the efficiencies of transformation up to 30.4% (treating Agrobacterium with acetosyringone), 31.8% (treating explants with cytokinin and auxin), 34.9% (paclobutrazol) and 38.6% (lipoic acid), respectively. When the three treatments were combined, we observed that the transformation efficiency was enhanced from 11.5% to 52.3%. The improvement of genetic transformation efficiency mediated by these three simple treatments may facilitate more efficient applications of transgenic and gene editing technologies for functional characterization of citrus genes and for genetic improvement of citrus cultivars

A novel two-step method for screening shade tolerant mutant plants via dwarfism

When subjected to shade, plants undergo rapid shoot elongation, which often makes them more prone to disease and mechanical damage. Shade-tolerant plants can be difficult to breed; however, they offer a substantial benefit over other varieties in low-light areas. Although perennial ryegrass (Lolium perenne L.) is a popular species of turf grasses because of their good appearance and fast establishment, the plant normally does not perform well under shade conditions. It has been reported that, in turfgrass, induced dwarfism can enhance shade tolerance. Here we describe a two-step procedure for isolating shade tolerant mutants of perennial ryegrass by first screening for dominant dwarf mutants, and then screening dwarf plants for shade tolerance. The two-step screening process to isolate shade tolerant mutants can be done efficiently with limited space at early seedling stages, which enables quick and efficient isolation of shade tolerant mutants, and thus facilitates development of shade tolerant new cultivars of turfgrasses. Using the method, we isolated 136 dwarf mutants from 300,000 mutagenized seeds, with 65 being shade tolerant (0.022%). When screened directly for shade tolerance, we recovered only four mutants from a population of 150,000 (0.003%) mutagenized seeds. One shade tolerant mutant, shadow-1, was characterized in detail. In addition to dwarfism, shadow-1 and its sexual progeny displayed high degrees of tolerance to both natural and artificial shade. We showed that endogenous gibberellin (GA) content in shadow-1 was higher than wild-type controls, and shadow-1 was also partially GA insensitive. Our novel, simple and effective two-step screening method should be applicable to breeding shade tolerant cultivars of turfgrasses, ground covers, and other economically important crop plants that can be used under canopies of existing vegetation to increase productivity per unit area of land

Transcriptome Analysis Reveals Differential Gene Expression and a Possible Role of Gibberellins in a Shade-Tolerant Mutant of Perennial Ryegrass

The molecular basis behind shade tolerance in plants is not fully understood. Previously, we have shown that a connection may exist between shade tolerance and dwarfism, however, the mechanism connecting these phenotypes is not well understood. In order to clarify this connection, we analyzed the transcriptome of a previously identified shade-tolerant mutant of perennial ryegrass (Lolium perenne L.) called shadow-1. shadow-1 mutant plants are dwarf, and are significantly tolerant to shade in a number of environments compared to wild-type controls. In this study, we treated shadow-1 and wild-type plants with 95% shade for 2 weeks and compared the transcriptomes of these shade-treated individuals with both genotypes exposed to full light. We identified 2,200 differentially expressed genes (DEGs) (1,096 up-regulated and 1,104 down-regulated) in shadow-1 mutants, compared to wild type, following exposure to shade stress. Of these DEGs, 329 were unique to shadow-1 plants kept under shade and were not found in any other comparisons that we made. We found 2,245 DEGs (1,153 up-regulated and 1,092 down-regulated) in shadow-1 plants, compared to wild-type, under light, with 485 DEGs unique to shadow-1 plants under light. We examined the expression of gibberellin (GA) biosynthesis genes and found that they were down-regulated in shadow-1 plants compared to wild type, notably gibberellin 20 oxidase (GA20ox), which was down-regulated to 3.3% (96.7% reduction) of the wild-type expression level under shade conditions. One GA response gene, lipid transfer protein 3 (LTP3), was also down-regulated to 41.5% in shadow-1 plants under shade conditions when compared to the expression level in the wild type. These data provide valuable insight into a role that GA plays in dwarfism and shade tolerance, as exemplified by shadow-1 plants, and could serve as a guide for plant breeders interested in developing new cultivars with either of these traits

Agave REVEILLE1 regulates the onset and release of seasonal dormancy in Populus

Deciduous woody plants like poplar (Populus spp.) have seasonal bud dormancy. It has been challenging to simultaneously delay the onset of bud dormancy in the fall and advance bud break in the spring, as bud dormancy, and bud break were thought to be controlled by different genetic factors. Here, we demonstrate that heterologous expression of the REVEILLE1 gene (named AaRVE1) from Agave (Agave americana) not only delays the onset of bud dormancy but also accelerates bud break in poplar in field trials. AaRVE1 heterologous expression increases poplar biomass yield by 166% in the greenhouse. Furthermore, we reveal that heterologous expression of AaRVE1 increases cytokinin contents, represses multiple dormancy-related genes, and up-regulates bud break-related genes, and that AaRVE1 functions as a transcriptional repressor and regulates the activity of the DORMANCY-ASSOCIATED PROTEIN 1 (DRM1) promoter. Our findings demonstrate that AaRVE1 appears to function as a regulator of bud dormancy and bud break, which has important implications for extending the growing season of deciduous trees in frost-free temperate and subtropical regions to increase crop yield