Plant regeneration from the root-derived embryonic tissues of Rosa hybrida L. cv. Charming via a combined pathway of somatic embryogenesis and organogenesis

This study describes culture conditions for a plant regeneration system via a combined pathway of somatic embryogenesis and organogenesis in root explant cultures of the commercial rose cultivar 'Charming'. Root explants formed white calluses at a frequency of 30% after 6 weeks of culture on Schenk and Hildebrandt (SH) medium supplemented with 11 mg l⁻¹ 2,4-dichlorophenoxyacetic acid. After 6 weeks of transfer to SH medium without growth regulators, initial white calluses gave rise to globular somatic embryos at a frequency of 2.8%, which were subsequently dedifferentiated to embryonic tissues. Somatic embryos or embryonic tissues initially derived from root explants did not undergo development beyond cotyledonary stage. To produce adventitious shoots, embryonic tissues were sliced and cultured on SH medium with 0.5 mg l⁻¹ 6-benzyladenine. After 4 weeks of culture, 28% of embryonic tissue explants formed adventitious shoots. Regenerated shoots were rooted on half strength SH medium with 0.1 mg l⁻¹ α-naphthalaneacetic acid and subsequently grown to maturity. Root-derived embryonic tissues were proliferated by subculture, while retaining the capacity for shoot production for a few years.     

Rapid selection of mutants of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> for carbohydrate and fatty acid metabolism by fourier transform infrared spectroscopy and gas chromatography combined with multivariate analysis

Transgene-tagged mutants of Chlamydomonas reinhardtii were generated by random insertional mutagenesis for screening of mutants of carbohydrate and fatty acid metabolism. Approximately 2,500 insertion mutants tagged with the aph7″ gene were produced from one mutagenesis in three weeks. To establish a rapid screening system for numerous insertional lines, whole cell extracts of 100 insertional lines were subjected to Fourier transform infrared spectroscopy (FT-IR) and gas chromatography (GC) analysis combined with multivariate analysis. Mutant lines 28, 67, and 90 showed dramatic differences in the carbohydrate (1,000∼1,200 cm⁻¹) and amide (1,500∼1,700 cm⁻¹) regions of the FT-IR spectrum compared to wild type strain CC-124. Separate GC analysis also showed that 16:0 iso, palmitic acid (16:0), and oleic acid (18:1) were the major fatty acids in the wild type strain. In mutant 80, the relative content ratio of 16:0 iso in total fatty acids was significantly lower than in wild type, whereas the ratios of palmitic acid and oleic acid to 16:0 iso were higher. In mutant 95, the ratio of 16:0 iso to total fatty acids was increased, whereas ratios of palmitic acid and oleic acid to 16:0 iso were decreased. In particular, mutant 57 showed remarkably different fatty acid patterns with novel peaks of long-chain fatty acids having more than 20 carbon atoms. The results of this study show that FT-IR and GC combined with multivariate analysis enable rapid selection of mutants of carbohydrate and fatty acid metabolism in C. reinhardtii.     

Caenimonas terrae sp. nov., isolated from a soil sample in Korea, and emended description of the genus Caenimonas Ryu et al. 2008

A white-coloured bacterium, SGM1-15^T, was isolated from a paddy soil sample from Suwon, Republic of Korea. The cells were strictly aerobic, Gram-negative and curved rod-shaped. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SGM1-15^T was closely related to Curvibacter delicatus LMG 4328^T (97.6% similarity) and Caenimonas koreensis EMB320^T (97.5% similarity). The major respiratory quinone system was Q-8 and the predominant cellular fatty acids were C_16:0 (39.9%), summed feature 3 (C_16:1 ω7c and/or iso-C_15:0 2-OH; 24.3%) and C_17:0 cyclo (22.7%). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major polyamines were 2-hydroxypurescine, purescine and spermidine. The DNA G+C content was 68.7 mol%. On the basis of the phylogenetic, physiologicl and chemotaxonomic data, stain SGM1-15^T represents a novel species of the genus Caenimonas, for which the name Caenimonas terrae sp. nov. is proposed. The type strain of Caenimonas terrae is SGM1-15^T (=KACC 13365^T =NBRC 106341^T).     

Antagonistic regulation of transmembrane 4 L6 family member 5 attenuates fibrotic phenotypes in CCl(4) -treated mice

The development of liver fibrosis from chronic inflammation can involve epithelial-mesenchymal transition (EMT). Severe liver fibrosis can progress to cirrhosis, and further to hepatocellular carcinoma. Because the tetraspanin transmembrane 4 L6 family member 5 (TM4SF5) induces EMT and is highly expressed in hepatocellular carcinoma, it is of interest to investigate whether TM4SF5 expression is correlated with EMT processes during the development of fibrotic liver features. Using hepatic cells in vitro and a CCl(4) -mediated mouse liver in?vivo model, we examined whether TM4SF5 is expressed during liver fibrosis mediated by CCl(4) administration and whether treatment with anti-TM4SF5 reagent blocks the fibrotic liver features. Here, we found that TM4SF5 expression was induced by the transforming growth factor (TGF)β1 and epidermal growth factor signaling pathways in hepatocytes in vitro. In the CCl(4) -mediated mouse liver model, TM4SF5 was expressed during the liver fibrosis mediated by CCl(4) administration and correlated with α-smooth muscle actin expression, collagen I deposition, and TGFβ1 and epidermal growth factor receptor signaling activation in fibrotic septa regions. Interestingly, treatment with anti-TM4SF5 reagent blocked the TM4SF5-mediated liver fibrotic features: the formation of fibrotic septa with α-smooth muscle actin expression and collagen I deposition was attenuated by treatment with anti-TM4SF5 reagent. These results suggest that TM4SF5 expression mediated by TGFβ1 and growth factor can facilitate fibrotic processes during chronic liver injuries. TM4SF5 is thus a candidate target for prevention of liver fibrosis following chronic liver injury.     

Expressional diversity of wheat nsLTP genes: evidence of subfunctionalization via <Emphasis Type="Italic">cis</Emphasis>-regulatory divergence

Previously, the wheat non-specific lipid transfer proteins (TaLTP), members of a small multigene family, were reported to evidence a complex pattern of expression regulation. In order to assess further the expression diversity of the TaLTP genes, we have attempted to evaluate their expression profiles in responses to abiotic stresses, using semi-quantitative RT-PCR. The expression profiles generated herein revealed that the TaLTP genes in group A evidenced highly similar responses against abiotic stresses, whereas differential expression patterns among genes in each group were also observed. A total of seven promoters were fused to a GUS reporter gene and the recombinants were introduced into Arabidopsis, while three promoters evidenced non-detectible GUS activity. The promoters of TaLTP1, TaLTP7, and TaLTP10 included in group A drove strong expressions during plant development with overlapping patterns, in large part, but also exhibited distinct expression pattern, thereby suggesting subfunctionalization processing over evolutionary time. However, only trace expression in cotyledons, young emerged leaves, and epidermal cell layers of flower ovaries was driven by the promoter of TaLTP3 of group B. These results indicate that their distinct physiological functions appear to be accomplished by a subfunctionalization process involving degenerative mutations in regulatory regions.     

Exogenous Glutamate Inhibits the Root Growth and Increases the Glutamine Content in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Previously, our work with ginseng hairy root shows that the tissue of low-branching and slow-growing phenotype contains high level of glutamine. In order to check if the high glutamine concentration inhibits the root growth, we applied exogenous glutamine or glutamate into growth medium and check the root growth of Arabidopsis. While glutamine did not affect root growth, over 0.1 mM glutamate inhibited severe root growth. However, when the amino acid solution was adjusted to pH 5.7 and added into medium, Arabidopsis seedlings show normal growth pattern on medium containing glutamate or aspartate. These results demonstrated that inhibition of the root growth by high concentration of exogenous glutamate was a result of the low pH toxicity caused by acidic amino acid, although low concentration (0.05 mM) of glutamate has an inhibitory effect on the primary root growth. The application of exogenous glutamine or glutamate increases glutamine concentration within root tissue about 3- to 4-fold. However, concentration of glutamate is not significantly increased. The KO mutant on each of the Gln1_1, Gln1_2, or Glu2 gene was little effective on the root growth. These results indicate that high concentration of endogenous glutamine observed in root tissue does not affect root growth.     

High frequency plant regeneration system for <Emphasis Type="Italic">Nymphoides coreana</Emphasis> via somatic embryogenesis from zygotic embryo-derived embryogenic cell suspension cultures

Culture conditions were established for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Nymphoides coreana. Zygotic embryos formed pale-yellow globular structures and calluses at a frequency of 85.6% when cultured on half-strength Murashige and Skoog (MS) medium supplemented with 0.3 mg l⁻¹ of 2,4-D. However, the frequency of pale-yellow globular structures and white callus formation decreased slightly with an increasing concentration of 2,4-D up to 10 mg l⁻¹ with the frequency rate falling to 16.7%. Cell suspension cultures were established from zygotic embryo-derived calluses using half-strength MS medium supplemented with 0.3 mg l⁻¹ of 2,4-D. Upon plating onto half-strength MS basal medium, over 92.3% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted into potting soil and achieved full growth to an adult plant in a growth chamber. The high frequency plant regeneration system for Nymphoides coreana established in this study will be useful for genetic manipulation and cryopreservation of this species.