An efficient micropropagation procedure via adventitious shoot proliferation was developed for Aglaonema using the popular red cultivar ‘Lady Valentine.’ Aseptic culture was initiated by culturing stem nodal segments on Murashige and Skoog (MS) medium supplemented with 32 mg·l⁻¹ gentamicin, 8 mg·l⁻¹ tetracycline and 4 mg·l⁻¹ chloramphenicol. The growth of the axillary buds performed the best when 10 mg·l⁻¹ 6-benzyladenine (BA) was incorporated into the medium, and neither gibberellic acid (GA₃) nor dark exposure could improve the elongation of the axillary shoots. The single stem nodal segments excised from the elongated shoots were treated with different combinations of α-naphthaleneacetic acid (NAA) and thidiazuron (TDZ) and an average of 10.9 adventitious shoots per stem segment was produced with 0.5 mg·l⁻¹ NAA and 2 mg·l⁻¹ TDZ. Small shoot clusters were subsequently incubated with different concentrations of BA and GA₃ and results showed that 0.5–5 mg·l⁻¹ BA treatments were more effective for shoot proliferation and elongation than 0.5–1 mg·l⁻¹ GA₃ treatments. The longest shoots (reaching 2.69 cm after three months) were obtained on medium containing 5 mg·l⁻¹ BA. Up to 80% of the elongated shoots successfully rooted ex vitro with the application of 1 and 2 mg·l⁻¹ indole-3-butyric acid (IBA) and 92.5% of these rooted shoots survived following transfer to the greenhouse.

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Microarray analysis was used to identify candidate genes that are involved in the formation of secondary cell walls and hydroxycinnamoyl-arabinoxylan (AX) in rice. In order to identify genes involved in secondary cell wall formation, gene expression was compared between wild-type whole internodes that contain cells with thickened secondary cell walls, such as vascular and cortical fiber cells, and wild-type internode parenchyma cells without secondary cell walls. In addition, gene expression was compared between the internode parenchyma of Fukei71 (F71), a rice dwarf mutant that accumulates large amounts of hydroxycinnamoyl-AX in pith parenchyma cells, and wild-type pith parenchyma cells to identify hydroxycinnamoyl-AX-related genes. To address the significant expression of candidate genes, gene lists were prepared for the phenylpropanoid pathway, major carbon metabolism, and cell wall biosynthesis, which is a useful platform to analyze cell wall formation in rice. The data indicated that a number of rice genes are potentially associated with secondary cell wall formation, such as the up-regulation of genes encoding cellulose synthase subunit A and ferulate 5-hydroxylase in wild-type whole internodes. Similarly, for hydroxycinnamoyl-AX synthesis, the expression of several genes changed, such as the down-regulation of genes encoding cinnamoyl-CoA reductase and the up-regulation of the phosphoenolpyruvate carboxykinase gene in F71 pith parenchyma.

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To explore the chemical basis of xylogenesis in bamboo cells, we compared secondary metabolite compositions between xylogenic and non-xylogenic suspension cell cultures of bamboo (Phyllostachys nigra), which we developed previously. Two compounds, one major and one minor, showed large-scale increases in the cells cultured under two lignification (xylogenic) conditions, compared with cells cultured under proliferation (non-xylogenic) conditions. Based on spectroscopic analyses, the major compound was identified as feruloylputrescine (FP) and the minor compound was identified as p-coumaroylputrescine (pCP). We examined the accumulation profiles of these hydroxycinnamic acid amides during a 16-day culture period. When cells were kept in proliferation conditions for 16 days, the contents of FP and pCP peaked at 2 days (0.32 and 0.25 nmol mg⁻¹ fresh weight, respectively) and decreased to trace levels thereafter. In contrast, the FP content increased throughout the 16-day culture period, reaching maximum levels of 4.3 and 6.8 nmol mg⁻¹ fresh weight in the two xylogenic conditions. The pCP content was lower than that of FP under both xylogenic conditions. The pattern of FP accumulation resembled that of lignin accumulation, as monitored by phloroglucinol-HCl staining. It is likely that FP plays a role in xylogenesis in suspension-cultured bamboo cells.

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A system for engineering plant chromosomes has been developed to facilitate the introduction of novel genes into the plant genome. The system is based on the establishment of a unique genetic locus within the ribosomal DNA (rDNA) region of the host chromosome to provide a permissive environment for expression of the introduced genes of interest (GOI). The genetic locus can exist within an independent, fully functional “minichromosome” (MC) or as a segment of a modified host chromosome (termed Engineered Trait Locus or ETL). The site-specific integration of transgenes to the rDNA locus isolates them from other endogenous genes, an advantage over conventional transformation in which foreign genes are inserted randomly into the host genome. Furthermore, MCs or ETLs can confer stability and high expression of the transgenes, as demonstrated in mammalian systems. To evaluate this system in plants, several MC and ETL lines have been generated in soybean, an important crop used worldwide for protein and oil consumption. The characterization of a soybean line containing an MC demonstrates that 1) the MC is stable over multiple generations as well as in field conditions, 2) maintaining the MC has no adverse phenotypic consequences, and 3) the MC can provide high-level expression of the introduced GOI.

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cis-Cinnamoyl glucosides are the allelochemicals in Thunberg’s meadowsweet (Spiraea thunbergii). The essential chemical structure responsible for the bioactivity of cis-cinnamoyl glucosides, cis-cinnamic acid (cis-CA), strongly inhibits the root growth of several plant species; however, its mode of action has not been characterized at the gene expression level. We conducted a time–course microarray analysis of gene expression in Arabidopsis in response to 20 µM cis-CA. Comparison of the microarray profiles revealed a 10-fold upregulation of several auxin-responsive GRETCHEN HAGEN-3 (GH3) genes and LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD) genes from 2 h to 6 h post-treatment. Two early auxin-responsive gene families, the Aux/IAA family (IAA1, IAA5) and the GH3 family (GH3.1, GH3.2, GH3.3), and an LBD gene (LBD16) were markedly upregulated at 2 h after treatment in the roots, but not in the shoots, of Arabidopsis and remained highly expressed for 4 h. The influence of an exogenous application of cis-CA on the indole-3-acetic acid pathway strongly suggests that a root-targeted induction of auxin-responsive genes is involved in the cis-CA-mediated plant growth inhibition.

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An efficient method of plant regeneration from seed-derived callus was established in ravenna grass [Erianthus ravennae (L.) Beauv.], which is an ornamental tall grass as well as an important biomass crop. For callus induction, mature seeds were cultured on media containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5, 1, 2 and 4 mg l⁻¹) and 6-benzyladenine (BA) (0, 0.1 and 0.2 mg l⁻¹) on callus induction using MS medium (Murashige and Skoog 1962) supplemeted with 1 g l⁻¹ casamino acids, 30 g l⁻¹ maltose and 8 g l⁻¹ agar as a basal medium. The highest callus induction was obtained on medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ BA, where 96% of explants formed callus. During the subculture of the calli on the same medium for 4 months, 3 types of calli showing different growth rate, color and morphology were differentiated. By using these 3 types of calli, effects of different concentrations of BA or 6-furfurylaminopurine (kinetin) (0, 0.1, 0.5, 1, 2 and 4 mg l⁻¹) and naphthaleneacetic acid (NAA) (0, 0.25, 0.5 and 1 mg l⁻¹) as plant growth regulators on shoot regeneration were evaluated using MS medium with 1 g l⁻¹ casamino acids as a basal medium. The highest frequency of shoot formation was obtained in type A callus, which had white and compact nature, on medium containing 30 g l⁻¹ sucrose, 1 mg l⁻¹ BA and 0.1–0.25 mg l⁻¹ NAA, where 89.3% of the calli formed shoots.

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A wide range of genotypes suitable for a plant regeneration system is needed when utilizing genetic transformation techniques to develop new crop cultivars. We examined the regeneration efficiencies for somatic embryogenesis in 61 genetically diverse sugar beet (Beta vulgaris L.) breeding lines developed in Japan using a previously reported procedure. Frequencies of embryogenic callus formation from seedling leaf and petiole explants ranged from 0 to 89% and those of somatic embryo formation from the calli ranged from 0 to 99%. There was no clear correlation between the two frequencies, suggesting that different genetic backgrounds are involved in the two formation mechanisms. To evaluate each breeding line’s capability for somatic embryogenesis, we proposed a somatic embryogenesis capability index based on four parameters. Based on the values of this index, only 21 breeding lines were suitable materials for regeneration via somatic embryogenesis. These breeding lines include four lines with moderate or better resistance to three major sugar beet diseases in Japan and one line with strong resistance to these diseases, and these lines will be useful materials with superior characteristics for developing transgenic breeding lines. To increase the regeneration efficiencies in the other 40 breeding lines, we examined other types of explants from seedlings and the effects of various plant growth regulators. We found that cotyledon explants generally showed improved callus formation and that using thidiazuron instead of N⁶-benzylaminopurine improved somatic embryo formation in some lines.

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The heat shock response of rice, including expression of heat shock transcription factors (Hsfs), was investigated to elucidate the molecular regulation of its high-temperature tolerance. In silico analysis revealed that the rice genome encodes more than 19 species of Hsf genes that can be organized into three classes, A, B, and C. Rice seedlings treated with high temperature, express three class A Hsf genes (HsfA2a, HsfA2c, and HsfA2d) and two class B Hsf genes (HsfB2b and HsfB2c) at significantly elevated levels. Transgenic rice plants overexpressing these three class A Hsf genes controlled by the rice actin 1 promoter or the wheat cold response (WCR) promoter expressed the transgenes, but did not express heat shock response genes such as small HSPs, whose expression is controlled by Hsfs. Treatment with geldanamycin, an inhibitor of HSP90, elevated the expression of HSPs in the WCR::Hsf transformant. Therefore, transgenic rice co-expressing WCR::Hsfs and a dominant negative HSP90 mutant were generated in which the heat shock response could be induced under WCR promoter-activating conditions. Successful induction of the heat shock response under cool conditions in this co-expression line suggests that HSP90 controls the heat shock response via the activity of Hsf in rice cells.

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Leaf epidermal stomata play pivotal roles in gas exchange and transpiration in higher plants. The regulatory mechanisms of the opening and closing of stomata (stomatal movement) and stomatal morphogenesis have been intensively studied. Such studies require the collection of detached epidermal cell layers (“peels”) for the detailed observation of stomata under the microscope. However, there are drawbacks to the conventional techniques for preparing and handling epidermal peels, i.e., a certain level of skill or an apparatus (e.g., a Waring blender) is required. In this report, we present a simple and rapid method for preparing epidermal peels called Perforated-tape Epidermal Detachment (PED). For PED, Time Tape is adhered to the adaxial epidermis of an Arabidopsis leaf, and the abaxial epidermal layer is detached using Scotch Tape perforated with a small hole. The area inside the hole is suitable for microscopic observation because the Scotch Tape does not mask the detached epidermal layer there. PED can also be used to prepare epidermal peels from tobacco and legume leaves. The crucial advantage of PED over the conventional blender method is that the epidermal cells prepared by PED are rarely damaged, and we demonstrate that PED can be used to obtain material for physiological assays of stomatal responses to blue light and externally applied ABA. We thus believe that the PED method is suitable for preparing epidermal cell layers for physiological studies.

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We previously characterized Arabidopsis Tóxicos en Levadura54 (ATL54), a ubiquitin ligase associated with secondary cell wall formation. The ATL54 gene is co-expressed with secondary wall-associated genes, and the knock-out of ATL54 up-regulates the expression of cellulose, lignin, and xylan biosynthetic genes in apical stem portions of four-week-old plants. Here, we report the tissue-level localization patterns and the regulation of ATL54 expression. The β-glucuronidase (GUS) reporter gene driven by the ATL54 promoter was significantly expressed in interfascicular fibers, xylary fibers, and vessels in inflorescence stems. A transient transfection assay using Arabidopsis T87 cells showed that the expression of the firefly luciferase gene driven by the ATL54 promoter was activated by MYB46, which is a key transcriptional activator of secondary wall formation. In addition, the electrophoretic mobility of ATL54 promoter fragments was shifted by a recombinant MYB46 protein. These results indicate that ATL54 expression is regulated by MYB46, and support the view that ATL54 has a role in secondary wall formation.

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Oryza sativa heavy metal P-type ATPase 3 (OsHMA3) plays a major role in the hyperaccumulation of cadmium (Cd) by the rice cultivars ‘Cho-Ko-Koku (CKK)’ and ‘Anjana Dhan.’ On the other hand, mutants that accumulate Cd in the shoots at a very low level have suggested the function of several genes such as OsHMA2 (which is neccesary to load Cd into xylem) or O. sativa natural resistance-associated macrophage protein 5 (OsNRAMP5, which regulates the uptake of Cd from soil into the roots). Although many studies have examined metal transporters, few have investigated the function of combination of multiple gene in plant. Herein, first, we used an RNA interference (RNAi) method to down regulate OsHMA3 and recovered plants in which the expression of OsHMA3 was strongly suppressed. Second, crossing of OsHMA3 RNAi plants with oshma2 mutants revealed a functional relationship between OsHMA3 and OsHMA2 in plant. We concluded that a complete loss of function of OsHMA2 is crucial to minimize Cd translocation from the roots to the shoots. The current findings also implied that the activity of OsHMA2 can be accelerated in plant in the roots without the C-terminal region of the protein.

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The circadian clock is an autonomous oscillator that produces endogenous biological rhythms with a period of approximately 24 h. A number of circadian clock-associated factors have been intensely studied in the model plant Arabidopsis thaliana (At), including pseudo-response regulators (PRRs), which are key regulators of the circadian clock. In Populus trichocarpa (Pt), seven orthologs of the AtPRR genes have been identified. Here, the PtPRR family of genes, PtPRR1, PtPRR37, PtPRR5a, PtPRR5b, PtPRR73, PtPRR9la, and PtPRR9lb, were analyzed for circadian expression at the transcriptional level. These genes were expressed diurnally in the following order: PtPRR9la/PtPRR9lb→PtPRR37/PtPRR73→PtPRR5a/PtPRR5b and PtPRR1, with the PtPRR mRNAs starting to accumulate sequentially in 2–3-h intervals. These sequential transcriptional events, termed ‘circadian waves of PtPRR,’ were not significantly affected by the photoperiod conditions. All PtPRR genes were shown to be primarily expressed in mature leaves. These results suggest that members of the PtPRR family play important roles in mechanisms underlying the poplar circadian clock.

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To develop a more efficient genetic transformation system for Cryptomeria japonica (Japanese cedar), the present study evaluated the effects of culture support during co-cultivation and the use of β-lactam antibiotics (meropenem and carbenicillin) to eliminate Agrobacterium on the transformation efficiency. The co-cultivation of embryogenic tissues and bacterium on filter paper wicks prevented the excess growth of Agrobacterium compared with that observed on solid medium, leading to an increased number of gene-transferred cells. Meropenem successfully eliminated the bacterium at low concentrations (10 mg l⁻¹) and had no phytotoxic effect. The transformed tissues were screened based on kanamycin resistance and green fluorescent protein (GFP) fluorescence, and the integration of the transgene into the plantlet genome was confirmed by Southern blotting. We successfully generated a mean of 105.3±9.02 independent transgenic lines per gram of embryogenic tissues using a combination of filter paper wicks and meropenem. The transformation efficiency of the improved method was approximately 30-fold higher than that observed using the conventional method.

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