There are two types of embryogenesis in plants: zygotic and somatic. Recently, some new tissue culture systems of somatic embryo formation have been established in Arabidopsis thaliana. Studies that use these somatic embryo induction systems together with classical tissue culture systems of carrot somatic embryos have shown that some embryogenesis-related genes (LEA genes, SERK, AGL15, BBM, LEC1, FUS3 and ABI3) were expressed in the same manner in both zygotic and somatic embryogenesis. ABA signal transduction in the late stage of embryogenesis was also shown to be common in both types of embryos. These findings showed that somatic and zygotic embryogeneses are physiologically and molecularly similar, and that tissue culture systems for somatic embryogenesis can be practically used to study the mechanism of plant embryogenesis. The somatic embryo tissue culture systems will enable the mechanism of plant embryogenesis to be understood, thereby providing much knowledge in this field.
The use of heavy ion beams is an effective method for inducing mutations in plants. After irradiation with beams of Nitrogen (N) or Neon (Ne), the frequency of flower color mutants increased through the regeneration of stem or leaf without lateral meristem in an interspecific hybrid of Torenia. From anthocyanin assay results, mutants were divided into two groups. One group involved the deletion of the blue gene (DFR). The other group involved the deletion or duplication of a gene related to pigment production. The results show that heavy ion beams are effective in obtaining artificial mutants.
Transgenic potato lines of cv. Desiree containing the DREB1A gene driven by the rd29A promoter were generated using Agrobacterium-mediated transformation. The morphological appearance of the 120 transgenic lines was classified into three categories as determined by in vitro test-tube evaluation. Southern blot analyses of genomic DNA were conducted using the restriction enzymes HindIII and DraI. There were significant differences between the transgenic lines and DSC in the quantitative salinity-tolerance evaluations at 1 M NaCl. Two transgenic lines were recognized as highly tolerant to salinity based on Duncan multiple range testing. Furthermore, there was a significant correlation between the mean tolerance level of the transgenic lines and the DREB1A copy number estimated from the Southern hybridization experiments. Northern hybridization experiments were subsequently done using a DREB1A cDNA probe and transgenic lines with different levels of salinity tolerance. Salt-tolerant transgenic lines expressed substantially more of the transgene at 2 to 5 h of salt treatment, after which the expression returned to basal levels. These observations suggest that the gene transfer of rd29A::DREB1A can be used to increase the salt tolerance of important agricultural crops, such as tetrasomic polyploid potatoes, as occurs in diploid model species, such as Arabidopsis.
Five potato (Solanum tuberosum L.) cultivars were transformed with Agrobacterium tumefaciens strain EHA101 harboring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary plasmid vector, pEKH1. The infected tuber explants co-cultivated for 3 days resulted in higher transformation efficiency (7–50% higher) for all cultivars than 2 days co-cultivation. PCR analysis showed an amplified fragment of wasabi defensin gene and the selectable marker, nptII gene in the genomic DNA of all clones rooted on MS medium supplemented with 100 mgl−1 kanamycin, suggesting their transgenic nature. Southern blot analysis confirmed that transgenic plants integrated 1–6 copies of wasabi defensin gene into their genome. Expression of wasabi defensin protein was confirmed in the leaf extracts of independent transgenic clones by Western blot analysis. Antifungal assay of detached leaves from non-transformed control and transgenic plants indicated that transgenic plants were partially resistant to the fungal pathogen, Botrytis cinerea (gray mold).
The long range goal of the present study is to practically utilize Ipomoea aquatica (water spinach) for phytoremediation of polluted water with sulfuric compounds. In higher plants, the sulfate assimilation pathway consists of 5 key enzymes, among which serine acetyltransferase (SAT) and cysteine synthase (CS) constitute one of the rate limiting steps. Subsequently we have attempted to improve the sulfur assimilation capacity of I. aquatica using genes encoding these two enzymes. Cotyledon segments of seedlings were transformed with ArabidopsisSAT and rice CS genes under the control of the cauliflower mosaic virus 35S promoter. Among 3,245 cotyledon explants, 325 regenerated shoots, and two showed a high tolerance to hygromycin, designated as SR3 and SR10. In transgenic lines, the SAT activity was over 2-fold, and the CS was 3-fold higher than those in the wild type control. The cysteine and glutathione contents were also 6- and 2-fold higher than the control, respectively. When cultured in the presence of 1 g l−1 (7 mM) sulfate, they accumulated sulfate as much as 20 mg g−1 fresh weight, being 5-fold higher than the control. Under standard culture conditions, transgenic lines grew faster than the control, showing a 20% increase in fresh weight within 5 weeks cultivation. These results suggested that strengthening of SAT and CS resulted in increase not only in sulfate uptake, but also in total biomass.
Transgenic plants over-expressing wasabi defensin gene were successfully produced in Phalaenopsis orchid by Agrobacterium-mediated transformation method. Embryogenic cell suspension culture of Phalaenopsis Wataboushi ‘#6.13’ was infected with A. tumefaciens strain EHA101 carrying a plasmid containing wasabi defensin gene and selectable marker nptII, hpt genes. Plantlets were regenerated through somatic embryogenesis from the calli selected on hygromycin-containing medium. Transformation of plantlets with wasabi defensin gene was confirmed by PCR analysis. Southern blot analysis confirmed successful integration of 1–4 copies of the gene. Production of the 5 kDa wasabi defensin protein with varying levels was confirmed in the leaf extracts of different transgenic clones using Western blot analysis. Most of the transgenic plants showed strong resistance to Erwinia carotovora, which causes soft rot disease in the control plant. These results suggest the usefulness of this gene for conferring the resistance to various diseases of Phalaenopsis and possibly other orchids.
The miniature cultivar Micro-Tom has attracted much attention as a model of tomato (Solanum lycopersicum) because it has small size (10–20 cm in height), a short life cycle (70–90 days), and grows well in ordinary laboratory spaces. Recently, expressed sequence tag data and full-length cDNA sequences have been accumulated for Micro-Tom. To provide genomic information resource for Micro-Tom, we constructed the MiBASE database, which can be access via the Internet at http://www.kazusa.or.jp/jsol/microtom/. In addition to sequence information, this database contains information on simple sequence repeats, single nucleotide polymorphisms between other tomato inbred lines, nonredundant sequence sets, gene ontology terms, metabolic pathway names, and gene expression data.
We prepared DNA array nylon filters using 10,905 cDNA clones selected from fruit and leaf cDNA libraries of the miniature tomato (Solanum lycopersicum) cultivar, Micro-Tom, as being representative of non-redundant sequences of 37,972 Micro-Tom expressed sequence tags (ESTs). Tomato gene expression during fruit maturation was analyzed using the array filters. Graphs of gene expression patterns were arranged into a 4×4 array using a self-organizing map algorithm. Using non-redundant sequences generated from 188,024 tomato ESTs, we assigned the cDNA clones on the array filters to 1151 genes that had no counterparts in the Arabidopsis thaliana genome using stringent conditions for e-values of BLAST searching. We found that the expression patterns of these non-Arabidopsis genes were evenly distributed in the self-organizing map, with no statistically significant difference with the distributions of whole genes. These findings suggested that the non-Arabidopsis genes participate in a wide variety of diverse functions during fruit maturation.
DcMYB1, a carrot MYB protein, acts as a transcriptional activator of the stress-inducible carrot PAL gene DcPAL1, and is induced by several environmental cues such as UV-B irradiation and elicitor treatment. Here, the DcMYB1 promoter region was isolated, and its putative cis-elements were characterized using several deletion mutants of the DcMYB1 gene promoter. This was undertaken with transient expression experiments using carrot protoplasts. The results indicated that GCC-box and one MYC binding sequence might participate in the up-regulation of DcMYB1 promoter activity induced by UV-B irradiation, and that the AG-motif-like and/or Box-P might undertake this by elicitor treatment. MYB and/or another MYC binding site neighboring the TATA-box might take part in the negative regulation of promoter activity, and the W-box near the TATA-box might be necessary for the up-regulation of promoter activity by both UV-B irradiation and elicitor treatment.
Cyclin-dependent kinases (CDKs) are central players that control the cell cycle. In plants, A- and B-type CDKs are directly involved in cell cycle regulation. B-type CDK (CDKB) is unique to plants; however, only limited information on this kinase has been accumulated thus far. In the present study, we identified a rice cDNA encoding CDKB1;1 and studied its expression in suspension-cultured cells and plant tissues. We found that this enzyme was expressed in actively dividing cells in suspension cultures and was downregulated by the depletion of sucrose from the medium. In plants, the CDKB1;1 transcripts were highly expressed in the shoot and root apical meristems, but not in mature plant organs. These results suggested that CDKB1 is mainly involved in mitotic cell division during plant development.
Five distinct genomic DNA fragments (EcPT1, EcPT2, EcPT3, EcPT4 and EcPT5) encoding phosphate transporters were isolated from Eucalyptus camaldulensis. EcPT2 and EcPT3 were exclusively expressed in the root, but were not enhanced by phosphate deprivation. The transcript level of EcPT2 was much higher than that of EcPT3. The EcPT2 is present as a single copy gene in Eucalyptus genome, and phylogenetic analysis and deduced amino acid sequence revealed that EcPT2 can be classified into the Pht1 family, a high-affinity phosphate transporter. These results suggest that EcPT2 functions in phosphate transport in root tissues not only under low-phosphate conditions as with typical Pht1 family members, but also under high-phosphate conditions.
DcMYB1 is a member of the R2R3-type MYB protein family in carrot and can activate expression of the DcPAL1 gene induced by stresses such as the dilution effect, UV-B irradiation and elicitor treatment. To understand the regulatory mechanism of DcMYB1 we screened cDNA for proteins interacting with DcMYB1 as bait in a yeast two-hybrid system. Several positive cDNA, which were isolated as prey, encoded DcMYB1, indicating that DcMYB1 could interact with itself. The interaction of full-length DcMYB1 protein with partial DcMYB1 amino acid sequences derived from DcMYB1 cDNA deletion mutants was observed in yeast and carrot protoplasts, suggesting the self-interaction of R2R3-type MYB protein of DcMYB1 in vivo.
We generated three types of herbicide-tolerant Yumenohatamochi rice plants (Oryza sativa L cv. ‘Yumenohatamochi’) transformed with pIES1A1, pIJ2B6, or pIKBACH that express human cytochrome P450 gene CYP1A1 or CYP2B6 or co-express CYP1A1, CYP2B6, and CYP2C19, respectively. The transformed plants were screened by a combination of hygromycin resistance, PCR, and herbicide tolerance. We compared the tolerance towards various herbicides with different modes of action. The herbicides tested include photosynthesis inhibitors (chlorotoluron, diuron), very long-chain fatty acid (VLCFA) synthesis inhibitors (acetochlor, alachlor, metolachlor, and mefenacet), a carotenoid biosynthesis inhibitor (norflurazon), and microtubule assembly inhibitors (pendimethalin, trifluralin, and amiprophos-methyl). The pIES1A1- or pIJ2B6-transformed Yumenohatamochi showed tolerance towards five and four different chemical classes of herbicides, respectively. The spectrum of herbicide tolerance of pIKBACH-transformed Yumenohatamochi was broader compared to transformants expressing the single human P450 gene. Transformed rice plants expressing P450s involved in xenobiotic metabolism may become useful tools for the breeding of herbicide-tolerant crops.
A protocol for regeneration and genetic transformation was established for Curcuma alismatifolia Gagnep. ‘Chiang Mai Pink’ using retarded shoots as explants. In vitro retarded shoots were cut into 0.5×0.5×0.5 cm blocks and co-cultivated with Agrobacterium tumefaciens strain AGLO harboring the binary vector, pBI121 or pBI121-Ca-ACS1. The explants were incubated in the bacteria suspension for 30 min. The explants and bacteria were cultured on MS medium for 2 days in darkness at 25°C for co-cultivation. Then, the explants were transferred onto MS medium containing 0.1 mg l−1 IAA, 4 mg l−1 IMA, 0.5 mg l−1 TDZ, 50 mg l−1 kanamycin and 500 mg l−1 vancomycin. The explants were subcultured every 2 weeks. After 4 weeks in culture, the explants with small shoot buds were transferred onto MS medium containing 50 mg l−1 kanamycin. Within 4 weeks, the shoots were separated and subcultured every 2 weeks on MS medium containing 0.1 mg l−1 IAA and 50 mg l−1 kanamycin. PCR analysis, histochemical GUS assay and Southern blotting of the regenerated plants confirmed transformation events. We obtained transformed plants within 3 months after co-cultivation with the bacteria and the transformation frequency exceeded 14%, which is suitable for practical use.
Microscopic observations of Kihada (Phellodendron amurense) callus revealed a strong correlation between starch accumulation and organ development. The starch accumulation started before any observable organ development and was prominent in cells which ultimately gave rise to organ primordia. A large amount of starch accumulation was observed in the three- or four-celled proembryo-like structure (PLS) organization of callus cultured on MS media supplemented with BAP plus 2,4-D. Two-celled PLS showed low performance for accumulation of starch. No starch accumulation was observed in globular and heart-shaped embryo-like structure ELS organization. During shoot primordium development, a decrease in the starch content of the cultured tissues was found, indicating the utilization of the glucan in the organogenic process. Starch granules disappeared with the development of the meristematic dome and leaf primordium and also started to disappear gradually with the development of cell walls and the tracheary elements.