DNA sequences of transgenic loci in the transgenes of tobacco and Arabidopsis were analyzed. Almost all transgenic loci studied contained cis elements characteristic of nuclear matrix attachment regions (MARs). A MAR sequence isolated from one such locus of tobacco cells was cloned into the original transformation vector, and introduced into tobacco cells. The presence of the MAR sequence resulted in a five- to tenfold increase in the transformant yields, and it appeared to stimulate both the expression of the transgene and its integration into the host genome. A search using the BLAST program revealed that the transgenic loci contained junction region sequences that may have originated from different chromosomes of the nuclear genome and from the chloroplast genome.
Transient and stable transgene expression was maximized in in vitro and greenhouse-derived chrysanthemum ‘Lineker’ and ‘Shuhou-no-chikara’ stem explants. In Agroinfection, Agrobacterium tumefaciens LBA4404-with either pBI121 or pKT2-or AGL0 (with pKT3) were utilized, while in particle bombardment pSKGN1 was utilized. Transformation efficiency was affected by both the gene introduction method and its experimental parameters and the origin and developmental state of the explant. There was a decrease in GUS focal points with a simultaneous increase in the number of blue staining areas for both ‘Lineker’ and ‘Shuhou-no-chikara’ in both Agroinfection (pKT2 and pKT3) and particle bombardment (pSKGN1) over time (0, 24, 48, 72 h and 1, 2 and 4 weeks). GUS transformation efficiency was 6.6, 26.89, 25.0 and 11.84% for Agroinfection, bombardment, sonication and Agrolistics, respectively for ‘Lineker’, and 0, 2.17, 0 and 7.69%, respectively for ‘Shuhou-no-chikara’, the highest level of stable GUS transgene expression occurring in thevenation.
We established a microprojectile-bombardment-mediated transformation system in embryogenic calli of Italian ryegrass (Lolium multiflorum Lam.). Bombardment conditions with a PDS-1000/He helium-driven biolistic device were optimized by transient expression assays of a chimeric β-glucuronidase (GUS) gene construct. The highest number of transient GUS expression events was observed with 24 h of high osmotic pretreatment and a bombardment pressure of 7584 kPa. Diploid and tetraploid Italian ryegrass calli were transformed under optimal conditions with two plasmids harboring the gene for antisense expression of cinnamyl alcohol dehydrogenase (CAD) or hygromycin phosphotransferase (HPT). Transformed calli were obtained via microprojectile bombardment followed by selection of transformants on media containing hygromycin at 50mg l-1 for 1 week and then 100mg l-1 for 5 weeks. A total of 33 transgenic plants were produced. The transformation frequencies in cultivars Waseaoba (diploid) and Meritra (tetraploid) were 3.8% and 2.3%, respectively. The total cointegration frequencies of the HPT and antiCAD genes (antisense orientation of CAD gene) were 55.6% in Waseaoba and 73.3% in Meritra. The expression of the antiCAD gene in the co transformed plants was confirmed by RT-PCR.
A cDNA clone, designated PsDof1, encoding a novel member of the Dof DNA-binding protein family was isolated from pea. Random-binding-site selection and gel retardation experiments showed that the GST-PsDof1 recombinant protein bound to specific DNA sequences with an AAAG core. A metal-chelating agent, 1, 10-phenanthroline, abolished binding of GST-PsDof1 to DNA, but the addition of Zn2+ restored it, indicating that zinc ions are required for its DNA-binding activity. Interestingly, there are possible sites for binding PsDof1 in its promoter sequence. Indeed, GST-PsDof1 binds to the AAAG-containing promoter sequence of the PsDof1 gene with higher affinity. These results suggest that PsDof1 is a novel transcription factor that is involved in its own transcriptional regulation.
In pollen culture methods developed for Nicotiana rustica and N. tabacum, it is possible to induce embryogenic dedifferentiation, i.e., transformation process from immature pollen to embryogenic cells. As biochemical markers for the dedifferentiation of pollen attention has been focused on several phosphoproteins which characteristically appeared in the two-dimensional gel electrophoretogram of total protein from pollen fed with 32Pi in both species. Here, the six phosphoproteins of N. rustica, NrEP1 to 6, were partially purified and their N-terminal amino acid sequences were identified. The sequences were similar to each other and also to those of the phosphoproteins (NtEPs) previously identified in N. tabacum. This suggests that a protein family possessing a common structure plays an important role in the induction process of pollen embryogenesis in Nicotiana.
Interspecific hybrids of Nicotiana debneyi × N. tabacum L. show hybrid lethality, which is one of the mechanisms for reproductive isolation. Apoptotic changes were detected in the cells of hybrid seedlings expressing lethality at 28°C, but not at high temperature (34°C) indicating lethality was suppressed. When hybrid seedlings cultured at 34°C were transferred to 28°C, lethal symptoms appeared. Chromatin condensation and nuclear fragmentation were observed in leaf protoplasts isolated from hybrid seedlings expressing lethality. Agarose gel analysis of DNA extracted from leaves of hybrid seedlings revealed a specific ladder pattern, suggesting nucleosomal fragmentation. Nuclear fragmentation was correlated with lethal symptoms in hybrid seedlings, as confirmed by flow cytometry. These results were observed in hybrid seedlings transferred to 28°C from 34°C but not in hybrid seedlings maintained at 34°C. This phenomenon suggests that apoptosis in hybrid seedlings from the cross N. debneyi×N. tabacum is temperature dependent.
The immature seeds were harvested from self-pollinated miniature rose cv.‘Shortcake’. When the immature seeds were cultured on the Murashige and Skoog’s (MS) medium without phytohormone for 3 months, the pale yellow and friable embryogenic calli were induced. The calli were subcultured on the MS medium with 1mg l-1 6-benzyladenine (BA) every 2 to 3 weeks for 3 months, many adventitious shoots were differentiated and average number of shoots per callus was 7.7. These shoots were easily separated and rooted on the halfstrength MS medium with 0.25mg l-1 IBA. It was clarified that both original and regenerated plants were tetraploid by the chromosome observation and ploidy analysis All regenerated plants showed genetic variations from the original plants, such as flower size and color, petal number, simple leaf shape and prickle number. Polymorphic DNA differences were also observed between the original and regenerated plants by the RAPD analysis.
We have isolated a cDNA clone encoding the 28.3 kDa polypeptide homologous to tomato ASR (abscisic acid, stress and ripening) proteins from Calystegia soldanella. The protein, designated CSASR (C. soldanella ASR), had not only homologous regions to ASR, but also a unique insert consisting of 139 amino acids containig eight repeating motifs of 11- to 12-mer amino acids near the N-terminus. The strongest expression of the CSASR gene was observed in anther, but no expression was found in the other parts of the flower in C. soldanella. The gene was also expressed in the leaf, but little in the stem or the root. Drought, NaCl, abscisic acid, and cold induced the gene expression in the shoot, but only drought and cold induced expression in the root. The findings suggested that CSASR might be involved in the reproduction and survival of C. soldanella in the seashore.
In Polish plants of Aldrovanda vesiculosa growing in a half-strength B5 medium with different KNO3 concentrations, the medium with 1000mgl-1 KNO3 was the best in all characteristics. The addition of 100, 300 and 1000mgl-1 peptone to half-strength B5 medium with 500mgl-1 KNO3 for Polish plants led to a step-wise increase of all growth characteristics but usually only the effect of 300 and 1000mgl-1 peptone was statistically significant. No clear relationship was found between growth characteristics and KNO3 concentration in a half-strength B5 for Japanese plants. The addition of 100, 300 and 1000mgl-1 peptone to a half-strength B5 medium with 500mgl-1 KNO3 led to a two- to threefold increase in the number of shoot apices. pH values below 3.0 in used B5 media were not toxic for the growth of Aldrovanda in vitro. In both strains of Aldrovanda tested, the full-strength B5 medium with 2500mgl-1 KNO3 appeared to be excessively concentrated for optimal growth.
A 38 kDa peptide in microsomal fraction prepared from oligogalacturonide elicitor-treated carrot cell culture showed an affinity to GTP after the separation by SDS-PAGE. Antibodies raised against the related alpha subunits of heterotrimer GTP-binding protein, anti-Gαs and anti-Gαolf, were found to crossreact with this peptide. In contrast, anti-G-proteins against other classes of α subunits of the trimeric complexes and low molecular weight monomers did not show the affinity to the peptide. These results suggest that a heterotrimer GTP-binding protein complex of which α subunit immunologically resembles Gαs and Gαolf may be involved in the early stage of oligouronide elicitor-induced phytoalexin biosynthesis in cultured carrot cells.