The expression of S8-RNase was confirmed in pistils of two Japanese pear cultivars, ‘Ichiharawase’ (S1S8) and ‘Heiwa’ (S4S8). The complete sequence of the S8-RNase gene was determined connecting the nucleotide sequences of partial cDNA and 5' terminal genomic DNA fragments amplified by RT-PCR and genomic PCR. The S8-RNase has an open reading frame of 684 nucleotides encoding 228 amino acid residues. A hypervariable region (HV) of S8-RNase, which is quite different from those of S1- to S7-RNases, includes an intron of 234 bp. The similarity of deduced amino acid sequences between S8-RNase and the seven S-RNases of Japanese pear ranged from 56.7% (S3-RNase) to 70.2% (S7-RNase). Based on its nucleotide sequence, we selected NruI as S8-RNase specific restriction endonuclease and established the PCR-RFLP system for discriminating S1- to S8-alleles.
Cysteine synthase and alliinase (alliin-lyase) are terminal enzymes responsible for synthesis of cysteine and degradation of alliin (S-alk(en)yl-cysteine sulfoxide), respectively, in Allium plants. We determined the intercellular localization of cysteine synthase and alliinase using antibodies against these proteins. Cysteine synthase was predominantly localized in bundle sheath and phloem cells of three Allium plants, A. tuberosum, A. cepa and A. sativum. Lower amounts could be detected in mesophyll cells. Alliinase was almost exclusively localized in bundle sheath cells of the three Allium species. These results suggest the importance of bundle sheath cells for both synthesis and degradation of sulfur-containing compounds in Allium plants.
Understanding the molecular mechanisms of making leaves from the shoot apical meristem (SAM) is one of the important issues in plant developmental biology. In this paper, we report on the isolation and characterization of the OsRS2 gene, which corresponds to the maize ROUGH SHEATH2 (RS2). In situ mRNA localization of OsRS2 has revealed that OsRS2 is preferentially expressed at future vascular regions and the abaxial side of lateral organ primordia around SAM. Rice has several advantages in studying on the molecular basis of the phyllotaxis because of its relatively simple phyllotactic pattern. Thus, OsRS2 will be useful for analyzing the mechanism of the leaf initiation in SAM as a molecular marker of primordium of lateral organs in SAM.
To search for strong promoters that confer constitutive expression of transgenes, we evaluated the promoters of an Arabidopsis tryptophan synthase protein β subunit gene (PTSB1) and a phytochrome B gene (PPHYB) as alternatives to the 35S RNA promoter (P35S) of Cauliflower mosaic virus. Characteristics of the Soybean chrolotic mottle virus promoter (PNCR) were also studied for comparison. In transgenic calli, GUS gene fused with PTSB1, PPHYB and PNCR showed 50% or more of the activity of P35S. To drive the NPTII marker gene, the four promoters were similarly useful. In generated transgenic tobacco plants, both PTSB1 and PPHYB were active in all tissues tested, and superior to P35S in the leaves. The four promoters differed slightly in their tissue-specific expression, but were expressed constitutively, indicating that PTSB1 and PPHYB as well as PNCR are useful as strong and constitutive promoters as alternatives to P35S for genetic manipulation of plants.
To maintain and to differentiate into various plastid lineages, replication of the plastid DNA (ptDNA) and division of the plastid must take place. However, replication initiation of the ptDNA has been less understood. The present study describes identification of the initiation region (origin) of ptDNA replication in the rice cultured cells. RNA-primed newly replicated DNA strands pulse-labeled with bromodeoxyuridine were isolated and size-fractionated. Locations of these nascent strands on the ptDNA determined the two major origin regions around the 3' region of each 23S rDNA in the inverted repeats (IRA and IRB). Two-dimensional agarose gel electrophoresis of the replication intermediates suggested that replication from each origin proceeds bidirectionally. This contrasted to replication by the double D-loop mechanism.
Genetic alterations, including somaclonal and culture variations, occur during the culture of plant cells. In this study, we established a colored cell line synthesizing anthocyanin as a variant of a colorless cell line. We examined the insertions of transposable elements belonging to the Tdc1 family in both the colored and colorless cell lines by inverse polymerase chain reaction (IPCR) using the inverted repeat sequence of Tdc1 as a primer. Most amplified DNA fragments differed in length between the colored and colorless cell lines. This reflected the different insertions of transposable elements belonging to the Tdc1 family in each of these cell lines, proposing that the movement of Tdc1 elements may constitute one of the events causing genetic alteration in somaclonal and culture variations. One of the insertion sites was located in the gene encoding an amino acid sequence similar to (S)-reticuline oxidase.
Systemic endopolyploidy during development of radish (Raphanus sativus L.) is described. Flow cytometric measurements of relative nuclear DNA contents revealed that a majority of somatic cells in radish plants undergoes several rounds of endoreduplication, resulting in tissues that contain cells with multiple ploidy levels (also called ‘endopolyploidy’ or ‘polysomaty’). The distribution of endopolyploid cells is organ-specific. Endopolyploidy was not present in the embryos in dry seeds. Rapid and extensive endoreduplication occurred in the radicle and the hypocotyl of the embryos during germination of seeds. Endoreduplication events were detected in all tissues except shoot tip tissues. In vitro grown plants contained cells with five ploidy levels that correspond to 2C, 4C, 8C, 16C and 32C. Tuberous root tissues of in vivo grown plants also showed highly polysomatic nature. It is probably that growth of radish plants does not depend on cell proliferation alone. Under normal developmental programs, most somatic cells undergo a transition from proliferating state to endoreduplicating state. The biological significance of endoreduplication in radish plants is discussed.
Tissue cultures of six lichens of Ramalina species were induced on malt-yeast extract medium supplemented with various concentrations of sodium chloride. The growth of mycobionts and photobionts from thallus fragments inoculated was affected by the concentrations of NaCl. Inhibitory effects of NaCl on the growth of tissue cultures were not different between maritime and non-maritime species of tested Ramalina lichens.
An efficient and simple in vitro regeneration protocol has been successfully developed from three inbred lines of Portulaca sp. cv. ‘Jewel’ that contain a lot of betalain. The present report describes the effect of thidiazuron [N-phenyl-N'(-1, 2, 3-thidiazol-5-yl) urea] (TDZ) on in vitro shoot regeneration from intact hypocotyl explant of seedlings. The optimum level of TDZ supplementation to the culture medium was 5μM for a 3 week-induction period followed by subculture of hypocotyl-derived callus on MS basal medium (MSO). Hypocotyls excised from 7 day-old seedling gave significantly higher number of regenerated shoots than those from 14 or 21 day-old seedling. The efficacy of thidiazuron was compared with N6-benzylaminopurine (BAP) and N6-furfurylaminopurine (KIN) (purine based cytokinins) on in vitro organogenesis. TDZ was found to be more effective than BAP and KIN as an inductive signal of regeneration. TDZ-supplemented medium with indole-3-acetic acid was not effective for the regenerative response. Transfer of the TDZ-stimulated shoots to growth regulator free MSO medium containing 3% sucrose resulted in the rapid and prolific growth of plantlets. In vitro shoots were rooted after culture on half-strength MS medium without growth regulator or in the presence of 2.5 or 5.0μM NAA and successfully acclimatized in greenhouse condition. The procedure developed in this study may be useful toward improvement and development of betalain through genetic manipulation.
A simple and efficient culture for plant regeneration was established from the leaflet tissue of Panicum maximum. The vegetative leaflets were excised from terminal tillers arising at the end of stolons and cultured for callus formation on Murashige and Skoog (MS) medium supplemented with 2, 4-dichlorophenoxyacetic acid. After 30 days of culture, compact callus formed. And after 60 days, white embryogenic calli were induced with wave-like-shapes. When the embryogenic calli were transferred onto MS medium supplemented with kinetin and giberellic acid for regeneration, they developed and formed multiple shoots. The rooted plants were obtained within 4 months. All of the regenerants were successfully grown to maturity.