Tomoya Akihama

Combination of thidiazuron and basal media with low salt concentrations increases the frequency of shoot organogenesis in soybeans [Glycine max (L.) Merr.]

Abstract A successful, efficient system for multiple soybean shoot induction of soybean [Glycine max (L.) Merr.] is reported. Multiple shoots were induced from cotyledonary nodes and hypocotyl segments cultured on media supplemented with 2 mg/l thidiazuron (TDZ) or 1.15 mg/l benzyladenine (BA). It was found that TDZ induced adventitious shoots more efficiently than BA and that hypocotyl segments promoted more adventitious shoots than cotyledonary nodes. The optimal TDZ concentrations for shoot organogenesis from hypocotyl segments were between 1 and 2 mg/l. Basal media also influenced the efficiency of shoot organogenesis. The frequency of adventitious shoot formation tended to increase when the salt concentration in the basal media supplemented with 2 mg/l TDZ was reduced. Two media (1/2B5 and 1/2L2) stimulated shoot organogenesis efficiently from hypocotyl segments. This method can thus be advantageously applied in the production of transgenic soybean plants.

Incongruence between RFLPs of chloroplast DNA and morphological classification in east Asian pear (Pyrus spp.)

Polymorphism of chloroplast DNA of 106 accessions of Pyrus (mainly east Asian accessions) was examined. Four haplotypes were observed with the combination of three independent restriction site mutations digested by EcoO109 I, Sal I, and Xba I, respectively. In the occidental pear accessions only the most plesiomorphic type was observed, whereas all four types appeared in the oriental pear accessions. This suggests that the oriental species of Pyrus and occidental ones may have evolved independently. The distribution of four haplotypes in the east Asian pear was quite incongruent with the species or infrageneric classification using mainly morphological characters. Considering the high crossability and much occurrence of suspected interspecific hybrids in wild populations, this disaccord is inferred to be the results of the hybridization and introgression between species.

Cloning and molecular analysis of cDNAs encoding three sucrose phosphate synthase isoforms from a citrus fruit (Citrus unshiu Marc.).

Three partial cDNA clones (pSPS1, pSPS2 and pSPS3) encoding sucrose phosphate synthases (SPS) were isolated by Reverse Transcription (RT)-PCR using first-strand cDNA prepared from the leaf or fruit of citrus (Citrus unshiu Marc.). The nucleotide and deduced amino acid sequences of the three clones showed significant similarities to SPS previously isolated in other plants. A full-length, cDNA clone, CitSPS1, was isolated from a fruit (juice sacs and pulp segment) cDNA library using one (pSPS1) of the three partial clones as a probe. The 3539-bp CitSPS1 clone coded for a 1057-amino acid polypeptide with a predicted molecular mass of 117.8 kDa. The amino acid sequence deduced from the CitSPS1 clone showed homology with SPS from maize (55.8% identity) and spinach (74.0% identity). Genomic Southern blot analysis suggested that CitSPS1 clone represents a low-copy-number gene. RNA blot analysis of leaf, flower and fruit showed that CitSPS1 and pSPS2 were expressed in all organs. However, the levels of expression of CitSPS1 in young leaves, flowers and immature fruits were low, but high in mature leaves, and fruit. pSPS2 transcripts were barely detectable in young leaves and immature fruits, low in mature leaves, and high in flowers and mature fruits. pSPS3 transcripts were only detected in young and in mature leaves.

Cryopreservation of in vitro grown shoot tips of cherry and sweet cherry by one-step vitrification

Abstract In vitro grown shoot tips of cherry (Prunus jamasakura Seib. cv. Sendaiya) were successfully cryopreserved by one-step vitrification. After the last subculture, 3-week-old shoots were coldhardened at 5°C for 45 days. Excised 3-mm shoot tips were precultured on solidified Murashige and Skoog medium supplemented with 0.7 mol 1−1 sucrose for 1 day at 5°C. Following preculture, the shoot tips were transferred to a 2-ml plastic cryotube, and the highly concentrated cryoprotective solution PVS2 was then added (vitrification method). After treatment with PVS2 at 25°C for 105 min, the shoot tips were directly plunged into liquid nitrogen. After rapid thawing, the shoot tips were plated on agar medium. The survival rate was about 80% regardless the time of storage in liquid nitrogen from 1 day up to 10 months. This vitrification method was successfully applied to two cherry cultivars and five sweet cherry cultivars.

Differential expression of three sucrose-phosphate synthase isoforms during sucrose accumulation in citrus fruits (Citrus unshiu Marc.)

Abstract The role of sucrose-phosphate synthase (SPS) in sucrose metabolism during citrus fruit development was investigated through sucrose content and enzyme activity in relation to the transcription of three SPS cDNA isoforms. The profiles of sucrose accumulation in edible tissue (juice sacs and segment epidermis) of satsuma mandarin (Citrus unshiu Marc.) agreed with those of SPS activity, that is, an increase in sucrose content accompanied high SPS activity. However, sucrose accumulation in edible tissue preceded the increase in SPS activity. The sucrose accumulation and the activity of SPS in peel tissue (albedo and flavedo) were lower than those in edible tissue. The expressions of three SPS genes (previously isolated and designated as CitSPS1, CitSPS2, and CitSPS3) were analyzed during the fruit developmental stage. Transcripts of the CitSPS1 in edible tissue were expressed at all stages analyzed and the transcript level was highest at the mature stage (223 days after flowering (DAF)). No transcripts of the CitSPS2 were found in edible tissue before 187 DAF, but thereafter transcripts were detected and increased until the mature stage. However, transcripts of CitSPS3 were undetected in edible tissue throughout the developmental stage. The expression of CitSPS1 in peel tissue was low in young fruit, then increased notably at 148 DAF, retaining its level until the mature stage. Transcripts of CitSPS2 in peel tissue showed the same pattern as that of edible tissue. The expression of CitSPS3 in peel tissue was detected at 148, 187 and 223 DAF, although its level was weak compared with CitSPS1 and CitSPS2. The activity of SPS in edible tissue paralleled the levels of transcription of CitSPS1 and CitSPS2, suggesting that the expression of these two genes might have an important role in determining the sucrose composition or accumulation in citrus fruit. In peel tissue, the patterns of SPS activity mostly agreed with the transcripts of CitSPS1, CitSPS2 and CitSPS3. These results also indicate that SPS genes are independently regulated, resulting in distinct temporal and spatial expression patterns.

Analysis of mitochondrial genomes among Citrus plants produced by the interspecific somatic fusion of `Seminole' tangelo with rough lemon

Interspecific somatic fusion was performed between ‘Seminole’ tangelo (Citrus reticulata Blanco xC. paradisi Macf.) protoplasts isolated from embryogenic callus and rough lemon (C. jambhiri Lush.) mesophyll protoplasts. Eight plants out of ten randomly selected regenerants had 18 chromosomes and the same nuclear rDNA fragment patterns as that of the mesophyll parent. The remaining two plants showed rDNA fragment patterns from both parents and had 36 chromosomes. For the analysis of mitochondrial DNA (mtDNA),rrn26 derived from pea was used to probeBamHI digests of the regenerants. All plants showed mtDNA band patterns identical to that of the callus parent, suggesting that eight plants were cybrids and the remaining two plants were somatic hybrids. In addition to the callus parent band patterns, additional fragments from the mesophyll parent and/or a novel band fragment were revealed in some of the putative cybrids by peaatpA probe after digestion withDraI andPstI. These results suggest the occurrence of mtDNA recombination/rearrangement inCitrus cybrids produced by somatic fusion in this interspecific combination.

A novel preculture method for the induction of desiccation tolerance in gentian axillary buds for cryopreservation

Abstract Axillary buds of in vitro-grown gentian (Gentiana scabra Bunge var. buergeri Maxim.) were successfully desiccated and cryopreserved using a novel two-step preculture method: preculturing axillary buds excised from in vitro-grown gentian plants on Murashige and Skoog (MS) semi-solid medium containing 0.1 M sucrose for 8–13 days at 25°C under a 16 h-photoperiod (first step) and the subsequent short period incubations on 0.4 M sucrose (MS agar medium) for 1 day followed by 0.7 M sucrose (MS agar medium) for 1 day (second step). After preculturing, the buds were air-dried down to about 10% water content (fresh weight basis) without encapsulation, and then immersed in liquid nitrogen (LN). With this cryogenic protocol, we could obtain 78–90% of survival of axillary buds following desiccation and LN exposure. Most of the buds that survived showed normal shoot and root development. The most important part of this protocol was the preculture which circumvents the conventionally used lengthy cold-hardening treatment. The results show that induction of desiccation tolerance by preculturing was specific to a few species of sugars used in the medium and not merely caused by osmotic effects. The efficient desiccation–cryopreservation protocol shown here using the new preculture method appears promising for routinely cryopreserving gentian germplasm.

The rice mitochondrial nad3 gene has an extended reading frame at its 5' end: nucleotide sequence analysis of rice trnS, nad3, and rps12 genes.

SummaryThe nucleotide sequences of the tRNASer (trnS), pseudo-tRNA, NADH dehydrogenase subunit 3 (nad3), and ribosomal protein S12 (rps12) genes from rice mitochondrial DNA (mtDNA) were determined. Both trnS and nad3 were confirmed to be single copy genes by Southern blot analysis. The nad3 and rps12 genes were arranged in tandem, and the two were co-transcribed. The order of the above four genes in rice mtDNA differed from the linear order observed for the wheat and maize genes. In rice mitochondria, the trnS and pseudo-tRNA genes were found upstream of the cytochrome c oxidase subunit I gene, instead of the nad3 and rps12 genes as observed in maize and wheat. Additionally, while the rice nad3 and rps12 genes remain paired, they too are in a different sequence environment from the wheat and maize genes. The apparent split of the two pairs of genes indicates the occurrence of a mitochondrial intramolecular recombinational event. Another peculiarity is that the sequence upstream of the translational initiation codon of the rice nad3 gene is different from that of the wheat and maize versions. The ATG initiation codon of wheat and maize nad3 is replaced by TTG in the rice nad3. A subsequent deduction of the amino acid sequence, accompanied by a primer extension analysis, indicates that the predicted rice NAD3 protein has an additional 37 amino acid residues at its N-terminus compared to the wheat and maize NAD3 proteins. cDNA sequence analysis showed no introns or the occurrence of RNA editing at the newly replaced TTG codon.

Callus formation from protoplasts of peach cell suspension culture

Abstract Intact viable protoplasts were isolated from suspension culture cells of peach (Prunus persica (L) Batsch cultivar ‘Hakuho’). Isolation efficiency was influenced by the enzyme composition and the age of the cell culture. First cell division was observed after 7 days of culture. Growing cell colonies were transferred to an agar medium after 2 months of culture and callus formation was obtained.

Creation of an initiation codon by RNA editing in the coxl transcript from tomato mitochondria

Nucleotide-sequence analysis showed that the gene for cytochrome oxidase subunit I (coxl) from tomato mitochondrial DNA has an ACG codon at a conserved position corresponding to an ATG initiation codon in other higher-plant coxl genes. cDNA-sequence analysis of the coxl transcripts showed that 15 positions in the genomic DNA were converted from C to U in the transcripts by RNA editing. One of the editing events is observed at the indicated ACG codon, producing an ATG initiation codon. The nucleotide sequences of 37 cDNA clones showed that the initiation codon was created in 32 out of the 37 clones, while nucleotide positions 254 and 11 were edited in 37 and 34 of the 37 clones examined, respectively, suggesting that creation of the initiation codon is a post-transcriptional event. The BamHI site at nucleotide position 757–762 within the coxI genomic DNA was altered in all 97 cDNA clones examined, demonstrating that RNA editing at this site in the transcripts is very common. RNA editing takes place to a lesser extent at the initiation codon, compared with editing at internal position 254. This indicates that editing is either a random process or that it involves a mechanism favoring less RNA editing in the initiation codon than in internal sites.