Using the technique of amplified restriction fragment length polymorphism (AFLP) analysis approximately 500 polymorphic loci were screened on the bulked segregant pools from F2 progeny of the cross between Fagopyrum esculentum (pin) and F. homotropicum. The objective was to find those markers with tight linkage to the buckwheat homostylar locus, concerned with self-compatibility. Analysis of 123 F2 plants identified nine markers that show no recombination in 36 recessive homozygous plants. In the nine markers, two (N2 and N7) were confirmed to derive from a single region. The N2 sequence was present only in F. homotropicum and was absent in common buckwheat, F. esculentum. Nucleotide sequence information from each flanking region of the two single locus markers was used to design region-specific primers for PCR amplification. N2 region-specific primer amplified a single fragment in F. homotropicum #1 but not in common buckwheat, F. esculentum #284 (pin). Whereas N7 AFLP marker was converted into a co-dominant marker for both parents. However, N7 marker showed size polymorphism between the parent lines. These markers can be utilized for fine mapping of the Sh allele in buckwheat, and for positional cloning of the gene.
A new, efficient and reproducible in vitro propagation system of Telopea speciosissima has been established. Excised receptacle disks were cultured for inducing adventitious shoots on modified woody plant media (WPM) supplemented with 6-benzyladenine (BA) and 3-indole-butyric acid (IBA) at various concentrations. Efficient shoot regeneration was obtained when explants were cultured on media containing 5-10mg l-1 BA and 0-0.2mg l-1 IBA. Most of the shoots were regenerated directly from the epidermis of the lateral side, and in some cases, from the cut surface of the receptacle disk explants. Nodal explants taken from the regenerated shoots were cultured on the modified WPM supplemented with a wide range of BA concentrations. The number of shoots produced from the nodal explants increased linearly until 0.3mg l-1 BA, where it then plateaued. The shoot length and leaf number were negatively correlated with increased BA concentration. Shoots were rooted by dipping their base into a solution of 150mg l-1 IBA for 10 minutes and then inserting them into Kanuma soil.
Expression and localization of a 36-kDa peptide (VP36) derived from a 24-kDa vacuolar protein (VP24) precursor were investigated in anthocyanin-producing sweet potato cells (Ipomoea batatas). VP24 is one of the major proteins in the anthocyanin-containing vacuoles, and synthesized as a large precursor protein that contains a C-terminal region in addition to the mature domain [Xu et al., Plant Physiol. (2001) 125: 447]. The accumulation patterns of both VP36 and VP24 were closely correlated with the accumulation of anthocyanin in the vacuoles. The immunocytochemical analysis using antibodies against the fusion protein containing a portion of the C-terminal peptide showed that VP36 was localized in intravacuolar pigmented globules (cyanoplasts) in a manner similar to VP24. These results further suggest that VP36 is a peptide derived from the VP24 protein precursor. Both VP36 and VP24 peptides are probably involved in formation of cyanoplasts in the anthocyanin-producing sweet potato cells.
To establish a simple and constant suspension culture system for gene manipulation, the leaflets of “C1” sexual bahiagrass line were used for embryogenic callus formation on Murashige and Skoog’s medium (MS) supplemented with 2mg l-1 2, 4-dichlorophenoxyacetic acid (2, 4-D). The obtained embryogenic calli were broken into pieces for suspension culture in N6 liquid medium (Chu et al., 1975) supplemented with 1mg l-1 2, 4-D. After 2 weeks of cultures two kinds of bigger and smaller calli were obtained. Another 2 weeks later, the bigger (≥4mm diameter) became brown color and vacuolated, and the smaller (≤2mm diameter) showed uniform size and dense cytoplasm. The smaller calli were then selected and moved onto the solid medium for regeneration. When the cultures were Continued, shoots and plants were regenerated. The abilities of shoot regeneration from suspension culture could be kept at least for 12 months. This study provides a simple and constant plant regeneration system available for making transgenic apomictic plants based on embryogenic suspension culture derived from leaflets of sexual bahiagrass.
Flower longevity is an important character for cut flower quality. However it is hard to evaluate flower senescence objectively, and critical measurement method is required. To improve measurement methods of flower senescence, we tried to estimate the wilting process of freesia petals based on apoptotic cell death as an indicator. Apoptotic changes such as chromatin condensation, nuclear fragmentation and DNA fragmentation were observed in the petals undergoing senescence. A good correlation (r=0.84) was also observed between the rate of nuclear fragmentation detected by flow cytometry and the rate of cell death indicated by trypan blue staining in petals. These results suggested that the rate of nuclear fragmentation reflected flower senescence, and that it was possible to evaluate flower senescence using flow cytometry.
We developed new binary vectors, pPZP2H-lac and pPZP2Ha3, for the transformation of rice plants. These binary vectors contain multiple cloning sites and the hygromycin B phosphotransferase gene as a plant-selectable marker. After Agrobacterium-mediated transformation, transgenic plants containing a T-DNA could be tightly selected by hygromycin B. The vectors are powerful tools for the functional analysis of genes.
The cDNA encoding a putative mitochondrial aldehyde dehydrogenase (ALDH2) of barley (Hordeum vulgare L.) was characterized and its predicted amino acid sequence was compared with those of ALDH proteins from various plant species. Southern hybridization revealed that barley ALDH is encoded by more than two ALDH genes. The steady-state level of barley ALDH2 mRNA, unlike that of rice ALDH2a mRNA, decreased under submerged conditions.
Cell division of suspension cultures of Hemerocallis hybrida was partially synchronized by treating with hydroxyurea (HU), a DNA synthesis inhibitor, followed by treatment with colchicine (COL), a spindle toxin, as the first step toward an efficient preparation of microprotoplasts. During COL treatment, suspension cells were analyzed for relative DNA contents using flow cytometry (FCM) as well as mitotic index (MI) under microscopy. MI reached a peak after 20h of COL treatment. In FCM analyses, histogram of asynchronous cultures showed three peaks (2C, 4C and 8C nuclei), and the numbers of both 4C and 8C nuclei gradually increased until 18h after the initiation of COL treatment. After 20h of COL treatment, the numbers of 4C and 8C nuclei transiently decreased, indicating that a large number of cells were at the prometaphase to metaphase stages. These results indicate that FCM analyses offer a simple and rapid means of preliminary examination for arresting cell-cycle at metaphase in partially synchronized suspension cultures of H. hybrida.
The apical buds of lateral branches asexually multiplied by cutting were treated with some chemical mutagens, and the growth and differentiation or morphological changes of the mutagen-treated buds were traced in developed flowers. As a result, the variations in size, shape, color and number of petals were detected most frequently in the flowers that were developed from apical buds treated with N-methyl-N'-nitro-N-nitrosoguanidine at 100μg ml-1. The variant petals were cultured on MS medium supplemented with NAA and BAP for in vitro isolation and multiplication of morphologically altered rose plants. Embryogenic calli were obtained via adventitious roots induced from the petals and successfully differentiated to intact plants. Consequently, the regenerated plants produced the varied flower different from that originally used for tissue culture. Thus, the present study suggested that our approach would provide an effective method for easily and rapidly inducing variations in flowers of rose and for in vitro multiplication of their regenerants.