Wheat leaf base segments treated with 2, 4-D (10μM) for 24h developed somatic embryos without an intervening callus when cultured on basal Murashige and Skoog’s medium for less than 2 weeks. This response is auxin specific and genotype independent. In fact, vacuum infiltration of 2, 4-D, although at a higher concentration (50μM), in the basal leaf segments for 1 min, followed by culture on the basal medium, was sufficient to evoke the embryogenic response. In comparison to indirect somatic embryogenesis, this highly efficient system for direct embryogenesis not only curtails the time span for somatic embryogenesis by 15-20 days, it also obviates any secondary changes that may occur during dedifferentiation. The rapid induction of somatic embryogenesis in wheat leaf bases, under easily manipulable culture conditions, thus provides a unique opportunity to monitor the molecular events associated with auxin-mediated induction of somatic embryogenesis and also to study the associated developmental events.
Seed cuttings including zygotic embryo axis with its two cotyledon parts (2-5 mm2) were extracted from green-pre-mature cucumber fruit and used as seed explants, or they were used after dividing them (after 3 days culture) into embryo axis and cotyledon parts. Their embryogenic potential increased with the increase of fruit maturity, the maximum was obtained when 3 weeks old fruits were used. The most efficient explant type was the cotyledon segments. Efficient cucumber embryogenesis was obtained using a two steps procedure: 1-somatic embryos were initiated and multiplied on Murashige and Skoog (MS) medium supplemented with 4.5μM 2, 4-dichlorophenoxyacetic acid (2, 4-D), 90 mM sucrose and 0.8% agar (NMM medium), 2-embryo maturation and germination were accomplished using the same medium without 2, 4-D (MGM medium). The most important sign for successful transfer from the first to the second step was the appearance of slight greenish patches on the embryogenic mass (EMS), which appeared in 10 weeks.
Leaves derived from in vitro-grown apical buds were used to explore the conditions required for induction of adventitious buds and regeneration of shoots. High efficiency of shoot regeneration was obtained with half-strength MS medium (1/2MS) for Pyrus pyrifolia, and Nitsch-Nitsch (NN) medium for Pyrus communis cultivars. In both species, the basal region of the leaf was more suitable than the distal region for callus and shoot bud formation. High concentration (5 mg l-1) of thidiazuron efficiently induced callus formation and shoot bud initiation in both species. The size, colour and shape of calli were the important factors for successful shoot regeneration. Higher frequencies of callus production and rooting from regenerated shoots were observed with increasing indolebutyric acid concentration in rooting medium. However, there was a significant genotypic difference in adventitious shoot and root regeneration of each pear cultivar.
The immature seeds were harvested from self-pollinated miniature rose ‘Shortcake’. The pale yellow and friable embryogenic calli were induced from an immature seed. The calli showed high frequency (50-60%) of transient GUS expression in Agrobacterium-mediated transformation. The population density of Agrobacterium and the sonication assistance treatment had slight effects on the transformation frequency. Among 65 putative transgenic shoots grown in vitro, seven transgenic rose plants have been established and flowered in a contained greenhouse. The flowers on the primary shoots showed unstable and quite different characteristics from those on the 2nd or later emerged shoots, which were very similar to the flowers of non-transgenic regenerated plants. The integration of T-DNA fragment in transgenic plants was confirmed by both PCR-Southern blot analysis with a GUS probe and Southern blot analysis with a hygromycin phosphotransferase RNA probe.
The expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is involved in the determination of final fruit size of tomato (Lycopersicon esculentum Mill.). We developed transgenic tomatoes that over-expressed the melon (Cucumis melo L. reticulatus) Cm-HMGR-CD gene, which encodes the catalytic domain of HMGR. The derived transgenic plants expressed Cm-HMGR-CD. Flow cytometric analysis revealed that cell division and/or elongation in the fruit pericarp during early fruit development was induced more rapidly in the transgenic plant than in the wild-type control. The fruit size in transgenic plants was larger than that in controls. The growth of fruit pericarp also enhanced in transgenic plants. These results suggest that HMGR might stimulate pericarp development by activation of cell division and/or elongation (differentiation), and thereby affects fruit size. In addition, the constitutive expression of Cm-HMGR-CD affected plant morphology. Thus, transgenic plant growth was suppressed and the leaves accumulated higher levels of chlorophyll, as compared to wild-type control plants.
Cultivation of genetically modified (GM) crops has been banned in many countries because of fears about their effects on human health and the environment. One reason cited by critics of these crops is the use of bacterial antibiotic resistance genes or herbicide resistance genes as selectable markers. To avoid using such genes, I employed the Arabidopsis thaliana gene encoding UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) as a selection marker in transformation of Arabidopsis. GPT catalyzes the initial reaction for the synthesis of asparagine-linked glycans that is inhibited by tunicamycin. Using the GPT gene in combination with tunicamycin functioned efficiently in the selection of transformed Arabidopsis. In addition, this selection was able to identify transformants at a very early stage post germination compared with the selection by kanamaycin. Application of this strategy to produce GM crops may help their acceptance by the public.
The endopolyploidy during seedling development of Welsh onion (Allium fistulosum L.) is described. Flow cytometric measurements of relative nuclear DNA contents showed that certain cells became polyploid. Endopolyploidy was not present in the nuclei of embryos during imbibition. Endopolyploidization had taken place in all organs tested. Leaf segments of in vivo grown 3 month-old plants had elevated ploidy levels and contained nuclei of 2C, 4C, 8C, and 16C, where C is the haploid nuclear genome complement. The endopolyploid nuclei fall into clear ploidy series (2C, 4C, 8C, and 16C). Therefore, the process of endopolyploidy appears to correspond to endoreduplication.
In order to understand responses of the halophyte sea aster (Aster tripolium L.) to salt stress at the molecular level, we used a differential screening strategy. Nine cDNAs of genes induced by salt stress were cloned by this method from sea aster. The clones were collectively named SASR (sea aster stress responsive). These cDNAs were mainly associated with osmoprotectant synthesis, protein metabolism, protein degradation or signal transduction. All the mRNAs corresponding to the SASR genes were accumulated under high salt conditions and the transcripts of 3 out of 9 genes were induced by osmotic stress.
We determined partial genomic sequences including a hypervariable HVa (RHV) region and introns of the S6-, S12-, S13-, S14-, S17-, S19-, and S21-RNases of apple. Previously, it was suggested by Broothaerts (2003) that the S6 corresponded to S25 in ‘McIntosh’/‘Wijcik’ and ‘Tydeman’s Early Worcester’, and S19 corresponded to S28. However, S6 and S19 are different from the S25 and S28, respectively. The determined sequences of the S6 and S12, and S17 and S19 are identical, and these four alleles seem to act as the same allele. We newly assigned S6a in place of S6 and S12, and S6b in place of S17 and S19 from the results of sequence analyses, not pollination tests. The sequences of S13 and S14 were also identical. We also newly assigned S11 in place of S13 and S14. We had speculated that S21 would correspond to St by PCR-digestion analysis, however, the determined sequence of S21 was different from St.
Indole-3-butyric acid (IBA) at 8 mg l-1 induced vigorous rooting in Bupleurum falcatum L. (Saiko) root culture in B5 medium after about 2 weeks, when it was added at the time when detached roots were transferred to fresh medium. The rooting activity markedly decreased with delay of IBA addition after root 2transplantation, and was hardly observed when IBA was added at the 7th day. Stresses such as drought or heat markedly promoted the IBA-induced rooting. Methylviologen, which causes superoxide generation in cells, and hydrogen peroxide also strongly promoted the rooting. We also examined the effects of active oxygen scavengers. Supplementation of some sugars, thiotaurin and methionine caused the rooting time delayed in a concentration-dependent manner. Histidine suppressed rooting much more, but did not the elongative growth of transferred roots at that time. All these results suggest that active oxygen species caused by stress are involved in the process of rooting with IBA.
Water spinach (Ipomoea aquatica) has high nutritional value and is considered one of the most important vegetables in Southeast Asia. Because of its quick growth and efficient absorption of various substances, it has been suggested to be useful for sequestration of environmental pollutants as well as offering a source of medical materials. We have developed and established a system for stable genetic transformation by infecting cut cotyledons with Agrobacterium harboring the GUS gene as a model case after evaluating conditions of bacterial cell density, growth phase and concentrations of acetosyringone. The resulting transgenic plants grew normally to maturity, and exhibited stable GUS activity. Thus, genetic modification of I. aquatica can be readily achieved, thereby improving its quality for whatever traits desired.
Sense and antisense DNA constructs of rice (Oryza sativa L.) alternative oxidase (AOX) gene, OsAOX1a, under the control of maize Ubiquitin promoter were introduced into rice. Leaves of transgenic plants with sense OsAOX1a were shown to produce high levels of AOX protein, which was not usually observed in wild-type leaves. Transgenic plants with decreased levels of AOX protein were identified by the investigation of AOX protein in calli derived from mature seeds with antisense OsAOX1a. Transgenic plants with increased and decreased levels of AOX will be useful for studies on the role of AOX.
The shoot culture of potted carnation plants exhibited a poor growth on a medium used previously for the shoot culture of non-potted carnations. In the present study, we determined suitable concentrations of N6-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA) to improve the growth of shoot cultures of potted carnation plants. The optimum concentrations of BA and NAA applied together for in vitro shoot cultures of potted carnation plants were 0.4 and 0.1 mg l-1, respectively. The optimum concentration of BA was much lower than that (1 mg l-1) reported for the culture of most non-potted carnation plants.
Atractylodes ovata (Compositae) shoot cultures were put into cold storage and the regenerated plants then evaluated. All the in vitro shoot cultures of A. ovata DC. survived cold storage at 2°C for up to 24 months. The cold-stored shoots were then multiplicated and rooted under normal culture conditions. The regenerated plants were cultivated in the field for one or two years and various biological and biochemical characteristics were compared with a control plant. Statistical analysis of the data showed no significant differences between the two groups in most morphological traits and the contents of the atractylon, a sesquiterpene component of essential oil in the rhizome. These results demonstrate that non-frozen cold storage of in vitro shoot cultures can be used as a germplasm preservation system for short-or medium-duration without deterioration of their biological and biochemical characteristics.