Yoshito Asano

Stimulation of in vitro shoot organogenesis in Glycine max (Merrill.) by allantoin and amides

Abstract Efficient de novo shoot organogenesis, directly from cut quadrant explants of the epicotyl-hypocotyl area (near the cotyledonary nodal region) of Glycine max (soybean), was stimulated by organic products of biological nitrogen fixation (amides and the ureide, allantoin) in the presence of benzyladenine. These organic nitrogen compounds had a stimulatory effect on shoot organogenesis only when the inorganic nitrogen source, ammonium nitrate was lowered to 1 20 of the level present in standard Murashige and Skoog (MS) medium. All other nitrogen combinations resulted in poor multiple shoot formation and in many cases enhanced callus formation. Ureide in the form of allantoin and amides (glutamine and asparagine) were critical components which enhanced shoot organogenesis and reduced callus formation.

Cytokinin and thiamine requirements and stimulative effects of riboflavin and α-ketoglutaric acid on embryogenic callus induction from the seeds of Zoysia japonica Steud

Summary To enhance the embryogenic callus induction from the seeds of a recalcitrant turfgrass species Zoysia japonica , various supplements to the medium including cytokinins, vitamins, organic acids, and amino acids were tested. Both cytokinin and thiamine were found to be essential for stimulating embryogenic callus induction. Among the cytokinin and cytokinin-like compounds tested, BA and thidiazuron seemed to be the most effective. In the presence of cytokinin and thiamine, the addition of riboflavin or α-ketoglutaric acid proved to further enhance embryogenic callus induction. The addition of 0.1 mg/L BA, 4mg/ L thiamine and 100 mg/L α-ketoglutaric acid to the medium yielded a significandy increased embryogenic callus rate of up to 28.4 % per total calli induced. Thus, we show for the first time the necessity of thiamine and the stimulative effects of riboflavin and α-ketoglutaric acid on plant somatic embryogenesis.

Stimulation of benzyladenine-induced in vitro shoot organogenesis in Cucumis melo L. by proline, salicylic acid and aspirin

Abstract Proline stimulated benzyladenine-induced multiple shoots directly from cut cotyledons of Cucumis melo in standard Murashige and Skoog (MS) medium. Along with enhanced shoot formation, the extent of callus formation was reduced. Proline at 10 mM with 3% sucrose gave the best results. When the proline analog, thioproline was added to the medium at 0.5 mM, the extent of proline-enhanced multiple shoot formation was reduced with concomitant increase in callus formation. We hypothesize that proline may be the preferred source of reductant to generate energy as well as supply NADP for purine synthesis during in vitro shoot organogenesis in Cucumis melo. Reduction of shoot organogenesis in response to thioproline, a known inhibitor of proline dehydrogenase, would support this hypothesis. Salicylic acid or its derivative o-acetyl-salicylic acid (aspirin) at 50–200 μM also stimulated benzyladenine-induced shoot organogenesis in the above system. This stimulation was further enhanced with 10 mM proline. Addition of 0.5 mM thioproline reduced salicylic acid or aspirin stimulated shoot organogenesis. Shoot organogenesis was inhibited with no callus formation at higher levels (300–1000 μM) of salicylic acid or aspirin with and without 10 mM proline. It is likely that stimulation of shoot organogenesis by salicylic acid or aspirin may be through regulation of proline metabolism.

Electroporation-mediated and silicon carbide-fiber-mediated DNA delivery in Agrostis alba L. (redtop)

Abstract The results of DNA delivery experiments in Agrostis alba by electroporation into embryogenic suspension culture-derived protoplasts and by the silicon carbide fiber-mediated method into intact suspension cells are described, using the two plasmid constructs, pFF19G and wheat dwarf virus (WDV)-derived pWI-GUS. The β-glucuronidase (GUS) activity of electroporated protoplasts was detected fluorometrically. The optimal electrical conditions for DNA delivery into the protoplasts was estimated to be around 750 V/cm for 38 ms. When electroporated protoplasts were incubated for a prolonged period of time, the GUS expression of pWI-GUS treated cells increased progressively during 5 days after electroporation, indicating the replication of this vector in the cells. The GUS expression in the cells treated with silicon carbide fibers and plasmid DNA was detected histochemically. The frequency of GUS expression units tended to be highest when DNA was delivered to the cells on the 6th day after subculture, giving an estimated frequency of GUS-expressed cell colonies of about 1%.

Transgenic plants of Agrostis alba obtained by electroporation-mediated direct gene transfer into protoplasts

A system for genetic transformation of Agrostis alba plants by electroporation-mediated DNA transfer to protoplasts is described. The npt II gene was used as a selectable marker. Selection with 20 mg/1 G418 (geneticin) yielded a total of over 50 resistant cell colonies from three independent experiments. Overall frequency of resistant colony formation was 1–3 × 10−6 based on the number of protoplasts plated and 1–2 × 10−5 based on the number of cell colonies recovered. Subsequent subcultures led to the development of plants with an apparently normal morphology. DNA analysis (PCR and Southern hybridization) and enzymatic analysis showed that the G418 resistant plants carried the transgene and expressed it. This is the first successful genetic transformation of an economically important temperate grass, Agrostis.

The Influence of Organic Nitrogen Sources on the Induction of Embryogenic Callus in Agrostis alba L.

Summary We have studied the effect of certain organic nitrogen sources on the induction of embryogenic callus from mature seed of Agrostis alba. Among the compounds tested were amino acids that are present in hydroxy proline rich glycoproteins, polyamines, and osmolytes like proline, glycine betaine, and the glycine betaine precursor choline. Proline had a significant stimulatory effect on the induction of embryogenic callus. Among the other compounds tested, only glutamine was stimulatory, whereas most other compounds were inhibitory, particularly at higher concentrations.

Plant regeneration from suspension culture-derived protoplasts of Agrostis alba L. (Redtop)

Abstract Embryogenic calli were induced at a frequency of 4.3% from the seeds of Agrostis alba on an agarose-solidified Murashige and Skoog medium with 5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). Embryogenic sectors were excised and suspended in a liquid medium with 2 mg/1 2,4-D. After weekly subcultures for 3–4 months, a few cell lines that grew fast in a finely dispersed state and with plant regeneration ability were selected. Among the three media compared, N6AA medium including l -proline and four other amino acids as an organic nitrogen source was found to be the most suitable for the maintenance of suspension cultures. The protoplast yield of 1–2 × 10 7 /g fresh weight was obtained from a cell line by an enzyme mixture treatment after a series of preculture procedures. Protoplasts were cultured embedded in an agarose-solidified modified Kaos 8p medium at a density of 2–5 × 10 5 /ml. The plating efficiency based on colony formation after 3 weeks of culture was 0.5–0.6%. Agarose blocks including protoplast-derived colonies were then resuspended in liquid medium to induce embryonic growth. Plants developed within 4–6 weeks from the calli when transferred onto agarose-solidified MS medium with 1 mg/1 kinetin.

Improved regeneration response of creeping bentgrass and japonica rice by maltose and lactose

The effect of alternative carbohydrate sources to sucrose for plant regeneration from long-term cell cultures of creeping bentgrass (Agrostis palustris Huds.cv.Penncross) and japonica rice (Oryza sativa L.cv.Nipponbare) was studied. Both maltose and lactose supported a higher degree of regeneration compared to sucrose; in 8-and 19-month-old cultures of creeping bentgrass, the frequencies of regenerating calli remained at 76–93% and the numbers of plants regenerated were 8 to 36-fold higher. In 35-month-old cultures of japonica rice, 2–4% of the calli were capable of regeneration on maltose and lactose media. These results indicate that loss of plant regeneration in long-term cultures is caused, at least in part, by specific cultural conditions and not by genetic changes.

Herbicide-resistant transgenic creeping bentgrass plants obtained by electroporation using an altered buffer

Abstract Modification of an electroporation buffer using Ca(NO3)2 and elevated pH (9–10) appeared to have a favorable effect on gene transfer to creeping bentgrass (Agrostis palustris Huds.) Penncross protoplasts, resulting in an increase in the transformation frequency of about twofold. Following electroporation with the plasmid pARK22 containing the bar gene, a total of 278 bialaphos-resistant cell colonies were obtained from four experiments. The bialaphos-resistant regenerants proved to be transgenic by Southern hybridization of the amplified DNA. All the tested transgenic plants showed herbicide (HERBIE) resistance at the field rate of 0.5–1% (vol/vol). Ammonia contents in leaves after spraying with the herbicide increased less in transgenic plants than in untransformed control plants.

Specific selection of embryogenic cells lines in Agrostis alba L. using the proline analog thioproline

Abstract The proline analog, thioproline was used to specifically isolate cell lines of Agrostis alba L. with the capacity to initiate embryogenic callus from mature seed. During the selection in the presence of 2,4-dichlorophenoxy acetic acid (2,4-D) as the auxin in standard Murashige and Skoog (MS) medium, only cell lines forming highly embryogenic callus proliferated. On the other hand, growth of cell lines forming non-embryogenic callus were inhibited. A thioproline concentration of 1.0 mM was particularly effective in specifically selecting embryogenic callus cell lines which were not inhibited in growth. At the same time 1.0 mM thioproline effectively inhibited the growth of non-embryogenic callus cell lines. The possible physiological mechanism by which the embryogenic callus cell lines were tolerant to thioproline is discussed.