Katsuji Oosawa

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.

Highly frequent appearance of tetraploidy in regenerated plants, a universal phenomenon, in tissue cultures of melon (Cucumis melo L.)

Abstract The frequency of tetraploidy in melon plants ( Cucumis melo L.) regenerated from somatic embryos, adventitious shoots, shoot primordia and in plants propagated from axillary buds was compared. Tetraploids were found in plants regenerated from somatic embryos, adventitious shoots and shoot primordia, and the frequency of tetraploids in the three culture methods was 31%,30% and 4%, respectively. No tetraploidy was observed in plants propagated from axillary buds. The highly frequent appearance of tetraploidy in regenerated plants appears to be a universal phenomenon in tissue culture of melon.

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.

Ploidy of somatic embryos and the ability to regenerate plantlets in melon (Cucumis melo L.)

SummaryThe number of chromosomes in cells of callus, somatic embryos and regenerated plantlets during somatic embryogenesis were examined in two cultivars of melon (Cucumis melo L.). Somatic embryos were diploid (50.0%/32.1%), tetraploid (38.5%/57.5%) and octoploid (11.5%/10.4%) whereas in callus cells diploidy (41.9%/43.3%), tetraploidy (27.9%/25.8%), octoploidy (11.6%/15.5%) and a low frequency of other types of ploidy and aneuploidy were observed. Mixoploid somatic embryos were not observed. These results suggest that the somatic embryos were selectively differentiated from diploid, tetraploid and octoploid cells, and that endopolyploidization of cultured cells occurred before the start of cell division leading to somatic embryogenesis. The ratio of diploid to tetraploid (1.30/0.55) in somatic embryos was less than that in callus cells (1.50/1.68) while ratios of diploid to octoploid (4.35/3.09) and tetraploid to octoploid (3.35/5.52) in somatic embryos were greater than those in callus cells (3.61/2.80 and 2.40/1.67). Therefore, it appears that the ability of callus cell to differentiate into somatic embryos increases in the following order: octoploid < diploid < tetraploid. Regenerated plantlets were diploid (65.5%/55.1%) and tetraploid (34.5%/44.9%). No octoploid plantlets were observed. The ratio of diploid to tetraploid in regenerated plantlets (1.72/1.23) was greater than that in somatic embryos. Therefore, it appears that the ability of somatic embryos to develop into plantlets increases in the following order: octoploid < tetraploid < diploid.

Selection of somaclonal variants with low-temperature germinability in melon (Cucumis melo L.).

SummaryPlants were regenerated from adventitious buds and somatic embryos (R0) of melon (Cucumis melo L.), the cultivar Andes. Somaclonal variants of melon with low temperature germinability were selected from the progenies (R1) of R0 plants. Among 5,618 R1 seeds harvested from 23 R0 plants that were regenerated from adventitious buds 4 seeds germinated after 5 days of culture at 15 °C (selection rate; 0.07%). However, among 374 R2 seeds harvested from 2 R1 plants no seed germinated after 7 days of culture at 14 °C. Among 9,181 R1 seeds harvested from 50 R0 plants regenerated from somatic embryos 110 seeds germinated after 5 days of culture at 15 °C (selection rate; 1.20%). Among 3,717 R2 seeds harvested from 17 R1 plants 113 seeds germinated after 7 days of culture at 14 °C (selection rate; 3.04%). R3 seeds were collected from these R2 plants following self-pollination. Forty-five of the 47 lines (R3) originated from 10 R0 plants showed higher germination rates than that of the original cultivar. Selected lines with low-temperature germinability showed greater fruit growth rate than the original cultivar during the middle stage when they were cultivated in a greenhouse under low-temperature conditions. Of fruits harvested from 31 lines, 15 lines showed greater fruit volume than the original cultivar.

Production of aneuploid melon plants following in vitro culture of seeds from a triploid x diploid cross

Aneuploid melon plants (Cucumis melo L.) were obtained from in vitro cultured seed, which were produced by crossing triploid (3x=36) x diploid (2x=24) plants. Twenty-six fruits were obtained from pollination of 29 bisexual flowers of triploid plants. Seeds were collected from the fruits at 2, 3, 4, 5 and >7 weeks after pollination and germinated in vitro on Murashige & Skoogs (MS) medium. Embryos developed from 0.6 to 1.6% of the cultured seeds after three weeks in culture. Shoots developed from 0 to 47% of embryos after transfer to half-strength MS medium. Some (0 to 50%) of elongated shoots that rooted were subcultured on the same medium. Five rooted plantlets were obtained through culture of 5,353 seeds. Four of the plants were aneuploid, with chromosome numbers of 27, 35, 45 and 46, respectively, and the one was tetraploid (4x=48).

Cryopreservation of shoot primordia cultures of melon using a slow prefreezing procedure

Tissue-cultured shoot primordia of melon (Cucumis melo L. cv. prince melon) were successfully cryopreserved in liquid nitrogen (LN) using a slow prefreezing method. The highest survival and recovery were obtained with the following procedure. Three week-old shoot primordia clumps were dissected into pieces of 2-3 mm of diameter and precultured in standard medium for 3 days. They were directly soaked in CSP1 cryoprotective solution (10%w/v sucrose, 10%w/v dimethylsulfoxide and 5%w/v glycerol) and incubated at room temperature for 30 min. Samples were ice-inoculated at -8 °C and cooled at a rate of between 0.3 and 1 °C min−1 with a programmable freezer to -30 °C for prefreezing. They were then plunged into LN for storage. After rapid thawing in 40 °C water, the cryoprotective solution was slowly diluted 5 fold in a dropwise manner with 3% sucrose and the shoot primordia were transferred onto regeneration medium. Under optimal conditions, more than 80% of cryopreserved shoot primordia were viable and 50 to 80% regenerated shoots after one month of reculture. Cryopreserved shoot primordia could be used both for reproducing a shoot primordia culture and for regenerating plants.

Transgenic melon (cucumis melo L.) and potential for expression of novel proteins important to food industry

Abstract Melon (Cucumis melo L.) is an important fruit crop cultivated widely in every region of the world. Our laboratory is targeting this species for production of novel proteins important to food industry. Prior to expression of protein of interest in transgenic melon an efficient genetic transformation system has to be developed. In this context we are testing a wide variety of promoters fused to reporter gene for β‐glucuronidase (GUS) for expression specifically in melon fruits. In this study in melon, salicylic acid‐inducible promoter region of pathogenesis‐related protein gene (PR1a) of tobacco fused to β‐glucuronidase (GUS) gene was introduced into melon via Agrobacterium‐mediated gene transfer using a binary vector system. Gene transfer was effective when Agrobacterium virulence factors like acetosyringone (100 μM) and low pH (5.2) were provided during the co‐culture step. Transformed shoots were recovered from benzyladenine‐induced cut cotyledons using kanamycin gene as a selective marker. Rege...