Katsuaki Ishii

Determination of genetic stability in long-term micropropagated shoots of Pinus thunbergii Parl. using RAPD markers

Abstract Random amplified polymorphic DNA (RAPD) markers were used to determine the genetic stability of long-term (more than 10 years) micropropagated shoots of Japanese black pine (Pinus thunbergii Parl.). Thirty-six shoots consisting of three morphotypes (short, medium, and long needles) were randomly chosen from about 4,000 micropropagated shoots regenerated from the explants of a single nematode-resistant mother plant. Out of 126 primers screened, 30 gave 134 clear reproducible bands. A total of 4,824 bands obtained from these studies exhibited no aberration in RAPD banding patterns among the tested shoots. Our results show that regenerants from our plant micropropagation system are genetically stable.

Germplasm conservation of the tropical forest trees,Cedrela odorata L.,Guazuma crinita Mart., andJacaranda mimosaefolia D. Don., by shoot tip encapsulation in calcium-alginate and storage at 12–25°C

Germplasm conservation of the tropical forest trees,Cedrela odorata L.,Guazuma crinita Mart., andJacaranda mimosaefolia D. Don., at above-freezing temperatures following alginate-bead encapsulation was attempted. Shoot tips excised from in vitro plantlets were encapsulated in calcium-alginate beads and stored on different substrates at 12, 20, and 25 °C. Percent viability when encapsulated shoot tips were stored on substrate containing only water solidified with 1% (wt/vol) agar was 80% after 12 months at 12°C forC. odorata, 90% after 12 months at 25°C forG. crinita, and 70% after 6 months at 20°C forJ. mimosaefolia.

Cloning and Expression of a cDNA Encoding an Insoluble Matrix Protein in the Gastroliths of a Crayfish, Procambarus clarkii

Abstract In the crayfish Procambarus clarkii, the gastroliths are formed as a paired structure in the stomach during the premolt period, and contain calcium carbonate and a small amount of an organic matrix. In this investigation, a cDNA encoding an insoluble matrix protein was isolated from P. clarkii. The open reading frame encoded 505 amino acid residues including two unique repeated sequences. The N-terminal half of the amino acid sequence, which included 10-amino-acid repeats, exhibited a high degree of similarity to that of involucrin, a protein synthesized in human keratinocytes. Northern blot analysis revealed that mRNA encoding the matrix protein is specifically expressed in the gastrolith discs during the premolt period in which the gastroliths formed. In the gastrolith discs, levels of expression of this mRNA correlated increases in weights of the gastroliths concomitant with their formation. Organ culture of the gastrolith discs suggested that expression of mRNA in the discs is induced by molting hormone, 20-hydroxyecdysone. These results reinforced the relationship between the matrix protein and formation of the gastroliths. Functional analysis showed that the protein inhibits calcium carbonate crystallization in a solution system, suggesting that the protein plays a role in the calcification of the gastroliths.

Somatic embryogenesis in sawara cypress (Chamaecyparis pisifera Sieb. et Zucc.) for stable and efficient plant regeneration, propagation and protoplast culture

Somatic embryogenesis inChamaecyparis pisifera was initiated from immature seeds collected from the end of June to early July. We obtained initiation frequencies ranging from 12.5 to 33.3% using whole seed explants in liquid media. Embryogenic cultures were maintained and proliferated for more than a year in solid and liquid media. High maturation frequencies of ‘high quality’ embryos were obtained on maturation media containing abscisic acid (ABA), activated charcoal (AC), and polyethylene glycol (PEG) as osmotic agent. More than one thousand cotyledonary embryos on average per 100 mg initial fresh weight of embryogenic cells were attained on medium containing 100µM ABA, 2 gL−1 AC, and 150 gL−1 PEG. About 97% germination frequencies and 92% plant conversion rates were achieved without any pretreatment. Growing of plants regenerated from somatic embryos has been monitored in the field. Furthermore, a procedure for culture of protoplasts isolated from embryonal masses was also described.

Immunolocalization of gastrolith matrix protein (GAMP) in the gastroliths and exoskeleton of crayfish, Procambarus clarkii

Abstract Gastrolith matrix protein (GAMP) is a novel protein purified from gastroliths of the crayfish, Procambarus clarkii, and has been suggested to be associated with calcium carbonate deposition. In the present study, a specific antibody against GAMP was raised and distribution of GAMP immunoreactivity was studied in crayfish gastrolith and exoskeleton. Localization of calcium carbonate in the exoskeleton, determined by silver nitrate staining and energy dispersive X-ray microanalysis, was compared with that of GAMP immunoreactivity. Crystalline forms of calcium carbonate were also determined. SDS-PAGE and Western blotting revealed that the gastrolith extract contained a major band which was GAMP-immunopositive and showed the same mobility as that of purified GAMP. The exoskeleton extract showed smear bands, but no immunoreaction was detected. By using immunohistochemistry, the anti-GAMP antiserum reacted almost uniformly with gastrolith matrix irrespective of gastrolith size. Epithelial cells of the gastrolith disc were also immunopositive. In the exoskeleton, exocuticle was strongly GAMP-immunopositive, whereas the endocuticle and membrane layer was slightly positive. The epicuticle was immunonegative. Calcium carbonate was detected in exocuticle, endocuticle and a part of the membrane layer, but not in the epicuticle. Thus, the distribution of GAMP immunoreactivity roughly corresponded with that of calcium carbonate. X-ray diffraction study showed that calcium carbonate in the gastrolith was amorphous, whereas that in the exoskeleton consisted of calcite crystals. These data indicate that a GAMP-immunoreactive substance is commonly distributed in the mineralized tissues of the crayfish, but may exist in a chemically different form in other tissues.

Somatic embryo culture for propagation, artificial seed production, and conservation of sawara cypress (Chamaecyparis pisifera Sieb. et Zucc.)

Abstract Somatic embryogenesis in Chamaecyparis pisifera Sieb. et Zucc. was initiated from immature seeds collected from the end of June to early July. Mass propagation through adventitious shoot bud production from somatic embryo culture on Woody Plant (WP) medium and artificial seed production using sodium alginate was achieved. A high bud forming index value (25.8) was obtained on medium supplemented with 1 μM 6-benzylaminopurine. The conversion rates from artificial seeds under aseptic and nonaseptic conditions were 60%–100% and 10%–12%, respectively. For germplasm conservation, somatic embryos and embryogenic cells were successfully stored at 4°C (medium-term storage) and in liquid nitrogen for long-term storage.

Monitoring Genetic Stability in Quercus serrata Thunb. Somatic Embryogenesis Using RAPD Markers

Genetic stability of propagules regeneratedvia somatic embryogenesis is of paramount importance for its application to clonal forestry. Random amplified polymorphic DNA (RAPD) markers were used to determine the genetic stability in somatic embryogenesis ofQuercus serrata Thunb. (Japanese white oak). Forty samples from an embryogenic line, consisting of regenerated plantlets, somatic embryos, and embryogenic calli, were examined using 54 decanucleotide primers. A total of 6520 clear reproducible bands obtained from these studies exhibited no aberration in RAPD banding pattern among the tested samples. Our results show that somaclonal variation is absent in our plant propagation system. The genetic stability is discussed in terms of the origin of somatic embryos.

Somatic embryogenesis and evaluation of variability in somatic seedlings of Quercus serrata by RAPD markers

Ouercus serrata (Japanese white oak) (Fig. 1) is widely distributed as coppice and thicket forests in Japan and Korea. Q. serrata is diploid with 24 chromosomes. The acorns mature in one year. This species is the host of many insects like stag beetles (Allomyrina,Lucanus spp.). Dried log trees are used for log-bed for Shiitake mushroom (Lentinus edodis) production which has now become a big industry. Black rot is a common disease of logs, and the infected logs prevent fruitbody or mushroom production. Hypocrea nigricans,H. muroiana and H. schweinitzii are the main organisms isolated from black rotted logs. High quality charcoal produced from this species has been well known for a long time. However, Ouercus forest area has decreased recently because of newly urbanization. Many clones of Q. serrata were selected as candidates for plus trees with good growth and high mushroom production on these log-beds. It is generally known that the mushroom production of the logs with smooth bark is larger than that with rough bark. Up to now, 253 plus trees were selected in Q. serrata and they are important for clonal propagation for developing seed orchards. Micropropagation from limited amount seeds of seed orchards is also preferable. Other Quercus species such as Q. acutissima and O. mongolica var. grosseserrata. are used as log-beds for growing mushroom in Japan.

Rapid in vitro propagation of Matteuccia struthiopteris (L.) Todaro – an edible fern

Abstract A rapid and efficient in vitro plant regeneration method was developed for Matteuccia struthiopteris (L.) Todaro (Ostrich fern). Side shoots, originating in meristems of sectioned rhizomes, were used as explant material. A very high rate of meristem multiplication was achieved by culturing the explants in half-strength MS liquid medium supplemented with 2.0 mg/l N-(4-Pyridyl)-N′-phenylurea (4-PU) and 0.5 mg/l thidiazuron (TDZ). Multiplication of the shoot primordia was faster in suspension culture than on solid medium. Rhizogenesis and growth of regenerants were best achieved on hormone-free one-quarter-strength MS solid medium amended with 0.4% agar and 1.0% activated charcoal. Regenerated plantlets continued to grow after transfer to soil in a phytotron.

Somatic embryo production and plant regeneration of Japanese black pine (Pinus thunbergii)

Somatic embryogenesis in Pinus thunbergii was initiated from megagametophytes containing immature zygotic embryos. Embryogenic cultures were maintained and proliferated in medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. High maturation frequencies of somatic embryos were obtained on maturation media containing maltose, activated charcoal, abscisic acid, and polyethylene glycol as osmotic agent. The best result among the cell lines tested was achieved with the cell line T-205-3. More than 900 somatic embryos per petri dish, on average, were obtained after about 8 weeks of culture on maturation medium. Sixty percent of somatic embryos tested germinated after transfer to plant growth regulator-free medium and then 85% of them converted into plantlets.