Emilio Maruyama

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.

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.

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.

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.

Efficient plant regeneration of Hinoki cypress (Chamaecyparis obtusa) via somatic embryogenesis

This report describes the efficient plant regeneration of Chamaecyparis obtusa Sieb et Zucc. via somatic embryogenesis. Embryogenic cultures were initiated from megagametophytes containing immature zygotic embryos. Embryogenic cultures were maintained and proliferated by 2–3-week interval subcultures in medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. High maturation frequencies of cotyledonary embryos were obtained on maturation medium containing maltose, polyethylene glycol, activated charcoal, and abscisic acid. Somatic embryos germinated readily after transfer to plant growth regulator-free medium. Growth of regenerated emblings has been monitored in a greenhouse.

Somatic Embryogenesis of Japanese Conifers

ABSTRACT The present work focused on improving somatic embryogenesis of the Japanese conifer species by studying the factors affecting culture condition. They include Chamaecyparis obtusa Sieb. et Zucc., Chamaecyparis picifera Sieb. et Zucc., Cryptomeria japonica D. Don, Pirns densiflora Sieb. et Zucc. and Pinus thunbergii Pari. When the zygotic embryos in the explants were either proembryos or early embryos, initiation of somatic embryogenesis was successful. The medium significantly affected induction success, the medium with auxin (3 or 5 or 10 uM 2,4-dichlorophenoxyacetic acid) and cytokinin(l or 3 uM 6-benzylaminopurine) being better than the medium with cytokinin alone. Maltose instead of sucrose was better for the maturation of the somatic embryos. Sawara cypress (Chamaecyparis picifera) plantlets regenerated from somatic embryo and grown in a greenhouse were planted out in the field to compare with the control seedlings. Somatic embryogenesis and plant regeneration from shoots cultures of Hinoki cypress (Chamaecyparis obtusa) in vitro was also successful.

Micropropagation of Bolaina Blanca (Guazuma crinita Mart.), a Fast-Growing Tree in the Amazon Region

Rapid clonal propagation of Bolaina blanca (Guazuma crinita Mart.) was established by the subculturing of the shoot-tips from aseptically germinated seedlings on woody plant medium (WPM) supplemented with trans-zeatin [trans-6-(4-Hydroxy-3-methylbut-2-enylamino)purine] (ZEA). After 45 days of culture, a seven-fold multiplication rate was achieved on WPM containing 10 μM of ZEA. Obtained shoots were simultaneously elongated and rooted on WPM containing 1 μM of kinetin [6-furfurylaminopurine] (KIN). After 60 days of culturing the growth of shoots was evident, and high rooting percentages were obtained. The plantlets were transferred into pots with vermiculite and acclimatized successfully in plastic boxes with transparent cover inside the growth cabinet.

Germplasm conservation of Guazuma crinita, a useful tree in the Peru-Amazon, by the cryopreservation of in vitro-cultured multiple bud clusters

In vitro-cultured adventitious bud clusters of Guazuma crinita Mart. were successfully cryopreserved by the one-step vitrification method. Small segments (1.0-1.5 mm3) cut from adventitious bud clusters were exposed to a cryoprotectant mix solution containing (w/v), 25 glycerol, 15 sucrose, 15 ethylene glycol, 13 dimethyl sulfoxide, and 2 polyethylene glycol, at 25 °C for 15-60 min prior to storage in liquid nitrogen. After rapid warming (37 °C), the segments were treated with woody plant medium containing 40 (w/v) sucrose for 20 min at 25 °C, and then transferred to recovery-growth medium. High survival rates (about 80) were achieved without any cold hardening and/or pre-culturing treatments, and about 30 of the surviving cryopreserved explants regenerated plants.

Micropropagation of Guazuma crinita mart. by root and petiole culture

SummaryShoot organogenesis of Guazuma crinita Mart. from root and petiole explants was obtained via adventitious bud formation. Root segments and petiole explants excised from in vitro generated plantlets were cultured on woody plant medium (WPM) supplemented with [trans-6-(4-hydroxy-3-methylbut-2- enyl)aminopurine] (zeatin) or with [6-benzyladenine] (BA). After 45 d of culturing, clumps of green bulbous structures containing small adventitious buds (clusters) were generated in all explants cultured with 10 µM zeatin under a photon flux density of 65 µmol m−2 s−1. For subsequent shoot differentiation, clusters were transferred onto medium containing 1 µM zeatin. After 60 d of culturing, 30% of clusters generated from petiole explants developed into plants. The regenerated plantlets were successfully acclimatized and all survived and grew well. No morphological abnormalities were observed.

In Vitro Culture of Japanese Black Pine (Pinus Thunbergii)

In vitro culture from mature embryos of Japanese black pine (Pinus thunbergii) plus trees was developed. For initial adventitious buds induction, Wolter and Skoog (1966) medium with 3 mg/1 BAP was the best among screened media. For elongation of shoots, a half strength Lepoivre (LP) medium containing 5 g/1 activated charcoal was the best. Rooting percentage was 0–50 % on the modified Root Induction Medium of Abo El-Nil (RIM). However there were big clonal differences in rooting percentages. Habituated plantlets were potted out to the field and growth performance was checked until 8 years. In vitro plantlet regeneration from 6–7 years old black pine which were selected as resistant clones using pine wood nematode inoculation test was tried. Induced brachyblast buds obtained by topping the trees were the best for expiants and the half-strength LP medium containing 2.25 mg/1 BAP and half-strength LP medium containing 5 g/1 activated charcoal were used in turn repeatedly for subculture in about 1–2 months interval. After subculture of 2 years, more than 5000 shoots were obtained from one clone. Shoots rooted in RIM containing IBA. Somatic embryogenic cells were induced from immature embryos collected from the end of June to mid July in Tsukuba, Japan. The suitable media for induction, maturation and germination of somatic embryos were different in the constituents. For induction, modified 1/2LP or Smith (1996) medium containing BAP and 2,4-D was used. For maturation, EM-3 medium (original) was added with ABA and PEG. Maltose instead of sucrose was effective for somatic embryo maturation. For germination of the matured somatic embryos, hormone free medium was used with or without activated charcoal powder. Regenerated plantlets were habituated and potted out in the vermiculite.