Agrobacterium-mediated transformation of cork oak (<Emphasis Type="Italic">Quercus suber</Emphasis> L.) somatic embryos

A simple method is presented for the unreported genetic transformation of cork oak (Quercus suber L.). Pro-embryo masses (PEMs) were induced on immature zygotic embryos applied to medium supplemented with 2.3 M 2,4-dichlorophenoxyacetic acid. The established PEMs were inoculated with Agrobacterium tumefaciens LBA4404/p35S GUS INT/pCAMBIA 1301 strain. Transformants were selected on hygromycin (HYG) 94 M-supplemented medium. Viable embryos constituted 13% of those selected on HYG during 4 months. Expression of -glucuronidase at 4 months following co-cultivation confirmed transformation in 5.8% embryos selected on HYG. This method forms a basis for genetic transformation of cork oak somatic embryos.     

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.     

Tissue culture micropropagation of Douglas-fir

A procedure was developed for plantlet production from embryos of Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. The Medium for Conifer Morphogenesis, used at full strength, and supplemented with 10 M benzyladenine for 17 days produced an average of 6.8 shoots on more than 90% of the embryos. The percentage of shoot-forming embryos as well as the average number of shoots per embryo varied significantly among eight seedlots. For secondary multiplication, 89% of the adventitious shoots produced axillary buds on MCM with 5 M benzyladenine. However, 0.5 M BA was more suitable for the elongation of axillaries. Rooting ranged from 0–87% depending upon the treatment. The highest percentage was obtained with a 7-week incubation in peat:perlite containing 1/5-strength medium, 1% sucrose and 2.7 M naphthaleneacetic acid, followed by 5 weeks on peat:perlite with 1/5-strength major and minor salts, full iron and organics, 1% sucrose and 0.1% charcoal.     

Micropropagation of western hemlock (Tsuga heterophylla [Raf.] Sarg.) from embryonic explants

An in vitro propagation protocol is described for western hemlock, an important forestry species in Canada. For shoot bud induction, embryonic explants were placed initially on one-third strength Schenk and Hildebrandt medium containing 5 M N⁶-benzyladenine (BA) or 5 M BA in combination with either 5 M kinetin or 5 M 2-isopentenyl adenine for 14 days. The explants were transferred to basal medium without cytokinins for 3 weeks, and then to basal medium containing 0.05% activated charcoal. Elongating shoots were subcultured every 4 weeks on charcoal medium. Shoots, 10 mm in stem height, were rooted either in agar or sterilized peat/perlite (1:1). Up to 70% of the shoots formed roots when they were transferred to the latter, moistened with 1/2 strength Gresshoff and Doy medium containing 5 M -naphthaleneacetic acid. About 90% of the plantlets survived transfer to greenhouse conditions.     

Maturation and plant recovery from embryogenic cells of Japanese larch: Effect of abscisic acid in relation to their morphology

Two embryogenic cell lines characterized by different morphology and color, white and red, were obtained from an immature zygotic embryo of Japanese larch (Larix leptolepis Gord.). Mature somatic embryos with cotyledons and regenerated plants were obtained from both cell lines. However optimal concentration of abscisic acid (ABA) for maturation varied depending on morphology of ECs. From the white ECs which intermingled with somatic embryos of very early stage, mature somatic embryos were induced on maturation medium containing 50 μM of ABA. On the other hand, mature somatic embryos with cotyledons were observed from the red ECs which consisted of somatic embryos of more developed stage on hormone-free medium or 0.1 μM ABA containing-medium.     

Five heavy metals in propagules of ten mangrove species of China

Five heavy metals in the propagules of 10 mangrove species in China have been investigated. The results revealed that the levels of five heavy metals in most of the propagules were lower than the background levels of the soil. The levels of copper, manganese, zinc, cadmium, and lead in the propagules varied at about 2.1–7.8 g/g, 3.9–28.0 g/g, 5.7–60.0 g/g, 0.014–0.057 g/g, and 0.018–0.038 g/g, respectively. On average, the levels of five heavy metals were in the order Zn > Mn > Cu > Cd > Pb. The biological absorption coefficients were 0.02–1.30 for copper, manganese, zinc, and cadmium and about 0.0007–0.0020 for lead in propagules, showing the following order: Zn > Cd > Cu > Mn > Pb. The above results indicate that the five heavy metals have low accumulation in most of the propagules of 10 mangrove species and are at safe levels for the effective utilization of mangrove resources.     

High-efficiency somatic embryogenesis and morphohistology and histochemistry of somatic embryo development in Larix leptolepis Gordon

A high-efficiency somatic embryogenesis protocol of Japanese larch (Larix leptolepis Gordon) has been established in our investigation. Calli were induced from immature zygotic embryos of female cones of L. leptolepis and then subcultured regularly on to a modified Gupta and Durzan (DCR) basal medium for 5 years. Embryogenic tissues showed distinct morphological changes dur-ing somatic embryo development when they were transferred to a maturation medium supplemented with abscisic acid (ABA) com-pared with the morphology in a medium lacking ABA. Histological observations indicated that polyembryony was a characteristic feature during early embryogeny and somatic embryos at later stages showed normal histodifferentiation. In addition, histochemical analysis revealed that abundant starch granules and proteins accumulated in mature embryos, indicating that they played important roles in the development and regeneration of normal plantlets from somatic embryos on hormone-free germination media     

Cultural factors influencing adventitious shoot and plantlet formation from slash pine cotyledons

Cultural factors affecting in vitro shoot and subsequent plantlet formation of slash pine (Pinus elliotti Engelm.) cotyledons were investigated. Basal media composition, N⁶-benzylaminopurine (BAP) concentration and exposure time significantly influenced bud induction in cotyledons cultured under a continuous photoperiod of 35–40 mol m^–2 s^–1 at 24 ± 1 °C. The largest number of adventitious shoots was obtained after 28 days exposure to 66 M BAP-supplemented modified Gresshoff and Doy 1 (GD1) medium. Relatively high frequencies of large shoots were obtained after a 14-day exposure to 22 M BAP-supplemented Brown and Lawrence (BL) or 66 M BAP-supplemented GD1. Adventitious shoots derived from 21- or 28-day exposures to BAP developed more slowly and were smaller in size than those derived from a 14-day exposure to the cytokinin. Shoot differentiation and subsequent growth were also influenced by basal media, media concentration, and presence of activated charcoal in the medium. The percentage of cotyledons forming shoots was highest on half-strength GD1 medium containing activated charcoal. Rooting was achieved in vitro under a continuous photoperiod of 60–70 mol M^–2 S^–1. Roots were formed when excised shoots were planted on GD 1/2 medium supplemented with 2.68 M 1^–1 a-napthaleneacetic acid (NAA) with or without BAP for 14 days. The proposed technique of slash pine propagation using cotyledon explants can produce up to 100 seedlings per embryo.     

Influences of altered phytohormone use on endogenous ABA and mRNA populations during white spruce (Picea glauca) somatic embryo culture

We examined endogenous abscisic acid [(+)-ABA] amounts and mRNA populations in white spruce (Picea glauca (Moench) Voss) somatic embryos in liquid suspension culture during a pretreatment prior to induction of maturation with exogenous (+)-ABA. The pretreatment consisted of removal of 2,4-dichlorophenoxyacetic acid (2,4-D) from the multiplication medium for 7 days. The removal of 2,4-D resulted in a slight increase in endogenous ABA in pretreated tissues compared to nonpretreated tissues at the end of the 7-day pretreatment period (42.4 versus 20.0 pg mg(-1) lyophilized tissue). Altered gene expression patterns were observed as early as one day after the start of the pretreatment, with more than 17 new polypeptides found in pretreated tissues. The influence of pretreatment continued to be observed after tissues were transferred to ABA-containing maturation medium. By comparing mRNA populations in pretreated and nonpretreated tissues cultured on either ABA-containing or ABA-free maturation medium, at least 12 mRNAs were observed to be induced by ABA, among which three polypeptides were ABA inducible only in pretreated tissues.     

In vitro and ex vitro rooting of micropropagated shoots using three green ash (Fraxinus pennsylvanica) clones

Shoot multiplication using seedling materials was achieved by subculture on Murashige and Skoog salts with Gamborg's B5 vitamins (MSB5) medium containing a combination of 5 M 6-benzyladenine (BA), 5 M thidiazuron (TDZ), and 1 M indole-3-butyric acid (IBA) with three green ash (Fraxinus pennsylvanica) clones, SD1009 (South Dakota origin), SD2002 (South Dakota origin), and KA2018 (Kansas origin). Shoots were rooted using in vitro and ex vitro methods. For in vitro rooting studies, elongated shoots were transferred to rooting plugs supplied with liquid MSB5 medium containing a 3×3 factorial arrangement of two different auxins, -naphthaleneacetic acid (NAA) and IBA, at three concentrations (0, 5, and 10 M). The most effective treatment for in vitro root number, root length, and shoot height was 5 M IBA. The three clones also were tested for ex vitro rooting using a quick dip in 1 mM NAA, 1 mM IBA, or control (no auxin). The maximal ex vitro rooting response occurred when shoot explants of the three clones were dipped in 1 mM IBA. Significant clonal differences were noted in response to in vitro and ex vitro rooting treatments. Rooted plantlets were acclimated to the greenhouse.     

A study on some anatomical parameters of the piercing-sucking process in leaves and branches of<Emphasis Type="Italic"> Pittosporum tobira</Emphasis> L. (Pittosporaceae) infested by the cottony cushion scale,<Emphasis Type="Italic"> Icerya purchasi</Emphasis> Maskell (Homoptera: Coccina,Margarodidae)

Pittosporum tobira L. (Family Pittosporaceae) is an important plant species grown in parks and gardens in Turkey. The cottony cushion scale, Icerya purchasi Maskell (Homoptera: Coccina, Margarodidae), is an important pest of P. tobira, and causes damage mainly to the leaves, branches and stems of the host plant. In spite of the great economic importance of I. purchasi to its host plant P. tobira, information has been limited on some basic anatomical parameters of the nature of the damage to leaves and branches. The present study was, therefore, initiated to investigate the insertion and penetration of stylets into the leaves and branches of P. tobira, the length of stylets and depth of penetration in both of these tissues, and penetration of stylets into the cortex, xylem and phloem tissues and into the secretory and non-secretory canals of the leaves and branches. The results showed great variations between the leaves and branches in all the criteria observed in I. purchasi infesting P. tobira. The study found 20 whole and 23 broken mouthparts in the leaves as compared to 3 whole mouthparts and 1 broken in the branches. Length of the stylets inserted into the leaves ranged from 33.83 m to 540.93 m and into the branches from 202.85 m to 340.8 m. The stylets reached greater depths in the leaves, at 540.93 m, as compared to 498.67 m in the branches. The stylets in the leaves were associated with 12 secretory canals, but with none in the branches. It was found that three stylets reached xylem in the leaves as compared to one in the branches. Similarly, 17 stylets in the leaves reached to the phloem, but only 2 in the branches. No stylets were found in the cortex of the leaves, but one was found in the branches. In the leaves, 11 stylets reached and remained in the non-secretory canals, but none in the case of the branches.     

Somatic embryogenesis from immature and mature zygotic embryos ofCryptomeria japonica I: Embryogenic cell induction and its morphological characteristics

Embryogenic cells (ECs) of sugi (Cryptomeria japonica D. Don) were induced from immature and mature zygotic embryos cultured on different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D)-containing modified Campbell and Durzan medium. The rate of induction of ECs varied depending on the stage of embryos collected. The highest percentage of induction (35%) was obtained with immature zygotic embryos collected July 18 and July 30, 1997, when 1 M of 2,4-D was added to the induction medium. The ECs easily proliferated when subcultured in a medium of the same composition as the induction medium within 3 weeks. Morphological characteristics of nonembryogenic cells and embryogenic cells of different developmental stages were studied under an inverted fluorescence microscope.Part of this work was presented at the 109th annual Japanese Forestry Society meeting at Tochigi, April 1998     

Oak somatic and gametic embryos maturation is affected by charcoal and specific aminoacids mixture

? Development of both somatic and gametic embryogenesis has many applications in clonal forestry and genetic improvement, for instance as mass-propagation of genetically improved plants and production of pure lines through doubled-haploid plant regeneration from gametic embryos.

? The goal of this work was to improve growth, maturation and plantlet regeneration of cork oak (Quercus suber L.) embryos from both somatic and gametic origin.

? Activated charcoal promoted a significant increase in growth in terms of relative size and weight of both somatic and doubled-haploid embryos, as well as a more efficient control of secondary somatic embryogenesis during development. A significant interaction was also observed with amino acid nutrition. While some amino acids (i.e., glutamine, arginine or asparagine) did not show significant differences with the controls, a mixture of these three amino acids or gamma amino butyric acid stimulated embryo growth. The highest survival rate during acclimation of plantlets from both somatic and doubled-haploid origin was obtained when the embryos had been previously cultured on basal medium with 3% sucrose and 1% activated charcoal for two months at 4 °C and germinated on medium supplemented with 6-benzylaminopurine and indole-3-butyric acid.

? We obtained more than 900 cork oak plantlets acclimated from several embryogenic lines, with a high survival rate, demonstrating that this methodology is applicable for large scale plantlet production. We also report the first regeneration of doubled-haploid plantlets in cork oak.

     

Zygotic and somatic embryo morphogenesis in Pinus pinaster: comparative histological and histochemical study

We compared morphogenesis and accumulation of storage proteins and starch in Pinus pinaster Ait. zygotic embryos with those in somatic embryos grown with different carbohydrate sources. The maturation medium for somatic embryos included 80 microM abscisic acid (ABA), 9 g l(-1) gellam gum and either glucose, sucrose or maltose at 44, 88, 175 or 263 mM in the presence or absence of 6% (w/v) polyethylene glycol (PEG) 4000 MW. Maturation medium containing 44 or 88 mM of a carbohydrate source produced only one or no cotyledonary somatic embryos per 0.6 g fresh mass of culture. The addition of PEG to the basal maturation medium resulted in a low yield of cotyledonary somatic embryos that generally showed incomplete development and anatomical abnormalities such as large intercellular spaces and large vacuoles. High concentrations of maltose also induced large intercellular spaces in the somatic embryonic cells, and 263 mM sucrose produced fewer and less developed cotyledonary somatic embryos compared with 175 mM sucrose, indicating that the effect of carbohydrate source is partially osmotic. Zygotic embryos had a lower dry mass than somatic embryos at the same stage of development. Starch granules followed a similar accumulation pattern in zygotic and somatic embryos. A low starch content was found in cotyledonary zygotic embryos and in somatic embryos developed in the presence of 175 mM maltose or 263 mM glucose. In zygotic embryos and in PEG-treated somatic embryos, protein bodies appeared later and were smaller and fewer than in well-developed somatic embryos grown without PEG. We propose that storage protein concentration might be a marker of embryo quality.     

Field measurements of gas exchange in Avicennia marina and Bruguiera gymnorrhiza

Diurnal gas exchange characteristics were measured simultaneously in two mangrove species, Avicennia marina and Bruguiera gymnorrhiza, over 7 d in summer (February–March), to compare their productivity. The study was undertaken in the Beachwood Mangroves Nature Reserve, Durban, South Africa, using fully expanded leaves of young and mature trees at the top of the canopy. Gas exchange was strongly influenced by photosynthetic photon flux density (PPFD), leaf temperature and the accompanying leaf to air vapour pressure deficit ( w). Carbon dioxide exchange was saturated at a PPFD of about 600 mol m^-2s^-1 in B. gymnorrhiza compared to 800 mol m^-2s^-1 in A. marina. Maximal CO₂ exchange occurred between 12h00 and 14h00 and was consistently greater in A. marina (8.8 mol m^-2s^-1) than in B. gymnorrhiza (5.3 mu;mol m^-2s^-1). Mean internal CO₂ concentrations ( c_i) were 260 l l^-1 in A. marina and 252 l l^-1 in B. gymnorrhiza. Photorespiratory activity was 32% in A. marina and 30% in B. gymnorrhiza. Mean water use efficiency (WUE) was 8.0 mol mmol^-1 in A. marina and 10.6 mol mmol^-1 in B. gymnorrhiza. Diurnal leaf water potentials ranged from –0.8 to –3.5 MPa and were generally lower in A. marina.     

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.     

Waldschutz gegen Borkenkäfer: Der Beitrag von Duftstoffmeteorologie und Populationsdynamik

Zusammenfassung Mit theoretischen Modellen kann die Effektivität verschiedener Ansätze (z. B. Fallenzahl und-abstand, Ködermenge und-abgaberate) für den Einsatz von synthetischen Pheromonen in Pest-Management-Systemen verglichen werden. Das GAUSS-sche Modell, das von einer Normalverteilung der Konzentrationsabnahme um eine Lockstoffquelle ausgeht, wurde benutzt, um fürT. lineatum die lockwirksame Reichweite und die Konkurrenzsituation von Naturköder und Falle abzuschätzen. Bei 100 000 /ha und 2 Wochen Flugdauer ergeben sich für (+)-Lineatin (Reaktionsschwelle 0,1 ng/m³, Produktion 6,43 ng//d, Produktionsdauer 3 d/) mittlere Tagesraten von 128,6 g/ha bzw. eine Gesamtproduktion von 1,929 mg/ha. Die lockwirksame Reichweite x_max beträgt je nach Wetterlag für 1T. lineatum- 20–40 cm, für Fallen mit 1 CONREL-Faser (Abgaberate 10 g/d [+]-Lineatin) 13–16m. Für eine 10fach höhere lockwirksame Reichweite sind nach dem Modell etwa 100fach, in der Praxis nach verschiedenen Autoren sogar 200fach stärkere Köder erforderlich.In Freilandversuchen über die Relation von Fangleistung und Beifängen unterschiedlicher Fangsysteme waren die Coleoptera (bes. Rhizophagidae und Staphylinidae) am häufigsten. In Trichterfallen lagen die Beifänge mit 9,2% deutlich höher als in Schlitzfallen (1,2%). Das Giftpolter hatte demgegenüber das größte Beifangspektrum; hier traten auch Ordnungen auf, die von anderen Fangsystemen nicht betroffen waren.Massenfang kan prinzipiell in Holzhöfen und Waldbeständen erfolgreich durchgeführt werden. Allerdings ist das Problem der Beifänge noch ungelöst und die Relations von potentieller Vermehrung der Borkenkäfer und maximal möglicher Populationsreduzierung durch Fallenfang unbekannt. Zudem kann eine nach Massenfang geringere Populationsdichte im Folgciahr durch zunehmende Reproduktionsraten oder durch ersatzweise Einnischung (competive displacement) anderer Borkenkäferarten ausgeglichen und damit der Forstschutzeffekt zunichte gemacht werden.

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Bark beetle control: contribution of pheromone meteorology and population dynamics

The use of semiochemicals in pest management systems of bark beetles is viewed in relation to optimization of resources investment, comparing beetle numbers in univoltine, requisitegoverned woodboringTrypodendron lineatum (Oliv.) with multigeneration bark inhabitingIps typographus (L.). The efficiency of different numbers of and distance between traps and pheromone load of dispensers was analysed using steady state models and comparing results with published and own field data. Competition between traps and beetle-infested logs, attractive distance and overlap of pheromone plume was compared forT. lineatum using models which assume a GAUSS distribution of concentration decrease around a pheromone source. For (+)-lineatin (reaction threshold 0.1 ng/m³, production 6.43 ng//d, duration of production 3 d/), 100,000 /ha and a flight period of 2 weeks, daily mean production is 128.6 g/ha. Attractive distance x_max is 20–40 cm for 1T. lineatum- and 13–16 m for a trap with 1 CONREL-dispenser (mean release rate 10 g/d (+)-lineatin). A 10-fold increase of attractive distance x_max needs a 100-fold increase of pheromone release rate according to the model, and 200-fold according to field experiments.In field experiments, Coleoptera (esp. Staphylinidae and Rhizophagidae) were the most abundant non-target trap catches. Insecticide-treated trap logs had the widest range of non-target insects including orders that were not affected by the non-insecticide trapping systems. Mass trapping can be successful in logging areas and/or forests. However, the exclusion of non-target insects still is an unsolved problem and we still have only limited knowledge on potential population increase of beetles and maximum reduction that can be achieved by trapping. Also, population levels reduced by mass trapping can be compensated by increasing reproductive effort of the succeeding generation and/or competitive displacement by secondary bark beetle species.

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Mit 5 Abbildungen und 2 Tabellen

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.

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Nach einem Vortag bei der Fortbildungstagung des Hessischen Forstvereins in Solms/Lahn am 23. Mai 1991.

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Herrn Prof. Dr. Dr.G. Wellenstein zum 85. Geburtstag.     

In vitro somatic embryogenesis in callus cultures of Azadirachta indica A. Juss.—a multipurpose tree

Plant regeneration via somatic embryogenesis was achieved in embryogenic callus cultures derived from immature zygotic embryos 40 days after anthesis of Azadirachta indica A. Juss. on semisolid basal Murashige and Skoog (MS) salts and vitamins supplemented with 1.11 µM 6-benzylaminopurine (BA) and 4.52–6.78 µM 2,4-dichlorophenoxyacetic acid (2,4-D). The globular-stage embryos were induced when the callus was transferred to medium with 1.11 µM BA and 0.45 µM 2,4-D. The highest average number of somatic embryos per 200 mg of callus was 152.8 after 8 weeks of culture on the medium. Maturation and germination of the somatic embryos were achieved on half-strength MS salts and vitamins supplemented 0.38–0.94 µM abscisic acid (ABA) and 2% (w/v) sucrose. The maximum percentage (64.2%) of germination was obtained with 0.94 µM ABA within 2 weeks of culture. Somatic embryo-derived plantlets were acclimatized in a greenhouse and subsequently showed normal growth. This efficient protocol will be helpful for propagating elite clones on a mass scale and will also be useful for genetic transformation study.     

Eine neue Virose beim Schwammspinner,Lymantria dispar L. (Lep., Lymantriidae)

A new virosis in caterpillars of Lymantria dispar L. (Lep., Lymantriidae) Caterpillars ofL. dispar found in the Apennin-mountains near Genua, Italy, showed a new virosis in a low percentage. The pathogen is characterized by the enormous dimensions (max. 20 ) and the cubic form of the polyhedra. It causes a nuclear hypertrophie particularly within the haemocytes,     

Somatic embryogenesis in sawara cypress (<Emphasis Type="Italic">Chamaecyparis pisifera</Emphasis> 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. This work was supported in part by the Japan Science and Technology Corporation and in part by a Grant for Research for the Future Program from the Japan Society for Promotion of Science.