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.     

培养基成分对黑松体胚发育成熟的影响

【目的】对日本抗性黑松体细胞胚成熟的影响因子进行研究,探究各因素在体细胞胚发生过程中的作用,为进一步提高体细胞胚发生的质量和数量提供理论依据。【方法】胚性细胞团在固体增殖培养基上继代生长10天,使用灭菌(75%乙醇擦拭、紫外灭菌30 min)的电子天平称量1 g胚性细胞团,采用50 mL无菌量筒(紫外灭菌30 min)量取30 mL培养液中于100 mL的三角瓶中,将1 g胚性细胞团转至100 mL的三角瓶中,手动搅拌至细胞团分散,置于90 r·min^-1摇床上,25℃黑暗培养7～8天。随后,取3 mL沉淀悬浮细胞进行继代增殖,每星期继代增殖一次至胚性细胞全部均匀散落于培养液。将继代4次的胚性细胞充分摇匀,取2 mL(鲜质量约200 mg)胚性悬浮细胞喷洒在不同组分麦芽糖(30、45、60 g·L^-1)、脱落酸(ABA)(0、5、10、20、30、50 mg·L^-1)、聚乙二醇(PEG8000)(0、50、75、100、125、150 g·L^-1)、活性炭(AC)(1、2、3 g·L^-1)、琼脂(琼脂6、8、10、12 g·L^-1,植物凝胶2、2. 5、3、3. 5 g·L^-1)以及麦芽糖、ABA、PEG 3因素的组合固体成熟培养基上。【结果】以#1337为材料的体细胞胚成熟发育过程中,麦芽糖45 g·L^-1,获得的体细胞胚数量显著增多。ABA浓度为5～20 mg·L^-1,体细胞胚数量呈显著上升趋势,ABA为20 mg·L^-1时,获得的体细胞胚最多。125 g·L^-1PEG8000为体胚发生最佳浓度; AC 2 g·L^-1时,胚性细胞形成结构完整、数量较多的成熟体胚;成熟培养基中添加琼脂粉,培养基不能凝固,植物凝胶更适合作为成熟培养基凝固剂。3 g·L^-1植物凝胶为抗性黑松体胚发育成熟的最佳浓度。在设计的9种成熟培养基组合中(胚性细胞#1337、#1537、#1637),胚性细胞产生体细胞胚最多的成熟培养基组合为麦芽糖45 g·L^-1、ABA 10 mg·L^-1、PEG8000 125 g·L^-1。在不同的麦芽糖、ABA和PEG组合中,不同无性系生产体细胞胚数量呈现出不同的变化趋势:胚性细胞系#1337体胚发育成熟的最佳组合为麦芽糖45 g·L^-1+ABA 10 mg·L^-1+PEG 125 g·L^-1;#1537和#1637体胚发育成熟的最佳组合为麦芽糖30 g·L^-1+ABA 10 mg·L^-1+PEG 125 g·L^-1;在3个无性系中PEG的极差最大,对抗性黑松体细胞胚发育成熟的影响最大。【结论】抗性黑松体胚发育成熟过程中,麦芽糖45 g·L^-1、ABA20～30 mg·L^-1、PEG8000 125 g·L^-1、AC 2 g·L^-1和植物凝胶3 g·L^-1对体细胞胚成熟都具有促进作用。30 g·L^-1麦芽糖、10 mg·L^-1ABA、125 g·L^-1PEG为抗性黑松体胚发育成熟的最佳组合。     

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L. ＇Moldova＇

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.     

Micropropagation of red sanders (Pterocarpus santalinus L.) using mature nodal explants

In vitro clonal multiplication of Pterocarpus santalinus L. was achieved using mature nodal explants of a 10-year-old elite quality tree. Combinations of serial transfer technique and incorporation of antioxidants (250 mg/l L-ascorbic acid and 50 mg/l citric acid) into the culture medium helped to minimize medium browning and improve explant survival during shoot sprouting. About 70% of explants were sprouted on Murashige and Skoog (MS) liquid medium containing 4.4 μM 6-benzyladenine (BA). The explant harvest period also influenced the bud break and shoot sprouting in nodal explants. The combination of 4.4 μM BA and 2.2 μM thidiazuron (TDZ) was found to be the most suitable growth regulator for obtaining the highest percentage of nodal segment sprouting (74%–75%), the number of secondary shoots per primary shoot (two or three), the shoot length (5–6 cm), the number of new nodal segments generated per active explant (four or five), and the multiplication coefficient (3.5) within 6 weeks. Repeated subculturing of nodal explants obtained from shoot cultures enabled continuous production of healthy axillary shoots. At the end of the sixth passage, about 90% of nodal explants produced five or six healthy green shoots, each being about 6.6 cm long with six or seven nodes. Multiplication coefficient was also increased from the first subculture (5.4) to the sixth subculture (8.3). The best rooting response was achieved on solidified half-strength MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 70% of the micropropagated plantlets were established successfully in 20-cm pots containing a mixture of soil and farmyard manure (4 : 1 ratio) and formed new leaflets.     

Somatic embryo culture for propagation,artificial seed production,and conservation of sawara cypress (<Emphasis Type="Italic">Chamaecyparis pisifera</Emphasis> Sieb. et Zucc.)

 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. Received: December 21, 2001 / Accepted: August 1, 2002 Acknowledgments 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 the Promotion of Science. Correspondence to:E. Maruyama     

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 performance potential of a somatic interior spruce seedlot

This study defined the field performance potential for 34 genotypes that composed a somatic seedlot of interior spruce (Picea glauca (Moench) Voss × Picea engelmannii Parry ex. Engelm.) crosses, white spruce (P. glauca (Moench) Voss) crosses, or a mixture. Each genotype was measured for morphological attributes: height, diameter, shoot dry weight, root dry weight, shoot-to-root ratio, height-to-diameter ratio at time of lifting. Each genotype was also measured for physiological attributes of cuticular transpiration (TFD_cut), osmotic potential at turgor loss point (Ψ_tlp) and freezing tolerance (index of injury at −6°C and −4°C; II_@−6 & II_@−4) during inactive and active shoot growth phases. Shoot growth potential (SGP = length of new leader elongation) and root growth potential (RGP) were conducted under four environmental regimes: nutrient-rich/well-watered, nutrient-poor/well-watered, low root temperature, and planting stress conditions. The somatic seedlot met target height, diameter and RGP standards for a plantable seedling crop in British Columbia, Canada, though genotypes differed in morphology at time of lifting. These genotypes also differed in their measured physiological attributes (TFD_cut, Ψ_tlp, II_@−6 and II_@−4) at time of lifting and during active shoot growth. Genotypic differences were also found for SGP and RGP under different testing environments. A stock quality assessment program describing elite genotypes within a seedlot can aid foresters in applying benefits of clonal forestry. Raymund S. Folk is now self employed.     

Regeneration of <Emphasis Type="Italic">Melia volkensii</Emphasis> Gürke (Meliaceae) through direct somatic embryogenesis

Experiments were conducted to study plant regeneration through direct somatic embryogenesis using mature zygotic embryo and cotyledonary explants from seeds of Melia volkensii stored for <3 and >12 months. Explants were cultured on Murashige and Skoog (MS) medium supplemented with BAP, NAA and 2,4-D (0.5, 1.0 and 2.0 mg l⁻¹) alone, and BAP (0.5, 1.0, 2.0 and 4.0 mg l⁻¹) in combination with 2,4-D or NAA (0.2 and 0.5 mg l⁻¹). After 4 weeks in culture, up to 60% of cotyledonary explants from the seeds stored for <3 months produced direct somatic embryos on BAP (0.5–4.0 mg l⁻¹) in combination with 2,4-D (0.2 mg l⁻¹). The number of somatic embryos ranged from 5 to 14 per explant in BAP (0.5 mg l⁻¹) and 2,4-D (0.2 mg l⁻¹) combination. Only 20% of cotyledonary explants from seeds stored for >12 months produced somatic embryos. Mature zygotic embryos failed to produce any somatic embryos. Subcultures of somatic embryos from cotyledonary explants of seeds stored for <3 months formed clusters of shootlets on semi solid MS and 1/2 MS media. After 6 weeks of subculture on multiplication MS media augmented with BAP (0.5 mg l⁻¹) and IAA (0.2 mg l⁻¹), 70% of the shoot tips formed 4–7 shoots per explant. Up to 33% of the multiplied shoots were rooted in MS medium supplemented with 2.0 mg l⁻¹ IBA. Plantlets developed normally into seedlings in the greenhouse.     

Effect of priming on the germination of<Emphasis Type="Italic">Peltophorum dubium</Emphasis> seeds under water stress

Peltophorum dubium seeds provided by Anhembi, SP were scarified in 98% H2SO4 for 15 rain to overcome mechanical dormancy. Seeds were primed in solutions of 0.2% Captan at 10℃ and 27℃, PEG 6000 -1.0 MPa at 10℃ and 27℃, 0.5 mol KNO3, 0.75 Mol KNO3, 1.0 Mol KNO3. Eight treatments including the primed seeds and nonprimed seeds, five replicates with 100 seeds for each treatment, were set to 15-cm-Petri dish with double filter paper moistened with testing solution PEG in refrigerator at 27℃. For the experiments of all the groups, osmotic potential were 0.0, -0.2, -0.4, -0.6, -0.8, -1.0, -1.2, and -1.4 MPa. P. dubium seeds were also set to water stress experiment in rolled paper with PEG solutions from 0.0 to -1.0 MPa.Germination percentage decreased with the increase of PEG concentration. Control group had a better germination percentage than other groups. Germination hardly occurred in PEG -1.4 MPa.     

Growth pattern analysis and stemwood production in an unmanaged old plantation of hiba, Thujopsis dolabrata, in northern Japan

We analyzed the growth patterns of Thujopsis dolabrata var. hondai trees in an old plantation (161 years old), where no silvicultural treatments (e.g., thinning) have been conducted since the initial planting. The analysis focused on understanding individual growth under a long-term self-thinning process, and the stand-level stemwood production at the mature stage was evaluated. Nine canopy-layer trees and one suppressed tree were used for the analysis of annual increments in stem diameters, heights, and stemwood volumes for a given past year using the ring-width data. Both the diameter (at basal portion) and height of all the canopy-layer trees increased at similar rates during the early stage (i.e., 60–70 years after planting); however, after this period, only the height growth rates declined sharply. The annual growth rates of stemwood volume also simultaneously leveled off at the stand age of 40–60 years. Subsequently, the patterns diverged conspicuously, e.g., the growth rates were maintained or increased in some individuals, while it gradually decreased in the case of others until the present year. The divergence of growth pattern was likely to be triggered by intertree competition at several decades after the onset of canopy closure. The current stemwood production of the sample trees, including the suppressed one, was positively correlated with certain size parameters such as stem diameter at breast height and sapwood area at a height of 4 m. Based on the diameter-base allometry, the total stand stemwood production was estimated to be about 12.8 m³ ha⁻¹ year⁻¹. This estimate was higher than those of some old natural T. dolabrata forests (2.0–8.6 m³ ha⁻¹ year⁻¹) that have been well managed by repetitive selection thinning. Furthermore, individual mean stemwood production of the study plantation (0.03 m³ tree⁻¹ year⁻¹) was within the range of these natural stands (0.01–0.05 m³ tree⁻¹ year⁻¹). These comparisons suggested that the old T. dolabrata plantation still maintained a relatively high stemwood production potential despite the absence of artificial controls of tree density in the past. In terms of timber production, this fact implied that a rather long rotation (>100 years) can be applicable in the management of T. dolabrata plantations.     

Somatic embryogenesis and plant regeneration inAcanthopanax sciadophylloides

Somatic embryos ofAcanthopanax sciadophylloides Franch. et Sav. were differentiated from both zygotic and somatic embryos and calli, and plants were regenerated from these somatic embryos. A zygotic embryo, enclosed within a small portion of the endosperm, was incubated on Murashige and Skoog (MS) media supplemented with various combinations (range 0–10.0 mg/l) of 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D). After 4 months, swelling of the zygotic embryos and callus formation was observed. When the swollen embryos were transferred to MS medium supplemented with 0.5 mg/l of 2,4-D, somatic embryos were formed in one to two months. After subculture on the same medium, new embryos were differentiated from various parts of the older somatic embryos. The calli were cultured on medium supplemented with 2.0 mg/l of 2,4-D and BAP for three weeks. Proliferated calli were transferred to medium supplemented with 1.0 mg/l of 2,4-D and BAP. Somatic embryos were differentiated from the calli within one to two months. Somatic embryos were germinated on half-strength MS medium without plant growth regulators and the plantlets were grown in soil. A part of this paper was presented at the 106th Annual Meeting of the Japanese Forestry Society (1995) & First Asia-Pacific Symposium on Forest Tree Genetic Improvement (Beijing).     

Germination,osmotic adjustment,and antioxidant enzyme activities of gibberellin-pretreated <Emphasis Type="Italic">Picea asperata</Emphasis> seeds under water stress

Germination of dragon spruce (Picea asperata Mast.) seeds pretreated with gibberellin (GA) in response to water stress and changes in the levels of osmotic adjustments as well as in activities of antioxidant enzymes were investigated. With decreasing water potential caused by increasing concentrations of PEG 6000, germination percentage and germination index decreased gradually; the decrease was especially prominent under the serious water stress from PEG −0.6 MPa. In contrast, osmotic regulation substances (free proline and soluble sugar contents), lipid peroxidation (MDA), and activities of antioxidant enzyme (ascorbate peroxidase, catalase, and peroxidase) increased markedly with decreased water potential. Similarly, the values in all parameters under −0.6 MPa PEG treatment were markedly higher than the control and −0.2 MPa PEG treatment. These results suggested that P. asperata seed germination was insensitive to water stress. In addition, seeds pretreated with GA had increased tolerance to water stress as measured by germination percentage and germination index, osmotic regulation substance, and antioxidant enzyme activities.     

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.     

Dissolved organic matter originating from the riparian shrub Salix gracilistyla

To estimate the importance of leaching of dissolved organic matter (DOM) as a pathway through which organic matter is supplied to stream ecosystems, we examined the amount of leachate over time and chemical properties of DOM leached from leaves in different conditions. The samples used were green leaves, yellow senescent leaves, and leaf litter of Salix gracilistyla Miq., which is the dominant riparian plant species in the middle reaches of rivers in western Japan. We analyzed dissolved organic carbon (DOC), total sugar, and polyphenol in the leachate of leaf samples collected from a fluvial bar in the middle reaches of the Ohtagawa River in Hiroshima Prefecture, Japan. Considerable leaching of DOC from senescent leaves [37.3 mg g⁻¹ dry weight (dw) leaf] and leaf litter (8.1 mg g⁻¹ dw leaf) occurred within 24 h after immersion. In contrast, DOC leached from green leaves was negligible until 1 week after leaf immersion. Carbon loss of leaves by leaching within 24 h after leaf immersion was estimated to be less than 8%, suggesting that leaching of DOC from S. gracilistyla leaves is a minor pathway through which organic matter is supplied to stream ecosystems. DOM leached from the leaves included sugar and polyphenol, which were among the major chemical forms of DOM leached from the leaves (based on the molecular mass). In a laboratory experiment in which the difference in the stability of DOM between the chemical forms was examined, sugar decomposed more rapidly than polyphenol.     

Modeling to assess growth respiration of stems inPinus densiflora trees

Multiple regression analyses were applied to the respiration data obtained by an excision method to distinguish between maintenance and growth respiration in stems ofPinus densiflora. Among several types of regression models, a few models showed marked stability of coefficient of growth related respiration that are independent of degrees of correlation between predictors and any combinations of predictors. These models predicted growth respiration as 0.45 g CO₂ g (dry weight)⁻¹. At 15°C, sapwood maintenance respiration rate was estimated to 0.72 mg CO₂ g⁻¹ day⁻¹. These estimates were not different from the results obtained with standing trees.     

Thidiazuron-induced somatic embryos,their multiplication,maturation, and conversion in <Emphasis Type="Italic">Cinnamomum pauciflorum</Emphasis> Nees (Lauraceae)

Thidiazuron (TDZ) induced somatic embryogenesis from immature zygotic embryos in Cinnamomum pauciflorum Nees while 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA) or picloram only induced callus and/or adventitious buds. The highest induction frequency for somatic embryogenesis was achieved with MS medium (Murashige and Skoog in Physiol Plant 15:473–497 1962) supplemented with 2.5 μM TDZ using torpedo-shaped embryos (3–5 mm in length) as explants. In addition, induction medium was supplemented with 0.8 g l⁻¹ casein, 0.4 g l⁻¹ glutamine, and 10 g l⁻¹ sucrose. Somatic embryos (SEs) initiated from root tips or hypocotyls without callus formation. SEs were maintained and multiplied via secondary somatic embryogenesis. Embryo maintenance medium was similar to induction medium except that TDZ was reduced to 0.5 μM. Secondary embryogenesis was enhanced by supplementation of 5 g l⁻¹ activated charcoal in the culture. The best medium for embryo maturation was MS medium containing 30 g l⁻¹ sucrose and 5 g l⁻¹ Phytagel without plant growth regulators. A typical mature SE consisted of two large cotyledons and a short embryo proper. Approximately 82% of selected mature SEs were able to germinate and 63% could convert into plantlets on germination medium that was composed of half strength MS medium salts, 10 g l⁻¹ sucrose, 3 g l⁻¹ Phytagel, and 5 g l⁻¹ activated charcoal.     

Biomass and net primary production of a <Emphasis Type="Italic">Pinus densiflora</Emphasis> forest established on a lava flow of Mt. Fuji in central Japan

We assessed the above- and below-ground biomass and net primary production (NPP) in a mature (85-year-old) Pinus densiflora forest established on a lava surface of Mt. Fuji in central Japan. The nitrogen (N) concentration of the forest soil was low (1.25%), and the mean soil carbon/nitrogen (C/N) ratio was 34.2; therefore, both plants and microorganisms would compete for N in our research forest. The total biomass was 192.62Mgha^–1, of which 67.28% was in the stems and 25.71% was in the roots. The fine-root biomass was 1.12% of the total biomass. The total NPP of the forest reached 11.89Mgha^–1 year^–1, which fell within the values reported for other cool temperate P. densiflora forests established on non-volcanic-related substrata. The below-ground production was about 39% of the total NPP; the value was relatively small under the conditions of low total N concentration and high soil C/N ratio. Our study suggested that P. densiflora could recruit and grow on geologically new substrata without increasing the allocation of its annual carbon budget to below-ground organs (i.e., roots).     

Effect of major inorganic nutrients on <Emphasis Type="Italic">β</Emphasis>-thujaplicin production in a suspension culture of <Emphasis Type="Italic">Cupressus lusitanica</Emphasis> cells

 The optimum conditions for β-thujaplicin production in a Cupressus lusitanica cell suspension culture were investigated. The conditions required for β-thujaplicin production were clearly different from the conditions for cell growth. The initial phosphate concentration and pH did not affect β-thujaplicin production. A total nitrogen source concentration higher than 3.2 mM suppressed production due to the presence of the ammonium ion. β-Thujaplicin production was observed at 95 mg/l without adding the ammonium ion to the medium. Strict control of major inorganic nutrients was not necessary to produce β-thujaplicin. This finding seems to be favorable for future automated production of β-thujaplicin in commercial cell culture plants.     

Development of transgenic hybrid sweetgum (<Emphasis Type="Italic">Liquidambar styraciflua</Emphasis> × <Emphasis Type="Italic">L. formosana</Emphasis>) expressing γ-glutamylcysteine synthetase or mercuric reductase for phytoremediation of mercury pollution

Using Agrobacterium-mediated gene transfer, we generated transgenic hybrid sweetgum (Liquidambar styraciflua × L. formosana) overexpressing two types of genes to enhance plant remediation of mercury-contaminated soil and water: bacterial γ-glutamylcysteine synthetase gene (ECS), the first and most important enzyme in phytochelatin synthesis, or various genes encoding a mercuric ion reductase (merA9, merA18, merA77). Hybrid sweetgum proembryogenic masses (PEMs) constitutively overexpressing ECS were able to grow in the presence of 50 μM HgCl₂, which inhibited wild-type PEMs, but plantlets regenerated from the PEMs had abnormal form and did not survive for more than a few weeks following germination. In contrast, mature somatic embryos generated from PEMs constitutively overexpressing merA9 and merA18 converted to normal plantlets on germination medium containing 25 μM HgCl₂, while control embryos were killed on 25 μM Hg(II)-medium. Transgenic merA plantlets displayed enhanced resistance to Hg(II) and released Hg(0) two to three times more efficiently than the wild-type plantlets.