Stereochemistry of matairesinol formation by <Emphasis Type="Italic">Daphne</Emphasis> secoisolariciresinol dehydrogenase

Secoisolariciresinol dehydrogenase activity was detected for the first time from Daphne odora and Daphne genkwa (Thymelaeaceae), which are known to produce optically pure (+)-matairesinol. In sharp contrast, (–)-matairesinol was formed selectively over the (+)-antipode by the secoisolariciresinol dehydrogenase preparation from both D. odora callus and D. genkwa shoots.     

Isolation and enzymatic formation of lignans ofDaphne genkwa andDaphne odora

Four lignans — pinoresinol, lariciresinol, secoisolariciresinol, matairesinol — were isolated from each ofDaphne odora andDaphne genkwa (Thymelaeaceae). Matairesinol isolated from both plants was optically pure (>99% e.e.) and dextrorotatory. Pinoresinol and lariciresinol isolated from the plants were not optically pure, and their enantiomeric compositions ranged from 88% to 95% e.e. in favor of (–)-enantiomers. As for secoisolariciresinol, the one fromD. odora was optically pure [(+)-enantiomer, >99% e.e.], and that fromD. genkwa was 97% e.e. in favor of the (+)-enantiomer. Lignan-synthesizing enzyme activity was detected from a Thymelaeaceae plant for the first time; cell-free extracts fromD. genkwa catalyzed the formation of (–)-lariciresinol (23% e.e.) from racemic (±)-pinoresinols. The stereochemistry of the enzymatic reaction is discussed in relation to the stereochemical features of the isolated lignans.Parts of this report were presented at the 42nd Lignin Symposium, Sapporo, October 1997; 48th Annual Meeting of the Japan Wood Research Society, Shizuoka, April 1998; and 49th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999     

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.     

Establishment of cell suspension line of <Emphasis Type="Italic">Populus tomentosa</Emphasis> Carr.

Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L^-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subcultured into a MS liquid medium supplemented with 1.5 mg·L^-1 2,4-D. The best subculture medium was MS ＋ 0.8 mg＇L-1 2,4-D ＋ 30 g·L^-1 sucrose with a subculture cycle of seven days.     

Biological activity of extracts fromCupressus lusitanica cell culture

Ethyl acetate extracts ofCupressus lusitanica suspension cell cultures were examined for their biological activities (viz., tyrosinase inhibitory, antioxidant, and antimicrobial activities). The extract from elicitor-treated cells showed all of the biological activities, whereas the extract from cultures without elicitor was not bioactive to a discernible extent. The biological activities shown by the cell culture extracts were almost solely accounted for by-thuj aplicin contained in the extracts. These results suggest that the ethyl acetate extract ofC. lusitanica cells treated with the elicitor is a valuable bioactive source without isolation or purification of-thujaplicin.     

Peat carrier increases inoculation success with Frankia on red alder (Alnus rubra Bong.) in fumigated nursery beds

Inoculation trials were set up in fumigated nursery beds for red alder (Alnus rubra Bong.) bare-root seedling production. Frankia inoculum was applied either as nodule homogenate or as pure culture (strain ArI5). The plots were laid out in 4 blocks of 8 treatments consisting of: control, nodule suspension, and three levels each of cell suspension and cells applied with a peat carrier. Numbers, height, and percentage nodulation on the seedlings were determined at mid-season. Numbers, size, dry weights, and degree of nodulation were determined at lifting. The peat inoculum treatment produced larger seedlings than the other treatments, both at mid-season and at lifting. The other treatments had little effect on growth. Size differences paralleled differences in degree of nodulation. Differences in percentage of seedlings nodulated were most pronounced at midseason, indicating that inoculation confered primarily an early-season advantage.     

Assessment of rhizospheric microorganisms of transgenic Populus tomentosa with cowpea trypsin inhibitor ( CpTI ) gene

To have a preliminary insight into biosafety of genetically transformed hybrid triploid poplars (Populus tomentosa × P. bolleana) × P. tomentosa with the cowpea trypsin inhibitor (CpTI) gene, two layers of rhizospheric soil (from 0 to 20 cm deep and from 20 to 40 cm deep, respectively) were collected for microorganism culture, counting assay and PCR analysis to assess the potential impact of transgenic poplars on non-target microorganism population and transgene dispersal. When the same soil layer of suspension stock solution was diluted at both 1:1 000 and 1:10 000 rates, there were no significant differences in bacterium colony numbers between the inoculation plates of both transgenic and non-transgenic poplars. The uniform results were revealed for both soil layer suspension solutions of identical poplars at both dilution rates except for non-transgenic poplars at 1:10000 dilution rates from the same type of soil. No significant variation in morphology of both Gram-positive and Gram-negative bacteria was observed under the microscope. The potential transgene dispersal from root exudates or fallen leaves to non-target microbes was repudiated by PCR analysis, in which no CpTI gene specific DNA band was amplified for 15 sites of transgenic rhizospheric soil samples. It can be concluded that transgenic poplar with the CpTI gene has no severe impact on rhizospheric microorganisms and is tentatively safe to surrounding soil micro-ecosystem. [Supported by the National Project in Transgenic Plant and Application (Grant No. J2002-2003)]     

Liquid culture and transformation of hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.)

Hinoki cypress (Chamaecyparis obtusa) is one of the most important timber resource forest trees in Japan. Because seed production from a seed orchard of hinoki cypress is not constant every year, micropropagation from a limited amount of material is useful. Up to now, the conventional tissue culture method using solid medium has been used. Here a new method using liquid culture in tubes rotated vertically is described. Shoot primordium of hinoki cypress was inoculated in Campbell and Durzan’s (CD) liquid medium containing different cytokinins (6-benzylaminopurine (BAP), Zeatin, thidiazurone (TDZ)), and the container tubes were rotated vertically around the axis at 2 times / min. Culture room temperature was 25°C and light condition was 16 h photoperiod per day of fluorescent lamps. Zeatin at 1μM concentration was the best for maintaining the shoot primordium production and TDZ induced callus on the surface of the shoot primordia. After shoot primordium multiplication in the liquid culture, they were transplanted to agar medium for shoot elongation. A high concentration of agar (up to 16 g/L) or AVF (anti vitrification factor from Dr. Nairn, 1995) was effective to prevent vitrification of the shoots. Transformation of shoot primordium was done using particle bombardment with vectors containingβ-glucuronidase (GUS) gene or herbicide resistance gene (bar). Positive result for transient transformation was observed with the histo-chemical study for transformation with GUS. Integration of a useful herbicidebar gene into the shoot primordium culture system was also tried and stably transformed plants were obtained. This is the first report of stable transformation of Japanese conifer using practically useful gene. The generous supply of AVF-B from Dr. B.J. Nairn, Tasman Forestry, NZ is also appreciated.     

南方红豆杉细胞组织培养技术

介绍南方红豆杉细胞组织培养方法,包括外植体选择、诱导培养、继代培养和生根培养等技术环节。悬浮细胞培养可有效提高南方红豆杉紫杉醇产量。通过添加激素、前体物质和诱导子能很好的解决愈伤组织褐化问题并且是提高紫杉醇产量的关键。     

野生唐古特白刺悬浮细胞系的建立及生长特性

以野生唐古特白刺的茎段、叶片为外植体,研究了NaClO对外植体的消毒效果和激素2,4-D、NAA和6-BA对愈伤组织诱导及愈伤组织形态的影响,并利用疏松型愈伤组织建立了白刺悬浮细胞系,对其生长特征进行了分析。结果表明:对茎段和叶片用2%的NaClO消毒10 min最适宜,时间过长或过短均会影响成活率;3种激素对愈伤组织的相对生长量和形态均有影响,其中,2,4-D是主要影响因子,且茎段诱导出的愈伤组织相对生长量比叶片的高;根据愈伤组织的生长速度和形态,适宜用浅黄色疏松型愈伤组织构建唐古特白刺悬浮细胞系,最佳试验组合为MS+2,4-D 1.5 mg·L^-1+NAA 0.2 mg·L^-1+6-BA 0.4 mg·L^-1,最佳继代接种量为7.5 mL母液,生长曲线呈"S"型,培养第7天细胞分裂指数达最大值(5.1%),培养第3天细胞活力最强(吸光值为0.69),存活率在细胞生长延迟期和稳定期较对数期下降略快,但均保持在84% 93%间。     

In vitro culture of immature embryos from Koelreuteria bipinnata var. integrifoliola

For the mass production of Koelreuteria bipinnata var. integrifoliola with selected, hybrid or genetically engineered genotypes, one potentially desirable propagation strategy is based on embryo culture. The immature embryo development in vitro from K. bipinnata var. integrifoliola was studied under different conditions of embryo age, basic culture media and plant growth regulators. The results show that: 1) germination rate of grade 3 embryos in immature seeds with 0.6–0.8 cm diameter was 98.9%. The germination rate of grade 2 embryos in immature seeds with 0.4–0.6 cm diameter was 77.8% and the germination rate of grade 1 embryos in immature seeds with 0.4 cm diameter was 15.6%. 2) The amounts of macroelements in MS medium had no clear effect on the germination rate of immature grade 3 embryos and had a modest effect on plantlet growth, where the best medium was MS or 1/2 MS. The rates were all greater than 90%. 3) The germination rate of grade 3 embryos was greater than 87% when the medium contained a low concentration of NAA or no plant growth regulators at all and decreased markedly when BAP alone or BAP and NAA together were added to the media. We suggest that in vitro culture of immature embryos from K. bipinnata var. integrifoliola can be enhanced when a small amount of plant growth regulators is added. The addition of BAP has an adverse reaction to the germination and development of immature embryos.     

伊氏杀线真菌与苏云金芽孢杆菌对松材线虫的联合毒力研究

[目的]探究伊氏杀线真菌与苏云金芽孢杆菌(Bacillus thuringiensis,Bt)对松材线虫的联合毒力。[方法]使用Esteya vermicola孢子悬浮液与Bt发酵液处理松材线虫,通过线虫的形态变化、死亡率及死亡速度3个方面测定E.vermicola与Bt联合对松材线虫的影响。[结果]经过E.vermicola孢子悬浮液处理过的线虫会出现内容物渗漏,体腔收缩、弯曲、断裂的现象;高浓度的E.vermicola孢子悬浮液与Bt发酵液联合处理松材线虫可以明显地提高线虫的死亡率,最高可以达到100%;联合处理的线虫死亡速度较快,随着处理时间的延长,死亡率会一直呈上升的趋势。[结论]应用这两种生防微生物在合适的浓度下混配能够在较短时间内提高松材线虫的死亡率。     

黄竹细胞悬浮培养和原生质体分离*

以牡竹属黄竹（ＤｅｎｄｒｏｃａｌａｍｕｓｍｅｍｂｒａｎｃｅｕｓＭｕｎｒｏ）的竹节及无菌苗根颈为外植体，培养于含有５ｍｇ／Ｌ２，４－Ｄ、０．２ｍｇ／ＬＫＴ和２００ｍｇ／ＬＬＨ的ＭＳ培养基上诱导愈伤组织，然后移至相同成份或不同浓度２，４－Ｄ的液体培养基中，进行悬浮培养，以建立分散性良好的悬浮细胞系。利用悬浮细胞和无菌苗叶片经酶解获大量原生质体，其产量分别约为每克鲜重２．５×１０５个和５×１０５个，活力均可达８０％。     

Constituents from the roots of <Emphasis Type="Italic">Taxus cuspidata</Emphasis>

The known propelargonidin, afzelechin-(48)-afzelechin (1), the known lignans 7-hydroxynortrachelogenin (2), epinortrachelogenin (3), nortrachelogenin (4), hydroxymatairesinol (5), allohydroxymatairesinol (6), matairesinol (7), oxomatairesinol (8), and isotaxiresinol (9), and the known taxoids taxinine M (10), taxayuntin (11), and 10-deacetyltaxol (12), and 10-deacetylbaccatin III (13) were isolated from the roots of Taxus cuspidata (Japanese yew, Taxaceae). The propelargonidin was isolated from Taxus spp. for the first time, and was detected in the roots, bark, and twigs.     

Reutilization of culture wastes ofPleurotus ostreatus andPhollota nameko for cultivation ofLyophyllum decastes

Possible reutilization of fresh and aged culture wastes of mushrooms for cultivatingLyophyllum decastes was investigated, although bark compost has commonly been used as a substrate for cultivating this fungus. The culture wastes studied were obtained after harvestingPleurotus ostreatus andPholiota nameko mushrooms. Mycelia ofL. decastes grew in the media containing both the fresh culture waste ofP. nameko and bark compost. However, it did not grow in the medium containing only the fresh culture waste ofP. nameko or in any media containing the fresh culture waste ofP. ostreatus. The mycelial growth inhibition in the fresh culture wastes ofP. ostreatus might be caused by the water-soluble inhibitors present. Mycelia ofL. decastes grew in all the media with aged culture wastes of bothP. ostreatus andP. nameko, which had been left outdoors for 6 months, regardless of whether bark compost was mixed. Fruit bodies were produced on all the tested media with aged culture wastes of both mushrooms, which had been left outdoors for a year. The aged culture waste ofP. nameko gave greater yields than the bark compost. This investigation shows that the aged culture wastes ofP. ostreatus andP. nameko could be reutilized for producingL. decastes mushrooms.This study was presented in part at the 47th annual meeting of Japan Wood Research Society, Kochi, April 1997     

Development, manufacture and application of Beta-Cypermethrin aqueous capsule suspension

The optimal formula of Beta-Cypermethrin aqueous capsule suspension was screened out through the experiment,preparation and manufacture of basic formulas, which has no sedimentation, suspended drops of oil, peels and agglomerating and the diameter of capsule is in range of 10-30μm, conformed to the technical requirement. The aftereffect of the 3.3% Beta-Cypermethrin aqueous capsule suspension which was manufactured according to the optimal formula was up more than 25 days. The experiments on controlling the larvae of Dendrolinmus superans Butler and the adults of Xylotrechus rusticus L were carried out with different concentrations of this chemical. The death rate reached 80% when 250 times solution of the chemical was sprayed on stem to control the larvae of D. superans. For control of the adults of X. rusticus, 200, 400 and 600 times solution of the chemical were applied and their control effects (death rate) reached 85.23%, 74.21% and 66.59% respectively. Two kinds of solution (200 times and 300 times) of the chemical were used to control the larvae of D. superans in large area, and the control effect for both concentrations was over 90%.     

Bio-control trials of Chaetomium spirale ND35 against apple canker

A new endophytic antagonistic fungus, Chaetomium spirale ND35 from Populus tomentosa, was reported. The bio-control trials of C. spirale ND35 against the Valsa Canker of apple were preliminarily investigated. The results of dual culture on PDA plate showed that C. spirale ND35 was capable of strong antagonism against Valsa ceratosperma, and for inhibiting the mycelial growth of V. ceratosperma,.the crude extract of liquid culture of corn steep powder broth was more effective than that one of malt extract broth (MEB). The results of bio-control in greenhouse and field indicated that the disease incidence of apple tree treated with C. spirale ND35 was lower significantly than that treated by other methods. The re-isolation experiment suggested that C. spirale ND35 could colonize in stems and branches of apple trees successfully, and the ND35 colonization rate of the treatment with solid wheat bran culture was higher than that of corn steep powder broth, but the field experiment result the control effect of liquid culture of C. spirale ND35 was better than that of solid culture.     

Large electro-fused protoplasts ofPopulus alba selected by a micromanipulator: Techniques and some characteristics of cells and their regenerants

An improved method for selection of large electro-fused protoplasts ofPopulus alba by a micromanipulator was developed. The conditions for electric cell fusion treatment were optimized. For the best result, protoplasts with a cell density of 5 × 10⁵/mL were treated with an alternate current (1 MHz, 200 V/cm) and pulsed with a direct current (2 kV/cm) for 100μs in 2.5 mM CaCl₂ and 0.55 M mannitol. The electo-fused protoplasts were cultured in NH₄NO₃-free Murashige and Skoog’s medium containing 0.6 M of mannitol, 0.09 M sucrose, 1μM of 2,4-dichlorophenoxyacetic acid and 0.1μM of benzyladenine, the same medium used for protoplast culture, but at a very low cell density of 5–10 × 10²/mL in a well of a 96-well culture plate. When cell aggregates derived from individual fused protoplasts were transferred to fresh medium with 0, 0.3 or 0.6 M mannitol, large colonies developed. In the shoot differentiation medium, the reaction of the calluses derived from large fused protoplasts towards the growth regulators differed from the non-fused ones. In medium containing 1μM each of naphthalene acetic acid andN-(2-chloro-4-pyridyl)-N′-phenylurea, growth of callus from electro-fused ones was not reduced by much compared to the control, but shoot differentiation was inhibited. Gibberellic acid (0.1–10μM) was beneficial to shoot regeneration; however, irregularly shaped leaves appeared at high gibberellic acid concentrations. Shoots regenerated were rooted in Murashige and Skoog’s medium containing 4μM of indole-3-butyric acid. Some plantlets obtained had a varied morphology. Based on the characteristics of growth, some cells derived from electro-fused protoplasts appear to be physiologically different from the non-fused one.     

Effect of elicitors,precursors and metabolic inhibitors on paclitaxel production by <Emphasis Type="Italic">Taxus cuspidata</Emphasis> cell culture

In this study, using Taxus cuspidata as a raw material, we obtained stable high-yielding cell lines by subculturing and quantified paclitaxel content using ultrasonic extraction combined with TLC–UV spectrophotometry. In single factor and multiple factors tests to optimize design and study the effects of elicitors, precursors, and metabolic inhibitors on paclitaxel production by Taxus cuspidata cells, paclitaxel production reached 4.32 mg/L when 100 μmol/L methyl jasmonate, 20 mg/L salicylic acid, 400 mg/L phenylalanine and 2 mg/L gibberellin (GA₃) were added to the culture medium of suspension cells. When adding metabolic adjustment factors on the 7th day of culture, extra- and intracellular paclitaxel production was the highest at 4.855 mg/L, paclitaxel release rate was 10.48 %, fresh mass and paclitaxel production of cell increased, respectively, by 6.08 and 11.57 %. By controlling the anabolism of paclitaxel, paclitaxel yield was significantly improved.