Cytological and genetical studies on male sterility inCryptomeria japonica D. Don

Genetic male sterility is a useful trait in plant breeding, especially in angiosperm crops such as corn, onion and carrot. We found a male sterile sugi (Cryptomeria japonica D. Don) tree in Toyama, Japan. Pollen of sugi is one of the major causes of pollinosis in Japan. We carried out this research in an attempt to make clear the characteristics and inheritance of this male sterility. Microsporogenesis of the male sterile tree proceeded meiosis, however, the microspores collapsed after they were separated from pollen tetrads in locules, resulting in complete male sterility. Most likely, ethylene evolution was responsible for male sterility expression. Full seed setting in the male sterile tree indicated normal macrosporogenesis. Seeds obtained from crossing between male sterile and normal lines showed relatively high level of germination and their seedlings grew vigorously. The somatic chromosome numbers of 241 germinated seeds, derived from the male sterile tree, were mostly 22, euploid. These results indicated that male sterile tree was different from other similar previously reported trees with low pollen fertility, resulting from triploid or trisomics. Probably, male sterility in sugi is either nuclear genetic male sterility or cytoplasmic male sterility. The study was partially supported by Program for Promotion of Basic Research Actives for Innovative Biosciences.     

Individual variations in monoterpenes released from Cryptomeria japonica and Pinus thunbergii needles

Variations in the compositions of low boiling point (LBP) monoterpenes in needle samples from 99 sugi (Cryptomeria japonica) and 100 kuromatsu (Pinus thunbergii) trees were investigated using a headspace technique. Considerable variations in the proportions of monoterpenes were revealed in both species. In sugi, the proportions of sabinene and α-pinene in the total LBP monoterpenes, ranging from 8.8% to 73.3% and from 14.5% to 73.3%, respectively, showed enormous variations among nine monoterpenes. The proportions of 3-carene and limonene, ranging from 0.1% to 29.5% and from 0.2% to 20.4%, respectively, also showed very specific variations. In kuromatsu, the proportions of β-pinene and α-pinene in the total LBP monoterpenes, ranging from 26.5% to 66.3% and from 18.7% to 46.9%, respectively, showed considerable variations among ten monoterpenes. The proportions of myrcene and 1,8-cineole, ranging from 0.9% to 18.5% and from 0.8% to 12.3%, respectively, also showed specific variations. Part of this article was presented at the 50th Annual Meeting of the Japan Wood Research Society, Kyoto, April, 2000, and the 13th Annual Meeting of the Chugoku Shikoku Branch of the Japan Wood Research Society, Okayama, September 2001     

Influences of genetic and environmental factors on the concentration of the allergen Cry j 1 in sugi (Cryptomeria japonica) pollen

Pollen from sugi (Japanese cedar, Cryptomeria japonica D. Don), a forest tree species that is widely grown in Japan, causes serious allergic disease. The major allergens from sugi pollen, Cry j 1 and Cry j 2, have been isolated and characterized. It has been reported that Cry j 1 concentration in pollen varies considerably among trees. If Cry j 1 concentration is genetically controlled, the planting of trees with low Cry j 1 concentrations would reduce pollinosis. We investigated genetic and environmental effects on Cry j 1 concentration in eight clones growing at four sites. Concentrations of Cry j 1 in pollen were measured with a monoclonal antibody-based Enzyme Linked Immunosorbent Assay (ELISA). The Cry j 1 concentrations differed significantly among clones and sites, but the site x clone interaction was not significant, suggesting that the Cry j 1 concentration is controlled primarily by genetic factors. We examined correlations between Cry j 1 concentration and temperature and precipitation from July through February. Temperature was not significantly related to Cry j 1 concentration, whereas cumulative precipitation during the 8 months and mean daily precipitation in September showed significant negative correlations with Cry j 1 concentration.     

Spatial and seasonal variations in photosynthetic properties within a beech (Fagus crenata Blume) crown

The light response curve and the intercellular CO₂ concentration response curve of CO₂ assimilation rate were investigated together with the light conditions at the four different heights within the beech crown from 1995 to 1997 on Mt. Fuji in Japan. On the seasonal fluctuation, the CO₂ assimilation rate at light saturated condition increased rapidly in May, and attained to the maximum between the end of June and July, thereafter, slightly decreased until the middle of August and rapidly decreased in September and October. The daily sum of photosynthetic photon flux density attenuated with deeping within the crown, and particularly, the relative value on 2nd position dropped to only 30%. TheA _max decreased from 10 to 5μmol m⁻² s⁻¹, approximately, with deeping within the crown. The light saturation point, quantum yield, light compensation point and dark respiration rate also varied with deeping. These results suggest that the photosynthetic properties vary gradually from sun to shade leaves along the light attenuation within a beech crown. At light saturated condition, the stomatal conductance and mesophyll conductance were strongly correlated withA _max among the four different heights (r > 0.96, respectively). TheC _i/C _a ratio was around 0.8, and there were no remarkable differences among the four different heights. These results suggest that the vertical gradient ofA _max depends on the variation of mesophyll conductance. The stomatal conductance may be also one of the major factor in the vertical gradient ofA _max. However the intercellular CO₂ concentration doesn’t influence the vertical gradient ofA _max within the crown. This work is supported by the Sasagawa Scientific Research Grant from The Japan Science Society and Grant-in-Aid for Scientific Research (C).     

Cultivation of <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> on umbrella plant (<Emphasis Type="Italic">Cyperus alternifolius</Emphasis>) substrate

Mycelial growth and mushroom yields of three strains of Pleurotus eryngii produced on wheat bran-supplemented umbrella plant (Cyperus alternifolius) substrate were assessed using surface brightness, bromophenol blue color reactions, ergosterol and glucosamine contents, and water potential as indicators of strain performance. Mycelial growth was 31%–46% greater, depending on strain, on the umbrella plant substrate compared with the mushroom industry standard sugi (Cryptomeria japonica) substrate. Mushroom yields on the first flush were 20%–23% higher, depending on strain, on the plastic bottle-contained umbrella plant substrate. However, yields on the second break were lower from the umbrella plant substrate. Because many growers in Japan only harvest one flush, production of P. eryngii on umbrella plant substrate may offer commercial producers an alternative basal ingredient to diminishing supplies of sugi sawdust.Part of this report was presented at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, April 2002.     

Molecular cloning of a cellulose synthase gene PtoCesA1 from Populus tomentosa and its genetic transformation in tobacco

A 3 125 bp cellulose synthase gene, PtoCesA1, which has a 98% identity to PtrCesA1 from Populus tremuloides, was cloned from cDNA prepared from secondary xylem of P. tomentosa. Four anti-expression vectors with different fragments of PtoCesA1, named as pBIPF, pBICC1, pBIPR and pBIBR, were constructed. Some traits of transformed tobacco of pBICC1, pBIPR and pBIBR differed from wild types, such as small leaves, “dwarf” phenotype and thinner xylem and fiber cell walls than wild plants consistent with a loss of cellulose. It indicated that the growth of transgenic tobacco was restrained by the expression of anti-PtoCesA1. Transgenic tobacco was obtained and the contents of cellulose and lignin were analyzed as well as the width and length of fiber cells, and xylem thickness for both transgenic and control plants. Transformed tobacco showed a different phenotype from control plants and it implied that PtoCesA1 was essential for the cellulose biosynthesis in poplar stems. [Supported by the Hi-Tech Research and Development Program of China (863) (2001AA244060 and 2003AA244020) and National Basic Research Program of China (973) (J1999016003)]     

Potential fecundity of the bamboo gall maker,Aiolomorphus rhopaloides (Hymenoptera: Eurytomidae), and its inquiline,Diomorus aiolomorphi (Hymenoptera: Torymidae), in relation to gall size and body size

I examined the adult potential fecundity of the bamboo gall maker,Aiolomorphus rhopaloides Walker (Hymenoptera: Eurytomidae), and its inquiline,Diomorus aiolomorphi Kamijo (Hymenoptera: Torymidae) by dissection just after emergence, and at death after rearing. Emerging females ofA. rhopaloides hadca. 80 mature eggs in their ovaries. The number of eggs at emergence and after death at rearing was not significantly different. However, emerging females ofD. aiolomorphi had no eggs in their ovaries but hadca 20 eggs at death after rearing. These results suggest thatA. rhopaloides is pro-ovigenic andD. aiolomorphi is synovigenic. Female body sizes of both species were positively correlated with the size of galls from which each emerged. Number of eggs in each species was also correlated with female body size, suggesting that gall size might be a good predictor of potential fecundity. This study was supported in part by a Grant-in-Aid for scientific Research (No. 11460068) from the Ministry of Education, Science, Sports, and Culture, Japan.     

Polymorphism analysis of Fagaceae and DNA-based identification ofFagus species grown in Japan based on therbcL gene

Fagaceae species in Japan were identified by restriction fragment length polymorphism (RFLP) and sequence comparison of a region ofrbcL. Of nine restriction endonucleases used for digestion, three (MspI,RsaI,HaeIII) produced different restriction patterns in Fagaceae. Digestion byMspI yielded four patterns: Fagus species,Castanea crenata, Pasania glabra, and others. Digestion byRsaI andHaeIII afforded two patterns:Fagus species and others. These facts indicate thatCastanea crenata andPasania glabra can be identified byMspI restriction patterns ofrbcL. Sequence comparison of a region of therbcL gene among 20 species of Fagaceae showed that: (1) they could be divided into seven groups; (2) there is a site mutation betweenFagus crenata andF. japonica. The latter indicates that the wood of both Fagus species are identifiable at the species level, which is not the case using conventional methods. This result indicates the possibility of wood identification based on DNA polymorphism in Fagaceae at the intrageneric level.Part of this paper was presented at the 46th annual meeting of the Japan Wood Research Society, Kumamoto, April 3–5, 1996 and the 47th annual meeting of the Japan Wood Research Society, Kochi, April 3–5, 1997     

Bark thickness of host pine trees and female body size affecting ovipositional success ofSirex nitobei matsumura (hymenoptera: Siricidae)

We examined the effect of bark thickness of host trees on the ovipositional success ofSirex nitobei in relation to female body size by analyzing the relationships between female body-weight and ovipositor length and between bark thickness ofP. densiflora andP. thunbergii trees and oviposition proportion (the proportion of eggs laid to the potential female lifetime fecundity) of the woodwasp. The ovipositor length ranged from 6 to 14 mm and was correlated positively with the fresh body-weight of females. The bark thickness of host trees had no significant correlation with the oviposition proportion of large females of more than 100 mg f.wt, whereas it was correlated negatively with that of small females of less than 100 mg f.wt. Thus,P. densiflora, which has thinner bark as a whole thanP. thunbergii, may be more suitable for oviposition, especially for smaller females. Research Fellow of the Japan Society for the Promotion of Science. This study was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, and Culture (No. 80003292).     

Isolation and sequence analysis of the promoter and an allelic sequence of the iron-sulfur protein subunit gene from the white-rot fungusPleurotus ostreatus

We have isolated a structural gene ofsdil, which encodes the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (EC 1.3.99.1), from a white-rot basidiomycete,Pleurotus ostreatus. Here we report isolation of the promoter region ofsdil and an allelic sequence encoding the second-type cDNA fragment isolated in the former experiments. The nucleotide sequence analysis of the promoter region revealed the existence of putative CAAT and TATA boxes, which permits us to develop an expression system in this species. The Southern blot analysis and the restriction fragment length polymorphism assay using monokaryotic strains demonstrated that no family genes tosdil exist in the haploid genome ofP. ostreatus. Moreover, a genetic analysis to detect a linkage between thesdil genotypes and flutolanil resistance in the mutantP. ostreatus strains was also developed.     

Inheritance and cross-resistance of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> insecticidal crystal protein Cry1Ac resistance in cotton bollworm <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> Hubner population from Tamil Nadu,India

Cotton bollworm, Helicoverpa armigera Hubner selected for five generations with Bacillus thuringiensis insecticidal crystal protein Cry1Ac in the laboratory developed 12.98-fold resistance. Resistance and susceptible populations were mass crossed to study the dominance of resistance. The Cry1Ac—selected (BCR) population showed 5.8-fold resistance to Cry1Aa and 5.04-fold resistance to Cry1Ab. The degree of dominance (D) was 0.34 and 0.40 for the R × S and S × R hybrids, respectively, which indicates incomplete recessive character of Cry1Ac resistance in the population. The estimated realized heritability (h ²) and response quotient (Q) of resistance for Cry1Ac were 0.52 and 0.15, respectively. This indicated the lower phenotypic variation in the selected population. The resistance risk assessment based on h ² indicated that the resistance would increased tenfold after <9 generations for Cry1Ac in the resistant population. The results show the ability of H. armigera to develop resistance against Cry1Ac and cross-resistance to Cry1Aa and Cry1Ab.     

Antisense expression of a caffeic acidO-methyltransferase ofStylosanthes humilis in transgenic poplar: Effect of expression onO-methyltransferase activity and lignin composition

Poplar (Populus tremula) was transformed with a construct carrying an antisense caffeic acidO-methyltransferase (COMT) cDNA (pOMT8) from a tropical pasture legume,Stylosanthes humilis. pOMT8 shows 83% overall homology to the corresponding COMT gene (pPCLA) of poplar. Of the 200 putatively-transformed plants regenerated on selective media after co-cultivation of poplar stem explants withAgrobacterium tumefaciens harbouring a CaMV 35S-antisensepOMT8 construct, a subset of 20 plants were randomly chosen for further analysis. PCR and Southern blot analysis demonstrated the stable integration of T-DNA into the genome of these plants. Antisense expression ofpOMT8 resulted in reductions in total COMT activity in the majority of the transgenic plants with the lowest total COMT activities (61–70% of untransformed control plants) being observed in four transgenic plants. The composition of lignin in transgenic plants was also changed, as detected by reductions in the content of syringyl units using infrared spectroscopy. However, no changes were found in the amount of insoluble lignin in transgenic plants as compared to untransformed control plants. These results indicate the potential of thepOMT8 gene to partially suppress COMT activity and modify the composition of lignin in transgenic poplar. This work was partly supported by General Management of Turkish Pulp and Paper Mills.     

Growth response ofMelaleuca cajuputi to flooding in a tropical peat swamp

We investigated the growth response ofMelaleuca cajuputi Powell to flooding at 3 sites in a tropical swamp in southern Thailand. The relative growth rate (RGR) in height tended to be higher where the water level was higher. The maximumRGR in height was achieved during the flood period at the 2 sites where water levels were higher than at the other site. The height growth ofM. cajuputi was not reduced by flooding, but enhanced. No decline in growth due to post-anoxic injury was observed after flooding.Melaleuca cajuputi may have mechanisms to tolerate rhizospheric oxygen deficiency and to avoid post-anoxic injury. This study was supported by a Grant-in-Aid for Basic Research (No. 09NP0901) from the Ministry of Education, Science, Sports, and Culture of Japan.     

Boring activity on coniferous tree branches byXylotrechus villioni (Villard) larvae (Coleoptera: Cerambycidae)

In anAbies homolepis Sieb. et Zucc. forest at Matsumoto, Nagano Pref., cumulative damage byXylotrechus villioni (Villard) (Coleoptera: Cerambycidae) was observed on tree branches. Of the 14 major branches in one sample tree, six had cumulative damage by boring larvae, of which 3 had been bored by multiple larvae. No adult emergence holes were found on the whole tree, suggesting very low survivorship from boring larva to emerging adult. Galleries made by single larvae were often observed to be terminated by a depression apparently carved by a picid woodpecker, suggesting that they are important predators of the cerambycid. The manner of cicatrix formation with phloem tissue over the larval gallery in anAbies veitchii Lindl. branch at Mt. Fuji, Yamanashi Pref. is presented. Studies onXylotrechus villioni (Villard) (Coleoptera: Cerambycidae), a primary borer of coniferous trees in Japan (III). This work was presented at the 104th Annual Meeting of Japanese Forestry Society at Morioka (April, 1993) and 20th International Congress of Entomology at Firenze (August, 1996). This work is supported by a Grant-in-Aid for Scientific Research (No.02660163) from the Ministry of Education, Science, Sports, and Culture of Japan.     

Rapid degradation of carbaryl by two novel strains ofArthrobacter spp. isolated from forest soil

Carbaryl is one of the carbamate insecticides widely used in agricultural fields and forests. To restore the environment contaminated with carbaryl application, it is suggested that microbial degradation of carbaryl could be an effective method. Two novel bacterial strains degrading rapidly carbaryl were isolated from forest soil. These strains designated as 19B and 19C were identified as novel species ofArthrobacter, respectively based on main bacterial properties including GC-content, major fatty acid composition, major menaquinone isoprenologue and 16S rRNA gene sequence analysis. Strain 19B started to degrade carbaryl within several hours and completely degraded 50μg/ml carbaryl in a mineral salt medium within a day. Similarly, strain 19C showed a high degradation ability. Gas chromatography-mass spectrometry analysis showed that the major degradation metabolite is 1-naphthol (M=144) and minor metabolites are 1,4-naphthalenedione (M=158) and 1,4-naphthalenediol (M=160), suggesting a pathway of carbaryl degradation by both strains. This research was supported by a Grant-in-Aid from the Ministry of Agriculture, Forestry and Fisheries, Japan.     

Chemical changes in terpenes of sugi (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis>) wood during steam drying in kiln at high temperature

Sawdusts of sugi (Cryptomeria japonica) wood prepared before and after steam drying at 120°C in a kiln were extracted with n-hexane and ethyl acetate to give n-hexane extracts and ethyl acetate extracts. From gas chromatography-mass spectrometry analysis of the ethyl acetate extracts from woods before and after steam drying, the components of 4-epi-cubebol, cubebol, and 2,7(14),10-bis-abolatrien-1-ol-4-one, which existed in the raw sugi wood, were proved to disappear in the steam-dried wood. These components were also absent in the ethyl acetate extract of the steam-condensed solution of waste steam from the kiln outlet. When these three components were treated with 0.2% (v/v) acetic acid solution at 120°C, δ-cadinene was produced as a major product from both 4-epi-cubebol and cubebol by dehydration and cleavage of the cyclopropane ring, and cryptomerone from 2,7(14),10-bisabolatrien-1-ol-4-one by hydration. The chemical changes of the three components presumably occur during steam drying of the sugi wood. This study was presented in part at the 85th Spring Meeting of the Chemical Society of Japan, Kanagawa, Japan, March 26–29, 2005     

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.     

Effect of water potential on fruit body formation ofLentinula edodes in sawdust-based substrate

The influence of substrate water potential () on the growth and fruiting of three genotypes of shiitake (Lentinula edodes) was investigated. A slight reduction of (–0.5MPa) stimulated mycelial and colony growth on liquid, agar, and sawdust-based substrates.L. edodes has been found to grow well at a around –0.5 MPa, which corresponds to a moisture content around 55%. A small decrease in at the final vegetative growth phase had positive effects on flush quantity. The substrate was significantly affected by the interaction between genotypes and spawn run time. The of well-colonized mature substrate was –0.7MPa before and –4.OMPa after the fruiting. The rose again to –0.7MPa during rapid absorbance of water by soaking, and this rise was repeated during the second and third flushes. It is suggested that the water-holding capacity of a substrate is related to culture maturity. Excellent water-providing capacity (higher) is expected in the substrate of well-matured cultures with a high density of mycelial colonization.Part of this paper was presented at the 46th annual meeting of the Japan Wood Research Society, Kumamoto, April 1996     

Induction of the biosynthesis of agatharesinol, a norlignan, in sapwood sticks of Cryptomeria japonica under humidity-regulated circumstances

To establish an experimental approach for studying the biosynthetic pathway of agatharesinol, a norlignan, induction of the formation of agatharesinol in Cryptomeria japonica (Japanese cedar, sugi) was attempted. Wood sticks were prepared from the sapwood immediately after cutting a sugi tree down, and they were allowed to stand in desiccators in which the humidity was adjusted to 76% and 88%, and in a room. When the wood sticks were allowed to stand in 76% humidity and in a room, they dried rapidly and no formation of agatharesinol was demonstrated by gas chromatography-mass spectrometry. These results suggest that in the sapwood dried rapidly, the cells that were biologically active in the sapwood, probably ray parenchyma cells, die rapidly and completely before the biosyntheses of secondary metabolites including agatharesinol was wholly established. The wood sticks allowed to stand in 88% humidity, on the other hand, dried gradually compared with the sticks placed in 76% humidity and in a room, and agatharesinol was formed as one of the predominant ethyl acetate extractives. These results suggest that delaying the drying of the sapwood sticks can control the dying period to an appropriate length for the ray parenchyma cells, during which the biosynthesis of agatharesinol is induced. Part of this study was presented at the 54th Annual Meeting of the Japan Wood Research Society, Sapporo, August 2004