Cytology of Callus of Cryptomeria japonica

Callusing has been initiated from the cotyledons of 4-week-old seedlings of Cryptomeria japonica on Murashige and Skoog's medium supplemented with 22.8 μM IAA. Callus was grown successfully for more than 2 years on the same medium without any decline in growth vigour. The callus has shown differentiation of tracheids with reticulate thickenings but differentiation of root, shoot or whole plant has not yet been observed. Cytology of 2-month-old callus showed the presence of cells containing normal diploid chromosome complement of 2n = 22 and normal mitosis. However, the 6-month-old callus revealed majority of cells with normal diploid complement and the usual karyotype, but a few cells were noticed with aneuploid numbers ranging from 2n = 15–17. Anaphase bridges and lagging chromosomes have also been encountered.     

Phytosulfokine stimulates somatic embryogenesis in Cryptomeria japonica

Phytosulfokine (PSK), which has been identified as a plant growth factor, had a dramatic stimulatory effect on the formation of somatic embryos of sugi (Cryptomeria japonica) in the presence of polyethylene glycol. The resultant somatic embryos germinated with synchronous sprouting of cotyledons, hypocotyls and roots, and most of the seedlings grew normally. A cDNA clone for the precursor to the PSK peptide of C. japonica was identified in an expressed sequence tags database. Our results support the existence of a PSK signaling pathway in C. japonica.     

Germination inhibitor from the Japanese cedar Cryptomeria japonica

(1S,6R)-2,7(14),10-Bisabolatrien-1-ol-4-one was identified as a germination inhibitor from the methanol extract of Japanese cedar wood, Cryptomeria japonica. The occurrence of this compound in 1 g fresh wood was 2.0 mg, and showed a maximum of 60% germination inhibition at the dose of 20 mg/filter paper (157 microg/cm(2)) against both of lettuce and rice seeds for 4 d. A selective activity between Dicotyledoneae and Monocotyledoneae seeds was not observed.     

AFLP and CAPS linkage maps of Cryptomeria japonica

We have used two DNA marker systems, AFLP and CAPS, in a two-way pseudo-testcross strategy applied to an F₁ population to construct genetic linkage maps of two local sugi cultivars. The AFLP markers detected about eight polymorphisms per parent per primer combination. Using 38 primer combinations, 612 AFLPs were detected in ’Haara 4’ and ’Kumotooshi’, of which 305 segregated in a 1:1 ratio (P>0.05). A total of 91 markers (83 AFLP and 8 CAPS) in ’Haara 4’ and 132 (123 AFLP and 9 CAPS) in ’Kumotooshi’ were distributed among 19 and 23 linkage groups, respectively, each of which included 2–17 markers. Maps of ’Haara 4’ and ’Kumotooshi’ spanned 1266.1 cM and 1992.3 cM, and covered approximately 50% and 80% of the sugi genome, respectively. Sequences derived from cDNA, which were previously used to construct a sugi linkage map, were also placed on our linkage maps as CAPS markers. Where a ’two-way pseudo-testcross’ is used, more than half of the sugi CAPS developed can be used to construct linkage maps for each parental family. The saturation of mapped markers, and the integration of several linkage maps derived from different mapping populations, is anticipated in the near future. Received: 15 August 1999 / Accepted: 27 August 1999     

Development and characterization of microsatellite markers for Cryptomeria japonica D.Don

Thirty four microsatellite markers for Cryptomeria japonica D. Don were developed by searching three types of library: a database of C. japonica cDNA sequences, a standard non-enriched genomic DNA library and a microsatellite-enriched library using magnetic particles. The enrichment of microsatellite sequences using magnetic particles is very efficient compared to the other two methods both in terms of the numbers of markers generated, and in the polymorphism they detect. The microsatellites developed from the genomic DNA library generally have longer repeat sequences and are more polymorphic than those from cDNA. All the developed microsatellite markers in this study showed polymorphism among 28 plus trees selected from locations scattered throughout Japan. The mean number of alleles per locus (MNA) detected in the 28 plus trees ranged from 2 to 21 with an average of 7.5. The Polymorphism Information Content (PIC) ranged from 0.160 to 0.936 with an average of 0.666. Co-dominant segregation of alleles in a three-generation pedigree of C. japonica was demonstrated at 34 microsatellite loci, and the segregation was not distorted from Mendelian expectation for all loci. In 12 out of 34 loci, a null allele was detected. Key relationships between polymorphic parameters, such as MNA and PIC, and the characteristics of microsatellite sequences, such as the longest repeat number, total repeat number and total number of nucleotides, were investigated using rank correlation coefficients, Kendall's tau. A positive correlation was found between repeat lengths and polymorphisms. The markers provide sufficient resolution for investigating gene flow within forests and seed orchards, and for genome mapping.     

In vitro establishment of Cryptomeria japonica (Taxocidaceae)

In this work, it was achieved to establish in vitro shoots of Cryptomeria japonica from 20 year old trees. The shoots were disinfected and treated with six different concentrations of kinetin and belciladenine in order to induce their development and budding. It was evaluated the effect of quality and lighting intensity on these using for this orange light at 20 microEs-1 m-2 and white light at 30 microEs-1 m-2. For shoots rooting it was used different concentrations of NAA (naftalen acetic acid) and IBA (indolbutiric acid) alone or combined. BA and KIN induced the bud formation in Cryptomeria but it was observed the best budding with BA at 9.1 microM. White light and orange light promoved the growth of explants as well as the growth of new buds but it was higher with orange light. The bud rooting was observed but it was not possible to find the best auxin concentration for rooting because of the plentiful callus formation on the base of explants and the root formation was very sporadic. The rooted shoots were placed on a substrate for their acclimation in greenhouse conditions.     

Pollen wall development in Cryptomeria japonica (Taxodiaceae)

Pollen wall development in Cryptomeria japonica was observed by scanning and transmission electron microscopy. The pollen of C. japonica is characterized by a non-saccate, projecting papilla. The exine of C. japonica consists of the outer granular ectexine and the inner lamellated endexine. At the tetrad stage, the initial granular layer of the pro-ectexine first forms on the microspore plasma membrane. The tripartite lamellae of the pro-endexine form under the pro-ectexine. The prosporopollenin material is deposited on the pro-ectexine and pro-endexine at the free spore stage. The ectexine granule increases its volume and the endexine lamellae thicken. The papilla protrudes during the tetrad stage. The tip of the papilla bends laterally where the exine is thinner. Exine construction in C. japonica is similar to that of Cunninghamia; however, the amount and size of the granular ectexine and lamellated endexine differ. The conspicuous papilla protrudes and bends during the tetrad period.     

Volatile components in cell suspension cultures of Cryptomeria japonica

The cell suspension culture of Cryptomeria japonica contains volatile oils, the yield of which was 0.005–0.01% of the fresh cells. In the volatiles, five aldehydes, ten fatty acids and their esters, and two diterpenes of abietatriene and ferruginol have been found. Of these, palmitic acid is present as the most predominant component, amounting to ca 40% of the volatiles.     

Two new sesquarterpenoids from the bark of Cryptomeria japonica

Two new sesquarterpenoids, i.e. ferrugicadinol A (1) and ferrugicryptomeridiol (3), and one known sesquarterpenoid, ferrugicadinol (2) were isolated from the bark of Cryptomeria japonica D. Don. Their structures were identified by extensive spectral analysis and comparison with the data of known analogues.     

A diterpene quinone from the bark of Cryptomeria japonica

A diterpene, cryptoquinone, was isolated from the bark of Cryptomeria japonica, the structure, 7,11,14-trioxoabieta-8,12-diene, was established by spectral analyses and X-ray crystallography. This diterpene quinone showed moderate antifungal activities against Pyricularia orizae and Alternaria alternata, and cytotoxic activity against mouse lymphoid neoplasm (P388) cells with an IC(50) of 0.26 microg/ml.     

Detection of Cryptomeria japonica roots with ground penetrating radar

Abstract

Coarse tree roots, which are responsible for most root carbon storage, are usually measured by destructive methods such as excavation and coring. Ground penetrating radar (GPR) is a non-destructive tool that could be used to detect coarse roots in forest soils. In this study, we examined whether the roots of Cryptomeria japonica, a major plantation species in Japan, can be detected with GPR. We also looked for factors that impact the analysis and detection of roots. Roots and wooden dowels of C. japonica were buried 30 cm deep in sandy granite soil. From GPR measurements with a 900 MHz antenna, the distribution and diameter of samples in several transects were recorded. The buried roots were detected clearly and could be distinguished at diameters of 1.1–5.2 cm. There were significant positive relationships between root diameter and parameters extracted from the resultant GPR waveform. The difference in water content between roots and soil is a crucial factor impacting the ability to detect roots with GPR. We conclude that GPR can be used as a non-destructive tool, but further investigation is needed to determine optimal conditions (e.g. water content) and analytical methods for using GPR to examine roots in forest sites.     

Development and characterization of chloroplast microsatellite markers for Cryptomeria japonica D. Don

We developed 17 chloroplast microsatellite markers, which consisted of seven mononucleotide microsatellites with a minimum repeat number of 10 and 10 dinucleotide microsatellites with a minimum repeat number of six, from the complete chloroplast genomic sequence of Cryptomeria japonica. A survey of 45 C. japonica individuals showed the number of alleles ranging from two to 11 alleles and a diversity index ranging from 0.085 to 0.895. Consequently, the 45 C. japonica individuals were divided into 39 haplotypes. These markers will be useful genetic markers in the gene flow analysis and population genetics of C. japonica.     

Physical map of chloroplast DNA in sugi,Cryptomeria japonica

Summary To investigate the evolution of conifer species, we constructed a physical map of the chloroplast DNA of sugi, Cryptomeria japonica, with four restriction endonucleases, PstI, SalI, SacI and XhoI. The chloroplast genome of C. japonica was found to be a circular molecule with a total size of approximately 133 kb. This molecule lacked an inverted repeat. Twenty genes were localized on the physical map of C. japonica cpDNA by Southern hybridization. The chloroplast genome structure of C. japonica showed considerable rearrangements of the standard genome type found in vascular plants and differed markedly from that of tobacco. The difference was explicable by one deletion and five inversions. The chloroplast genome of C. japonica differed too from that of the genus Pinus which also lacks one of the inverted repeats. The results indicate that the conifer group originated monophyletically from an ancient lineage, and diverged independently after loss of an inverted repeat structure.     

Photoprotective role of rhodoxanthin during cold acclimation in Cryptomeria japonica

To examine the role of rhodoxanthin in long‐term acclimation to low temperatures, we monitored seasonal changes in pigment composition, photosynthesis, chlorophyll fluorescence and the level of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) in needles of wild‐type and mutant forms of Cryptomeria japonica. In winter, rhodoxanthin accumulated in sun‐exposed needles of wild‐type plants, but not in those of the mutant. The level of chlorophyll decreased in both types of plant in winter. In contrast, the level of the xanthophyll cycle pool increased in both cases. The level of the pool in the mutant was twice that in the wild type in winter, on a Chl basis, even though the levels in both were similar in summer. The synthesis of rhodoxanthin might be triggered by photo‐inhibitory conditions, as suggested by the sustained elevated levels of zeaxanthin (Z) and antheraxanthin (A). In the wild type and the mutant, the quantum yield of CO₂ fixation (φ), the photosynthetic capacity, the photochemical efficiency of photosystem II (PSII), the photochemical quenching and the level of Rubisco in summer were similar. However, all these values for the wild type were higher than those for the mutant in winter. The non‐photochemical quenching (NPQ) in the mutant in winter increased rapidly even under low light conditions due to the high sustained levels of Z and A. In contrast, in the wild type, the conversion of Z via A to rhodoxanthin prevented the rapid increase in NPQ to maintain the relatively high level of φ. These findings suggest that rhodoxanthin might play an important photoprotective role in long‐term acclimation to cold. The dynamic regulation of the amount of rhodoxanthin relative to the level of the xanthophyll cycle pool might act to maintain an appropriate balance between light absorption, photosynthesis and the thermal dissipation of energy due to excess absorbed light in winter.     

Formation and vertical distribution of sapwood and heartwood in Cryptomeria japonica D. Don

The formation and vertical distribution of sapwood and heartwood were studied with a 45-year-old Cryptomeria japonica D. Don. The tree was grown at a plantation with 1.5 m × 3.0 m spacing near Miao-Li, Taiwan and was felled on 27 February 1992. The thickness of sapwood and heartwood was expressed by a ring count and a linear measurement. The east-west (E-W) wood strips were collected from 0.3 m above ground upwards to the top of the tree at 2.5 m intervals. The sapwood thicknesses from the base to the 10.3 m tree level height are around 20–22 growth rings and 42±2 mm. At the top of the tree, the sapwood thickness is narrower. The heartwood, which decreases in thickness with increasing tree level heights is not found at the top of the tree. The heartwood appears as a conical shape in the tree trunk. There is no statistical difference in sapwood/heartwood thickness between E-W aspects. Tree level heights and the tree level age were found to be important parameters in determining the thickness of sapwood/heartwood.     

Geographical distribution of myxomycetes living on Cryptomeria japonica bark in Japan

There is little available data on the biogeography of myxomycetes at the regional scale within any given climate zone. To investigate the geographical distribution of these protozoans, we focused on corticolous myxomycetes associated with the bark of Cryptomeria japonica trees, which we sampled extensively throughout Japan. Myxomycete sporophores developed in 73% of 2244 moist-chamber cultures of 188 bark samples from 24 regions, including 31 species. The most abundant species were Paradiacheopsis rigida and Cribraria confusa, which accounted for over 20% of all myxomycetes sampled. Non-metric multidimensional scaling was used to analyze the distribution of myxomycetes in relation to geographical variables and bark pH. The distribution of myxomycetes was influenced by altitude, temperature and bark pH. Temperature gradients and relative abundance of species were negatively correlated in P. rigida and Comatricha laxa and positively correlated in C. confusa, Macbrideora cornea, and Diderma chondrioderma. Bark pH was also positively correlated with the relative abundance of C. confusa, D. chondrioderma, and Physarum nutans and negatively correlated with that of P. rigida, P. cribrata, Enerthnema papillatum, and E. melanospermum. Geographical distribution of corticolous myxomycete communities was determined based on temperature and bark pH, which acted as local barriers in our study area.     

Identification of QTLs associated with male strobilus abundance in Cryptomeria japonica

It has become increasingly important to understand the factors controlling the abundance of male strobili in Cryptomeria japonica since allergic reactions to the species?? pollen have become prevalent in Japan. There is considerable inter-annual variation in the abundance of male strobili, so it is important to investigate and validate quantitative trait loci (QTL) for strobili abundance across different years. Data on third generation families derived from an individual that produces abundant male strobili were studied using Kruskal?CWallis tests and Bayesian regression analysis, resulting in the detection of a single major QTL that was observed over multiple years. This QTL explained between 10.4 and 26.5?% of the phenotypic variation in male strobilus abundance. The consistency and significance of this QTL??s effects suggest that it has a major role in male strobilus production in C. japonica, although we also detected other QTLs, indicating that strobilus production is subject to complex and multifactorial regulation. These QTL may also affect precocity because we were unable to separate their effects from the genetic variability in male strobilus production. The major QTL that we detected occurs in a region homologous to a QTL detected in a previous study examining trees with a different genetic heritage following artificial induction of male strobilus production. This result further supports the importance of the QTL detected in the present study. Identifying the important genes in this QTL will therefore significantly increase our understanding of the genetic mechanisms of pollen production in C. japonica.