Search Results (14)

This study aimed to establish a CRISPR/Cas9 gene-editing system for Larix kaempferi (Lamb.) Carr. (Japanese larch). We screened L. kaempferi U6 promoters and used them to drive sgRNA expression in the CRISPR/Cas9 gene-editing system. The L. kaempferi embryogenic callus was used as the receptor material for genetic transformation, and the frequency and types of gene editing were then analyzed. The results showed various mutations in the transgenic materials, including base substitutions and deletions, and the editing frequency ranged from 5% to 14.29%. In summary, we established a CRISPR/Cas9 gene-editing system for L. kaempferi. Our results demonstrate that the CRISPR/Cas9 system can efficiently edit genes in L. kaempferi, with significantly higher editing frequencies observed when sgRNA expression is driven by endogenous LaU6 promoters compared to the exogenous promoter ProAtU6-26. This work provides technical support for the study of L. kaempferi gene functions and genetic improvement. Full article

Promoters are powerful tools for breeding new varieties using transgenic technology. However, the low and unstable expression of target genes is still a limiting factor in Larix kaempferi (Lamb.) Carr (Japanese larch) genetic transformation. In this study, we analyzed L. kaempferi transcriptome data, screened out highly expressed genes, cloned their promoters, and constructed plant expression vectors containing the β-glucuronidase (GUS) reporter gene driven by these promoters. Recombinant vectors were introduced into the L. kaempferi embryogenic callus by means of the Agrobacterium-mediated transient or stable genetic transformation method, and the promoter activity was then determined by measuring GUS expression and its enzyme activity in the transformed materials. Four highly expressed genes were identified: L. kaempferi Zhang Chen Yi-1 (LaZCY-1), Zhang Chen Yi-2 (LaZCY-2), Translationally Controlled Tumor Protein (LaTCTP), and ubiquitin (LaUBQ). The 2000 bp fragments upstream of ATG in these sequences were cloned as promoters and named pLaZCY-1, pLaZCY-2, pLaTCTP, and pLaUBQ. Semi-quantitative and quantitative RT-PCR analyses of transient genetic transformation materials showed that all four promoters could drive GUS expression, indicating that they have promoter activities. Semi-quantitative and quantitative RT-PCR analyses and the histochemical staining of stable genetic transformation materials showed that the pLaUBQ promoter had higher activity than the other three L. kaempferi promoters and the CaMV35S promoter. Thus, the pLaUBQ promoter was suggested to be used in larch genetic transformation. Full article

SCARECROW-LIKE6 (SCL6) plays a role in the formation and maintenance of the meristem. In Larix kaempferi (Lamb.) Carr., an important afforestation tree species in China, SCL6 (LaSCL6) has two alternative splicing variants—LaSCL6-var1 and LaSCL6-var2—which are regulated by microRNA171. However, their roles are still unclear. In this study, LaSCL6-var1 and LaSCL6-var2 were transformed into the Arabidopsis thaliana (L.) Heynh. genome, and the phenotypic characteristics of transgenic A. thaliana, including the germination percentage, root length, bolting time, flower and silique formation times, inflorescence axis length, and branch and silique numbers, were analyzed to reveal their functions. It was found that LaSCL6-var1 and LaSCL6-var2 overexpression shortened the root length by 41% and 31%, respectively, and increased the inflorescence axis length. Compared with the wild type, the bolting time in transgenic plants was delayed by approximately 2–3 days, the first flower and silique formation times were delayed by approximately 3–4 days, and the last flower and silique formation times were delayed by about 5 days. Overall, the life cycle in transgenic plants was prolonged by approximately 5 days. These results show that LaSCL6 overexpression inhibited the transitions from the vegetative meristem to inflorescence meristem and from the flower meristem to meristem arrest in A. thaliana, revealing the roles of LaSCL6-var1 and LaSCL6-var2 in the fate transition and maintenance of the meristem. Full article

Two integrated harvesting methods have been primarily applied to increase the opportunity for forest biomass utilization. In Korea, small shovels with a carrier for cut-to-length harvesting (CTL system) and tower yarders for whole-tree harvesting (WT system) are commonly used for the transportation of tree assortments (i.e., sawlogs and logging residue). No previous studies are available in South Korea that have compared and highlighted the operational performance and yield of logging residues between the CTL and WT harvesting systems. Thus, our study’s main objectives were to (1) evaluate the productivity and costs of the two harvesting systems through a standard time study method and (2) estimate the amount of harvesting logging residue at the landing. The productivities of the CTL and WT systems were 1.45 and 2.99 oven-dried tons (odt)/productive machine hour (PMH), at a cost of 86.81 and 45.41 USD/odt, respectively. In the WT system, the amount of logging residue (2.1 odt/ha) collected at the landing was approximately four-times larger than that of the CTL system (0.5 odt/ha). Our results suggested that the WT system is a less expensive and more suitable system when there are markets demanding logs and biomass, whereas the CTL system remains a less expensive option for stem-only harvesting. Furthermore, these results are important for estimating the economic and environmental amount of residue that could be potentially recovered and utilized from the forest types included in the study. Full article

A simple and efficient protocol for the genetic transformation of Japanese larch (Larix kaempferi (Lamb.) Carr.) was developed by altering the infection method and duration and the bacterial removal process. More than 600 hygromycin-resistant embryonal masses with the vector pCAMBIA1301 were obtained, with an average of 20 transgenic lines per gram of fresh weight. Nine hygromycin-resistant transformation events (designated P1–P9) were analyzed using PCR, quantitative real-time PCR, and histochemical β-glucuronidase (GUS) assays. The GUS transcript abundance in each transformed cell line ranged from 10¹ to 10³ magnitudes, with a maximum abundance of 2.89 × 10³. In addition, the pLaTCTP::GUS vector, which contains GUS under the control of the L. kaempferi LaTCTP promoter, led to constitutive expression of GUS in embryonal-suspensor mass and somatic embryos. The transcript abundance of the exogenous genes HPT and GUS, driven by the CaMV 35S or LaTCTP promoter, ranged from 10¹ to 10⁴, which was equivalent to genes with moderate and low abundances in Japanese larch. The relatively low expression levels of exogenous genes in transformants might reflect the large genome of Japanese larch. Additional transgenic cell lines need to be screened to obtain transformants with higher expression levels of foreign genes for further functional research in Japanese larch. Full article

Extreme climate events such as heat waves, drought, and heavy rainfall are occurring more frequently and are more intense due to ongoing climate change. This study evaluated the early growth performance of one-year-old Larix kaempferi (Lamb.) Carr. seedlings under open-field extreme climate conditions including experimental warming and different precipitation regimes. We recorded the survival rate, root collar diameter, height, biomass, shoot-to-root ratio, and seedling quality index using nine treatments (three temperature levels, i.e., control, warming by 3 °C and by 6 °C, × three precipitation levels, i.e., control, drought, and heavy rainfall) in July and August 2020. The survival rate of seedlings did not differ between treatments, showing high values exceeding 94% across treatments. The measured shoot height was largest under warming by 3 °C and high rainfall, indicating that moderate warming increased seedling height growth in a moist environment. Heavy rainfall decreased stem volume by 21% and 25% under control and warming by 6 °C treatments, respectively. However, drought manipulation using rain-out shelters did not decrease the growth performance. Overall, extreme climate events did not affect the survival rate, biomass, shoot-to-root ratio, and seedling quality index of L. kaempferi. We thus conclude that, regarding growth responses, L. kaempferi seedlings may be resistant to short-term extreme warming and drought events during summer. Full article

Shear strength is important for the application of Larix kaempferi (Lamb) Carr. The structural difference between earlywood and latewood of Larix kaempferi affects its mechanical properties, especially shear strength. The microstructures of earlywood and latewood in Larix kaempferi, however, are different. In this study, we investigated the shear strength and shear failure mode in the RL direction of 40 Larix kaempferi specimens. The results demonstrated that the initial crack appears in any location of a growth ring, whereas shear failure is concentrated in earlywood, as well as the junction between earlywood and latewood. The destruction of earlywood is the tear destruction, whereas when the destruction happened in the junction of earlywood and latewood, one to three earlywood cells usually adhered to latewood. At the cell wall level, the shear failure of earlywood was mostly observed in the direction of the microfibril angle (MFA). When the crack occurs in latewood, the destruction of latewood also occurs in the intercellular layer and preserves the complete morphology of tracheids. When destruction occurs in the wood ray, the ray cells detach intact from the tracheids. The failure mode is determined by the microstructure of earlywood and latewood. Our research suggests that the density, cell wall thickness, and MFA have significant differences between earlywood and latewood. The earlywood was found to have an MFA of 25.4°, a cell wall thickness of 6.36 µm, and a density of 0.39 g/cm³. The MFA, cell wall thickness, and density of latewood density were 17.60°, 12.37 µm, and 0.78 g/cm³, respectively. However, there was no significant difference found in the crystallinity between the earlywood (43.97%) and latewood (42.79%). The correlation between the microstructures and shear strength showed that earlywood with a thin cell wall, large MFA, and low density had poor shear performance, while the latewood with a thicker tracheid, smaller MFA, and higher density had better shear performance. Therefore, when shear failure occurred, it occurred in earlywood. We thus deduced that the MFA, cell wall thickness, and density of earlywood synergically affect the shear strength in the RL direction of L. kaempferi. Full article

The study of somatic embryogenesis can provide insight into early plant development. We previously obtained LaMIR166a-overexpressing embryonic cell lines of Larix kaempferi (Lamb.) Carr. To further elucidate the molecular mechanisms associated with miR166 in this species, the transcriptional profiles of wild-type (WT) and three LaMIR166a-overexpressing transgenic cell lines were subjected to RNA sequencing using the Illumina NovaSeq 6000 system. In total, 203,256 unigenes were generated using Trinity de novo assembly, and 2467 differentially expressed genes were obtained by comparing transgenic and WT lines. In addition, we analyzed the cleaved degree of LaMIR166a target genes LaHDZ31–34 in different transgenic cell lines by detecting the expression pattern of LaHdZ31–34, and their cleaved degree in transgenic cell lines was higher than that in WT. The downstream genes of LaHDZ31–34 were identified using Pearson correlation coefficients. Yeast one-hybrid and dual-luciferase report assays revealed that the transcription factors LaHDZ31–34 could bind to the promoters of LaPAP, LaPP1, LaZFP5, and LaPHO1. This is the first report of gene expression changes caused by LaMIR166a overexpression in Japanese larch. These findings lay a foundation for future studies on the regulatory mechanism of miR166. Full article

With the increasing popularity of cross-laminated timber (CLT) constructions around the world, there have been attempts to produce CLT using local wood species in different countries, such as Japanese larch (Larix kaempferi (Lamb.) Carr.) in China. Thus, the need to investigate the connection performance also increases to support the design and construction of CLT buildings using local wood species. In this study, the withdrawal properties of three different types of self-tapping screws (STS), with a diameter of 6 mm, 8 mm, and 11 mm, were tested with Japanese larch CLT. The results revealed that the withdrawal strength of STS increased with increasing density and effective length, but decreased with an increasing diameter. With a density increment of 0.05 g/cm³, the withdrawal strength increased by an average of 9.4%. With an effective length increment of 24 mm, the withdrawal strength increased by an average of 1.4%. An empirical regression model was adopted to predict the withdrawal strength of Japanese larch CLT based on the results, which can be used for potential engineering design of CLT connections using STS. Full article

Somatic embryogenesis is an ideal model process for studying early plant development. Embryonic cell lines of Larix kaempferi (Lamb.) Carr overexpressing LaMIR166a were obtained in our previous study. Here, a combination of de novo transcriptomics and extensively targeted metabolomics was used to study the transcriptional profiles and metabolic changes in wild-type and LaMIR166a-overexpressed embryonic cell lines. A total of 459 metabolites were found in the wild-type and transgenic cell lines. Compared to those in the wild-type cell lines, transcripts and metabolites were significantly altered in the LaMIR166a-overexpressed cell lines. Among differentially expressed genes (DEGs), phenylalanine and flavonoid synthesis genes were significantly enriched, and among differentially accumulated metabolites (DAMs), phenolic acids and flavonoids accumulated in particularly high amounts. Thus, the flavonoid biosynthetic pathway seems to be the most abundant pathway in response to LaMIR166a overexpression. Based on the Kyoto Encyclopedia of Genes and Genomes database, the association analysis of metabolome and transcriptome data showed that flavonoid biosynthesis and plant hormone signal transduction processes were significantly changed in miR166a-overexpression lines, suggesting that miR166 might be involved in these processes. The present study identified a number of potential metabolites associated with LaMIR166a overexpression, providing a significant foundation for a better understanding of the regulatory mechanisms underlying miR166. Full article

Simple sequence repeats (SSRs) are widely used as markers for the assessment of genetic diversity and marker-assisted breeding. In a previous study, two SSRs (GCA and CCA), were found in the genomic sequence of Larix (La) SCL6, which plays important roles in the growth and development of Larix kaempferi (Lamb.) Carr. In this study, we analyzed the polymorphisms of these two SSRs in the L. kaempferi population. We found that each SSR had five different polymorphisms, among which (GCA)₇ and (CCA)₇ were predominant. In addition, 12 haplotypes were detected, with (GCA)₇(CCA)₇ having the highest frequency. Furthermore, we detected the haplotypes of LaSCL6 in mature trees and their seeds and analyzed the relationships between parents and offspring. The expression patterns of five LaSCL6 alleles were analyzed and they showed balanced expression during vegetative development. Taken together, these findings not only provide more genetic information on LaSCL6, but also provide a candidate marker for genetic studies and breeding. Full article

Forests in Korea are mainly located in steep mountainous areas, where small-shovel-based extraction technology is widely used, with the level of mechanization undoubtedly low due to financial limitations. On this steep terrain, a better approach may be to use cable yarders, which can offer high revenues through cable-based extraction. Therefore, improving the efficiency of cable yarding activities in good-quality timber forests is necessary. The main objectives of this study were to (1) evaluate the productivity and cost of a cable yarder operation for tree-length clearcut treatment of a Larix kaempferi (Lamb.) Carr. stand and (2) compare the productivity efficiency of two yarder (K301-4 and HAM300) types. The productivity rates of the K301-4 ranged from 10.2 to 12.5 m³/productive machine hours, with corresponding costs of US $12.6–15.4 /m³. The productivity of the HAM300 was 26% lower than that of the K301-4 for a 30% lower cycle log volume while yarding and a comparable lateral distance. This study provides insights to support production and management decisions in the forest supply chain associated with planning cable-yarding operations. Full article

To ensure sustainable forestry, it is important to establish an efficient management procedure for improving the seed production capacity of seed orchards. In this study, we evaluated the effects of girdling and increasing light intensity on female cone production in an old L. kaempferi (Lamb.) Carr. seed orchard. We also evaluated whether there is a genotype-specific reproductive response to these factors among clones. The results showed that female cone production was augmented by girdling and increasing light intensity. There was a difference in the effectiveness of girdling treatment levels, and the probability of producing female cones increased markedly at higher girdling levels. At light intensities where the relative photosynthetic photon flux density was higher than 50%, more than half of the trees tended to produce female cones, even in intact (ungirdled) trees, and the genotype-specific response to light intensity was more apparent in less-reproductive clones. These findings suggested that girdling less-reproductive trees combined with increasing light intensity was an effective management strategy for improving cone production in old seed orchards. Full article

Forecasts of prices can help industries in their risk management. This is especially true for Japanese logs, which experience sharp fluctuations in price. In this research, the authors used an exponential smoothing method (ETS) and autoregressive integrated moving average (ARIMA) models to forecast the monthly prices of domestic logs of three of the most important species in Japan: sugi (Japanese cedar, Cryptomeria japonica D. Don), hinoki (Japanese cypress, Chamaecyparis obtusa (Sieb. et Zucc.) Endl.), and karamatsu (Japanese larch, Larix kaempferi (Lamb.) Carr.). For the 12-month forecasting periods, forecasting intervals of 80% and 95% were given. By measuring the accuracy of forecasts of 12- and 6-month forecasting periods, it was found that ARIMA gave better results than did the ETS in the majority of cases. However, the combined method of averaging ETS and ARIMA forecasts gave the best results for hinoki in several cases. Full article