Thinning is an important silvicultural practice for improving the productivity and wood production in plantation forest. Different intensities of thinning management can affect tree growth and alter soil nutrient effectiveness, thus affecting soil fungal community structure and diversity. Our objective is to determine the soil factors and their regulatory mechanisms that influence stand growth by thinning, and to provide data to support the establishment of large diameter timber cultivation technology for Picea koraiensis. In this study, we conducted medium- and high-intensity thinning in 43a P. koraiensis plantation middle-aged forests and investigated the growth indexes, soil physicochemical properties, and fungal community diversity in rhizosphere and non-rhizosphere soils of the stands after thinning at different densities (904 plants/ha for control, 644 plants/ha for 30% thinning intensity, and 477 plants/ha for 50% thinning intensity). The results showed that all growth indicators (annual growth of tree height, diameter at breast height, height under live branches and crown width) of the plantation after high-intensity thinning (477 plants/ha) were higher than those of the control (no thinning, significant) and medium-intensity thinning (644 plants/ha). Mycorrhizal infection rate was higher at the beginning of the growing season than at the end of the growing season, and increased slightly with decreasing stand density. Compared to the control, all medium- and high-intensity thinning treatments significantly improved soil nutrient content (P < 0.05), including total carbon, total nitrogen, total phosphorus, total potassium, Available phosphorus and Available potassium. Fungal diversity was higher but lower in abundance than the control in both rhizosphere and non-rhizosphere soils after thinning. The number of OTUs and fungal richness and diversity indices of non-rhizosphere soil fungi were higher than those of rhizosphere soil fungi. In conclusion, this study provides new evidence that reasonable intercalation can increase the radial and vertical growth of P. koraiensis plantation forests and promote the diversity of subsurface soil fungal communities. It is shown that thinning intensity regulates biogeochemical cycles in P. koraiensis plantation ecosystems by affecting soil nutrients and fungal community structure. Therefore, 50% thinning intensity can be used to increase timber production in plantation forests during large diameter timber cultivation of P. koraiensis and improve predictions associated with achieving long-term forest management strategies.

Korean pine is an important afforestation tree species in Northeast China, which has a high ecological and economic value. Although regeneration of somatic embryogenesis using immature zygotic embryos of Korean pine as explants has been successful, it cannot be applied to automation and large-scale production. Therefore, we urgently need a method that can increase the output of somatic embryos (SEs) to meet the needs of large-scale production. We used Korean pine 1-1 and 1-100 cell lines as research materials to evaluate the effects of inoculum-density, culture time, orbiting speed, vessel volume, plant growth regulator (PGR) concentration, and carbon source on the proliferation of embryogenic tissue (ET). The somatic embryogenesis ability of ET cultured in different liquid suspension media was also evaluated. We found that during liquid suspension culture of Korean pine ET, the sedimented cell volume (SCV), fresh weight (FW) and dry weight (DW) were affected by inoculumdensity, culture time, orbiting speed, 2 ,4-D concentration, 6-BA concentration and carbon source type. Fourty mg center dot mL-1 ET were transferred to a 200 mL Erlenmeyer flask containing 20 mL liquid medium, and cultured at 100 rpm/min for 14 days to obtain the maximum proliferation. In addition, we also found that SCV, FW and DW were higher when PGRs were reduced in the liquid suspension medium. The substitution of maltose for sucrose resulted in slow growth of cultures and limited SE yield (13 SEs g-1 FW). Although culture proliferation was high at 50 rpm, SE yield was inhibited by 48% compared with 100 rpm (50 rpm = 33 SEs g-1 FW; 100 rpm/min = 70 SEs g-1 FW). Cultivation in low-concentration PGR(1.15 mu M center dot L-1 2,4-D, 0.25 mu M center dot L-1 6-BA) and sucrose liquid medium at 100 rpm/min (80 SEs g-1 FW) could not only promote culture proliferation but also increase SE yield. The determination of the suspension culture scheme of Korean pine ET provides a reference for further expansion to bioreactor culture in the future and lays a foundation for the automation and scale of somatic embryogenesis of Korean pine.

Many cell lines in the embryogenic callus cannot produce somatic embryos (SEs) even if they meet the optimal SE maturation culture conditions during conifer somatic embryogenesis. This phenomenon hinders the progress of the industrial-scale reproduction of conifers. Therefore, there is an urgent need to obtain morphological and physiological markers to screen embryogenic calli in response to SE maturation conditions. To detect cell lines with high somatic embryogenesis potential during the proliferation process, we counted the number of pro-embryos and early SEs (ESEs) in different cell lines and storage substances, endogenous hormones, and polyamine contents. The results showed that the yield of P. koraiensis SEs was heavily dependent on genotype (p = 0.001). There were high levels of PE III (pro-embryo III) number, ESE number, and soluble protein content, in the response cell lines (R cell lines), which were 1.6-, 3-, and 1.1-fold those of the obstructive cell lines (B cell lines), respectively. The B cell line had high levels of starch, auxin (IAA), Put, Spd, and putrescine: spermine (Put: Spm) compared to the R cell line. In addition, the numbers of PE III, ESEs, and soluble protein content were significantly positively correlated with SE yield. In contrast, the contents of starch, abscisic acid (ABA), Put, Spm, and Spd were significantly negatively correlated with SE yield. To ensure the accuracy of the results, we used nine cell lines to test the results. The PE III and ESE numbers and the Spm and Spd contents were positively correlated with SE yield, while the levels of starch, ABA, IAA, Put: Spd, and Put: Spm were negatively correlated with SE yield. Thus, we recommend using high PE III and ESEs as morphological indicators and low levels of starch, IAA, ABA, and Put: Spm as physiological markers to screen cell lines with a high somatic embryogenesis potential. In addition, we also found that the relationship between Spd, Spm, and SE yield was opposite in the two experimental results. Therefore, we speculate that the differences in Spd and Spm content are mainly affected by genotype. In conclusion, this study obtained the morphological and physiological markers of some high-somatic embryogenic cell lines by comparing the differences between nine somatic embryogenic cell lines. Our results can guide the improvement of conifer somatic embryogenesis technology and can provide a theoretical basis for accelerating the application of biotechnology in large-scale artificial breeding.

Programmed cell death occurs in browning explants of Fraxinus mandshurica during somatic embryogenesis, but the underlying mechanism is unclear. In this study, single cotyledons of zygotic embryos of F. mandshurica were used as explants. Mitochondrial structure and function, caspase-3-like protease activity, hydrogen peroxide metabolism, and nitric oxide accumulation induced by high concentrations of sucrose and plant growth regulators were studied. The results show that plant growth regulators induced somatic embryogenesis and also promoted explant browning. High sucrose concentrations had similar effects. High concentrations of sucrose and plant growth regulators led to the accumulation of hydrogen peroxide and nitric oxide which induced changes in mitochondrial structure and function such as modifications in mitochondrial morphology, increased membrane permeability, decreased membrane potential, and the release of cytochrome c into the cytoplasm. An increase in caspase-3-like protease activity triggered programmed cell death in some browning explant cells. During somatic embryogenesis there were increased activities of superoxide dismutase, peroxidase, and catalase, which are associated with hydrogen peroxide metabolism and jointly maintain reactive oxygen species levels. Intracellular nitric oxide synthase and nitrate reductase activities were not significantly correlated with nitric oxide content. Instead, intracellular nitric oxide may be derived from non-enzymatic reactions. Our results indicate that hydrogen peroxide and nitric oxide may function as signals, playing key roles in somatic embryogenesis and programmed cell death of explant cells of F. mandshurica . The interaction between nitric oxide and reactive oxygen species determines the occurrence of programmed cell death in explant cells; somatic embryogenesis and programmed cell death are positively regulated by hydrogen peroxide. However, the regulation of nitric oxide is complex.

The breaking of dormancy mediated by reactive nitrogen species (RNS) is related to the accumulation of reactive oxygen species (ROS) in germinating embryos but the underlying mechanism is unclear. The objectives of this study were: (1) to explore the relationship between RNS-mediated dormancy release and ROS accumulation in germinating embryos of Sorbus pohuashanensis ; and, (2) to investigate the relationships among germination time, ROS metabolism, and endogenous hormone synthesis. We studied the effects of exogenous nitric oxide (NO) donor sodium nitroprusside (SNP), the NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), abscisic acid (ABA), the exogenous ethylene donor ethrel, and the ethylene receptor inhibitor 2,5-norbornadien (NBD) on embryo germination and seedling growth. Embryos were released from dormancy by pretreatment with NO or ethylene, which was related to increased ethylene biosynthesis and decreased ABA levels. Breaking of dormancy by SNP was related to increased levels of ethylene, hydrogen peroxide, and glutathione, increased activities of superoxide dismutase and glutathione peroxidase, and decreased levels of ABA, superoxide anions, and malondialdehyde. These effects of nitric oxide were especially significant in seedling hypocotyls and radicles. These results demonstrate that NO can break S. pohuashanensis embryo dormancy by inducing ethylene biosynthesis, and that this signalling pathway is closely related to ROS accumulation and the antioxidant defence response.

The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm. Callus can be induced from immature embryos of Korean pine ( Pinus koraiensis Sieb. et Zucc.; Pinaceae) as explants, but there are problems, such as low proliferation efficiency, loss of embryogenicity, poor vigor; thus, best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear. To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation, we varied hormone concentration, subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages, then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design. The results showed that the family origin and collection date of explants significantly affected callus induction (induction rate reached 1.67%). Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L −1 6-benzyl adenine, 1 mg L −1 naphthaleneacetic acid, 30 g L −1 sucrose, 500 mg L −1 , l -glutamine, 500 mg L −1 casein hydrolysis and 6.5 g L −1 agar. In addition, the combination of 2,4-dichlorophenoxyacetic acid + 6-benzyl adenine also had a better proliferative effect on callus. The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different. Transfer to new medium every 13–15 days not only maintained robust callus vigor, but also yielded a larger proliferation coefficient. The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine.

Korean pine broadleaf mixed forest is an important ecosystem for maintaining biodiversity in Northeast China. Korean pine is also an important species for the production of timber and nuts in the mountainous areas of Northeast China. In this study, we compared three types of Korean pine callus and found that embryogenic callus had high amounts of storage substances (protein, sugar and starch). Non-embryonic callus had high levels of polyphenols and polyphenol oxidation, while callus that lost somatic embryogenesis potential had lower levels of storage substances (protein, sugar and starch) and higher contents of peroxidase and catalase. These results indicate that high contents of storage substances (protein, sugar and starch), and low levels of polyphenols and polyphenol oxidase can be used as physiological markers of callus with somatic embryogenic potential. During the development process of Korean pine somatic embryos, fresh weight and dry weight gradually increased, while water content gradually decreased. Soluble protein, starch, soluble sugar and superoxide dismutase also increased during development, while peroxidase and catalase levels reduced over time. These results indicate that somatic embryogenesis involves energy storage, and antioxidant enzymes cooperate to regulate the occurrence and development of embryos. These results provide physiological markers for identification of embryogenic callus with somatic embryogenesis, to evaluate callus suitable for somatic embryogenesis, and provide basis for further research on the molecular mechanisms of somatic embryogenesis.

Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo (SE) yield, unsynchronized SE development, and a high percentage of deformed SEs. We aimed to improve F. mandshurica SE production by synchronizing SE development, improving SE quality, and inducing root formation to obtain complete regenerated plants. Cotyledons of immature zygotic embryos of F. mandshurica were induced to form callus and then SEs. The SE induction percentage from explants differed among 32 mother trees, and the one with the highest SE induction percentage (29.8%) was used for further experiments. The highest callus induction percentage was 94.2% on ½-strength Murashige and Skoog medium (MS½) supplemented with 0.15 mg·L−1 naphthalene acetic acid. The highest callus proliferation coefficient (240.5) was obtained on McCown’s Woody Plant Medium containing 0.1 mg·L−1 6-benzyl adenine and 0.15 mg·L−1 2, 4-dichlorophenoxyacetic acid. The highest number of SEs (1020.5 g−1 fresh weight) was obtained on MS½ medium supplemented with 1 mg·L−1 6-benzyladenine. The highest number of cotyledon embryos (397/g fresh weight) was obtained by incubating materials on medium containing 1 mg·L−1 abscisic acid and then applying a drying treatment. The cotyledon embryos were milky white, uniformly sized (average length 4.7 mm), and 80% of them were normal. The SE rooting percentage on ½MS medium containing 0.01 mg·L−1 NAA was 37.5%. Overall, the germination percentage of SEs was 26.4%, and complete regenerated plants were obtained after transplanting and acclimation. These results provide more possibilities for the preservation and breeding of F. mandshurica.

Korean pine is the dominant species of Korean pine forests. It is an economically valuable species that yields oil, high-quality timber and nuts, and it offers great prospects for further development. Complete regenerated plants of Korean pine were obtained via somatic embryogenesis using megagametophytes as the explant. The seeds of 27 families of Korean pine were collected to induce embryogenic lines. We compared the effects of explant collection time, family and medium components (concentrations of sucrose, plant growth regulators and acid-hydrolyzed casein) on embryogenic lines induction. The effects of plant growth regulators and L-glutamine contents on the proliferation and maturation of embryogenic cell lines were studied, and the germinating ability of different cell lines was evaluated. The embryogenic lines induction percentage of Korean pine reached 33.33%. When 4.52 μmol·L−1 2,4-D and 2.2 μmol·L−1 6-BA were added to the medium of embryogenic lines proliferation, the ability of embryo maturation was the best (cell line 001#-100 was 135.71·g−1 fresh weight). Adding 1–1.5g L−1 L-glutamine to the proliferation medium can improve the ability of embryo maturation (cell line 001#-100 was 165.63·g−1 fresh weight). The germination percentage of the three cell lines tested was significant, and the highest was 66%. We report on successful regeneration and cryopreservation methods for somatic embryos of Korean pine. This technology could be used to propagate the excellent germplasm resources of Korean pine and to establish multi-varietal forestry.

Background Puccinellia tenuiflora is the most saline-alkali tolerant plant in the Songnen Plain, one of the three largest soda saline-alkali lands worldwide. Here, we investigated the physicochemical properties of saline-alkali soils from the Songnen Plain and sequenced the transcriptomes of germinated P. tenuiflora seedlings under long-term treatment (from seed soaking) with saline-alkali soil extracts. Results We found that the soils from Songnen Plain were reasonably rich in salts and alkali; moreover, the soils were severely deficient in nitrogen [N], phosphorus [P], potassium [K] and several other mineral elements. This finding demonstrated that P. tenuiflora can survive from not only high saline-alkali stress but also a lack of essential mineral elements. To explore the saline-alkali tolerance mechanism, transcriptional analyses of P. tenuiflora plants treated with water extracts from the saline-alkali soils was performed. Interestingly, unigenes involved in the uptake of N, P, K and the micronutrients were found to be significantly upregulated, which indicated the existence of an efficient nutrition-uptake system in P. tenuiflora . Compared with P. tenuiflora , the rice Oryza sativa was hypersensitive to saline-alkali stress. The results obtained using a noninvasive microtest techniques confirmed that the uptake of NO 3 - and NH 4 + and the regulatory flux of Na + and H + were significantly higher in the roots of P. tenuiflora than in those of O. sativa . In the corresponding physiological experiments, the application of additional nutrition elements significantly eliminated the sensitive symptoms of rice to saline-alkali soil extracts. Conclusions Our results imply that the survival of P. tenuiflora in saline-alkali soils is due to a combination of at least two regulatory mechanisms and the high nutrient uptake capacity of P. tenuiflora plays a pivotal role in its adaptation to those stress. Taken together, our results highlight the role of nutrition uptake in saline-alkali stress tolerance in plants.

The improvement and development of saline–alkali land is of great significance for promoting food production and sustainable development. It is necessary to study the mechanism of saline–alkaline tolerance and breed saline–alkaline tolerant crops to improve the utilization of saline–alkali land. For this study, we conducted a three-year pot experiment to screen the saline–alkaline tolerant germplasm of 72 rice genotypes from hundreds of elite cultivars during the whole growth period using a certain proportion of soda saline–alkali soil. The selected salt-tolerant variety was combined with a salt-sensitive variety to analyze the saline–alkaline tolerance mechanism by using the saline–alkaline soil leachate. We eliminated 36 genotypes with low seedling survival rates under salt–alkali stress, and the salt-tolerant Jiudao-66 (D68) variety had a higher survival rate than most varieties. The membership degree of Jiudao-66, according to the salt tolerance index of multiple agronomic traits, is higher than that of 34 varieties, with a higher survival rate except when compared to D36. The survival rate and these salt tolerance indexes of Jiudao-66 were significantly higher than those of Kitaake (salt-sensitive). Under the stress of leachate, the content of proline and soluble sugars in the shoots of Jiudao-66 were higher than that of Kitaake, and the total antioxidant capacity was stronger than that of Kitaake. However, the content of malondialdehyde was lower than that of Kitaake. Additionally, the Na+/K+ ratios in shoots and roots were not significantly differently between Kitaake and Jiudao-66. The results showed that Jiudao-66, as a salt-tolerant variety, is more tolerant to salt and alkali in a near-natural state due to its stronger tolerance of osmotic stress, and it can accumulate more proline and soluble sugars under stress. At the same time, Jiudao-66 also has a stronger antioxidant capacity. Its ion regulation ability has no obvious advantage.