Four freshly established cell lines of blue spruce (Picea pungens Engelm.), with different capacities for somatic embryogenesis (SE), were studied to assess their SE formation and maturation, aiming at identifying key factors responsible for the variation in SE potential. We examined dynamic cytological and physiological changes, including morphology and structure, storage substances, antioxidant enzyme activities and endogenous hormone levels. The results showed that cell lines 202, 201, 211 with SE capacity exhibited active metabolic processes and significant increases in superoxide dismutase (SOD) and peroxidase (POD) activities, total protein (TP) and malondialdehyde (MDA) contents across developmental stages. In contrast, the cell line lacking SE capacity showed low or unchanged enzyme activities. These findings suggest that SOD, POD, MDA and TP can serve as physiological markers for identifying SE capacity. During SE development and maturation, endogenous hormone levels (GA, IAA, ABA) remained unchanged in the non-embryogenic cell line. However, in highly embryogenic line, hormone levels generally increased and then decreased, while lines with intermediate and weak SE showed a steady increase. Our findings provide critical insights into optimizing culture media formulations to enhance blue spruce SE maturation, thereby improving efficiencies of elite genotype selection and large-scale clonal propagation. Such advancements are essential for meeting the growing demand for high-quality planting materials in landscape applications and forestry production. Nevertheless, the underlying molecular mechanisms by which reactive oxygen species (ROS) influence SE maturation capacity, as well as the synergistic regulatory interactions among endogenous hormones during different embryo developmental stages, remain insufficiently understood and warrant further investigation. Furthermore, the technological framework and regulatory patterns identified in this study are not limited to blue spruce; the physiological markers and developmental cues elucidated herein may serve as valuable references for SE research in coniferous species. To validate the practical applicability of this approach, comprehensive field trials are required to evaluate the growth performance, adaptability, and stability of SE-derived plants under natural environmental conditions.

Cranberry(Oxycoccos)has strong cold resistance,and its fruit has high health value and economic value.In this stud-y,the tissue cultured seedlings of cranberry were used as cuttings to explore the soaking and quick dipping tests of cranberry cuttings under different indole-3-acetic acid(IAA),1-naphthlcetic acid(NAA)and 3-indolebutyric acid(IBA)concentrations,and the effects of different hormone types and concentrations on rooting rate,seedling height growth,biomass and root growth characteristics(including the longest root length,root surface area,average root diameter and average root number).The results showed that,when NAA soaking treatment was 100 and 200 mg/L,the root system grew well,but the longest root length was only(30.77±0.18)cm(NAA 100 mg/L),and the average root diameter was(0.3±0.02)mm.However,the longest root length of cuttings treated with IBA 1000 mg/L per hour was(30.13±3.22)cm,the average root diameter was(0.39±0.04)mm,the root surface area was(1.27±0.01)cm2,and the average number of roots was(128.22±2.38).From this point of view,there was little difference in root growth between the soaking treatment and the rapid soaking treatment,but in general,when the IBA concentration was 1000 mg/L,the rooting rate,was 85% at the highest.Therefore,the quick dipping method was more suitable for the rooting of cranberry tissue culture seed-lings.In this study,the cutting propagation technology of tissue culture seedlings of cranberry was established for the first time,which laid the foundation for large-scale propagation of cranberry.

Porcine pluripotent stem cells had been derived from different culture systems. PeNK6 is a porcine pluripotent stem cell line that we established from an E5.5 embryo in a defined culture system. Signaling pathways related with pluripotency had been assessed in this cell line, and TGF-β signaling pathway–related genes were found upregulated significantly. In this study, we elucidated the role of the TGF-β signaling pathway in PeNK6 through adding small molecule inhibitors, SB431542 (KOSB) or A83-01 (KOA), into the original culture medium (KO) and analyzing the expression and activity of key factors involved in the TGF-β signaling pathway. In KOSB/KOA medium, the morphology of PeNK6 became compact and the nuclear-to-cytoplasm ratio was increased. The expression of the core transcription factor SOX2 was significantly upregulated compared with cell lines in the control KO medium, and the differentiation potential became balanced among three germ layers rather than bias to neuroectoderm/endoderm as the original PeNK6 did. The results indicated that inhibition of TGF-β has positive effects on the porcine pluripotency. Based on these results, we established a pluripotent cell line (PeWKSB) from E5.5 blastocyst by employing TGF-β inhibitors, and the cell line showed improved pluripotency.

Embryogenic tissue (ET) is important for genetic modification and plant re-generation. The proliferation ability and vigor of ET are crucial for plant propagation via somatic embryogenesis. In this study, ET was induced from mature zygotic embryos in blue spruce (Picea pungens Engelm.). There were significant differences in ET induction between two provenances, i.e. 78.8 ± 12.5% and 62.50 ± 12.8% respectively. Effects of 2,4-Dichlorophenoxy acetic acid (2,4-D), 6-Benzyl amino-purine (6-BA) and/or sucrose on ET proliferation and somatic embryo (SE) maturation were further investigated with four cell lines. The highest ET proliferation rate reached 1473.7 ± 556.0% biweekly. Concentrations of 2,4-D or 6-BA applied at tissue proliferation stage impacted SE maturation among the cell lines, whereas sucrose showed less effects. The highest rate, 408 ± 230 mature SEs/g FW, was achieved in SE maturation cultures. This research demonstrated that the culture conditions, i.e. the specific concentrations of 2,4-D and BA, at ET proliferation stage affected not only ET growth, but also the quality of ET for SE maturation. This study revealed the necessity and benefit in developing both the general and the genotype-specific protocols for efficient production of mature SEs, or somatic plants in blue spruce.

以蔓越莓组培苗为插穗,探究8 种基质对扦插生根的影响.结果表明:全泥炭土不适于组培苗扦插生根,生根率只有22％;总体来看全苔藓基质最适宜组培苗扦插生根,生根率最高,为 86％,苗高增长量为 4.62±0.25 cm,生物量最高(鲜重2.33±0.18 g,干重0.51±0.04 g),生根状况也最好.

Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low induction and proliferation rates, hindering the production of improved plant materials. We investigated the effects of the explant sterilization method, 4 °C cryopreservation, basal medium, ethylene removal, liquid medium supplementation, and a combination of PGRs on embryogenic callus (EC) induction of Korean pine, using immature embryos of Korean pine as explants. The effects of sucrose and maltose on EC proliferation and maturation were investigated. The differences in the maturation ability of EC somatic embryos before and after cryopreservation were evaluated using the induced embryonic cell lines. The results showed that zygotic embryos (ZEs) performed better than megagametophytes (MGs) as explants. The induction rate of EC was significantly increased after 28 days of cryopreservation at 4 °C. The induction rate of EC in the #5 family increased from 10.00% to 62.8%. The EC induction rate of the five families cultured with the DCR basal medium was higher than that with the mLV basal medium. Among them, the induction rate of the #5 family cultured with the mLV basal medium was 23.3%, while that with the DCR basal medium was 60.9%, an increase of 2.6 times. There was no significant difference in the maturation ability of EC somatic embryos before and after cryopreservation. In conclusion, this study provides a method to improve the EC induction rate and maturation ability of Korean pine.

Cellular redox state and hormone levels have important regulatory roles in the process of somatic embryogenesis (SE), but experimental data on the topic are limited in conifer SE. This study investigated changes in redox pairs (reduced glutathione (GSH) vs oxidized glutathione (GSSG) and ascorbic acid (ASA) vs dehydroascorbate (DHA)), activities of enzymes (glutathione reductase (GR), DHA reductase (DHAR), and ascorbate peroxidase (APX)), and levels of endogenous hormones (indole-3-acetic acid (IAA), zeatin-riboside (ZR), gibberellin (GA), and abscisic acid (ABA)) following exogenous supplements of 2,4-dichlorophenoxyacetic acid (2,4-D), GSH, and L-buthionine sulfoximine (BSO) to the proliferation medium of Picea pungens SE and linked the changes with proliferation efficiencies. While 2,4-D promoted proliferation and maturation in a concentration-dependent manner, the joint application of 2,4-D (2 mg/L) and GSH (0.5 mmol/L) was the most effective. Such beneficial roles were not observed when GSH alone was applied and exogenous BSO (1 mmol/L) prohibited proliferation. Supplements of 2,4-D and GSH, individually or jointly, enhanced the GR activity, decreased the DHAR activity and increased amounts of redox compounds, yet more in GSH and ASA than in GSSG and DHA, creating reduced environments. Exogenous BSO had reversal effects, creating an oxidized environment, and so was the joint use of 2,4-D and BSO, although at a less extent. 2,4-D and GSH reduced while BSO increased IAA content. The alternations in redox state as well as in IAA content from exogenous 2,4-D or BSO supplements coincided with the corresponding changes in proliferation. The notable finding was that exogenous GSH, regardless its substantial roles in reducing environments or IAA content, did not improve proliferation. Thus, the role of GSH on proliferation varied, depending on 2,4-D existing, and could not be fully accounted for by dynamic changes in redox state or hormone level. Alternations in APX and hormones ABA, ZR, GA, and IAA/ABA ratio were also found not highly involved in altering proliferation.

Exogenous glutathione (GSH) promotes the proliferation of embryogenic callus (EC) cells in Korean pine in the course of somatic embryogenesis, and reactive oxygen species (ROS) may play an important role in regulating the proliferation of EC cells by exogenous GSH. However, the concrete metabolic response of ROS is unclear. In this study, two cell lines of Korean pine with high proliferative potential 001#-001 (F, Fast proliferative potential cell line is abbreviated as F) and low proliferative potential 001#-010 (S, Slow proliferative potential cell line is abbreviated as S) were used as test materials. The responses of ROS-related enzymes and substances to exogenous GSH and L-Buthionine-sulfoximine (BSO) were investigated in EC cells. The results showed that the exogenous addition of GSH increased the number of early somatic embryogenesis (SEs) in EC cells of both F and S cell lines, decreased the amount of cell death in both cell lines. Exogenous addition of GSH promoted cell division in both cell lines, increased intracellular superoxide dismutase (SOD) and catalase (CAT) activities, inhibited intracellular hydrogen peroxide (H 2 O 2 ), malondialdehyde (MDA) and nitric oxide (NO) production, and increased NO/ROS ratio. In conclusion, the exogenous GSH promoting the proliferation of Korean pine EC cells, the activity of intracellular antioxidant enzymes was enhanced, the ROS level was reduced, and the resistance of cells to stress was enhanced.

Picea pungens (Engelm.), known for its blue-green needles, has become a likable ornamental species in northeast China since 2000. Nonetheless, a lack of propagation methods that can maintain genetic fidelity and develop seedlings at a large scale prevents the further expansion of the species. Somatic embryogenesis (SE), paired with cryopreservation technologies, may provide a valid alternative. Picea pungens SE is not new, but its practical application has been limited due to low efficiencies in SE initiation and maturation as well as a lack of effective cryopreservation technology. In this study, experiments were carried out to overcome the limitations by modifying culture media. For initiation, the efficiency was enhanced by adjusting concentrations of 2.4-dichlorophenoxy acetic acid (2,4-D), 6-benzyl amino–purine (6-BA) or sucrose supplemented to the induction medium. The concentrations of 4.0 mg/L 2,4-D, 2 mg/L 6-BA, and 5 to 10 g/L sucrose were found optimal in maximizing initiation efficiency. For maturation, the efficiency, expressed as the number of mature somatic embryos per gram of fresh mass cultured (E/gFM), varied greatly with the choices of the basal medium and concentration of abscisic acid (ABA) of the maturation medium. Based on our results, the judicial choices were using the DCR medium as the basal medium and 10 mg/L ABA. The maturation efficiency could also be improved by adjusting the maturation medium’s osmotic pressure by manipulating the concentrations of carbohydrate and Gelrite and culture density. While the maturation medium, using sucrose as carbohydrate source or supplemented with a low (<8 g/L) Gelrite concentration, facilitated maturation, optimal selections were truly genotype-dependent. Our results also suggest that, while the optimal culture density varied with genotype, in general it is needless to culture more than 100 mg embryogenesis tissues per dish (size: 10 × 1.5 cm). Based on this study, the optimum pretreatment for embryogenesis tissue cryopreservation was culturing the tissues on the proliferation medium with 0.4 mol/L sorbitol for 24 h, followed by treatment with 5% Dimethyl sulfoxide. This study significantly improved the initiation (achieved a frequency of 0.56) and embryo maturation efficiencies (achieved 1030 E/gFM) and established an effective preculturing protocol for cryopreservation (recovered 1354 E/gFM) for the species. The protocols developed here, paired with the available ones for other SE steps in the literature, form a well-refined SE technology intended for commercial application to Picea pungens.

Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low proliferation rates, hindering the production of improved plant materials. One way of improving proliferation rates is achieved by improving the redox status of the culture medium. In this study, we investigated the effects of exogenous glutathione (GSH) and L-buthionine sulfoximine (BSO, the inhibitor of glutathione synthase) on the EC proliferation rate in Korean pine (Pinus koraiensis), using cell lines with both high (F: 001#-001) and low (S: 001#-010) proliferation potential. We found that exogenous GSH promoted cell proliferation in both cell lines, while exogenous BSO inhibited proliferation in both cell lines. At 35 d with exogenous GSH treatment, the fresh weight of F and S cell lines increased by 35.48% and 48.39%, respectively, compared with the control. The exogenous application of GSH increased the intracellular levels of GSH, total GSH (T-GSH), oxidized glutathione (GSSG), ascorbic acid (ASA), total ASA (T-ASA), and the ratios of GSH:T-GSH and ASA:T-ASA in both F and S cell lines. Furthermore, exogenous GSH increased the activity of both glutathione reductase (GR) and dehydroascorbate reductase (DHAR) while decreasing the activity of ascorbate peroxidase (APX) in both cell lines. It appears that the application of exogenous GSH promotes a reducing cultural environment, which is conducive to EC proliferation in Korean pine. By helping to reveal the mechanism whereby GSH regulates redox homeostasis in Korean pine EC cells, we have laid the foundation for a large-scale breeding of Korean pine somatic embryogenesis technology system.

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.

The embryogenesis capacity of conifer callus is not only highly genotype-dependent, but also gradually lost after long-term proliferation. These problems have seriously limited the commercialization of conifer somatic embryogenesis (SE) technology. In this study, the responsive SE cell line (R-EC), the blocked SE cell line (B-EC), and the loss of SE cell line (L-EC) were studied. The morphological, physiological, transcriptomic, and metabolomic profiles of these three types of cells were analyzed. We found that R-EC had higher water content, total sugar content, and putrescine (Put) content, as well as lower superoxide dismutase (SOD) activity and H2O2 content compared to B-EC and L-EC. A total of 2566, 13,768, and 13,900 differentially expressed genes (DEGs) and 219, 253, and 341 differentially expressed metabolites (DEMs) were found in the comparisons of R-EC versus B-EC, R-EC versus B-EC, and B-EC versus L-EC, respectively. These DEGs and DEMs were mainly found to be involved in plant signal transduction, starch and sugar metabolism, phenylpropane metabolism, and flavonoid metabolism. We found that the AUX1 and AUX/IAA families of genes were significantly up-regulated after the long-term proliferation of callus, resulting in higher auxin content. Most phenylpropane and flavonoid metabolites, which act as antioxidants to protect cells from damage, were found to be significantly up-regulated in R-EC.

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.

[目的]通过对红皮云杉(Picea koraiensis)体细胞胚胎发生条件的筛选和超低温保存技术研究,建立其完整的体胚发生体系及超低温保存条件,为红皮云杉优良种质资源的大量繁殖及保存提供基础.[方法]以红皮云杉合子胚为外植体,以两种基本培养基、6种植物生长调节剂组合为培养条件,筛选胚性愈伤组织诱导的适宜条件.并将获得的胚性愈伤组织进行超低温保存和体胚发育与成熟萌发试验,以3种脱落酸(ABA)和Gelrite浓度以及4种基本培养基为培养条件,筛选出体胚成熟和萌发的适宜培养条件.[结果]①两种基本培养基相比较,改良RJW(诱导率为30.00％)优于1/2 LV(诱导率为22.50％)基本培养基.当萘乙酸(NAA)质量浓度为3.0 mg/L、6-苄氨基腺嘌呤(6-BA)质量浓度为0.5 mg/L时胚性愈伤组织诱导率最高为50.00％;②胚性愈伤组织增殖培养3个月后进行超低温保存试验,经恢复培养10 d左右可以清晰地观察到成活的胚性愈伤组织,3个细胞系的胚性愈伤组织均可成活;③体胚发育阶段,改良RJW培养基添加Gelrite 6.0 g/L和ABA 20 mg/L时,3个细胞系体胚数量均较高,但是畸形胚比例也偏高,特别是HY-1细胞系畸形胚比例高达77.14％;当Gelrite质量浓度增加到8.0 g/L时,3个细胞系畸形胚比例均降低,其中HY-2细胞系体胚数量为396.00个/g,畸形胚比例为10.33％;④红皮云杉体胚成熟培养两个月进行体胚萌发,在1/2 LM上体胚萌发能力最弱,其次是LM基本培养基,改良RJW体胚萌发能力最佳.不同细胞系体胚萌发能力也不同,体胚萌发能力最高为65.00％.[结论]① 胚性愈伤组织诱导的适宜培养基为改良RJW基本培养基,适宜的激素组合为NAA 3.0 mg/L+6-BA 0.5 mg/L;②本试验进行超低温保存的细胞系均可成活;③体胚发育与成熟适宜的ABA质量浓度为20 mg/L,适宜Gelrite质量浓度为8.0 g/L;④体胚萌发的适宜基本培养基为改良RJW基本培养基.本研究建立了红皮云杉体胚发生体系并成功进行胚性愈伤组织的超低温保存条件,该技术体系可用于红皮云杉优良种质的快速繁殖.

本文对从俄罗斯引进的24个花楸品种,采取嫁接和组织培养方式进行保存.初步分析各品种的保存和生长情况,认定花楸品种可采用嫁接和组织培养方法进行保存和繁殖,为今后花楸品种的筛选和繁殖提供方法.

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.

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.

以红皮云杉作为砧木,北美蓝云杉当年生枝条为接穗,探究不同嫁接方法和时间对嫁接效果的影响.结果表明:5月中旬以切接的嫁接方法更适合于北美蓝云杉异砧嫁接,嫁接成活率达81％.

[目的]探讨黑果枸杞的组织培养技术,为实现规模化高效离体快繁提供技术依据.[方法]以黑果枸杞当年生嫩茎为外植体,研究激素组合对黑果枸杞组培苗初代、增殖以及生根培养的影响.[结果]适合黑果枸杞初代培养的培养基为MS+6-BA1.0 mg/L+NAA0.2 mg/L,腋芽生长良好,萌芽率达88.61％,且玻璃化情况较少;适合微枝增殖培养的培养基为MS+6-BA0.1mg/L+NAA0.2mg/L,微枝生长速度快,增殖倍数可达4.8倍,少见玻璃化现象,且苗生长健壮,叶片嫩绿;适合微枝生根的培养基为1/2MS+IBA0.2mg/·L,生根率达98％.适宜的移栽基质为营养土:河沙:蛭石=3:1:1(体积比).[结论]黑果枸杞嫩茎离体培养和茎芽增殖可以获得再生植株实现离体快繁,本研究结果为黑果枸杞优良种质规模化繁殖提供了技术支持.