Long-term subculture of embryogenic callus leads to a decline in somatic embryogenesis and germination capacity, which may be associated with increased methylation levels. 5-Azacytidine (5-AzaC), a methylation inhibitor, modulates DNA methylation and is widely involved in regulating plant growth, development, and metabolism. In order to investigate the effect of 5-AzaC on somatic embryogenesis and germination in Larix olgensis, we supplemented the proliferation medium with different concentrations of 5-AzaC. The results showed that the addition of 5-AzaC inhibited the proliferation of embryogenic callus, with the proliferation of embryogenic line N2 completely inhibited at 100 μM, while that of embryogenic line N4 ceased at 20 μM. In contrast, treatment with 10 μM and 20 μM of 5-AzaC significantly increased the somatic embryo yield in both embryogenic lines, with the peak yield observed at 20 μM for embryogenic line N2 and at 10 μM for embryogenic line N4. Furthermore, the addition of 10 μM 5-AzaC effectively reduced the deformity rate during somatic embryo germination in embryogenic line N2 and N4, by 15.91% and 13.53%, respectively. These findings demonstrate that 5-AzaC can partially restore the somatic embryogenesis potential of embryogenic callus in L. olgensis under long-term subculture. Additionally, these results suggest that its effects may be both concentration-dependent and genotype-specific. The results provide a potential approach to mitigating the decline in embryogenic competence, while also demonstrating significant potential for large-scale propagation.

Global forests face increasingly severe drought events, which suppress the growth of larch. Transcription factors (TFs), acting as regulators, can increase plant stress tolerance by modulating gene expression networks. This study integrated RNA-seq, WGCNA and physiology to systematically analyze the adaptation mechanisms of Larix olgensis under drought stress. Among the 50,110 unigenes identified via RNA-seq, 1,391 unigenes were annotated as TFs. These TFs were clustered into four distinct clusters based on expression profiles. TFs within Cluster 1 exhibited significant differential expression during visible wilting of L. olgensis (72 h and 96 h). Ten TFs were selected as candidate TFs from Cluster 1 based on fold-change rank (p < 0.05). Subsequent WGCNA clustered the 50,110 unigenes into 19 co-expression modules. Notably, the firebrick4 module displayed a strong positive correlation with hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents, whereas the darkolivegreen module showed a strong positive correlation with peroxidase (POD) activity. Three TFs in the firebrick4 module (TRINITY_DN13923_c0_g2, TRINITY_DN3004_c0_g1 and TRINITY_DN1230_c0_g1) and two TFs in the darkolivegreen module (TRINITY_DN1156_c1_g3 and TRINITY_DN1156_c1_g1) were identified by co-expression network analysis. The findings of this study contribute to the knowledge of drought-responsive TFs in L. olgensis, establishing a foundational resource for future functional studies and molecular breeding.

Viburnum opulus L. ‘Roseum’ is a highly valuable ornamental plant for landscaping, but it has a long propagation cycle and low propagation coefficient. In this study, stem segments with axillary buds from Viburnum opulus L. ‘Roseum’ were used as explants. We systematically analyzed the use of sodium hypochlorite for the sterilization of explants, as well as the effects of different plant growth regulator combinations and concentrations on shoot bud induction, shoot proliferation, the rooting of tissue-cultured shoots, and the transplanting of the tissue-cultured shoots. A complete rapid propagation technology system for Viburnum opulus L. ‘Roseum’ was established. The results showed that a disinfection method using 75% ethanol for 30 s and soaking in 5% sodium hypochlorite for 5 min was the most suitable for disinfecting the stem segments of Viburnum opulus L. ‘Roseum’, which showed low contamination and a 73.33% survival rate. The ideal medium for primary bud induction was WPM (Woody Plant Basal Medium) + 2.0 mg·L−1 6-benzylaminopurine (6-BA) + 0.15 mg·L−1 indole-3-butyric acid solution (IBA) + 25 g·L−1 sucrose. The optimal medium for shoot proliferation was WPM + 1.0 mg·L−1 6-BA + 0.15 mg·L−1 IBA + 25 g·L−1 sucrose, achieving an induction rate of 7.17. For the rooting of tissue-cultured shoots, the most suitable formulation was 1/2 WPM + 0.3 mg·L−1 naphthaleneacetic acid (NAA) + 0.3 mg·L−1 activated charcoal (AC) + 25 g·L−1 sucrose, which induced robust and developed root systems. This study provides a technical basis for the establishment of a fast propagation system for the industrial production of Viburnum opulus L. ‘Roseum’.

Nandina domestica ‘Firepower’ is one of the most popular colorful foliage species in landscaping. However, it is currently propagated mainly by seeding and cuttings, with a low reproduction coefficient, hindering the cultivation of this species. Therefore, establishing an in vitro regeneration system would be beneficial for the industrialized production of Nandina domestica ‘Firepower’. In this study, an ex vivo regeneration system was established using the direct organogenesis pathway. In early April, the new shoots of Nandina domestica ‘Firepower’ were selected, and the stem segments of 1~2 cm were cut as the disinfection materials for the explants. The optimal formulation for inducing axillary shoots was 1/2 MS + 1.5 mg L−1 6-benzylaminopurine (BA) + 0.3 mg L−1 indole-3-butric acid (IBA). The optimal formulation for the differentiation and proliferation of axillary shoots was 1/2 MS + 1.5 mg L−1 BA + 0.01 mg L−1 IBA with a multiplicity of proliferation of 9.22. We determined that the rooting of axillary shoots required a combination of IBA, naphthalene acetic acid (NAA), and activated carbon (AC). The optimal formulation for rooting was 1/2 MS + 0.2 mg L−1 NAA + 0.3 mg L−1 IBA + 0.2 mg L−1 AC. After a two-day hardening period for tissue-cultured plantlets, a substrate consisting of peat soil, vermiculite, and perlite at a ratio of 2:2:1 was determined to be the optimal cultivation formulation. This system provides a framework for the industrialized production of Nandina domestica ‘Firepower’.

Larix olgensis is a valuable timber species in northern China, typically propagated through somatic embryogenesis (SE). However, long-term subculture can lead to a loss of embryogenic potential. This study aimed to establish a simple and stable protocol for the cryopreservation and regeneration of L. olgensis embryogenic callus (EC) that preserves its SE potential and regenerative capacity. The slow-freezing method was employed for cryopreservation. A cryopreservation protocol for L. olgensis EC was developed by optimizing the preculture duration and conditions, cryoprotectant composition and thawing temperature. The results showed that optimal outcomes were achieved using a 24 h stepwise preculture on medium containing 0.2 and 0.4 mol∙L−1 sucrose, followed by cryoprotectant treatment with 0.4 mol∙L−1 sucrose, 2.5% (v/v) dimethyl sulfoxide (DMSO) and 10% polyethylene glycol 6000 (PEG6000), and thawing at 37 °C. EC cryopreserved using this protocol achieved a 100% recovery rate. Moreover, the cryopreserved recoverable EC successfully underwent SE, progressing through germination and rooting. Cryopreservation duration (storage duration in liquid nitrogen) did not affect cell viability and proliferation rate, confirming the protocol’s suitability for long-term cryopreservation of L. olgensis EC. This study provides a valuable reference for the cryopreservation and regeneration of L. olgensis EC, with potential applications for other coniferous species. It establishes a robust foundation for the large-scale propagation of conifers.

Pinus koraiensis is classified as a second-class protected wild plant in China, recognized for its considerable economic and ecological importance. However, progress in functional research and breeding applications for this species has been hindered by the lack of an effective genetic transformation system. The purpose of this study was to develop a reliable and efficient genetic transformation system for a Pinus koraiensis embryonic callus using somatic embryogenesis technology. The Pinus koraiensis embryonic callus and β-glucuronidase (GUS) were employed as the reporter gene in an Agrobacterium-mediated transformation to investigate critical transformation factors, including antibiotic type and concentration, Agrobacterium bacterial solution concentration, infiltration, and co-cultivation times. The findings indicated that the proliferation of the Pinus koraiensis embryonic callus was substantially inhibited by 10 mg·L−1 of Hygromycin (Hyg), and a remarkable 93.42 ± 2.13% efficiency was achieved with an OD600 absorbance value of 0.6 during transformation. Two days of optimal co-cultivation yielded a transformation rate of 82.61%, with the resistant embryonic callus exhibiting a high GUS staining rate of 88.89%. Resistant somatic embryos were effectively obtained following the optimized protocol. This research contributes to the advancement of seed resource breeding and genetic enhancement for Pinus koraiensis, establishing a solid foundation for the investigation of gene functions specific to this species.

Larch is a cold-temperate tree species native to the northern hemisphere and tolerant to low temperatures. It is one of the most significant timber species in Northeast China. This study examined growth changes in hybrid larch seedlings from five lines to explore the physiological responses of these seedlings to low-temperature stress. Using 8-month-old hybrids of larch seedlings, we subjected the plants to cold stress at 4 °C and freezing stress at −20 °C over three periods of 6, 12, and 24 h, and treatment at 25 °C was used as a control. Results showed that significant correlations were found among the growth indicators, with larch line 1306 having the lowest incremental growth indicators, the largest root-to-crown ratio, and better cold tolerance than the other larch lines. The levels of soluble sugars (SSs), soluble proteins (SPs), malondialdehyde (MDA), and relative electrolyte leakage (REC) increased significantly in all lines under low-temperature stress. The activities of superoxide dismutase (SOD) and catalase (CAT) showed variation over time. Significant correlations were found between MDA and REL, SS, SR, Pro, CAT, and SOD in most of the lines; no significant correlation was found between MDA and the other indices in lines 1301 and 1309; and significant correlations were found between most of the physiological indices in line 1306.

Rhododendron aureum is identified as a vulnerable species in China. The establishment of an in vitro regeneration system assists significantly in species protection. Here, an in vitro regeneration system was developed using both direct and indirect organogenesis pathways. The role of thidiazuron (TDZ) in different developmental stages was also investigated. The leaves of wild-harvested R. aureum plants were used for callus induction after hydroponic cultivation. The optimal formula was found to be woody plant basal medium (WPM) supplemented with 0.5 mg·L−1 TDZ and 0.5 mg·L−1 3-indolebutyric acid (IBA), while the optimal formula for the subculture and induction of adventitious buds was WPM containing 0.1 mg·L−1 TDZ and 0.5 mg·L−1 IBA. The leaves from tissue-cultured seedlings were then used for the induction of bud clusters. An association was observed between the differentiation of bud clusters and the ratio of auxin and cytokinin. The optimal formula for the induction of bud clusters was WPM containing 0.5 mg·L−1 TDZ and 0.1 mg·L−1 IBA, yielding a 50% induction rate and the maximum number of buds. Higher concentrations of TDZ were found to be beneficial for bud proliferation, while a lower concentration was conducive to stem elongation. The optimal formula for subculture was WPM containing 0.1 mg·L−1 TDZ and 0.5 mg·L−1 IBA, while that for stem elongation was WPM supplemented with 0.002 mg·L−1 TDZ and 0.5 mg·L−1 IBA. Only IBA was found to be necessary for rooting, with increased IBA concentrations leading to an increased number of roots and earlier root formation, with larger callus areas; thus, an IBA concentration of 1.0 mg·L−1 was found to be optimal for the rooting of R. aureum. After hardening the seedlings for two days, a substrate composed of vermiculite and peat soil in a 10:1 ratio was identified as a transplantation formula. This system provides directions both for the protection of endangered species and the promotion of industrial development.

Fraxinus mandshurica is a widely used greening and ornamental tree species. However, its genetic transformation system has been hampered by problems such as low transformation efficiency, among others, which can hinder research related to molecular breeding and the analysis of functional genes. Thus, in this study, a novel genetic transformation method for efficient transformation of the embryonic callus of Fraxinus mandshurica was investigated. The method was optimized in terms of factors such as antibiotics, infection solution concentrations, co-culture time, and somatic embryo maturation. The results indicated that the optimal antibiotic concentration was 10 mg·L−1 of hygromycin (Hyg). At this point, the callus proliferation multiple was only 0.12. The highest transformation efficiency was found to be 93.93% when the absorbance of the infection solution concentration at OD600 was 0.4. Interestingly, transformation efficiency was found to be highest (77.9%) at 48 h of co-culture, with a GUS staining rate of 88.23%. The medium for somatic embryo maturation of transformed callus was half-strength MS medium (MS 1/2) containing 60 g·L−1 polyethylene glycol, 1 mg·L−1 abscisic acid, 400 mg·L−1 casein enzymatic hydrolysate (CH), 20 g·L−1 sucrose, 1 g·L−1 activated charcoal, and 5 g·L−1 gellan gum. The medium for somatic embryo germination was MS ½, containing 0.2 mg·L−1 of N-(Phenylmethyl)-9H-purin-6-amine(6-BA) and 5.0 mg·L−1 of gibberellin (GA). These results are of significance for the verification of the gene function and future genetic improvement of Fraxinus mandshurica.

为了解转录因子bHLH在长白落叶松(Larix olgensis)中的功能,探究该基因在长白落叶松不同组织中及不同逆境胁迫下的表达特性,从长白落叶松根、茎和叶3个不同部位的转录组数据中获得bHLH34基因全长序列,并设计引物,克隆得到长白落叶松bHLH34基因,其完整的开放阅读框(ORF)长度为696 bp,共编码231个氨基酸.构建亚细胞定位表达载体,瞬时转化毛果杨(Populus trichocarpa)原生质体,在激光共聚焦显微镜下观察显示,Lob-HLH34基因定位在细胞核内.系统进化树分析结果显示,长白落叶松与云杉(Picea asperata)、卷柏(Selaginella tamariscina)树种该基因的亲缘关系较近.利用qRT-PCR技术分析了bHLH34基因在长白落叶松中的组织表达特异性和应对非生物胁迫的表达.结果 表明LobH34基因在长白落叶松的根、茎、叶中均有表达,其中在茎部表达量最低,在叶中相对表达量最高.LobHLH34基因在NaC1、PEG和ABA处理时,不同器官中的表达量也有所不同.推测长白落叶松bHLH34基因参与了植物的生长、发育、响应逆境胁迫的过程,且在不同器官中具有特异性.

为了解毛果杨中固有无序蛋白PtrIDP1(Potri.010G161200.1)基因的相关信息,探究该基因在毛果杨的不同组织、不同逆境胁迫下的表达特性,本研究根据Phytozome数据库中得到的基因全长序列设计引物,克隆得到该基因的目的片段.该基因完整的CDs区序列长度为423 bp,共编码140个氨基酸.构建亚细胞定位表达载体,瞬时转化洋葱表皮细胞,在激光共聚焦显微镜下观察显示,PtrIDP1定位在细胞核内.利用实时定量RT-PCR技术分析了PtrIDP1基因在毛果杨不同组织中的表达特异性和应对非生物胁迫的表达特性.结果 表明:PtrIDP1基因在毛果杨的根、茎、叶中均有表达,其中在根部表达量最低,在茎和叶中相对表达量较高.PtrIDP1基因在高盐和干旱胁迫诱导时其表达量的变化模式不同,初步分析认为PtrIDP1基因参与了毛果杨非生物逆境胁迫的响应过程.

Larix olgensis or larch is an economically important coniferous tree species with rapid growth in the early stages, strong adaptability, and a short time to harvest. The genetic improvement of larch has garnered considerable attention in recent years for reclaiming timber forests. However, traditional breeding methods are largely ineffective for achieving rapid genetic improvement of L. olgensis. Studies show that the efficiency of plant regeneration can be improved by optimizing somatic embryogenesis. On this basis, we devised a stable, fast and efficient Agrobacterium-mediated genetic transformation method using suspended embryogenic calluses as explants and β-glucuronidase as the reporter. We evaluated the effects of the Agrobacterium load, co-culture period, and addition of acetosyringone and transformant screening antibiotic on the transformation efficiency. In addition, we tested the pCAMBIA 1300-PtHCA 2-1 promoter-GUS binary expression vector, which contains the GUS gene ORF under the control of Populus trichocarpa high cambial activity PtHCA 2-1 promoter, and observed the tissue-specific expression of the GUS gene in the somatic embryos of transgenic larch. This novel technique can not only accelerate the generation of superior transgenic strains of L. olgensis but also aid in future gene functional studies.

为了解长白落叶松过氧化氢酶（CAT）基因的相关信息,探究该基因在长白落叶松不同组织中及不同逆境胁迫下的表达特性,本研究根据长白落叶松转录组数据库中获得的CAT1基因全长序列设计引物,克隆得到长白落叶松CAT1基因,命名为LoCAT1。该基因完整的开放阅读框（ORF）长度为954 bp,共编码317个氨基酸。系统进化树分析结果显示,LoCAT1基因与北美云杉、银杏等CAT基因亲缘关系较近。利用实时定量RT-PCR技术分析了LoCAT1基因在长白落叶松中的组织表达特异性和应对非生物胁迫的表达模式。结果表明:LoCAT1基因在长白落叶松的根、茎、叶中均有表达,其中在茎部表达量最低,在叶中相对表达量最高。在非生物胁迫下,LoCAT1基因在长白落叶松根、茎、叶中的表达均发生了变化,但表达模式不同。在NaCl处理后,根和茎中LoCAT1基因均表现为下调表达,在12 h时表达量最低,而叶中LoCAT1基因表达在24 h明显受抑制,随后被上调表达,胁迫96 h时表达量最高。PEG 6000 处理后,根和茎中LoCAT1基因的表达在胁迫早期被明显抑制,随后被上调表达。而叶中LoCAT1基因的表达在所有时间点均表现为上调表达。本研究推测长白落叶松LoCAT1基因可能参与了植物响应逆境胁迫的应答。

The interspecific hybridizations of Fraxinus mandshurica Rupr. × Fraxinus americana L. (MA) and Fraxinus mandshurica × Fraxinus velutina Torr. (MV) were conducted to solve the problems of poor cold adaption associated with the introduction of Fraxinus in Heilongjiang province. High-voltage electrostatic field (HVEF) treatment to pollen was performed to overcome the prefertilization barriers. The hybrids adapted more strongly and grew better than the pure species (heterosis over higher parent (HHP) of the 9-year volume index was 5.5% for MA and 23.1% for MV) in Heilongjiang province. HVEF treatment greatly improved the number of seeds (0.25- to 5.52-fold) and seedlings (1.63- to 8.71-fold) of the hybrids. Additionally, three excellent female parents (nos. 15, 16, and 17) and seven hybrid combinations of MA (D94, D70, and D100) and MV (D103, D116, D105, and D104) with excellent growth traits were selected. The HHPs of volume index were 39.1%–112.5% for selected hybrids. Additionally, predicted growth trends of the hybrids showed that the hybrids will maintain a 7.7% to 9.3% height advantage over F. mandshurica through ages 10 to 15 years in Mao Ershan. These findings will accelerate the genetic breeding process of Fraxinus species.

【目的】以落叶松胚性系为研究对象,建立和优化适合落叶松胚性愈伤组织增殖的悬浮培养体系,在此基础上对悬浮组织进行体细胞胚的成熟诱导。旨在为实现落叶松胚性愈伤组织的快速增殖及体细胞胚的规模化繁育奠定基础。【方法】对已诱导获得的长白落叶松、兴安×日本杂种落叶松及日本×长白杂种落叶松胚性愈伤组织进行继代培养,取新鲜增殖的组织进行悬浮培养。采用L9（34）正交试验设计,以胚性愈伤组织的增殖量及增殖率为响应值对悬浮培养条件进行筛选和优化,并对选出的最适培养条件进行验证。以悬浮增殖的胚性组织进行体细胞胚成熟培养,统计体胚发生量。【结果】在落叶松胚性愈伤组织的悬浮培养过程中,接种量、震荡强度及培养时间对胚性组织增殖具有显著的影响。在一定范围内,落叶松胚性组织的增殖量及增殖率随初始接种量的增加而降低,随震荡强度的升高呈先增加后下降,而随培养周期的延长而增加。以4 g·L -1 接种于含2,4-D 0.15 mg·L -1 、6-BA 0.05 mg·L -1 及KT 0.05 mg·L -1 的BM培养基（SCM）,在120 r·min -1 避光条件下悬浮培养,3个落叶松胚性系的胚性组织均能快速、稳定地增殖,培养15天的增殖率分别为2 569.42%、4 189.96%及3 001.67%,表现出较明显的种间差异。成熟培养方式对落叶松胚性悬浮组织的体胚发生量影响极显著（P=0.000）。悬浮培养获得的胚性组织接种到含琼脂6.0g·L -1 的固体增殖培养基（PCM）上继代15天后,转接到添加肌醇10 g·L -1 且无生长调节剂的1/4BM过渡培养基（TCM）上培养14天,再转入含有ABA 20 mg·L -1 、Ag NO35 mg·L -1 、PEG400080 g·L -1 的体胚成熟培养基上培养8周,体胚发生量显著提高（P=0.000）。在此条件下,3个落叶松胚性系OO-A1、GK-F1及KO-H的体胚发生量分别为（101.69±11.19）、（93.09±9.34）及（5.78±1.47）个·g -1 。【结论】悬浮培养能在短期内获得大量分散均匀、质量较高的落叶松胚性愈伤组织,且不会影响体细胞胚的发生和成熟。在BM液体培养基（SCM）中,接种量为4 g·L -1 、震荡强度为120 r·min -1 、暗培养15天,落叶松胚性组织的鲜质量可增加26.99~42.90倍。悬浮培养获得的胚性组织经固体增殖培养基（PCM）继代15天及过渡培养基（TCM）预培养14天后,再进行体胚成熟诱导可明显提高其体胚发生量。

With embryogenic callus of Larix olgensis is, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo. Three dominant factors emerged, and we developed a response surface model based on the Box–Behnken design. We defined the optimal conditions for the maturation of somatic embryos. The contents of abscisic acid, silver nitrate, sucrose and casein hydrolysis significantly affected the amount of maturing embryos, but inositol, maltose and glutamine had no effect. By establishing a response surface model with multiple factors, we predicted that the optimal number of L. olgensis somatic embryos was 204 ± 4 g −1 on basal medium, containing 18.28 mg L −1 abscisic acid, 5.46 mg L −1 silver nitrate and 82.67 g L −1 sucrose. In the verification experiments, the addition of 20 mg L −1 abscisic acid, 5 mg L −1 silver nitrate and 80 g L −1 sucrose to BM yielded an average of 202.06 somatic embryos per gram. These results should guide large-scale breeding of L. olgensis .

采用正交试验设计,研究不同激素配比对鸡树条荚蒾茎段和叶片组培再生的影响,建立鸡树条荚蒾组培再生体系.结果表明:适于茎段分化的培养基为:WPM+2 mg/L 6-BA+0.2 mg/L IBA+20 g/L蔗糖,分化率为93.66％;适于叶片愈伤组织诱导的培养基为:WPM+2 mg/L 6-BA+0.1 mg/L IBA+2 mg/L 2,4-D,诱导率为93.33％,添加50 mg/L维生素C、50 mg/L活性炭可使叶片的褐化现象减轻;适于生根的培养基为:WPM+1.5 mg/L IBA+1 g/L活性炭,生根率为94.62％.

Genotype Nisqually-1 is the first model woody plant with an available well-annotated genome. Nevertheless, a simple and rapid transformation of Nisqually-1 remains to be established. Here, we developed a novel shoot regeneration method for Nisqually-1 using leaf petiole and stem segment explants. Numerous shoots formed in the incision of explants within two weeks. The optimized shoot regeneration medium (SRM) contained 0.03 mg l −1 6-benzylaminopurine, 0.02 mg l −1 indole-3-butyric acid and 0.0008 mg l −1 thidiazuron. Based on this, Agrobacterium -mediated genetic transformation of stem explants was examined using the vector pBI 121 that contains the β-glucuronidase (GUS) as a reporter gene. Consequently, factors affecting transformation frequency of GUS-positive shoots were optimized as follows: Agrobacteria cell suspension with an OD 600 of 0.4, 20 min infection time, 2 days of co-cultivation duration and the addition of 80 µM acetosyringone into Agrobacteria infective suspension and co-cultivation SRM. Using this optimized method, transgenic plantlets of Nisqually-1 – with an average transformation frequency of 26.7% – were obtained with 2 months. Southern blot and GUS activity staining confirmed the integration of the foreign GUS gene into Nisqually-1. This novel transformation system for Nisqually-1 was rapid, efficient, and simple to operate and will improve more genetic applications in this model tree.

With the larch embryogenic callus,through the Design Box-Behnken design test,we established a response surface model based on the somatic embryogenesis.The culture conditions of somatic embryo early maturation was optimized in Larix olgensis.The relative ion concentration (P =0.007),the inositol concentration(P =0.000),the culture period (P =0.000) and the interaction between inositol content and culture period (P =0.007) effect on the number of somatic embryos of L.olgensis were extremely significant.Cultivation of Larix somatic embryo maturation was 337.04 ind · g-1 at the early stage of the most optimum conditions for relative ion content of 26.767 7％ BM basic culture medium,inositol 10.454 5 g · L-1,culture time 12.656 6 d,and the number of somatic embryos average.In order to facilitate the practical operation,the culture condition was changed to the relative ion concentration of 25％ BM,inositol 10 g · L-1,incubation period of 13 d,then the average number of somatic embryos was 336.46 ind · g-1,that was consistent with the predicted value 336.29 ind · g-1.Compared with previous test,the quantity and quality of L.olgensis somatic embryos were increased significantly.

Objective]Immature zygotic embryos of Larix olgensis were used as explants to study induction, proliferation and somatic embryogenesis of embryogenic callus in order to reveal the key factors affecting Larix olgensis embryogenic callus induction,and at the same time,to explore the effects of growth regulators of different types and concentrations on proliferation and somatic embryogenesis of embryogenic callus, in subculture process. [Method]Embryogenic callus was induced using immature seeds from superior individuals of three families of Larix olgensis,and the effects of seed collection time,family,concentration of 2,4-D and basic medium on the induction of embryogenic callus&nbsp;were studied. Subsequently,the embryogenic callus subculture on the proliferation medium containing different kinds and concentrations of growth regulators,and the somatic embryos were obtained by the process of somatic embryogenesis. Finally,morphologically normal somatic embryos were selected and germinated,and transplanted after rooting of the somatic embryos. [Result]There was significant differences in induction rate of embryonic callus of immature embryos with different collection times. The induction rate was 5. 61% 63 days after pollination,the induction rate was 22. 35%70 days after pollination,and the induction rate was zero 80 days after pollination. In this experiment,the average induction rate of embryogenic callus from the families of‘77-22’,‘77-37 ’ and‘73-50 ’ was 6. 69%,11. 17% and 3. 11%,respectively. The concentration of 2,4-D had a certain effect on the induction of embryogenic callus,the induction rate was increased with the proliferated of 2,4-D concentration in a certain range. It was up to 11. 11% when the concentration of 2 ,4-D reached 1. 5 mg·L -1 . The induction rate began to decrease when the concentration of 2 ,4-D exceeded 1. 5 mg·L -1 . The medium of BM,MS and S were able to induce embryogenic callus,the induction rate in BM medium was the highest,followed by S medium,and the induction rate in MS medium was the lowest. Embryogenic callus culturing for 15 days on BM medium which containing 2 ,4-D 0. 3 mg·L -1 ,6-BA 0. 1 mg·L -1 and KT 0. 1 mg·L -1 can obtain more embryogenic callus,the proliferation rate was up to 345. 93%. Culturing for 14 days on BM medium which containing 2,4-D 1. 5 mg·L -1 ,6-BA 0. 5 mg·L -1 and KT 0. 5 mg·L -1 ,the number of somatic embryos per gram embryogenic callus was up to 179. 87 on average. The germination rate of somatic embryo and regeneration rate of plantlets was up to 75% and 66. 67%,respectively. The survival rate of transplanting was 27. 08%.[Conclusion]The embryos of L. olgensis seeds collected 70 days after open-pollination was suitable to induce embryogenic callus,the basic medium BM contains 2 ,4-D 1. 5 mg·L -1 . Embryogenic callus was prone to lose the ability of somatic embryogenesis in the medium with high concentration of exogenous hormones,and the proliferation speed was slow,but the number and the germination rate of somatic embryo were relatively high. An appropriate decrease of the concentration of the growth regulator was beneficial to keep the ability of somatic embryogenesis and proliferation of callus,but the quantity and the germination rate of somatic embryogenesis were somewhat decreased. It was not conducive to the proliferation of callus when the concentration of the growth regulator in the medium was low,but the ability of somatic embryogenesis was maintained for a long time. The proliferation of embryogenic callus can be improved by using NAA 0. 15 mg·L -1 instead of 2,4-D 0. 5 mg·L -1 ,and the germination rate of somatic embryos can be improved to a certain level. Therefore,according to different purposes at different stages,it was necessary to select the medium containing different types and concentrations of exogenous hormones for subculture.