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。

【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 .

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.