High stability of nuclear microsatellite loci during the early stages of somatic embryogenesis in Norway spruce

Somatic embryos of Norway spruce (Picea abies (L.) Karst.) differentiate from proembryogenic masses (PEMs), which are subject to autodestruction through programmed cell death. In PEMs, somatic embryo formation and activation of programmed cell death are interrelated processes. We sought to determine if activation of programmed cell death in PEMs is caused by genetic aberrations during somatic embryogenesis. Based on the finding that withdrawal of auxin and cytokinin induces programmed cell death in PEMs, 1-week-old cell suspensions were cultured in medium either with or without auxin and cytokinin and then transferred to maturation medium containing abscisic acid. We analyzed the stability of three nuclear simple sequence repeat (SSR) microsatellite markers at successive stages of somatic embryogenesis in two cell lines. There were no mutations at the SSR loci at any of the successive developmental stages from PEMs to cotyledonary embryos, irrespective of whether or not the proliferation medium in which cell suspensions had been cultured contained auxin or cytokinin. The morphologies of plants regenerated from the cultures were similar, although withdrawal of auxin and cytokinin significantly stimulated the yield of both embryos and plants. We conclude, therefore, that the high genetic stability of somatic embryos in Norway spruce is unaffected by the induction of programmed cell death caused by withdrawal of auxin and cytokinin.     

Storage lipid dynamics in somatic embryos of Norway spruce (Picea abies): histochemical and quantitative analyses

Adequate storage compounds are a prerequisite for successful development during the later stages of somatic embryogenesis; however, the critical amount of reserves below which somatic embryos fail to mature and germinate has not been determined. We analyzed storage lipids during Norway spruce (Picea abies (L.) Karst.) somatic embryogenesis. As maturation progressed, lipids, which were stored as lipid bodies in the cytoplasm, were localized first in suspensor cells of the early embryos, and later in the embryonic root pole, superficial layers of the hypocotyl and in cotyledons. The concentration of total lipids exhibited marked variation, with values peaking during cotyledon development and then decreasing during maturation. Linoleic (18:2), oleic (18:1), palmitic (16:0) and 5,9-octadecenic (5,9-18:2) acids were the most abundant fatty acids in embryos. As embryos developed, linoleic acid concentration increased slightly, whereas oleic acid concentration decreased. Oleic acid was the most prominent component of the fatty acid spectrum in isolated dormant zygotic embryos and megagametophytes. Addition of 5% polyethylene glycol to the medium during somatic embryo maturation caused a shift in the fatty acid spectrum toward that of zygotic embryos. During maturation, changes in the exogenous carbohydrate supply had no significant effect on total lipid concentration in mature embryos. A marked decrease in lipid concentration was detected during desiccation, indicating the importance of adequate lipid reserves during this developmental stage. The lipid content of zygotic embryos differed considerably with harvest year and location, suggesting that zygotic embryo data cannot be an indicator of somatic embryo quality.     

黑松合子胚和体细胞胚发育阶段的形态特征比较

为掌握黑松合子胚和体细胞胚发育进程,以未成熟合子胚为外植体,建立了黑松体细胞胚胎发生体系。对合子胚和体细胞胚发育阶段的形态特征进行观察和比较,结果表明,合子胚和体细胞胚的发育进程均可划分为8个阶段,两者同一阶段的形态特征大致相同,只有第2阶段和第4阶段有细微差别。     

Critical Factors Affecting Ex Vitro Performance of Somatic Embryo Plants of Picea abies

The potential to use somatic embryos for large-scale propagation of elite genotypes, for integration into breeding programmes and for connecting breeding and mass propagation, is receiving much attention. However, before the methods are applied it is important that the plants regenerated via somatic embryogenesis grow as expected, i.e. as seedlings or cuttings. Growth of somatic embryo plants is under a cumulative influence of a number of treatments given during the in vitro phase and during the ex vitro establishment phase. The aim of this study was to identify treatments with a negative influence on the subsequent growth of somatic embryo plants of Norway spruce (Picea abies L. Karst.). Based on the results, the time of contact with abscisic acid during somatic embryo maturation and the length of continuous light treatment (CLT) during the first growth period strongly affect the height growth during two successive growth periods. In both cases longer treatments exerted negative effects. Based on these results a new method was set up, which includes: (1) prematuration treatment of the suspension culture in a growth regulator-free medium, by which the maturation step is synchronized and contracted; and (2) a two-phase germination treatment, first on a solidified medium and then in a liquid medium. This treatment avoids extended CLT during the first growth period. Another advantage of the two-phase germination treatment is a better root-system development. Somatic embryo plants produced according to this method can be transferred directly from in vitro conditions to the greenhouse.     

Early selection improves clonal performance and reduces intraclonal variation of Norway spruce plants propagated by somatic embryogenesis

Height growth during the first and second growth periods (i.e., the June-September period in consecutive years) and intraclonal variation were assessed in 13 Norway spruce (Picea abies (L.) Karst.) clones propagated by somatic embryogenesis. The plants were acclimatized and grown in a greenhouse until mid-July and then transferred outdoors. The clonal mean heights after the first and second growth periods were lower for somatic embryo plants than for seedlings from corresponding families sown at the time of somatic embryo plant ex vitro transfer, because a large proportion of somatic embryo plants were small. We determined whether certain selection criteria at ex vitro transfer can be used to identify somatic embryo plants with height growth characteristics comparable with those of seedlings. Epicotyl length and presence of lateral roots proved to be important parameters for selection, whereas main root length was less useful. A combined selection for somatic embryo plants with lateral roots and with an epicotyl length exceeding 8 mm resulted in taller plants and reduced intraclonal variation after the first and second growth periods. The growth of somatic embryo plants selected in this way was similar to that of seedlings from the corresponding families. We conclude that selection according to these criteria at ex vitro transfer can result in improved performance of clonal stock propagated by somatic embryogenesis.     

Potential Effects of Temperature on Early Reproductive Development and Progeny Performance in Picea abies (L.) Karst

In order to enhance cone production in Norway spruce [Picea abies (L.) Karst] seed orchards have been established in more southerly, warmer sites in Norway. This has led to concern and some evidence that seedlings obtained from parent trees grown at warmer sites may not be well adapted if planted in northern regions. Selective events during sexual reproduction in the warmer climate may select for traits not well adapted for cold climates. This study describes ovule and male gametophyte development, fertilization and proembryo and early embryo development under six different temperature regimens of warm or cold, warm-cold or cold-warm, and compares development in relation to temperature sums in degree days (dd) and calendar date. The outside treatment in a cold climate in Norway served as the control, and normal development and few ovule abortions and abnormalities were observed. In no treatments were abnormalities in male gametophyte development observed. In treatments in which trees were kept in the greenhouse throughout the study or moved during the study from the outside to inside the greenhouse, a higher incidence of megagametophyte, egg or embryo abortion was generally observed.     

Fluorometrische Beurteilung der relativen SO2-Resistenz von Fichte (Picea abies [L.] Karst.) anhand niedermolekularer Metabolite aus Nadelextrakten

Fluorometric examination of the relative insistence to SO₂ of Norway spruce (Picea abies [L.] karst.) using low molecular metabolites from needle extracts. Relatively resistant and “normal” spruce grafts were shown by a fluorometric method to possess different concentrations of fluorescent needle metabolites. The described method could be useful in verifying resistence qualities of Norway spruce.     

Defence reactions of developing somatic embryos of Algerian fir (Abies numidica)

Defence reactions of embryonal suspensor mass (ESM), precotyledonary, cotyledonary and desiccated cotyledonary somatic embryos of Abies numidica were tested by dual cultures with Phaeolus schweinitzii. Defence reactions were expressed at a very early stage of somatic embryo development. Both ESM and early somatic embryos inhibited mycelial growth, but the strongest defence was shown by the precotyledonary somatic embryos. The cotyledonary and desiccated cotyledonary embryos also showed defence reactions but with less intensity. Eight major components of soluble proteins, already present in the ESM, increased in concentration during subsequent developmental stages. Synthesis of two low‐molecular components of 6 and 3 kDa appeared in desiccated embryos. Probable regulation of defence reactions by auxins in early somatic embryos, as well as by abscisic acid content and storage proteins in subsequent developmental stages, is discussed.     

Storage protein changes during zygotic embryogenesis in interior spruce

The major storage proteins isolated from protein bodies of embryo tissues of interior spruce Picea glauca (Moench) Voss/Picea engelmanii Parry had apparent molecular weights of 41, 35, 33, 24 and 22 kD. Minor proteins of 30 and 27.5 kD were also observed. Based on their solubility characteristics, the 41 kD protein was identified as a water and buffer-soluble albumin, and the 35, 33, 24 and 22 kD proteins were characterized as buffer-insoluble, high salt-soluble globulins. Two-dimensional electrophoresis revealed each protein was composed of several isoelectric variants. Developmentally specific accumulation of storage proteins was observed during embryogenesis. The 41 kD protein only accumulated during the later stages of cotyledon maturation, whereas the other storage proteins began to accumulate during the early stages of embryo development. All storage proteins showed major accumulations during cotyledon maturation.     

Norway spruce emblings as cutting donors for tree breeding and production

In the Nordic countries, Norway spruce (Picea abies) is a major species in tree breeding. In order to facilitate breeding work and availability of highly bred forest regeneration material, the time required for breeding and implementation of results should be shortened. This could be done by accelerating production of clonal material for field testing, and possibly for planting stock, by combining production of rooted cuttings with somatic embryogenesis (SE). This would allow efficient production of numerous plants of the same genotype, with equal age and propagation history between genotypes. In the present work, we studied the rooting potential of cuttings from Norway spruce emblings. Altogether 36 clones from 12 families representing elite breeding materials and ornamental forms were examined under different rooting conditions (container type and rooting media) in 2015 and 2016. Our results show that Norway spruce emblings are good donors for cuttings. Best combination (peat–vermiculite mixture and Plantek 81f containers) resulted in 91% rooting, variation among the tested clones of elite breeding materials being 55–100% per treatment. The rooting variation between families is acceptable for breeding purposes. High rooting (87–96%) of ornamental forms indicates propagation potential with the combination of SE and rooted cuttings.     

Colonization patterns and topochemical aspects of sap streak caused by Stereum sanguinolentum in Norway spruce

Fresh sapwood blocks of Norway spruce (Picea abies) were exposed to 12- and 60-day attack by Stereum sanguinolentum in pure culture and were examined with respect to structural, topochemical and colour alterations. Final stages of degradation by 5. sanguinolentum were studied on naturally decayed spruce wood after 1 year of fungal growth. The development of discoloration in the main anatomical directions of wood was assessed. Furthermore, microspectrophotometrical studies on the decomposition of cell wall polyphenols were performed. Initial signs of sap streak appeared already 8 days after fungal infection. Within the three-dimensional decay pattern, mycelial aggregations associ-ated with the ray tissue proved to be the major cause of discoloration. UV extinction measurements taken at 280 nm of different parts of cell wall transverse sections revealed a specific pattern of lignin distribution and its derivatives during fungal degradation.     

落叶松体胚发育中12个针叶树特异microRNAs表达分析

以落叶松体细胞胚胎发育8个阶段的材料构建小RNA文库,进行了Solexa测序、miRNA鉴定、靶基因预测、qRT-PCR定量分析等研究。结果表明:发现的87个miRNAs中,29个为针叶树特异的,来自12个家族,分别为miR946、miR947、miR950、miR951、miR1311、miR1312、miR1313、miR1314、miR1316、miR3701、miR3702和miR3704,其中25个长度为22nt,且在小RNA文库中的最低拷贝数为18(miR1311),最高拷贝数为33 009(miR950)。发现12个miRNA家族的34个靶基因可能参与生长发育、抗病、AGO反馈调节等多方面遗传调控;qRT-PCR分析表明:72%miRNAs表达次高峰在后期单胚或早期子叶胚,在中期子叶胚最低,最高峰在后期子叶胚,分别与子叶形成、胚胎后熟、胚胎休眠的生理活动相吻合,而miRNA前体的表达模式不同于miRNA,前体变化幅度小于成熟体。     

水曲柳体细胞胚与合子胚发生的细胞学研究

水曲柳(Fraxinus mandshurica)属木犀科(Oleaceae)白蜡树属,是我国东北重要珍贵硬阔树种之一,主要分布于小兴安岭、长白山、辽宁东部山地等地区, 以材质优良而著称.由于长期不合理的采伐利用,目前可利用的资源急剧减少,已被列为国家三级保护植物(傅立国,1992).进行水曲柳体细胞胚胎发生的研究,在资源保护、树种快繁和基因工程育种上有其重要的现实意义.     

Genetic aspects of age dynamics of a natural Picea abies (L.) Karst. population in the Białowieża Primeval Forest, Poland

We studied temporal changes in the genetic structure and diversity of a naturally regenerated Picea abies (L.) Karst. (Norway spruce) population in the Bia?owie?a Primeval Forest, Poland. The analysis included five age classes of trees: newly germinating seedlings (in laboratory conditions), seedlings up to 3 years old, young trees 4–10 years old, middle-aged trees 11–100 years old, and trees older than 100 years. We conducted genetic analyses of 26 allozyme loci using dormant buds from 405 individuals and 100 embryos. The results showed that the naturally regenerating Norway spruce population is genetically heterogeneous across the studied age classes. As determined by Chi squared tests, there were statistically significant differences in frequencies of alleles and genotypes as well as Wright’s index values (F) among the five age classes. The level of genetic differentiation (pairwise F_ST = 0.5–2.3 %) among the age class groups was equal to levels previously determined for various populations of this species. The Ewens-Watterson test for neutrality showed that one or two loci across different age classes of Norway spruce were affected by the selection process. The distinctiveness of embryo and seedling classes was confirmed by the number of detected alleles, the number of private alleles, the level of observed heterozygosity, and Wright’s index value. The results obtained in the present study indicate there are interesting dynamics of adaptation processes occurring in the natural age-diverse population of Norway spruce in the Bia?owie?a Primeval Forest.     

挪威云杉及杂种落叶松稳定胚性细胞系及体胚再生系统的建立

本文报道了挪威云杉(Picea abies)和杂种落叶松(Larix deciduas× L. kaempferi)人工授粉种子诱导胚性细胞系及植株的再生.挪威云杉在德国Saxon Ore 山区被选为对空气污染有抗性的树种并种植在位于Waldsieversdorf的林木遗传育种研究所的种植园中.对挪威云杉测试了几个不同亲本组合的种子形成胚性细胞系的能力.这些云杉种子于1992-01采收,含有干燥的成熟合子胚.作为对照,相同的亲本组合云杉种子于1993-08和1998-08采收,合子胚处于成熟中.杂种落叶松则只有一个人工授粉组合,大田试验证实幼苗生长良好.分别于1995和1996年采收含有未成熟合子胚的未成熟种子.诱导愈伤形成用附加细胞分裂素(激动素和/或BAP)和生长素(NAA, 2,4-D)及有机氮源(水解酪蛋白和谷氨酰胺)的标准培养基(SPE和MSG),于黑暗下培养.尽管在建立培养体系后5～12周产生了大量胚性愈伤组织,诱导率超过25%,但1 a后只有超过1%的胚性愈伤组织稳定生长.挪威云杉稳定胚性细胞系的数量与成熟合子胚的采收季节及建立细胞系的时间(8月或1月)无关,但在8月收获的合子胚在挪威云杉5～12周后胚性愈伤组织诱导率要高些,然而能在悬浮培养中生长及随后成熟体胚能顺利再生植株的稳定胚性细胞系的数量有限.挪威云杉体胚未充分发育的根影响其移栽到土壤中的成活率.在建立的12个细胞系中只有一个在干燥保存后能从体胚再生正常植株.同时考察了影响挪威云杉形成成熟体胚的因素,如用PEG4 000(7.5%)及ABA[8mg·L-1(30.26μmol)-1]处理及ABA灭菌方式(高温高压灭菌或过滤灭菌)对成熟体胚形成的影响.总体来说,过滤灭菌的ABA增加了成熟体胚的数量.营养培养基中用淀粉部分取代蔗糖只导致2个细胞系中的一个增加了成熟体胚的数量.1995和1996年对人工授粉杂种落叶松合子胚发育与自然授粉(不套授粉袋)的相比较,发现人工授粉袋的使用延迟了合子胚的发育.我们的实验中成熟合子胚没有形成胚性细胞系,只有未成熟合子胚形成了胚性细胞系.1995年用落叶松未成熟合子胚建立的胚性细胞系没有形成体胚,而1996年建立的4个胚性细胞系得到了可以移栽到温室中的再生植株.1.5 a的连续继代培养以后,大多数的杂种落叶松和挪威云杉的胚性细胞系的再生能力都下降.导致这种现象可能有多种原因包括培养物的老化以及出现难以检测到的内生细菌.尽管一组实验从最初培养即用了抗生素,但没有收效.因此目前只得到少量的稳定胚性细胞系(<5%),长期继代后丧失再生能力及无法成功移栽到土壤都是植株再生的重要的限制因素,有待进一步研究克服这些困难.     

Maturation and plant recovery from embryogenic cells of Japanese larch: Effect of abscisic acid in relation to their morphology

Two embryogenic cell lines characterized by different morphology and color, white and red, were obtained from an immature zygotic embryo of Japanese larch (Larix leptolepis Gord.). Mature somatic embryos with cotyledons and regenerated plants were obtained from both cell lines. However optimal concentration of abscisic acid (ABA) for maturation varied depending on morphology of ECs. From the white ECs which intermingled with somatic embryos of very early stage, mature somatic embryos were induced on maturation medium containing 50 μM of ABA. On the other hand, mature somatic embryos with cotyledons were observed from the red ECs which consisted of somatic embryos of more developed stage on hormone-free medium or 0.1 μM ABA containing-medium.     

Endogenous ascorbic acid modulates meristem reactivation in white spruce somatic embryos and affects thymidine and uridine metabolism

Previously, we demonstrated that, in some cell lines, exogenous applications of ascorbic acid (ASC) enhance the conversion frequency of white spruce (Picea glauca (Moench) Voss) somatic embryos, by stimulating mitotic activity in the apical meristems. To examine this event in more detail, we investigated the effects of ASC on de novo, salvage and degradation pathways of pyrimidine metabolism by following the metabolic fate of (14)C-labeled orotic acid, thymidine, uridine and uracil in shoot and root poles of germinating embryos, after altering the cellular ASC content of the embryos. Alterations in endogenous ASC content did not affect the utilization of either orotic acid or uracil, but affected the metabolism of thymidine and uridine. Specifically, a lowering of endogenous ASC content by applications of lycorine (L), an inhibitor of the last enzyme of the ASC de novo biosynthetic pathway, resulted in a lower embryo conversion frequency, as well as a reduced percentage of thymidine and uridine incorporated into nucleotides and nucleic acids. The reduction in thymidine and uridine anabolism was mainly ascribed to the decreased activities of thymidine kinase (TRK) and uridine kinase (URK), the respective salvage enzymes of thymidine and uridine, measured in L-treated embryos. These effects were solely a result of a decrease in endogenous ASC content because applications of ascorbic acid plus lycorine (ASC + L) increased embryo conversion frequency, thymidine and uridine salvage activities, and TRK and URK activities to near control values. Inclusion of exogenous ASC in the germination medium did not affect the percentage of embryos able to convert to viable plantlets, although it increased thymidine and uridine utilization for nucleic acid synthesis in the shoot and root poles of the embryos. Taken together, these findings confirm that cellular ASC plays a key role in the reactivation of the apical meristems of germinating white spruce somatic embryos.     

Polyamines in embryogenic cultures of Norway spruce (Picea abies) and red spruce (Picea rubens)

Embryogenic cultures of red spruce (Picea rubens Sarg.) and Norway spruce (Picea abies (L.) Karst.) were initiated from dissected mature zygotic embryos. The tissues were grown on either proliferation medium or maturation medium. On proliferation medium, the embryogenic tissue continued to produce early stage somatic embryos (organized meristems attached to elongated, suspensor-like cells), whereas on maturation medium fully mature embryos developed from the embryonic tissue. Analysis of polyamines in tissues grown on these two media showed that: (1) both putrescine and spermidine concentrations were always higher in cultures grown on proliferation medium than in cultures grown on maturation medium; (2) in both species, spermidine concentrations declined with time in the tissues grown on maturation medium; and (3) spermine was present in only minute quantities and showed only a small change with time. The presence of difluoromethylornithine in the culture medium had little effect on polyamine concentration, whereas the presence of difluoromethylarginine caused a decrease in putrescine concentrations in both red spruce and Norway spruce tissues grown on proliferation medium or maturation medium.     

Initiation of somatic embryogenesis from immature zygotic embryos of oocarpa pine (Pinus oocarpa Schiede ex Schlectendal)

The focus of the current project was to establish somatic embryogenesis protocols for the tropical pine species Pinus oocarpa using immature zygotic embryos (ZEs) as explants. Somatic embryogenesis is best supported by mimicking natural seed-embryo developmental conditions, through a tissue culture medium formulation based on the mineral content of the seed nutritive tissue [megagametophyte (MG)]. A novel culture medium (P. oocarpa medium, PO) was tested in combination with different plant growth regulator (PGR) concentrations and compared with standard Pinus taeda media for the initiation of somatic embryogenesis from immature ZEs of P. oocarpa. Immature MGs containing immature ZEs of two mother trees were used with 12 and 8% extrusion rates for mother tree genotypes 3 and 5, respectively. In both mother trees the percentage capture was 2%. Multiplication of two captured cell lines (T5C2S01 and T5C1S12) was improved by lowering the concentrations of PGRs to 2.5 μM each 2,4-dichlorophenoxyacetic acid and abscisic acid (ABA) plus 1.0 μM each 6-benzylaminopurine and kinetin. Mature somatic embryos formed on 40 μM ABA, 6% (w/v) maltose, 12% (w/v) PEG 8000 and 0.6% (w/v) Phytagel. While PO medium appeared suboptimal for somatic embryo induction, it did exhibit potential for enhanced culture proliferation and subsequent improved maturation with cell line T5C2S01, where microscopic analysis revealed better embryo morphology on PO medium than on 1250 medium. However, this enhancement was not observed with cell line T5C1S12. Germination was preceded by partial desiccation for a period of 2-3 weeks before transferring the embryos to germination medium. Germination was observed after 7 days under low light, and apical primordia slowly expanded after transfer to ex vitro conditions. To our knowledge, this is the first report on the production of somatic seedlings in P. oocarpa.