Assessment of Age-Related Polyploidy in Quercus Robur L. Somatic Embryos and Regenerated Plants Using DNA Flow Cytometry

Flow cytometric analysis with 4,6-diamidino-2-phenylindole (DAPI) staining was used to screen for chromosomal changes in Quercus robur during in vitro culture. The initiated cell lines (1992 until 1999) were maintained via secondary embryogenesis on P24 medium with 0.9 M 6-benzylaminopurine (BAP) in regular subculture intervals of 6 weeks. Regenerated plants established in the greenhouse and in vitro plantlets derived from encapsulated somatic embryos were screened. The embryogenic cell lines were characterized as individual clones by isoenzyme analysis. Flow cytometric relative DNA content analysis of the first screening period revealed that somaclonal variation in form of tetraploidy occurred in two out of 26 tested somatic embryo clones (Alt and Jung). These two clones lost their ability to convert into plantlets. Intraspecific relative DNA content variation including technical variation was below 3 %. In the second screening period, however, 3 out of 37 clones (Alt, E4.31H9 and P3.27H) contained tetraploid cells leading to the assumption that the frequency of tetraploidy seems to be correlated with the duration of in vitro culture. No chromosomal differences were detected in regenerated plants. However, tetraploidy occurred in 8 % of the tested clones over a culture period of 7 years.     

栓皮栎体细胞胚胎发生的细胞组织学观察

以栓皮栎未成熟合子胚为外植体,在添加0.25mg/L 2,4-D和0.5mg/L 6-BA的MS培养基上6周可诱导产生2种类型的胚性愈伤组织,一种表面具光泽、白色;另一种表面光滑湿润具光泽,色泽淡黄或无色透明。组织切片表明,胚性愈伤组织的细胞体积小,细胞核大,细胞质浓,细胞排列紧密;非胚性愈伤组织细胞的体积大,细胞核小,细胞质稀薄。胚性细胞团培养在不含激素的培养基上可诱导产生体细胞胚。体细胞胚直接起源于胚性细胞团表皮或近表皮的单细胞,经历与合子胚相似的球形胚、心形胚、鱼雷胚和子叶胚发育阶段。所有发育时期的体细胞胚的胚轴、子叶均产生次生体胚,它们起源于细胞质较浓的表皮单细胞。     

Factors Influencing the Induction and Viability of Somatic Embryos of Quercus Robur L.

To induce somatic embryogenesis in Quercus robur L. immature zygotic embryos at different developmental stages were collected in weekly intervals from June until September in three consecutive years from four open pollinated trees at two Vienna sites. Acorns were surface sterilised and cultured firstly on P24 medium with 5μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 μM 6-benzylaminopurine (BAP) or on hormone-free P24 medium and secondly on P24 medium with 0.9 μM BAP. The formation of white-yellow globular structures of somatic embryos started during the fourth week after the induction treatment. High induction frequencies of 30 - 80 % were achieved on 2,4-D/BAP medium, whereas rates on hormone-free medium were below 20 %. The initiation of somatic embryogenesis was favoured in the heartshaped and early cotyledonary stage of the zygotic embryo in all three years and lasted until the acorns reached maximum size in August. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic embryogenesis in leaf callus from a mature Quercus suber L. Tree

Summary Somatic embryos were obtained from a 60-yr-old Quercus suber L. tree. Leaf explants were cultivated on Murashige and Skoog medium with 30 gl⁻¹ sucrose, 3 gl⁻¹ gelrite, pH adjusted to 5.8, and different growth regulator combinations. Callus induction took place at 24±1°C in the dark during the first 3 wk. After 3 mo, calluses that showed embryogenic structures were transferred to the same medium without growth regulators. Somatic embryogenesis was only observed in calluses induced on E3 medium (supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid and 9.0 μM zeatin). On average, 7.5% of the initial explants formed embryogenic calluses in this medium. Somatic embryo proliferation was high due to secondary embryogenesis. On average, 10% of the somatic embryos germinated and 40% of these germinated embryos converted into plants. Plants were elongated on the same medium without growth regulators and acclimated to greenhouse conditions.     

花楸合子胚诱导体细胞胚胎发生研究

分别以完整成熟胚、切去一个子叶的成熟胚和切下的子叶为外植体,以MS为基本诱导培养基、1/2MS为基本分化培养基,进行了花楸体细胞胚胎发生研究。结果表明:以完整合子胚作为外植体的体胚诱导率最高,为100%,最佳植物生长调节剂组合为5 mg.L-1NAA+2 mg.L-16-BA;NAA和6-BA浓度及二者的交互作用对愈伤组织和体胚诱导率的影响极显著;光照配合延长继代间隔时间有利于体胚发生。实体观察结果表明,花楸体胚发生方式有直接发生和间接发生两种;体胚发育经历了球形期、心形期、鱼雷形期和子叶期。组织学观察结果表明,体胚具有两极性,子叶期体胚结构完整。     

早熟棉体细胞胚胎发生和植株再生体系的建立

以8个早熟或特早熟棉花为材料,通过不同激素组合(1.0 mg/L IBA+0.5 mg/L KT;0.1 mg/L 2,4-D+0.1 mg/L KT)诱导其愈伤组织,为早熟棉花的遗传转化以及基因功能研究奠定基础.结果表明,2种激素组合均能诱导产生4种主要类型的愈伤组织:淡黄色颗粒状愈伤组织,褐化的愈伤组织,翠绿致密的愈伤组织,疯长型愈伤组织.4种愈伤组织转入增殖培养基中前2种愈伤组织能够分化出胚性愈伤组织,胚性愈伤组织再转到分化培养基上培养能够产生胚状体,即体细胞胚;体细胞胚进一步发育成为再生小苗.用该组织培养再生系统,成功地使晋棉5号、中棉27号和辽棉10号等3个早熟棉品种在5～7个月内通过体细胞胚胎分化获得再生小苗.     

黑龙江省不同地点蒙古栎林生态特点研究

通过对黑龙江省６个地点的天然蒙古栎林的结构和更新特点的分析,蒙古栎林可划分为不同特点的蒙古栎群落,即纯蒙古栎群落、蒙古栎桦树群落、蒙古栎槭树群落、蒙古栎红松群落和蒙古栎松混交林群落,其演替趋势如下：红蒙古栎群落、蒙古栎桦树群落至蒙古栎槭树落、蒙古栎红松群落,再至蒙古栎红松混交林群落,蒙古栎群落类型的多样性主要反应群落不同的演替阶段,造成蒙古栎群落多样的原因是人类活动和自然因素作用的结果,随着群落的演替,蒙古栎的优势地位逐渐被消弱,乔木种类丰富度增多,草本种类丰富度增多;蒙栎的相对密度下降,林内环境由于干燥逐渐变中性至较湿润,蒙古栎幼苗和幼树在总幼苗和幼树中所占的比例下降,耐荫物中色木槭等的幼苗和幼树所占比例上升;在演替过程中,蒙古栎分布格局－聚集分布的聚集程度逐渐降低,并向随机分布的方向发展。     

Somatic Embryogenesis from 20 Open-Pollinated Families of Portuguese Plus Trees of Maritime Pine

Immature zygotic embryos from 20 open-pollinated (OP) families of maritime pine (Pinus pinaster) plus trees were screened for their somatic embryogenic capacity. The best time for zygotic embryo collection was between 30th June and 16th July 1999 when most embryos were at a pre-cotyledonary stage of development. The somatic embryogenesis (SE) initiation frequency was highest on DCR basal medium with 13.6 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 µM 6-benzylaminopurine (BAP) supplemented with L-glutamine and casein hydrolysate. On this medium, initiation frequencies among OP families ranged from 4.6 to 49.1%. Initiation of embryogenic cell lines from all 20 OP families was possible only on DCR based medium, but the addition of L-glutamine and casein hydrolysate significantly increased the number of zygotic embryos producing SE. Most families showed a similar behaviour on different initiation media; however, a few exceptions were observed. Further development of somatic embryos on maturation medium, consisting of DCR with 120 µM abscisic acid (ABA), 100 g l^–1 polyethylene glycol (PEG) and 10 g l^–1 gellan gum, occurred in 29% of 896 embryogenic lines representing all 20 OP families. However, development into cotyledonary somatic embryos was observed in only 11% of the cell lines, but this still represented 18 OP families.     

Somatic Embryogenesis and Plantlet Regeneration from Callus of Mature Zygotic Embryos of Masson Pine

Embryogenic cultures were initiated from mature zygotic embryos of masson pine (Pinus massoniana Lamb. ) on DCR medium supplemented with 2, 4-D 10 mg/L, KT and BA each at 4 mg/L. Pale and translucent calli with early stage proembryos were maintained and multiplicated on DCR medium supplemented with 2, 4-D 1.0 mg/L KT and BA each at 0.4 mg/L. Robust late-stage proembryos were obtained when the calli were cultured on DCR medium containing 9000 mg/L myo-inositol. Abscisic acid and activated charcoal promoted the formation of cotyledonary embryos at the highest frequency of 35.1 %. Mature somatic embryos could germinate and develop further into plantlets when they were isolated and cultured on a hormone-free DCR medium.     

Efficient Somatic Embryogenesis in Alstroemeria

In Alstroemeria high frequencies of compact embryogenic callus (CEC) induction (40%) and friable embryogenic callus (FEC) induction (15%) were obtained from nodes with axil tissue cultured first on a Murashige and Skoog (MS) medium supplemented with 10 μM thidiazuron and 0.5 μM indole-3-butyric acid and after that on a Schenk and Hildebrandt (SH) medium supplemented with 9.1 μM 2,4-dichlorophenoxy acetic acid and 2.2 μM benzylaminopurine (BA). Both types of callus were maintained on modified MS medium supplemented with 20.8 μM picloram. CEC and FEC formed somatic embryos and subsequently plants when transferred to MS medium supplemented with 2.2 μM BA. Plants were produced after 12 weeks (CEC) or after 16 weeks (FEC) of culture. Regenerated plants were established in the greenhouse and flowered normally.     

Somatic Embryogenesis in Leguminous Plants

Abstract: This review examines recent advances in the induction and development of somatic embryos in leguminous plants. Emphasis has been given to identify the current trends and successful strategies for the establishment of somatic embryogenic systems, particularly in the economically important species. It appears that, in legumes, somatic embryogenesis can be realized relatively easily especially in young meristematic tissues such as immature embryos and developing leaves. In the majority of the species examined, chlorophenoxyacetic acids remained the most active inductive compounds; however, the new generation growth regulators such as thidiazuron are emerging as successful alternatives for high-frequency direct regeneration of somatic embryos, even from well differentiated explant tissues. Low-frequency embryo production, poor germination and conversion of somatic embryos into plantlets and somaclonal variation are the major impediments limiting the utility of somatic embryogenesis for biotechnological applications in legumes. These limitations, however, may be considerably reduced in the near future, as more newly developed growth regulators with specific morphogenic targets become available for experimentation. From the published data, it is apparent that more effort should be given to develop repetitive embryogenic systems with high frequency of germination and regeneration, since such systems will find immediate application in mass propagation and other crop improvement programmes. As our understanding of various morphogenic processes, including growth and differentiation of zygotic embryos, is fast expanding, it is conceivable that development of highly efficient somatic embryogenic systems with practical application can be anticipated, at least for the important leguminous crops, in the foreseeable future.     

Strong differences in Quercus robur-associated ectomycorrhizal fungal communities along a forest-city soil sealing gradient

Ectomycorrhizal fungi (EM) that associate with tree roots in a symbiotic relationship may be crucial in mediating tree health in urban environments, but research on the effects of urbanization on EM communities is very limited so far. Here, we compared EM communities of adult pedunculate oaks (Quercus robur) between urban and forest environments, and assessed the effect of soil sealing around the trees on their EM community composition and EM diversity. We sampled 32 oak individuals across 4 sampling classes (Street, Lane, Park and Forest), and we characterized their EM communities using 454 amplicon pyrosequencing. The EM communities were not nested but they were significantly different between all sampling classes, with a very strong community differentiation between forest and urban trees. There were indications that EM richness declined with increasing sealed soil surface, with a significant effect of sampling class on estimated EM richness and diversity. We also identified indicator EM of the different sampling classes. The most important soil factor affecting EM community composition was pH, followed by plant available phosphorus, total nitrogen content and organic matter. Our results may have important implications when developing EM inocula for managing tree health in urban environments.     

麻栎成熟合子胚外植体体胚发生和植株再生

为探索麻栎快速繁殖技术新途径,以成熟合子胚为外植体诱导体胚发生,进一步培养形成幼苗。结果表明:体胚诱导以MS+1.0mg.L-1 6-BA+1.0mg.L-1 IBA+1.0g.L-1谷氨酰胺+0.5g.L-1脯氨酸为最优,培养30d诱导率达70.0%;体胚成熟以1/2MS+2.0mg.L-1 6-BA+0.5mg.L-1 IBA+2.0mg.L-1 ABA+4.0g.L-1谷氨酰胺+2.0g.L-1脯氨酸为佳,培养60d,体胚完全成熟;体胚萌发最适培养基为1/2MS+0.2mg.L-1 6-BA+10g.L-1山梨醇,且冷处理有利于体胚的萌发,萌发率高达100%,萌发培养80d,形成再生植株。     

麻栎茎段体胚发生和组织学观察初报

分别于2010年4月和6月采集同一麻栎母株的成熟茎段,在培养基MS+6-BA 1 mg·L^-1+IBA 1 mg·L^-1+谷氨酰胺1 000 mg·L^-1+脯氨酸500 mg·L^-1上诱导体胚发生,选出较好的外植体采集时间。通过组织学和扫描电镜观察,研究麻栎体胚的起源和愈伤组织结构特征。结果表明,外植体较好的采集时间为4月。组织切片表明,麻栎体胚具有三种不同的起源方式,起源于愈伤组织内部、表皮或者初生胚性复合体表面;胚性愈伤组织的细胞较小,细胞质浓厚,染色较深,和非胚性组织细胞明显不同。扫描电镜(SEM)观察表明,胚性愈伤组织细胞呈球形,大小均一,多以细胞团形式存在;非胚性愈伤组织细胞无规则形状,细胞间隙较多,大多分散存在,这些可以作为区分两类愈伤组织的典型特征。     

Litter dynamics in post-fire successional forests of Quercus ilex

Forest floor regeneration after fire in a holm oak forest was studied in a chronosequence in Catalonia (NE Spain). The sampled area represented situations of high fire disturbance because of the frequency of wildfires and the high impact of erosive processes. Forest floor bulk samples from the L, F and H layers were taken in 5 burned plots, aging from 0 to 35 years. They were analyzed for dry standing weight, and organic carbon and N content.Plant cover reached almost 100% two years after the fire. During the first 20 years, shrubs and herbs had the highest surface covering, and thereafter, holm oaks became dominant.Holm oak litterfall stabilized by the fourth year after burning.The major shrub species which were present in the chronosequence had two different patterns with respect to nitrogen: 1) a low litter C/N ratio (i.e. nitrogen fixing species); 2) a high litter C/N ratio.The litter decay coefficients were relatively high, allowing for a rapid structuration and formation of the L and F layers after the fire. Indeed, 95% of the maximum steady standing weight accumulates in 8–9 years. The pattern of cover in the different strata influenced the lateral distribution and variable accumulation of the forest floor layers and determined different turnover rates.     

Dendroclimatic regions of pedunculate oak (Quercus robur L.) in Belarus

In this study, we present the boundaries of five dendrochronologically homogeneous regions of pedunculate oak (Quercus robur L.) and the master chronologies for them for the period 1703–2018 based on 27 tree-ring chronologies from different locations around Belarus. The average length of the master chronologies ranges from 70 to 310 years. Our research shows that the radial increment of a pedunculate oak in Belarus is primarily limited by the previous year’s August-September temperature, by the current year’s May temperature and by June precipitation. Oak growth in northern Belarus is mostly limited by air temperature, whereas precipitation plays more significant role in central and southern parts of the country. The most unfavorable years for oak growth in Belarus were 1940 and 1952, both of which were years when a very frosty winter was followed by a droughty summer. The results provide an important achievement in Belarusian dendrochronology, since they represent a significant breakthrough for Eastern Europe, where there is a considerable lack of dendroclimatic researches of broadleaved species.     

林木基因克隆研究进展

林木种质资源丰富, 种质间遗传差异大, 控制林木重要性状的基因克隆及转化对培育优良林木新品种具有很强的实用价值, 但许多具有潜在应用价值的林木基因未得到充分发掘和有效分离。近年来, 随着各种不同林木cDNA文库的建立, 大规模随机EST测序技术的运用以及克隆技术的不断完善, 特别是毛果杨(Populus trichocarpa)基因组测序计划的完成, 大量与林木重要性状相关的基因被分离和鉴定。这些重要基因的获得为利用转基因技术培育高产、优质、抗逆、抗病虫害的林木新品种奠定了一定的基础。该文综述了20多年来国内外林木基因克隆的研究进展, 对基因克隆及其应用过程中亟待解决的问题进行了讨论, 并对其发展趋势进行展望。     

Somatic Embryogenesis or Shoot Formation Following High 2,4-D Pulse-Treatment of Mature Embryos of Paspalum scrobiculatum

Mature zygotic embryos of Paspalum scrobiculatum L. cv. PSC 1 on MS or N₆ nutrient medium supplemented with various concentrations of 2,4-D (4.5 – 22.5 μM) formed embryogenic callus, which differentiated into somatic embryos within 5 weeks of culture. The somatic embryos after transfer to hormone-free regeneration medium germinated and formed plantlets. Of the two nutrient formulations, N₆ was relatively better than MS for somatic embryogenesis. A culture for 11 d on 100 μM 2,4-D was essential for the establishment of an embryogenic callus. Shorter duration, 4-d or 7-d culture on 2,4-D medium, supported some proliferation and subsequent differentiation into shoot-buds or multiple-shoots, in high-frequency cultures. This is first instance in monocots of a controlled regeneration response; either somatic embryogenesis or shoot formation. This revised version was published online in July 2006 with corrections to the Cover Date.     

High Frequency Somatic Embryogenesis in Cotton

A highly reproducible system for efficient somatic embryogenesis was developed to regenerate plantlets from cotton (Gossypium hirsutum L.) cultivars (Nazilli M-503 and Nazilli 143). Shoot apices, hypocotyls and nodes of 10-d-old seedlings were used as explants. High frequency (100 %) embryogenic calli was initiated from all tested explants on Murashige and Skoog (1962) (MS) media supplemented with 1 g dm^–3 polyvinylpyrrolidone (PVP), 1 mg dm^–3 6-benzylaminopurine (BAP), 0.5 mg dm^–3 kinetin for Nazilli M-503 and 1 g dm^–3 PVP, 2 mg dm^–3 BAP, 0.5 mg dm^–3 kinetin for Nazilli-143. Globular stage somatic embryos were produced 4 months after transfer to hormone-free MS medium supplemented with 1 g dm^–3 PVP. Subsequent subculture of globular embryos every 3 weeks on hormone-free MS medium led to the development of torpedo and cotyledonary stage embryos with the frequency of 75 and 83.2 % from hypocotyl explants of Nazilli M-503 and Nazilli-143, respectively. Afterwards, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination and development into plantlets. The highest germination frequency (42.9 %) for Nazilli M-503 somatic embryos were obtained on hormone-free MS medium after 5 months with hypocotyl explants, whereas germination frequencies of Nazilli-143 embryos from hypocotyl, node and apex explants varied between 22 – 30 %.     

棉花体细胞胚胎发生的研究进展

经过30多年的发展, 已经在多个不同的棉花品种中获得了体细胞胚, 并再生成苗。但由于体细胞胚胎发生往往受多种因素影响, 如何从愈伤组织高效率地转化为胚性愈伤组织依然是限制棉花组织培养与遗传转化的关键问题之一。本文概述了棉花体细胞胚胎发生的研究进展, 分别从棉花体细胞胚胎发生的起源、影响棉花体细胞胚胎发生的内外因素以及寻找棉花体细胞胚胎发生特异表达基因等几方面进行综述, 并对研究中存在的问题进行了讨论。