Protective enzymatic systems against activated oxygen species compared in normal and vitrified shoots of Prunus avium L. L. raised in vitro

Vitrification of shoots of Prunus avium L. L. was induced and expressed in a four week in vitro multiplication cycle simply by replacing agar by gelrite. The first vitrification symptoms were visible from the 7th day on. Enzymatic antioxidants were compared weekly in crude extract of normal (on agar) and vitrifying (on gelrite) shoots. The activity of superoxide dismutase was higher in vitrifying shoots. The other enzymes (gaîacol-peroxidase, catalase, ascorbate peroxidase, mono- and dehydro-ascorbate reductases, glutathione reductase) had lower activities. Increased superoxide dismutase activity might mean hydrogen peroxide accumulation and decreased activities of the other enzymes, deficiency in its detoxification. The question therefore is raised whether the hyperhydric morphological abnormalities result from the accumulation of toxic oxygen forms. Vitrification is often considered as a morphological response to several stresses. Contrary to most plants which adapt themselves to stresses by increasing all the above defence enzymes, in vitro shoots under vitrifying conditions appear unable to react in a similar manner.Abbreviations Apx ascorbate peroxidase - Gpx gaîacol peroxidase - CAT catalase - H₂O₂ hydrogen peroxide - SOD superoxide dismutase - MDHAR monodehydroascorbate reductase - DHAR dehydroascorbate reductase - GR glutathione reductase - MS Murashige and Skoog (1962) - IBA indolebutyric acid - BAP benzyladenine - GA₃ gibberellic acid     

Chloroplast DNA study in wild populations and some cultivars of Prunus avium L.

The PCR-RFLP technique was used to detect chloroplast DNA diversity in wild populations of Prunus avium from five European deciduous forests and some cultivars. A study of 10.8% of the total chloroplast genome detected eight insertion-deletion (indel) mutations, distributed over 12 haplotypes. Six haplotypes (H1, H2, H3, H4, H5 and H6) were found in wild populations and eight (H2, H6, H7, H8, H9, H10, H11 and H12) in the cultivars. Only two haplotypes (H2 and H6) are shared by the wild populations and the cultivars. The most-abundant and frequent haplotype in wild populations is H2 (frequency=78%). The wider geographical distribution along with the high frequency reflects its ancient origin. Of the five populations, three are polymorphic. Populations GA (Scotland) and KE (Germany) have unique haplotypes. The total cpDNA diversity in wild populations is h_T=0.40, and a major portion of it is within populations (h_S=0.37). The genetic differentiation among populations was low (G_STC=0.08) and no genetic structure among wild populations was observed. A minimum-length spanning tree, demonstrating relationships among the haplotypes in wild populations, indicated two possible chloroplast lineages. The ten identified cultivars were represented by seven haplotypes; this result proposes the possible utilisation of the PCR-RFLP technique for the characterisation of sweet cherry cultivars. The cpDNA diversity in P. avium should be considered carefully for phylogenetic studies involving this species. Received: 10 July 2000 / Accepted: 19 October 2000     

弱光对三年生油桃源叶韧皮组织超微结构的影响

以日光温室内栽培的三年生油桃"早红艳"(Prunus persica L. var. nectarine Ait "Zao Hong Yan")为试材,利用透射电子显微镜(TEM)技术首次对处于正常光照条件和弱光条件下的源叶的韧皮组织进行了超微结构观察,重点研究了叶片超微结构对弱光的响应.结果表明:在正常光照条件下,油桃源叶各级叶脉的筛管和伴胞的平均直径均比弱光条件下的大,表明光能影响细胞的发育,而弱光抑制了细胞的生长.在正常光照下的伴胞具有致密的细胞质,内含丰富的线粒体、内质网、多泡体、囊泡和质体,而弱光下的伴胞明显液泡化,同时含有少量的线粒体和内质网.在弱光条件下也观察到叶绿体基粒片层边缘紊乱,筛孔处于阻塞状态.在弱光下,邻近维管束鞘细胞的叶肉细胞内积累了大量的淀粉粒和少量的线粒体.淀粉粒的积累可能是由于光合同化产物的生产与输出之间的不平衡,这一观察结果有力地支持了大部分叶片是在光照条件下输出其大部分同化产物的观点.在筛管/伴胞、伴胞/韧皮薄壁细胞、韧皮薄壁细胞/韧皮薄壁细胞和韧皮薄壁细胞/维管束鞘细胞之间的胞间连丝密度都在弱光条件下下降,在正常光照强度下支脉筛管/伴胞和韧皮薄壁细胞/维管束鞘细胞之间可以观察到胞间连丝,而在弱光下几乎观察不到胞间连丝的存在,所以同化物的运输在弱光条件下可能以质外体运输为主,而在正常光照强度下,共质体运输可能是主要的运输方式.这些研究结果证明,日光温室内油桃通过源叶韧皮组织超微结构的变化对弱光做出适应性响应.     

Comparison between woody indexing and a rapid hybridisation assay for the diagnosis of little cherry disease in cherry trees*

Sweet cherry (Prunus avium) and sour cherry (Prunus cerasus) trees from orchards in the Kootenay and Okanagan Valleys of British Columbia, Canada were assayed for the presence of little cherry disease by three different methods: Northern blot analysis of double-stranded RNA, woody indexing for fruit symptoms on sweet cherry cv. Lambert, and woody indexing for foliar symptoms on cv. Canindex 1. Results of the three methods were in agreement for 85% of the samples. Of the 78 orchard trees tested, double-stranded RNA isolated from 48 trees hybridised with a radiolabeled cloned probe specific for little cherry disease. When the 48 trees were tested by woody indexing, buds from 41 trees induced fruit symptoms on cv. Lambert, but only 32 yielded foliar symptoms on cv. Canindex 1 under the conditions of the experiment. Of the 30 orchard trees that did not yield a positive response to the Northern blot analysis, 26 samples were negative on cv. Lambert and 26 were negative on cv. Canindex 1. Northern blot analysis of the 78 cv. Lambert indicator trees revealed that there was an absolute correlation between the presence of little cherry disease-associated double-stranded RNA and the development of typical little cherry disease symptoms on the indicator trees. Reliability of woody indexing of orchard samples was impaired by poor transmission of the disease from the inoculating bud to the indicator tree. Woody indexing with cv. Canindex 1 was particularly prone to a large number of apparently erroneous negative results. Of the three protocols used, diagnosis of little cherry disease by Northern blot analysis was found to be the most reliable and offered a greatly accelerated means of diagnosis.     

Effects of low light on phloem ultrastructure and subcellular localization of sucrose synthase in <Emphasis Type="Italic">Prunus persica</Emphasis> L. var. <Emphasis Type="Italic">nectarina</Emphasis> Ait. fruit

Using electron microscopy, the ultrastructure of phloem unloading zone was examined in the Prunus persica L. var. nectarina Ait. fruit. Our study showed that, in the SE/CC (sieve element/companion cell) complexes, CC developing under low light had a thin cytoplasm layer with few mitochondria and numerous small vacuoles, and not clearly seen nuclei. The cytoplasm vacuolation indicated that the cytoskeleton was destroyed at low light. The effects of low light on CC development suggest that unloading evidently linked to the low accumulation of soluble sugars by fruit. At the young fruit stage, flesh parenchyma around the phloem tissue had no starch grains in the plastids in fruit developing under low light. This is a further indication that less photoassimilates was translocated from source leaves to fruit sinks under low light during the young fruit developmental stage. The activity of sucrose synthase (SuSy), the key enzyme of sucrose metabolism in fruit, increased dramatically during fruit maturation. The highest SuSy activity during the rapid fruit growth phase suggests that sink strength could be correlated with the SuSy activity. The high SuSy activity under normal light possibly indicates that fruit had a capacity to utilize sucrose irrespective of their site of phloem unloading. Immunogold electron microscopy showed that SuSy was localized mainly in the vacuole of flesh parenchyma cells. The vacuole-localized SuSy can hydrolyze sucrose imported from the phloem, which may explain the apparent correlation between SuSy activity and phloem unloading. The double sieve element (SE/SE) complexes occurred in a greater number and had thicker cell walls under normal light intensity than under low light intensity. These data demonstrate clearly that low light decreased SuSy activity in the control of phloem unloading. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 509–517. This text was submitted by the authors in English.     

弱光对三年生油桃源叶韧皮组织超微结构的影响

以日光温室内栽培的三年生油桃“早红艳”(Prunus persica L．var．nectarine Ait“Zao Hong Yan”)为试材,利透射电子显微镜(TEM)技术首次对处于正常光照条件和弱光条件下的源叶的韧皮组织进行了超微结构观察,重研究了叶片超微结构对弱光的响应。结果表明：在正常光照条件下,油桃源叶各级叶脉的筛管和伴胞的平均直均比弱光条件下的大,表明光能影响细胞的发育,而弱光抑制了细胞的生长。在正常光照下的伴胞具有致密的胞质,内含丰富的线粒体、内质网、多泡体、囊泡和质体,而弱光下的伴胞明显液泡化,同时含有少量的线粒体和质网。在弱光条件下也观察到叶绿体基粒片层边缘紊乱,筛孔处于阻塞状态。在弱光下,邻近维管束鞘细胞的肉细胞内积累了大量的淀粉粒和少量的线粒体。淀粉粒的积累可能是由于光合同化产物的生产与输出之间的平衡,这一观察结果有力地支持了大部分叶片是在光照条件下输出其大部分同化产物的观点。在筛管／伴胞、伴／韧皮薄壁细胞、韧皮薄壁细胞／韧皮薄壁细胞和韧皮薄壁细胞／维管束鞘细胞之间的胞间连丝密度都在弱光条下下降,在正常光照强度下支脉筛管／伴胞和韧皮薄壁细胞／维管束鞘细胞之间可以观察到胞间连丝,而在弱光几乎观察不到胞间连丝的存在,所以同化物的运输在弱光条件下可能以质外体运输为主,而在正常光照强度下,质体运输可能是主要的运输方式。这些研究结果证明,日光温室内油桃通过源叶韧皮组织超微结构的变化对弱．做出适应性响应。     

Structural basis of the rind disorder oleocellosis in Washington navel orange (Citrus sinensis L. Osbeck)

Oleocellosis, a physiological rind disorder of citrus fruit, is an unattractive surface blemish caused by phytotoxic effects of released rind oils. The development of oleocellosis in Washington navel orange (Citrus sinensis L. Osbeck) was examined by following a time sequence of surface symptoms and microscopic rind changes. The two natural causes of oleocellosis were simulated: mechanical damage to the fruit and transfer of rind oil between fruit. Mechanical fruit injury resulted in rupture of the epidermis above oil glands. Released surface oil appeared to infiltrate the rind via the ruptured epidermis resulting in rapid degeneration of cortical, but not epidermal, cell contents. Oil application to the rind surface produced a more severe blemish than did mechanical damage. The oil appeared to diffuse through the cuticle causing degeneration of the contents of all cell layers, including the epidermis. Loss of membrane integrity was detected within 30 min, followed by cell content degeneration and cell collapse. The resulting blemish, characterized by rind collapse and darkening, developed substantially within 3 d and was attributed to the cellular damage.     

In vitro plant regeneration from leaves and internode sections of sweet cherry cultivars (Prunus avium L.)

Regeneration of adventitious shoots from leaves and, for the first time, from internode sections were compared and optimized for five economically important sweet cherry cultivars, i.e. Schneiders, Sweetheart, Starking Hardy Giant, Kordia and Regina (Prunus avium L.). The influence of basal media, carbon source, combination and dosage of phytohormones, ethylene inhibitor such as silver thiosulfate and a 16 h:8 h light:dark photoperiod versus complete darkness were evaluated. Both, DKW/WPM (1:1) and Quoirin/Lepoivre (QL) basal media stimulated organogenesis more than QL/WPM (1:1), Chee and Pool (CP), Murashige Skoog (MS), Driver and Kuniyuki (DKW) or woody plant (WPM) media did. An induction phase in darkness resulted in lower or zero regeneration rates. The best regeneration efficiencies were generally obtained with thidiazuron in combination with indole-3-butyric-acid. The addition of silver thiosulfate resulted in a similar or reduced regeneration efficiency. Significant genotypic variability in adventitious bud formation was evident for both explant sources, leaf and internode section. Adventitious shoots were obtained from 11% of leaf explants and 50% of internode sections indicating that shoot regeneration from internodes was significantly more efficient than from leaves.     

费菜和长药八宝叶形态结构及其与耐旱性关系的研究

利用光学显微镜观察比较了费菜（Sedum aizoon L.）和长药八宝（S. spectabilis Boreau）的叶表皮形态及解剖结构,并对其进行不同程度的水分胁迫,研究干旱逆境条件对其叶片显微结构的影响。结果表明：两种景天的叶形态、解剖结构与耐旱性之间存在较密切的相关性。两种植物成熟叶均具较薄的角质层和发达的储水组织,叶肉组织中没有栅栏组织和海绵组织的明显分化。二者的维管组织较发达,且具较发达的孔下室,维管束周边发现一些吸水能力较强的异细胞,这些特征是植物耐旱的关键。     

宁夏沙区沙拐枣属和柽柳属的引选及抗逆造林

宁夏分布有大面积的流动沙地和盐碱地,因此,引进适生的植物对治沙造林、改善生态环境有重大意义。本文报道沙拐枣属(Calligonum L.)和柽柳属(Tamarix L.)的引选及抗逆性造林技术方面的结果。     

Eimeria crotalviridis sp. n. from Prairie Rattlesnakes, Crotalus viridis viridis, in New Mexico with Data on Excystation of Sporozoites and Ultrastructure of the Oocyst Wall

SYNOPSIS Oocysts of Eimeria crotalviridis sp. n. are described from prairie rattlesnakes, Crotalus viridis viridis in New Mexico on the basis of light and electron microscopy and in vitro excystation of sporozoites. Sporulated oocysts of E. crotalviridis are elliptical, 26.4 × 22.3 (23–29 × 20–24) μm with ovoid sporocysts 11.7 × 8.1 (11–13 × 7–9) μm. A micropyle, micropyle cap and polar bodies are absent, but oocyst and sporocyst residua and Stieda and substieda bodies are present. Excysted sporozoites are 12.4 × 2.8 (11–13 × 2–3) μm and have 1 large posterior refractile body and a nucleus with a prominent nucleolus. Ultrastructurally, the oocyst wall has 2 layers, a thick, electron-dense, highly sculptured outer layer composed of a fine granular matrix and a thin, granular, osmiophilic inner layer, separated from the outer layer by at least one unit membrane. These layers are 441 (353–510) and 21.6 (19–29) nm thick, respectively. Within 15 min after exposure to a trypsin-sodium taurocholate fluid, sporozoites of E. crotalviridis excysted from 5-month-old sporocysts.     

The effect of temperature and ageing on root apical meristems during seedling growth of Triticum aestivum L.: a specific effect on nucleoli

Abstract. Growth and cytology of root apical meristems of Chinese Spring wheat were studied in relation to temperature. The maximum rate of growth increased with temperature, a marked rise occurring between 10°C and 12°C. At all temperatures studied nucleolar volume increased to a maximum and then declined. The maximum nucleolar size achieved showed particular temperature sensitivity, higher volumes being attained at lower temperatures. The peak at 5°C was 70% higher than at 20°C. However, in comparison, cell and nuclear volumes were only 38% and 47% larger, respectively. Ultrastructural analysis of the nucleoli revealed a temperature-dependent relationship between the proportion of granular component and dense fibrillar component. The results are discussed in relation to the regulation of ribosome synthesis and function during growth and development at different temperatures.     

A barcode analysis of the genus Lobophora (Dictyotales,Phaeophyceae) in the western Atlantic Ocean with four novel species and the epitypification of L. variegata (J.V. Lamouroux) E.C. Oliveira

In the western Atlantic Ocean, the brown algal genus Lobophora is currently represented by a single species, L. variegata, with a type locality designated by Lamouroux as ‘Antilles’. In this study, we used molecular-assisted alpha taxonomy (MAAT) to assess species diversity of Lobophora in Bermuda, the Florida Keys, St. Croix and Guadeloupe (Lesser Antilles). Using cox1 and cox3 sequences as barcode markers, five species of Lobophora, four of them novel, were delineated, all previously having been identified in the area as L. variegata. Our morphological and habitat studies, made possible by abundant sampling, have revealed unique characters for each of these western Atlantic species, including distinct cellular arrangements, as well as different depth ranges for certain species. Observations made from Lamouroux’s holotype of Dictyota variegata (= Lobophora variegata) allowed us to assess the anatomy of this species, which enabled us to easily align this early taxon to one of our genetic species from the western Atlantic. As the type was unavailable for genetic analysis, we selected a recent St. Croix (Virgin Is., Antilles) specimen as the epitype to support it with molecular sequence data.     

Morphometrics and SEM analysis of the species pair Lithobius mutabilis L. Koch, 1862 and L. glacialis Verhoeff, 1937 (Chilopoda: Lithobiomorpha)

The sets of morphological characters of two nominal species in the genus Lithobius Leach, 1814, Lithobius mutabilis L. Koch, 1862 and Lithobius glacialis Verhoeff, 1937, were studied in order to test specific dissimilarity. Morphometrics were examined and analysed statistically. In addition, a comparative SEM analysis of external characters was made. Morphometrically significant differences between the species were found concerning body length, the width ratio of the head to the 5th tergite, the number of antennal articles, and the length/width proportions of certain articles (e.g. the femur) of the 15th legs of males. Because of these and several additional differences, such as in tergite surface structure, modifications to the 15th legs of males, the structure of the female gonopod claws, and between the ecological profiles, L. glacialis, which is found in the high Alps at altitudes above timberline (1300 m), can be distinguished clearly from L. mutabilis. This study demonstrates another example for a cryptic species pair, a common phenomenon between lowland and alpine populations of small invertebrates. The combination of morphometrics and SEM offers a powerful methodology for resolving previously uncertain questions in the species-level taxonomy of centipedes.     

宁夏枸杞根和茎的化学成分及抗炎活性研究

研究宁夏枸杞(Lycium barbarum L.)根部和茎部的化学成分。采用硅胶柱、ODS开放柱、Sephadex LH-20葡聚糖凝胶柱及半制备反相高效液相等色谱手段,对宁夏枸杞根和茎部乙醇提取物的石油醚部位及乙酸乙酯部位化学成分进行分离纯化,根据其理化性质以及波谱数据鉴定得到12个化合物,分别为N-[2-(3,4-dihydroxyphenyl)-2-hydroxyethyl]-3-(4-methoxyphenyl)prop-2-enamide(1)、3-(4-hydroxy-3-methoxy phenyl)-N-2-(4-hydroxyphenyl)-2-methoxyethyl]acrylamide(2)、N-trans-coumaroyloctopamine(3)、(E)-2-(4,5-dihydroxy-2-{3-[(4-hydroxyphenethyl)amino]-3-oxopropyl}phenyl)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(4-acetamidobutyl)acrylamide(4)、1,2-dihydro-6,8-dimethoxy-7-hydroxy-1-(3,4-dihydroxy-phenyl)-N1,N2-bis[2-(4-hydroxyphenyl)ethyl]-2,3-naphthalene dicarboxamide(5)、(+)-syringaresinol(6)、zhebeiresinol(7)、(±)-eriodictyol(8)、isovanilin(9)、5,5′-dimethoxybiphenyl-2,2′-diol(10)、p-hydroxyphenethyltrans-ferulate(11)、E-ferulic acid hexacosyl ester(12),所有化合物均为首次从该植物中分离得到。此外,采用MTT法和抑制一氧化氮(NO)生成实验,从细胞毒活性和抗炎活性两方面评估了化合物的生物活性。结果表明,化合物2具有显著的抗炎活性,其IC50值(17.00±1.11μmol/L)小于阳性对照药槲皮素的IC50值(17.21±0.50μmol/L)。     

水烛的花部形态观察——兼论与长苞香蒲的分类学界定

通过腊叶标本研究、野外观察和文献考证,结合栽培试验,研究了香蒲科水烛（Typha angustifolia L.）花部结构特征,补充描述了该种丝状毛在子房柄上的着生方式、小穗不孕雌花数目等性状特征,观察了在成熟期不同阶段其孕性雌花柱头与小苞片的长度变异。结果显示,水烛孕性雌花小苞片呈宽披针形、匙形或条形,先端褐色,短于柱头,或与柱头近等长或稍长于柱头;子房柄上的丝状毛除少数散生外,多数基部合生呈鞘状或束状,在子房柄下部呈1～4轮排列;小穗不孕雌花常3（～4）枚。研究材料在7月中旬前后雌花小苞片明显短于柱头,随果穗成熟小苞片与柱头近等长。长苞香蒲（T.domingensis Pers.）子房柄上的丝状毛形态和着生方式与水烛中的情况基本一致,但小苞片白色透明,小穗不孕雌花常1（～2）枚。这表明水烛孕性雌花小苞片和柱头的长度比例与不同成熟阶段有关系,不宜作为与长苞香蒲的区别特征,而小穗不孕雌花数目和小苞片颜色等特征对两物种的划分有较重要的意义。     

茶皂素对菜青虫的拒食作用方式及机制

采用注射、熏蒸、点滴、饲喂、内吸等方法测定了茶皂素对菜粉蝶Ｐｉｅｒｉｓ　ｒａｐａｅ　Ｌ．５龄幼虫的拒食活性。结果表明,茶皂素对５龄菜青虫具有很强的饲喂拒食活性,２４小时选择性拒食中浓度ＡＦＣ_(５０)为４０．８８ｍｇ／Ｌ,非选择性拒食中浓度为３２１．９２ｍｇ／Ｌ;并具有一定的内吸拒食作用,尤其是根部施药法内吸拒食活性较明显。茶皂素对菜青虫无显著的注射、熏蒸、点滴拒食作用。     

中华芦荟组培苗与正常苗某些理化特性的比较研究

对中华芦荟（Aloe vera L.var.chinensis) 组培苗与正常苗的干重,含水量,可溶性糖含量,叶绿素及类胡萝卜素含量,蛋白质含量及蛋白质电泳,光合速率,呼吸速率,超氧化物歧化酶活性等理化指标进行了比较研究,结果表明,中华芦荟组培苗与正常苗的上述各项指标均差异不显著,证明植物组织培养方法是中华芦荟快繁的一条有效途径,可用组培苗来代替正常的扦插苗。     

药用植物刺山柑愈伤组织诱导及细胞生长代谢特征研究

本文研究了不同外植体及激素对刺山柑愈伤组织诱导的影响,不同激素配比对愈伤组织增殖培养以及悬浮细胞的生长与代谢特征.结果表明:以刺山柑叶片作为诱导愈伤组织的材料,效果较佳;愈伤组织诱导和继代的适宜培养条件是分别是MS+0.5 mg/L 2,4-D+1.0mg/L6-BA和MS+1.0mg/L2,4-D+1.5mg/L6-BA.刺山柑悬浮培养细胞的生长周期为30天左右,细胞生长曲线呈"S"形,生物量增长2.8倍左右;细胞生长周期内,碳源消耗规律表现为蔗糖和可溶性总糖的浓度持续降低,而还原糖则表现为先升高后降低;过氧化物酶活测定显示酶活水平与蔗糖浓度的高低呈一定程度的正相关.