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.     

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。     

凯特杏花粉的离体培养及影响因子分析

采用离体培养法,在不同培养基组分含量、pH值、温度及植物生长调节物质的培养条件下,对凯特杏(Prunus armeniacaL.cv Katy)花粉的离体萌发和花粉管生长状况进行观察研究。结果表明:(1)凯特杏花粉离体萌发及花粉管生长的适宜培养基为20%蔗糖 0.04%硼酸 0.01?Cl2,最适pH为6.0,最适温度为20℃,培养20 h后,花粉的萌发率达68.76%,花粉管长度达1 083.53μm。(2)不同植物生长调节物质对花粉萌发和花粉管生长作用不同,赤霉素浓度为5~8 mg/L、矮壮素浓度为10~150 mg/L、多效唑浓度为5~10 mg/L时对凯特杏花粉萌发和花粉管生长都有促进作用,但国光丁酰肼对凯特杏花粉萌发和花粉管生长均有抑制作用。     

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.     

黄独脱毒苗叶片和茎段再生体系的建立

以黄独茎尖再生苗为试材,研究不同因素对黄独脱毒苗叶片和茎段再生体系的影响,以期对黄独脱毒苗的再生体系进行优化。结果表明,叶片和茎段诱导愈伤组织的最佳培养基是MS+KT 2 mg·L^-1+2,4-D 2 mg·L^-1;叶片和茎段诱导愈伤组织的最佳蔗糖浓度分别为30和50 g·L^-1;叶片和茎段在黑暗中较容易诱导出愈伤组织;叶片和茎段愈伤组织分化的最佳培养基是MS+KT 4 mg·L^-1+NAA 0.1 mg·L^-1;继代2次的叶片和茎段愈伤组织较容易分化;黄独不定芽生根的最佳培养基是1/2MS+IBA 0.1 mg·L^-1+NAA 0.5 mg·L^-1+PP₃₃₃ 1 mg·L^-1。本实验成功建立了黄独脱毒苗叶片和茎段的再生体系,为黄独脱毒苗的工厂化生产奠定了技术基础。     

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.     

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。     

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.     

Pollen Tetrads in Lilium with a Discussion on the Delimitation Between L.semer vivoideum and Its Ally

In the course of our palynological study on the Chinese Lilium, pollen tetrads are found in two taxa of the genus: L. sempervivoideum and L. amoenum. The latter is distributed in NW. and S. Yunnan Province. It was established by Wilson, but reduced later by himself to a synonym of L. sempervivoideum which occurs from S. Sichuan to C. Yunnan. However, it is regarded as a species distinct from the former by many authors, such as Sealy (1949), Woodcock and Stearn (1950) and S. Y. Liang (1980). Their pollen tetrads are similar in aperture appearance and sexine patterns, but different in the shape, size and arrangement of sculptural elements, as shown by LM and SEM examination. Their leaf shape and flower colour are different, though there are some transitions between them. Based on the facts mentioned above, it is reasonable to regard the two taxa as two subspecies in L. sempervivoideum.     

遮荫对黑茶藨子叶片形态结构和光合特性的影响

以黑茶藨子为试验材料,通过人工遮荫的方式,研究了不同遮荫强度（全光照、20%和60%遮荫）和遮荫时间（初期、中期和后期）对黑茶藨子叶片形态解剖结构和光合特性的影响。结果表明：（1）20%、60%遮荫处理的叶片厚度均显著低于全光照处理,上下表皮厚度则高于全光照处理;随着遮荫强度的加大,栅栏组织与海绵组织厚度比值大小均呈减少趋势,随着遮荫时间的延长,呈增加趋势。（2）遮荫初期和中期,20%遮荫处理的叶片净光合速率（P_n）比全光照和60%遮荫处理的值略高,但差异并不显著（P>0.05）,其蒸腾速率（T_r）、水分利用效率（WUE）也略有增加;但是,60%遮荫处理的叶片净光合速率（P_n）显著低于全光照处理（P<0.05）。遮荫后期,60%遮荫处理的叶片净光合速率（P_n）显著高于全光照处理,且水分利用效率（WUE）、气孔导度（G_s）、蒸腾速率（T_r）均显著高于全光照和20%遮荫处理。综上所述,遮荫处理导致叶片厚度减少,海绵组织比例增加,有利于减少透射光中的光量子丧失,提高光能利用效率;生长早期和中期作20%轻度遮荫处理,后期作60%中度遮荫处理能提高植株对光和水分的利用效率、维持较高的光合速率、延缓衰老。     

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.     

Melanogenesis‐Inhibitory and Cytotoxic Activities of Chemical Constituents from the Leaves of Sauropus androgynus L. Merr. (Euphorbiaceae)

A new steroid, 20‐hydroxyisofucosterol (stigmasta‐5,24(28)‐diene‐3β,20β‐diol) ( 7 ), along with six known compounds 1 – 6 were isolated from the MeOH extract of the leaves of Sauropus androgynus L. Merr . (Euphorbiaceae). The structure of new steroid was determined by HR‐APCI‐MS and various NMR techniques in combination with literature data. Subsequently, their anti‐inflammatory, cytotoxic activities against five human cell lines, as well as inhibitory activities against the α‐MSH induced melanogenesis on the B16 cell line were evaluated. As the results, steroid compounds, 6 and 7 exhibited moderate cytotoxic to HL60, AZ521, SKBR3, and A549 tumor cell lines (IC₅₀ 26.9 – 45.1 μm ) with high tumor selectivity for A549 relative to WI38 cell lines (SI 2.6 and 3.0, resp.). And, flavonoid compounds, 4 and 5 exhibited superior inhibitory activities against melanogenesis (67.0 – 94.7% melanin content), even with no or low toxicity to the cells (90.1 – 99.6% cell viability) at the concentrations from 10 to 100 μm . Furthermore, Western blot analysis suggested that compound 5 could inhibit melanogenesis by suppressing the protein expressions of MITF, TRP‐1, TRP‐2, and tyrosinase.     

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.     

花椰菜BobACT基因的克隆及其作为内参基因的研究

实时荧光定量PCR技术是探索植物基因功能和调节机理的有效手段。选择合适的内参基因是获得实时荧光定量PCR准确性数据的必备条件。ACT基因高度保守且表达稳定,常作为内参基因被广泛应用。为了获得花椰菜ACT基因,以转录组测序和RT-PCR方法为手段克隆得到花椰菜肌动蛋白基因Actin。该基因等电点为5.395,理论分子量为41.77 kD;其cDNA开放阅读框长1134 bp,编码氨基酸377个,GenBank登录号为MG598643。Wolf Psort分析发现,BobActin蛋白亚细胞定位于细胞质基质中。Motif Scan分析显示,BobActin蛋白质的氨基酸序列4～377位为Actin保守结构域。进化分析表明,同源序列基因编码的蛋白质与同为十字花科的甘蓝、芜菁和油菜同源蛋白的相似性达到90%以上,具有高度的保守性。在此基础上,设计了1对荧光定量PCR引物,分析显示,该引物具有较高的特异性和扩增效率,在花椰菜根、茎、花、花球、叶片等不同组织和低温、高温、盐处理、干旱处理、ABA处理等胁迫处理下均能稳定表达,适合在花椰菜基因表达研究中作为内参基因,为开展花椰菜重要功能基因的挖掘、表达模式以及调控机理的研究提供参考。花椰菜在内参基因方面的研究还处于初步阶段,今后可继续克隆其他内参基因,丰富花椰菜的内参基因库,从而进一步提高花椰菜基因表达分析研究的稳定性、重复性和准确性。