Moderation of morphogenetic and oxidative stress responses in flax in vitro cultures by hydroxynonenal and desferrioxamine

Hypocotyl segments of 7-day old seedlings of flax (Linum usitatissimum L.) cultivars Atalante, Flanders, Jitka, Szegedi 30 and Super were screened for organogenesis (shoot and root induction) and embryo-like structure production. A non-destructive assay for hydroxyl radicals (*OH), utilising DMSO as a radical trap, was used to determine *OH formation during tissue culture and morphogenesis. Desferrioxamine, an inhibitor of Fenton reaction, and 4-hydroxy-2-nonenal, a cytotoxic Lipid peroxidation product, were exogenously applied to flax cultures to determine the effect of antioxidative and prooxidative status on morphogenetic responses induced through the exogenous application of plant growth regulators. Flax genotypes varied in their response to treatments after exposure to different plant hormones. Hydroxyl radical (*OH) formation correlated with morphogenetic responses and this was affected by plant hormones. Desferrioxamine and 4-hydroxy-2-nonenal also moderated morphogenetic responses and influenced hydroxyl radical formation during in vitro propagation.     

海南蛇足石杉株龄及年生长量研究

蛇足石杉因含有丰富的石杉碱甲而引起广泛关注,需求巨增,但该植物生长缓慢,野生资源濒临灭绝,且人工培植相关技术尚未成熟.为保护野生资源及今后开展繁育研究工作,需进一步了解蛇足石杉的生物学特性.据此开展了对海南蛇足石杉天然群体的调查,调查内容主要涵盖芽胞层数、层间距、株高及环境条件等.结果表明,芽胞逐年产生,集中分布于分枝...     

疣壶藓尖叶变种的芽胞形成及其生态意义

该研究以疣壶藓尖叶变种(Gymnostomiella vernicosa var.acuminata)为实验材料,在人工培养条件下观察并记录其芽胞产生及脱落过程,以揭示该分类群及相关类群的芽胞形成过程和机制,为疣壶藓尖叶变种的分类提供参考性特征指标,明确苔藓植物芽胞的进化和生态学意义。结果显示,疣壶藓尖叶变种的芽胞形成过程划分为3个阶段:(1)茎表皮细胞的外切向壁局部向外隆起,外突的疣壶藓尖叶变种表皮细胞富含叶绿体,可进行光合作用。(2)芽胞起始细胞横向分裂,形成一个基细胞和一个顶细胞;基细胞经数次横向分裂,下部形成柄状结构,上部的细胞常呈喇叭形的“柄托”;顶细胞经多方向细胞分裂,形成椭球状至圆球状芽胞体,形成初期为富含叶绿体的厚壁细胞,成熟后叶绿体数目逐渐减少,最终变成深棕色至红棕色。(3)由薄壁的柄细胞随机破损使芽胞从母株上脱落。研究表明,在无法预测的多变栖息环境中,相对于有较高的能量、遗传和生态消耗的有性生殖,疣壶藓尖叶变种产生芽胞是一种风险分担策略,可以提高生存几率和有效利用资源,可视为一种扩大和维持种群的有效途径。     

Transgenic manipulation of plant embryo sacs tracked through cell-type-specific fluorescent markers: cell labeling, cell ablation, and adventitious embryos

Expression datasets relating to the Arabidopsis female gametophyte have enabled the creation of a tool set which allows simultaneous visual tracking of each specific cell type (egg, synergids, central cell, and antipodals). This cell-specific, fluorescent labeling tool-set functions from gametophyte cellularization through fertilization and early embryo development. Using this system, cell fates were tracked within Arabidopsis ovules following molecular manipulations, such as the ablation of the egg and/or synergids. Upon egg cell ablation, it was observed that a synergid can switch its developmental fate to become egg/embryo-like upon loss of the native egg. Also, manipulated was the fate of the somatic ovular cells, which can become egg- and embryo-like, reminiscent of adventitious embryony. These advances represent initial steps toward engineering synthetic apomixis resulting in seed derived wholly from the maternal plant. The end goal of applied apomixis research, fixing important agronomic traits such as hybrid vigor, would be a key benefit to agricultural productivity.     

High frequency androgenesis from isolated microspores of maize

Anthers from a highly androgenic genotype of maize (139/39-02), when cultured in a modified, liquid YP medium, dehisced within 2–7 days resulting in a stationary suspension of microspores. After 12–15 days, the microspore suspension was found to contain multicellular masses which went on to produce macroscopic embryo-like structures within 20–25 days of culture initiation. Embryogenic callus could be obtained by transferring microspore-derived embryos onto a modified N6 medium supplemented with 2.5 mg/l dicamba and 0.1 mg/l 2,4-D. Subculture onto hormone-free medium resulted in plant regeneration. Over 400 embryo-like structures per 100 anthers cultured have been obtained from liquid induction medium as compared to 55 embryos per 100 anthers cultured on an agar-solidified medium. Approximately 5–25% of these embryo-like structures went on to produce callus from which plants could be recovered. Mechanical isolation of microspores from anthers precultured for 0, 3, and 7 days also resulted in embryo production and plant regeneration. This represents the first report of plant recovery from isolated maize microspores. The use of a liquid induction medium applied to a highly androgenic genotype allows for the production of large numbers of microspore-derived plants and provides a single, haploid cell regeneration system for maize.     

Accumulation,localisation, and toxic effects of cadmium in the liverwort Lunularia cruciata

Summary. Accumulation, tissue and intracellular localisation, and toxic effects of cadmium were investigated in the liverwort Lunularia cruciata. The results of analyses carried out by atomic absorption spectrometry on single plants showed that the cadmium accumulation was dose- and time-dependent. Cadmium localisation was assessed by X-ray scanning electron microscopy microanalysis in gemmalings and in the different tissues of the thallus and by X-ray transmission electron microscopy microanalysis at the cellular level. The metal preferentially accumulated in the hyaline parenchyma and at the base of the gemma cups. Inside the cell, cadmium accumulated in the vacuoles and the cell wall. Metal accumulation was accompanied by a concomitant increase in sulphur content within the vacuoles of stressed cells. Gel-permeation chromatography showed that most of the cadmium was associated with a low-molecular-mass fraction eluting at a ratio of elution volume to void volume corresponding to that of phytochelatins. The excess of sulphur deposited in the vacuoles may well have been caused by the stress-induced synthesis of phytochelatins. At the ultrastructural level, sublethal concentrations of cadmium caused alterations of the fine structure of the cells, inducing marked alterations of the chloroplast structure. Cadmium also induced a dose-dependent inhibition of apical thallus growth and gemma germination.Correspondence and reprints: Department of Plant Biology, University Federico 11, via Foria 223, 80132 Naples, Italy.     

ABA诱导毛酸浆下胚轴体细胞胚状结构的直接发生

研究了ABA与2,4-D等植物激素对毛酸浆组织培养的影响。其中,以浓度为1.0mg/L的2,4-D对毛酸浆下胚轴愈伤组织诱导结果最好。在子叶、子叶柄、下胚轴以及胚根四者当中,下胚轴对2,4-D的反应状况最好。结合ABA与2,4-D在MS培养基上能够有效地诱导出高质量的球形胚状结构。经过4周的培养,在2.0mg/L2,4-D和1.0mg/L ABA组合的情况下,球形胚状结构的诱导频率可达96.7％,每块外植体平均能产生6.7个球形胚状结构。球形胚状结构可以至少在5代以内反复发生。进一步讨论了ABA对毛酸浆体细胞胚发生的影响以及体细胞胚发生与环境胁迫的可能关系。     

From axenic spore germination to molecular farming

The first bryophyte tissue culture techniques were established almost a century ago. All of the techniques that have been developed for tissue culture of seed plants have also been adapted for bryophytes, and these range from mere axenic culture to molecular farming. However, specific characteristics of bryophyte biology—for example, a unique regeneration capacity—have also resulted in the development of methodologies and techniques different than those used for seed plants. In this review we provide an overview of the application of in vitro techniques to bryophytes, emphasising the differences as well as the similarities between bryophytes and seed plants. These are discussed within the framework of physiological and developmental processes as well as with respect to potential applications in plant biotechnology.     

Microscopic analysis and seasonality of gemma production in the freshwater red alga Hildenbrandia angolensis (Hildenbrandiales, Rhodophyta)

The development and release of the unique vegetative propagules of the freshwater encrusting alga Hildenbrandia angolensis Welwitsch ex West et West, gemmae, were studied using several different microscopic and histochemical techniques. In addition, the seasonality of gemma production was monitored bimonthly over a 12‐month period in two spring‐fed streams in Texas, USA. Gemmae differentiate within the thallus and are subsequently released from the surface of the crust. Release of the gemmae most likely occurs by digestion of surrounding cells, as suggested by the presence of starch granules and lipid globules in the region between the released gemma and the thallus. The initial separation of the gemmae from the thallus occurs from the sides of the gemma or the bottom, or possibly simultaneously. Contrary to previous studies, we have observed that gemma production occurs endogenously within the thallus of freshwater Hildenbrandia, rather than on the surface of the crust in raised structures. Histochemical tests and electron microscopic examination indicate that the cells of the gemmae contain a large amount of floridean starch. The starch granules frequently form rings surrounding the nuclei of both gemma and thallus cells; a feature infrequently reported for florideophyte red algae. Our seasonality investigations indicate that large fluctuations in gemma production occur over 1 year, but at least some gemma production continues year‐round in the streams examined.     

喀斯特山区粗裂地钱风兜亚种冬季芽胞杯的形态多样性特征及芽胞传播观察

芽胞杯是地钱属特有的无性繁殖器官,关于其冬季形态特征及繁殖传播的行为研究较少。现以贵州喀斯特山区常见的粗裂地钱风兜亚种（Marchantia paleacea subsp.diptera）为代表,在最冷的冬季1月份,对其芽胞杯、杯内芽胞产量及传播方式进行野外定点观察和采样分析。结果显示：（1）冬季芽胞杯形态多样。根据其颜色和杯内芽胞特点将其划分为4个生长时期：未成熟期（透明）、成熟期（绿色）、衰退期（紫色）和衰亡期（紫黑色）,反映了冬季芽胞杯生长发育的不同阶段。（2）各生长时期的芽胞杯数量不同,表现出有序的凋亡特征。在统计的708个芽胞杯中,4个时期芽胞杯数量分别为62、209、254和183个,且不同时期的芽胞杯内芽胞的平均产量明显不同,不同时期单杯芽胞的平均产量分别为42、131、87和0 个;（3）冬季芽胞杯及芽胞在配子体上的密度较高,每平方米分别达到10 139和754 889个;（4）除春夏季常见的被雨滴敲打传播外,通过重力作用传播是冬季芽胞的一种重要传播方式。冬季粗裂地钱风兜亚种配子体上的芽胞杯处在不同的生长时期,形成的芽胞仍十分丰富,这对该物种适应喀斯特山区最冷月严苛环境条件具有积极的意义。     

Serial Development of Foliar Gemmae in Tortula (Pottiales, Musci), an Ultrastructural Study

The development and liberation mechanism of foliar gemmae havebeen studied by electron microscopy in two mosses, Tortula latifoliaBruch and Tortula papillosa Wils. The gemmae develop on theadaxial surface of mature leaves from single initial cells onboth the lamina and costa in T. latifolia but only on the costain T. papillosa . Elongation of the initial cell is associatedwith the deposition of a highly extensible new wall whilst theold wall and cuticle in the apical dome rupture. The first divisionis transverse and separates a short basal cell embedded in thefoliar tissue and a distal cell, or gemma primordium, protrudingfrom the leaf surface. Subsequent divisions of the gemma primordiumgive rise to a six-to-eight-celled globose gemma with mucilaginousouter walls. During gemma development the basal cell producesa new wall and elongates again whilst the common wall with thegemma splits apart centripetally along the boundary betweenthe old and new wall in the basal cell; plasmodesmal connectionsare gradually severed and eventually the young gemma remainsconnected to the basal cell only by mucilage. After separationof the first-formed gemma, the basal cell may expand and producea second gemma by the same mechanism. The whole process maybe repeated several times resulting in the formation of a chainof gemmae stuck together by mucilage and which are liberatedonly when the leaves are fully hydrated. Accumulation of abundantlipid deposits in the gemmae after symplasmic isolation reflectsconsiderable photosynthetic autonomy. Abscission; bryophytes; cell wall formation; plasmodesmata; vegetative reproduction     

In vitro morphogenesis of sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis>) hypocotyl protoplasts: the effects of protoplast density,haemoglobin and spermidine

Sunflower, as one of the most important oil-producing crops, represents an important target for genetic improvement through gene transfer or somatic hybridization. Unfortunately, sunflower is recognized as recalcitrant to in vitro culture. The aim of our paper was to improve sunflower protoplast regeneration. Three cultivars (Romanian hybrids) and one inbred line were used for protoplast isolation from etiolated hypocotyls. Isolated protoplasts were embedded in alginate disks and cultured in two plating densities, using two culture regimes as indicated by previous authors. Plating efficiency, callus development and plant regeneration were evaluated as well as old callus histology. In cv. ‘Select’, the effects of 1:50 haemoglobin and 1 mM spermidine were assayed on asymmetric division and/or plating efficiency. Plant regeneration from hypocotyl protoplasts was achieved for two cvs., ‘Florom 328’ and ‘Turbo’, with the former proving once more its totipotency. The best culture regime proved to be as recommended by Krasnyanski and Menczel (1993), but the best density in the culture medium was the highest ever tested, 8 × 10⁵ pp ml⁻¹. Moreover, the histology of old green compact protoplast-derived callus revealed a very well organized structure suggesting senescence. In the non-responsive cv. ‘Select’, haemoglobin was found to stimulate protoplast asymmetric division and the development of heart-shaped embryo-like structures, while spermidine stimulated overall protoplast plating efficiency.     

Factors inducing regeneration response in oat (Avena sativa L.) anther culture

The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’. Despite the high resistance of oat to the process of androgenesis, all tested cultivars produced embryo-like structures and only two of them, ‘Akt’ and ‘Chwat’, produced fertile doubled haploid plants. A strong cultivar dependency was observed during induction of androgenesis. Further, cold pretreatment together with high temperature shock enhanced the efficiency of this technique. The highest number of embryo-like structures and haploid plants was obtained from cv. ‘Chwat’ (3.6% and 0.8%, respectively). Embryo-like structure formation also depended on the distance from the base of the flag leaf to the penultimate leaf of the panicle. Most of them were observed on anthers harvested from panicles of which the distance from the base of the flag leaf to the penultimate leaf was less than 4 cm. The presence of the induction medium supplemented with different plant growth regulators was essential for the induction of embryo-like structures but did not increase the production of haploid plants and doubled haploid lines. The highest number of embryo-like structures and plants was obtained on W14 medium with the addition of 2.0 mg/dm³ 2,4-dichlorophenoxyacetic acid and 0.5 mg/dm³ kinetin (2.7%). The low haploid plant regeneration rate (from 0.03 to 0.05%) still limits the practical application of anther culture for the production of doubled haploid lines in oat.

     

Effects of resource availability, and fruit and ovule position on components of fecundity in Alliaria petiolata (Brassicaceae)

In four field and glasshouse experiments designed to alter the supply of resources through manipulation of nutrients, root tissue, leaf area and fruit number in Alliaria petiolata (Brassicaceae), more than 99% of ovules per plant showed signs of fertilization, suggesting that seed production in this plant was not pollen limited. However, in all treatments a significant proportion of fruits and seeds did not develop to maturity. Total fruit and seed production did not differ significantly from controls when plants were given nutrient supplements at flowering. Removal of 50–75% of the root tissue in 1-yr-old plants significantly reduced fruit set, but had no effect on individual seed development. Removal of cauline leaves significantly reduced most measures of fruit and seed production, suggesting that current photosynthate is critical for fruit and seed filling. Seed maturation was significantly affected by both fruit position within an infructescence and ovule position within a fruit. Basally located fruits and ovules (within fruits) developed more mature seeds than distally positioned fruits and ovules. Plants responded to removal of basal fruits by re-allocating resources to distal fruits that would normally have aborted. Our results suggest that fruits and seeds act as reproductive sinks competing for parental photosynthate. Patterns of resource allocation within infructescences and fruits were also modified by our experiments.     

The role of abscisic acid in plant tissue culture: a review of recent progress

Abscisic acid (ABA) plays a significant role in the regulation of many physiological processes of plants. It is often used in tissue culture systems to promote somatic embryogenesis and enhance somatic embryo quality by increasing desiccation tolerance and preventing precocious germination. ABA is also employed to induce somatic embryos to enter a quiescent state in plant tissue culture systems and during synthetic seed research. Application of exogenous ABA improves in vitro conservation and the adaptive response of plant cell and tissues to various environmental stresses. ABA can act as anti-transpirant during the acclimatization of tissue culture-raised plantlets and reduces relative water loss of leaves during the ex vitro transfer of plantlets even when non-functional stomata are present. This review focuses on the possible roles of ABA in plant tissue culture and recent developments in this area.     

Photoperiod modulates growth,morphoanatomy, and linalool content in Lippia alba L. (Verbenaceae) cultured in vitro

Biotechnology provides valuable tools to support conservation of plant species, especially in case of threatened taxa or when dealing with seed unavailability, low viability or sterility. However, plant cell culture methods have often to face problems associated with tissue recalcitrance to in vitro systems. Recalcitrance can be related to a variety of triggering factors, involving many efforts and manipulations within one or more of the micropropagation stages before obtaining successful results. An in vitro propagation protocol was developed for Zelkova sicula, a very rare and endangered relict tree, endemic to Sicily (Southern Italy). The species revealed extremely recalcitrant to in vitro culture approaches, but after many trials throughout a number of years an effective micropropagation protocol was completed. The rooting rate was about 84% of the treated explants, 8% of which were successfully acclimatized outdoor and reintroduced in the wild within a comprehensive conservation project. The technique allowed to overcome the problems of sexual sterility of this species, hence contributing concretely to contrast the problems connected with its conservation. However, additional efforts need to be carried out in order to refine the acclimatization step and further improve the whole process effectiveness.

     

真藓科（Musci：Bryaceae）芽孢形态特征及其分类学意义

在光学显微镜（LM）和扫描电子显微镜（SEM）下对6属15种真藓科（Bryaceae）植物的芽孢进行了形态特征观察。结果显示,真藓科芽孢形态多样,可以作为该科种间分类鉴定的重要标准。芽孢在不同发育时期,形态特征差异较大。无性芽孢在环境条件许可的情况下才会产生,产生芽孢的植株一般不易发生有性生殖。     

植物比较解剖学在中国50年的进展和展望

系统与进化植物学是当今植物科学中的主流学科之一,植物比较解剖学是该学科的重要组成部分。根据近50年国内主要期刊统计,我国植物学者重点在种子植物的维管组织结构、叶及其表皮结构、花的结构和发育、种子及种皮特征、分泌组织以及一些原始种子植物等方面开展了比较解剖研究,为一些植物科、属的系统分类提供了解剖学依据。但在研究的植物种类、内容和方法尚存在不足,发表的论文相对较少。根据我国植物种质资源丰富,研究基础较好等特点,认为我国植物比较解剖学发展前景宽广,并对进一步发展提出一些建议。     

Aggregated seed arrival alters plant diversity in grassland communities

Aims Species aggregation is commonly seen in plant communities and may increase diversity by causing intraspecific competition to exceed interspecific competition. One potential source of this spatial aggregation is seed dispersal but it is unclear to what extent aggregated seed distributions affect plant diversity in real communities. Using a field experiment, I tested whether uniform or aggregated seed arrival alters community structure and whether these effects vary with sowing density.Methods The experiment consisted of two spatial seeding treatments (uniform and aggregated) that were fully crossed with three seed density treatments. Sixty, 3 × 4-m plots were arrayed in a low-diversity grassland located in Kansas, USA. Each plot was divided into forty-eight, 0.5 × 0.5-m patches. For aggregated seeding treatments, each of the 15 species was sown into three randomly selected patches within the plot (3×15 = 45). To create a uniform species arrival but control for the seed addition method, all 15 species were sown into 45 individual patches (with three patches remaining unsown) within each plot. Seed mass for each species was held constant at the plot scale between uniform or aggregated treatments within a given level of the sowing density treatment. After two growing seasons, plant density was quantified for all sown species in 15 randomly selected patches from each plot.Important findings I found evidence for shifts in community structure in response to the different spatial seeding patterns. The evenness of added species was higher under aggregated than uniform sowing patterns. There was no detectable effect of aggregated seed sowing on species richness at 3.75 m 2 scale. However, when species richness was extrapolated to larger scales (11.25 m 2), aggregated sowing was predicted to have greater richness than uniform sowing. Effects of seed aggregation on community structure were apparent only at moderate to high sowing rates, yet the latter are within the range of measured seed dispersal in similar grasslands. Additionally, as sowing density increased, seed mass became an increasingly effective predictor of relative abundances for added species, but only under uniform sowing patterns supporting the idea that aggregated dispersal may buffer weaker (smaller seeded) species from competition during colonization. This is the first experiment to show that aggregated seed dispersal patterns can increase at least some components of plant diversity in undisturbed grasslands and suggests that previous seed dispersal experiments, which utilize uniform seed sowing, may underestimate the potential effect of dispersal on plant community structure.     

The biochemistry and biology of extracellular plant lipid-transfer proteins (LTPs)

Plant lipid-transfer proteins (LTPs) are abundant, small, lipid binding proteins that are capable of exchanging lipids between membranes in vitro. Despite their name, a role in intracellular lipid transport is considered unlikely, based on their extracellular localization. A number of other biological roles, including antimicrobial defense, signaling, and cell wall loosening, have been proposed, but conclusive evidence is generally lacking, and these functions are not well correlated with in vitro activity or structure. A survey of sequenced plant genomes suggests that the two biochemically characterized families of LTPs are phylogenetically restricted to seed plants and are present as substantial gene families. This review aims to summarize the current understanding of LTP biochemistry, as well as the evidence supporting the proposed in vivo roles of these proteins within the emerging post-genomic framework.