New record for Halophila decipiens Ostenfeld in Kenya based on morphological and molecular evidence

The seagrass Halophila decipiens Ostenfeld was recorded for the first time in Kenya in 2003. It was growing in subtidal mixed meadows with H. ovalis in protected bays at a depth of 3 m. DNA sequence analysis and morphological characteristics confirmed the identification. It is possible that this species is more widely distributed in the east African region. There was very little sequence differentiation between the Kenyan H. decipiens and the other samples in the Indo-West Pacific, and between this region and the Atlantic Ocean, further validating from an evolutionary perspective recent dispersal of this species.     

Localization and antioxidant capacity of flavonoids from intertidal and subtidal Halophila johnsonii and Halophila decipiens

In this study, flavonoid localization, content and total antioxidant capacity in leaves of subtidal Halophila decipiens were compared to intertidal and subtidal Halophila johnsonii. H. johnsonii leaves had significantly higher flavonoid content (3.5 and 3.8 nmol quercetin equivalent mm⁻² leaf for intertidal and subtidal H. johnsonii, respectively) and antioxidant capacity (101.7 and 224.2 nmol Trolox equivalent mm⁻² leaf for intertidal and subtidal H. johnsonii, respectively) than H. decipiens leaves (1.4 nmol quercetin equivalent mm⁻² leaf and 21.0 nmol Trolox equivalent mm⁻² leaf). Flavonoid content did not significantly differ between intertidal and subtidal H. johnsonii, however, antioxidant capacity was significantly higher in subtidal plants. Confocal laser scanning microscopy of fresh leaf cross sections indicated that both species contained flavonoids in the cuticle, but only H. johnsonii contained intracellular flavonoids. Intracellular flavonoids are better situated to perform antioxidant functions in planta. These results suggest that flavonoid compounds in H. johnsonii are capable of sunscreen and antioxidant functions while an antioxidant role for flavonoids within H. decipiens is not supported.     

The distribution and expansion of the invasive seagrass Halophila stipulacea in Dominica, West Indies, with a preliminary report from St. Lucia

The seagrass Halophila stipulacea Forsskål, native to the Red Sea, is an invasive species in the Mediterranean that was recently observed offshore Grenada, in the Caribbean. Here, we document the presence of this seagrass in Dominica and St. Lucia, demonstrating it has spread across part of the eastern Caribbean. H. stipulacea in Dominica was present in seven locations along the west coast covering more than 22.9 ha of the benthos, at depths from 2 to 18 m. Populations were concentrated in or adjacent to bays frequented by recreational or commercial boats, likely vectors for the introduction. Morphological features varied from bed to bed, with depth being the predominant driving factor. H. stipulacea had a rapid mean lateral bed expansion rate of 0.5 cm d⁻¹, with a maximum rate of >6 cm d⁻¹. H. stipulacea patches often occurred exclusive of the otherwise dominant seagrasses of the Caribbean. The potential for the expansion of H. stipulacea, combined with its tolerance for a wide spectrum of environmental conditions, positions it as a potential threat to local and regional biodiversity.     

Wavelength-specific photosynthetic responses of Halophila johnsonii from marine-influenced versus river-influenced habitats

The seagrass Halophila johnsonii Eiseman grows from the upper intertidal to 3 m depths in habitats ranging from near-marine inlets to tidal riverine. These habitats have distinct optical characteristics, primarily due to variable concentrations of watershed-derived chromophoric dissolved organic matter (CDOM), which increases the attenuation of short-wavelength (blue and UV) light. H. johnsonii contains a suite of flavonoids that are thought to serve as UV-protective pigments (UVP). In this study, photosynthetic responses at specific wavelengths were compared between plants from a river-influenced high-CDOM habitat (Oleta River) and those from an adjacent marine, low-CDOM environment (Haulover Inlet) in Florida. Oxygen flux was used to measure dark respiration and photosynthesis under near-constant radiant energy at nine specific wavelengths from 340 to 730 nm. Riverine plants had higher gross photosynthetic rates and quantum efficiencies than inlet plants at the shortest wavelengths (350, 400 and 450 nm), while inlet plant photosynthetic rates and quantum efficiencies were higher at the two longest wavelengths measured (694 and 730 nm). Riverine plants also exhibited greater variation in photosynthetic responses across the spectrum and more variable pigment levels among replicates. Chlorophyll a and b concentrations were significantly greater in riverine plants suggesting that they were more shade-acclimated compared to the marine populations. Differences in wavelength-specific photosynthetic responses and chlorophyll levels indicate that the riverine plants were blue-shade acclimated. The higher and more variable UVP levels in the riverine population were not consistent with shade acclimation; however, these flavonoid pigments may protect chloroplasts from photodamage during short-term, high intensity irradiance conditions that occur over the course of tidal cycles at this highly fluctuating riverine site.     

Seed bank,biomass, and productivity of Halophila decipiens, a deep water seagrass on the west Florida continental shelf

One of the largest contiguous seagrass ecosystems in the world is located on the shallow continental shelf adjacent to the west coast of Florida, USA and is comprised of seasonally ephemeral Halophila decipiens meadows. Little is known about the demography of the west Florida shelf H. decipiens, which may produce 4.56 × 10⁸ g C day⁻¹ or more during the peak growing season. We documented seagrass distribution, biomass, and productivity, and density of sediment seed reserves, seedlings, flowers and fruits on the southeastern portion of the west Florida shelf by sampling along a transect at three stations in 10, 15, and 20 m water depth. Biomass, flower, fruit, seedling, and seed bank densities tended to be highest at stations in 10–15 m water depth and lowest at 20 m. Flowers and fruit were most prevalent during summer cruises (June and August 1999, July 2000). Seedling germination occurred during summer, fall (October 1999), and winter (January 2000) sampling events, with the highest seedling densities present during the winter. Seed bank density remained consistent through time. A Category I hurricane with sustained winds of 120 km h⁻¹ passed over the stations, but only limited impact on H. decipiens biomass was observed. The presence of a persistent seed bank provides for recovery after storm disturbance, annual reestablishment of populations, and continual maintenance of the 20,000 km² of deep water seagrass habitat present on the west Florida shelf.     

A practical method for overcoming the dormancy of <Emphasis Type="Italic">Taxus baccata</Emphasis> isolated embryos under <Emphasis Type="Italic">in vitro</Emphasis> conditions

Conditions for obtaining an efficient mass propagation procedure to overcome isolated Taxus baccata embryo dormancy were investigated. The protocol herein described was efficient for overcoming the dormancy of T. baccata isolated embryos under in vitro conditions, enabling the conservation and propagation of this species. T. baccata seeds were unable to germinate directly after collection under in vitro conditions. Very good sterility and germination was achieved by soaking seeds in distilled water at a low temperature (+4°C) at least for 48 h instead of leaching them for 7 d under running water, followed by maintaining isolated embryos on the Murashige and Skoog medium (MS) supplemented with 5 g l⁻¹ activated charcoal. That treatment allowed one to shorten the time of the experiment and gave almost 100% sterility. The best germination was observed in darkness, but to obtain worthy seedlings, it was necessary to place cultures in a 16-h photoperiod after a 2-wk incubation. There was no significant difference in germination between seeds collected from different populations of Southern Poland.     

濒危海草贝克喜盐草在海南东寨港的分布及其群落基本特征

贝克喜盐草( Halophila beccarii)是当前全球面临灭绝风险的10种海草之一,被国际自然保护联盟( IUCN)列为易危( VL)种。由于贝克喜盐草生境的特殊性(受涨退潮影响的潮间带)及个体十分纤小且易被沉积物覆盖,加之种群更新速率极快,导致通常难以被发现。据报道,贝克喜盐草仅在我国的广西、广东、台湾、香港、福建和海南6个省(区)有小面积的分布。该研究首次报道了位于海南东寨港的贝克喜盐草海草的分布、群落基本特征以及生境概况。结果表明：与国内其它地区的贝克喜盐草相比,调查期间东寨港的贝克喜盐草面积较大,为15.4 hm2;密度适中,为(10394±576)茎.m-2;覆盖度较低,为~5％;生物量较低,总生物量仅(9.224±1.151) gDW.m-2,地上生物量为(2.339±0.245) gDW.m-2,地下为(6.886±1.055) gDW.m-2,地上与地下生物量之比值较低(0.34);叶片较小,叶长为(4.83±0.87) mm,叶宽为(1.44±0.21) mm,调查时在植物体上未发现有花或果,但在沉积物中发现有一定数量的贝克喜盐草种子(2105±664)粒.m-2。该研究区的海水受到一定程度的水体污染。建议海南东寨港国家级自然保护区在今后的管理工作中,应把海草也作为重点保护对象。     

Northern range extension of the seagrasses Halophila johnsonii and Halophila decipiens along the east coast of Florida,USA

The northern geographic limit for Halophila johnsonii and Halophila decipiens has been reported as Sebastian Inlet, within the Indian River Lagoon, Florida. Surveys conducted in August 2007 determined the new northern limit to be 21.5 km north of the previously known limit. This new northern limit is a 10% range extension for H. johnsonii, a federally threatened species. We conclude that these range extensions are recent, based on (1) the small size of patches; (2) unusually good water clarity conditions due to a recent drought; (3) recent mild/warmer winters; and (4) a recent mechanism for transporting propagules, the numerous hurricanes of 2004. Although this recent range extension is considered ephemeral, similar range extensions may have occurred in the past and may occur again in the future under favorable conditions given the high capacity of these two species for dispersal to favorable sites. The northern limits of these species should not be viewed as static locations; rather, they must be considered dynamic features.     

In vitro germination of some Western European orchids

Several germination factors were studied in 23 Western European orchids in order to obtain a germination percentage which approximates the maximum percentage of coloured embryos after an adaptation of the tetrazolium test. The duration of seed pretreatment with Ca(OCl)₂+ Tween-80 was one of the most important factors controlling the germination of Western European orchids. Best germination occurred in continuous darkness at 23°C on dilute media. The nitrogen source had a species related influence. The best results were obtained with casein hydrolysate, although it could nearly be replaced by one amino acid, L-glutamine. A cytokinin was required by Cypripedium calceolus and Epipactis hellborine but was not necessary for Lisiera ovata and Dactylorhiza maculata.
Experiments with 3 species have shown that the objective, to obtain a germination percentage, which approximated the maximum percentage of coloured embryos, was achieved. This has led to the elaboration of a basic sowing method for European orchids. The application of this method gave good germination responses with 21 of the 23 species tested.     

In vitro culture of embryonic cells from the shrimp, Penaeus chinensis

Studies were carried out in our laboratory to develop cell cultures from embryonic tissue of Penaeus chinensis. Good yields of dissociated, uncontaminated, viable cell suspensions were obtained by physical disruption of harvested embryonic tissues. Primary cultures in the form of proliferating foci of cells were readily initiated using MPS medium (osmolality: 2.4%) with 20% heat-inactivated fetal bovine serum. Insulin-like growth factor-II (IGF-II) and basic fibroblast growth factor (bFGF) at different concentrations were also added into the culture medium. Passage of primary cultures resulted in rapid proliferation and good adherence of lymphocytes in the presence of IGF-II at 80 ng/ml and bFGF at 20 ng/ml. Cells maintained in subculture for up to 10 passages still had good cellular morphology and division rates.     

丁香(Syringa L.)种间杂交幼胚离体培养研究

采用幼胚离体培养方法克服幼胚败育从而直接获得杂种实生苗,对影响丁香幼胚培养成功的各种因子作了较系统的研究.结果表明,丁香幼胚培养的最适培养基为Monnier;最佳糖浓度为50g·L^-1;幼胚在50～60d胚龄时培养最容易成功;加入适量的椰乳、谷氨酰胺或谷氨酸及活性炭可以促进幼胚的萌发和生长.低浓度(0.01mg·L^-1)BA的加入可以提高幼胚的萌发率;NAA浓度以0.01mg·L^-1为最佳.     

The photosynthetic performance of the tropical seagrass Halophila ovalis in the upper intertidal

Some ecophysiological adaptation strategies of the tropical seagrass Halophila ovalis were investigated with respect to this plant's ability to grow in the upper intertidal in either monospecific pools (but not together with other intertidal species) or emergent and exposed to high temperatures and irradiances during several hours every day. It was found that Halophila ovalis could raise the pH in simulated pools to 8.6, while the two other major (biomass wise) intertidal seagrasses raised the pH to 8.8 (Cymodocea rotundata) and 9.2 (Thalassia hemprichii). In situ, midday pH values of 8.5, 8.7 and 9.0 were recorded in pools inhabiting the three species, respectively. It was further found that photosynthetic electron transport rates (ETR) measured continuously in situ for one leaf during a diurnal cycle followed the irradiance up to a daily maximal value of 2200 μmol photons m⁻² s⁻¹, but dropped by ca. 50% when the leaf became air exposed just before noon. This drop in ETR upon emergence was verified by “point measurements”, while ETRs of leaves that grew in small pools formed as the tide receded followed the irradiance more closely. Similarly, F_v/F_m measured after 15 min of dark adaptation decreased by ca. 50% in emergent leaves during midday, but maintained higher daily values in the submerged leaves.     

In vitro culture of lemon juice vesicles

This study explores the possibility of culturing Citrus limon (L.) Burm. f. cv. Eureka (lemon) juice vesicles as isolated intact tissues capable of retaining their unique growth structure in vitro. Isolated juice vesicles either attached to or detached from the endocarp/mesocarp (albedo) tissue of origin were grown on various nutrient media using several physical environments. Various growth responses achieved in vitro from cultured vesicles are described. Intact vesicles attached to endocarp/mesocarp tissue were found to grow up to 6 months in culture as a distinct tissue with a minimum of adverse callus proliferation. Callus formation from some cultured explants occurred on all media and physical environments tested. Callus production eventually obliterated and irreversibly altered the normal development of juice vesicles. Inherent vesicle physiology rather than the tissue culture environment was the major determining factor affecting culture growth. Reducing the concentration of carbohydrates (fructose, glucose or sucrose) added to media from 3 to 0.01% or 0.0% reduced, but still did not totally prevent, callus production. Treatment of vesicles with 1000 mg/l ascorbic acid or citric acid, or 0.1 mg/l indole-3-acetic acid or abscisic acid enhanced the occurrence of normal appearing vesicles.Mention of a trade name or proprietary product or vendor does not constitute approval or guarantee of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.     

Influence of agar and activated charcoal on uptake of gibberellin and plant morphogenesis In vitro

Summary Agar and activated charcoal (AC) are commonly used in tissue culture. However, their deeper actions and functions are largely unknown. This experiment investigated the effect of agar and AC, singly and jointly, on gibberellin (GA) uptake by corn shoots. Corn seeds were germinated on Murashige and Skoog medium (MS). Shoot excised from 1-wk-old seedlings were cultured on liquid (0.0 g l⁻¹ agar) or solid (8 g l⁻¹ agar) MS containing 3 μM indole-3-acetic acid, 13.3 μM N⁶-benzyladenine, and 6000 CPM ml⁻¹ [³H]GA₄ as tracer. Both liquid and solid media had two treatments, one without AC and one supplemented with 5 g l⁻¹AC. Uptake of [³H]GA₄ and morphogenesis of corn shoots were recorded after 2 wk of culture. Corn explants cultured in AC-free media acquired high levels of [³H]GA₄, while explants from AC-containing media showed only traces of [³H]GA₄. Explants cultured in AC-free liquid medium contained about twice the amount of [³H]GA₄ as those from AC-free solid medium. Addition of agar reduced shoot length, while addition of AC increased both shool and root length. It is concluded that: (1) agar reduced the uptake of GA₄; and (2) GA₄ was irreversibly adsorbed by AC, and thus became unavailable to corn explants.     

In vitro production of specialized reproductive torulose hyphae byFrankia strain ORS 021001 isolated fromCasuarina junghuhniana root nodules

Summary AFrankia strain (ORS 021001) isolated fromCasuarina junghuhniana root nodules was shown to produce four type of structuresin vitro: vegetative hyphae, sporangiospores within sporangia, N₂-fixing vesicles, and a fourth type of structure which is described in detail in this report. Structures of this latter type which we propose to call reproductive torulose hyphae: (RTH) result from enlargement and multiple segmentation of vegetative hyphae into torulose chaions of spore-like cells. RTH differ from sporangia in three major aspects: morphology, morphogenesis and outgrowth. RTH play an important role in survival and reproduction ofFrankia strain ORS 021001. Adding activated charcoal to the nutrient medium promotes the formation ofFrankia colonies orginating from RTH.     

In vitro culture of Plasmodium berghei using a new suspension system

Attempts were made to develop techniques for the continuous in vitro culture of Plasmodium berghei. The candle jar method (Trager &; Jensen, 1976) proved to be insufficient for the culture of this rodent malaria parasite. Parasitaemia decreased rapidly after the first schizogonic cycle in culture. A simple suspension technique was developed using a newly designed culture apparatus which can be placed in the laminar-flow. All manipulations necessary for the refreshment of medium and cells can be made with almost no disturbance of the culture conditions. With this system it was possible to culture P. berghei repeatedly for more than a week, completing at least four schizogonic cycles with considerable mcrozoite invasion and a 2–6-fold multiplication. Infection rates of up to 6.0% were achieved and cultures were maintained for 9 days. Several specific properties of P. berghei and the differences between the candle jar method and the new suspension method are discussed to explain the results obtained in both systems.     

海洋固氮菌和解磷菌的分离鉴定及发酵条件优化

【目的】从西沙喜盐草根际沉积物中分离纯化得到具有高效固氮能力及解磷能力的菌株。优化其发酵培养条件,研究其制备海洋微生物菌剂的可能性。【方法】从形态学特征、生理生化、16S rDNA及功能基因水平进行鉴定,通过乙炔还原法、钼锑抗显色法检测菌株的固氮酶活性和解磷能力,单因素法和响应面法优化其发酵培养条件,溶血试验和急性毒性实验鉴定菌株的安全性。【结果】结果表明,菌株AZ16属于星箭头菌(Sagittula stellate),革兰氏阴性菌,选择性固氮培养基中菌落呈黄圆形黏稠状,固氮酶活性达34.63 nmol C2H2/(mL·h),最适生长条件为:盐度25‰、pH 7.5、温度33°C、接种量5.0%;菌株XT37为海洋芽孢杆菌(Bacillus sp.),革兰氏阳性菌,选择性固氮培养基中菌落呈深黄色圆形褶皱,植酸酶活性达239.49μg/L,最适合生长条件为:盐度25‰、pH 6.7、温度28°C、接种量5.0%。溶血实验和急性毒性实验证明两株菌属实际无毒级别。【结论】两株菌具有高效的固氮解磷功能,以及抗高盐、强碱等环境的能力,安全无毒,因此有潜力应用于多功能混合微生物菌剂的研制。     

Genetic variation in populations of the threatened seagrass Halophila beccarii (Hydrocharitaceae)

Halophila beccarii (Hydrocharitaceae), a small monoecious seagrass, has been listed as a threatened species. In this study, a total of 106 samples were collected from four Chinese populations located at the northern limit of its distribution range. Using six polymorphic microsatellites, we found low genetic variation in this species, in which the mean number of alleles per locus was 2.8, and 16 multi-locus genotypes were revealed. In the four populations, the mean number of alleles per locus ranged from 1.2 to 2, one to eight genotypes were found, and clonal diversity ranged from 0 to 0.23; observed and expected heterozygosity ranged from 0.17 to 0.40 and from 0.09 to 0.24, respectively. Strong genetic differentiation was found among the populations, and the standardized fixation index (F′_ST) was 0.787. Species traits (i.e., clonal growth) and bottleneck effects due to drastic population fluctuation may contribute to the observed low genetic variation.     

Chemical analysis of flavonoid constituents of the seagrass Halophila stipulacea: First finding of malonylated derivatives in marine phanerogams

The flavonoid fraction from the butanol extract of a Mediterranean sample of the seagrass Halophila stipulacea was chemically analyzed. A new malonylated flavone glucoside, genkwanin-4′-O-(6“-malonyl-glucopyranoside) (3), was isolated together with known flavone glucosides 4-9, previously reported only from terrestrial sources. The structure of 3 was established by means of spectroscopic techniques, mainly NMR methods.