In vitro Post-germination Growth and Development of Embryos of Alectra (Scrophulariaceae)

In vivo, seeds of the obligate root parasite Alectra vogelii Benth. (Scrophulariaceae) germinate only after being soaked in water for a period of time (pretreatment) followed by stimulation by certain factors exuded from a host root. Germinated seedlings do not develop beyond radicle emergence, and finally die, unless their radicles make contact with and penetrate into a host root conductive system. In vitro, germinated embryos obtained by exposing sterilized and pretreated seeds to root exudate of Vigna unguiculata were aseptically cultured on Knop's, White's and Murashige and Skoog's media. The embryos grew into seedlings with shoots and roots on a medium containing mineral salts and sucrose, but not on mineral salts alone. Seedling performance was generally not improved when the mineral salts-sucrose media were supplemented with vitamins. Shoot extension growth was better on Murashige and Skoog's mineral salts-sucrose medium than on Knop's or White's medium. However, seedling development was greatly boosted when cultivated on White's minerals salts-sucrose medium supplemented with coconut milk. Seedlings turned green on transfer to light but did not flower. The successful culture of these embryos and seedlings on a simple, chemically defined medium of mineral salts and sugar suggests that these nutrient components are the minimal external requirements for stimulation and support of normal seedling growth. These may be obtained in vivo by the parasite's tapping of the host root conductive system.     

Regeneration capacity of oceanic-montane liverworts: implications for community distribution and conservation

Abstract

Scotland’s mountains are home to a rare and unique liverwort community, ‘the oceanic-montane liverwort-rich heath’, but its component species are absent from regions where they could potentially thrive. Many biological characteristics of these species are unknown, making it impossible to explain the reasons for their rarity; however, they have not been observed to produce sporophytes within Britain. We use ex situ cultivation of whole liverworts and fragments, and in situ cultivation of fragments, to assess the growth rate and the potential for vegetative reproduction of several species. Most of the species grew from both fragments and as whole plants, indicating that the rarity of the liverwort heath is not due to poor powers of regeneration. We propose that growth rate and the potential to regenerate from fragments are important factors structuring the liverwort heath community, at least locally. Furthermore, this study demonstrates that there is potential for ex situ conservation of rare liverwort species, in situ enhancement of existing populations, and creation of new ones.     

Mnioloma fuscum (Marchantiopsida: Calypogeiaceae), an unexpected addition to the indigenous flora of New Zealand

Abstract

Mnioloma fuscum, a liverwort species known to date primarily from tropical-montane regions, is reported for New Zealand for the first time. Its occurrence in New Zealand is unexpected as the next nearest known site is believed to be the Solomon Islands, some 3500 km distant. The occurrence of Mnioloma fuscum in New Zealand highlights the contribution tropical regions have made to the composition of New Zealand's hepatic flora.     

The Effects of Using Trehalose as a Carbon Source on the Proliferation of Phalaenopsis and Doritaenopsis Protocorm-Like-Bodies

In this communication, the effects of trehalose and culture system on protocorm-like body (PLB) growth were investigated. PLB derived from Phalaenopsis and Doritaenopsis cultivars, which were grown on solidified trehalose amended NP medium showed higher proliferation rate than on NP medium containing sucrose. For P. ‘Hwa Feng Red Jewel’ and Dtp. ‘Mount Beauty×Su’s Red Lip’, the proliferation rates on solidified trehalose media were almost two times higher than those on sucrose media after 8-week culture. However, Knudson C (KC) medium did not reveal the similar results between trehalose and sucrose. In liquid culture system, both trehalose amended NP and KC media brought about better results than sucrose containing media for PLB proliferation. For culture system test, solidified media showed higher proliferation rate than liquid media under the same medium composition. Roller bottles were more suitable than flask-shaking cultures in liquid systems for PLB proliferation.     

Athalamia hyalina (Sommerf.) S.Hatt. in Scotland,new to the British Isles

Abstract

The liverwort Athalamia hyalina (Sommerf.) S.Hatt. and the family Cleveaceae are reported as new to the British Isles from the eastern Scottish Highlands. The Scottish plants are described and illustrated and the significance of the discovery is assessed. In Scotland, Athalamia hyalina occurs on thin soil on eroding limestone ledges in a montane habitat with a continental climate. It is in active growth in the winter months, producing spores in spring. Its range is restricted but it occurs within a protected area and is not threatened at present. The species is widespread in the northern hemisphere.     

Nutrient utilization and requirement under photoheterotrophic growth of Marchantia polymorpha: Improvement of the culture medium

Previously we reported on a suspension culture of chlorophyllous cells of a liverwort, Marchantia polymorpha L., under photoheterotrophic conditions. The chemically defined medium was a modified Murashige and Skoog's medium, which contained, besides glucose, inorganic salts, a growth regulator (2,4-D), and twenty-four organic compounds as micro constituents. Because of this complexity, we undertook a simplification of the medium. Having examined the utilization of the major nutrients and the requirements for the micro constituents, we have succeeded in improving the medium. The new medium contains phosphate at 3.13 mM and only eight out of the twenty-four original micro organic constituents. In this new medium, the cells grow under a well-balanced nutritional condition, with richer chlorophyll and at a higher rate during the exponential phase than in the original medium.     

STUDIES ON STRIGA SENEGALENSIS III. IN VITRO CULTURE OF SEEDLINGS. ESTABLISHMENT OF CULTURES

Striga senegalensis seeds were cultured on Knop's and modified Murashige-Skoog's medium. An aqueous root exudate/extract of the host (Sorghum vulgare) was found essential for the germination of seeds in vitro. The seedlings grew better on a medium containing sucrose than on mineral salts alone. Only limited growth was achieved on Knop's medium. Better growth was supported by Murashige-Skoog's medium. By transfer to larger culture vessels seedlings were kept in the latter for several months. The seedlings developed chlorophyllous shoots in light and flowered after 4–4½ months. The successful culture of these seedlings in a simple medium of only mineral salts and sugar indicates that the parasitic seedling depends on the host for only water, mineral salts and sugar and not for more elaborate substances.     

Influence of growth medium on antifungal activity of neem oil (<Emphasis Type="Italic">Azadirachta indica</Emphasis>) against <Emphasis Type="Italic">Lagenidium giganteum</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

The inhibition of mycelial growth of Lagenidium giganteum by neem oil was lower than that of Metarhizium anisopliae in PYG and Emerson’s YpSs agar media. However, neem oil did not inhibit the mycelial growth of L. giganteum in sunflower seed extract agar medium, but did it inhibit the mycelial growth of M. anisopliae. The minimum inhibitory concentration of neem oil for L. giganteum was higher than that for M. anisopliae. The minimum fungicidal concentration of neem oil in PYG medium was lower than in YpSs for both fungi. The spores of L. giganteum grown in SFE medium could be used with neem oil for vector control.     

Media for the detection of Bacillus larvae spores in honey

Bacillus larvae, the causative agent of American foulbrood in honey bees completes its life cycle of germination, outgrowth and sporulation in young honey bee larvae by killing them and often bringing about the destruction of the entire hive. While B. larvae germinates and outgrows on complex organic media in vitro, the literature suggests, for reasons that are not at all clear, that a relatively large number of spores of B. larvae are required to yield each visible colony (colony forming units, CFU) on media. Various researchers have reported that from 16 to 3,000 or more spores of B. larvae are required to yield a single colony on an agar plate. HANSEN in Denmark designed a useful method of spreading approximately 80 mg of honey directly on the surface of a PETRI plate containing “J” agar medium to determine if B. larvae spores are present in the honey. In the present study, selected media were tested for the ability to recover B. larvae spores in honeys in the form of visible colonies (CFU) using HANSEN's strek method. A modification of a medium (TMYGP) developed by DINGMAN and STAHLY, (T-HCL-YGP agar), recovered considerably more viable B. larvae spores in the form of visible colonies (CFU) than HANSEN's “J” medium. When “J” medium was fortified with 0.1% sodium pyruvate, it was comparable to modified T-HCL-YGP medium in its recovery of B. larvae spores. Brain heart infusion agar (BHIA) with the addition of thiamine recovered more spores in the form of viable colonies than did “J” medium but it was not as efficient as T-HCL-YGP medium. Serial dilution from 100 to 10,000 times of weighed samples of honey with deionized water led to higher spore counts (CFU per g honey) than that indicated by undiluted honeys plated at 80 mg levels directly onto the surface of media by the HANSEN procedure.     

华北落叶松树皮表面细菌群落多样性及其分布格局

附生在树皮表面的微生物,对于宿主植物的健康和环境适应性发挥重要作用,树皮微生境中微生物群落结构和多样性及其维持机制有待关注。【目的】本文对庞泉沟自然保护区不同海拔梯度的华北落叶松树干的背阴面与向阳面的树皮表面细菌群落的分布特征和适应机制进行了研究。【方法】通过PCR-DGGE和高通量测序技术研究细菌群落特征,对树皮表面细菌群落的空间分布特征进行了非度量多维尺度(NMDS)排序分析,通过冗余分析(RDA)研究细菌群落与树皮表面理化性质的关系,单因素方差分析(One-way ANOVA)比较树皮背阴面与向阳面细菌群落组成,基于零偏差分析研究背阴面与向阳面细菌群落构建的驱动因素。【结果】不同海拔梯度间的细菌群落结构有显著性差异(ANOSIM;P<0.05),树皮表面的微环境因子p H和总碳(TC)与群落结构显著相关(P<0.05);向阳面树皮表面的光合自养型细菌(隶属于蓝细菌门的未命名的目)的相对丰度显著高于背阴面,而根瘤菌目的相对丰度呈相反的趋势,光照可能是引起细菌群落结构发生变化的驱动因子;零偏差分析结果表明,华北落叶松树皮背阴面、向阳面细菌群落的多样性格局主要受环境过滤的影响。【结论】环境因子主导的确定性过程是驱动该地区华北落叶松树皮表面细菌群落结构和多样性的主导因素。     

Fungal associates of the tree-killing bark beetle,Ips typographus,vary in virulence,ability to degrade conifer phenolics and influence bark beetle tunneling behavior

The bark beetle Ips typographus carries numerous fungi that could be assisting the beetle in colonizing live Norway spruce (Picea abies) trees. Phenolic defenses in spruce phloem are degraded by the beetle's major tree-killing fungus Endoconidiophora polonica, but it is unknown if other beetle associates can also catabolize these compounds. We compared the ability of five fungi commonly associated with I. typographus to degrade phenolic compounds in Norway spruce phloem. Grosmannia penicillata and Grosmannia europhioides were able to degrade stilbenes and flavonoids faster than E. polonica and grow on minimal growth medium with spruce bark constituents as the only nutrients. Furthermore, beetles avoided medium amended with phenolics but marginally preferred medium colonized by fungi. Taken together our results show that different bark beetle-associated fungi have complementary roles in degrading host metabolites and thus might improve this insect's persistence in well defended host tissues.     

Coralloid root regeneration on Macrozamia megagametophites

Abstract

Megagametophytes of Macrozamia communis were incubated in White's Basal Medium and in White's Basal Medium modified with 2,4-D and kinetin. On the medium enriched with growth substances, regeneration of coralloid roots was induced. These are morphologically identical to sporophytic coralloid roots, without any endosymbiont and displaying negative geotropism. These results confirm the fact that coralloid roots represent an inherent feature of the root system of the Cycadales rather than being the result of induction by microbial factors. Therefore it is possible to suggest that coralloid roots represent vestigial pneumatophores.     

Discovery of bacteria in the cell wall of the aquatic liverwort Jungermannia vulcanicola Steph. in an acid stream with pH 4.2-4.6

Abstract

T.E.M.s of the leaves of the aquatic liverwort Jungermannia vulcanieola showed the existence of rod-shaped bacteria and numerous holes in the cell walls. This suggests that bacteria playa role in the decomposition of the liverwort in an aquatic ecosystem.     

葡萄糖对小球藻(Chloralla sp. HN08)光合作用和生长的影响

【目的】探讨葡萄糖作为外加碳源对热带海洋小球藻(Chloralla sp.HN08)生物质生产和脂、光合色素、碳水化合物及可溶性蛋白等细胞主要成份含量的影响。【方法】分析比较小球藻HN08在光合自养和兼养(添加10 g/L葡萄糖)2种营养方式下的生长速率、细胞密度、光合放氧速率、油脂相对含量,以及可溶性总糖、淀粉和可溶性蛋白的含量。【结果】结果表明,在光照条件下葡萄糖(10 g/L)能促进小球藻(Chloralla sp.HN08)生长,提高细胞终密度,而异养条件下藻细胞逐渐衰亡。兼养条件下,细胞相对生长速率及细胞终密度分别是自养条件下的6.8倍和1.3倍。兼养藻细胞中可溶性糖、淀粉、油脂含量显著高于(P0.05)光合自养细胞,然而可溶性蛋白质和光合色素含量显著低于(P0.05)光合自养细胞。添加葡萄糖的小球藻液的光饱和点和呼吸速率均高于光自养条件下的细胞,但2种培养条件下藻液的净光合速率无显著差异(P0.05)。【结论】光照条件下,添加葡萄糖可显著提高小球藻HN08相对生长速率和细胞终密度,促进油脂与淀粉的积累。     

The Cultivation of Blastocrithidia triatomae Cerisola et al., 19711

Blastocrithidia triatomae, a species very resistant to cultivation in conventional media, was successfully grown in association with cultured lepidopteran cells. The cultured flagellates were identical to those in the insect host, including the presence of characteristic cysts on the flagellum. Growth rate was better for cultures in the Heliothis zea medium than in IPL-45 medium or modified McConnell's medium.     

Human mesenchymal stromal cells preserve their stem features better when cultured in the Dulbecco's modified Eagle medium

Background aimsThe human mesenchymal stromal cell (hMSC), a type of adult stem cell with a fibroblast-like appearance, has the potential to differentiate along the mesenchymal lineage and also along other cell lineages. These abilities make hMSC a promising candidate for use in regenerative medicine. As the hMSC represents a very rare population in vivo, in vitro expansion is necessary for any clinical use. hMSC characterization is commonly carried out through the expression of specific markers and by the capability of differentiating toward at least adipo-, osteo- and chondrocytic lineages. Commitment processes also result in significant changes in the ultrastructure in order to acquire new functional abilities; however, few studies have dealt with the ultrastructural characteristics of hMSC according to the time of incubation and type of media.MethodsThe immunophenotype, functional characteristics and ultrastructural features of bone marrow (BM) hMSC cultured in two different media were investigated. The media chosen were Iscove's modified Dulbecco's medium (IMDM) and the Dulbecco's modified Eagle medium (DMEM). The latter has been recommended recently by two international transplantation and cytotherapy societies, the International Society of Cellular Therapy (ISCT) and European Group for Blood and Bone Marrow Transplantation (EBMT), for hMSC expansion for clinical applications.Results and ConclusionsThe present results indicate that culture conditions greatly influence hMSC ultrastructural features, proliferation, growth and differentiation. In particular, our findings demonstrate that DMEM preserves the hMSC stem features better. Furthermore, the results obtained in IMDM suggest that a small size does not always correlate with conditions of cell immaturity and a greater proliferative potential.     

Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii

Methods for efficient growth and manipulation of relatively uncharacterized bacteria facilitate their study and are essential for genetic manipulation. We report new growth media and culture techniques for Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium known. A low osmolarity defined growth medium (LOD) was developed that avoids problems associated with precipitates that form in previously reported media allowing the monitoring of culture density by optical density at 680 nm (OD₆₈₀) and more efficient DNA transformation by electroporation. This is a defined minimal medium and does not support growth when a carbon source is omitted, making it suitable for selection of nutritional markers as well as the study of biomass utilization by C. bescii. A low osmolarity complex growth medium (LOC) was developed that dramatically improves growth and culture viability during storage, making it a better medium for routine growth and passaging of C. bescii. Both media contain significantly lower solute concentration than previously published media, allowing for flexibility in developing more specialized media types while avoiding the issues of growth inhibition and cell lysis due to osmotic stress. Plating on LOD medium solidified by agar results in ~1,000-fold greater plating efficiency than previously reported and allows the isolation of discrete colonies. These new media represent a significant advance for both genetic manipulation and the study of biomass utilization in C. bescii, and may be applied broadly across the Caldicellulosiruptor genus.     

树兰原球茎内生细菌群落多样性及功能分析

【目的】通过分析树兰原球茎内生细菌群落组成、多样性特征和促生功能,探究树兰种子萌发相关的核心细菌类群及生物学功能。【方法】以树兰原球茎(树兰种子在树皮基质上共生萌发、在树叶基质上共生萌发、在MS1培养基上非共生萌发)和共生萌发基质(松树皮、腐熟树叶)共5个样本为研究材料,采用高通量测序技术分析不同萌发条件下原球茎内生细菌的16S rRNA基因多样性,比较分析细菌群落多样性和物种组成特征,通过传统的内生细菌分离方法获得共生萌发原球茎内生细菌菌株,并进行促生潜力评价。【结果】从5个研究样本中共获得2 735个可操作分类单元(operational taxonomic unit, OTU),属于41门453科876属,其中变形菌门(Proteobacteria)和放线菌门(Actinobacteria)为优势门。主坐标分析(principal coordinates analysis, PCoA)结果表明,树兰原球茎与萌发基质细菌群落结构存在差异,非共生萌发原球茎与在树皮基质上共生萌发原球茎内生细菌群落结构最为接近。功能预测表明,在树叶基质上共生萌发的原球茎内生细菌固氮功能显著高于其他萌发条件。通过分离培养,共获得内生细菌19株,分属12属16种,其中鞭毛膨胀芽孢杆菌(Tumebacillus flagellatus)、Bradyrhizobium cenepequi和人参腐殖土魏茨曼氏菌(Weizmannia ginsengihumi)为共生萌发原球茎共有种;韩国假单胞菌(Pseudomonaskoreensis)和 W. ginsengihumi兼具有溶磷、产吲哚乙酸(indole-3-acetic acid, IAA)和铁载体的潜在能力。【结论】在不同环境中萌发的树兰原球茎均有丰富的内生细菌群落定殖;从共生萌发原球茎中分离的内生细菌具有固氮、溶磷、产IAA和铁载体等促生功能。本研究为兰科植物种子萌发相关微生物资源挖掘及兰科植物与微生物互作研究提供科学依据。     

Biomass and nutritive value of Spirulina (Arthrospira fusiformis) cultivated in a cost-effective medium

Introduction

Cultivation of spirulina at commercial-scales relies on analytical grade–based media, which are expensive and so are the product.

Purpose

This study assessed the biomass, proximate composition, and other useful compounds in Spirulina (Arthrospira fusiformis) produced with a cost-effective culture medium (LCMA), and the results were compared with those from a standard Zarrouk medium–grown spirulina.

Methods

The LCMA medium was formulated by using a commercial NPK10-20-20 fertilizer as a source of the three major nutrients for spirulina growth, and other three ingredients from Zarrouk medium. The experiment was conducted for 28 days in the glass aquaria under indoor conditions. Standard analytical methods were applied for the determination of proximate composition, chlorophyll, minerals, and vitamins in the spirulina biomass.

Result

The LCMA medium showed the best growth conditions by accumulating higher chlorophyll content (0.99 ± 0.02%) and dry weight (0.75 ± 0.01 g/100 ml) as well as attaining higher optical density (2.06 at day 15) earlier than the Zarrouk medium. The results of the proximate analysis for spirulina cultured in the LCMA medium were of good quality, with the protein contributing more than 50% of its dry matter. It was further noticed that the LCMA was an ideal medium for optimization of vitamins and some minerals since it recorded a significant amount of most of the analyzed vitamins together with the minerals sodium and potassium compared with the Zarrouk medium.

Conclusion

It is suggested that LCMA medium could be used as the alternative and cheap medium for maximization of biomass and production of useful biochemical compounds in spirulina species.

     

Embryonic development of an endoparasitoid,Microplitis croceipes (Hymenoptera: Braconidae) in cell line-conditioned media

Summary Embryos of the parasitoidMicropolitis croceipes develop from pregerm band stage to first larval instar in cell culture medium conditioned by a cell line (IPLB-LdFB) derived from fat body from an atypical hostLymantria dispar. However, the percentage of eggs that develop normally to the first larval instar stage is significantly less than for those maintained in IPL-52B medium conditioned with host fat body tissue. Therefore, we examined the capacity of five insect cell lines to promote growth and development of pregerm band eggs in five media, IPL-52B, TC-199, TC-100, Grace’s, and ExCell 400. The developmental response ofM. croceipes was dependent both on the cell line and the cell culture medium used. TC-100, TC-199, and Grace’s media promoted development to the germ band stage without the need for conditioning with host tissue. IPL-52B supported development to the germ hand stage when a defined lipid concentrate was added. In IPL-52B medium, the IPLB-LdFB cell line promoted a significantly higher number of eggs developing to germ band relative to the other cell lines; however, none of the cell line-conditioned IPL-52B medium significantly stimulated egg hatch relative to the control medium. None of the cell line-conditioned Grace’s media had a significant effect on eggs attaining germ band stage compared with the Grace’s control medium. However, Grace’s medium conditioned with the IAL-TND1 and IPLB-LdFB cell lines promoted development beyond germ band, resulting in a significantly higher percentage of hatching eggs than the Grace’s control medium. Although the BCIRL-HZ-AMI cell line, which is derived from the parasitoid’s typical host, did not induce hatch in either IPL-52B medium or Grace’s medium, it promoted hatch in TC-199 and Excell 400 media. Fat body taken from the same species that the cell lines were derived from was a better predictor of a cell line’s embryotrophic activity in Grace’s medium rather than in IPL-52B medium. Thus, the composition of the medium and the species and tissue type of the cell line source must be evaluated interactively to determine optimal conditions for promoting development ofM. croceipes in vitro.