Artemisia judaica L.: micropropagation and antioxidant activity

Artemisia judaica L., an Egyptian medicinal plant used in the treatment of gastrointestinal disorders, was mass-propagated and grown using solid, paper-bridge-support liquid, liquid-flask and bioreactor cultures. The liquid-flask culture using 50 ml MS liquid medium in 250 ml flask yielded significantly greater shoot proliferation than either solid cultures or paper-bridge-support liquid cultures. Increasing flask capacity from 100 to 500 ml improved shoot proliferation and growth. Mass-propagation efficiencies of various bioreactor systems, viz. temporary immersion reactors and bubble column reactors, were also compared. The temporary immersion bioreactor was found to have significant advantages for A. judaica shoot proliferation. The shoot cultures from the temporary immersion reactor formed complete plantlets when subcultured onto a medium containing 1 micromoll(-1) indole-3-butyric acid (IBA), and mature plants were established, acclimatized and thrived in standard greenhouse conditions. Assays of antioxidant activity and total flavonoid content of in vitro and in vivo grown tissues were evaluated as gross parameters of medicinal efficacy. Significantly higher antioxidant activity and flavonoid contents were observed in the tissues of mature greenhouse-grown plants. The efficient in vitro production systems developed in this study provided sterile, consistent tissues for investigation of bioactivity and germplasm conservation of A. judaica.     

Lactobacillus plantarum; a deleterious contaminant of plant tissue cultures

Lactobacillus plantarum was isolated from in vitro plant cultures of Hemerocallis Stella d'ora, Catherine Woodbury and Stafford. Infected cultures deteriorated rapidly during three subcultures (15 weeks) when grown in vitro , showing chlorotic white shoots and grey calli. Weaned plants developed normally when transferred to the soil. The drop in multiplication rate of plant cultures coincided with a decrease in pH of the growth media. Uninfected plants of Hemerocallis Stella d'ora showed the same symptoms after inoculation with L. plantarum. Lactobacillus plantarum was reisolated from inoculated plant cultures that showed symptoms, thus fulfilling Koch's postulates. In plants inoculated with L. plantarum both the amount of dl-lactic acid formed and the number of plants showing symptoms increased with increasing numbers of bacteria in the inoculum, wheras plant multiplication rate and pH decreased. These effects could be reproduced by adding dl-lactic acid to the multiplication medium of plants free from L. plantarum , suggesting that the bacterial production of lactic acid was responsible for the changes in infected plants.     

The rare occurrence of plant tissue culture contamination by Methylobacterium mesophilicum

A pink-pigmented, facultative methylotrophic (PPFM) bacterium, Methylobacterium mesophilicum, which is found on the leaf surface of most plants, has been reported to be a covert contaminant of tissue cultures initiated from Glycine max (soybean) leaves and seeds by Holland and Polacco (1992). The bacteria can be detected as pink colonies when leaves are pressed or tissue culture homogenates are plated on a medium with methanol as the sole carbon source. Since the presence of contaminating bacteria can confound any biochemical results obtained with such cultures (Holland and Polacco 1992), we wanted to determine the extent of the contamination of our tissue cultures of soybean and other species. No PPFMs were detected in any soybean culture we have, and previous results describing the biochemical characteristics of ureide utilization by one of our soybean suspension cultures (27C) also indicates that PPFM bacteria were not present. Analysis of about 200 other strains of 11 different species maintained in this lab showed that only three of about 160 callus cultures, recently initiated from Datura innoxia leaves, contained PPFMs. The D. innoxia leaves did have PPFMs on their surface but in most cases they did not survive the surface disinfestation and culture regimes. Thus PPFM bacterial contamination should not be a serious problem in most plant tissue cultures.Abbreviations AMS ammonium mineral salts medium - PPFM pink-pigmented facultative methylotrophic bacteria     

Ecology of Microbial Saprophytes and Pathogens in Tissue Culture and Field-Grown Plants: Reasons for Contamination Problems In Vitro

This review compares published surveys of microbial populations in plant tissue and cell cultures with the microbial saprophytes and pathogens found on field grown plants and microbial populations in the laboratory environment. From this comparison and the measured reduction in contamination after improvements in working practices in the laboratory, conclusions can be drawn about the importance of the explant and the laboratory as sources of contamination.

Mechanisms of pathogenicity in vitro are described to explain why bacteria, fungi, and yeasts that are not pathogenic to plants in the field become pathogens in plant tissue cultures. Conversely, plant metabolism and its effect on the tissue culture environment are described to explain why prokaryotes, viruses, and viroids that cause disease in the field can stay latent in vitro.

Detection methods for latent contaminants in plant tissue cultures are summarized, and the strategies and methods for prevention or treatment of contamination are discussed.     

克隆整合和石油污染对芦苇湿地土壤理化性质的影响

克隆整合是克隆植物重要的性状之一。它不仅能够提高分株对环境胁迫的耐受能力,而且可能影响分株周围的土壤属性。为检验克隆整合对土壤属性的影响,在黄河三角洲芦苇湿地开展克隆整合和石油污染的两因子的野外实验。每年将0、5或10 mm厚的原油添加到直径为60 cm的圆形芦苇群落样方内来模拟无污染、轻度或重度石油污染,并通过保留或切断样方内外芦苇根状茎的连接来实现克隆整合的有或无。实验开始于2014年,并于2016年10月采集样方内土壤样品,测定土壤团聚体组成、pH值、电导率、总碳、总氮、总磷和有机碳含量。石油污染显著增加了土壤粗大团聚体(粒径:2 mm)、pH值、总氮和有机碳含量,降低了土壤微团聚体(粒径:0.053—0.25 mm)占比以及电导率。克隆整合显著降低了土壤pH值,提高了土壤电导率和氮磷比。克隆整合和石油污染的交互作用仅对电导率有显著效应。因此,石油污染和克隆整合都可以影响湿地土壤的理化性质,而克隆整合对分株周围土壤理化性质的影响可能进一步影响克隆植物的优势度。     

Establishment of arbuscular mycorrhizal plants originating from xerothermic grasslands on heavy metal rich industrial wastes–new solution for waste revegetation

Industrial waste substrata, rich in heavy metals, are poorly suited for plant growth. Efforts are made to establish an appropriate plant cover to reduce erosion and further contamination. Grasses are the usual solution, as they grow fast, thrive on poor substrata and have well-developed root systems. Some of them are also highly dependent on mycorrhizal symbiosis that supports their growth especially on poor and polluted soils. However, the commercially available grasses often meet a lack of well established mycorrhiza on the site and the introduced plant populations dramatically decrease with time, despite large financial input including covering the substratum with soil and intensive watering. The aim of this paper was to select proper plants together with mycorrhizal fungi that could accelerate the establishment of the vegetation and improve its diversity under these extreme conditions, minimizing the financial costs of the reclamation (no use of soil layering and watering). The experiments were carried out under field and laboratory conditions. The plant seeds used originated from dry calcareous grasslands. The seeds were germinated under field conditions or in pots filled with soil supplemented with substratum from the industrial wastes. The seedlings were inoculated with AM fungi and introduced on the field plots a few weeks after germination. The inoculum consisted of either crude inoculum harvested from the dry calcareous grasslands or strains originating from polluted areas. Plants colonized by mycorrhizal fungi established well in the experimental plots. The results suggest that inocula from dry calcareous grasslands are potentially useful in revegetation of industrial wastes. Although in several cases the photosynthetic activity of plants was lower than at the natural sites, almost all plants survived and formed seeds. In all experiments the plant vitality was estimated on the basis of chlorophyll a fluorescence and was useful to show differences between waste substrata, inocula and coexisting plant species. The interactions between mycorrhizal and non-mycorrhizal plants were studied under greenhouse conditions and at least no negative effect of this coexistence was found.     

The effect of 2,4-dichlorophenoxyacetic acid and donor plant environment on plant regeneration from immature inflorescence-derived callus of Lolium perenne L. and Lolium multiflorum L.

Callus cultures were obtained from immature inflorescences of perennial ryegrass (Lolium perenne) and Italian ryegrass (Lolium multiflorum). Inflorescence segments were cultured on Murashige and Skoog medium (MS) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The response in culture with regard to compact callus induction, embryogenesis and plant regeneration was determined for different varieties. The in vitro response was compared for explants from field-grown plants and explants from greenhouse-grown plants. The effect of different 2,4-D concentrations on the in vitro response was also investigated in one L. perenne variety and one L. multiflorum variety. The percentage of explants that formed compact callus and embryogenic callus differed strongly with the cultivar. There was no consistent effect of the growth conditions of the donor plants or the 2,4-D concentration of the medium on this response. Green plants were regenerated from all the cultivars tested. Explants from field-grown plants showed a higher tendency to form albino shoots than explants from greenhouse-grown plants. In the L. perenne variety tested higher 2,4-D concentrations (up to 15 mg/l) resulted in a lower regeneration frequency of green shoots and a higher regeneration frequency of albino shoots (up to 12.5 mg/l). In the L. multiflorum variety tested the effect of 2,4-D on regeneration was less pronounced.     

Elemental composition of arbuscular mycorrhizal fungi at high salinity

We investigated the elemental composition of spores and hyphae of arbuscular mycorrhizal fungi (AMF) collected from two saline sites at the desert border in Tunisia, and of Glomus intraradices grown in vitro with or without addition of NaCl to the medium, by proton-induced X-ray emission. We compared the elemental composition of the field AMF to those of the soil and the associated plants. The spores and hyphae from the saline soils showed strongly elevated levels of Ca, Cl, Mg, Fe, Si, and K compared to their growth environment. In contrast, the spores of both the field-derived AMF and the in vitro grown G. intraradices contained lower or not elevated Na levels compared to their growth environment. This resulted in higher K:Na and Ca:Na ratios in spores than in soil, but lower than in the associated plants for the field AMF. The K:Na and Ca:Na ratios of G. intraradices grown in monoxenic cultures were also in the same range as those of the field AMF and did not change even when those ratios in the growth medium were lowered several orders of magnitude by adding NaCl. These results indicate that AMF can selectively take up elements such as K and Ca, which act as osmotic equivalents while they avoid uptake of toxic Na. This could make them important in the alleviation of salinity stress in their plant hosts.     

Microbial hazards in plant tissue and cell cultures

Summary A wide range of microorganisms (filamentous fungi, yeasts, bacteria, viruses and viroids) and micro-arthropods (mites and thrips) have been identified as contaminants in plant tissue cultures. Contaminant may be introduced with the explant, during manipulations in the laboratory or by micro-arthropod vectors. Contaminants may express themselves immediately or can remain latent for long periods of time. This often makes it difficult to identify the source of contamination. Disinfection protocols have now been developed for a wide range of plant species including those infected with viruses/viroids or endophytic bacteria. They may include the selection of pathogen-free donor plants or donor plant treatments such as thermotherapy. Also microbiological quality assurance systems (e.g. Hazard Analysis Critical Control Point; HACCP procedures) have been adapted to the needs of commercial plant tissue culture laboratories. These are aimed at, preventing the introduction of pathogens, into tissue cultures at establishment and in the laboratory. In established in vitro cultures preventative strategies have proved to be essential, since it is extremely difficult to eliminate environmental bacterial and fungal contaminants using, antibiotics and fungicides. In many cases anti-microbial treatments only inhibit contaminants and low levels of contamination persist. In particular, the use of antibiotics against Gram-negative bacteria (including plant pathogenic bacteria and Agrobacterium tumefaciens vector systems used in genetic engineering) has been shown frequently to be extremely difficult or unsuccessful. Detection of latent contamination may involve the use of general and semi-selective microbial growth media or serological and PCR-based molecular techniques for specific pathogens. However, it is often difficult to detect low numbers of latent bacterial contaminants (e.g. levels present following antibiotic treatment or when acidified plant media are used). This poses a particular risk in the production of transgenic plants where the elimination or detection of Agrobacterium tumefaciens-based vector systems cannot be guaranteed with the currently available methodologies. Recent research has also shown that there is a risk of the transmission of human pathogens in plant tissue cultures.     

High frequency direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood (Populus deltoides)

Simple, reproducible, high frequency, improved plant regeneration protocol in Eastern Cottonwood (Populus deltoides) clones, WIMCO199 and L34, has been reported. Initially, aseptic cultures established from axillary buds of nodal segments from mature plus trees on MS liquid medium supplemented with 0.25 mg l⁻¹ KIN and 0.25 mg l⁻¹ IAA. Nodal and internodal segments were found to be extra-prolific over shoot apices during course of aseptic culture establishment, while 0.25 mg l⁻¹ KIN concentration played a stimulatory role in high frequency plant regeneration. Diverse explants, such as various leaf segments, internodes, and roots from in vitro raised cultures, were employed. Direct plant regeneration was at high frequency of 92% in internodes, 88% in leaf segments, and 43% in root segments. This led to the formation of multiple shoot clusters on established culture media with rapid proliferation rates. Many-fold enhanced shoot elongation and growth of the clusters could be achieved on liquid MS medium supplemented with borosilicate glass beads, which offer physical support for proliferating shoots leading to faster growth in comparison to semi-solid agar or direct liquid medium. SEM examination of initial cultures confirmed direct plant regeneration events without intervening calli. In vitro regenerated plants induced roots on half-strength MS medium with 0.15 mg l⁻¹ IAA. Rooted 5- to 6-week-old in vitro regenerated plants were transferred into a transgenic greenhouse in pots containing 1:1 mixture of vermicompost and soil at 27 ± 2°C for hardening and acclimatization. 14- to 15-week-old well-established hardened plants were transplanted to the field and grown to maturity. The mature in vitro raised poplar trees exhibited a high survival rate of 85%; 4-year-old healthy trees attained an average height of 8 m and an average trunk diameter of 25 cm and have performed well under field conditions. The regeneration protocol presented here will be very useful for undertaking genetic manipulation, providing a value addition to Eastern Cottonwood propagation in future.     

Micropropagation of mature <Emphasis Type="Italic">Cornus mas</Emphasis> ‘Macrocarpa’

Sprouting axillary buds sampled from a mature 27-year-old shrub of Cornus mas ‘Macrocarpa’ were used as starting material for in vitro culture establishment. Multiple shoot cultures, grown on basal woody plant medium with the pH adjusted to 5.6–5.7 and supplemented with 6-benzylaminopurine in combination with 1-naphthaleneacetic acid, were capable of continuous axillary and adventitious shoot proliferation up to 1 year. Later on, growth ceased, shoot tip necrosis appeared and shoot cultures died. Transfer of living shoots onto modified woody plant medium with the pH adjusted to 6.8–7.0 led to vigorous growth of multiple shoot cultures without any loss of multiplication rates or decreased vitality for several years. The use of 6-benzylaminopurine in combination with 1-naphthaleneacetic acid proved superior to the application of thidiazuron which induced a frequent formation of short and fasciated shoots. 1-naphthaleneacetic acid promoted in vitro adventitious rooting frequency up to 73.3%, whereas indole-3-butyric acid was not effective. Ex vitro acclimatized plants did not show any visually detectable morphological variation.     

Effect of phenotypic plasticity on epiphytic survival and colonization by Pseudomonas syringae.

The bacterial epiphyte Pseudomonas syringae MF714R was cultured on agar or in broth or collected from colonized leaves; it was then inoculated onto greenhouse-grown bean plants incubated in a growth chamber at low relative humidity or in the field or onto field-grown bean plants. Cells cultured in liquid medium survived the least well after inoculation of leaf surfaces under all conditions. Cells cultured in solid medium exhibited the highest percent survival and desiccation tolerance in the growth chamber but generally survived less well in the field than did cells harvested from plants. Cells harvested from plants and inoculated onto plants in the field usually exhibited the highest percent survival, started to increase in population earlier, and reached a higher number than did cells cultured in vitro. Differences in field survival were apparently not attributable to differential UV tolerance. The observed effects of phenotypic plasticity on epiphytic survival and colonization should be considered in risk assessment studies, in studies of bacterial epidemiology, and in the use of microbial antagonists for biological pest control.     

植物离体培养中微生物污染的鉴定与控制(综述)

本文综述植物离体培养过程中微生物污染的鉴定与控制的研究进展,包括通过指示培养和菌种鉴别以鉴定污染菌;从保护条件下生长的植株上取材以及材料的预处理,以便有效地控制附生菌和应用抗生素控制内生菌.     

Plant community establishment in a restored wetland: Effects of soil removal

Question: This study investigated the establishment of wetland plant assemblages following soil removal and restored hydrology in a former agricultural field. The following questions were posed. Does plant community composition differ as a result of soil removal? Does soil removal reduce the frequency of non-wetland plants? Does soil removal reduce the frequency of non-native invasive plants? Location: The Panzner Wetland Wildlife Reserve (PWWR) in Summit County, northeastern Ohio, USA. Methods: During 2000–2001, restoration was conducted on two adjoining fields (3.9 ha total) by excavating the upper 40–50 cm of soil layer and establishing 12 10 m × 10 m undisturbed control plots. Preliminary data included seed bank composition and soil organic matter, estimated from three different soil depths on the control plots. In spring 2004, a 10 m × 10 m soil-removed plot was established adjacent to each control plot. Plant percent cover of all species was estimated within the center 5 m × 5 m of every plot. Above-ground biomass of all species from three 0.25-m² quadrats was collected. Environmental water measurements included water depth, temperature, dissolved oxygen, pH, and conductivity. Results: The top 10 cm of soil contained the most seeds, the highest species diversity, the greatest proportion of annual to perennial plants, and the lowest organic content. Obligate and facultative wetland plants were found in soil-removed plots while facultative upland and upland plants were found in control plots. The only plots with arable weeds were the control plots. However, plant communities on soil-removed plots in the North field, which had a higher elevation (ca. 15–20 cm), had a different species composition than soil-removed plots in the South field. Conclusions: The results of a controlled, replicated large-scale study on the effects of soil removal showed that soil removal altered both the biotic and abiotic environment, but that the proximity to the water table was the primary controlling factor in the assembly of plant communities.     

In vitro selection and identification of sweetpotato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) plants tolerant to NaCl

In vitro selection of sweetpotato (Ipomoea batatas (L.) Lam.) plants tolerant to NaCl was achieved using embryogenic suspension cultures of sweetpotato cv. Lizixiang and gamma-ray induced mutation. Cell aggregates from embryogenic suspension cultures of Lizixiang were irradiated with 80 Gy gamma-ray, and 1 week after irradiation they were cultured in a selective medium containing 342 mM NaCl for in vitro selection. A total of 276 plants were regenerated from the irradiated 2,783 cell aggregates by a two-step in vitro selection procedure. After the regenerated plants were propagated into plant lines on the basal medium, they were cultured on the medium supplemented with 86, 171, 257 and 342 mM NaCl, respectively, in order to evaluate their in vitro salt tolerance. Of them 18 plant lines showed significantly higher in vitro salt tolerance than control plants. Proline and superoxide dismutase (SOD) were more accumulated in these 18 plant lines than in control plants when both were exposed to NaCl. Salt tolerance of the 18 plant lines was further evaluated with Hoalgland solution containing different concentrations of NaCl in a greenhouse. The results indicated that 3 of them had significantly better growth and rooting ability than the remaining 15 plant lines and control plants at 171 mM NaCl.     

Conservation and Propagation of High Altitude Medicinal and Aromatic Plant: Hedychium spicatum

The micropropagation of H.spicatum, a medicinal and aromatic plant was investigated as an option for conservation and propagation, as wild populations are fast depleting. The source of raw material is rhizomes of plants that are collected from the wild. There is no planned cultivation of the plant. Multiple shoot cultures were established on MS medium supplemented with BAP and IAA from the pre-existing buds on the rhizome. Prolonged cultivation on the same medium or transfer to hormone free medium induced roots/rhizome formation; liquid medium proved more suitable. Greenhouse hardened plants were transferred to field. A successful protocol with 99% root formation and 80–85.5% field survival has been formulated.     

Influence of waterlogging on the emergence and growth ofLolium perenne L. shoots from seed coated with calcium peroxide

Summary The emergence and seedling growth of cv. S. 24 perennial ryegrass plants was significantly reduced by two waterlogging treatments in a glasshouse environment. These adverse effects of temporary waterlogging were alleviated by coating the seed with calcium peroxide. This oxygenating agent did not enhance or reduce seedling establishment when the soil medium was not waterlogged. Calcium peroxide did not affect the germination of coated seeds when placed on wet filter paper.The establishment of plants in contrasting soils, which experienced water tables close to ground level at two upland field sites, was also improved by coating perennial ryegrass seed with calcium peroxide. Alleviation of the detrimental effects of excessively high water tables was not observed in plants which were established from seed coated with calcium carbonate.     

一种土壤中大豆疫霉菌分离新方法*

由于腐霉菌的干扰,土壤中大豆疫霉菌的分离十分困难。利用大豆疫霉菌的致病性和大豆对病原菌的选择作用排除腐霉菌,我们建立了一种简单、有效的土壤中大豆疫霉菌的分离方法。该方法用不含抗大豆疫霉根腐病基因的大豆叶碟诱钓大豆疫霉菌的游动孢子,将诱钓叶碟直接接种不含抗大豆疫霉菌基因的大豆植株,再对病株进行选择性或非选择性分离获得大豆疫霉菌。此方法能十分有效地排除腐霉菌干扰和细菌的污染,直接获得纯化菌株。应用该方法我们在以前未报道有大豆疫霉根腐病发生的山东、河南、安徽、江苏和浙江分离到大豆疫霉菌。     

Ubiquitous presence of fastidious endophytic bacteria in field shoots and index-negative apparently clean shoot-tip cultures of papaya

This study establishes the widespread prevalence of fastidious or viable but non-culturable endophytic bacteria in field shoots and in unsuspicious shoot-tip cultures of papaya (Carica papaya L.) against the norm of asepsis in vitro. A total of 150 shoot-tips (approximately 10 mm) were inoculated on MS-based culture medium after surface sterilization of field-derived axillary shoots of cv. Surya during November or January (100 and 50, respectively) when 35–50% cultures showed endophytic microbial growth on culture medium. Indexing of apparently clean cultures using bacteriological media helped in detecting and removing additional 14–17% stocks with covert bacteria during the first two passages. The rest of the stocks stayed consistently index-negative during the first eight subculture cycles, but appeared positive in PCR-screening undertaken thereafter employing universal bacterial 16S rRNA gene primers indicating the association of non-cultivable bacteria. Direct sequencing of the PCR product yielded overlapping nucleotide data signifying mixed template or the presence of diverse endophytic microorganisms. This was confirmed by light microscopy of tissue sap revealing viable bacteria in considerable numbers, which were detected under phase contrast or with negative staining. Planting tissue segments or applying homogenate from these stocks on diverse bacteriological media did not induce the organisms to grow in vitro. The shoot cultures displayed variation in growth and rooting potential, the onus of such variation was solely attributable to the associated microorganisms. The findings were confirmed with additional field shoots and fresh in vitro stocks established subsequently. The observations have implications in micropropagation and all other applications involving plant cell, tissue, organ, and protoplast culture.     

Judicious use of kinetin to improve growth and yield of rice in nickel contaminated soil

The present study was conducted to evaluate the effect of kinetin on growth and yield of rice in the presence and absence of nickel contamination. Rice seedlings were dipped in kinetin solution (10^?3, 10^?4 and 10 M^?5) for 2 hours and transplanted in pots having soil contaminated with nickel sulfate @ 130 mg kg^?1. Experiment was laid out according to completely randomized design with four replications. Results revealed that kinetin significantly improved growth and yield of rice grown in nickel contamination. Kinetin @ 10^?4 M showed maximum improvement in plant height, paddy yield, 1000 grain weight, number of tillers and panicles up to 9.76, 15.72, 11.77, 11.87, and 10.90%, respectively, as compared to plants grown in contaminated soil without kinetin. Kinetin also improved the uptake of nutrients (NPK) in straw and grain of plants grown in Ni contaminated soil. Plants treated with kinetin had more concentration of Ni in shoot but less in grain compared to plants grown in Ni contaminated soil without application of kinetin. The application of kinetin can reduce stress effect on plants through improvement in the biomass of plant. This strategy could be used to increase the phytoextraction of Ni from the contaminated soil.