Sterile Mixing Research Articles

Acclimatization of Micropropagated Stevia rebaudiana Bertoni to ex-vitro Conditions: Comparative Ecophysiology and Leaf Anatomy

In vitro propagation has been extensively used for the rapid multiplication of many medicinal plants. However, its wider use is often restricted by the high percentage of plant loss or damage when transferred to ex vitro conditions. In the present study, a successful attempt has been made to acclimatize the tissue culture raised plants of Stevia rebaudiana Bert. under climate controlled conditions with a 95% survival rate. Well rooted cultures were carefully taken out of the medium, washed thoroughly and pre-incubated in coco natural growth medium (Canna-Continental, Los Angeles, CA, www. canna-hydroponics.com) in small jiffy pots for 10 days at 25oC and 18 hours under cool florescent lights. The pots were covered with polythene bags to maintain humidity. These plants were then transferred to a sterile potting mix, fertilome (Canna-Continental, Los Angeles, CA), in larger pots. All these plantlets were kept under controlled environmental conditions (Temperature, 25±3oC; light, ˜800µmol m-2s-1 and RH, 55±5%) with mother plants in the indoor cultivation facility at Coy-Waller laboratory, University of Mississippi. Well grown tissue culture raised plants were then evaluated for their ecophysiological (photosynthesis, transpiration, stomatal conductance, internal CO2 concentration and water use efficiency) and leaf anatomical (stomatal length, width, frequency and index; leaf transverse section; epidermal cells; palisade cells and spongy parenchyma cells) characteristics and were compared with the mother plant. Our results reveal no statistical differences in gross morphology, ecophysiology and anatomical characteristics of the two types of plants. Acknowledgements: This research was partially funded by the USDA, Agricultural Research Service Specific Cooperative Agreement No. 58–6408–7-012.

Comparative micropropagation efficiency of diploid and triploid mulberry (Morus alba cv. S1) from axillary bud explants

Micropropagation ability of two cytotypes (2n = 2x = 28 and 2n = 3x = 42) of mulberry (variety S 1) was tested from axillary buds through organogenesis. Both shoot and rootlets development periods varied considerably with ploidy variations. The triploid shoots generally grew more vigorously in Murashige and Skoog (MS) medium fortified with 6-benzyl amino purine (6-BAP) than those of diploids. Maximum shoot initiation frequency (80%) was obtained with 4.4 µM of 6-BAP on 4.2 days of culture in triploid; whereas, the axillary buds sprouted within 3.5 days with 100% shoot initiation frequency in diploid cultures. Highest shoot length (4.8 and 5.6 cm per explant) was obtained at 8.8 and 4.4 µM 6-BAP supplementations in diploid and triploid cytotypes, respectively. Subsequently, regenerated shoots were transferred to auxin rich rooting media supplemented with either the different doses of indole acetic acid (IAA) or naphthalene acetic acid (NAA). Root initiation was more vigorous in diploid than triploids with both the tested growth regulators. Maximum number of rootlets per shoot (15) and root length (4.2 cm) were observed in MS basal medium supplemented with 4.0 µM NAA after 21 days of culture of diploid plants; while in triploid cytotype, maximum rootlets per shoot (8.3) were noticed with 4.4 µM NAA after 21 days of culture. Rooted plantlets were hardened in plastic cups containing sterile sand and soil mix (1:1; w/w) for 21 days and subsequently transferred to clay pots (l x b x h: 12 x 12 x 12 cm) in shade with a survival of 65 and 52% for diploid and triploid cytotypes, respectively. The time required for field establishment of micro-propagated triploid was about 60 days.

EfficientIn vitroplant regeneration through leaf base derived callus cultures of abiotic stress sensitive popular AsianIndicarice cultivar IR 64 (Oryza sativaL.)

A simple and efficient protocol has been developed for high frequency plant regeneration through callus cultures derived from leaf bases of abiotic stress sensitive Asian indica rice variety IR 64. Leaf base segments (4-5 mm diameter) were obtained from 6-day-old dark grown seedlings germinated on halfstrength Murashige and Skoog medium and cultured on MS medium supplemented with different concentrations of 2,4-Dichlorophenoxyacetic acid (2.2-18 μM) and Kinetin (0.2-1.7 μM). Among the various combinations, 13.5 μM 2,4-D and 1.3 μM Kn resulted in high callus induction frequency (87.5%) with a maximum fresh weight of 0.22 g per segment. The regeneration frequency was 75.5% with multiple shoots within 3 weeks of transfer on MS medium supplemented with 13.3 μM 6-benzylamino purine and 8 μM Naphthaleneacetic acid. The shoots readily rooted on half-strength MS medium without any hormonal supplements. In vitro regenerated plantlets with multiple shoots and roots were transferred to sterile soil and vermiculite mix and maintained in shade house for 30 days. Complete plantlets were then transferred to nursery and acclimatized to the external environment until seed set. RAPD profile reveals monomorphism and thus confirming the genetic stability of the regenerated plants. This method has the potential for both direct as well as indirect method of transformation for the production of genetically modified plants.

A laboratory investigation and validation of methods for sampling contaminated uniforms and work-wear

This study was designed to investigate and validate methods for sampling and retrieval of micro-organisms from contaminated uniform/work-wear. Recent guidance in the United Kingdom states there is no conclusive evidence that work-wear poses an infection transmission risk in the healthcare environment. Although some studies have identified the presence of pathogenic organisms on uniforms, the link between healthcare associated infection (HCAI) and work-wear has not been established in the current literature. A key aspect of such investigations is the ability to reproducibly recover and detect the number of micro-organisms on the garments under investigation. Therefore, in order to undertake further research into contamination levels of uniforms, the methods used to retrieve organisms need to be validated for this particular purpose. In this study swatches of standard, sterile work-wear polyester mix material were inoculated with Staphylococcus aureus, Bacillus subtilis and Bacillus atrophaeus to represent potentially pathogenic organisms likely to be implicated in HCAI. Following incubation, four sampling methods were tested in a laboratory setting (swabbing, carpet sampler, Sartorius air sampler, Casella slit sampler) against the reference method (stomaching) and the numbers of colony forming units (cfu) recovered from the swatches were then recorded. The carpet sampler was the most efficient method in recovering microbiological contamination that had been applied dry to the sterile swatches. At the higher inoculum levels, the carpet sampler retrieved 51% of the challenge organisms compared to the Sartorius air sampler (6%), Casella slit sampler (10%) and swabbing (6%). The reference method of stomaching recovered 104%. The results demonstrated that wet contamination of certain materials can lead to significant binding of micro-organisms to the test fabric. Advantages of the carpet sampler compared to other methods include the requirement for less equipment, ease of use in a clinical environment and the capacity to test large numbers rapidly.

First Report of Crown Rot of Grafted Cucumber Caused by Fusarium solani in China

Grafting has been widely and effectively used in cucumber (Cucumis sativus) cultivation for approximately 30 years in China to avoid Fusarium wilt caused by Fusarium oxysporum Schl. f. sp. cucumerinum Owen. In greenhouses, 90% of cucumbers are grafted onto pumpkin (Cucurbita moschata) rootstock. However, in March 2009, a severe crown rot causing yellowing and wilting of the leaves was observed on grafted cucumber in a large number of greenhouses in Lingyuan, western Liaoning Province in China. Symptoms consisted of dark brown, water-soaked lesions and a dense, white mycelial mat at the base of the stem. Lingyuan is the largest district for cucumber cultivation using grafting techniques in solar greenhouses in China. In 30 surveyed greenhouses in Sanshijiazi Village in the city of Lingyuan, the incidence of affected plants ranged from 10 to 40%, which caused serious economic losses. Fusarium spp. were isolated from the surface-sterilized basal stems of symptomatic plants on potato dextrose agar and incubated on potato sucrose agar for 4 days at 25°C. Colonies of the isolates produced a brown pigmentation and sparse, aerial mycelia, with a cream color on the underside. Conidiophores were elongated and branched or unbranched. Microconidia were abundant, hyaline, ellipsoid to ovoid, and 6 to 14 × 2.5 to 3.5 μm. Macroconidia were cylindrical, abundant, mostly two to six septate, 22 to 63 × 3.2 to 5.0 μm, with the apical cell rounded and blunt, and the basal cell rounded. On the basis of morphological characteristics, the fungus was identified as F. solani (C. Booth). For confirmation, the internal transcribed spacer region of rDNA was amplified and sequenced. A 449-bp sequence shared 99% homology with that of a F. solani GenBank accession previously reported from Japan (No. AF150473.1). The new sequence was deposited in GenBank (Accession No. HM015882). Pathogenicity of three isolates was determined in two experiments using different methods of inoculation. In one, 30 seedlings of pumpkin (C. moschata) with one true leaf each were inoculated by dipping their roots in a suspension of 106 spores ml-1, while control plants were mock inoculated with sterile water. Plants were then potted in a sterile mix of peat moss and vermiculite (2:1 vol/vol). In the other, pregerminated pumpkin seeds were sown in the same medium with a conidial suspension added at a rate of 106 spores ml-1, while other seeds were sown in sterile soil as controls. Plants for both experiments were maintained in a greenhouse at 25°C. Twelve days after inoculation, inoculated plants in both experiments showed a cortical rot on the crown and stem base with a brown, water-soaked appearance. Twenty-one days later, inoculated plants developed wilting and yellowed leaves. Disease incidence was 100%. No symptoms occurred on the control plants. Both experiments were repeated once with the same results. The pathogen was recovered from symptomatic tissue, confirming Koch's postulates. F. solani has been previously reported to cause root rot on cucurbit in California (2) and crown rot on grafted cucumber in the Netherlands (1). To our knowledge, this is the first report of crown rot of grafted cucumber caused by F. solani in China.

Signatures of heavy sterile neutrinos at long baseline experiments

Sterile neutrinos with masses $\ensuremath{\sim}0.1\text{ }\text{ }\mathrm{eV}$ or higher, allowed with the current oscillation data, can potentially play an important role in astrophysics and cosmology. We explore possible signatures of such sterile neutrinos at long baseline experiments. We determine the neutrino conversion probabilities analytically in a 4-neutrino framework, including matter effects, treating the sterile mixing angles ${\ensuremath{\theta}}_{14}$, ${\ensuremath{\theta}}_{24}$, ${\ensuremath{\theta}}_{34}$, the deviation of ${\ensuremath{\theta}}_{23}$ from maximality, as well as ${\ensuremath{\theta}}_{13}$ and the ratio $\ensuremath{\Delta}{m}_{\ensuremath{\bigodot}}^{2}/\ensuremath{\Delta}{m}_{\mathrm{atm}}^{2}$ as small parameters for a perturbative expansion. This gives rise to analytically tractable expressions for flavor conversion probabilities from which effects of these parameters can be clearly understood. We numerically calculate the signals at a neutrino factory with near and far detectors that can identify the lepton charge, and point out observables that can discern the sterile mixing signals. We find that clean identification of sterile mixing would be possible for ${\ensuremath{\theta}}_{24}{\ensuremath{\theta}}_{34}\ensuremath{\gtrsim}0.005$ ($3\ensuremath{\sigma}$) and ${\ensuremath{\theta}}_{14}\ensuremath{\gtrsim}0.06\text{ }\text{ }\mathrm{rad}$ ($3\ensuremath{\sigma}$) with the current bound of ${\ensuremath{\theta}}_{13}<0.2\text{ }\text{ }\mathrm{rad}$; a better ${\ensuremath{\theta}}_{13}$ bound would allow probing smaller values of sterile mixing. We also generalize the formalism for any number of sterile neutrinos, and demonstrate that only certain combinations of sterile mixing parameters are relevant irrespective of the number of sterile neutrinos. This also leads to a stringent test of the scenario with multiple sterile neutrinos that currently is able to describe all the data from the short baseline experiments, including LSND and MiniBooNE.

Sterile neutrinos, lepton asymmetries, primordial elements: How much of each?

We investigate quantitatively the extent to which having a primordial leptonic asymmetry $({n}_{\ensuremath{\nu}}\ensuremath{\ne}{n}_{\overline{\ensuremath{\nu}}})$ relaxes the bounds on light sterile neutrinos imposed by BBN and LSS. We adopt a few assumptions that allow us to solve the neutrino evolution equations over a broad range of mixing parameters and asymmetries. For the general cases of sterile mixing with the electron or muon neutrino, we identify the regions that can be reopened. For the particular case of a LSND-like sterile neutrino, soon to be rejected or confirmed by MiniBooNE, we find that an asymmetry of the order of ${10}^{\ensuremath{-}4}$ is needed to lift the conflicts with cosmology.

Corrigendum to: A novel in vitro rooting method employing an aerobic medium

The beneficial influence of an aerobic propagation medium for in vitro cultures during the rooting phase was found for 28 Australian species and genotypes from the families Liliaceae, Haemodoraceae, Myrtaceae, Thymelaeaceae, Proteaceae, Goodeniaceae and Rutaceae. Microcuttings from established shoot cultures were pulsed for 7 days in the dark on a high-auxin (40 µM indole-3-butyric acid, IBA), agar-solidified medium. The microcuttings were then transferred either to an agar-solidified medium without plant-growth regulators (M1) or a sterile propagation mix. The protocol utilising propagation mix used is referred to as IVS (in vitro soil-less medium). The pulsed cuttings in agar or IVS were placed in the culture room under standard light and temperature regimes and allowed to root. When compared over two harvest times, the use of IVS as a rooting medium gave consistent improvements over the use of M1 medium for percentage rooting, average total root length and root number per microcutting. In total, 27 of the 28 species tested rooted in IVS medium at equal or better rates than in M1. In three cases, Actinodium cunninghamii, one of the genotypes of Pimelea physodes and one of the genotypes of Eriostemon australasis shoots did not root in M1 but showed good root development in IVS medium. With few exceptions, average root length and root number in microcuttings rooted in IVS were superior to the lengths and numbers recorded in agar medium. The materials handing advantages and the application of IVS are discussed.

Effects of sterile neutrinos on the ultrahigh-energy cosmic neutrino flux

We investigate the effect of sterile neutrinos that are nearly degenerate with active ones on the flux of ultrahigh-energy cosmic ray neutrinos at earth. This offers a way to probe neutrino oscillations in the mass-squared range 10−16 eV2≲δm2≲10−11 eV2 which maybe hard to detect by any other means. Taking into account the present experimental uncertainties of the active–active mixing angles and by allowing any values for the active–sterile mixing angles we find that the ratio of the electron and muon neutrino fluxes may change by −40% to 70% in comparison with the ratio in the absence of active–sterile mixing.

The influence of medium aeration on in vitro rooting of Australian plant microcuttings

Media with different air filled porosity were compared with standard agar medium for root induction and root elongation for two Australian plants Grevillea thelemanniana and Verticordia plumosa×Chamelaucium uncinatum. Microcuttings from shoot cultures were pulsed for 7 days on a high auxin (40 μM IBA), agar-solidified medium in the dark. The rooting of the microcuttings was then compared on standard agar medium (M1, 1/2 MS, no hormones) and on three experimental treatments: – porous-agar medium (1/2 MS, no hormones, 30 g agar l−1, solidified then blended to provide aeration); – white sand, or white sand wet with M1 medium; and – a sterile propagation mix. The protocol using the propagation mix is referred to as IVS (In Vitro Soil). A separate experiment involved flushing the IVS soil profile with low or normal oxygen. The controls on M1 medium showed low and variable rooting percentages. The rate of root induction and the average total root length per microcutting at final harvest was significantly higher using the IVS protocol, porous-agar or white sand, while addition of agar medium to sand suppressed the percentage rooting and elongation as did flushing the air space in the IVS rooting medium with low oxygen. Other species tested on M1 medium and IVS including Pimelea physodes, Conospermum eatoniae, Verticordia grandis, and a Chamelaucium megalopetalum×C. uncinatum hybrid all showed a significant improvement on the IVS system. The IVS culture technique reduces plant-handling costs.

Reaction of seedling roots of 14 crop species to Fusarium graminearum from wheat heads

To increase our understanding of the epidemiology of fusarium head blight of wheat and barley, a study was conducted under controlled conditions to determine whether Fusarium graminearum Schwabe from wheat can cause seedling blight or root rot in various crop species. Inoculum of F. graminearum, consisting of a wheat floret infected with the pathogen, was placed adjacent to surface-sterilized seed of each crop in a sterile potting mix. Wheat, barley, oat, rye, triticale, canaryseed, flax, canola (Brassica napus L. and Brassica rapa L.), mustard, bean, field pea, lentil, and chickpea were included in the study. Seedling emergence and root rot severity were scored at 3–4 weeks after seeding. The effect of temperature on seedling blight severity was also tested in barley cv. Brier. Inoculation reduced emergence in all crops, except canola, mustard, and field pea, and increased root rot severity in most crops. Emergence of seedlings was not affected at the lowest temperature (10:5°C day:night) and no root infection occurred. However, as the temperature increased from 10 to 30°C, seedling emergence and establishment were reduced and root rot severity increased. Infection of roots, crowns, and seedlings of the crops grown in rotation with wheat indicates that these crops may act as alternative hosts to F. graminearum.

Fate of the sterile neutrino

In light of recent Super-Kamiokande data and global fits that seem to exclude both pure ν μ → ν s oscillations of atmospheric neutrinos and pure ν e → ν s oscillations of solar neutrinos (where ν s is a sterile neutrino), we reconsider four-neutrino models to explain the LSND, atmospheric, and solar neutrino oscillation indications. We argue that the solar data, with the exception of the 37Cl results, are suggestive of ν e → ν s oscillations that average to a probability of approximately 1 2 . In this interpretation, with two pairs of nearly degenerate mass eigenstates separated by order 1 eV, the day-night asymmetry, seasonal dependence, and energy dependence for 8B neutrinos should be small. Alternatively, we find that four-neutrino models with one mass eigenstate widely separated from the others (and with small sterile mixings to active neutrinos) may now be acceptable in light of recently updated LSND results; the 37Cl data can be accommodated in this model. For each scenario, we present simple four-neutrino mixing matrices that fit the stated criterion and discuss future tests.

346 Ex Vitro Rooting of Micropropagated Hamamelis

Hamamelis cultivars are typically propagated by grafting onto H. virginiana rootstock. Grafting is labor-intensive and the understock frequently suckers which can lead to the loss of the scion. A cultivar growing on its own root system would eliminate this problem. Our research was undertaken to develop a successful method of rooting micropropagules. The source material was established cultures of H. × intermedia `Diane,' H. virginiana, and a H. vernalis selection. The rooting treatments consisted of four concentrations of K-IBA (0, 5, 10, and 20 μM) in 0.02% Tween 80. Three replicates of eight cuttings each were taken from the three sources for each of the four treatments. The cuttings were placed in 50-mL beakers, cut-end down, with 10 mL of the treatment solution. The beakers were sealed with Parafilm, and cuttings were soaked for 24 h. After treatment, the cuttings were randomly stuck into Kadon flats prepared in advance with a sterile mix of 1 peat: 1 perlite and were watered-in. Cuttings were misted, and flats were covered with plastic and Remay. They were kept in a warm (19-24 °C) greenhouse. Cuttings rooted in 3 to 4 weeks and were subsequently fertilized weekly with Peter's 20N-20P-20K at 150 ppm. At 12 weeks, data were collected for the rate of survival, height, branching, number of nodes, and root mass, and the plants were transplanted to quart pots. Ninety percent of the cuttings rooted; the most favorable response was with 10 μM K-IBA, although all treatments produced >80% rooting. This method was time and labor efficient. Moreover, micropropagation is not dependent on the season, and production of new plants could proceed on a continuous basis, making this a viable alternative to commercial grafting.

Application method and growing medium affects the response of cucumber seedlings to inoculation with Trichoderma harzianum

Cucumber seeds were inoculated at sowing with a mixture of seven isolates of Tvichoderma harzianum, previously observed to confer plant growth promotion in commercial horticulture. The Trichodevma mixture was applied by three different methods: spore-coated organic pellets, a dried biomass powder, or seed coating. These were added to three different growing media: sterile potting mix, field soil (naturally containing a Pythium sp.) or field soil drenched with captan. The number of healthy cucumber seedlings was recorded after 8 days. There was no effect of inoculation in potting mix. The number of healthy seedlings was lower in the untreated field soil than in the soil treated with captan. The numbers of healthy seedlings were lower in the pellet and dried biomass treatments than in untreated field soil, but with seed coating, the numbers of healthy seedlings were not significantly affected. The effect of seed coating was tested again, with the isolates of T havzianum applied individually and as a inixture to field soil. There was no significant effect on the number of healthy seedlings compared with controls in any Trichoderma treatment.

MICROPROPAGATION OF CHAMOMILLA RECUTITA (L.) RAUSCHERT

Chamomile (Chamomilla recutita (L.) Rauschert) is a Compositae medicinal plant, cross-pollinated, able to be cultivated also on mountain lands, mainly for the production of dry flowers, rich of essential oils, which are known for their beneficial effects. The cultivation in Italy is not diffused enough to satisfy the internal demand; furthermore, the reduced availability of selected varieties does not stimulates cultivation. The research is directed towards breeding and fast propagation of newly selected varieties. Aim of the reported experiments was to find a protocol for micropropagating selected varieties of chamomile. Seeds of the varieties Italia Minardi (diploid, 2n=2x=18), BK2 and Lutea (tetraploids, 2n=4x=36) were used to start the aseptic culture. After surface sterilization and germination of the seeds, selected shoots were transferred to the proliferation phase. At the end of 2 subsequent 30-day subculture multiplication rates, length of proliferated shoots and presence of roots were evaluated using MS basal medium and comparing 4 different growth regulator compositions. Repeated subculturing was also tested. A substratum made of a sterile soil mix was used for the rooting phase. The best medium for the proliferation phase of all genotypes contained 0.5 mg/l of kinetin; it induced an average multiplication rate of 3.2 and a shoot length ranging from 1.7 to 3.5 cm. Rooting on soil mix was above 90% in most cases, depending upon genotype and original proliferation medium. Micropropagation of different genotypes of chamomile was successfully achieved.

Simple devices for sterile aeration and mixing in NMR cell metabolism studies

In the past, difficulty in performing plant and animal cell metabolism studies in NMR samples has often been experienced. Thus, without continuous agitation, cell aggregations formed, leading to inhomogeneous mixtures which excluded the substrate, thereby vitiating kinetic analysis of the experiments. Again, contamination by foreign microorganisms (which in most instances proved to be more vigorous in replication and metabolism than the cells of interest) also occurred. Early attempts to use gases bubbled from the bottom of the sample tube as a mixing device were partially successful but bubbles in the sampling volume seriously degraded field homogeneity and further added to contamination problems. Santos and Turner (1) have described a system which used bubbles rising in an open-ended inner tube to pump the culture, thus overcoming the mixing problems without degrading field homogeneity. A later, somewhat more complicated design based on the same principles has been presented by Boume (2). We have recently modified the design for 10 and 5 mm sample tubes and have also prevented contamination of the initially sterile incubation mixtures by constant flushing with gases which have passed through 0.1 grn filters, the tube itself being maintained at slight positive pressure. The 10 and 5 mm pumping devices as shown in Figs. 1 and 2, respectively, have been successfully utilized in studies in which human breast cancer cell metabolism is followed over 12 hour periods. However, on attempting to use these designs with plant callus cell cultures which are much heavier and more viscous than cancer cells or other mammalian and bacterial cells, we found that the pumping action provided by bubbling was insufficient to prevent cell clumping. Attempts to solve this problem by changing inner tube dimensions, bubble size and rate, and sample tube diameter were unsuccessful. A more powerful agitation system was needed, and we therefore developed the mechanically stirred mixing and aerating system shown in Fig. 3, which uses the normal NMR sample spinner operating at approximately 15 cps as energy source, so that the sample solution is spun over a stationary propeller producing vigorous mixing. This system is somewhat more difficult to sterilize, but we find that, with care, contamination problems are not encountered. The filter and Teflon needle are attached to the Teflon rod above the sample tube cap with tape. Efficient mixing and aeration of plant callus cell cultures with this system was achieved, and samples which had previously died within an hour were stable for days. Our original design utilizes a copper stirrer blade which could be replaced by a blade made of Teflon or glass. This proved unnecessary for the 20 mm sample tube stirrer, however, since no significant problems with sample