Semisolid Agar Medium Research Articles

Use of rocker bioreactor for propagation of the Vaccinium corymbosum L.

Plant biotechnology has the potential to meet the market demand for a significant number of seedlings. The current state of the industry is characterised by exemplary results in the field of micropropagation, with notable achievements observed in the cultivation of diverse plant crops, particularly berries. Nevertheless, certain aspects of this technology remain imperfect at the industrial level. The multiplication of microshoots of berry crops remains a costly process. The transition of numerous laboratories from semi-solid agar medium to liquid has resulted in a reduction in the costs associated with propagation. However, the majority of standard bioreactors are costly and frequently challenging to maintain. In the present study, a rocker-type bioreactor system for micropropagation of blueberries was tested using Vaccinium corymbosum L. ‘Pink Lemonade’ as an example. In comparison to the traditional method of micropropagation on a gel medium, the rocker-type bioreactor demonstrates superior outcomes. The multiplication coefficient of blueberry shoots was found to be approximately twofold higher when the liquid WPM medium was supplemented with 0.2 mg/l zeatin within the bioreactor, in comparison to the control variant cultivated on a semi-solid medium of identical composition. The shoots obtained in the bioreactor successfully completed the stages of rooting and acclimatisation to post-aseptic conditions.

Soil Microorganisms Increase Mobility of Iron to Rice (<em>Oryza sativa</em> L.) Plants

Understanding the mechanisms involved in iron toxicity tolerance of rice would help develop technologies to close yield gaps. A study was conducted aiming to understand the role of rhizosphere microorganisms in tolerance of iron toxicity in rice plants. Three varieties with different iron toxicity tolerance capacities were selected [BW 267-3 (tolerant), BW 363 (moderately tolerant) and BW 272-6b (susceptible)]. A pot experiment was conducted in a three factor factorial design including soil treatment with microorganisms (presence and absence), three iron levels in growth medium (2, 75 and 300 mg/L) and three rice varieties as grouping factors. At 42 days after germination dry matter yield and total Fe, K and P in shoots were determined. Rhizosphere oxidizing potential of each variety was determined by visualizing oxidizing power of rice roots embedded in a semisolid agar medium containing iron sulfide. Results indicated that presence of soil microorganisms significantly (p<0.05) reduced dry matter yield but increased Fe uptake of all three varieties. However, in presence of microorganisms, Fe uptake remained more consistent irrespective of the Fe concentration in growth medium. Root oxidizing power had a positive relationship with iron toxicity tolerance. The highest oxidizing potential was observed in BW 267-3. Plant-microbial interactions on growth and uptake of Fe, K and P were more prominent in BW 363 and BW 272-6b compared to BW 267-3. We conclude that microorganisms mobilize Fe to rice plants. Further, remarkable differences exist between rice varieties in root oxidizing power and plant-microbial interactions depending on their iron toxicity tolerance.

Rapid Isolation and Purification of Secreted Bacteriocins from Streptococcus mutans and Other Lactic Acid Bacteria.

Bacteriocins are small ribosomally synthesized antimicrobial peptides produced by some microorganisms including lactic acid bacteria (LAB), a group of Gram-positive bacteria (cocci, rods) expressing high tolerance for low pH. Bacteriocins kill bacteria rapidly and are biologically active at very low concentrations. Bacteriocins produced by LAB are primarily active against closely related bacterial species. Many bacteriocins have been investigated with respect to their potential use in promoting human, plant, and animal health, and as food biopreservatives. Bacteriocins produced by LAB are particularly interesting since several LAB have been granted GRAS (Generally Recognized as Safe) status. Because it is not always possible to extract active bacteriocins secreted from cells grown in liquid medium, we developed a simple and inexpensive peptide extraction procedure using a semi-solid nutrient-rich agar medium. We hereby present a detailed procedure that leads to the rapid extraction of secreted bioactive bacteriocin peptides from the oral species Streptococcus mutans, a prolific bacteriocin-producing species, and its potential application for bacteriocin extraction from other LAB (e.g., Streptococcus, Lactococcus, Enterococcus). We also present a simple method for the detection of bacteriocin activity from the purified extracellular peptide extract.

Cell Culture and Gene Expression Studies in Relation to Biomineralization in the Black-lip Pearl Oyster, Pinctada margaritifera

Nacre is composed of aragonite platelets and organic material formed by molluscs as inner shell layer through biomineralization process, and mantle epithelial cells control nacre formation. A primary culture of granulated epithelial cells was established from mantle tissue of Pinctada margaritifera, through a process of continuous sub-culturing at 28°C in yeast supplemented sea water culture medium. Nuclear beads were placed in culture wells containing semi-solid agar medium and incubated in vitro with cultured granulated epithelial cells in order to evaluate the nacre secretion. On visual observation, a brown coloration was observed on the surface of the bead after 7-10 days. Evaluation of the surface of the nuclear beads by scanning electron microscopy (SEM) after 60 days of incubation revealed a good brick and mortar pattern, characteristic of nacreous layer formation. A lustrous hue was also seen to develop on bead surfaces after this stage. SEM images of a cross section of the nacre-coated bead showed a pattern of arrangement of aragonite tablets similar to that seen in cross sections of the nacre layer of shell of molluscs. The functional ability of cultured granulated epithelial cells was further confirmed by detecting gene expression of two matrix proteins, nacrein and amorphous calcium carbonate binding proteins (ACCBP), which play an important role in formation of the nacreous layer, in both cultured cells and in native mantle tissue. Amplification products for nacrein (480 bp) and ACCBP (500 bp) genes were obtained in both native mantle tissue and in vitro cultured mantle epithelial cells. There was good correlation between the expression patterns of the two genes in in vitro cultured cells and in native mantle tissue, signifying that cultured mantle epithelial cells retain their functional characteristics of biomineralization.

Connecting single-cell properties to collective behavior in multiple wild isolates of the Enterobacter cloacae complex.

Some strains of motile bacteria self-organize to form spatial patterns of high and low cell density over length scales that can be observed by eye. One such collective behavior is the formation in semisolid agar media of a high cell density swarm band. We isolated 7 wild strains of the Enterobacter cloacae complex capable of forming this band and found its propagation speed can vary 2.5 fold across strains. To connect such variability in collective motility to strain properties, each strain’s single-cell motility and exponential growth rates were measured. The band speed did not significantly correlate with any individual strain property; however, a multilinear analysis revealed that the band speed was set by a combination of the run speed and tumbling frequency. Comparison of variability in closely-related wild isolates has the potential to reveal how changes in single-cell properties influence the collective behavior of populations.

The overlapping continuum of host range among strains in the Pseudomonas syringae complex

Pseudomonas syringae is the most frequently emerging group of plant pathogenic bacteria. Because this bacterium is ubiquitous as an epiphyte and on various substrates in non-agricultural settings, there are many questions about how to assess the risk for plant disease posed by strains in the environment. Although P. syringae is considered to have discrete host ranges in defined pathovars, there have been few reports of comprehensive comparisons of host range potential. Here we present results of host range tests for 134 strains, representing eight phylogroups, from epidemics and environmental reservoirs on 15 to 22 plant species per test conducted in four separate tests to determine the patterns and extent of host range. We sought to identify trends that are indicative of distinct pathotypes and to assess if strains in the P. syringae complex are indeed restricted in their host range. We show that for each test, strains display a diversity of host ranges from very restricted to very broad regardless of the gamut of phylogroups used in the test. Overall, strains form an overlapping continuum of host range potential with equal representation of narrow, moderate and broad host ranges. Groups of distinct pathotypes, including strains with currently the same pathovar name, could not be identified. The absence of groupings was validated with statistical tests for pattern recognition. The extent of host range was positively correlated with the capacity of strains to swarm on semi-solid agar medium and with the abundance of genes in biosynthetic clusters and was inversely correlated with the abundance of genes for proteins with transmembrane domains in their genomes. Our results are consistent with the current paradigm that disease symptoms are the result of multiple molecular interactions between P. syringae and its plant host that are modulated by abiotic and biotic conditions. This leads us to propose that pathovar denominations do not correspond to the underlying biology of P. syringae. A new concept of pathogenicity that accounts for the continuum of pathogenic potential in P. syringae would open new perspectives to understand the evolution of pathogenicity in this bacterium and new insights to anticipate disease and to manage plant health.

Environment-directed activation of the Escherichia coliflhDC operon by transposons.

The flagellar system in Escherichia coli K12 is expressed under the control of the flhDC-encoded master regulator FlhDC. Transposition of insertion sequence (IS) elements to the upstream flhDC promoter region up-regulates transcription of this operon, resulting in a more rapid motility. Wang and Wood (ISME J 2011;5:1517-1525) provided evidence that insertion of IS5 into upstream activating sites occurs at higher rates in semi-solid agar media in which swarming behaviour is allowed as compared with liquid or solid media where swarming cannot occur. We confirm this conclusion and show that three IS elements, IS1, IS3 and IS5, transpose to multiple upstream sites within a 370 bp region of the flhDC operon control region. Hot spots for IS insertion correlate with positions of stress-induced DNA duplex destabilization (SIDD). We show that IS insertion occurs at maximal rates in 0.24 % agar, with rates decreasing dramatically with increasing or decreasing agar concentrations. In mixed cultures, we show that these mutations preferentially arise from the wild-type parent at frequencies of up to 3×10-3 cell-1 day-1 when the inoculated parental and co-existing IS-activated mutant cells are entering the stationary growth phase. We rigorously show that the apparent increased mutation frequencies cannot be accounted for by increased swimming or by increased growth under the selective conditions used. Thus, our data are consistent with the possibility that appropriate environmental conditions, namely those that permit but hinder flagellar rotation, result in the activation of a mutational pathway that involves IS element insertion upstream of the flhDC operon.

Ensifer meliloti overexpressing Escherichia coli phytase gene (appA) improves phosphorus (P) acquisition in maize plants.

The Escherichia coli phytase gene appA encoding enzyme AppA was cloned in a broad host range plasmid pBBR1MCS2 (lac promoter), termed pVA1, and transformed into the Ensifer meliloti 1020. Transformation of pVA1 in Ensifer meliloti {E. m (pVA1)} increased its phosphatase and phytase activity by ∼9- and ∼50-fold, respectively, compared to the transformants containing empty plasmid as control {E. m (pBBR1MCS2)}. The western blot experiments using rabbit anti-AppA antibody showed that AppA is translocated into the periplasm of the host after its expression. Ensifer meliloti harboring AppA protein {E. m (pVA1)} and {E. m (pBBR1MCS2)} could acidify the unbuffered phytate minimal media (pH 8.0) containing Ca-phytate or Na-phytate as sole organic P (Po) source to below pH 5.0 and released P. However, both {E. m (pVA1)} and {E. m (pBBR1MCS2)} neither dropped pH of the medium nor released P when the medium was buffered at pH 8.0 using Tris-Cl, indicating that acidification of medium was important for the enzymatic hydrolysis of phytate. Further experiments proved that maize plants inoculated with {E. m. (pVA1)} showed increase in growth under sterile semi solid agar (SSA) medium containing Na-phytate as sole P source. The present study could be helpful in generating better transgenic bioinoculants harboring phosphate mineralization properties that ultimately promote plant growth.

Evaluation of endophytic bacteria from some rice varieties for plant growth promoting activities

Eighteen endophytic bacteria were isolated from different rice varieties by using Jensen’s nitrogen free medium. According to morphological and biochemical tests, isolates S1, R1, R2, R5 and R6 may be Rhizobium spp. and the remaining isolates may be Azospirillum spp. Fourteen out of isolates had plant growth hormone (Indole Acetic Acid) producing activity. R7 was the best Indole Acetic Acid producer among the isolated strains at two days incubation period in Jensen’s nitrogen free medium with L-tryptophan 0.5mg /ml at 37˚C and the concentration produced was 120.55 ppm. In nitrogen free semi-solid agar medium containing trace amount of bromothymol blue as indicator, all isolates showed nitrogen fixing activity during seven days incubation period. All isolates except S6 could solubilize phosphate on Pikovskaia’s medium supplemented with (0.5% Ca3(PO4)2). S5 was the best P- solubilizer at 5 days incubation period both in plate screening showing zone diameter 45mm and 539.78 ppm quantitatively. Fourteen isolates except (LS, R2, S4, S8) could also decompose potassium (ranging from 2-30mm in diameter) on K- decomposing medium with 0.12% Kmica. R3 was the best K-decomposer by plate screening (30mm in diameter) and 19.84 ppm quantitatively. S3 and R3 had good antagonistic activities against Rhizotonia solani and LS and R5 to Phythium spp. and S7 to Fusarium oxysporum respectively.

Transient high level mammalian reovirus replication in a bat epithelial cell line occurs without cytopathic effect

Mammalian reoviruses exhibit a large host range and infected cells are generally killed; however, most studies examined only a few cell types and host species, and are probably not representative of all possible interactions between virus and host cell. Many questions thus remain concerning the nature of cellular factors that affect viral replication and cell death. In the present work, it was observed that replication of the classical mammalian reovirus serotype 3 Dearing in a bat epithelial cell line, Tb1.Lu, does not result in cell lysis and is rapidly reduced to very low levels. Prior uncoating of virions by chymotrypsin treatment, to generate infectious subviral particles, increased the initial level of infection but without any significant effect on further viral replication or cell survival. Infected cells remain resistant to virus reinfection and secrete an antiviral factor, most likely interferon, that is protective against the unrelated encephalomyocarditis virus. Although, the transformed status of a cell is believed to promote reovirus replication and viral “oncolysis”, resistant Tb1.Lu cells exhibit a classical phenotype of transformed cells by forming colonies in semisolid soft agar medium. Further transduction of Tb.Lu cells with a constitutively active Ras oncogene does not seem to affect cell growth or reovirus effect on these cells. Infected Tb1.Lu cells can produce low-level of infectious virus for a long time without any apparent effect, although these cells are resistant to reinfection. The results suggest that Tb1.Lu cells can mount an unusual antiviral response. Specific properties of bat cells may thus be in part responsible for the ability of the animals to act as reservoirs for viruses in general and for novel reoviruses in particular. Their peculiar resistance to cell lysis also makes Tb1.Lu cells an attractive model to study the cellular and viral factors that determine the ability of reovirus to replicate and destroy infected cells.

Role of Type IV Pili in Virulence of Pseudomonas syringae pv. tabaci 6605: Correlation of Motility, Multidrug Resistance, and HR-Inducing Activity on a Nonhost Plant

To investigate the role of type IV pili in the virulence of phytopathogenic bacteria, four mutant strains for pilus biogenesis-related genes were generated in Pseudomonas syringae pv. tabaci 6605. PilA encodes the pilin protein as a major subunit of type IV pili, and the pilO product is reported to be required for pilus assembly. The fimU and fimT genes are predicted to produce minor pilins. Western blot analysis revealed that pilA, pilO, and fimU mutants but not the fimT mutant failed to construct type IV pili. Although the swimming motility of all mutant strains was not impaired in liquid medium, they showed remarkably reduced motilities on semisolid agar medium, suggesting that type IV pili are required for surface motilities. Virulence toward host tobacco plants and hypersensitive response-inducing ability in nonhost Arabidopsis leaves of pilA, pilO, and fimU mutant strains were reduced. These results might be a consequence of reduced expression of type III secretion system-related genes in the mutant strains. Further, all mutant strains showed enhanced expression of resistance-nodulation-division family members mexA, mexB, and oprM, and higher tolerance to antimicrobial compounds. These results indicate that type IV pili are an important virulence factor of this pathogen.

The Influence of Microgravity on Invasive Growth inSaccharomyces cerevisiae

This study investigates the effects of microgravity on colony growth and the morphological transition from single cells to short invasive filaments in the model eukaryotic organism Saccharomyces cerevisiae. Two-dimensional spreading of the yeast colonies grown on semi-solid agar medium was reduced under microgravity in the Σ1278b laboratory strain but not in the CMBSESA1 industrial strain. This was supported by the Σ1278b proteome map under microgravity conditions, which revealed upregulation of proteins linked to anaerobic conditions. The Σ1278b strain showed a reduced invasive growth in the center of the yeast colony. Bud scar distribution was slightly affected, with a switch toward more random budding. Together, microgravity conditions disturb spatially programmed budding patterns and generate strain-dependent growth differences in yeast colonies on semi-solid medium.

Effect of Transgenic Rhizobacteria Overexpressing Citrobacter braakii appA on Phytate-P Availability to Mung Bean Plants

Rhizosphere microorganisms possessing phytase activity are considered important for rendering phytate-P available to plants. In the present study, Citrobacter braakii phytase gene (appA) was over-expressed in rhizobacteria possessing plant growth promoting (PGP) traits for increasing their potential as bioinoculants. AppA was cloned under the lac promoter in the broad host-range expression vector pBBR1MCS2. Transformation of the recombinant construct pCBappA resulted in high constitutive phytase activity in all of the eight rhizobacterial strains belonging to genera Pantoea, Citrobacter, Enterobacter, Pseudomonas (two strains), Rhizobium (two strains) and Ensifer that were studied. Transgenic rhizobacterial strains were found to display varying level of phytase activity, ranging from 10 folds to 538 folds higher than the corresponding control strains. Transgenic derivative of Pseudomonas fluorescens CHA0, a well-characterized plant growth promoting rhizobacterium, showed highest expression of phytase (~8 U/ mg) activity in crude extracts. Although all transformants showed high phytase activity, rhizobacteria having ability to secrete organic acid, showed significantly higher release of P from Ca-phytate in buffered minimal media. AppA over-expressing rhizobacteria showed increased P content, dry weight (shoot) or shoot/ root ratio of mung bean (Vigna radiata) plants, to different extents, when grown in semi solid agar (SSA) medium containing Na-phytate or Ca-phytate as the P sources. This is the first report of over-expression of phytase in rhizobacterial strains and its exploitation for plant growth enhancement.

Diversity of cold-active protease-producing bacteria from arctic terrestrial and marine environments revealed by enrichment culture

A new approach for enrichment culture was applied to obtain cold-active protease-producing bacteria for marine and terrestrial samples from Svalbard, Norway. The method was developed for the enrichment of bacteria by long-term incubation at low temperatures in semi-solid agar medium containing meat pieces as the main source of carbon and energy. ZoBell and 0.1x nutrient broth were added for marine and terrestrial microorganisms, respectively, to supply basal elements for growth. One to three types of colonies were observed from each enrichment culture, indicating that specific bacterial species were enriched during the experimental conditions. Among 89 bacterial isolates, protease activity was observed from 48 isolates in the screening media containing skim milk. Good growth was observed at 4 degrees C and 10 degrees C while none of the isolates could grow at 37 degrees C. At low temperatures, enzyme activity was equal to or higher than activity at higher temperatures. Bacterial isolates were included in the genera Pseudoalteromonas (33 isolates), Arthrobacter (24 isolates), Pseudomonas (16 isolates), Psychrobacter (6 isolates), Sphingobacterium (6 isolates), Flavobacterium (2 isolates), Sporosarcina (1 isolate), and Stenotrophomonas (1 isolate). Protease activity was observed from Pseudoalteromonas (33 isolates), Pseudomonas (10 isolates), Arthrobacter (4 isolates), and Flavobacterium (1 isolate).

Antibacterial Activity of an Atmospheric Pressure Plasma Jet Against Relevant Wound Pathogens in vitro on a Simulated Wound Environment

AbstractThe aim of the study was to test the efficacy of a hand‐held atmospheric pressure plasma jet (APPJ) toward typical wound pathogens in vitro simulating antisepsis on wound surfaces. The plasma jet has been proved to be highly effective in vitro against the most commonly encountered pathogenic species of acute and chronic wounds reaching nearly the power of antiseptics. The following bacteria and fungi were treated on half rigid media (agar) imitating wound colonization: methicillin‐sensitive Staphylococcus aureus ATCC 1924 (MSSA), Enterococcus faecium ATCC 6057 (EF), Pseudomonas aeruginosa ATCC 15442 (PA), Candida albicans ATCC 10231 (CA), and β‐hemolyzing Streptococci of the Lancefield serogroup A (HSA). Highest reduction factor (RF) was obtained treating PA (RF 4.0) followed by HSA (3.2), MSSA (2.7), CA (2.0), and EF (1.9). Consequently, simulating wound surfaces with moist environment using semisolid agar media, the APPJ allowed bactericidal treatment of highly contaminated surfaces of 55 cm2 imitating skin and wound colonization within 6 min. This antibacterial reduction power together with its handsome flexibility of the APPJ could be a suited therapeutic option in the therapy of infected or colonized wounds.magnified image

Organic-acid-producing, phytate-mineralizing rhizobacteria and their effect on growth of pigeon pea ( Cajanus cajan)

Phytates represent a significant pool of organic phosphorus (Po) that is largely unavailable to plants. This study deals with phytate-mineralizing (PM), organic-acid-producing (OAP) rhizobacterial isolates, their characterization and their effect on plant growth. Their genetic diversity was assessed by 16S rRNA amplified ribosomal DNA restriction analysis (ARDRA) and selected isolates were identified by partial sequencing of 16S rRNA gene. Na-phytate and Po rich poultry farm manure (PFM) used as sources of phosphorus in semi-solid-agar (SSA) medium and soil respectively, for plant inoculation studies, where Cajanus cajan (pigeon pea) used as plant. Of thirty-nine rhizobacterial isolates, nineteen were proficient at releasing phosphate (Pi) (up to 85 μg/ml) from sparingly soluble calcium (Ca)-phytate and concomitantly decreasing the pH of minimal medium with 100 mM glucose from 8.0 to below 5. When the medium contained glycerol in place of glucose, Ca-phytate remained undissolved with no significant Pi released and no decline in pH. Genetic diversity of phytate-mineralizing (PM) rhizobacterial isolates suggests that the isolates mainly fall in two populations: acid-producing (AP) population (mainly represented by members of Enterobacteriaceae) and non-AP population. OAP-PM rhizobacterial isolates were identified as Citrobacter, Pantoea, Klebsiella and Enterobacter species. Organic acids (OAs) secreted by PM isolates were detected by HPLC, showed secretion of gluconic and acetic acids. Importance of OAs in Ca-phytate dephosphorylation was demonstrated in vitro using A. ficuum phytase. Gluconate and acetate additions enhanced phytase catalyzed dephosphorylation of Ca-phytate in vitro. Sonicated cell lysates of isolates showed significant Pi release from Ca-phytate compared to whole cells, indicating inaccessibility of Ca-phytate due to poor solubility. Selected isolates showed that they possess cell-associated acid phytase and modulators of phytase activity suggested that the enzymes are histidine acid phosphatase (HAP) type of phytase. OAP-PM isolates PP1 and DHRSS showed significant increase in dry shoot/root ratio and P content of shoot in Na-phytate containing semi-solid agar (SSA) medium, but isolate DHRSS did not increase dry shoot/root ratio in soil experiments containing poultry farm manure as source of P, although it significantly increased shoot P content of plants. The inoculation of isolates enhanced the shoot P content and dry shoot/root ratio, but did not increase the dry weight in SSA medium. It may be concluded that some OAP-PM rhizobacterial isolates that release P from Ca-phytate show increase in shoot P content in phytate containing SSA medium and in soils.

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.

In vitro leaf anatomy, ex vitro photosynthetic behaviors and growth of Calathea orbifolia (Linden) Kennedy plants obtained from semi-solid medium and temporary immersion systems

In vitro bud clusters of Calathea orbifolia (Linden) Kennedy were obtained and subcultured in semi-solid (agar) medium and temporary immersion system (TIS) for 12 weeks. Uniform young plants were selected and transferred to soilless mix in a growth chamber for ex vitro acclimatization during 35 days, followed by growing in a shaded greenhouse for 65 days. Comparison of in vitro leaf anatomy, ex vitro photosynthetic behaviors and growth was made between two cultural systems. Plants in TIS produced thicker leaf chlorenchyma and aquiferous parenchyma, lower stomatal frequency and more epicuticular wax than did those in semi-solid medium. Plants from semi-solid medium had consistently lower leaf Fv/Fm values than plants from TIS. Leaf Fv/Fm value in plants from TIS decreased to 0.65 at day 7 after transfer and increased soon up to 0.76 thereafter. In contrast, leaf Fv/Fm value in plants from semi-solid medium reduced to 0.27 at day 7 after transfer and increased slowly up to 0.68 at day 35. During ex vitro acclimatization, plants in TIS had substantial higher photosynthetic rates than plants in semi-solid medium. Plants from TIS had subsequent higher leaf area, fresh and dry weights than plants from semi-solid medium.

Split-dose exposures versus dual ion exposure in human cell neoplastic transformation

Since radiation fields of space contain many-fold more protons than high atomic number, high energy (HZE) particles, cells in astronaut crews will experience on average several proton hits before an HZE hit. Thus radiation regimes of proton exposure before HZE particle exposure simulate space radiation exposure, and measurement of the frequency of neoplastic transformation of human primary cells to anchorage-independent growth simulates an initial step in cancer induction. Although previous investigations indicated a synergistic increase in transformation yields in the cells exposed to protons followed by HZE particles, these experiments did not differentiate between the effect of splitting of the dose into two fractions and that of changing the ion beams. To test this, we irradiated cells with split doses of either protons or HZE particles, then measured clonogenic survival and neoplastic transformation, as measured by colony formation in semi-solid soft agar medium. The data show that the split dose of 20 cGy plus 20 cGy of either H or HZE ions gave about the same effect as the 40 cGy uninterrupted dose, quite different from the effect of the mixed ion beam H + HZE irradiation. We also asked if lower proton doses than 20 cGy followed 15 min later by 20 cGy of HZE ions gave greater than additive transformation frequencies. Substantial increases in transformation levels were observed for all proton doses tested, including 1 cGy. These results point to the signal importance of protons in affecting the effect of space radiation on human cells.

Expression of the Candida albicans Morphogenesis Regulator Gene CZF1 and Its Regulation by Efg1p and Czf1p

The ability of Candida albicans to transit between different cellular morphologies is believed to be important for virulence. Morphological transitions occur in response to a variety of environmental signals. One such signal is encountered when cells are grown in a semisolid matrix. An important regulator of cellular morphology is the putative transcription factor CZF1. Here we demonstrate that transcription of CZF1 is responsive to growth parameters such as the temperature, carbon source, growth phase of cells, and the physical environment. In wild-type cells, a CZF1 transcript of about 4 kb was expressed when cells were grown embedded in semisolid agar medium, as well as in late exponential phase when cells were grown in liquid medium. Deletion of EFG1, a key regulator of morphogenesis, abolished CZF1 expression. Overexpression of CZF1 revealed that this gene also autoregulates its expression. Efg1p and Czf1p were shown by chromatin immunoprecipitation to act by binding to the promoter of CZF1. The coupling of environmental cues to the expression of a morphogenetic transcription factor may allow C. albicans to coordinate morphogenesis in response to specific conditions encountered in the human host.