Agar-solidified Medium Research Articles

Arsenic tolerance and accumulation through a hypersaline food web

A has been historically used as a pesticide and wood preservative, and was also produced as industrial effluent. The Laguna Madre (Texas, USA) is hyper-saline estuary impacted by arsenic and other anthropogenic compounds. The Laguna Madre has few freshwater inputs; however, one input, the Arroyo Colorado, is a man-made waterway for agricultural runoff and may contribute to arsenic influx into the Laguna Madre. We determined arsenic levels in water, sea grass, bacteria, and insects from the Arroyo Colorado and/or the Laguna Madre using Inductively-Coupled Plasma (ICP) spectroscopy and other methods. The objective was to investigate the connectivity between the aquatic and terrestrial shoreline food web. Furthermore, arsenic tolerance was tested in bacteria isolated from Laguna Madre sediments to determine the maximum concentration tolerated by microorganisms in the ecosystem. Bacteria were isolated on solid agar media containing 100 M sodium arsenate (AsV) or sodium arsenite (AsIII) then subsequently transferred to media with higher levels of arsenic. Overall, bacteria tolerated arsenate more readily than arsenite; maximum tolerated arsenic concentrations were 60 mM and 1 mM for sodium arsenate and sodium arsenite, respectively. Biotic accumulation of arsenic in sea grass, bacteria, and insects was also observed. However, arsenic values varied among trophic level, exposure pathway, and organism.

Double-phase culture system for large scale production of pineapple

We have developed an efficient and simplified method for shoot proliferation of the pineapple (Ananas comosus L.), using a Double-Phase Culture System (DPS) (semi-solid medium with a layer of liquid medium on the top). Under these conditions, up to 430 microshoots were produced from 5 initial shoots within 5 months, and without shoot manipulation. Thus, >50% shoots proliferated as compared to that on agar-solidified medium. Shoots taken from DPS were rooted within 4 weeks at a frequency of up to 100%, in full-strength or half-strength Murashige and Skoog medium containing 0.5–2.5 μM indole-3-butyric acid. The plantlets were then transferred into soil, and they survived acclimatization with 100% success. Thus, DPS could be useful for large-scale micropropagation of a range of pineapple cultivars, with high production of plantlets at the end of process, and reduced labor costs during the subcultures.

AnagrQuorum Sensing System That Regulates Granulose Formation and Sporulation in Clostridium acetobutylicum

The Gram-positive, anaerobic, endospore-forming bacterium Clostridium acetobutylicum has considerable biotechnological potential due to its ability to produce solvents as fermentation products, in particular the biofuel butanol. Its genome contains a putative agr locus, agrBDCA, known in staphylococci to constitute a cyclic peptide-based quorum sensing system. In staphylococci, agrBD is required for the generation of a peptide signal that, upon extracellular accumulation, is sensed by an agrCA-encoded two-component system. Using ClosTron technology, agrB, agrC, and agrA mutants of C. acetobutylicum ATCC 824 were generated and phenotypically characterized. Mutants and wild type displayed similar growth kinetics and no apparent differences in solvent formation under the conditions tested. However, the number of heat-resistant endospores formed by the mutants in liquid culture was reduced by about one order of magnitude. On agar-solidified medium, spore formation was more strongly affected, particularly in agrA and agrC mutants. Similarly, accumulation of the starch-like storage compound granulose was almost undetectable in colonies of agrB, agrA, and agrC mutants. Importantly, these defects could be genetically complemented, demonstrating that they were directly linked to agr inactivation. A diffusible factor produced by agrBD-expressing strains was found to restore granulose and spore formation in the agrB mutant. Furthermore, a synthetic cyclic peptide, designed on the basis of the C. acetobutylicum AgrD sequence, was also capable of complementing the defects of the agrB mutant when added exogenously to the culture. Together, these findings support the hypothesis that agr-dependent quorum sensing is involved in the regulation of sporulation and granulose formation in C. acetobutylicum.

English

Due to the production of abundant capsular polysaccharides and or exopolysaccharides, some bacteria form mucoid and opaque colonies on solid agar medium. Polysaccharides interfere with the isolation and characterization of DNA. Extraction of polysaccharide-free DNA from the mucoid bacteria is required for genetic and biochemical studies. In this study, mucoid Vibrio parahaemolyticus and Klebsiella pneumonia were used as model organisms for comparing the effectiveness of two DNA extraction methods. The two methods were proved reproducible and cost-effective for extracting high-purity genome DNA from mucoid bacteria for further applications. Key words: Mucoid bacteria, Vibrio parahaemolyticus, Klebsiella pneumonia, DNA isolation, polysaccharides.

Development of Flexible Antimicrobial Packaging Materials against Campylobacter jejuni by Incorporation of Gallic Acid into Zein-Based Films

In this study, antimicrobial films were developed against Campylobacter jejuni by incorporation of gallic acid (GA) into zein-based films. The zein and zein-wax composite films containing GA between 2.5 and 10 mg/cm(2) were effective on different C. jejuni strains in a concentration-dependent manner. Zein and zein-wax composite films showed different release profiles in distilled water but quite similar release profiles at solid agar medium. Depending on incorporated GA concentration, 60-80% of GA released from the films, while the remaining GA was bound or trapped by film matrix. The GA at 2.5 and 5 mg/cm(2) caused a considerable increase in elongation (57-280%) of all zein films and eliminated their classical flexibility problems. The zein-wax composite films were less flexible than zein films, but the films showed similar tensile strengths and Young's modulus. Scanning electron microscopy indicated different morphologies of zein and zein-wax composite films. This study clearly showed the good potential of zein and GA to develop flexible antimicrobial films against C. jejuni.

Clonal propagation of triploid Acorus calamus Linn. Using dual-phase culture system

Acorus calamus is an important medicinal plant which has been used in Indian traditional medicine since time immemorial. Various bioactive molecules, viz., acorin, α- and β-asarone, asaryldehyde, caryophylene, isoasarone, methylisoeugenol, and safrol have been isolated from this plant. However, the use of this plant for medicinal purpose has been recently banned due to the high toxic property of β-asarone. The triploid Acorus calamus is reported to be low in β-asarone content and thus found to be the ideal raw material for medicinal use. The present investigation represents our finding for successful in vitro clonal propagation of the elite triploid accessions of Acorus calamus for mass propagation. In the dual-phase culture system consisting of agar-solidified Murashige and Skoog medium overlaid by liquid fraction of the same medium, maximum multiple shoot induction was favored by supplementation of α-naphthaleneacetic acid (0.5 mg L−1) and 6-benzylaminopurine (2.0 mg L−1). In vitro rooting of the microshoots was maximum in the medium supplemented with indolebutyric acid at 2.0 mg L−1. The well-rooted microshoots could be successfully hardened and transplanted in the field. This result can be reproduced and is a viable protocol for successful clonal propagation of the seedless triploid Acorus calamus for conservation and sustainable development.

Evaluation of normal ocular bacterial flora with two different culture media.

The purpose of this study was to determine if the use of broth culture medium is efficient in investigating bacterial flora of the normal eyelid and conjunctiva. Samples from the conjunctiva and eyelid of healthy patients of various ages who were undergoing ocular surgeries were obtained and cultured at 3 periods: before topical antibiotic prophylaxis, in the postoperative period during topical antibiotic treatment, and 15 days after discontinuation of antibiotic use. Samples were inoculated into both brain heart infusion broth and blood agar plate, and the growth results of both media were analyzed. Brain heart infusion broth medium showed a significantly higher bacterial growth of gram-positive cocci in most periods. The solid blood agar medium had a higher recovery of gram-positive bacilli before prophylaxis only in the older patients. Our results show that a more complete analysis of eyelid and conjunctival flora can be obtained using both liquid and solid media to increase the chances of isolate recovery. The inclusion of liquid media in this analysis was even more relevant in the period of concomitant use of antibiotic treatment.

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.

Zinc Compounds Combined With Cyclodextrin Inhibit Growth of Helicobacter pylori In-vitro

Zinc compounds have anti-gastric ulcer activity in animal models and in man. Since Helicobacter pylori is a major aetiologic agent in peptic ulcer disease this raised the possibility that part of the anti-ulcer activity of zinc could be due to its effects on the growth of H. pylori. We therefore investigated the in-vitro effects of zinc sulphate and a slow-release zinc compound, zinc monoglycerolate (ZMG), on the growth of H. pylori cultured in either solid agar or liquid phase media (in microtiter plates). Growth media comprised buffered peptone adjusted to pH 5·1 and pH 7·2; the lower pH mimicks that in the stomach without unduly affecting the growth of H. pylori. A total of 26 strains comprising clinical isolates and standards were cultured with the zinc compounds for one to three days. The results showed that Zn2+ and ZMG alone had little effect on the growth of H. pylori in solid or liquid phase media at either pH. However, incorporating β-cyclodextrin (β-CyD, 1 mg mL−1) with both zinc compounds produced marked inhibition of growth in all strains at concentrations of 22·0–77·5 μg mL−1, with more pronounced inhibition at the lower pH. Zinc compounds inhibit growth of H. pylori when combined with β-CyD; the latter may aid penetrance of zinc compounds into the bacteria.

Inducible trehalase enzyme activity of Morchella conica Persoon mycelium

Morchella conica Pers. strains of the study were isolated from fruit bodies collected in ash-mixed forests. At first, the strains were cultured on potato dextrose agar (PDA), then on modified Murashige and Skoog (MS) solid agar media. A normal-growing strain was chosen for the trehalase induction experiments. During the trehalase induction treatment, mycelia were grown in liquid culture containing different concentrations of trehalose. After the induction period of trehalase enzymes, physiological state of the mycelium and the oxidative stress were monitored in the vegetative mycelia by measuring the change of the malondialdehyde content, superoxide dismutase enzyme activity, the fresh and dry weight. The examined Morchella conica strain utilized the trehalose properly. The rising amount of the trehalose triggered the increase of the mycelial trehalase enzyme activity. Our results clearly proved that both neutral and acidic trehalase isoenzyme activity of the Morchella conica mycelium are inducible and are playing important role in the utilization of external trehalose.

Organogenesis and encapsulation of in vitro-derived propagules of Carrizo citrange [Citrus sinensis (L.) Osb. × Poncirius trifoliata (L.) Raf

Due to widespread polyembryony, Citrus rootstocks are usually propagated by open-pollinated seed germination, although micropropagation offers many advantages. Encapsulation technology has recently attracted the interest of researchers in the field of plant propagation because it combines the advantages of zygotic or gamic seeds with those of micropropagation. In this study, we examined the encapsulation of Carrizo citrange uninodal microcuttings (3–4 mm long) and evaluated the influence of the calcium alginate coating, a short time storage at cold temperature, and different sowing substrates on the viability and regrowth of the explants. A secondary aim was to develop an efficient protocol to induce root formation in the microcuttings. The results showed that encapsulation did not negatively affect the viability, providing a satisfactory regrowth, and storage potential for 30 days at low temperature. No differences in viability and regrowth were detected between the two different sowing substrates tested (agar-solidified medium and paper filter). To optimize the production of microcuttings required to perform the encapsulation experiments, in a preliminary experiment we assessed different factors affecting the in vitro shoot regeneration from epicotyl segments (obtained from seeds of Carrizo citrange germinated in vitro), including the influence of in vitro organogenesis, explant orientation, cut surface contact with the medium, treatments with different growth regulators, and distance of the organogenic explants from the cotyledonary node. The highest organogenic response was obtained from segments horizontally cultured (particularly from the basal portion), from segments with the cut surface in contact with the medium and from explants cultured on medium supplemented with 6-benzyladenine. No significant difference in regeneration efficiency was found in response to the distance of the epicotyl portions from the cotyledonary node.

White-rot Fungal Cultures for Degradation of Water Soluble Pollutants: The Aspect of Bacterial Stress

Potential of ligninolytic fungi to degrade organopollutants including synthetic dyes has been proven and the use of immobilized fungal cultures in semisolid state-, trickling-bedand rotating disk reactors for efficient biodegradation of textile dyes repeatedly shown. The effect of bacteria on fungal biofilms and their biodegradation efficiency treated in this study is a factor important in application of fungal cultures under nonsterile conditions. The results will demonstrate tolerance of highly degradative fungi Pleurotus ostreatus, Dichomitus squalens and Irpex lacteus to growth inhibition by coliform and soil bacteria on solid agar media. In static, liquid-medium cultures of I. lacteus working at pH 4.5 and 6.0 for 10 days, the capacity of decolorization of anthraquinone dye Remazol Brilliant Blue R (RBBR) was significantly inhibited by 105106 CFU of Escherichia coli but not Pseudomonas aeruginosa. No specific effect on ligninolytic enzyme production was observed. The presence of 109 CFU of E. coli in semisolid-state cultures of I. lacteus during a 4-week cultivation did not negatively influence the capacity of the fungus to decolorize RBBR. Bacterial counts increased during cultivation but fungal growth was not affected by the presence of the bacterium. Manganesedependent peroxidase was the main enzyme present. The enzyme levels exhibited variations attributable to the presence of the bacterium. Electron microscopy showed that fungal biofilms were dense and stable during long-term runs even in the presence of E.coli. Sustainability and efficiency of fungal biofilms in long-term biodegradation experiments was demonstrated. The work was supported by the projects IAAX00200901 and AV0Z50200510. Biography C. Novotny obtained Ph.D. at Charles University, Prague (1976). He is senior scientist at the Institute of Microbiology of the ASCR, v.v.i., Prague (1976-2010). He was head of Laboratory of Yeast Physiology (19811990) and Laboratory of Experimental Mycology (2002-2006). Topics of research are: microbial physiology, bioremediation and environmental biotechnologies. He has published 53 papers in impacted journals and is assistant editor of Folia Microbiologica.

The extreme xerophilic mould Xeromyces bisporus — Growth and competition at various water activities

Little is known about the mould, Xeromyces bisporus, unique in its strong xerophilicity and ability to grow at water activity ( a w) 0.62, lower than for any other known organism. The linear growth rates of one fast and one slow-growing strain of X. bisporus were assessed at 20, 25, 30 and 37 °C on solid agar media containing a mixture of glucose and fructose to reduce a w to 0.94, 0.88, 0.84, 0.80, 0.76 and 0.66. Growth rates of xerophilic species closely related to X. bisporus, viz. Chrysosporium inops, C. xerophilum and Monascus eremophilus, were also assessed. Optimal conditions for growth of both X. bisporus strains were approx. 0.84 a w and 30 °C, despite FRR 2347 growing two- to five-fold faster than CBS 185.75. X. bisporus FRR 2347 even grew well at 0.66 a w (0.48 mm/day). C. inops and C. xerophilum were more tolerant of high a w than X. bisporus, and could be differentiated from each other based on: the faster growth of C. xerophilum; its preference for temperatures ≥ 30 °C and a w ≥ 0.94 ( c.f. ≤ 25 °C and ~ 0.88 a w for C. inops); and its ability to grow at 0.66 a w, which is the lowest a w reported to date for this species. M. eremophilus grew slowly (max. 0.4 mm/day) even in its optimal conditions of ~ 0.88 a w and 25 °C. To investigate the competitive characteristics of X. bisporus at low a w, both X. bisporus strains were grown in dual-culture with xerotolerant species Aspergillus flavus and Penicillium roqueforti, and xerophilic species A. penicillioides, C. inops, C. xerophilum and Eurotium chevalieri, on glucose–fructose agar plates at 0.94, 0.84, 0.80 and 0.76 a w and at 25 °C. Growth rates and types of interactions were assessed. Excretion of inhibitory substances acting over a long-range was not observed by any species; inhibitors acting over a short-range that temporarily slowed competitors' growth or produced a protective zone around the colony were occasionally observed for A. penicillioides, C. inops and C. xerophilum. Instead, rapid growth relative to the competitor was the most common means of dominance. The xerotolerant species, A. flavus and P. roqueforti were dominant over X. bisporus at 0.94 a w. E. chevalieri was often dominant due to its rapid growth over the entire a w range. At a w < 0.80, X. bisporus was competitive because it grew faster than the other species examined. This supports the concept that its ideal environmental niche is sugary foods with low a w.

Sequence analysis of two different subtypes of thioredoxin gene of Helicobacter pylori

To analyze the gene sequences of Trx1 and Trx2 of Helicobacter pylori (HP) from different gastric diseases, define the amino acid sequences and investigate the relationship with these diseases. The HP strains were isolated from gastric mucosa of 25 patients with chronic gastritis, peptic ulcer and gastric cancer respectively and cultured on solid blood agar medium. The primers of Trx1 and Trx2 were designed according to the sequences of GenBank. The Trx1 and Trx2 were respectively amplified by PCR (polymerase chain reaction) and were sequenced by the bioinformatics method. The sequences were compared with those of the international standard HP strains. The whole sequence of Trx1 and the first 295 bp of Trx2 were successfully amplified to allow for sequence comparison by BioEdit. The Trx1 from different HP strains contained 321 bp encoding 106 amino acids. The mutation ratio was 13.4% (43/321). All amino acids contained the same active motif Cys-Gly-Pro-Cys. The homology of Trx1 amino acids from 25 strains was 97.2% (103/106). The mutation ratio for first 295 bp of Trx2 was 18.6% (55/295). All amino acids encoded by Trx2 contained one CXXC zone while the Cys-Gly-Pro-Cys motif was not found. The homology of Trx2 amino acids was 84.7% (83/98). Two different subtypes of Trx from clinically isolated Hp strains have mutation sites. But the homology of encoded amino acids is relatively high because of invalid mutations. Trx1 of HP strains from different diseases all contains a redox active motif Cys-Gly-Pro-Cys while Trx2 contains only a CXXC zone. The above results will provide valuable rationales for future studies of the biological function and pathogenic mechanisms of HP Trx1 and Trx2.

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.

HIGH TEMPERATURES DURING BLOOM CAN INHIBIT POLLEN GERMINATION AND TUBE GROWTH, AND ADVERSELY AFFECT FRUIT SET IN THE PRUNUS DOMESTICA CULTVARS 'IMPROVED FRENCH' AND 'MUIR BEAUTY'

ISHS IX International Symposium on Plum and Prune Genetics, Breeding and Pomology HIGH TEMPERATURES DURING BLOOM CAN INHIBIT POLLEN GERMINATION AND TUBE GROWTH, AND ADVERSELY AFFECT FRUIT SET IN THE PRUNUS DOMESTICA CULTVARS 'IMPROVED FRENCH' AND 'MUIR BEAUTY'

Influence of gelling agent and cytokinins on the control of hyperhydricity in Aloe polyphylla

Shoot regeneration and occurrence of hyperhydricity in Aloe polyphylla were greatly affected by the type of gelling agent. The use of gelrite resulted in a significantly lower multiplication and almost four times higher hyperhydricity (65%) compared to agar-solidified medium. Gelrite was further selected to evaluate if hyperhydricity can be overcome by altering the physical properties of the gel, as represented by increasing gelrite concentrations. Four concentrations of gelrite (0, 2.4, 6 and 16 g l−1) were tested in combination with zeatin, N6-benzyladenine (BA) or thidiazuron (TDZ). Almost all explants grown in liquid media in the presence of cytokinins became hyperhydric and lost their ability to regenerate. The greatest shoot formation was obtained on media with 2.4 g l−1 gelrite and 5 μM zeatin or BA, however hyperhydricity was very high. Satisfactory reduction in hyperhydricity was achieved only at 16 g l−1 gelrite, under which conditions the multiplication also decreased. The use of TDZ resulted in very low shoot regeneration and high hyperhydricity irrespective of the gelrite concentration.

Fitness-Associated Sexual Reproduction in a Filamentous Fungus

Sex is a long-standing evolutionary enigma. Although the majority of eukaryotes reproduce sexually at least sometimes [1-3], the evolution of sex from an asexual ancestor has been difficult to explain because it requires sexually reproducing lineages to overcome the manifold costs of sex, including the destruction of favorable gene combinations created by selection [4, 5]. Conditions for the evolution of sex are much broader if individuals can reproduce either sexually or asexually (i.e., facultative sex) and allocate disproportionately more resources to sex when their fitness is low (fitness-associated-sex or FAS [6-10]). Although facultatively sexual organisms have been shown to engage in more sex when stressed [11], direct evidence for FAS is lacking. We provide evidence using 53 genotypes of the filamentous fungus Aspergillus nidulans in a reciprocal transplant experiment across three environments. Different genotypes achieved highest fitness in different environments and genotypes invested relatively more in sex in environments in which their fitness was lower, showing that allocation to sexual reproduction is a function of how well-adapted a genotype is to its environment. FAS in A. nidulans is unlikely to have evolved as a strategy to resist or avoid stress because asexual spores are more dispersive and equally resistant [12, 13].

Differential Susceptibility of Escherichia coli Cells toward Transition Metal-Doped and Matrix-Embedded ZnO Nanoparticles

Dependence of the antibacterial behavior on ZnO (TM-doped and surface-modified) nanoparticles on Escherichia coli cells has been investigated. ZnO nanoparticles that differ in size, activator ion and in the microenvironment in which these nanoparticles are embedded were used. Comprehensive antibacterial studies of these ZnO nanoparticles owing to their size and surface defects are carried out against E. coli cells. These studies have been carried out both in Luria-Bertani medium and on solid agar medium in the presence and absence of light. The differences in antibacterial effect have been quantified in terms of minimum inhibitory concentration, minimum bactericidal concentration, colony forming unit counts, and qualitatively evaluated by growth curves and disk diffusion tests. The difference in antibacterial activities of the ZnO nanoparticles may be attributed to the enhanced or reduced oxygen vacancies and defect states and could be attributed to the increased or decreased surface defects.

Features of anthocyanin biosynthesis in pap1-D and wild-type Arabidopsis thaliana plants grown in different light intensity and culture media conditions

The number of different anthocyanin molecules potentially produced by Arabidopsis thaliana and which anthocyanin molecule is the first product of anthocyanidin modification remain unknown. To accelerate the understanding of these questions, we investigated anthocyanin biosynthesis in rosette leaves of both pap1-D and wild-type (WT) A. thaliana plants grown in nine growth conditions, which were composed of three light intensities (low light, middle light, and high light) and three media derived from MS medium (medium-1, 2, and 3). These nine growth conditions differentially affected the levels of anthocyanins and pigmentation patterns of rosette leaves, which were closely related to the diversification levels of cyanin structures. The combined growth conditions of high light and either medium-2 or medium-1 induced the most molecular diversity of anthocyanin structures in rosette leaves of pap1-D plants. Twenty cyanin molecules, including five that were previously unknown, were characterized by HPLC-ESI-MS and HPLC-TOF-MS analyses. We detected that the A. thaliana anthocyanin molecule A11 was most likely the first cyanin derived from the multiple modification steps of cyanidin. In addition, in the same growth condition, rosette leaves of pap1-D plants produced much higher levels and more diverse molecular profiling of cyanins than those of WT plants. The transcript levels of PAP1, PAL1, CHS, DFR, and ANS cDNAs were much higher in pap1-D rosette leaves than in WT ones. Furthermore, on the same agar-solidified medium, an enhancement of light intensity increased levels and molecular diversity of cyanins in both pap1-D and WT rosette leaves. In the same light intensity condition, the responses of anthocyanin levels and profiling to medium alternation were different between pap1-D and WT plants.