Molecular analysis of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> isolated from Brazil nuts

Brazil nuts are an important export market in its main producing countries, including Brazil, Bolivia, and Peru. Approximately 30,000 tons of Brazil nuts are harvested each year. However, substantial nut contamination by Aspergillus section Flavi occurs with subsequent production of aflatoxins. In our study, Aspergillus section Flavi were isolated from Brazil nuts (Bertholletia excelsa), and identified by morphological and molecular means. We obtained 241 isolates from nut samples, 41% positive for aflatoxin production. Eighty-one isolates were selected for molecular investigation. Pairwise genetic distances among isolates and phylogenetic relationships were assessed. The following Aspergillus species were identified: A. flavus, A. caelatus, A. nomius, A. tamarii, A. bombycis, and A. arachidicola. Additionally, molecular profiles indicated a high level of nucleotide variation within β-tubulin and calmodulin gene sequences associated with high genetic divergence from RAPD data. Among the 81 isolates analyzed by molecular means, three of them were phylogenetically distinct from all other isolates representing the six species of section Flavi. A putative novel species was identified based on molecular profiles.     

An insight into the distribution,genetic diversity,and mycotoxin production of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> in soils of pistachio orchards

In the present study, 193 Aspergillus strains were isolated from a total of 100 soil samples of pistachio orchards, which all of them were identified as Aspergillus flavus as the most abundant species of Aspergillus section Flavi existing in the environment. Approximately 59%, 81%, and 61% of the isolates were capable of producing aflatoxins (AFs), cyclopiazonic acid (CPA), and sclerotia, respectively. The isolates were classified into four chemotypes (I to IV) based on the ability to produce AFs and CPA. The resulting dendrogram of random amplified polymorphic DNA (RAPD) analysis of 24 selected A. flavus isolates demonstrated the formation of two separate clusters. Cluster 1 contained both aflatoxigenic and non-aflatoxigenic isolates (17 isolates), whereas cluster 2 comprised only aflatoxigenic isolates (7 isolates). All the isolates of cluster 2 produced significantly higher levels of AFs than those of cluster 1 and the isolates that produced both AFB₁ and AFB₂ were found only in cluster 2. RAPD genotyping allowed the differentiation of A. flavus from Aspergillus parasiticus as a closely related species within section Flavi. The present study has provided for the first time the relevant information on distribution and genetic diversity of different A. flavus populations from nontoxigenic to highly toxigenic enable to produce hazardous amounts of AFB₁ and CPA in soils of pistachio orchards. These fungi, either toxigenic or not-toxigenic, should be considered as potential threats for agriculture and public health.     

Survey of Vietnamese Peanuts,Corn and Soil for the Presence of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>

Aspergillus flavus and Aspergillus parasiticus cause perennial infection of agriculturally important crops in tropical and subtropical areas. Invasion of crops by these fungi may result in contamination of food and feed by potent carcinogenic aflatoxins. Consumption of aflatoxin contaminated foods is a recognised risk factor for human hepatocellular carcinoma (HCC) and may contribute to the high incidence of HCC in Southeast Asia. This study conducted a survey of Vietnamese crops (peanuts and corn) and soil for the presence of aflatoxigenic fungi and used microsatellite markers to investigate the genetic diversity of Vietnamese Aspergillus strains. From a total of 85 samples comprising peanut (25), corn (45) and soil (15), 106 strains were isolated. Identification of strains by colony morphology and aflatoxin production found all Vietnamese strains to be A. flavus with no A. parasiticus isolated. A. flavus was present in 36.0% of peanut samples, 31.1% of corn samples, 27.3% of farmed soil samples and was not found in virgin soil samples. Twenty-five per cent of the strains produced aflatoxins. Microsatellite analysis revealed a high level of genetic diversity in the Vietnamese A. flavus population. Clustering, based on microsatellite genotype, was unrelated to aflatoxin production, geographic origin or substrate origin.     

Microbiological and physicochemical factors affecting <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> incidence in Cavendish banana (<Emphasis Type="Italic">Musa cavendishii</Emphasis>) chips production in Southern Philippines

Microbiological and physicochemical factors affecting the incidence of Aspergillus section Flavi in dried Cavendish banana (Musa cavendishii) chips production in Southern Philippines were examined. The average counts of Aspergillus section Flavi (AFC) in fresh and dried Cavendish bananas from 10 production batches of the Philippine Agro-Industrial Development Cooperative in Davao del Norte, Southern Philippines were 1.2 × 10² and 1.6 × 10² cfu/g, respectively. Isolates from both samples were identified to be Aspergillus flavus based on spore type and conidial structure of isolates. An increasing trend in the AFC of Cavendish bananas was observed during dried banana chips processing. Variability in the AFC between production batches was attributed to differences in aerobic and fungal populations and physicochemical characteristics of the fruits, peel damage of the raw materials, concentration of AFC in the air and food-contact surfaces of the production area, and temperature and relative humidity (RH) conditions of the environment during production and storage. Physicochemical characteristics of Cavendish bananas from the receipt of raw materials up to the first day of drying were within the reported range of values allowing growth and toxin production by aflatoxigenic fungi. Air-borne AFC varied depending on the section of the production area examined. The close proximity of the waste disposal area from the production operation to the preparation, drying and storage areas suggests that cross-contamination, probably air-borne or insect-borne was a likely occurrence. The hands of workers were also identified as AFC sources. Results of this study highlight the need for the development of strategies to control aflatoxigenic fungi and aflatoxin contamination in Philippine dried Cavendish bananas.     

Environmental and developmental factors influencing aflatoxin production by <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>

Aflatoxins are carcinogenic mycotoxins formed by a number of fungi in the genus Aspergillus. The major fungi responsible for aflatoxin formation in crop seeds in the field and in storage are Aspergillus flavus and A. parasiticus. This review emphasizes developmental, environmental, biological, and chemical factors that influence aflatoxin formation by A. flavus and A. parasiticus.     

Effect of acid lactic bacteria isolated from faeces of healthy dogs on growth parameters and aflatoxin B<Subscript>1</Subscript> production by <Emphasis Type="Italic">Aspergillus</Emphasis> species in vitro

The aim of the present study was to evaluate the inhibitory effect of Enterococcus faecium and Lactococcus lactis subsp. lactis isolated from faeces of healthy dogs on (i) lag phase, (ii) growth rate, and (iii) aflatoxin B₁ production by Aspergillus section Flavi on in vitro assays. Thirteen lactic acid bacteria (LAB) isolates were used as antagonist microorganisms. Antagonistic activity was assayed against four potentially aflatoxigenic Aspergillus section Flavi isolates: A. flavus (AF210 and AF281), A. parasiticus (AP245) and A. parasiticus (NRRL 2999). In general, the longest lag phases of Aspergillus isolates were obtained with E. faecium GJ40. Respecting the growth rate, no significant reduction was found in this parameter in the interaction assays with A. flavus and antagonist isolates respecting the control. While in A. parasiticus a significant reduction in growth rate was only observed in the interaction among reference strain and E. faecium MF5 isolate (p < 0.05). In general, AFB₁ production was reduced by most of the LAB isolates assayed, except for E. faecium GJ18, GJ20, MF3 and MF4. This study provides the first data about the antiaflatoxigenic activity of autochthonous LAB isolated from dog faeces.     

Investigation of stored wheat mycoflora,reporting the<Emphasis Type="Italic">Fusarium</Emphasis> cf.<Emphasis Type="Italic">langsethiae</Emphasis> in three provinces of Iran during 2007

Wheat is the most important cereal produced in Iran. A mycological survey was carried out for the first time, on the stored wheat samples in Tehran, East Azarbayejan and Mazandaran provinces in 2007. Exogenous and endogenous fungi, were isolated by the method of flotation with Malachite green agar (MGA 0.25) and Freeze blotter techniques respectively. In this study, 46 species belonging to 23 different genera were isolated.Cladosporium spp. (57.1–89.2%) andAlternaria spp. (82.4–100%) species were the predominant fungal species identified as endogenous mycoflora. The predominant exogenous fungi werePenicillium spp. (78.4–92.8%) andAspergillus spp. (71.4–85.7%) species.Fusarium proliferatum was the most prevalent species ofFusarium isolates.Aspergillus niger (39.4%) andAspergillus flavus (36.7%) were the predominantAspergillus species identified as exogenous mycoflora.Aspergillus flavus (26.6%) was the predominantAspergillus species identified as endogenous mycoflora. Flotation method with MGA 0.25 recommended for isolating of hyaline fungi from wheat cereals. In this study one isolate fromFusarium species was isolated on the basis of morphology and ribosomal internal transcribed spacer classified asFusarium langsethiae but on the basis of partial translation elongation factor-1alpha gene grouped withFusarium sporotrichioides. To our knowledge, this is the first report aboutF. cf.langsethiae in Iran and Asia.     

Fungal load in <Emphasis Type="Italic">Bradysia agrestis</Emphasis>, a phytopathogen-transmitting insect vector

Larvae of Bradysia agrestis, a phytopathogen-transmitting insect vector in East Asia, were sampled from geographically (ecologically) segregated regions to identify their intestinal fungal flora. A total of 24 fungal strains were isolated from the insect vectors and selected based on morphological differences. In addition, 38 fungal strains were isolated from the ulcerated parts of invaded host plants by the same method, revealing the impact of vector fungal flora on their host plants. For molecular identification of the fungi, internal transcribed spacer (ITS) regions were amplified and sequenced. Their sequences were compared with sequences of other fungal strains obtained from NCBI GenBank, and their phylogeny was determined. The dominant fungal genera in the insect vector were Penicillium (25%), Aspergillus (21%), and Cladosporium (13%). In plant scar lesions, most fungal isolates belonged to the genera Fusarium (31.6%), Phoma (7.8%), Didymella (7.8%), and Epicoccum (7.8%). Fungal genera in vectors or host plant lesions differed by study site. Furthermore, diversity indices by study site showed clear differences based on Margalef’s richness (2.06, 2.40, 3.04), and Menhinick’s (1.89, 2.12, 2.53), and Simpson’s indices (0.14, 0.07, 0.07). In addition, common fungal strains in insect vectors were found to be closely related to members of the genera Cladosporium, Penicillium, or Aspergillus. Among these strains, those showing the highest homology with Aspergillus terreus, which regarded as beneficial fungal genera could be considered ideal paratransgenesis candidates. Some other fungal strains from vectors or ulcerated plant parts from each study site after B. agrestis invasion may be harmful in terms of plant disease or agrifood safety. This study provides information on the fungal microbiota of B. agrestis, an emerging problem in East Asia, and proposes paratransgenesis candidates to control this insect vector. Furthermore, potential transferable pathogens or commensal fungi were revealed by comparing the fungal biota between the insect gut and the ulcerated parts of the invaded host plants.     

Molecular identification of <Emphasis Type="Italic">Aspergillus</Emphasis> and <Emphasis Type="Italic">Eurotium</Emphasis> species isolated from rice and their toxin-producing ability

Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp, infection by direct plating on malt extract salt agar (MESA). All Aspergillus holomorphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. nigef) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it does not work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2.     

Clavatol and patulin formation as the antagonistic principle of <Emphasis Type="Italic">Aspergillus clavatonanicus</Emphasis>, an endophytic fungus of <Emphasis Type="Italic">Taxus mairei</Emphasis>

Many endophytic fungi are known to protect plants from plant pathogens, but the antagonistic mechanism has rarely been revealed. In this study, we wished to learn whether an endophytic Aspergillus sp., isolated from Taxus mairei, would indeed produce bioactive components, and if so whether (a) they would antagonize plant pathogenic fungi; and (b) whether this Aspergillus sp. would produce the compound also under conditions of confrontation with these fungi. The endophytic fungal strain from T. mairei was identified as Aspergillus clavatonanicus by analysis of morphological characteristics and the sequence of the internal transcribed spacers (ITS rDNA) of rDNA. When grown in surface culture, the fungus produced clavatol (2′,4′-dihydroxy-3′,5′-dimethylacetophenone) and patulin (2-hydroxy-3,7-dioxabicyclo [4.3.0]nona-5,9-dien-8-one), as shown by shown by NMR, MS, X-ray, and EI-MS analysis. Both exhibited inhibitory activity in vitro against several plant pathogenic fungi, i.e., Botrytis cinerea, Didymella bryoniae, Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani, and Pythium ultimum. During confrontation with P. ultimum, A. clavatonanicus antagonized its growth of P. ultimum, and both clavatol as well as patulin were formed as the only bioactive components, albeit with different kinetics. We conclude that A. clavatonanicus produces clavatol and patulin, and that these two polyketides may be involved in the protection of T. mairei against attack by plant pathogens by this Aspergillus sp.     

Antimicrobial potentials of endophytic fungi residing in <Emphasis Type="Italic">Quercus variabilis</Emphasis> and brefeldin A obtained from <Emphasis Type="Italic">Cladosporium</Emphasis> sp.

Among 67 endophytic fungi isolated from Quercus variabilis, 53.7% of endophytic fungal fermentation broths displayed growth inhibition on at least one test microorganism, such as pathogenic fungi (Trichophyton rubrum, Candida albicans, Aspergillus niger, Epidermophyton floccosum, Microsporum canis) and bacteria (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens). Moreover, 19.4% of strains showed a broader antimicrobial spectrum, such as Aspergillus sp., Penicillium sp., Alternaria sp., 20.9% of strains showed strong inhibition (+++) to pathogenic bacteria, while only 7.5% displayed that to test fungi. The most active antifungal strain I(R)9-2, Cladosporium sp. was selected and fermented. From the broth, a secondary metabolite, brefeldin A was obtained. This is the first report on the antimicrobial potentials of endophytic fungi residing in Q. variabilis and isolation of brefeldin A produced by Cladosporium sp.     

A Review Molecular Typing Methods for <Emphasis Type="Italic">Aspergillus flavus</Emphasis> Isolates

Aspergillus flavus is the second most important Aspergillus species causing human infections. The importance of this fungus increases in regions with a dry and hot climate. Small phylogenetic studies in Aspergillus flavus indicate that the morphological species contains several genetically isolated species. Different genotyping methods have been developed and employed in order to better understand the genetic and epidemiological relationships between environmental and clinical isolates. Understanding pathogen distribution and relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. Typing techniques can also give us a deeper understanding of the colonization pattern in patients. Most of these studies focused on Aspergillus fumigatus because it is medically the most isolated species. To date, there has not been any publication exclusively reviewing the molecular typing techniques for Aspergillus flavus in the literature. This article reviews all these different available methods for this organism.     

Efficient production of <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-transformed roots and composite plants in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Recalcitrance of most large-seeded legumes, such as peanut, to regeneration and genetic transformation has hampered studies on gene function and efforts for genetic improvement. Agrobacterium rhizogenes-mediated transformation provides a system for rapid and efficient transformation of plant tissues. In this study, embryonic axes along with cotyledons of peanut were injected with a suspension culture of A. rhizogenes using microliter syringes. The influence of several factors such as plant genotype, A. rhizogenes culture stage, co-culture period of A. rhizogenes, and acetosyringone concentration in the co-cultivation medium have been evaluated. It is found that A. rhizogenes-mediated transformation of peanut is genotype-independent. Up to 61% transformation was recorded when embryonic axes were co-cultivated with 5 × 10⁷ A. rhizogenes cells from logarithmic phase for 2 days on co-culture medium containing 50 μmol l⁻¹ acetosyringone. Composite plants with transgenic roots were harvested after 45 days of treatment. Furthermore, this method was applied to assess the insecticidal activity of a synthetic cry8Ea1 gene against Holotrichia parallela in transgenic roots of peanut.     

Biosynthesis of silver nanoparticles by a new strain of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. compared with <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">fumigatus</Emphasis>

Locally isolated strains of a thermoalkalotolerant Streptomyces sp. and Aspergillus fumigatus were used for the in vitro biosynthesis of silver nanoparticles from AgNO₃ solutions. An autolysed cell-free culture filtrate from each strain was used, indicating that the formation mechanism depends on intra-cellular components for both organisms, since culture broths had no significant nanoparticle formation potential. Nanoparticle formation was indicated by a change of the solution from colourless or light brown to dark brown after 24 h or more, and UV–visible spectroscopy and x-ray diffraction analysis confirmed the formation by both organisms. The initial formation kinetics were faster with Aspergillus, but formation continued for a longer period with Streptomyces, resulting in higher concentrations after 48 h. Transmission electron microscope images revealed well dispersed nanoparticles with diameters ranging from 15 to 45 nm from A. fumigatus, while those from Streptomyces sp. had a narrower size distribution of 15–25 nm. The higher productivity and preferred narrower size distribution of Streptomyces, together with its well established industrial use, may make it the preferred choice for further optimization studies.     

Antimicrobial Activity and Biodiversity of Endophytic Fungi in <Emphasis Type="Italic">Dendrobium</Emphasis><Emphasis Type="Italic">devonianum</Emphasis> and <Emphasis Type="Italic">Dendrobium thyrsiflorum</Emphasis> from Vietman

Endophytic fungi are rich in orchids and have great impacts on their host plants. 53 endophytes (30 isolates from Dendrobium devonianum and 23 endophytic fungi from D. thyrsiflorum) were isolated, respectively, from roots and stems of Dendrobium species. All the fungi were identified by way of morphological and/or molecular biological methods. 30 endophytic fungi in D. devonianum were categorized into 11 taxa and 23 fungal endophytes in D. thyrsiflorum were grouped into 11 genera, respectively. Fusarium was the dominant species of the two Dendrobium species in common. Antimicrobial activity of ethanol extract of fermentation broth of these fungi was explored using agar diffusion test. 10 endophytic fungi in D. devonianum and 11 in D. thyrsiflorum exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among 6 pathogenic microbes (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus). Out of the fungal endophytes isolated from D. devonianum and D. thyrsiflorum, Phoma displayed strong inhibitory activity (inhibition zones in diameter >20 mm) against pathogens. Epicoccum nigrum from D. thyrsiflorum exhibited antibacterial activity even stronger than ampicillin sodium. Fusarium isolated from the two Dendrobium species was effective against the pathogenic bacterial as well as fungal pathogens. The study reinforced the assumption that endophytic fungi isolated from different Dendrobium species could be of potential antibacterial or antifungal resource.     

Effect of auxins on indirect <Emphasis Type="Italic">in vitro</Emphasis> morphogenesis and expression of <Emphasis Type="Italic">gusA</Emphasis> transgene in a lectinaceous medicinal plant, <Emphasis Type="Italic">Euphorbia nivulia</Emphasis> Buch.-Ham.

Summary The types of auxin in Murashige and Skoog (MS) medium containing N ⁶-benzyladenine (BA) determined indirect morphogenesis, i.e. development to bipolar somatic embryos or monopolar shoots in Euphorbia nivulia Buch.-Ham. Indirect in vitro morphogenesis depended on growth regulators, explant excision period, and light. Calli induced from explants collected in March–April were superior in the induction of indirect morphogenesis to those collected in July–August. Light enforced in vitro morphogenesis, while darkness was inhibitory. The presence of kinetin in the medium also inhibited morphogenesis. Calli developed on explants collected in March–April grown on MS medium fortified with α-naphthaleneacetic acid (NAA) and BA facilitated indirect organogenesis, while those developed on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and BA underwent somatic embryogenesis. MS medium with 13.3 μM BA and 2.69 μM NAA was the best for induction of shoots from callus, which developed a mean of 15.7 shoots. Shoots were best rooted on half-strength MS medium enriched with 2.46 μM indole-3-butyric acid with a mean of 5.1 roots per shoot. MS medium supplemented with 2.26 μM 2,4-D and 4.44 μM BA induced the highest number (mean of 13.4) of somatic embryos. Of the embryos transferred on half-strength MS medium containing 2.89 μM gibberellic acid, 78% of embryos developed to the cotyledonary stage. Most cotyledonary embryos (80%) underwent conversion to plantlets upon being transferred to half-strength MS basal medium in light. The survival rate of organogenesis and embryo-derived plants was 80 and 90%, respectively. Calli transformed with Agrobacterium tumefaciens showed expression of the gusA transgene and resistance to kanamycin, but did not undergo morphogenesis.     

Camptothecin-resistant fungal endophytes of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Concentrations of the DNA topoisomerase I inhibitor camptothecin were determined in different tissues of Camptotheca acuminata (Nyssaceae). The data were dependent on tissue type and developmental stage. Despite the presence of the toxic compound camptothecin, nine endophytic fungi were isolated from healthy C. acuminata plants and were tested for their camptothecin sensitivity. Two isolates showed almost no inhibition at a camptothecin concentration of 10 μg/ml. Even at a concentration of 100 μg/ml the inhibition was moderate.     

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> NRRL 3357 and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> SRRC 143

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B₁ and B₂ biosynthesis, while A. parasiticus cultures had significantly increased B₁ and G₁ biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.     

<Emphasis Type="Italic">In vitro</Emphasis> culture studies on <Emphasis Type="Italic">Stevia rebaudiana</Emphasis>

Summary Shoot apex, nodal, and leaf explants of Stevia rebaudiana Bertoni can regenerate shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 8.87 μM) and indole-3-acetic acid (5.71 μM). Rooting of the in vitro-derived shoots could be achieved following subculture onto auxin-containing medium. A survival rate of 70% was recorded at the hardening phase on the substrate cocopeat. The presence of the sweet diterpene glycosides, viz. stevioside and rebaudioside, was confirmed in the in vitro-derived tissues of Stevia using HPTLC techniques. Callus cultured on agar-solidified MS medium supplemented with BA (8.87 μM) and indole-3-butyric acid (9.80 μM) showed the highest sweetener content.     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.