Optimal Co-culture Research Articles

Erythromycin mediates co-flocculation between cyanobacterium Synechocystis sp. PCC 6803 and filamentous fungi in liquid cultivation without organic compounds

Photoautotrophic cyanobacteria assimilate the greenhouse gas carbon dioxide as their sole carbon source for producing useful bioproducts. However, harvesting the cells from their liquid media is a major bottleneck in the process. Thus, an easy-to-harvest method, such as auto-flocculation, is desirable. Here, we found that cyanobacterium Synechocystis sp. PCC 6803 co-flocculated with a natural fungal contamination in the presence of the antibiotic erythromycin (EM) but not without EM. The fungi in the co-flocculated biomass were isolated and found to consist of five species with the filamentous Purpureocillium lilacinum and Aspergillus protuberus making up 71% of the overall fungal population. The optimal co-cultivation for flocculation was an initial 5 mg (fresh weight) of fungi, an initial cell density of Synechocystis of 0.2 OD730, 10 µM EM, and 14 days of cultivation in 100 mL of BG11 medium with no organic compound. This yielded 248 ± 28 mg/L of the Synechocystis-fungi flocculated biomass from 560 ± 35 mg/L of total biomass, a 44 ± 2% biomass flocculation efficiency. Furthermore, the EM treated Synechocystis cells in the Synechocystis-fungi flocculate had a normal cell color and morphology, while those in the axenic suspension exhibited strong chlorosis. Thus, the occurrence of the Synechocystis-fungi flocculation was mediated by EM, and the co-flocculation with the fungi protected Synechocystis against the development of chlorosis. Transcriptomic analysis suggested that the EM-mediated co-flocculation was a result of down-regulation of the minor pilin genes and up-regulation of several genes including the chaperone gene for pilin regulation, the S-layer protein genes, the exopolysaccharide-polymerization gene, and the genes for signaling proteins involved in cell attachment and abiotic-stress responses. The CuSO4 stress can also mediate Synechocystis-fungi flocculation but at a lower flocculation efficiency than that caused by EM. The EM treatment may be applied in the co-culture between other cyanobacteria and fungi to mediate cell bio-flocculation.

Establishment of Agrobacterium tumefaciens – mediated genetic transformation of apple pathogen Marssonina coronaria using marker genes under the control of CaMV 35S promoter

Premature leaf fall of apple caused by Marssonina coronaria is economically very important apple disease and all the commercially available apple cultivars are susceptible to this disease. The non-availability of an efficient transformation system for this fungus hinders the functional genomics research. Herein, we report for the first time, the successful Agrobacterium-mediated transformation in apple leaf blotch fungus M. coronaria by transferring T-DNA harbouring the genes for hygromycin phosphotransferase (hpt), β-glucuronidase (uidA) and green fluorescent protein (gfp) under the control of CaMV 35S promoter. The key factors that affect the transformation efficiency including type of recipient fungal material, acetosyringone concentration, the conditions for co-cultivation, Agrobacterium concentration, Agrobacterium strains and membrane types as support were investigated. The present results have recommended that 250 μM concentration of acetosyringone, 24 °C temperature and 48 h time, 0.5 OD600 of A. tumefaciens, EHA105 Agrobacterium strain and Whatman filter paper were the optimal co-cultivation conditions for the transformation of M. coronaria by using fragmented mycelia suspension and mycelial plugs. We observed that conidia were tedious to transform as compared to the fragmented mycelia and mycelial plugs of this slow growing fungus. These optimized parameters yielded 54 and 70 average transformants per 60 mycelial plugs and 104 fragmented mycelia, respectively. Fungal transformants were analysed for T-DNA integration, gus gene expression and gfp gene expression. Strong gus histochemical staining and green fluorescence expression indicated that the CaMV 35S promoter can drive gene expression in M. croronaria. Some mutants showed difference in the morphology of the colony as compared to the wild type control. This report will be very useful to inspect molecular basis of apple-M. coronaria interactions by deciphering the functional roles of various genes in this pathogenic fungus.

Co-cultivation of Euglena gracilis and Pseudoalteromonas sp. MEBiC 03607 for paramylon production

Depending on the culture conditions, Euglena gracilis can produce a variety of macromolecules such as vitamins, lipids, proteins, and polysaccharides. In particular, the ability of E. gracilis to store a lot of β-glucans implies it has significant biotechnological potential. In this study, the growth of E. gracilis and its paramylon content were improved through co-cultivation with the bacterium Pseudoalteromonas sp. MEBiC 03607. To determine the optimal co-cultivation strategy, the effects of the algae-to-bacterial inoculum ratio and growth stage of E. gracilis were examined. Under optimal conditions, the growth of E. gracilis and its paramylon content were increased by more than 17 and 25%, respectively, as a direct result of the cultivation with bacteria. Through qRT-PCR analysis, it was confirmed that when the bacteria were inoculated, the expression of gene related to β-glucan synthase was increased, while the expression of wax ester synthase-related gene was decreased. This indicates that bacteria promote the synthesis of β-glucans of E. gracilis and inhibit the conversion of β-glucan to wax ester. These results demonstrate a novel approach to improve the growth of E. gracilis and its paramylon productivity.

A protocol for Agrobacterium-mediated transformation of Japanese cedar, Sugi (Cryptomeria japonica D. Don) using embryogenic tissue explants.

Sugi (Cryptomeria japonica D. Don) is the most important afforestation coniferous tree in Japan. Coniferous trees normally have a long juvenile period and require a long cultivation time for breeding. Through a traditional breeding project that began in the 1950s, first generation plus trees with excellent traits were selected primarily from artificial forests and used as seedlings. Recently, the second generation plus trees obtained by crossing between plus trees have been selected. In light of this situation, the improvement of Sugi by a transgenic approach is effective in terms of shortening the breeding period compared with conventional crossing-dependent approaches. There are three key points to an efficient Agrobacterium-mediated transformation system: (1) establishment of explants with high regeneration ability, (2) optimal co-cultivation conditions for explants and Agrobacterium, and (3) efficient elimination of Agrobacterium. Here we describe a protocol for Agrobacterium-mediated transformation of Sugi that meets the above criteria using embryogenic tissues as explants isolated from immature seeds obtained by crossing.

Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.).

Stable transformation of common bean (Phaseolus vulgaris L.) has been successful, to date, only using biolistic-mediated transformation and shoot regeneration from meristem-containing embryo axes. In this study, using precultured embryo axes, and optimal co-cultivation conditions resulted in a successful transformation of the common bean cultivar Olathe using Agrobacterium tumefaciens strain EHA105. Plant regeneration through somatic embryogenesis was attained through the preculture of embryo axes for 12 weeks using induced competent cells for A. tumefaciens-mediated gene delivery. Using A. tumefaciens at a low optical density (OD) of 0.1 at a wavelength of 600 nm for infection and 4-day co-cultivation, compared to OD600 of 0.5, increased the survival rate of the inoculated explants from 23% to 45%. Selection using 0.5 mg L-1 glufosinate (GS) was effective to identify transformed cells when the bialaphos resistance (bar) gene under the constitutive 35S promoter was used as a selectable marker. After an 18-week selection period, 1.5% -2.5% inoculated explants, in three experiments with a total of 600 explants, produced GS-resistant plants through somatic embryogenesis. The expression of bar was confirmed in first- and second-generation seedlings of the two lines through reverse polymerase chain reaction. Presence of the bar gene was verified through genome sequencing of two selected transgenic lines. The induction of regenerable, competent cells is key for the successful transformation, and the protocols described may be useful for future transformation of additional Phaseolus germplasm.

Agrobacterium rhizogenes-transformed roots of Astragalus membranaceus (Fisch. Ex Link) Bunge as a source of valuable secondary metabolites

In this study the results of agrobacterial transformation of Astragalus membranaceus (Fisch. Ex Link) Bunge, a medicinal plant from Mongolia and China with a wide spectrum of pharmacological action were presented. As explants for transformation, in vitro derived microshoots as well as hypocotyls, cotyledons and primary shoots isolated from seedlings were used. The most effective from two tested Agrobacterium rhizogenes strains (A4-RT, 15834 SWISS) was 15834 SWISS, causing the transformation of various types of explants, including primary shoots as a more responsive type. The optimal co-cultivation time of explants with bacterial suspension (48 hours) and the highest transformation rate of primary shoots (36.4%) were established. PCR analysis confirmed the transgenic nature of the roots transformed with strain 15834 SWISS and the absence of bacterial contamination.

Efficient Transformation of the White Jelly Mushroom Tremella fuciformis (Tremellomycetes) and Its Companion Fungus Annulohypoxylon stygium (Ascomycetes) Mediated by Agrobacterium tumefaciens.

Tremella fuciformis is an edible white jelly mushroom with medicinal qualities. The formation of T. fuciformis fruiting bodies is highly dependent on the presence of Annulohypoxylon stygium under natural conditions and during artificial cultivation. A lack of efficient transformation systems restricts the ability of researchers to functionally characterize the genes in these two interacting fungi. In this study, we tested the utility of the Agrobacterium tumefaciens-mediated transformation of T. fuciformis and A. stygium protoplasts. A. tumefaciens strain EHA105 cells harboring the pTrGEH plasmid, which contains genes for enhanced green fluorescent protein (egfp) and hygromycin B phosphotransferase (hph), was co-cultured with T. fuciformis protoplasts. Meanwhile, EHA105 cells harboring the pAnGRH plasmid, which contains the red fluorescent protein (rfp) and hph genes, was co-cultured with A. stygium protoplasts. The egfp, rfp, and hph genes were under the control of the promoter for gpd, which encodes glyceraldehyde-3-phosphate dehydrogenase. Optimal co-cultivation was achieved with a 1:1 mixture of bacteria (OD600 0.4-0.6) and fungal protoplasts (105/mL) incubated at 25°C in a medium containing 200 μM acetosyringone. The subsequent selection on hygromycin B-containing medium yielded 45 and 187 stable transformants per 105 protoplasts for T. fuciformis and A. stygium, respectively. The integration of the transformed DNA into the two fungal genomes was confirmed by polymerase chain reaction (PCR), Southern blot analysis, fluorescence imaging, and a quantitative real-time PCR. All results confirmed the feasibility of our transformation approach, which may facilitate future functional analyses of T. fuciformis and A. stygium genes.

Agrobacterium tumefaciens-Mediated Transformation of the King Tuber Medicinal Mushroom Lentinus tuber-regium (Agaricomycetes).

Lentinus tuber-regium is a sclerotium-forming basidiomycetous mushroom. It has increasingly aroused people's attention for its medicinal effects. In this study, we investigated the applicability of the Agrobacterium tume-faciens-mediated transformation (ATMT) method in L. tuber-regium. A. tumefaciens strain GV 3101 harboring the vector pPEH was used to transform the mycelium of L. tuber-regium strain ACCC50657. The genes for hygromycin B phosphotransferase (hph) and enhanced green fluorescent protein (egfp) under the control of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene promoter of Pleurotus ostreatus were employed as the selection marker and reporter gene, respectively. The optimal cocultivation temperature and time for transformation were 3 days and 4 days at 25°C and 20°C, respectively. Southern blot analysis showed the variation in the copy number and position of hph, which indicated random integration of hph. Polymerase chain reaction and fluorescence microscopy indicated that the P. ostreatus gpd promoter can drive the fused hph-egfp gene expression in L. tuber-regium. This is the first report that the ATMT method was successfully applied to L. tuber-regium. This reliable and efficient transformation method could be a powerful tool for strain genetic improvement and gene function study in L. tuber-regium.

Generation and Screening of T-DNA Insertion Mutants Mediated by Agrobacterium tumefaciens in the Garden Asparagus Stem Blight Pathogen Phomopsis asparagi

The garden asparagus stem blight caused by filamentous fungus Phomopsis asparagi exposes a serious threat on asparagus production globally. However, to present, we understand poorly about the molecular mechanisms of fungal pathogenicity. To facilitate functional genomics research of P. asparagi, here we developed a highly efficient and stable Agrobacterium tumefaciens-mediated transformation approach which yielded 150-200 transformants per 1×106 conidia. Our results indicated that 25°C, acetosyringone concentration of 150μmol/L, and 72h were recommended as optimal co-cultivation conditions for the transformation. Using this transformation approach, we constructed a T-DNA insertion mutant library containing 1253 strains. Twenty randomly selected T-DNA insertion mutants were able to grow on 0.2×PDA selective media after five successive subcultures without selective pressure, indicating that the exogenous T-DNA was stably integrated into the P. asparagi genome. We confirmed several randomly selected mutants using PCR with primers specific to the hph gene. Southern blots suggested that three out of the five selected mutants have a single T-DNA insertion. Interestingly, multiple mutant candidates with growth defects were obtained from the growth assay. Moreover, several mutants were selected for further analysis on the T-DNA flanking sequences through TAIL-PCR analysis. A sequence comparison of total junction fragments implied that the insertion of T-DNA within P. asparagi genome appeared to be a random event. The transformation technology and genetic resources developed here will facilitate studies of pathogenic mechanisms in this devastating filamentous fungal pathogen of garden asparagus.

Identification of pathogenicity-related genes in the rice pathogen Ustilaginoidea virens through random insertional mutagenesis

Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming a popular effective system as an insertional mutagenesis tool in filamentous fungi. To gain more insight into the cellular and molecular mechanisms in the pathogenesis of Ustilaginoidea virens, the causal agent of rice false smut disease, a T-DNA insertion mutant library of U. virens was established using ATMT. We optimized a range of conditions to improve the transformation efficiency. Transformants were most effectively obtained when the optimal co-cultivation time is 72h, with 50μM AS in medium and 100μl A. tumefaciens for co-cultivation, leading to the production of 160–185 hygromycin B resistant transformants per 1×105 conidia. Southern blot analysis indicated that 58.14% of transformants had a single T-DNA copy. Among 5600 transformants tested for virulence, 37 mutants with reproducible pathogenic defects were obtained. The flanking sequences of three avirulent tranformants (B20, B1015 and B1465) and two pathogenicity-reduced transformants (B726 and B785) were amplified by high-efficiency thermal asymmetric interlaced PCR. Sequence analyses revealed that single T-DNA insertion in mutant B20 targeted the coding region of a gene encoding a protein highly similar to SUN family protein, and in mutant B726 targeted upstream of a gene with unknown function. The two T-DNA insertion sites in mutant B785 were found in the coding region of a gene encoding C6 transcription factor, but failed in amplified flanking sequence of another T-DNA. Chromosomal rearrangement occurred in the genome of mutant B1016 and B1465 with single T-DNA insertion. Among avirulent mutants, B20 showed altered colony growth and pigmentation. The T-DNA insert in B20 was detected in the coding region of a gene named UvSUN2. Morphophysiological characterization analysis suggested that UvSUN2 might be a virulence factor, and possibly required for proper fungal growth, cell wall construction, and stress responses in U. virens. This study optimize and validate the transformation procedure, maximizing the number of single copy transformants and developing an efficient procedure for rescuing adjacent host sequences along with the inserted T-DNA. This is the first report of identification several candidate virulence factors and validated one in U. virens. Together with identification of avirulent mutants and their associated genes, results suggested that ATMT can effectively be used to identify genes associated with pathogenicity in U. virens, and provided novel insights into molecular mechanisms underlying virulence of this pathogen.

Can oysters control eutrophication in an integrated fish-oyster aquaculture system?

is a serious problem occurring in ponds or estuaries where fish culture is practised. A possible solution to this problem is to raise oysters in an integrated culture with fish to improve water clarity and to help remove excess nutrients. In order to determine the effects that cultured oysters have on the environment, an experimental study was performed in the Moothakunnam region of the Vembanad lake in the Ernakulam District during February-November, 2012. The study was performed in four cages (1× 1× 1 m) referred to as treatments T1, T2, T3 and T4. The four treatments used fish ( Etroplus suratensis ) and oysters (Crassostrea madrasensis) in the ratio of 1:0.3, 1:0.5, 1:0.7 and 1:0 respectively. Several parameters were measured and compared between the four treatments, and these included temperature, pH, salinity, dissolved oxygen, biochemical oxygen demand, chemical oxygen demand, total suspended solids, ammonia, nitrite, nitrate and orthophosphate. Results showed that the parameters, DO, BOD, COD, TSS, ammonia and orthophosphate varied with different treatments and months. However, the contents of nitrite and nitrate showed variation only with respect to months. The eutrophication index (E) values calculated for the four treatments were 9.45 ± 4.41, 5.25 ± 3.23, 11.32 ± 6.17 and 11.52 ± 5.45 respectively. The lower E value obtained was for T2 which supports the conclusion that oysters can control eutrophication in an integrated aquaculture system. The optimal co-cultivation proportion of fish to oysters obtained from the present study was 1:0.5.

Comparative proteomic analysis of primary hepatocytes co-cultured with bone marrow mesenchymal stem cells in vitro

Objective To identify the differentially expressed proteins in hepatocytes within the optimal co-cultivation system with bone marrow mesenchymal stem cells (MSCS).Methods The optimal co-cultivation of MSCS and primary hepatocytes was generated in vitro.CD44-CD45-cell population (hepatocytes) was sorted from co-culture system by flow cytometry.Protein samples were isolated by twodimensional (2D) gel electrophoresis.Bioinformatics was employed to analyze the relevance between the altered proteins.The protein expression levels of interest were confirmed by Western blotting.Results Thirty-four altered protein spots were unambiguously identified with high confidence,which are likely associated with hepatic metablic functions,cell cycle,cellular proliferation and apoptosis,as well as immune modulation.Conclusion Several differentially expressed functional proteins within co-cultured hepatocytes have been revealed by comparative proteomics,which may be associated with enhanced hepatic functionality,prolonged cellular survival,and immune modulation. Key words: Porcine hepatocytes ; Bone marrow mesenchymal stem cells ; Co-culture ; Proteome

Grapevine (Vitis vinifera L.).

Grapevine (Vitis) is considered to be one of the major fruit crops in the world based on hectares cultivated and economic value. Grapes are used not only for wine but also for fresh fruit, dried fruit, and juice production. Wine is by far the major product of grapes, and the focus of this chapter is on wine grape cultivars. Grapevine cultivars of Vitis vinifera L. have a reputation for producing premium quality wines. These premium quality wines are produced from a small number of cultivars that enjoy a high level of consumer acceptance and are firmly entrenched in the market place because of varietal name branding and the association of certain wine styles and regions with specific cultivars. In light of this situation, grapevine improvement by a transgenic approach is attractive when compared to a classical breeding approach. The transfer of individual traits as single genes with a minimum disruption to the original genome would leave the traditional characteristics of the cultivar intact. However, a reliable transformation system is required for a successful transgenic approach to grapevine improvement. There are three criteria for achieving an efficient Agrobacterium-mediated transformation system: (1) the production of highly regenerative transformable tissue, (2) optimal cocultivation conditions for both grapevine tissue and Agrobacterium, and (3) an efficient selection regime for transgenic plant regeneration. In this chapter, we describe a grapevine transformation system that meets these criteria. We also describe a protocol for the production of transformed roots suitable for functional gene studies and for the production of semi-transgenic grafted plants.

Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line.

An improved Agrobacterium -mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds. This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T0 plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T0 plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.

Experimental study on bone marrow mesenchymal stem cells-induced tolerance of hepatocytes to the cytotoxicity of sera from the patients with liver failure in vitro

Objective To clarify the tolerance of hepatocytes co-cultured with bone marrow mesenchymal stem cells (BMSCs) to the cytotoxicity of sera from the patients with liver failure induced,and the supporting effects on liver failure sera by co-culture.Methods Serum samples from 18 patients with acute-on-chronic liver failure due to hepatitis B were collected with informed consent.Normal serum was obtained from 18 normal volunteers.The optimal co-cultivation of BMSCs and primary hepatocytes was generated.Four groups were creased as follows:hepatocyte alone with liver failure serum (Hep-F),co-culture with liver failure serum (Co-F),hepatocyte alone with normal serum (Hrp-N),and co-culture with normal serum (Co-N).The additional serum concentration was set as 10％,20％,40％,60％,80％ and 100％,respectively.The albumin expression and cell cycle of hepatocytes after 24 h culture were examined to determine the optimal liver failure serum concentration.The morphology and viability were also monitored.The indices of hepatic functions in every group were tested.Subsequently,the cells were inoculated in complete medium containing 10％ fetal bovine serum for another 24 h,and hepatocyte specific functions of Hep-FB,Co-FB,Hep-NB,and Co-NB were examined,respectively.Results A well attachment and self-organization of islet-like hepatocyte aggregates were encouraged in Co-F group with 60％ liver failure serum.It also achieved the best albumin secretion and largest population of hepatocytes in G2-S phase.The cell ultrastructure remained similar to that of hepatocytes in vivo.Meanwhile,hepatocyte viability assay showed a metabolically active,viable cell population in Co-F group instead of quite a few dead cells in Hep-F group.The activities of superoxide dismutase (SOD),lactate dehydrogenase (LDH),glutamine (GLN) and CHE in Co-F group were all significantly increased as compared with those in Hep-F group (P ＜ 0.05).The similar trends were observed between Co-FB group and Hep-FB group except SOD.Conclusion The co-cultured hepatocytes with bone marrow MSCSs could counteract the cytotoxicity of midhigh concentration serum with acute-on-chronic liver failure,which would manifest a well hepatic supporting function. Key words: Hepatocytes; Biological artificial liver; Bone marrow mesenchymal stem cell; Co-culture

Bioremediation efficiency of Gracilaria verrucosa for an integrated multi-trophic aquaculture system with Pseudosciaena crocea in Xiangshan harbor, China

The red algae, Gracilaria verrucosa, was co-cultivated with the fish Pseudosciaena crocea in an integrated multi-trophic aquaculture (IMTA) system for nearly two months in summer at coastal waters of Xiangshan harbor in the East China Sea. The nutrient reduction efficiency of ammonium and phosphorus was determined. Prior to the G. verrucosa co-cultured, the eutrophication index ( E) values of cage areas ranged from 4.2 to 32.0, indicating serious eutrophication conditions, and the high ratio of N/P and relative high P concentration indicated a nitrogen surplus. During the co-culture period, G. verrucosa showed efficiently at removing inorganic nitrogen (IN) and inorganic phosphate (IP), and maintained a more stable dissolved oxygen (DO) and chlorophyll a (Chl a) level in the IMTA system. The maximum reduction efficiency of PO 4–P, NO 2–N, NH 4–N and NO 3–N was 58%, 48%, 61% and 47%, respectively. Based on the DIN balance in the IMTA system, the optimal co-cultivation proportion of P. crocea to G. verrucosa in this area was 1 cage, 144.95 m 2 or 1 kg, 7.27 kg, respectively. Results of the present study indicated that environmental advantage could be achieved by integrating the red alga G. verrucosa co-cultured with fish at the open coastal waters in the East China Sea.

Eutrophication assessment and bioremediation strategy in a marine fish cage culture area in Nansha Bay, China

On the basis of field data measured during four cruises from January to November 2007, variations in the characteristics of dissolved inorganic nitrogen and phosphate were analyzed in Nansha marine fish cage culture area, Ningbo City, China. Dissolved inorganic nitrogen (DIN) was selected as the parameter to balance seaweed absorption and fish DIN production. The contents of DIN and phosphate varied with different seasons, and eutrophication index (E) value ranged from 2.41 to 15.99, indicating serious eutrophication conditions; the annual average value of N/P of 32.95 indicates a nitrogen surplus in this system. The eutrophication condition in Nansha Bay was mainly caused by the fish cage culture activities. Based on their biological characteristics, Laminaria and Gracilaria were selected as the bioremediation species in winter and spring and summer and autumn, respectively. The optimal co-cultivation proportion of fish cage to Laminaria and Gracilaria in this bay was 1 cage, 450 m2 and one cage, 690 m2, respectively.

A reliable protocol for transformation of Catharanthus roseus through Agrobacterium tumefaciens.

Proliferation of axillary shoot buds and multiple shoot formation in Catharanthus roseus was obtained in 96 % explants on MS medium (3 % sucrose) containing NAA + BA. 2,4-D induced callusing in both, the nodal as well as in leaf segments. Leaf-derived callus was used for transformation with Agrobacterium tumefaciens LBA4404/pBI-S1. Bacterial cell concentration, duration of co-cultivation and acetosyringone concentration influenced transformation efficiency. Under optimal co-cultivation conditions, 98 % of the explants showed GUS expression. PCR based amplification of the transformed and subsequently selected callus tissue indicated the presence of uidA, Gly I and nptII genes.

Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori

The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants. The results indicate that the Agrobacterium-mediated transformation of A. awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important. Index descriptors: Agrobacterium tumefaciens; T-DNA; Virulence proteins; DNA-transformation; Aspergillus awamori; Filamentous fungi

Agrobacterium tumefaciens -mediated transformation for random insertional mutagenesis in Colletotrichum lagenarium

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 106 conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.