Agrobacterium tumefaciens-mediated transformation (AtMT) has been widely used for generation of fungal transformants and recently applied to Beauveria bassiana. In this study to comprehend how the AtMT promoter influences on the expression of selection marker (hygromycin B resistance gene; hph), two different Ti-Plasmids were constructed: pCeg (gpdA promoter-based) and pCambia-egfp (CaMV 35S promoter-based). Putative transformants were subjected to the PCR, RT-PCR and qRT-PCR to inspect the T-DNA insertion rate and gene expression level. In conclusion, more than 80% of the colonies succeeded in AtMT transformation and the hph expression level of AtMT/pCeg colonies was higher than that of AtMT/pCambia-egfp colonies. This result can provide useful information on the AtMT of B. bassiana, especially antibiotics susceptibility and promoter-dependant expression level.

Beauveria bassiana JEF-007 with strong virulence against Riptortus pedestiris was selected for the Agrobacerium tumefaciens-mediated transformation(AtMT). AtMT generated two transformants, B1-06 and C1-49, showed significantly reduced virulence against R. pedestris. To identify the virulence-related genes, thermal asymmetric interlaced(TAIL) PCR and flanking region analysis were performed. From the analysis, two genes, Complex I intermediated-associated protein 30(CIA30) and Autophagy protein 22(Atg22), possibly related virulence in B. bassiana JEF-007. For the analysis of two putative virulence-related genes in JEF-007, hairpin RNA interference (hpRNAi) is under consideration. This work can provide the functional roles of the virulence-related genes in B. bassiana JEF-007.

Bean bug, Riptortus pedestris (Hemiptera: Alydidae) is an agriculturally serious pest in East Asian countries. Chemical pesticides have been contributed to the management of the pest, but nowadays insect resistance limits the use of chemical pesticides, thus alternatively new pesticides with different mode of actions such as entomopathogenic fungi are considered. Herein entomopathogenic Beauveria bassiana JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by contact-exposure and spray methods. The Agrobacterium tumefaciens-mediated transformation of B. bassiana JEF-007 generated random transformants and some mutants showed reduced virulence against Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and R. pedestris nymph. Compared to the wild-type, the two transformants showed remarkably different morphology, conidial production, and thermotolerance. To figure out pathogenicity-related genes, thermal asymmetric interlaced (TAIL) PCR of the random transformants was performed and possibly some virulence-related genes were predicted. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.

Bean bug, Riptortus pedestris is an agriculturally serious pest in East Asian countries, reducing the value of crop quality and loss of income in agribusiness. Chemical pesticides have contributed to the management of the pest, but nowadays insect resistance limits the use of chemical pesticides, thus alternatively new pesticides with different mode of actions such as entomopathogenic fungi are considered. Beauveria bassiana and Metarhizium anisopliae JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by spray and contact-exposure methods. Supernatant showed different level of enzyme activity including chitinase, Pr1 protease and lipase. The Agrobacterium tumefaciens-mediated transformation generated random transformants and some mutants had reduced virulence. TAIL-PCR of the random transformants revealed virulence-related genes. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.

The entomopathogenic fungus, Beauveria bassiana is widely used in integrated pest management (IPM), however its successful application is often limited by the little effort to explore its functions of unknown genes. In this work, egfp-expression cassette was randomly integrated into B. bassiana using Agrobacterium tumefaciensmediated transformation, and the general features of the mutants with unusual characteristics and the localization of the integrated genes were explored. To construct a transformation vector, egfp-expression cassette including gpdA promoter and trpC terminator was cut from pBARKS1-egfp using SacI and HindIII and integrated into pCAMBIA containing hygromycin B resistant hygR gene, designated as pCAMBIA-egfp. Transformed B. bassiana isolates were grown on quarter strength-Sabouraud dextrose agar containing 150 μg hygromycinB ml-1. Expression of egfp was investigated by RT-PCR and a fluorescent microscope (400×). Through the genome walking of the transformants using adaptor primers and gene specific primers, unique bands were detected on the egfp-expressing transformants, which were sequenced to figure out the flanking regions. This work provides a platform of methodology to figure out unknown functional genes of B .bassiana and possibly suggest an improved strategy to use the entomopathogen in IPM.

Enhanced green fluoresce protein gene (egfp) was expressed in Beauveria bassiana ERL836 based on the Agrobacterium tumefaciens-mediated transformation (AtMT) method in this study. The ERL836 transformants were generated with pCambia-egfp binary vector. Ten transformants were randomly selected and analyzed for the T-DNA insertion and gene expression. The results revealed that 60% of the fungal putative transformants were inserted by the T-DNA fragment. Of these transformants, 33.33% (2 transformants) expressed the egfp gene. The egfp transformants showed strong green fluorescence with different expression levels. The results of this study could provide a reference for foreign protein expression in B. bassiana by using the AtMT method.

Human GH (hGH) has been available over 40 years for the treatment of children with GH deficiency. Human growth hormone (hGH) is mainly produced in the somatotrophic cells of the pituitary in brain and is the product of the GH-N gene. Among the edible mushrooms, the king oyster mushroom (Pleurotus eryngii ) is one of the most popular mushrooms in Asia, Europe and North America. The increasing popularity of P. eryngii among consumers is due to its flavor, texture and shelf life. We report a modified Agrobacteriummediated method for the efficient transformation of hGH2 in Pleurotus eryngii . The binary vector pCAMBIA1304 was used for the initial transformation and detected by GUS and GFP. Infiltrated samples transformed with pCAMBIA1304 showed a wider GUS response than the co-cultivated in the 50㎍/㎖ hygromycin selection medium. This transformation technique offers new prospects for the production of useful protein in the genetically improved mushroom.

A protocol for the production of transgenic Panax ginseng C.A. Meyer was established via Agrobacterium tumefaciens-mediated genetic transformation of direct somatic embryos. A number of conditions related to the co-cultivation were tested with respect to maximizing transformation efficiency. The results showed that pH of the co-cultivation medium (5.7), the bacterial growth phase (optical density; OD600 = 0.8), co-cultivation period (3 days), and acetosyringone concentration (100 μM) had positive effects on transformation. Selected plantlets were cultured on the medium at an elevated hygromycin level(30 mg/l). Integration of the transgenes into the P. ginseng nuclear genome was confirmed by PCR analysis using hpt primers and by Southern hybridization using hpt-specific probe. The transgenic plantlets were obtained after 3-month cultivation and did not show any detectable variation in morphology or growth characteristics compared to wild-type plants.

The cost of conventional cultivation of ginseng (Panax ginseng C.A. Meyer) is very expensive, because shadow condition should be maintained during cultivation periods owing to inherently weak plant for high-temperature. Therefore, application of plant biotechnology may be possible to overcome these difficulties caused by conventional breeding of ginseng. Transgenic plants were produced via Agrobacterium tumefaciens Gv3101, both carrying the binary plasmid pBI121 mLPISO with nptII and Iso (isoprene synthase) gene. Integration of the transgenes into the P. ginseng nuclear genome was confirmed by PCR analysis using nptII primers and Iso primers. RT-PCR result also demonstrated the foreign isoprene synthase gene in three transgenic plant lines (T1, T3, and T5) which was expressed at the transcriptional level. When whole plants of transgenic ginseng were exposed to high temperature at 46℃ for 1 h, a non-transformed plant was wilted from heat shock, whereas a transgenic plant appeared to remain healthy. We suggest that the introduction of exogenous isoprene synthase is considered as alternative methods far generating thermotolerance ginseng.

apical meristem using Agrobacterium tumefaciens-mediated transformation method. The plant expression vector used in the studycontained GUS gene and NPTI for selection under the control of CaMV35S promoter. Transformed calli and plantlets wereselected on G