Establishment of Agrobacterium tumefaciens-mediated Genetic Transformation System for Aspergillus awamori

[ Objective ] This study aimed to establish Agrobacterium tumefaciens-mediated genetic transformation system for Aspergillus awamori and investigate the feasibility of expressing heterologous proteins in A. awamori. [ Method] Appropriate A. awamori host strains were determined according to the secretory protein profile. Selectable marker was selected for genetic transformation by drug sensitivity analysis. The established A. awamori genetic transformation system was used for transformation and expression analysis of Rhizomucor miehei lipase （RML）. The feasibility of using A. awamori to express heterologous proteins was investigated by identification of transformants and property analysis. [ Result~ Based on the analysis of secretory protein profile, A. awamori strains CBS115.52 and CICC2257 were determined as the host strains for heterologous protein expression ; drug sensitivity analysis shows that hygromycin B resistance gene （ HygBr ） is an effective ge- netic seleetable marker; by using Agrobacterium tumefaciens-mediatcd transformation （ATMT） method, the plasmid pHGW-amdS containing HygBr was successfully transformed into A. awamori strain CBS115.52 to establish the genetic transformation system ofA. awamorl with HygBr as selectable marker. RML was transformed into A. awamori and its expression was validated by substrate hydrolysis test, SDS-PAGE and Western blot. [ Conclusion] This study demonstrates that the genetic transformation system of A. awamori mediated by Agrobacterium tumefaciens has potential feasibility for expression of heterologous proteins.

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum

Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alter-natively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.

Optimization of Genetic Transformation System of Tobacco K326 Mediated by Agrobacterium

[Objective]The aim was to optimize genetic transformation system in tobacco K326 mediated by Agrobacterium.[Method]The leaf of tobacco aseptic seedling was taken as explants to study the optimization of Agrobacterium-mediated genetic transformation system.[Result] The highest transformation efficiency was obtained when the explants were pre-cultured in the medium of MS ＋ 2 mg/L 6-BA ＋ 0.2 mg/L IAA for 2 d,and then infected with Agrobacterium GV3101（OD600 =0.6） for 5 min.The PCR detection proved that npt II gene had been integrated into the regenerated tobacco plants.[Conclusion]A highly efficient genetic transformation system of tobacco leaf mediated by Agrobacterium was established.

Meropenem as an Alternative Antibiotic Agent for Suppression of Agrobacterium in Genetic Transformation of Orchid

A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies （PLBs） has been reported in this article. The in vitro activities of meropenem and four comparator antibacterial agents against three Agrobacterium tumefaciens strains, LBA4404, EHA101, and GV3101, were assessed. In addition, the effect of meropenem on the growth of Dendrobium phalaenopsis PLBs was determined. Compared with other commonly used antibiotics （including ampicillin, carbenicillin, cefotaxime, and cefoperazone）, meropenem showed the highest activity in suppressing all tested A. tumefaciens strains （minimum inhibitory concentration [MIC] 〈 0.5 mg L^-1, which is equal to minimum bactericidal concentration [MBC]）. Meropenem, at all tested concentrations, except for 10 mg L^-1 concentration, had little negative effect on the growth of orchid tissues. The A. tumefaciens strain EHA101 in genetic transformation with vector plG121Hm in infected PLBs of the orchid was visually undetectable after a two-month subculture in 1/2 MS medium with 50 mg L^-1 meropenem and 25 mg L^-1 hygromacin. The expression and incorporation of the transgenes were confirmed by GUS histochemical assay and PCR analysis. Meropenem may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.

Development of Alfalfa (Medicago sativa L.) Regeneration System and Agrobacterium-Mediated Genetic Transformation

The regeneration ability of four alfalfa （Medicago sativa L.） cultivars, Xinjiang Daye, Longdong, Gannong 1 and Gannong 3, was studied, and the effects of various cultivars, explant sources and medium recipes on regeneration were compared. The better callus forming frequency obtained from hypocotyls of Xinjiang Daye is 88.5% and regeneration frequency is 9.8% in our initial experiments. To further optimize regeneration system for genetic transformation, we therefore changed concentrations of plant growth regulators and supplemented with glutamine into callus-induction and shoot-regeneration media. Callus forming frequency and shoot differentiation frequency were increased to 100%. The time taken to generate transgenic plants （16 weeks） was shorter than that for previouse procedure （25 weeks） and regeneration frequency was promoted to 15.1%. The results show that addition of glutamine is particularly important for shortening period of regeneration and promoting regeneration frequency. For study of genetic transformation of alfalfa, Agrobacterium tumefaciens-mediated transformation of Xinjiang Daye was developed based on this optimized regeneration system. The plant expression vector carrying two glutamine synthetases （GS 1 and GS2） and △1-pyrroline-5-carboxylate synthetase （P5CS） gene was used for alfalfa in vitro transformation. Six transgenic alfalfa plantlets with resistance to PPT were obtained. The introduction of foreign genes into plants was assessed in the transformants by PCR analysis and Southern hybridizations.

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize(Zea mays L.)

Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.

Establishment of A Simple and Efficient Agrobacterium-mediated Genetic Transformation System to Chinese Cabbage(Brassica rapa L.ssp.pekinensis)

Chinese cabbage,belonging to Brassica rapa species,is an important vegetable in Eastern Asia.It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low.The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies.In this study,we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors,with 93.50%regeneration rate on average.Based on this,a simple and efficient Agrobacteriummediated genetic transformation methodwas established,without pre-culture procedure and concentration adjustment of hormone and AgNO_(3) in co-cultivation and selection media.Using this system,transformants could be obtained within 3.5–4.0 months.Average transformation frequency is up to 10.83%.The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage.

Production of Transgenic Tall Fescue Plants with Enhanced Stress Tolerances by Agrobacterium tumefaciens-Mediated Transformation

In order to improve stress tolerances of turf-type tall fescue （Festuca arundinacea Schreb.）, Agrobacterium tumefaciens strain EHA105 carrying plasmid pCMD containing stress tolerance-related CBF1 gene from Arabidopsis thaliana was used to transform mature seeds-derived embryogenic calli of four cultivars. A total of 112 transgenic plants were regenerated from 32 independent lines and verified by histochemical detection of GUS activity, PCR assay and Southern hybridization analysis. The transformation frequency ranged from 0.92 to 2.87% with apparent differences among the cultivars. Stress tolerances of transgenic plants were enhanced, which was shown by the facts that transgenic plants had distinct growth superiority and significantly higher survival rate than non-transformed ones under high salinity and high osmosis stresses, and that relative electronic conductivity of in vitro leaves treated with low and high temoeratures, dehvdration and high salinity stresses was 25-30% lower in transgenic plants than in control plants.In addition,it was observed that growth of transgenic plants was inhibited due to constitutive overexpression of CBF1 gene under normal environmental conditions.

Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of (-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of (-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.

Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani AG-1 IA

To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system of Agrobacterium tumefaciens-mediated transformation(ATMT) for R.solani AG-1 IA was established successfully.The optimal conditions for this ATMT system were as follows:the concentration of hygromycin B at 30 μg/mL for transformant screening,8 h of pre-induction,20 h of co-culture,200 μmol/L of AS in ISM,co-culture at 25 ℃ and pH 5.6 to 5.8 of ISM at the co-culture phase.The transformants still displayed high resistance to hygromycin B after subculture for five generations.A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene,and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants.Moreover,PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A.tumefaciens,with four strains of A.tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A.tumefaciens.An expected band of 730 bp was amplified from the four strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10 transformants.The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118.

Scarabaeid Larvae- and Herbicide-Resistant Transgenic Perennial Ryegrass (Lolium perenne L.) Obtained by Agrobacterium tumefaciens-Mediated Transformation of cry8Ca2, cry8Ga and bar Genes

Insect pest and weeds are two major problems for forage and turf grasses. In this study, scarab larvae- and herbicide-resistant transgenic perennial ryegrass （Lolium perenne L.） was obtained by transforming it with cry and bar genes simultaneously via the Agrobacterium-mediated method. To optimize the callus induction and plant regeneration conditions, various concentrations of 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine were assayed. The transformation efficiencies of different Agrobacterium suspension media, used during Agrobacterium-mediated transformation, were compared. Then, plasmids of pCAMBIA3301 containing cry gene （cry8Ca2 or cry8Ga） and bar gene, driven by ubiquitin promoter, were transformed into perennial ryegrass. The transformants were generated and confirmed by both Southern hybridization analysis and Western hybridization analysis. Further, the resistance of transgenic perennial ryegrass plants to scarab larvae and herbicide were analyzed. After 30 d of co-cultivation with scarab larvae, the damage to the root system of transgenic plants was less than that of non-transgenic control plants. Additionally, the leaves of transgenic plants were resistant to Basta, while leaves of the wild plants wilted after Basta spraying. These results show that cry gene and bar gene were successfully transferred into perennial ryegrass by the Agrobactgerium-mediated method, and convey resistance to scarab larvae and herbicide in transgenic perennial ryegrass plants.

Agrobacterium tumefaciens-mediated transformation of modern rose(Rosa hybrida)using leaf-derived embryogenic callus

Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for rose using Agrobacterium tumefaciens-mediated transformation of embryogenic callus.Expanding rose leaves were used as explants to induce somatic embryos,which were subjected to transformation with A.tumefaciens strain GV3101 using Green Fluorescence Protein(GFP)as a marker gene.It took about 8 months to generate transgenic shoots from embryogenic callus.PCR,RT-PCR,Southern and Western blotting,as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome.According to our data,a transformation efficiency of up to 6%can be achieved by following this optimized protocol.

Adventitious shoot regeneration of Platanus acerifolia Willd. facilitated by Timentin,an antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation

The effects of Timentin and cefotaxime （Cef） on shoot regeneration of the London plane tree （Platanus acerifolia Willd.） and their use for the suppression ofAgrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were compared. Shoot regeneration was significantly reduced on the media with Cef at concentrations from 100 to 500 mg·L^-1. Timentin showed negative effect on plant regeneration at concentrations of 100 and 500 mg·L^-1; however, 300 mg·L^-1 Timentin was shown to facilitate shoot regeneration significantly and the regeneration frequency increased from 64% （control） to 88%. Effective suppression of A. tumefaciens could be obtained with 500 mg·L^-1 Cef, but plant regeneration was completely inhibited at this level. The A. tumefaciens on infected P. acerifolia leaf tissues was visually undetectable after three subcultures on a medium with 300 mg·L^-1 Timentin. Considering the effect of Cef and Timentin on plant regeneration and suppression of Agrobacteria, Timentin at 300 mg·L^-1 is the preferred application in .4. tumefaciens-mediated transformation ofP acerifolia.

The Factors of Genetic Transformation for Indica Rice Kasalath

Objective The aim was to explore conditions of genetic transformation for Indica rice Kasalath and laid a foundation for further study on molecular biology. Method With callus of Kasalath as transformation receptor, Agrobacterium tumefaciens-mediated method was used to conduct genetic transformation. The genetic transformation system was optimized from several aspects, including co-culture mode, co-culture time and the affertreatment method of co-culture. Result The results showed that two days is the best co-culture time for genetic transformation, the acquisition rate of resistant callus was up to 84.1%, and transformation rate was up to 73%. Whether callus contact to the culture medium directly or indirectly has no significant effect on transformation. [ Conclusion] Genetic transformation successfully transferred exogenous gene OsMAPk2 into the rice genome.

Study on the Genetic Transformation Conditions of Begonia wallichiana L.with Leaf Disc Method and Corresponding Identification Techniques

[Objectives]This study was conducted to improve the Agrobacterium-mediated genetic transformation system of Begonia wallichiana.[Methods]With sterilized tube seedling leaves as the recipient material and GFP as the reporter gene,optimization experiments were carried out in terms of infection time and method,co-cultivation time and method,and PCR detection technology.[Results]The transformation effect was better under the conditions of shaking Agrobacterium liquid,infection time of 1-2 h,and co-cultivation on sterilized filter paper for 2 d.After co-cultivation,the recipient material was first subjected to recovery culture,and then used for Hyg gradient screening,which was conducive to obtaining resistant transformants.The designed specific PCR detection technology could quickly identify false positives in resistant regenerated plants,and the proportion of transgenic plants was 16.7%.[Conclusions]The research results provide a new technical reference for the genetic transformation of ornamental plants.

Preliminary Studies on Establishment of Genetic Transformation System in Loblolly Pine

A transformation system was established for loblolly pine (Pimus taeda L ) mature zygotic embryos using Agrobacterium tumefaciens.The gene coding for the glucuronidase (GUS) gene was introduced into loblolly pine tissues andits transient expression was detected with histochemical staining. The influences of different genotypes. Agrobacterium concentrations. and cocultivation time on GUS expression and Kanamycin resistant callus and shoot regeheration were investigated. The results showed that the highest `GUS expression frequency (1 6.3%) and shoot regencration frequency(7.8%) wereobtained from genotype 9-1003 with, Agrobactemm concentration decreased 9 times and cocultivation time of 56 hours.respectively GUS expression was, obtained in all genotypes tested The successtul expression of the GUS gene in differentgenotypes suggested that it will be a useful transformation system for loblolly pine

Advances in Genetic Transformation of Tea(Camellia sinensis)

Long-term fertility cycle,low self-compatibility and short flowering period limited the tea breeding by conventional method.It is necessary to apply biotechnology,especially genetic transformation method,to obtain new mutants and to accelerate tea breeding.This paper reviews the status and progress in genetic transformation and related influencing factors.It has been noticed that tea genetic transformation has advantages in getting useful target mutants.The genetic transformation by Agrobacterium-mediated and gene gun methods are usually used.However,further research should be focused on improvement of plant regeneration.

Transient Expression of VHB and GLUT1 in the Green Alga Haematococcus pluvialis Using Agrobacteriummediated Transformation

The first successful Agrobacterium-mediated transformation of the green alga Haematococcus pluvialis using the vectors hosting the genes coding for VHB （ Vitreoscilla hemoglobin） and GLUT1 （glucose transporter 1 ） is reported here. The greenish yellow fluorescence of EGFP was observed when the zeocin-resistant cells were viewed with a fluorescent microscope. The functional expression of EGFP showed that the Agrobacterium-mediated transformation could efficiently transfer the exogenous gene into H. pluvialis. RT-PCR was used to successfully amplify the mRNA of VHB and GLUT1 genes from transformed cells, while Southern blots indicated the integration of VHB and GLUT1 genes into the genome of H. pluvialis. Transferring VHB and GLUT1 genes into this alga would pave the way for manip- ulation of many important pathways relevant to the food, pharmaceutical, and nutraceutical industries.

Transformation CodA Gene to Lily Plants by Agrobacterium tumefaciences Mediated

Glycinebetaine(GB),an osmotic substance,could improve some stress tolerance in plants.CodA gene,originating from bacteria,could translate choline oxidase which stimulates the synthesis of GB in plants.To create lily lines resistant to heat,Belladonna lily and Yelloween lily had been transferred CodA gene through Agrobacterium tumefaciens.The bacteria harbored a binary vector carrying the hygromycin phosphotransferase,choline oxidase(CodA)and intron-containingβ-glucuronidase(Gus)genes were co-cultivated with lily bulb scales slides.The result showed that most the bulb scales had developed into bulblets in a regulator-free growth medium,while some expressed the hygromycin-resistance,heat tolerance and Gus gene expression.Among them,one line demonstrated primarily the transcription level expression through reverse transcription polymerase chain reaction(RT-PCR).Moreover,they were tested with the accumulation of GB which was evident that the transferred line had four times of GB volumes higher than that of wild type.The original evidence could open a right approach to enhance stress tolerance in lily plants.

Agrobacterium-Mediated Transformation of Embryogenic Calli of Anliucheng and Regeneration of Plants Containing the Chimaeric Ribonuclease Gene

Anliucheng (Citrus sinensis Osbeck), a very seedy and widely spread acidless sweet orange cul-tivar in south of China, was transformed by the strain of Agrobacterium Tumefaciens EHA105 carrying pTA29-barnase gene, which will induce pollen sterility in transgenic plants. The embryogenic calli of Anliucheng were co-cultivated with Agrobacterium tumefaciens for 3 days, and then transferred to selective medium containing 50 mg I/1 basta (a kind of herbicide) for 5 weeks. The resistant calli were recovered and regenerated 118 embryoids. A total of 13 entire plants were obtained after micro-grafted on trifoliate orange. These regenerated plants were verified by PCR amplification and confirmed by PCR-Southern blotting analysis.