Enemies atpeace:Recentprogressin Agrobacterium-mediated cereal transformation

Agrobacterium tumefaciens mediated plant transformation is a versatile tool for plant genetic engineering following its discovery nearly half a century ago.Numerous modifications were made in its application to increase efficiency,especially in the recalcitrant major cereals plants.Recent breakthroughs in transformation efficiency continue its role as a mainstream technique in CRISPR/Cas-based genome editing and gene stacking.These modifications led to higher transformation frequency and lower but more stable transgene copies with the capability to revolutionize modern agriculture.In this review,we provide a brief overview of the history of Agrobacterium-mediated plant transformation and focus on the most recent progress to improve the system in both the Agrobacterium and the host recipient.A promising future for transformation in biotechnology and agriculture is predicted.

Genetic Transformation of Aloe barbadensis Miller by Agrobacterium tumefaciens

Despite the importance of aloe in cosmetic and pharmaceutical industries, improvement of aloe （Aloe barbadensis Miller） by genetic engineering was seldom reported previously. In this study, regeneration and transformation conditions, including explant selection and surface sterilization, use of different Agrobacterium strains, and co-culture processing, are optimized. The use of 20.0% sodium hypochloride （25 rain） for sterilization was less detrimental to the health of explant than 0.1% mercuric chloride （10 min）. Regeneration frequency from stems was much higher than that from leaves or sheaths. Explants were infected by Agrobacterium （30 rain） in liquid co-cultural medium, and this was followed by three days co-culture on sterile filter papers with light for 10 h per day at 24℃. Histochemical data demonstrated that the transient expression of GUS gene in the stem explants of aloe infected with Agrobacterium strains EHAI05 and C58CI was 80.0% and 30.0%, respectively, suggesting the higher sensitivity of the explants to EHAI05 than to C58C1. Infected tissues were selected using G418 （10.0-25.0 mg/L） to generate transformants. Sixty-seven G418 resistant plantlets were generated from the infected explants. Southern blotting, PCR, and ELISA analyses indicated that the alien gene were successfully transferred into aloe and was expressed in the transgenic plants. This newly established transformation system could be used for the genetic improvement of aloe.

Study on Agrobacterium tumefaciens-mediated Transformation of Brassica campestris L. with Fusion Gene Ycoil-bFGF

[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed （ Brassica campestris L. ） by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [ Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows： explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [ Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.

Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaffolder and striped stem borer

Immature embryos of rice varieties "Xiushui11" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant cant were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56%-72% measured as numbers of Geneticin (G418)-resistant calli produced and 36%-60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38%-61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16%-75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.

Improvement of Plant Regeneration from Immature Embryos of Wheat Infected by Agrobacterium tumefaciens

Wheat, one of the most important food crops, has been extensively studied with respects to plant regeneration and transformation employing the immature embryos as recipient tissues. However, the transformed tissues often become severely necrotic after co-cultivation with Agrobacterium, which is one of the major obstacles in gene delivery. In this study, wheat varieties CB037, Kenong 199, Xinchun 9, Lunxuan 987, and Shi 4185 showed desirable culture potential or high infection ability in Agrobacterium-mediated transformation. Similarly, optimal regeneration conditions were determined by testing their ability to inhibit the cell necrosis and cell death phenotype. Two auxins of 2,4-dichlorophenoxyacetic acid （2,4-D） and 3,6-dichloro-o-anisic acid （dicamba） were evaluated for highly significant effect on both callus and plantlet production, although they were genotype-dependent in wheat. Substitution of 2,4-D by dicamba enhanced the growth and regeneration ability of callus from the immature embryos of most genotypes tested. The callus growth state couldn’t be modified by adding antioxidants such as ascorbic acid, cysteine, and silver nitrate or organic additives such as thiamine HCl and asparagine to the media. On the contrary, the best tissue statement and plant regeneration was achieved by employing the media containing the simplest MS （Murashige and Skoog） components and dicamba without organic components and vitamins. Thereby, our results are thought to inhibit wheat cell necrosis effectively and suggested to be used for more wheat genotypes.

Regular Production of Transgenic Wheat Mediated by Agrobacterium tumefaciens

Wheat is the number one crop both in acreage and total yield in the world. Therefore, it is very important to improve wheat by gene engineering techniques even though it belongs to the plants insensitive to gene transformation, especially to Agrobacterium-mediated transformation. Wheat immature embryos of 1.0 - 1.5mm in size, C58cl of Agrobacterium strain harboring pPTN249, pPTN270, pPTN254, and pSIS-GFP respectively (all the vectors contain the aphA selectable gene driven by enhanced 35S promoter and a target gene controlled by ubi promoter or E35S promoter), AB medium for Agrobacterium activate culture, WCC medium for co-culture, were used in this study. The embryos with 4 days of pre-culture were transferred onto selection medium with 10mg/L geneticin, 50mg/L ticarcillin, 50mg/L vancomycin, and 50mg/L cefotaxine after 30 minutes of infection and 2 days of co-cultivation with Agrobacterium. Followed callus production, shoot regeneration on selection medium, 114 resistant plantlets were obtained from 10 transformation experiments of four genotypes. By nptll ELISA (nptII enzyme assay), PCR, Southern blot and leaf bleach, 29 positive plants were identified from two genotypes of Bobwhite and Yangmai 10, with an average transformation efficiency of 0.82%. The result tested by Southern blot also showed that the transgenic plants with single- copy integration of target gene took 65.52% among total positive plants. The ELISA value of transgenic plant was also related to the copies of alien DNA integrating into wheat chromosomes, the transgenic plants with single copy integration giving higher ELISA value than the ones with 2 or 3 copies integration.

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.

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.

Optimization of the uidA Gene Transfer of Rosa hybrida via Agrobacterium tumefaciens: an Assessment of Factors Influencing the Efficiency of Gene Transfer

To develop a transformation protocol of Rosa hybrida Samantha via Agrobacterium tumefaciens, the authors examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing -glucuronidase gene. The results indicate that explant, light condition, salt concentration and acetosyringone (AS) concentration in co-culture medium are the most important factors, and factors like co-culture temperature, co-culture period and bacteria density have a strong effect on the growth of bacteria and then T-DNA transfer. Optimized co-cultivation was performed by inoculation of embryogenic callus with bacteria at a density of OD600= 0.50.8 for 20 min and co-culture in darkness under 23 C on medium with 1/2 MS salts and 300 mol稬1 AS for 3 d.

Reliable and Efficient Agrobacterium tumefaciens-Mediated Genetic Transformation of Dianthus spiculifolius

Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant.In this study,seeds of D.spiculifolius were used as explants for callus induction,adventitious bud differentiation,and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D),6-benzyl aminopurine(6-BA),and naphthaleneacetic acid(NAA)to Murashige and Skoog medium.The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min,and transgenic regenerated plants were obtained by kanamycin(30mg·L^−1)screening.RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D.spiculifolius.Theβ-glucuronidase(GUS)histochemical staining confirmed GUS gene expression in transgenic calli,adventitious buds,and regenerated plants of D.spiculifolius.The green fluorescent protein(GFP)visual analysis showed GFP gene expression in transgenic calli.Furthermore,subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D.spiculifolius callus cells.These results indicated a successful establishment of a reliable and efficient A.tumefaciens-mediated genetic transformation system,which will contribute to functional gene research and genetic improvement of D.spiculifolius.

Computational prediction of over-annotated protein-coding genes in the genome of Agrobacterium tumefaciens strain C58

Agrobacterium tumefaciens strain C58 is a type of pathogen that can cause tumors in some dicotyledonous plants.Ever since the genome of A. tumefaciens strain C58 was sequenced, the quality of annotation of its protein-coding genes has been queried continually, because the annotation varies greatly among different databases. In this paper, the questionable hypothetical genes were re-predicted by integrating the TN curve and Z curve methods. As a result, 30 genes originally annotated as ＂hypothetical＂ were discriminated as being non-coding sequences. By testing the re-prediction program 10 times on data sets composed of the function-known genes, the mean accuracy of 99.99% and mean Matthews correlation coefficient value of 0.9999 were obtained. Further sequence analysis and COG analysis showed that the re-annotation results were very reliable. This work can provide an efficient tool and data resources for future studies of A. tumefaciens strain C58.

Trehalose Synthase Gene Transfer Mediated by Agrobacterium tumefaciens Enhances Resistance to Osmotic Stress in Sugarcane

Trehalose synthase gene (TSase) from Grifola frondosa was transferred into sugarcane (Sac-charum hybrid L.) using Agrobacterium-mediated method to improve sugarcane drought-tolerance. The results indicated that embryogenic callus of sugarcane was sensitive to A. tumefaciens EHA105 strain in the transformation system employed. The high frequency PPT-resistant plants were obtained from transformated with 3 weeks callus after incubation, which reached 4.5% on average. The transgenic plants were confirmed by PCR and Southern analysis. Some transgenic plants showed multiple phenotypic alterations and some plants demonstrated improvement tolerance to osmotic stress.

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.

Agrobacterium tumefaciens-mediated GUS gene transfer to Sophora japonica L.

Agrobacterium-mediated genetic transformation of Sophora japonica was standardized using the Agrobacterium tumefa- ciens strain LBA4404 that harbored the binary vector pBI121 containing genes for fl-glucuronidase （GUS） and neomycin phos- photransterase （npt II）. S. japonica transformants were selected by the ability of the leaf explants to produce kanamycin-resistant calli that regenerated into kanamycin-resistant plantlets. Successful transformation was confirmed by histochemical assay for GUS activity, PCR analysis and Southern blot. The period of nearly two months was required for the regeneration of transgenic plantlets fi＇om the explants. The transformed plants resembled their parents in morphology.

Regeneration and gene transformation systems of Robima pseudoacacia 'Idaho' mediated by Agrobacterium tumefaciens

Robinia pseudoacacia ‘Idaho＇ is one of several multi-purpose trees used in ornamental, soil and water conservation, fodder and nectar sources. Plant abiotic stress tolerance transformed by genes could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration. For this paper, we studied the effects of auxin and cytokine on Idaho locust in vitro regeneration and the establishment of gene transformation systems for plants mediated by Agrobacterium tumefaciens. Results showed that the ratios of cytokinin and auxin were the major factors affecting adventitious bud differentiation on a MS medium; the concentration of 0.5 mg·L^-16-BA benefitted callus proliferation and 0.25 mg·L^-1 IBA promoted shoot rooting; however, a higher IBA concentration will inhibit rooting. The most effective antitoxin for screening transgenic Idaho locust shoots was G418 and the most sensitive concentration of it was 8 mg·L^-1.

The Efficient Transformation of Wheat in Planta by Agrobacterium tumefaciens

Transformation of wheat was performed by pipetting spikelets with Agrobacterium tumefaciens which contained expression vectors using Npt II as reporter gene. Transformants were identified through kana-mycin resistance, PCR and Southern blot. The results showed that transformation efficiency was within 2.0 to 3. 2% in all tested varieties of wheat. Then the simple and efficient protocol of wheat transformation by Agrobacterium tumefaciens in planta was primarily established.

Agrobacterium tumefaciens-mediated GUS gene transformation of Robinia pseudoacacia 'Idaho'

Based on the plant regeneration system, a GUS gene transformation system to Idaho locust （Robinia pseudoacacia ‘Idaho＇） mediated by Agrobacterium tumefaciens was established. The successful transformation was confirmed by regenerating the shoots fi＇om the infected leaves in the presence of hygromysin; by histochemical X-gluc assays of 15-glucuronidase （GUS） and by PCR and PCR-Southern blotting analysis. The ratio of positive transgenic plants is 5.8% （5 out of 86 plants）. With this system, the target gene DREB was introduced into the leaves of Idaho locust. The transgenic plants regenerated, which was verified by PCR-Southern blot- ting. It is suggested that the transformation system could be a new, simple, reliable and practical route to gene transformation of R. pseudoacacia ＇Idaho＇ mediated with A. tumefaciens.

Factors Influencing Induction of Agrobacterium tumefaciens Virulence Genes

Agrobacterium species are routinely employed for plant genetic modification due to the relatively simple procedures, cost-competitiveness, low copy num- ber, independence to vector DNAs, and targeted integration into transcriptionally active regions of plant chromosomes with defined T-DNA. However, to date, there are still a great number of plant species reluctant to Agrobacterium-mediated transformation. Evidence suggests that the infection capability of Agrobacterium is deter- mined by virulence （vir） genes of Ti plasmid outside ofA. tumefaciens chromosome. Among all v/r genes, virA and virG are constitutively expressed, while the ex- pression of other vir genes is induced by phenolic compounds. In addition, carbohydrates can enhance vir induction mediated by phenolic compounds, while low phosphate and acidic pH conditions may also enhance the induction of vir genes. To improve Agrobacterium-mediated transformation efficiency for potential applica- tions in research and industry, molecular mechanisms of vir induction by factors such as phenolic compounds, carbohydrates, low phosphate, acidic pH and incuba- tion temperature are discussed in this review.

Characterization of Avirulent TnphoA Mutants in <i>Agrobacterium tumefaciens</i>to Enhance Transformation Efficiency

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane protein. Agrobacterium strain A6007 was mutagenized with E. coli strain mm294A plasmid pRK609 having TnphoA to obtain mutants defective in virulence. Because alkaline phosphatase activity is only detected when the PhoA gene product from the transposon is secreted out of the protoplasm, the virulence mutants are located in genes that code for transmembrane or periplasmic proteins. Attempts were made to obtain the sequences adjacent to the TnphoA inserts through several different approaches including Inverse PCR, Cloning, and Tail PCR. Transposon-adjacent sequence was obtained from one membrane anchor subunit in Bradyrhizobium japonicum i.e. succinate dehydrogenase which has enhanced transformation efficiency.