Agrobacterium-mediated Transformation Procedure Research Articles

An Improved Procedure for Agrobacterium-Mediated Transformation of 'Carrizo' Citrange.

Although several protocols for genetic transformation of citrus have been published, it is highly desirable to further improve its efficiency. Here we report treatments of Agrobacterium cells and citrus explants prior to and during co-cultivation process to enhance transformation efficiency using a commercially used rootstock ‘Carrizo’ citrange [Citrus sinensis (L.) Osb. × Poncirius trifoliata (L.) Raf.] as a model plant. We found explants from light-grown seedlings exhibited higher transformation efficiency than those from etiolated seedlings. We pre-cultured Agrobacterium cells in a 1/10 MS, 0.5 g/L 2-(N-morpholino) ethanesulfonic acid (MES) and 100 µM acetosyringone liquid medium for 6 h at 25 °C before used to infect citrus explants. We incubated epicotyl segments in an MS liquid medium containing 13.2 µM 6-BA, 4.5 µM 2,4-D, 0.5 µM NAA for 3 h at 25 °C prior to Agrobacterium infection. In the co-cultivation medium, we added 30 µM paclobutrazol and 10 µM lipoic acid. Each of these treatments significantly increased the efficiencies of transformation up to 30.4% (treating Agrobacterium with acetosyringone), 31.8% (treating explants with cytokinin and auxin), 34.9% (paclobutrazol) and 38.6% (lipoic acid), respectively. When the three treatments were combined, we observed that the transformation efficiency was enhanced from 11.5% to 52.3%. The improvement of genetic transformation efficiency mediated by these three simple treatments may facilitate more efficient applications of transgenic and gene editing technologies for functional characterization of citrus genes and for genetic improvement of citrus cultivars.

Optimization of Agrobacterium-mediated transformation procedure for an indica rice variety-Khang Dan 18

In addition to traditional methods, advanced biotechnologies, especially CRISPR/Cas9-mediated genome editing, have emerged as effective tools for improving important agronomic traits in rice. However, the critical step for utilizing these systems is to develope an effective system for rice transformation and regeneration. This study was performed to establish procedures for Agrobacterium-mediated transformation and regeneration of rice cultivar Khang Dan 18 (KD18) – a popular indica variety in the North of Vietnam. The tissue cultures procedure with optimized medium compositions showed high frequencies of callus induction and shoot regeneration, at 94.7% and 45.3% respectively. We found that 30 mg/L of hygromycin was an effective concentration for transgenic KD18 selection. Light-yellow friable calli were used as the starting material for transformation mediated by A. tumefaciens strain AGL1 harbouring pHUE411 vector containing gus intron and hygromycin resistance gene (hptII). Important factors related to transformation procedure had been optimized in this study. The high transformation frequency (12.8%) was achieved by using the optimized procedure for KD18 cultivar. In which, bacterial density (OD600), infection time and co-cultivation period were performed at 0.1, 20 minutes and 3 days, respectively. PCR analysis using specific primers and the histological GUS staining demonstrated the presence of hptII and gus genes in transgenic rice lines. This result provides a potential protocol to transfer genes of interest into KD18 as well as other indica rice cultivars.

Rapid delivery of Cas9 gene into the tomato cv. ‘Heinz 1706’ through an optimized Agrobacterium-mediated transformation procedure

Solanum lycopersicum ‘Heinz 1706’ is a pioneer model cultivar for tomato research, whose whole genome sequence valuable for genomics studies is available. Nevertheless, a genetic transformation procedure for this cultivar has not yet been reported. Meanwhile, various genome editing technologies such as transfection of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) ribonucleoprotein complexes into cells are in the limelight. Utilizing the Cas9-expressing genotype possessing a reference genome can simplify the verification of an off-target effect, resolve the economic cost of Cas9 endonuclease preparation, and avoid the complex assembly process together with single-guide RNA (sgRNA) in the transfection approach. Thus, this study was designed to generate Cas9-expressing ‘Heinz 1706’ lines by establishing an Agrobacterium tumefaciens-mediated transformation (ATMT) procedure. Here, we report a rapid and reproducible transformation procedure for ‘Heinz 1706’ by finetuning various factors: A. tumefaciens strain, pre-culture and co-culture durations, a proper combination of phytohormones at each step, supplementation of acetosyringone, and shooting/rooting method. Particularly, through eluding subculture and simultaneously inducing shoot elongation and rooting from leaf cluster, we achieved a short duration of three months for recovering the transgenic plants expressing Cas9. The presence of the Cas9 gene and its stable expression were confirmed by PCR and qRT-PCR analyses, and the Cas9 gene integrated into the T0 plant genome was stably transmitted to T1 progeny. Therefore, we anticipate that our procedure appears to ease the conventional ATMT in ‘Heinz 1706’, and the created Cas9-expressing ‘Heinz 1706’ lines are ultimately useful in gene editing via unilateral transfection of sgRNA into the protoplasts.

Development of Efficient, Reproducible and Stable Agrobacterium-Mediated Genetic Transformation of Five Potato Cultivars

SUMMARYThe developments in transformation technology have enabled the scientists to incorporate, mutate or substitute gene(s) leading to a particular trait; advancing it to a point where only few technical limitations remain. Genotype dependency and explant types are important factors affecting transformation efficiency in potato. In the present study, a rapid, reproducible and stable Agrobacterium-mediated transformation procedure in potato was developed by a combination of different plant growth regulators. Leaf discs and internodal explants of five cultivars of potato, i.e. Lady Olympia, Granola, Agria, Désirée and Innovator were infected with Agrobacterium tumefaciens strain LBA4404 containing pBIN19 expression vector with β-glucuronidase gusA gene under the control of 35S CaMV promoter. Kanamycin was used as plant selectable marker for screening of primary transformants at concentration of 100 mg/L. Both explants responded positively; internode being more suitable explant for better transformation efficiency. Based on GUS histochemical assay, the transformation efficiency was 22, 20, 18.6, 15 and 10% using the internodal explant, and 15, 12, 17, 8 and 6% using leaf discs as explant in Lady Olympia, Granola, Agria, Désirée and Innovator respectively. Furthermore, PCR assays confirmed the presence of gusA and nptII genes in regenerated plants. The molecular analysis in succeeding progeny showed proper integration and expression of both genes. The results suggest Lady Olympia as the best cultivar for future transformation procedures. Overall, the short duration, rapidity and reproducibility makes this protocol suitable for wider application of transgenic potato plants.

Host-induced gene silencing of Pectobacterium carotovorum quorum sensing gene enhances soft rot disease resistance in potato plants

Quorum sensing is a regulatory mechanism of gene expression in bacteria which regulated many physiological processes such as production of virulence factors in Pectobacterium carotovorum. In this research, the plant expression vector, pBI121, harbouring the anti-quorum sensing gene, aiiA, was transformed into Potato (Solanum tuberosum cv. Agria) explants by Agrobacterium mediated transformation procedure and putative transformants containing pBI121/aiiA were obtained. The results of bioassay test revealed that the expression of aiiA gene in potato plant induces the resistance to the early stage of bacterium pathogenesis. The soft rot symptoms such as wilting, yellowing and tissue maceration were not observed until 48 hr after inoculation of transgenic potatoes with P. carotovorum. Although in non-transgenic plants, the disease symptoms were appeared after 24 hr of plants infection. Hence, this study proves that the heterologous expression of aiiA gene enhances the resistance against potato soft rot disease.

Optimization ofAgrobacterium-mediated transformation procedure for grapevine ‘Kyoho’ with carrot antifreeze protein gene

We report an Agrobacterium-mediated transformation procedure optimized for ‘Kyoho’ that is a major table grapevine cultivar in Korea, and its transgenic plants with antifreeze protein gene of carrot (DcAFP). The full length of DcAFP coding region in accordance with the previous report was isolated from young leaves of carrot and recombined into a plant transformation vector. Ethylene inhibitors such as silver nitrate and aminoethoxyvinylglycin (AVG) supplemented in a co-cultivation medium distinctly increased frequency of shoot regeneration when explants were sub-cultured in a selection medium: particularly ten-fold higher in treatment with 0.1 mg/L AVG than one without ethylene inhibitor. Among various antibiotics and their concentrations, the combination of 150 mg/L cefotaxime plus 150 mg/L ClavamoxTM was selected for elimination of Agrobacterium cells in addition to minimization of adverse effect on shoot regeneration, while 50 mg/L kanamycin monosulfate effectively suppressed regeneration of nontransgenic shoots. Applying the elucidated culture condition, we finally obtained a total of 5 transgenic ‘Kyoho’ plantlets with DcAFP, of which integration with the grapevine genome and transcription was confirmed by nucleic acid analyses.

Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum)

BackgroundSwitchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection.ResultsAfter evaluating the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N6 macronutrient/B5 micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50–100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical β-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines.ConclusionThe process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.

Functional\xa0characterization of the PHT1 family transporters of foxtail millet with development of a novel Agrobacterium-mediated transformation procedure

Phosphate is an essential nutrient for plant growth and is acquired from the environment and distributed within the plant in part through the action of phosphate transporters of the PHT1 family. Foxtail millet (Setaria italica) is an orphan crop essential to the food security of many small farmers in Asia and Africa and is a model system for other millets. A novel Agrobacterium-mediated transformation and direct plant regeneration procedure was developed from shoot apex explants and used to downregulate expression of 3 members of the PHT1 phosphate transporter family SiPHT1;2 SiPHT1;3 and SiPHT1;4. Transformants were recovered with close to 10% efficiency. The downregulation of individual transporters was confirmed by RT-PCR. Downregulation of individual transporters significantly reduced the total and inorganic P contents in shoot and root tissues and increased the number of lateral roots and root hairs showing they have non-redundant roles. Downregulation of SiPHT1;2 had the strongest effect on total and inorganic P in shoot and root tissues. Complementation experiments in S. cerevisiae provide evidence for the ability of SiPHT1;1, 1;2, 1;3, 1;7 and 1;8 to function as high affinity Pi transporters. This work will aid development of improved millet varieties for global food security.

Efficient callus formation and plant regeneration are heritable characters in sugar beet (Beta vulgaris L.).

BackgroundObtaining dedifferentiated cells (callus) that can regenerate into whole plants is not always feasible for many plant species. Sugar beet is known to be recalcitrant for dedifferentiation and plant regeneration. These difficulties were major obstacles for obtaining transgenic sugar beets through an Agrobacterium-mediated transformation procedure. The sugar beet line ‘NK-219mm-O’ is an exceptional line that forms callus efficiently and is easy to regenerate, but the inheritance of these characters was unknown. Another concern was whether these characters could coexist with an annual habitat that makes it possible to breed short life-cycle sugar beet suitable for molecular genetic analysis.FindingsFive sugar beet lines including NK-219mm-O were crossed with each other and subjected to in vitro culture to form callus. F1s with a NK-219mm-O background generally formed callus efficiently compared to the others, indicating that efficient callus formation is heritable. The regeneration potential was examined based on the phenotypes of calli after placement on regeneration medium. Five phenotypes were observed, of which two phenotypes regenerated shoots or somatic embryo-like structures. Vascular differentiation was evident in regenerable calli, whereas non-regenerable calli lacked normally developed vascular tissues. In a half-diallel cross, the callus-formation efficiency and the regeneration potential of reciprocal F1s progeny having a NK-219mm-O background were high. Finally, we crossed NK-219mm-O with an annual line that had a poor in vitro performance. The callus-formation efficiency and the regeneration potential of reciprocal F1 were high. The regenerated plants showed an annual habitat.ConclusionsEfficient callus formation and the high plant regeneration potential of NK-219mm-O were inherited and expressed in the F1. The annual habitat does not impair these high in vitro performances.Electronic supplementary materialThe online version of this article (doi:10.1186/s41065-016-0015-z) contains supplementary material, which is available to authorized users.

Metabolic engineering of Dunaliella salina for production of ketocarotenoids.

Dunaliella is a commercially important marine alga producing high amount of β-carotene. The use of Dunaliella as a potential transgenic system for the production of recombinant proteins has been recently recognized. The present study reports for the first time the metabolic engineering of carotenoid biosynthesis in Dunaliella salina for ketocarotenoid production. The pathway modification included the introduction of a bkt gene from H. pluvialis encoding β-carotene ketolase (4,4'β-oxygenase) along with chloroplast targeting for the production of ketocarotenoids. The bkt under the control of Dunaliella Rubisco smaller subunit promoter along with its transit peptide sequence was introduced into the alga through standardized Agrobacterium-mediated transformation procedure. The selected transformants were confirmed using GFP and GUS expression, PCR and southern blot analysis. A notable upregulation of the endogenous hydroxylase level of transformants was observed where the BKT expression was higher in nutrient-limiting conditions. Carotenoid analysis of the transformants through HPLC and MS analysis showed the presence of astaxanthin and canthaxanthin with maximum content of 3.5 and 1.9µg/g DW, respectively. The present study reports the feasibility of using D. salina for the production of ketocarotenoids including astaxanthin.

Characterization of the 2,3-Oxidosqualene Cyclase Gene from Antrodia cinnamomea and Enhancement of Cytotoxic Triterpenoid Compound Production.

Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.

The procedure providing enhanced Agrobacterium-mediated transformation of wheat

The examinations of conditions for establishing a variety independent Agrobacterium-mediated transformation procedure for wheat are preferable since many of cultivars and breeding lines remain recalcitrant for biotechnological manipulation, mainly due to low efficiency of plant regeneration in vitro, which is highly genotype specific. This paper describes and discusses an improved protocol for enhanced and low-genotype dependent Agrobacterium-mediated transformation using a super-binary vector LBA4404/pTOK233 carrying reporter gus-intron gene and hygromycin (hpt) and kanamicyn (nptII) selectable marker genes. The protocol was optimized on highly responsive common wheat cv. Vesna. Transient expression monitored by the gus-intron on explants after 3, 6 and 25 days of co-cultivation, followed by GUS expression and hygromycin resistance in whole plants indicated the protocol including a co-cultivation of freshly isolated immature embryos in the presence of ascorbic acid, and acetosyringone added only in the bacteria-containing infection medium combined with a delayed and stepwise increasing hygromycin B selection procedure significantly enhanced the transformation efficiency in cv. Vesna that exceed 7% of treated explants from previously 0.41%. Explant pre-cultivation did not additionally improve transformation efficiency. The optimized protocol was successful in evoking satisfactory transformation efficiencies from 3.6% to 10.8% in 5 less-responsive wheat genotypes. All 57 T0 hygromycin-resistant and GUS-positive lines were phenotypically normal and fertile. Therefore, the conditions employed in this study may serve as a base to facilitate the transformation in other, particularly recalcitrant wheat cultivars.

Establishment of an Agrobacterium-mediated genetic transformation procedure for the experimental model orchid Erycina pusilla

The establishment of a genetic transformation system for Erycina pusilla (E. pusilla) would be a major step for orchid research and help establish the species as a potential experimental model. Here we describe the development of an efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation of E. pusilla protocorms. Plants were transformed with a binary plasmid containing the Arabidopsis thaliana gene Methionine sulfoxide reductase B7 (MSRB7) using the A. tumefaciens strain EHA105. Explants were selected on hygromycin-containing New Dogashima medium (Tokuhara and Mii in Plant Cell Rep 13(1):7–11, 1993) with 6-benzylaminopurine and α-naphthaleneacetic acid for 5 months to diminish the ratio of false positive explants, and it was determined that 3 month-old E. pusilla protocorms represented the optimal stage for transformation. Positive transformants were confirmed by Southern blot and PCR analyses, and by methyl viologen tolerance assays. Since E. pusilla has a shorter life cycle than most other orchids, self-pollinated T1 progeny was obtained within only 18 months. In addition, the results indicated that overexpression of MSRB genes can be used to avoid the effects of abiotic stress in economically important plants such as orchids.

Transgenic tomato line expressing modified Bacillus thuringiensis cry1Ab gene showing complete resistance to two lepidopteran pests

The modified truncated Bt-cry1Ab gene of Bacillus thuringiensis has been used for the development and selection of over expressing transgenic events in a commercially important variety of tomato (Solanum lycopersicum L.) by Agrobacterium-mediated leaf-disc transformation procedure. The integration and inheritance of cry1Ab gene in T0 transgenic plants and their progenies were determined by PCR, RT-PCR and Southern blot hybridization analysis. The toxin expression was monitored by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The transgenic line Ab25 E, expressing 0.47 ± 0.01% Cry1Ab toxin of total soluble protein (TSP) was finally selected in the T4 generation from the segregating population, showing 100% mortality to the second instar larvae of H. armigera and S. litura and minimal damages to leaves and fruits. Southern blot analysis data revealed single copy introgression of cry1Ab gene in highly-expressing Ab25 E transgenic line and expression of Cry1Ab toxin of molecular mass ~65 kDa was evident in Western blot analyses in transgenic plants of T4, T5 and T6 generation. Receptor binding assay performed with partially purified Cry1Ab protein from Ab25 E transgenic tomato line, confirmed efficient protein-protein interaction of Cry1Ab toxin with receptor(s) of both the insects. The higher level of Cry1Ab toxin (≈ 0.47 ± 0.01% TSP) did not affect the normal in vitro regeneration, plant development and fruit yield in this transgenic line. This high expressing Cry1Ab homozygous transgenic line can be a useful candidate in tomato breeding programmes for introgression of important agronomical traits.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-84) contains supplementary material, which is available to authorized users.

Agrobacterium-mediated transformation of Medicago truncatula cell suspension culture provides a system for functional analysis

Over the past decade, Medicago truncatula has been adopted as a model legume species for a range of “omic” studies. The availability of different transformation techniques has greatly advanced functional genomic studies in this species. In the present work, an efficient procedure for Agrobacterium-mediated transformation of M. truncatula cv. “Jemalong 2HA” through a cell suspension culture was developed. This procedure resulted in transformed single cells or cell clusters, giving rise to stable transgenic plants within 4 mo. Transformation experiments were performed with a vector carrying two marker genes: β-glucuronidase (GUS) and green fluorescent protein (GFP) under the control of endogenous gene promoters from LIKE AUX1 3 (LAX3) and GRAS transcription factor (named after GD3BERELLIC ACID INSENSITIVE [GAI], REPRESSOR OF GA1 [RGA], and SCARECROW [SCR]), as well as with a binary destination vector for overexpressing the cyclin-like F-box gene fused to GFP. Maximum transformation efficiency was achieved under the following experimental conditions: acetosyringone at a concentration of 25 μM, bacterial suspension with an optical density of 0.3 at 600 nm, inoculation under agitation at 100 rpm for 24 h, co-cultivation periods of 48 h, and an uninterrupted selection with 50 mg/L kanamycin. Selection of positive transformation events was imposed early in the regeneration stage (after 48 h co-cultivation), following a large-scale screening for GFP activity. Histochemical GUS and GFP reporter activity was detected in single cells, embryogenic zones, emerging embryos, in vitro plantlets, and T1 progeny seedlings. The transgenic nature of transformed plants was further confirmed by nptII-specific PCR amplification of T0 and T1 plant lines. The transgenic plants grown under standard greenhouse conditions displayed a wild-type phenotype and the obtained progeny segregated in a classical Mendelian manner. The fundamental steps in the transformation procedure are outlined and discussed.

Development of an efficient Agrobacterium-mediated transformation system and production of herbicide-resistant transgenic plants in garlic (Allium sativum L.).

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.

Efficient somatic embryogenesis and Agrobacterium-mediated transformation of pothos (Epipremnum aureum) ‘Jade’

Leaf and petiole explants of monocotyledonous pothos (Epipremnum aureum) ‘Jade’ were cultured on Murashige and Skoog basal medium supplemented with N-(2-chloro-4-pyridl)-N′-phenylurea (CPPU) or N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA). Somatic embryos appeared directly from explants after 4–8 weeks of culture; 9.1 μM TDZ with 1.1 μM NAA induced 61.1 % leaf discs and 94.4 % of petiole segments to produce plantlets through embryo conversion. Using this established regeneration method and an enhanced green fluorescent protein (GFP) gene (egfp) as a reporter marker, an Agrobacterium-mediated transformation procedure was developed. Leaf discs and petiole segments were inoculated with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pLC902 that contains novel bi-directional duplex promoters driving the egfp gene and hygromycin phosphotransferase gene (hpt), respectively. The explants were co-cultivated with strain EHA105 for 3, 5, and 7 days, respectively prior to selective culture with 25 mg l−1 hygromycin. A 5-day co-cultivation led to 100 % of leaf discs to show transient GFP expression and 23.8 % of the discs to produce stable GFP-expressing somatic embryos. A 7-day co-cultivation of petiole explants resulted in the corresponding responses at 100 and 14.3 %, respectively. A total of 237 transgenic plants were obtained, and GFP fluorescence was observed in all plant organs. Regular PCR and quantitative real-time PCR analyses confirmed the presence of 1 or 2 copies of the egfp gene in analyzed plants. The highly efficient regeneration and transformation systems established in this study may enable genetic improvement of this vegetatively propagated species through biotechnological means.

Establishment of Agrobacterium-mediated genetic transformation system in Dahlia

An efficient system for Agrobacterium-mediated transformation was established in dahlia (Dahlia×pinnata) ‘Yamatohime’. Mass of shoot primordia (MSP) induced on MS medium supplemented with 10 mg l−1 TDZ were inoculated with Agrobacterium tumefaciens strain EHA101 (pIG121-Hm) harboring both β-glucuronidase (GUS) and hygromycin resistant genes. After 2 days of co-cultivation, the MSP were transferred to a selection medium containing hygromycin with meropenem for bacterial elimination. Shoots were successfully regenerated from survived MSP on hormone-free medium without hygromycin and they rooted on hormone-free medium containing hygromycin. The hygromycin-resistant plants thus obtained showed histochemical blue staining for GUS. Transformation of plants was confirmed by PCR and Southern blot analyses. Transgenic ‘Kokucho’ were also produced by using the same transformation procedure, suggesting wide applicability of this Agrobacterium-mediated transformation procedure for other dahlia cultivars.

Genetically Transforming Russian Potato Cultivars for Resistance to Colorado Beetle

Using Agrobacterium-mediated transformation procedure, a total of 204 primary transformants of the Elizaveta, Lugovskoi and Nevski potato varieties carrying the cry IIIa gene were obtained. A number of biomolecular tests, including Southern hybridization, insert copy number screening, immunofluorescence analysis for assessment of the target gene expression levels, and PCR control for testing of the insert integrity, was carried out for these transformants, as well as the biosafety field trials for the transgenic lines selected during the biomolecular analysis. As a result, 3 transgenic lines (E2, N1, and L5) carrying one insert copy per genome with target gene expression level over 10 ppm were selected.

Expression and assembly of ApxIIA toxin of Actinobacillus pleuropneumoniae fused with the enterotoxigenic E. coli heat-labile toxin B subunit in transgenic tobacco

Actinobacillus pleuropneumoniae is a major etiological agent of swine pleuropneumonia, a highly contagious respiratory infection that causes severe economic losses in the swine production industry. ApxIIA, one of the virulence factors in A. pleuropneumoniae, is considered a candidate for the development of a vaccine against this bacterial infection. In this study, a fusion gene consisting of enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit (LTB) and an ApxIIA fragment (amino acids 619–801) (ApxIIA619–801) was fused to the endoplasmic reticulum (ER) retention signal (SEKDEL) and introduced into a plant expression vector under the control of a ubiquitin promoter. The plant expression vector was transformed into tobacco Nicotiana tabacum L. cv. MD609 using an Agrobacterium-mediated transformation procedure. The integration and transcription of the LTB fusion gene were confirmed by genomic DNA PCR amplification and Northern blot analysis, respectively. The synthesis and assembly of LTB fusion protein were evidenced by Western blot analysis and the GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). A quantitative ELISA used to measure the amount of LTB fusion protein produced in the transgenic plant revealed high levels of the fusion protein, up to 30 μg g−1, in lyophilized leaf tissue. These results demonstrate the feasibility of using a transgenic plant to express the LTB-ApxIIA619–801 fusion protein as a first step towards producing plant-based edible vaccines to elicit immune responses against A. pleuropneumoniae via an oral mucosal delivery system.