Agrobacterium-Mediated Transformation and Plant Regeneration of Triticum aestivum L.

The use of two Agrobacterium tumefaciens strains for transformation of Triticum aestivum L. cv. Vesna was studied. Immature embryos, isolated 15 d after pollination, were co-cultivated with the super-binary LBA4404/pTOK233 and the binary AGL1/pDM805 vectors. While the transient GUS-intron expression was high (69.9 and 80.0 %), the number of plants regenerated on selective media containing hygromycin or phosphinotricin did not exceed 0.4 and 0.13 %, respectively. Nevertheless, the regenerated plants were fertile and produced seeds. The T₀ plants, as well as the T₁ seedlings, displayed the activity in the β-glucuronidase histochemical assay and a positive signal in PCR analysis for the presence of uidA gene sequences in their genomes. The data suggest that the transformation of wheat cv. Vesna with both Agrobacterium strains is feasible. This revised version was published online in July 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of a recalcitrant grain legume,lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medik)

A simple and reproducible Agrobacterium-mediated transformation protocol for a recalcitrant legume plant, lentil (Lens culinaris M.) is reported. Application of wounding treatments and efficiencies of three Agrobacterium tumefaciens strains, EHA105, C58C1, and KYRT1 were compared for T-DNA delivery into lentil cotyledonary node tissues. KYRT1 was found to be on average 2.8-fold more efficient than both EHA105 and C58C1 for producing transient β-glucuronidase (GUS) gene (gus) expression on cotyledonary petioles. Wounding of the explants, use of an optimized transformation protocol with the application of acetosyringone and vacuum infiltration treatments in addition to the application of a gradually intensifying selection regime played significant roles in enhancing transformation frequency. Lentil explants were transformed by inoculation with Agrobacterium tumefaciens strain, KYRT1 harboring a binary vector pTJK136 that carried neomycin phosphotransferase gene (npt-II) and an intron containing gusA gene on its T-DNA region. GUS-positive shoots were micrografted on lentil rootstocks. Transgenic lentil plants were produced with an overall transformation frequency of 2.3%. The presence of the transgene in the lentil genome was confirmed by GUS assay, PCR, RT-PCR and Southern hybridization. The transgenic shoots grafted on rootstocks were successfully transferred to soil and grown to maturity in the greenhouse. GUS activity was detected in vegetative and reproductive organs of T₀, T₁, T₂ and T₃ plants. PCR assays of T₁, T₂ and T₃ progenies confirmed the stable transmission of the transgene to the next generations.     

Efficient <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of oilseed mustard [<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern.] using leaf piece explants

Leaf piece explants of five Brassica juncea (L.) Czern. cultivars were transformed with an Agrobacterium tumefaciens strain EHA105 harboring the plasmid pCAMBIA1301, which contains the β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of cauliflower mosaic virus 35S (CaMV35S) promoter. Transgenic plants were regenerated on Murashige and Skoog (MS) medium fortified with 8.87 μM 6-benzylaminopurine, 0.22 μM 2,4-dichlorophenoxyacetic acid, and 20 μM silver nitrate in the presence of 30 mg/l hygromycin. When co-culture took place in the presence of 100 μM acetosyringone, the efficiency of stable transformation was found to be approximately 19% in the T ₀ generation, with the transgenic plants and their progeny showing constitutive GUS expression in different plant organs. Southern blot hybridization of uidA and hpt genes confirmed transgene integration within the genome of transformed plants of each cultivar. Inheritance of hpt gene for single copy T-DNA inserts showed a 3:1 pattern of Mendelian segregation in progeny plants through germination of T ₁ seeds on MS medium containing 30 mg/l hygromycin. The protocol described here reports superior transformation efficiency over previously published protocols and should contribute to enhanced biotechnology applications in B. juncea.     

Genetic characterization of powdery mildew resistance in U.S. hard winter wheat

Powdery mildew significantly affects grain yield and end-use quality of winter wheat in the southern Great Plains. Employing resistance resources in locally adapted cultivars is the most effective means to control powdery mildew. Two types of powdery mildew resistance exist in wheat cultivars, i.e., qualitative and quantitative. Qualitative resistance is controlled by major genes, is race-specific, is not durable, and is effective in seedlings and in adult plants. Quantitative resistance is controlled by minor genes, is non-race-specific, is durable, and is predominantly effective in adult plants. In this study, we found that the segregation of powdery mildew resistance in a population of recombinant inbred lines developed from a cross between the susceptible cultivar Jagger and the resistant cultivar 2174 was controlled by a major QTL on the short arm of chromosome 1A and modified by four minor QTLs on chromosomes 1B, 3B, 4A, and 6D. The major QTL was mapped to the genomic region where the Pm3 gene resides. Using specific PCR markers for seven Pm3 alleles, 2174 was found to carry the Pm3a allele. Pm3a explained 61% of the total phenotypic variation in disease reaction observed among seedlings inoculated in the greenhouse and adult plants grown in the field and subjected to natural disease pressure. The resistant Pm3a allele was present among 4 of 31 cultivars currently being produced in the southern Great Plains. The genetic effects of several minor loci varied with different developmental stages and environments. Molecular markers associated with these genetic loci would facilitate incorporating genetic resistance to powdery mildew into improved winter wheat cultivars.     

Transformation of a recalcitrant grain legume, Vigna mungo L. Hepper, using Agrobacterium tumefaciens-mediated gene transfer to shoot apical meristem cultures

The efficiency of Vigna mungo L. Hepper transformation was significantly increased from an average of 1% to 6.5% by using shoot apices excised from embryonic axes precultured on 10 M benzyl-6-aminopurine (BAP) for 3 days and wounded prior to inoculation in Agrobacterium tumefaciens strain EHA105 carrying the binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a -glucuronidase (GUS) gene (gusA) interrupted by an intron. The transformed green shoots that were selected and rooted on medium containing kanamycin, and which tested positive for nptII gene by polymerase chain reaction, were established in soil to collect seeds. GUS activity was detected in whole T₀ shoots and T₁ seedlings. All T₀ plants were morphologically normal, fertile and the majority of them transmitted transgenes in a 3:1 ratio to their progenies. Southern analysis of T₁ plants showed integration of nptII into the plant genome.     

Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions.     

Genetic transformation of golden pothos (<Emphasis Type="Italic">Epipremnum aureum</Emphasis>) mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

To establish a procedure for Agrobacterium tumefaciens-mediated transformation of golden pothos (Epipremnum aureum) plants, the effects of selection antibiotics and the preculture period of stem explants before A. tumefaciens infection were examined. Explants were co-cultivated with A. tumefaciens EHA105, harboring the plasmid pGWB2/cGUS, on a somatic embryo-inducing medium supplemented with acetosyringone. Resulting transgenic somatic embryos were screened on an antibiotic selection medium, and the transgenic pothos plants were regenerated on a germination medium. Hygromycin was the optimum selection antibiotic tested. The preculture period significantly affected the transformation efficiency, with explants precultured for one-day showing the best efficiency (5–30%). Both transformed hygromycin-resistant embryos and regenerated plants showed β-glucuronidase activity. Southern blot analysis confirmed transgene integration into the pothos genome. This reproducible transformation system for golden pothos may enable the molecular breeding of this very common indoor plant.     

Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris alpha-amylase inhibitor-1 gene into mungbean Vigna radiata (L.) Wilczek using bar as selectable marker

Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and α-amylase inhibitor, have been developed for the first time. Cotyledonary node explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pKSB that carried bialaphos resistance (bar) gene and Phaseolus vulgaris α-amylase inhibitor-1 (αAI-1) gene. Green transformed shoots were regenerated and rooted on medium containing phosphinothricin (PPT). Preculture and wounding of the explants, presence of acetosyringone and PPT-based selection of transformants played significant role in enhancing transformation frequency. Presence and expression of the bar gene in primary transformants was evidenced by PCR-Southern analysis and PPT leaf paint assay, respectively. Integration of the Phaseolus vulgaris α-amylase inhibitor gene was confirmed by Southern blot analysis. PCR analysis revealed inheritance of both the transgenes in most of the T₁ lines. Tolerance to herbicide was evidenced from seed germination test and chlorophenol red assay in T₁ plants. Transgenic plants could be recovered after 8–10 weeks of cocultivation with Agrobacterium. An overall transformation frequency of 1.51% was achieved.     

Development of transgenic pearl millet (Pennisetum glaucum (L.) R. Br.) plants resistant to downy mildew

Transgenic pearl millet lines expressing pin gene—exhibiting high resistance to downy mildew pathogen, Sclerospora graminicola—were produced using particle-inflow-gun (PIG) method. Shoot-tip-derived embryogenic calli were co-bombarded with plasmids containing pin and bar genes driven by CaMV 35S promoter. Bombarded calli were cultured on MS medium with phosphinothricin as a selection agent. Primary transformants 1T₀, 2T₀, and 3T₀ showed the presence of both bar and pin coding sequences as evidenced by PCR and Southern blot analysis, respectively. T₁ progenies of three primary transformants, when evaluated for downy mildew resistance, segregated into resistant and susceptible phenotypes. T₁ plants resistant to downy mildew invariably exhibited tolerance to Basta suggesting co-segregation of pin and bar genes. Further, the downy mildew resistant 1T₁ plants were found positive for pin gene in Southern and Northern analyses thereby confirming stable integration, expression, and transmission of pin gene. 1T₂ progenies of 1T₀ conformed to dihybrid segregation of 15 resistant:1 susceptible plants.     

Microsatellite mapping of a Triticum urartu Tum. derived powdery mildew resistance gene transferred to common wheat (Triticum aestivum L.)

A powdery mildew resistance gene from Triticum urartu Tum. accession UR206 was successfully transferred into hexaploid wheat (Triticum aestivum L.) through crossing and backcrossing. The F₁ plants, which had 28 chromosomes and an average of 5.32 bivalents and 17.36 univalents in meiotic pollen mother cells (PMC), were obtained through embryos rescued owing to shriveling of endosperm in hybrid seed of cross Chinese Spring (CS) × UR206. Hybrid seeds were produced through backcrossing F₁ with common wheat parents. The derivative lines had normal chromosome numbers and powdery mildew resistance similar to the donor UR206, indicating that the powdery mildew resistance gene originating from T. urartu accession UR206 was successfully transferred and expressed in a hexaploid wheat background. Genetic analysis indicated that a single dominant gene controlled the powdery mildew resistance at the seedling stage. To map and tag the powdery mildew resistance gene, 143 F₂ individuals derived from a cross UR206 × UR203 were used to construct a linkage map. The resistant gene was mapped on the chromosome 7AL based on the mapped microsatellite makers. The map spanned 52.1 cM and the order of these microsatellite loci agreed well with the established microsatellite map of chromosome arm 7AL. The resistance gene was flanked by the microsatellite loci Xwmc273 and Xpsp3003, with the genetic distances of 2.2 cM and 3.8 cM, respectively. On the basis of the origin and chromosomal location of the gene, it was temporarily designated PmU.     

Stable transformation of sunflower (Helianthus annuus L.) using a non-meristematic regeneration protocol and green fluorescent protein as a vital marker

Stable transformation of sunflower was achieved using a non-meristematic hypocotyl explant regeneration protocol of public inbred HA300B. Uniformly transformed shoots were obtained after co-cultivation with Agrobacterium tumefaciens carrying a gfp (green fluorescent protein) gene containing an intron that blocks expression of gfp in Agrobacterium. Easily detectable, bright green fluorescence of transformed tissues was used to establish an optimal regeneration and transformation procedure. By Southern blot analysis, integration of the gfp and nptII genes was confirmed. Stable transformation efficiency was 0.1%. From 68 T₁ plants analyzed, 17 showed transmission of transgene DNA and 15 of them contained the intact gfp gene. Expression of gfp was detected in 10 T₁ plants carrying the intact gfp gene using a fluorimetric assay or western blot analysis. Expression of the nptII gene was confirmed in 13 T₁ plants. The transformation system enables the rapid transfer of agronomically important genes.     

Genetic transformation of <Emphasis Type="Italic">Agave salmiana</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and particle bombardment

Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (β-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the conditions for both shoot regeneration and rooting were optimized using leaves and embryogenic calli of Agave salmiana. The transgenes were detected by Polymerase Chain Reaction (PCR) in 11 month old plants. The transgenic nature of the plants was also confirmed using GUS histochemical assays. Transformation via co-cultivation of explants with Agrobacterium harbouring the pBI121 binary vector was the most effective method of transformation, producing 32 transgenic plants and giving a transformation efficiency of 2.7%. On the other hand, the biolistic method produced transgenic calli that tested positive with the GUS assay after 14 months on selective medium while still undergoing regeneration.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.National Research Council of Canada Publication No. 38003     

Exploration on the vacuum infiltration transformation of pakchoi

Agrobacterium tumefaciens mediated vacuum infiltration transformation in planta has been established in pakchoi, a kind of Chinese cabbage, but the transformation frequency in harvested seeds has varied in the range of 0.5 to 3.0 × 10⁻⁴ over several years and is much lower than the transformation frequency in Arabidopsis thaliana. To understand that, the distribution and vitality changes of A. tumefaciens in plant tissues were examined. Results revealed that there was a majority of A. tumefaciens in the flower compared with that in the stem and in the leaf at all times after infiltration. As fact of transformants in the upper part of the treated plant (T₀) stalk and fact of the survival of A. tumefaciens in the plant were proved, possibilities of optimizing the transformation conditions to increase the transformation frequency in pakchoi was discussed.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the medicinal plant <Emphasis Type="Italic">Centaurea montana</Emphasis>

An efficient transformation system was developed for Centaurea montana by co-cultivation of leaf explants with Agrobacterium tumefaciens strain AGL1 that contained a plasmid harboring the isopentenyl transferase gene under the control of the developmentally regulated Atmyb32 promoter of Arabidopsis thaliana and the gene encoding for hygromycin resistance under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter. A total of 990 explants were infected with Agrobacterium, and 18 shoots were regenerated resulting in an overall transformation efficiency of 1.8%. Molecular analyses, including PCR, Southern blotting and RT-PCR, were performed on T₀ and T₁ plants to confirm chromosomal integration and expression of the transgene in the phenotypically normal transformed plants. Transformation of C. montana was also performed using A. tumefaciens supervirulent strain EHA105 harboring the β-glucuronidase (GUS) reporter gene. Expression of the GUS gene in the putative transgenics was confirmed using a histochemical GUS assay.     

Chromosomal location of a gene suppressing powdery mildew resistance genes Pm8 and Pm17 in common wheat (Triticum aestivum L. em. Thell.)

The chromosomal location of a suppressor for the powdery mildew resistance genes Pm8 and Pm17 was determined by a monosomic set of the wheat cultivar Caribo. This cultivar carries a suppressor gene inhibiting the expression of Pm8 in cv Disponent and of Pm17 in line Helami-105. In disease resistance assessments, monosomic F₁ hybrids (2n=41) of Caribo x Disponent and Caribo x Helami-105 lacking chromosome 7D were resistant, whereas monosomic F₁ hybrids involving the other 20 chromosomes, as well as disomic F₁ hybrids (2n=42) of all cross combinations, were susceptible revealing that the suppressor gene for Pm8 and Pm17 is localized on chromosome 7D. It is suggested that genotypes without the suppressor gene be used for the exploitation of genes Pm8 and Pm17 in enhancing powdery mildew resistance in common wheat.     

Transgenic grapefruit plants expressing the PAPETALA3-IPT gp gene exhibit altered expression of PR genes

Transgenic grapefruit plants (Citrus paradisi cv. ‘Duncan’) with the isopentenyltransferase (ipt) gene under the control of APETALA3 promoter have been produced using Agrobacterium-mediated transformation. The relative expression level of the ipt gene was between 2.3 and 7 times higher in transformed plants than in the wild-type but despite the presence of a tissue-specific promoter, the expression was not limited only to flower tissue. Increased levels of trans-zeatin riboside between 9.4 and 32-fold found in transgenic grapefruit were considered the consequence of ectopic expression of the ipt gene. Chlorophyll levels in fully expanded uppermost leaves were also about 30% higher in transgenic than in wild-type plants. Involvement of cytokinins in control of expression of three pathogenesis-related protein genes: β-1,3-glucanase, a stress related PR gene 24P220, and an acidic chitinase, 24P262 was examined. Expression of β-1,3-glucanase, and 24P220 gene were significantly enhanced in transgenic plants while the expression of chitinase was reduced to low levels. Our results confirm the effect of cytokinins on expression of genes implicated in the response of grapefruit plants to pathogen attack and suggest a possible role of cytokinins in pathogen resistance.