L-Cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cotyledonary-node cells

A major limitation in producing transgenic soybeans [Glycine max (L.) Merrill] using the Agrobacterium-mediated cotyledonary-node method is low-frequency T-DNA transfer from Agrobacterium tumefaciens into cotyledonary-node cells. We increased Agrobacterium infection from 37% to 91% of explants in the cotyledonary-node region by amending the solid co-cultivation medium with L-cysteine, which resulted in a fivefold increase in stable T-DNA transfer in newly developed shoot primordia. Southern analysis detected greater than a twofold increase in transformation efficiency, as determined by the number of independent fertile, transgene plants per explants inoculated. Enzymatic browning on explant tissue was also reduced, which suggests cysteine may interact with wound- and pathogen-defense responses in the soybean explant, resulting in an increased T-DNA delivery into the cotyledonary-node cells.     

A simple and efficient method for obtaining transgenic soybean callus tissues

In the present study, a simple and efficient method for obtaining transgenic callus tissues of soybean [Glycine max (L.) Merr.] was developed based on Agrobacterium-mediated transformation. Hypocotyl segments of soybean were used as the starting material. Several factors such as soybean genotype, Agrobacterium concentration, inoculation time, co-cultivation period and addition of antioxidants in co-cultivation medium affecting the transformation efficiency were examined. The explants were cultured on callus induction medium containing 0.5 mg L^?1 6-benzylaminopurine and 2.0 mg L^?1, 2,4-Dichlorophenoxyacetic acid for callus induction. Callus tissues were induced at both the acropetal and basipetal ends. CaMV35S::GUS and CaMV35S::GFP transgenic callus tissues were obtained using the optimized protocol. The average transformation efficiency reached up to 87.7 % based on GUS detection. From inoculation with Agrobacterium to obtaining transgenic soybean callus will take about 3 weeks. In order to validate this method for gene function investigation, GVG::GmSARK transgenic soybean callus tissues were obtained and their senescence-associated phenotypes were assessed. To our knowledge, this is the first report using hypocotyl segments as starting materials to obtain transgenic callus, and this system provides a method for high-throughput screening of functional genes of interest in transformed soybean callus.     

Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars

For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacterium-mediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l^?1 citric acid, 100 µM acetosyringone, and 100 mg l^?1 l-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l^?1 hygromycin and the embryos were germinated in basal medium containing 20 mg l^?1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into soybean has been developed.     

Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants

A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions. Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM l-cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions. Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed in four R₁ progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R₁ progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes into the genome of finger millet in the future.     

Expression of the gus A gene in embryogenic cell lines of Sitka spruce following Agrobacterium-mediated transformation

Transformation of Picea sitchensis (Bong) Carr. was investigatedby incubating embryogenic cell lines, initiated from immatureand mature zygotic embryos, with a supervirulent strain of Agrobacteriumtumefaciens. The latter carried a gus A-intron gene. Transientgene expression was determined histochemically by recordingthe number of distinct areas of ß-glucuronidase (GUS)activity. Maximum expression of the gus gene was achieved witha bacterial suspension with an OD₆₀₀ of 0.8–1.1 dilutedwith an equal volume of MPM medium, Inoculation of cells withbacteria for 30 mm, 72 h co-cultivation period and exposureof Agrobacterium and plant cells to 50 µM acetosyrmngone.These results are discussed in relation to Agrobacterium-mediatedgene delivery for the stable transformation of Sitka spruceand other conifers. Key words: Sitka spruce, Agrobacterium, transformation, embryonal suspensor masses, GUS activity     

Factors affecting soybean cotyledonary node transformation

Cotyledonary node transformation efficiency was evaluated using a sonication assisted Agrobacterium-mediated transformation (SAAT) protocol, three dissimilar A. tumefaciens strains, and explants derived from 28 diverse cultivars and/or genotypes of soybean [Glycine max (L.) Merr.]. The explants were evaluated at 10 and 45 days after co-cultivation for transformation with a binary vector containing both a GUS-intron gene and an NPTII selectable marker. The best overall strain of A. tumefaciens was determined to be KYRT1 based on stable GUS transformation of soybean cotyledonary node explants measured at the terminal 45 day evaluation point. SAAT did not increase stable transformation at 45 days post co-cultivation. SAAT was determined to significantly decrease shoot proliferation of some genotypes, but it is unclear what effect this may have on the recovery of transformed shoots. Significant differences were also detected between genotypes for transformation and shoot proliferation frequency. Received: 9 March 1998 / Revision received: 9 July 1998 / Accepted: 28 July 1998     

Analysis of conditions forAgrobacterium-mediated transformation of tobacco cells in suspension

We have developed anAgrobacterium-mediated transformation system, using tobacco cell suspensions, that permits evaluation of factors affecting transformation within seven days of co-cultivation. Tobacco cell transformation was determined by monitoring -glucuronidase (GUS) activity detected in plant cell extracts. The use of a chimeric gene construct, 35S-GUS/INT, containing a portable intron in theuidA reading frame, assured only plant-specific GUS expression. During the co-cultivation period, induction of the bacterialvir-region was monitored using a heterologous gene construct composed of avirB promoter fragment from pTiC58 fused to the chloramphenicol acetyltranferase (CAT) gene ofTn9. Tobacco cell transformants were confirmed by antibiotic selection of transformed plant cells and by X-Gluc staining. Maximum transformation was obtained when plant suspension cultures were growing rapidly which also was coincidental with elevated levels of bacterialvir-region expression. One week after co-cultivation, the transformed cultures exhibited a stable pattern of GUS activity which remained constant without antibiotic selection. The system was used to compare the virulence of a number ofAgrobacterium strains. GUS activity of plant cells co-cultivated with a strain containing a cointegrate plasmid was 3-fold higher than that of one with a binary configuration of the T-DNA. When the co-cultivatingAgrobacterium strain also carried the plasmid used to monitorvir induction, the frequency of transformation was reduced by as much, as 97%.     

Plate flooding as an alternative <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for American chestnut somatic embryos

In an attempt to improve Agrobacterium-mediated transformation frequency of American chestnut somatic embryos, a novel method of inoculation/co-cultivation was developed. Plate flooding is a simple method where the Agrobacterium inoculum is poured onto the embryos while they remain on multiplication medium. This method tested the hypothesis that wounding tissues prior to co-cultivation was unnecessary or counterproductive. Two clones, WB296 and P1-1, were tested for differences in transformation efficiency as measured by the number of transformed embryogenic cell lines per Petri dish, the total number of transformed cell lines (embryos plus callus) and percentage of transformants that remained embryogenic. Plate flooding using clone WB296 produced significantly more transformed embryo cell lines and had a higher percentage of transformants remain embryogenic. The number of total transformed cell lines (embryos plus callus) was the same as obtained by other methods (desiccation, blot dry, sand abrasion, sonication and vacuum infiltration). With clone P1-1 there were no significant differences among the inoculation/co-cultivation treatments tested. Polymerase chain reaction and Southern hybridizations confirmed that the transgene of interest had been stably integrated into both American chestnut clones. Whole plants were regenerated from clone P1-1.     

A new GST-MAT vector containing both ipt and iaaM/H genes can produce marker-free transgenic tobacco plants with high frequency

. Agrobacterium-mediated transformation is highly dependent upon competency of the target plant tissues. It is important to develop the capacity of transformed cells to include cell proliferation and differentiation. A system which results in cell proliferation and differentiation only of transformed cells is highly desirable for plant transformation. We report here a new GST-MAT vector system (MATIMH), in which the ipt gene combined with iaaM/H genes was used as the selectable marker gene and the GST-II promoter was used as the promoter of the R gene in a site-specific recombination system. In tobacco transformation, the combination of the ipt gene and the iaaM/H genes can result in the production of both auxin and cytokinin in transformed tissues and induce regeneration of transgenic shoots exhibiting an ipt-shooty phenotype more efficiently than the ipt gene alone. When we transformed 20 tobacco leaf discs with the MATIMH vector, marker-free transgenic plants were produced from five (41.6%) out of 12 ipt-shooty lines. These results indicated that the combination of the iaaM/H genes and the ipt gene can more efficiently produce both transgenic plants and marker-free transgenic plants.     

Agrobacterium-mediated transformation of Campanula glomerata

A transformation system for Campanula glomerata 'Acaulis' based on the co-cultivation of leaf explants with Agrobacterium tumefaciens LBA4404 or EHA105 was developed. A. tumefaciens was eliminated when the explants were cultured on medium containing 400 mg/l vancomycin and 100 mg/l cefotaxime. Transgenic plants containing the uidA gene that codes for #-glucuronidase (gus) were obtained following co-cultivation with either strain of A. tumefaciens, LBA4404 or EHA105, both of which harbored the binary vector pGUSINT, coding for the uidA and neomycin phosphotransferase II (nptII) genes. While the transformation frequency (2-3%) was similar for both strains, A. tumefaciens LBA4404 was effectively eliminated from Campanula at a lower concentration of antibiotic as compared to EHA105. The concentration of individual antibiotics required to eliminate EHA105 resulted in a decreased rate (55-67%) of regeneration. The highest percentage of explants that regenerated plants (79%) and the highest regeneration rate was achieved with 100 mg/l cefotaxime combined with 400 mg/l vancomycin. Plants were also transformed with the isopentenyl transferase (ipt) gene using LBA4404 containing the 35S-ipt vector construct (pBC34).     

Assessment of factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of the unicellular green alga, <Emphasis Type="Italic">Chlorella vulgaris</Emphasis>

The successful establishment of an Agrobacterium-mediated transformation method and optimisation of six critical parameters known to influence the efficacy of Agrobacterium T-DNA transfer in the unicellular microalga Chlorella vulgaris (UMT-M1) are reported. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1304 containing the gfp:gusA fusion reporter and a hygromycin phosphotransferase (hpt) selectable marker driven by the CaMV35S promoter were used for transformation. Transformation frequency was assessed by monitoring transient β-glucuronidase (GUS) expression 2 days post-infection. It was found that co-cultivation temperature at 24°C, co-cultivation medium at pH 5.5, 3 days of co-cultivation, 150 μM acetosyringone, Agrobacterium density of 1.0 units (OD₆₀₀) and 2 days of pre-culture were optimum variables which produced the highest number of GUS-positive cells (8.8–20.1%) when each of these parameters was optimised individually. Transformation conducted with the combination of all optimal parameters above produced 25.0% of GUS-positive cells, which was almost a threefold increase from 8.9% obtained from un-optimised parameters. Evidence of transformation was further confirmed in 30% of 30 randomly-selected hygromycin B (20 mg L⁻¹) resistant colonies by polymerase chain reaction (PCR) using gfp:gusA and hpt-specific primers. The developed transformation method is expected to facilitate the genetic improvement of this commercially-important microalga.     

Transformation of Wall Deficient Cultured Tobacco Protoplasts by Agrobacterium tumefaciens

Attachment of virulent Agrobacterium tumefaciens to plant cells is required for transformation. To further study the components of the plant cell wall that may be involved in the attachment process, tobacco (Nicotiana tabacum L.) protoplasts were cultured in the presence of 2,6 dichlorobenzonitrile (DB), an inhibitor of cellulose biosynthesis, and then assayed for their ability to be transformed by Agrobacterium. The DB treated protoplasts were deficient in wall production. Nevertheless, they were transformable at high frequency by wild type Agrobacterium strains but not by mutant strains that lack the ability to bind to normal, walled cells. Small quantities of calcofluor white positive material present on DB treated cells were correlated with their competence to be transformed. Further, the plant:bacterial association that leads to transformation is shown to become stable within 5 hours after bacterial co-cultivation with either control or DB treated cells.     

RETRACTED ARTICLE: Transgenic ramie [<Emphasis Type="Italic">Boehmeria nivea</Emphasis> (L.) Gaud.]: factors affecting the efficiency of <Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation and regeneration

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for ramie [Boehmeria nivea (L.) Gaud.] based on the examinations of several factors affecting plant transformation efficiency. The effects of Agrobacterium cell density, acetosyringone, co-cultivation temperature, co-cultivation duration, co-cultivation photoperiod and pH on stable transformation were evaluated. Agrobacterium at a concentration of OD = 0.5–0.8 improved the efficiency of transformation. Concentration of acetosyringone at 50 mg/L during co-cultivation significantly increased transformation efficiency. Co-cultivation at 20°C, in comparison to 15, 25 and 28°C, consistently resulted in higher transformation frequencies. A relatively short co-cultivation duration (3 days) was optimal for ramie transformation. Co-cultivation medium at pH 5.9 and co-cultivation in darkness both improved the transformation efficiencies of ramie. An overall scheme for producing transgenic ramie is presented, through which an average transformation rate from 10.5 to 24.7% in five ramie varieties was obtained. Stable expression and integration of the transgenes were confirmed by histochemical GUS assay, kanamycin painting assay, PCR and Southern blotting. This optimized transformation system should be employed for efficient Agrobacterium-mediated transformation of ramie. An erratum to this article can be found at     

Influence of putrescine, silver nitrate and polyamine inhibitors on the morphogenetic response in untransformed and transformed tissues of Cichorium intybus and their regenerants

The addition of 40 mM putrescine (Put) to Murashige and Skoog's (MS) medium resulted in increased shoot multiplication and shoot growth in untransformed plants relative to transformed plants of Cichorium intybus L. Put at a concentration of 40 mM also resulted in flowering in both systems on the 28th day, with elevated titers of endogenous conjugated Put and spermine (Spm) in both untransformed and transformed plants. The addition of 40 µM AgNO₃ to untransformed axillary buds of C. intybus L. cultured on MS media resulted in increased shoot multiplication (36.9DŽ.63 shoots per culture) and increased shoot growth (7.82ǂ.76 cm) as compared to transformed ones (11.6ǂ.89 shoots per culture; 3.20ǂ.24 cm). Moreover, cultures treated with 40 µM AgNO₃ showed in vitro flowering on the 28th day in both systems, with the endogenous levels of conjugated spermine being higher in untransformed plants than in transformed ones. The morphogenetic response and the endogenous conjugated pool of polyamines were lower following !-DL-difluromethylarginine and !-DL-difluromethylornithine treatments; the addition of put (40 mM) and AgNO₃ (40 µM) restored these to normal levels. Under exogenous put feeding, ethylene production was lower in both the untransformed and transformed cultures. We believe that an interplay between polyamine and ethylene biosynthesis is involved in regulating the morphogenetic response in both transformed and untransformed shoots of C. intybus. The response to AgNO₃ and Put treatment was not altered by the transformation process.     

Lipoic acid—an unique plant transformation enhancer

Including lipoic acid (LA) in culture media during Agrobacterium transformation processes of four crop species has significantly improved the transformation methods of the crops, even for previously recalcitrant genotypes. Plant transformation efficiency of soybean was significantly increased from 0.6% to 3.7% and tomato from 29.8% to 87.0%. Transformation efficiency was doubled from 2.8% to 5.7% in wheat. The frequency of glyphosate-resistant embryos had a significant increase from 41.4% to 61.2% in cotton. Regeneration of non-transgenic shoots under selection (“shoot escapes”) was significantly reduced in tomato from 91.5% to 46.2% while in soybean from 92.0% to 72.0% under optimal conditions. This study also demonstrated that the increase of transformation efficiency in tomato was accompanied by as much as a significant 2-fold reduction in severity of browning of Agrobacterium-infected plant tissues and up to a significant 3-fold increase in the percentage of explants with a high level of transient gene expression. LA application in plant transformation has enabled the resolution of three common problems in plant transformation: browning or necrosis of the transformed cells or tissues, difficulty in regenerating transformed cells or tissues, and shoot escapes, which severely limit the number of transgenic plants that can be regenerated.     

An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars

Agrobacterium-mediated transformation of indica rice varieties has been quite difficult as these are recalcitrant to in vitro responses. In the present study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation system of rice (Oryza sativa L. ssp. indica) cv. IR-64, Lalat, and IET-4786. Agrobacterium strain EHA-101 harboring binary vector pIG121-Hm, containing a gene encoding for β-glucuronidase (GUS) and hygromycin resistance, was used in the transformation experiments. Manipulation of different concentrations of acetosyringone, days of co-culture period, bacterial suspension of different optical densities (ODs), and the concentrations of l-cysteine in liquid followed by solid co-culture medium was done for establishing the protocol. Among the different co-culture periods, 5 days of co-culture with bacterial cells (OD_600 nm?=?0.5–0.8) promoted the highest frequency of transformation (83.04 %) in medium containing l-cysteine (400 mg l^?1). Putative transformed plants were analyzed for the presence of a transgene through genomic PCR and GUS histochemical analyses. Our results also suggest that different cultural conditions and the addition of l-cysteine in the co-culture medium improve the Agrobacterium-mediated transformation frequencies from an average of 12.82 % to 33.33 % in different indica rice cultivars.     

Recovery of Basta-resistant Sedum erythrostichum via Agrobacterium-mediated transformation

Sedums are used as groundcover, in rock gardens and flower borders, and for greening the top floor of buildings, cottages, and thatched roofs. In this study, Agrobacterium-mediated genetic transformation of Sedum erythrostichum was studied by introducing a herbicide-resistant gene (phosphinothricin-N-acetyl-transferase) and a reporter gene (#-glucuronidase, GUS). Following co-cultivation with Agrobacterium on MS medium supplemented with 0.5 mg/l !-naphthaleneacetic acid (NAA) and 2 mg/l 6-benzylaminopurine (BA) for 3 days, leaf segments were transferred onto medium containing 300 mg/l cefotaxime. When adventitious shoots developed directly near the margins of explants after 3 weeks, they were transferred to selection medium with 25 mg/l kanamycin. Of a total of 640 infected leaf explants, 24 (3.75%) produced kanamycin-resistant adventitious shoots; of these, 2.5% were GUS-positive. Transgenic plantlets were confirmed using polymerase chain reaction, Southern, and Northern analyses. Ninety-four percent of the transgenic plantlets were successfully transferred to soil and produced flowers. All GUS-positive transgenic plants were strongly resistant to Basta (phosphinothricin at 200 mg/l) after spraying.     

Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line

Key message

An improved Agrobacterium -mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds.

Abstract

This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T₀ plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T₀ plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.     

Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene

Ponkan (Citrus reticulata Blanco), one of the most important commercial cultivars of mandarin, is very seedy. In this study, the chimeric ribonuclease gene (barnase) driven by an anther tapetum-specific promoter (pTA29) was introduced into embryogenic callus of Ponkan by Agrobacterium-mediated transformation using the bar gene as a selectable marker. In contrast to previous reports, embryogenic calluses were used as the explant for Agrobacterium infection and transgenic plant regeneration. Selection of transformed callus was accomplished using basta. After 3 days of co-culture, calluses were transferred to MT medium with 50 mg/l basta and 400 mg/l cefotaxime. Resistant calluses were recovered and proliferated after three to four subcultures and then regenerated plantlets. A total of 52 resistant plants were recovered, of which 43 were verified to be transformants by polymerase chain reaction amplification of a fragment of the transgene. Southern hybridization of seven randomly selected transformed plants further confirmed their transgenic nature. The potential of this strategy for breeding citrus seedless types is discussed.