Conditioned medium promotes the attachment ofAgrobacterium tumefaciens strain NT 1 to carrot cells

Summary Wild-typeAgrobacterium tumefaciens bind to carrot suspension culture cells. Avirulent strain NT 1 did not bind to carrot cells when they were incubated together in Murashige and Skoog medium. Conditioned medium was prepared by incubatingA. tumefaciens virulent strain C 58 with carrot cells and removing the bacteria and carrot cells using filter sterilization. This conditioned medium promoted the binding of NT 1 to carrot cells. Conditioned medium did not promote the nonspecific attachment ofEscherichia coli to carrot cells. These results suggest that when wild-typeA. tumefaciens are incubated with plant host cells, some substance(s) involved in bacterial attachment are released into the medium. Filter-sterilized medium from the incubation of the nonattachingchvB mutant A 1045 with carrot cells promoted the attachment of strain NT 1 even though A 1045 bacteria did not bind to the carrot cells. However, filter-sterilized medium from the incubation of the non-attachingatt mutant Att-B 123 with carrot cells was unable to promote the binding of strain NT 1. This suggests that nonattaching mutants ofA. tumefaciens can be divided into two groups on the basis of the properties of the substances released into the medium when the bacteria are incubated with carrot cells.Abbreviations MS Murashige and Skoog tissue culture medium Dedicated to the memory of Professor John G. Torrey     

Reduced rhizosphere colonization ability ofAgrobacterium tumefaciens chromosomal virulence (chv) mutants

Mutations in the chromosomal virulence (chv) region ofA. tumefaciens strain A723 reduce virulence, motility, and ability of the bacteria to bind to plant cells. We conducted experiments to assess the ability ofchv mutants to colonize the rhizosphere ofPisum sativum. The mutation had no effect on ability of bacteria to grow with a defined number of root cap cells as the sole carbon and nitrogen source. Ten days after inoculation, there were up to 103-fold more wild type thanchv mutant bacteria present in the rhizosphere of inoculated plants.     

Transformation of forage legumes using Agrobacterium tumefaciens

Summary Galls were induced in six species of forage legumes following inoculation with wild-type strains of A. tumefaciens. The plant species was more influential than the bacterial strain in determining the type of tumour produced. Inoculation of Medicago sativa resulted in small, disorganised tumours. The three Trifolium species, T. repens, T. hybridum and T. pratense, formed galls which tended to produce roots and both Onobrychis viciifolia and Lotus corniculatus produced teratomatous galls. The shoots elongated in the latter species only. In L. corniculatus, tissues that were infected by five bacterial strains were capable of shoot regeneration when cultured on a hormone-free medium. The transformed nature of these shoots was confirmed by their failure to root, the production of callus from leaves cultured on hormone-free medium and the presence of opines.     

Transformation of the wild tomatoLycopersicon chilense Dun. byAgrobacterium tumefaciens

Summary Leaf disc transformation-regeneration technique was applied to the drought tolerant wild relative of cultivated tomato,Lycopersicon chilense, using a plasmid construct which contained the coding sequences of neomycin phosphotransferase (NPTII) and chloramphenicol acetyltransferase (CAT) genes. The two genotypes used, LA2747 and LA1930, showed a distinct difference in their aptitude to transformation; a higher success rate was obtained for the first genotype in every stage of the process. Shoots were formed on the regeneration medium containing 100 g/ml kanamycin through direct or indirect organogenesis. Root formation became only possible when the concentration of kanamycin was reduced to 50 g/ml. Expression of chloramphenicol acetyltransferase gene was observed in all of the kanamycin-screened plants after they matured; the activity of the gene was absent or low in some of the young plants. The presence of the CAT gene in transgenic plants was further confirmed by Southern blot analysis. Although transgenic plants grew to maturity, they did not produce fruit, owing to the self incompatibility ofL. chilense. Abbreviations BAP 6-benzylaminopurine - CAT chloramphenicol acetyltransferase - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - LB Luria Broth - EDTA ethylenediamine-tetraacetic acid     

Multiple copies of virG enhance the transient transformation of celery,carrot and rice tissues by Agrobacterium tumefaciens

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical -glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.Current address: Department of Agronomy, Purdue University     

Adherence of Agrobacterium tumefaciens to the cells of immature wheat embryos

Agrobacterium attached to wheat embryos in vitro. This attachment was plasmid independent, and occurred on both wounded and unwounded cell surfaces. The pattern of attachment clearly demonstrated that bacterial attachment to cereal cells follows the same trends observed for dicotyledonous plants. During the inoculation period the bacterial cells attach to the plant cell walls either with lateral or polar orientation. Wounding (mechanical or enzymatic) preferentially promoted adherence of the bacteria at the wound site, however, attachment was not wound dependent.     

High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize

A high throughput genetic transformation system in maize has been developed with Agrobacterium tumefaciens mediated T-DNA delivery. With optimized conditions, stable callus transformation frequencies for Hi-II immature embryos averaged approximately 40%, with results in some experiments as high as 50%. The optimized conditions include N6 medium system for Agrobacterium inoculation, co-cultivation, resting and selection steps; no AgNo₃ in the infection medium and adding AgNo₃ in co-cultivation, resting and selection medium; Agrobacterium concentration at 0.5×10⁹ c.f.u. ml^–1 for bacterium inoculation; 100 mg l^–1 carbenicillin used in the medium to eliminate Agrobacterium after inoculation; and 3 days for co-cultivation and 4 days for resting. A combination of all of these conditions resulted in establishing a high throughput transformation system. Over 500 T₀ plants were regenerated and these plants were assayed by transgene expression and some of them were also analyzed by Southern hybridization. T₁ plants were analyzed and transmission of transgenes to the T₁ generation was verified. This represents a highly reproducible and reliable system for genetic transformation of maize Hi-II.     

Haploid transformation in Brassica napus using an octopine-producing strain of Agrobacterium tumefaciens

Summary Microspore-derived embryos of Brassica napus were transformed using the disarmed octopine-producing LBA4404 strain of Agrobacterium tumefaciens containing the binary vector pBin19. Octopine-producing strains have previously been reported to be ineffective in transforming Brassica. Four actively growing yellow/ green sectors were selected from the embryos on 50 mg/l kanamycin and plants regenerated. Analysis for NPT-II activity in these young plants initially indicated no expression of the bacterial NPT-II gene. The plants were nevertheless grown to maturity, selfed and S₁ seed was collected. Three of the S₁ plants produced microspores which were from 4 to 20 times more tolerant to kanamycin than the original parent. Southern analysis revealed that one plant (EC-1) had a single site of insertion and the other two plants (EC-2 and EC-6) had two sites of insertion with sequence homology to the bacterial NPT-II gene. Microspores from the EC-2 and EC-6 transgenics produced embryos on approximately five times the level of kanamycin tolerated by microspores from untransformed plants, while the EC-1 transgenic produced microspores with more than 20 times the tolerance to kanamycin. Analysis of S₁ progeny of the EC-1 transgenic indicated that 100% of the progeny exhibited the trait through both Southern analysis and by expressing tolerance to kanamycin in microspore-derived embryos.     

Cultivar dependence of transformation rates in moth bean after co-cultivation of protoplasts with Agrobacterium tumefaciens

Summary A simplified protoplast regeneration system for Vigna aconitifolia was developed. A plating efficiency of 60% was obtained using mesophyll protoplasts from 10-day-old seedlings. By co-cultivation of protoplasts with Agrobacterium tumefaciens containing the Ti plasmid derivative pGV 38501103 neo kanamycin-resistant colonies were obtained; 23% of the transformed lines showed expression of the nonselected co-transferred nopaline synthase gene. Transformation was confirmed by Southern blot analysis using a nonradioactive detection system. The plant cultivar used was an important factor in determining transformation frequencies since one of the cultivars had an 85 fold higher transformation rate than the other.On deputation from: Bhabha Atomic Research Centre, Bombay, India, under the Indo-FRG Bilateral Programme     

Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules

We report on a novel transformation procedure for barley by Agrobacterium infection of in vitro cultured ovules. Ovules of the cultivar Golden Promise were isolated a few hours after pollination and infected with the Agrobacterium tumefaciens strain AGL0 carrying the binary vector pVec8-GFP. The vector harboured a hygromycin resistance gene and the green fluorescence protein (GFP) gene. GFP-expressing embryos were isolated from the ovules, regenerated to plants and investigated by Southern blot analysis. Transformation frequencies amounted to 3.1% with hygromycin selection and 0.8% without selection. Mendelian inheritance and stable expression of the GFP gene was confirmed in 18 independent lines over two generations. We conclude that the described technique allows for the rapid and direct generation of high quality transgenic plants.Communicated by W. Harwood     

Genetic transformation of NERICA,interspecific hybrid rice between <Emphasis Type="Italic">Oryza glaberrima</Emphasis> and <Emphasis Type="Italic">O. sativa</Emphasis>, mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin. The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and segregation of the transgenes were demonstrated by molecular and genetic analyses in the T₀ and T₁ generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the frequency of transformation was lower and sterility was more frequent.     

Transformation of vitis tissue by different strains of Agrobacterium tumefaciens containing the T-6b gene

Summary Stem pieces and leaf disks of Vitis spp. were cocultured with Agrobacterium tumefaciens strains carrying the UidA (ß-glucuronidase = GUS) gene. The transformation efficiency was highly increased by using a modified T-6b gene (a gene from pTiTm4) which interferes with normal growth and allows regeneration of normal Nicotiana rustica plants (Tinland 1990). The strains first tested on stem segments were subsequently tested in a leaf explant system. On leaves the transformation efficiency of the strains was much lower than with stems. Both the T-6b gene and the hsp 70-T-6b gene (a modified T-6b gene under the control of a heat shock promoter) allowed the initiation of GUS-positive buds.Abbreviations GUS ß-glucuronidase - BAP benzylaminopurine - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide     

Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA

We introduced a plant selection marker, nptII, to the left of border A in the Agrobacterium Ti plasmid pTiA6. Infection of tobacco leaf discs with the modified Agrobacterium strain gave rise to kanamycin-resistant calli which grew in a hormone-dependent manner. Southern hybridization analysis of DNA isolated from four transformants indicated initiation of DNA transfer at or near border A and absence of T-DNA sequences. These results demonstrate that DNA transfer events starting at a left border on a native Ti plasmid and moving away from the T-DNA region occur and that they can be detected by designing a suitable selection strategy.     

Deviating T-DNA transfer fromAgrobacterium tumefaciens to plants

We analyzed 29 T-DNA inserts in transgenicArabidopsis thaliana plants for the junction of the right border sequences and the flanking plant DNA. DNA sequencing showed that in most lines the right border sequences transferred had been preserved during integration, corroborating literature data. Surprisingly, in four independent transgenic lines a complete right border repeat was present followed by binary vector sequences. Cloning of two of these T-DNA inserts by plasmid rescue showed that in these lines the transferred DNA consisted of the complete binary vector sequences in addition to the T-region. On the basis of the structure of the transferred DNA we propose that in these lines T-DNA transfer started at the left-border repeat, continued through the vector part, passed the right border repeat, and ended only after reaching again this left-border repeat.     

Effect of age and type of tissue on genetic transformation of Lotus corniculatus by Agrobacterium tumefaciens

Various experiments of Lotus corniculatus cv. Leo were infected with Agrobacterium tumefaciens strains C58 (wild-type) and GV3101 (control). A maximum of 83 per cent of cotyledons excised from 7 day old seedlings and 63 per cent of leaves excised from seedlings grown in vitro formed galls in culture. The genotype of the seedling had an effect on the response.     

Transformation of Solanum brevidens using Agrobacterium tumefaciens

Leaf pieces of in vitro-cultured plantlets of the wild potato species Solanum brevidens Phil. were cocultivated with Agrobacterium tumefaciens that contained nptII and uidA genes on the disarmed plasmid pBI121. Independent transgenic shoots were regenerated from solidified and liquid medium that contained 50 mg l^–1 kanamycin. Two Agrobacterium strains were investigated for transformation efficiency. GV2260, which contained p35SGUSINT, resulted in a 11% transformation frequency, compared with 1% using LBA4404. Transformation rates were 7% in liquid culture and 3% on solidified medium. All kanamycinresistant, putatively transformed plantlets were confirmed positive by histochemical GUS assays. GUS activity in 22 independently transformed plants was quantified by fluorometric assay. Southern analysis of randomly selected transgenic plants showed that each transgenic plant contained at least one copy of the uidA gene.Abbreviations GUS ß-glucuronidase - MS Murashige-Skoog medium - BA 6-benzylaminopurine - 2ip 6-(, -dimethylallylamino)purine - IAA indole-3-acetic acid - GA₃ gibberellic acid - npt II neomycin phosphotransferase II - NOS nopaline synthase - MUG 4-methyl umbelliferyl glucuronide - MU 7-hydroxy-4-methylcoumarin - X-gluc 5-bromo-4-chloro-3-indolyl ß-D-glucuronic acid     

Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens

A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T₀) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T₀ and T₁ generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved.     

Identifi cation of antibiotics that are effective 2 in eliminatingAgrobacterium tumefaciens

An assay was performed to identify the antibiotics that are most effective againstAgrobacterium tumefaciens strains EHA101 and LBA4404, and to determine if these antibiotics inhibited tobacco callus and shoot formation. We tested ten antibiotics: cefotaxime, carbenecillin, erythromycin, spectinomycin, polymixin B, chloramphenicol, methicillin, Augmentin 500, Augmentin 250, and moxalactam. The effectiveness of each antibiotic against the two strains was determined by measuring the zones of inhibition of bacterial growth in a disk-diffusion assay. The five antibiotics that generated the largest zones of inhibition for each strain were assayed to determine their effects on callus formation. Cefotaxime was the most active antibiotic tested against strain LBA4404, and moxalactam was the most effective antibiotic against EHA101. Both cefotaxime and moxalactam had little or no effect on callus development.     

Structural genesvirC1 andvirC2 of the host range determiningvirC operon are determinants of virulence inAgrobacterium tumefaciens

The host range determiningvir C operon ofAgrobacterium tumefaciens is known to consist of two open rea’ding frames designatedvirC1 andvirC2. Earlier work that employed insertional mutations invirC1 andvirC2 established the role of thevirC2 component in the determination of virulence. In this work a plasmid with an internal deletion invirCl was constructed. This deletion derivative restored virulence to bacteria carrying a mutation in thevirC2 region but not to bacteria carrying avirC1 mutation. This evidence establishes that bothvirC1 andvirC2 are required for efficient host plant transformation byAgrobacterium tumefaciens.