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.     

Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens

High frequency transformation of Arabidopsis thaliana leaf explants has been obtained using a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase gene (NPT II) as a selectable marker. The rate of transformation ranged from 55 to 63 percent when acetosyringone (AS), a natural wound response molecule, was added to an Agrobacterium tumefaciens culture prior to incubation with leaf segments. Without acetosyringone, the transformation rate was approximately 2 to 3 percent. Calli resistant to G418 were regenerated into mature flowering plants in the presence of 10 g/ml G418. Southern analysis and neomycin phosphotransferase assays confirmed the insertion and expression of the NPT II gene in regenerated Arabidopsis plants.     

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     

Agrobacterium tumefaciens-mediated transformation of recalcitrant crops

The most widely used technique for the introduction of new genetic information into plant cells is based on the natural gene transfer capacity ofAgrobacterium tumefaciens. Currently, this technique is routinely applicable in just a few model species, like tobacco and petunia. Thus far, the numerous efforts to apply the technique to crop species have had limited success. In this review, an attempt is made to survey all the research experience onAgrobacterium tumefaciens-mediated transformation of recalcitrant crops and to highlight the problems generally encountered. The main difficulty appears to be directing the gene transfer towards those plant cells that are amenable to regeneration. The various ways to reduce stress during the transformation and regeneration process are often beneficial. The influence of the developmental stage of the plant material and the host range of theAgrobacterium strain depends largely on the plant species used, which hampers the formulation of common procedures. However, some general guidelines for the development of a transformation protocol are discussed.     

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.     

Elucidation of the O-chain structure from the lipopolysaccharide of Agrobacterium tumefaciens strain C58

A linear homopolysaccharide built of 3-alpha-L-6dTalp residues, randomly acetylated at position C-4, is described for the O-specific polysaccharide of Agrobacterium tumefaciens strain C58. This structure, determined by spectroscopical and chemical methods, is strictly correlated to that of Rhizobium loti strain NZP2213, which differs for the degree and the position of O-acetylation.     

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.     

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     

A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes

Summary Binary Ti plasmid vector systems consist of two plasmids in Agrobacterium, where one plasmid contains the DNA that can be transferred to plant cells and the other contains the virulence (vir) genes which are necessary for the DNA transfer but are not themselves stably transferred. We have constructed two nononcogenic vectors (pARC4 and pARC8) based on the binary Ti plasmid system of Agrobacterium tumefaciens for plant transformation. Each vector contains the left and right termini sequences from pTiT37. These sequences, which determine the extent of DNA transferred to plant cells, flank unique restriction enzyme sites and a marker gene that functions in the plant (nopaline synthase in pARC4 or neomycin phosphotransferase in pARC8). After construction in vitro, the vectors can be conjugatively transferred from E. coli to any of several Agrobacterium strains containing vir genes. Using A. rhizogenes strain A4 containing the resident Ri plasmid plus a vector with the nopaline synthase marker, we found that up to 50% of the hairy roots resulting from the infection of alfalfa or tomato synthesized nopaline. Thus, vector DNA encoding an unselected marker was frequently co-transferred with Ri plasmid DNA to an alfalfa or a tomato cell. In contrast, the frequency of co-transfer to soybean cells was difficult to estimate because we encountered a high background of non-transformed roots using this species. Up to five copies of the vector DNA between the termini sequences were faithfully transferred and maintained in most cases suggesting that the termini sequences and the vir genes from the Ri and Ti plasmids are functionally equivalent.     

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     

High-frequency transformation ofArabidopsis thaliana byAgrobacterium tumefaciens

Incorporation of 5 mg/L silver thiosulphate into media for seed germination and callus induction, as used in the transformation protocol originally described by Valvekens et al. (1988), was found to increase the frequency of regeneration of transformants ofArabidopsis thaliana ecotypes C24 and Landsbergerecta by at least 10- to 100-fold. Other factors, such as density of the bacterial inoculation culture, density of the root explants and duration of bacteria-plant cocultivation period, were also found to influence the efficiency of recovery of transformants.     

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     

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.     

Presence of a plant cell wall is not required for transformation of Nicotiana by Agrobacterium tumefaciens

Agrobacterium has been used to transform zero to six-day-old cell wall nonregenerating (CWNR) and cell wall regenerating (CWR) leaf protoplasts of tobacco. Transformed cells were selected by phoytohormone autotrophic growth and were verified by detection of the presence of lysopine dehydrogenase. Transformation frequencies in CWNR protoplasts were at least as high as those in CWR protoplasts, indicating that a plant cell wall is not required for the process of crown gall tumorigenesis. Transformation frequencies were highest in two-day-old protoplasts. This age coincides with the onset of DNA synthesis and the first mitosis within the cell populations. We suggest that the initiation of cell cycle activity may be important for the transformation process.     

Genetic transformation of prickly-pear cactus (Opuntia ficus-indica) by Agrobacterium tumefaciens

A system for genetic transformation of an elite prickly pear cactus (Opuntia ficus-indica L., cultivar Villa Nueva) by Agrobacterium tumefaciens was developed. Beginning with direct bacterial infection by using a hypodermic syringe to the meristematic tissue termed areoles, transgenic plants were obtained by selection with 100 mg l⁻¹ kanamycin. Transient and stable GUS activities were monitored on kanamycin-resistant shoots and regenerated plants, respectively. Genetic transformation of regenerated plants growing under selection was demonstrated by PCR and Southern blot analysis; transgene copy number in the genome of transgenic plants ranged from two to six, while the transformation frequency obtained by the system reported here was of 3.2%. This method may be useful for routine transformation and introduction of several important genes in prickly pear cactus.     

Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots

Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes.     

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.     

Genetic transformation of Populus nigra by Agrobacterium tumefaciens

Two clones of Populus nigra L. were tested in vivo and in vitro for their susceptibility to three different Agrobacterium tumefaciens wild-type strains evaluating number and size of resulting calluses. Strain C58 proved to be the most virulent.Various parameters affecting Agrobacterium-mediated transformation of P. nigra clones were further analyzed using ß-glucuronidase gene transient expression. The clone Jean Pourtet proved to be more susceptible than the clone San Giorgio. A. tumefaciens strain A281 pKIWI105 proved to be the most virulent. The optimal procedure involved dipping of leaf discs into a bacterial suspension (7×10⁸ cells/ml) for 20 min, followed by a 48 h co-cultivation period on semi-solid regeneration medium.Leaf explants were co-cultivated with two disarmed A. tumefaciens strains. Plantlets of San Giorgio were regenerated, tested for ß-glucuronidase activity and rooted on selective medium containing kanamycin. Polymerase chain reaction analysis and Southern blot hybridization confirmed the integration of the neomycin phosphotransferase II gene into the poplar genome.Abbreviations BAP 6-benzyl-aminopurine - CaMV Cauliflower Mosaic Virus - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS and gus ß-glucuronidase - hpt hygromycin phosphotransferase - IBA indole-3-butyric acid - KIN kinetin - LB Luria Bertani - MS Murashige and Skoog - NAA ßnaphthaleneacetic acid - NOS Nopaline synthase - NPTII and nptII neomycin phosphotransferase II - PCR Polymerase chain reaction - PVC poly-vinyl-cloride - SDS sodium dodecyl sulfate - SSC sodium cloride-sodium citrate - Tris tris(hydroxymethyl)amino-methane - WPM Woody Plant Medium