Journal of Experimental Botany, Vol. 51, No. 353, pp. 1961-1968,
December 2000
© 2000 Oxford University Press
Original Papers |
Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones
Laboratoire Androgenèse et Biotechnologie, Université de Picardie Jules Verne, 33 rue Saint-Leu, 80039 Amiens cedex 01, France
Received 21 December 1999; Accepted 11 July 2000
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Abstract |
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Many plant species and/or genotypes are highly recalcitrant to Agrobacterium-mediated genetic transformation, and yet little is known about this phenomenon. Using several Arabidopsis genotypes/ecotypes, the results of this study indicated that phytohormone pretreatment could overcome this recalcitrance by increasing the transformation rate in the known recalcitrant genotypes. Transient expression of a T-DNA encoded ß-glucuronidase (GUS) gene and stable kanamycin resistance were obtained for the ten Arabidopsis genotypes tested as well as for the mutant uvh1 (up to 69% of petioles with blue spots and up to 42% resistant calli). Cultivation of Arabidopsis tissues on phytohormones for 2–8 d before co-cultivation with Agrobacterium tumefaciens significantly increased transient GUS gene expression by 2–11-fold and stable T-DNA integration with petiole explants. Different Arabidopsis ecotypes revealed differences in their susceptibility to Agrobacterium-mediated transformation and in their type of reaction to pre-cultivation (three types of reactions were defined by gathering ecotypes into three groups). The Arabidopsis uvh1 mutant described as defective in a DNA repair system showed slightly lower competence to transformation than did its progenitor Colombia. This reduced transformation competence, however, could be overcome by 4-d pre-culture with phytohormones. The importance of pre-cultivation with phytohormones for genetic transformation is discussed.
Key words: Agrobacterium tumefaciens, Arabidopsis thaliana ecotypes, transformation, uvh1 mutant, phytohormones.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Agrobacterium-mediated transformation is a well established method which is most widely used for production of transgenic plants in numerous dicots, and even in some monocot plant species (De Block, 1994
). Success in obtaining transgenic plants, however, depends upon the susceptibility of target cells to Agrobacterium and their subsequent ability to regenerate well-developed plants. In contrast to the progress made in the understanding of molecular events occurring within Agrobacterium prior to and during transformation, little is known about the corresponding molecular events inside the plant, for example, the basis of host cell competence and early cellular events during transformation (Potrykus, 1990; Zupan and Zambryski, 1995, 1997). In addition, as severe recalcitrance to transformation is often observed for certain genotypes, the absence of genotype-independent transformation methods have made practical applications of this system not easily adaptable for some crop species. Competence for transformation may either be absent or low in recalcitrant explants (or genotypes); however, it can be enhanced by phytohormone treatments (Valvekens et al., 1988; Sangwan et al., 1991, 1992; Geier and Sangwan, 1996; Villemont et al., 1997). The inoculation of hormone-resistant mutants have shown that Agrobacterium-mediated transformation seems to be dependent on the hormone sensitivity of the plants (Lincoln et al., 1992). An explant becomes susceptible to Agrobacterium when it is wounded or when pre-cultured on medium containing phytohormones (Potrykus, 1990; Valvekens et al., 1988; Sangwan et al., 1991, 1992).
Transformation of cells needs success in all the successive steps of T-DNA entry into the host cell and transgene expression. The molecular mechanisms of bacterial induction of virulence from activation of the vir genes (Winans, 1992) to production of T-DNA is relatively well documented (Kado, 1991; Zupan and Zambryski, 1995). However, little is known about plant host factors involved in transformation and the gene expression process. Only a few mutations affecting T-DNA transfer or integration have been identified in plants (Nam et al., 1997, 1999; Mysore et al., 2000). Previously, it was reported that the UV-hypersensitive mutant (uvh1) was defective in T-DNA integration (Sonti et al., 1995). This result suggested that a host gene is required for T-DNA integration. However, results from this laboratory and from other authors (Nam et al., 1998; Preuss et al., 1999) have convincingly shown that this mutant is highly susceptible to transformation.
Using well-defined tissue culture conditions for efficient transformation, a system was established that allowed the observation of the key role of phytohormones during transformation (Sangwan et al., 1992; Villemont et al., 1997). The main objective of this study was to identify tissue culture conditions, including phytohormone treatments, that were required to overcome the strong plant genotypic effect which is frequently observed in Agrobacterium tumefaciens-mediated transformation protocols.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant material
Seeds of various Arabidopsis thaliana ecotypes, and the uvh1 mutant were provided by the Nottingham Arabidopsis Stock Centre (Nottingham, UK). Seeds of Arabidopsis thaliana (L.) Heynh ecotype C24 (provided by M Van Lijsbettens, University of Ghent, Belgium), were surface-sterilized with 2% calcium hypochlorite for 20 min, and rinsed three to four times with sterile distilled water. Seeds were germinated in Petri dishes containing MS basal medium (Murashige and Skoog, 1962
), and were grown further under low light (20 µE m–2 s-1; 16/8 h light/dark period) at 24 °C. Roots, leaves and petioles were isolated from 3-week-old plants, before flower stem development (Fig. 1A). Table 1 presents the different genotypes/ ecotypes used; some of these are frequently used in transformation experiments.
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Bacterial strain
An Agrobacterium tumefaciens strain derived from C58C1Rif harbouring the binary plasmid pGSGluc1 (obtained from J Leemans, PGS, Ghent, Belgium) was used as described previously (Sangwan et al., 1991). Between the T-DNA borders, this plasmid harbours the nptII gene encoding neomycin phosphotransferase II, under the control of the TR1' promoter, and the GUS gene under the control of the TR2' promoter. The TR promoter is a bi-directional dual promoter from the T-DNA of an octopine-type Ti plasmid (Velten and Schell, 1985). Bacteria for transformation experiments were cultivated on Luria Broth medium supplemented with antibiotics (streptomycin 300 mg l-1, spectinomycin 100 mg l-1, carbenicillin 100 mg l-1, rifampicin 100 mg l-1) and stored at 4 °C. Bacterial suspensions used in transformation experiments were derived from overnight cultivation of one colony in 10 ml Luria Broth liquid medium at 28 °C on a rotary shaker (210 rpm).
Culture media
Petiole explants were cultured on MS-modified supplemented medium, with 20 g l-1 sucrose and 8 g l-1 Difco-Bacto-Agar. 0.25 g l-1 MES (2-N-morpholino) ethanesulphonic acid) was added to the medium, and the pH was adjusted to 5.7 with KOH. Phytohormones and antibiotics were filter-sterilized and added after autoclaving (120 °C for 20 min.): 2,4-dichlorophenoxyacetic acid (2,4-D), N6-furfurylaminopurine (kinetin), 6-benzylaminopurine (BA), 
-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), N6-(2-isopentenyl) adenine (2iP), and cefotaxime, kanamycin, streptomycin, spectinomycin, carbenicillin, and rifampicin.
Transformation procedure
Petioles were isolated from mature plants and immediately cultured on either liquid or solid MS medium. Pre-cultivation used MS medium containing 1 mg l-1 2,4-D, and 0.5 mg l-1 kinetin. Co-cultivation with bacteria was performed in 90 mm Petri dishes containing 20 ml MS liquid medium at room temperature. Approximately 2 ml bacterial suspension, containing 106–108 cells ml-1, was co-cultured with explants for 20 min in each Petri dish. Petioles were dried on a sterile filter paper in order to remove excessive bacterial suspension. Explants were co-cultivated for 2 d on a solid MS medium containing phytohormones. After this 2 d infection period, petioles were then washed with MS medium supplemented with cefotaxime to prevent further bacterial growth, blot-dried, and transferred to solid MS medium containing 1 mg l-1 2,4-D, 0.5 mg l-1 kinetin and 250 mg l-1 cefotaxime for 5 d to induce callus formation. For subsequent selection of transformed cells, tissues were transferred to MS medium containing 1 mg l-1 BA, 0.5 mg l-1 NAA and 100 mg l-1 kanamycin. Incubations in Petri dishes were under similar light conditions to ones used for seed germination.
Regeneration
All calli obtained were further cultivated to establish stable genetic transformation. Green shoots differentiated after 3–4 weeks and were transferred to root-inducing MS medium supplemented with 1 mg l-1 IAA and 50 mg l-1 kanamycin. Before flower stem formation, regenerated plants were transferred to the greenhouse.
Histological GUS assay
To score transformation frequencies, histochemical assays to reveal GUS expression in plant cells were performed and the number of petioles containing transformed cells was scored. ß-Glucuronidase enzyme activity was detected using the substrate 5-bromo-4-chloro-3-indolyl glucuronid (X-Gluc, Duchefa) (as described by Jefferson, 1987). This assay gives a blue coloration in the presence of enzyme. Explants were tested by incubating overnight with the substrate (1% v/v in phosphate buffer, pH 7.4, supplemented with Triton X-100 0.01%) at 37 °C, and then transferred to ethanol at 70 °C. This assay gives qualitative data scored as the percentage of explants with blue spots, and situates the transformed cells.
Enzymatic GUS assay
For quantitative data on transformation, ß-glucuronidase activity was measured as previously described (Jefferson, 1987). For each sample protein was extracted separately. The substrate used was 4-methylumbelliferyl-ß-D-glucuronid (MUG, Sigma), which gives a fluorescent product, 4-methylumbellferone (MU). A spectrofluorometer (Hitachi U-200) calibrated with known quantities of MU was used to quantify the amount of MU formed for each sample.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The main steps of transformation were pre-cultivation of tissues with phytohormones, co-cultivation with Agrobacterium, selection of calli, and plant regeneration on selective media. The effect of different parameters in the pre-cultivation phase have been studied for the early transformation rates as well as for the later events, i.e. callus induction and shoot regeneration on a selective media.
Effect of pre-cultivation on early expression of transferred genes
The early expression of transferred genes was measured just after the 2 d of co-cultivation with Agrobacterium. Explants were simply washed once in liquid medium and blot-dried before GUS assay. Control explants (pre-cultivated only, without contact with Agrobacterium) never showed endogenous GUS activity. Except for the ecotype Columbia, Arabidopsis thaliana ecotypes used in theses experiments produce relatively high transformation rates (Table 2). Despite the low number of genotypes tested, a genotypic influence on transformation rates could be observed. Qualitative and quantitative data demonstrated that the transformation rates of petioles increased in all genotypes within a period of 8 d of pre-cultivation.
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Table 2
shows GUS expression (Fig. 1E, F) in co-cultivated petioles which were pre-cultivated with phytohormones for different periods of time. Quantitative data on ß-glucuronidase activity for petioles in the same conditions gave similar results (data not shown). Both measurements suggested that a contact period with phytohormones before co-cultivation increases transformation frequencies of the petiole to 30–60%. Without pre-cultivation, only 5% to 15 or 20% of the co-cultured petioles show GUS expression. An optimal pre-cultivation time can be defined for each genotype studied.
Genotypes were grouped into three categories according to their response to pre-cultivation period with respect to early transformation frequency. Groups are separated by lines in Table 2. The genotypes Landsberg erecta responds after 2 d, Nossen and Columbia respond after 4 d and these three genotypes responded slowly to prolonged pre-cultivation and reached higher transformation rates only after 6–8 d of pre-cultivation. However, the genotypes Wassilewkija, Tossa de mar and Bensheim responded faster and reached their highest transformation rates after pre-cultivation for 4, 6 and 8 d. The third group, consisting of Rchew and C24, seemed to have 4 d optimal pre-cultivation period for transformation. Longer pre-cultivation of this group before co-cultivation led to lower frequencies of cultured petioles with GUS-expression.
Comparable studies with roots and leaves did not provide additional information. Roots, cultivated on medium supplemented with 2iP and IAA (Valvekens et al., 1988), showed very high transformation rates (nearly 80%) even without any pre-cultivation treatment. Differences between genotypes in the percentage of roots with blue spots were not statistically significant, although roots co-cultivated with Agrobacterium after a few days of pre-cultivation seem to produce more spots in this histological GUS assay. (Table 3). Leaves generally produced low transformation frequencies. Nevertheless, in general less than 10% of the leaves without pre-culture showed blue spots, while pre-cultivation on BA- and NAA-supplemented medium could increase the transformation frequencies to 25%. All these results suggest an increased efficiency in transformation for pre-cultivated explants. For further study of acquisition of capacity to be transformed with Agrobacterium tumefaciens, petiole segments, which are the most informative material, were chosen.
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Stable transformation: callus induction
Stable transformation frequencies were scored with petioles left on selective medium supplemented with kanamycin. After 5 d on the callus induction medium used for pre-cultivation and co-cultivation, petioles were transferred to shoot induction medium (Fig. 1B) for regeneration. Callus generally appeared at the cut ends of the petiole (Fig. 1C), however, it also formed from the middle of the petioles when wounded during preparation. Transformed cells were observed at the adaxial side as well as the abaxial side of petioles (Fig. 1E). Stable transformation rates, measured as the percentage of petioles with green calli after 3 weeks on selective medium, are shown in Fig. 2. Callus formation frequencies on the selective media increased with prolonged pre-cultivation time. In most of the genotypes, the highest frequencies of callus formation was obtained with the longest time of phytohormone pre-treatment (8 d). Some differences, however, appeared between these data and those obtained just after co-cultivation (Table 2), but the genotypes have been separated into the same three groups. The transformation rates from the genotypes WS-0, WS-2, C24, and Rschew at 8 d pre-cultivation period seemed to indicate that their response to phytohormone pretreatment differed from the response indicated by the results in Table 2. Early expression of the GUS gene gave a measure of both transient and stable transformation, and transformation events occurring at the end of the infection period could not be taken into account. Late expression of the nptII gene measured only stable transformation after a long culture period. Transformation rates obtained by these two different methods may differ not only in intensity (kanamycin resistance gave a higher percentage of positive explants than GUS expression), but also in their development with pre-cultivation duration.
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To confirm the results from selective media, shoots were excised (Fig. 1D
) and grown on root induction media supplemented with kanamycin. Plant leaves were tested using the GUS assay to assess their transformed nature. All the plants from one experiment with 50 tested tissues (roots or leaves) for each pre-cultivation time and genotype were positive. In particular, mature transformed plants were obtained from the uvh1 mutant (26% of transformed shoots from pre-cultivated root explants were regenerated on kanamycin medium). Genotypes C24, Rschew, Bensheim, Nossen, and Wassilewskija (WS-0) showed genotypic variation in the percentage of shoot regeneration, 32%, 32%, 9%, and 74% respectively, from pre-cultivated root explants on kanamycin medium.
Transformation of the uvhl mutant
The uvh1 mutant, which is hypersensitive to ultraviolet light and ionizing radiation, was described earlier (Harlow et al., 1994). It is derived from the Columbia ecotype and its sensitivity is inherited as a single recessive Mendelian trait, probably located on chromosome V (Jenkins et al., 1995). It was suggested that the uvh1 mutant is not defective in a UV protection system (Harlow et al., 1994), however, it is defective in protection from several types of DNA damage. The mutant had been described earlier as non-transformable by Agrobacterium (Sonti et al., 1995). Recently, however, the uvh1 mutant has been transformed successfully with Agrobacterium (Nam et al., 1998; Preuss et al., 1999) using both roots in vitro and flower bud inoculation in vivo. The intention of the authors was to use this mutant for a comparison with the genotypes as a negative control. In fact, it was found that the uvh1 mutant could be transformed and transgenic shoots obtained in vitro, indicating that stable transformation is possible with this genotype with petioles, roots and leaves. The uvh1 mutant, described as defective in a DNA repair system, could help in understanding factors for genetic transformation competence. Concerning its transformation frequency, the mutant is classified with the two genotypes C24 and Rschew (Table 2; Fig. 2). Four-day pre-cultivation gave the highest transformation frequency: 69% of GUS positive petioles and 5.8 nmol MU min-1 µg-1 protein. After 3 weeks on the selective medium, 46% petioles with 4 d pre-culture treatment showed green calli. Isolated roots from the uvh1 mutant could be transformed after 4 d and 6 d pre-culture treatment, resulting in 26% of roots regenerating transgenic shoots.
Acquisition of susceptibility to Agrobacterium transformation
All the experiments in this study demonstrated an increase in Agrobacterium transformation frequencies, expressed as transient and stable expression of marker genes, with pre-cultivation on medium containing phytohormones, which is in agreement with previous reports (Sangwan et al., 1991, 1992). Other studies with younger tissues (Akama et al., 1992) also indicated differences in susceptibility of Arabidopsis genotypes to Agrobacterium. The susceptibility of Arabidopsis genotypes to crown gall disease has been studied with root segments (Nam et al., 1997). One ecotype (UE1) was somewhat integration-deficient and others (B1-1 and Petergof) were transformation-deficient because of lack of bacterial attachment. Another hypothesis can now be suggested based on the different reactions of the ecotypes to the duration of pre-cultivation before transformation. Here, it is shown that the genotype effect could be overcome by increasing the duration of the phytohormone treatment. The response of Arabidopsis to phytohormones is important for transformation, and may have an effect at various steps of the transformation process, including T-DNA integration. Phytohormone treatment activates cell division and dedifferentiation in many tissues. The first dividing cells were visible in petioles after 2 d pre-cultivation and nearly all the explants showed actively dividing and dedifferentiated cells after the 4 d pre-cultivation or more (data not shown). It is known that the phase of the cell cycle influences stable transformation (Villemont et al., 1997), and that many other modifications occur in phytohormone-activated cells. The formation of new and thin cell walls and changes in microtubule organization (Sangwan et al., 1992) probably have effects on transformation efficiency and, for example, may have influence in specific attachment capacity to Agrobacterium. Genotypic effect on Agrobacterium-mediated transformation of Arabidopsis may be due to the differences in the cells response after phytohormone treatment. A 4 d pre-cultivation treatment induces cell division, and could overcome a low sensitivity to Agrobacterium. Modifications in cells and division capacity by pre-culture and its effect on Agrobacterium tumefaciens transformation should be further investigated.
Moreover, results concerning the uvh1 mutant suggest that an intact DNA repair system is not apparently necessary for transformation. Clearly the uvh1 mutant is not resistant to Agrobacterium-mediated transformation. However, the uvh1 mutant gives different transformation rates from its progenitor Colombia. The single locus mutation conferring sensitivity to ionizing radiation seems to affect competence to Agrobacterium tumefaciens transformation only slightly. Pre-treatment with phytohormones can apparently overcome this reduced transformation susceptibility in this mutant in the same way that it improves transformation in the other low responsive Arabidopsis genotypes. Further studies of this mutant and other mutants with changes in Agrobacterium tumefaciens transformation susceptibility or in the DNA repair system could be a very interesting strategy to follow. Such mutants have already been isolated (Nam et al., 1999). The rat5 mutant for example, described as deficient in root transformation, is affected in histone H2A gene dosage (Mysore et al., 2000), suggesting that histone H2A is involved in Agrobacterium-mediated root transformation. Comparison of the various Arabidopsis genotypes and these new mutants may provide useful tools for studying the Agrobacterium transformation process in plant cells and the nature of recalcitrance to Agrobacterium of some genotypes.
These data offer a substantially simplified protocol for overcoming genotypic effects during Arabidopsis transformation. This approach may assist in developing transformation protocols of recalcitrant and economically important genotypes in other plant species.
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Acknowledgments |
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Financial support was provided by the Biopôle Végétal, of Picardie, Amiens, France. We thank Miss Monsera Estopa for her participation in some experiments, and Professor Sven B Andersen for critical reading of the manuscript.
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Notes |
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1 To whom correspondence should be addressed. Fax: +33 3 22 827612. E-mail: Brigitte.Sangwan{at}sc.u\|[hyphen]\|picardie.fr
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