Plant Cell Reports
Plant Cell Reports (1991) 9:571-574
9 Springer-Verlag 1991
Direct gene transfer to protoplasts of Arabidopsis thaliana Hans Karesch, Roland Bilang, Ortrun Mittelsten Scheid, and Ingo Potrykus Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH), ETH-Zentrum, CH-8092 Zfirich, Switzerland Received October 8, 1990/Revised version received October 1, 1990 - Communicated by H. L6rz
ABSTRACT We performed a series of direct gene transfer experiments with protoplasts of Arabidopsis thaliana ecotype Z0rich. An average of more than 100 transformants were selected per 10s treated protoplasts. Stable transformation was confirmed by integration of the marker gene into high molecular weight DNA and by its genetic transmission to subsequent offspring generations.
Abbreviations: ATF: absolute transformation frequency; PEG: polyethyleneglycol; hpt : hygromycin phosphotransferase gene; CTAB: N-CetyI-N,N,N-trimethyl-ammonium bromide; MES: 2-(Nmorpholino)ethanesulfonic acid
INTRODUCTION Reliable and frequent regeneration of fertile plants from protoplasts is obligatory for transformation by the technique of direct gene transfer to plants. The availability of a culture system for Arabidopsis thaliana protoplasts (Damm and Willmitzer 1988; Karesch et al., this issue) encouraged us to perform transformation experiments with this species by adapting protocols developed for Nicetiana tabacum and Nicetiana plumbaginifolia (Paszkowski et al. 1984; Shillito et al. 1985; Negrutiu et al. 1987). Although transformation of Arabidopsis thaliana has been achieved by Agrobactenum tumefaciens infection of leaf discs (Lloyd et al. 1986; An et al. 1986; Sheikholeslam and Weeks 1987; Schmidt and Willmitzer 1988), germinating seeds (Feldmann and Marks 1987) or root explants (Valvekens et al. 1988), transformation by direct uptake of DNA may broaden the spectrum of experimental possibilities in this important plant model system (for review see Meyerowitz 1989), allowing to vary form and size of the exogenous DNA, to study transient gene expression or to perform large scale transformations towards mutation complementation or gene targeting. Here we report on transgenic Arabidopsis thaliana plants obtained Offprint requests to." I. Potrykus
from PEG mediated direct gene transfer to protoplasts of ecotype Z0rich. The experiments were done in parallel to work on ecotype Columbia which has been reported by Damm et al. 1989.
MATERIALS & METHODS Protoplast isolation and transformation. Protoplasts of Arabidopsis thaliana ecotype Z0rich were isolated as described (Karesch et al., Plant Cell Reports, this issue). After final washing in W5 (154 mM NaCI2, 125 mM CaCI=,5 mM KCI, 5raM glucose, pH 5.8) and pelleting they were suspended in mannitol-magnesium-solution (500 mM mannitol, 15 to 60 mM MgCI2,~0.1%MES, pH 5,6) at a density of 1.5 x 10S/ml.Five to 30 Izg plasmid pGL2 (Fig. 1) conferring hygromycin resistance (kindly provided by J. Paszkowski, our institute) and 30 ~g calf thymus DNA (Sigma, sheared to an average size of 510 kbp), both sterilized by EtOH-precipitation and dissolved in water at a concentration of 1.0 mg/ml, were added to 0.5 ml protoplast suspension in a 12 ml centrifuge tube. Control samples were either not treated with DNA or with calf thymus DNA alone. An equal volume of PEG-solution was added and mixed with the protoplasts by gentle agitation. The PEG-solution was prepared by dissolving 40 % (wN) polyethyleneglycol 4000 (Merck) in 400 mM mannitol, 100 mM Ca(NO~)2 and autoclaving. This sterilization method results in a significant drop of ca. 2 pH units, therefore the pH is adjusted to 8 prior to autoclaving. After 5 to 15 min incubation at room temperature the suspension was gradually diluted by addition of 9 x 1 ml W5 at one minute intervals. After sedimentation at 60 x g for 5 min the protoplasts were resuspended in 1 ml W5, followed by addition of 2 ml 400 mM mannitol-magnesium solution, pelleted once more and finally resuspended in 500 mM mannitol-magnesium at a density of 1.4 x 106/ml. Embedding and culture during the first days was essentially as described by Karesch et al. (this issue). Selection and regeneration. After 9 days the cultures were transferred to medium containing 25 mg/I hygromycin B (Calbiochem). The selection medium was replaced 8 times in weekly intervals and resistant calli were counted at the end of that period. Liberation of the colonies and regeneration of plants were as
572
~
BH1 0
",
BH1 1033
Hill 1277
hpt
Table 1: ATF obtained after transformation of 10~g of Hindlll linearized pGL2 per 0.7x 106protoplasts. Experi- Date ment (in 1989)
pGL2 (4458bp)
Fig. 1: Plasmid pGL2. Dark lines and boxes represent sequences of pDH51 (Pietrzak et aL 1986) with promoter and termination signals (T) of Cauliflower Mosaic Virus 35S transcript. Open box represents coding region ofthe hptgene (Gritz and Davies 1983), BHI=BamHf, Hill= Hindlll restriction sites.
described in the preceding paper, with the exception that all steps prior to root induction were done under selective conditions with 25 mg/I hygromycin. Analysis of plant genomic DNA. Genomic DNA from individual plants was obtained with a modified CTAB-extraction protocol (Murray and Thompson 1980). Freeze-dried plants were used as starting material. DNA yield (measured after RNase A treatment) was 200-600 y.g/g fresh weight. Endonuclease digestion, get electrophoresis and blotlJng on Hybond N membrane (Amersham) were done using standard techniques recommended by the suppliers. The radioactively labeled 1033 bp BamHI fragment of pGL2 (covering the ceding region of the hptgene, fig. 1) was used as a probe for hybridization. All filters were washed with high stringency. Resistance assay of progeny. Seeds harvested from self-pollinated transgenic plants were surface-sterilized by 10 rain incubation in a 7% calcium-hypochlorite solution containing a few drops of Tween 80, followed by extensive washing with sterile distilled water. Seeds were germinated in dim light on 0.5 MS-O (Karesch et al., this issue) containing 25 mg/I hygromycin.
RESULTS Selection system. The effect of hygromycin on protoplast-derived microcolonies was tested in concentrations of 0, 1, 2, 5, 10, 20 and 50 mg per liter culture medium. 20 mg/I was found to be reliable for total growth inhibition: the colonies turned brown and died after 5-10 days of selective growth conditions. The same concentration completely inhibited the development of seedlings after germination. A hygromycin concentration of 25 mg/I was therefore chosen for use in transformation experiments as well as in progeny analysis. Colony formation or growth of seedlings from non-transformed material was never observed under these selection conditions. Resistant colonies in transformed cultures were counted after 8 weeks (Fig. 3) prior to release from the embedding matrix. Transformation frequency. Eight out of 11 transformation experiments yielded more than 100 resistant colonies per one million protoplasts. The average absolute transformation frequency (ATF = ratio of transgenic calli to the number of initially plated protoplasts) from all experiments was calculated to be 1.2 x 104, with values ranging from
1 2 3 4 5 6 7 8 9 10 11
26.4. 09.5. 17.5. 26.5. 01.6. 15.6. 30;6. 07.7. 19.7. 25.8. 01.9.
Number of resistant colonies / 10~ protoplasts (2-4 replicas per experiment) 70, 72 122, 98 66, 40 40, 74, 46 114, 141 148, 157 153, 62, 149, 120 139, 229, 179, 188 283, 250 95, 213 133, 66
Average
71 110 53 53 128 153 121 184 267 154 100
50 to 260 colonies/106 protoplasts (Table 1). A relative transformation frequency describing the ratio between the number of colonies under selective and non-selective conditions could not be calculated due to the high plating density in our experiments. In general, it was found that the number of resistant colonies is correlated with t~e number of dividing protoplasts in the control cultures. This might be reflected by the variation of the ATF in experiments performed over a period of 6 months, showing a maximum in the summer months. A similar seasonal dependence was previously observed for the plating efficiency in protoplast cultures (Karesch, unpublished results). The transformation frequency was significantly influenced by the addition of carrier DNA: treatment with the plasmid pGL2 alone resulted in a 10 times lower ATF of only 105. The use of plasmid in its circular form decreased the ATE to 4075 % compared with the numbers obtained with plasmid DNA linearized outside the gene at the Hindlll site. Variation of the DNA amount in the range of 5-30 F.g linearized plasmid per 7 x 105 protoplasts did not change either ATF nor copy number of integrated sequences. The dependence of the transformation frequency on the concentration of magnesium ions described for Nicotiana tabacum (Negrutiu et al. 1987) was not found to be as obvious for Arabidopsis protoplasts: variation of the MgCI2-concentration in the mannitol-magneslum solution between 7.5 and 30 mM did not significantly alter the ATF. A decrease was only found with concentrations below and above this range. Regeneratlon of transformsnts. We seFected 211 hygromycinresistant calli randomly for further culture on solid medium. Their growth rate in the presence of the antibiotic was the same as for the wJldtype calli under non-selective conditions. The shoot formation frequency (percentage of calli regenerating at least one shoot) was also similar (44 %) compared to that of non-selected controls (46 %). In contrast, root growth is very sensitive to hygromycin even in transgenic shoots. Rooting was therefore performed without selection pressure. An example of a regenerated, transgenic plant is shown in Fig. 5. On average, ca. 500 seeds were obtained from plants kept in axenic conditions. Seed setting of plants transferred to soil was even better.
573 Fig. 2: Southern blot of ganomic DNA (5 p.g per lane) from regenerated plants, probed with the hpt coding region (BamHI fragment of pGL2) lane 1 + 2: wildtype of Arabidopsis thaliana ecotype Z0rich lane 3+4, 5+6, 7+8, 9+10, 11+12:5 different transformed plants lane 13: BamHI digested plasmid pGL2 lane 1,3,5,7,9,11: DNA not digested lane 2,4,6,8,10,12,13: DNA digested with BamHI
Molecular evidence for transformation. In order to confirm integration of the marker gene, genomic DNA of 20 single, resistant plants was subjected to Southern analysis. Hybridization with the coding region of the hpt gene was found in all resistant regenerants in high molecular weight DNA and with the expected 1033 bp fragment (see Fig. 1) in BamHI digests (Fig. 2). The strength of the hybridization signals suggests different copy numbers between 1 and 10. Some samples appear to have additional fragments of higher molecular weight with homology to the probe, indicating plasmid rearrangement prior to or during integration. (Fig. 4, lanes 10 and 12). Individual F1 progeny plants from these parental plant always showed identical hybridization patterns, suggesting tight linkage of the different hpt-sequences. Inheritance of the transgene. Genetic transmission of the integrated gene was investigated in the progeny of 28 regenerated plants obtained by self-pollination. Based on an average seed number of 57 per plant, numbers of resistant and sensitive seedlings were statistically analyzed by the X2 assay against a 3:1 segregation. The Fl-populations fall into 4 groups (Table 2). Plants of group 1 and 2 inherit the resistance marker according to the expected Mendelian segregation of a dominant character in the case of multiple, unlinked copies of the transgene (group 1) and single or multiple, but tightly linked copies (group 2, example shown in Fig. 4) respectively. Other plants had less than expected (group 3) or no resistant progeny (group 4). This is, however, not correlated with reduced seed setting or lower germination frequency. Seven seed populations derived from progeny of a group 2 plant segregated either 3:1 or 4:0, confirming that the introduced gene can be stably transmitted further to the F2 generation.
DISCUSSION Stable transformation of Arabidopsis thaliana ecotype ZOrich by PEG mediated direct gene transfer to protoplasts, followed by regeneration of fertile plants, has been confirmed by the expression of the resistant phenotype in regenerants, by transgene integration into
high molecular weight DNA and by genetic transmission of the marker gene to subsequent generations. The rate of transformation is as high as described for ecotype Columbia, using a similar protocol (Damm et al. 1989). This is in contrast to the poor response of other genotypes described in that report. The transformation procedure originally developed for tobacco protoplasts (Negrutiu et al., 1987) can therefore be regarded as a starting point for further application in different strains of Arabidopsis thaliana as soon as a protoplast regeneration system is available. Some parameters affecting transformation in other systems (Shillito et al. 1985; Negrutiu et al. 1985) appear to have a similar influence in the Arabidopsis system: the addition of carrier DNA, linearization of the plasmid DNA and use of a pH-stabilized PEG solution are recommended for maximal transformation frequency, whereas the amount of transforming plasmid DNA is not correlated with ATF. Nevertheless, there is a high variation of transformation efficiency (ATF 1.2 x 10.4 +/- 0.6 x 104) in independent experiments even under standardized conditions, suggesting that the source and the treatment of the pretoplasts rather than experimental conditions during the transformation procedure itself are the limiting factor. The influence of other parameters like the magnesium ion concentration may not be clearly visible as long as the overall plating efficiency cannot be increased. The selection system used to screen for transformants is highly reliable. Every analyzed clone surviving the described selective conditions proved to contain integrated hpt-homologous DNA, with a copy number range similar to that found in Nicotiana (Paszkowski et al. 1989). The lack of resistant seedlings in the progeny of some plants was therefore surprising and provoked DNA analysis of their FI plants grown under non-selective conditions. It was found that the hptgene is transmitted but not expressed in the next generation. Although the ratio of plants with a non-Mendelian inheritance of the resistant phenotype is remarkably high in our experiments, similar aberrant segregation ratios were found in other Arabidopsis transformation experiments (Damm et al. 1989; Feldmann et al. 1989; Valvekens et al. 1989). Further investigation of the hpt gene inactivation is in progress.
574
Fig. 3: Hygromycin resistant microcalli embedded in alginate beads. Squares = 1 mm 2
Fig. 4: Fl-seedlings of an untransformed plant (left) and a plant transformed with pGL2 (right) on medium containing 25 mg/I hygromycin B.
Fig. 5: Fertile transgenic plant
Table 2: Segregation of hygromycin resistance in the F1 genera-
tion 3roup
Acknowledgements
REFERENCES
An G, Watson BD, Chiang CC (1986) Plant Physiol.81:301-305 Datum B, Willmitzer L (1988) Mol.Gen.Genet.213:15-20 Datum B, Schmidt R, Willmitzer L (1989) Mol.Gen.Genet.217:6-12 Feldmann KA, Marks MD (1987) MoI.Gen.Genet. 208:1-9 Feldmann KA, Marks MD, Christianson ML, Quatrano RS (1989) Science 243:1351-1354 Gritz L, Davies J (1983) Gene 25:179-188 Karesch H, Bilang R, Potrykus I Plant Cell Rep., this issue Lloyd AM, Barnason AR, Rogers SG, Byrne MC, Fraley RT, Horsch RB (1986) Science 234:464-466 Meyerowitz EM (1989) Cell 56:263-269
Segregation (resistant: sensitive)
Germination No. of F1frequency populations (%)
1
83-100
>3 :1
29- 97
7
2
73- 81
3 :1
38-100
7
3
17- 47
Plant Cell Reports (1991) 9:571-574
9 Springer-Verlag 1991
Direct gene transfer to protoplasts of Arabidopsis thaliana Hans Karesch, Roland Bilang, Ortrun Mittelsten Scheid, and Ingo Potrykus Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH), ETH-Zentrum, CH-8092 Zfirich, Switzerland Received October 8, 1990/Revised version received October 1, 1990 - Communicated by H. L6rz
ABSTRACT We performed a series of direct gene transfer experiments with protoplasts of Arabidopsis thaliana ecotype Z0rich. An average of more than 100 transformants were selected per 10s treated protoplasts. Stable transformation was confirmed by integration of the marker gene into high molecular weight DNA and by its genetic transmission to subsequent offspring generations.
Abbreviations: ATF: absolute transformation frequency; PEG: polyethyleneglycol; hpt : hygromycin phosphotransferase gene; CTAB: N-CetyI-N,N,N-trimethyl-ammonium bromide; MES: 2-(Nmorpholino)ethanesulfonic acid
INTRODUCTION Reliable and frequent regeneration of fertile plants from protoplasts is obligatory for transformation by the technique of direct gene transfer to plants. The availability of a culture system for Arabidopsis thaliana protoplasts (Damm and Willmitzer 1988; Karesch et al., this issue) encouraged us to perform transformation experiments with this species by adapting protocols developed for Nicetiana tabacum and Nicetiana plumbaginifolia (Paszkowski et al. 1984; Shillito et al. 1985; Negrutiu et al. 1987). Although transformation of Arabidopsis thaliana has been achieved by Agrobactenum tumefaciens infection of leaf discs (Lloyd et al. 1986; An et al. 1986; Sheikholeslam and Weeks 1987; Schmidt and Willmitzer 1988), germinating seeds (Feldmann and Marks 1987) or root explants (Valvekens et al. 1988), transformation by direct uptake of DNA may broaden the spectrum of experimental possibilities in this important plant model system (for review see Meyerowitz 1989), allowing to vary form and size of the exogenous DNA, to study transient gene expression or to perform large scale transformations towards mutation complementation or gene targeting. Here we report on transgenic Arabidopsis thaliana plants obtained Offprint requests to." I. Potrykus
from PEG mediated direct gene transfer to protoplasts of ecotype Z0rich. The experiments were done in parallel to work on ecotype Columbia which has been reported by Damm et al. 1989.
MATERIALS & METHODS Protoplast isolation and transformation. Protoplasts of Arabidopsis thaliana ecotype Z0rich were isolated as described (Karesch et al., Plant Cell Reports, this issue). After final washing in W5 (154 mM NaCI2, 125 mM CaCI=,5 mM KCI, 5raM glucose, pH 5.8) and pelleting they were suspended in mannitol-magnesium-solution (500 mM mannitol, 15 to 60 mM MgCI2,~0.1%MES, pH 5,6) at a density of 1.5 x 10S/ml.Five to 30 Izg plasmid pGL2 (Fig. 1) conferring hygromycin resistance (kindly provided by J. Paszkowski, our institute) and 30 ~g calf thymus DNA (Sigma, sheared to an average size of 510 kbp), both sterilized by EtOH-precipitation and dissolved in water at a concentration of 1.0 mg/ml, were added to 0.5 ml protoplast suspension in a 12 ml centrifuge tube. Control samples were either not treated with DNA or with calf thymus DNA alone. An equal volume of PEG-solution was added and mixed with the protoplasts by gentle agitation. The PEG-solution was prepared by dissolving 40 % (wN) polyethyleneglycol 4000 (Merck) in 400 mM mannitol, 100 mM Ca(NO~)2 and autoclaving. This sterilization method results in a significant drop of ca. 2 pH units, therefore the pH is adjusted to 8 prior to autoclaving. After 5 to 15 min incubation at room temperature the suspension was gradually diluted by addition of 9 x 1 ml W5 at one minute intervals. After sedimentation at 60 x g for 5 min the protoplasts were resuspended in 1 ml W5, followed by addition of 2 ml 400 mM mannitol-magnesium solution, pelleted once more and finally resuspended in 500 mM mannitol-magnesium at a density of 1.4 x 106/ml. Embedding and culture during the first days was essentially as described by Karesch et al. (this issue). Selection and regeneration. After 9 days the cultures were transferred to medium containing 25 mg/I hygromycin B (Calbiochem). The selection medium was replaced 8 times in weekly intervals and resistant calli were counted at the end of that period. Liberation of the colonies and regeneration of plants were as
572
~
BH1 0
",
BH1 1033
Hill 1277
hpt
Table 1: ATF obtained after transformation of 10~g of Hindlll linearized pGL2 per 0.7x 106protoplasts. Experi- Date ment (in 1989)
pGL2 (4458bp)
Fig. 1: Plasmid pGL2. Dark lines and boxes represent sequences of pDH51 (Pietrzak et aL 1986) with promoter and termination signals (T) of Cauliflower Mosaic Virus 35S transcript. Open box represents coding region ofthe hptgene (Gritz and Davies 1983), BHI=BamHf, Hill= Hindlll restriction sites.
described in the preceding paper, with the exception that all steps prior to root induction were done under selective conditions with 25 mg/I hygromycin. Analysis of plant genomic DNA. Genomic DNA from individual plants was obtained with a modified CTAB-extraction protocol (Murray and Thompson 1980). Freeze-dried plants were used as starting material. DNA yield (measured after RNase A treatment) was 200-600 y.g/g fresh weight. Endonuclease digestion, get electrophoresis and blotlJng on Hybond N membrane (Amersham) were done using standard techniques recommended by the suppliers. The radioactively labeled 1033 bp BamHI fragment of pGL2 (covering the ceding region of the hptgene, fig. 1) was used as a probe for hybridization. All filters were washed with high stringency. Resistance assay of progeny. Seeds harvested from self-pollinated transgenic plants were surface-sterilized by 10 rain incubation in a 7% calcium-hypochlorite solution containing a few drops of Tween 80, followed by extensive washing with sterile distilled water. Seeds were germinated in dim light on 0.5 MS-O (Karesch et al., this issue) containing 25 mg/I hygromycin.
RESULTS Selection system. The effect of hygromycin on protoplast-derived microcolonies was tested in concentrations of 0, 1, 2, 5, 10, 20 and 50 mg per liter culture medium. 20 mg/I was found to be reliable for total growth inhibition: the colonies turned brown and died after 5-10 days of selective growth conditions. The same concentration completely inhibited the development of seedlings after germination. A hygromycin concentration of 25 mg/I was therefore chosen for use in transformation experiments as well as in progeny analysis. Colony formation or growth of seedlings from non-transformed material was never observed under these selection conditions. Resistant colonies in transformed cultures were counted after 8 weeks (Fig. 3) prior to release from the embedding matrix. Transformation frequency. Eight out of 11 transformation experiments yielded more than 100 resistant colonies per one million protoplasts. The average absolute transformation frequency (ATF = ratio of transgenic calli to the number of initially plated protoplasts) from all experiments was calculated to be 1.2 x 104, with values ranging from
1 2 3 4 5 6 7 8 9 10 11
26.4. 09.5. 17.5. 26.5. 01.6. 15.6. 30;6. 07.7. 19.7. 25.8. 01.9.
Number of resistant colonies / 10~ protoplasts (2-4 replicas per experiment) 70, 72 122, 98 66, 40 40, 74, 46 114, 141 148, 157 153, 62, 149, 120 139, 229, 179, 188 283, 250 95, 213 133, 66
Average
71 110 53 53 128 153 121 184 267 154 100
50 to 260 colonies/106 protoplasts (Table 1). A relative transformation frequency describing the ratio between the number of colonies under selective and non-selective conditions could not be calculated due to the high plating density in our experiments. In general, it was found that the number of resistant colonies is correlated with t~e number of dividing protoplasts in the control cultures. This might be reflected by the variation of the ATF in experiments performed over a period of 6 months, showing a maximum in the summer months. A similar seasonal dependence was previously observed for the plating efficiency in protoplast cultures (Karesch, unpublished results). The transformation frequency was significantly influenced by the addition of carrier DNA: treatment with the plasmid pGL2 alone resulted in a 10 times lower ATF of only 105. The use of plasmid in its circular form decreased the ATE to 4075 % compared with the numbers obtained with plasmid DNA linearized outside the gene at the Hindlll site. Variation of the DNA amount in the range of 5-30 F.g linearized plasmid per 7 x 105 protoplasts did not change either ATF nor copy number of integrated sequences. The dependence of the transformation frequency on the concentration of magnesium ions described for Nicotiana tabacum (Negrutiu et al. 1987) was not found to be as obvious for Arabidopsis protoplasts: variation of the MgCI2-concentration in the mannitol-magneslum solution between 7.5 and 30 mM did not significantly alter the ATF. A decrease was only found with concentrations below and above this range. Regeneratlon of transformsnts. We seFected 211 hygromycinresistant calli randomly for further culture on solid medium. Their growth rate in the presence of the antibiotic was the same as for the wJldtype calli under non-selective conditions. The shoot formation frequency (percentage of calli regenerating at least one shoot) was also similar (44 %) compared to that of non-selected controls (46 %). In contrast, root growth is very sensitive to hygromycin even in transgenic shoots. Rooting was therefore performed without selection pressure. An example of a regenerated, transgenic plant is shown in Fig. 5. On average, ca. 500 seeds were obtained from plants kept in axenic conditions. Seed setting of plants transferred to soil was even better.
573 Fig. 2: Southern blot of ganomic DNA (5 p.g per lane) from regenerated plants, probed with the hpt coding region (BamHI fragment of pGL2) lane 1 + 2: wildtype of Arabidopsis thaliana ecotype Z0rich lane 3+4, 5+6, 7+8, 9+10, 11+12:5 different transformed plants lane 13: BamHI digested plasmid pGL2 lane 1,3,5,7,9,11: DNA not digested lane 2,4,6,8,10,12,13: DNA digested with BamHI
Molecular evidence for transformation. In order to confirm integration of the marker gene, genomic DNA of 20 single, resistant plants was subjected to Southern analysis. Hybridization with the coding region of the hpt gene was found in all resistant regenerants in high molecular weight DNA and with the expected 1033 bp fragment (see Fig. 1) in BamHI digests (Fig. 2). The strength of the hybridization signals suggests different copy numbers between 1 and 10. Some samples appear to have additional fragments of higher molecular weight with homology to the probe, indicating plasmid rearrangement prior to or during integration. (Fig. 4, lanes 10 and 12). Individual F1 progeny plants from these parental plant always showed identical hybridization patterns, suggesting tight linkage of the different hpt-sequences. Inheritance of the transgene. Genetic transmission of the integrated gene was investigated in the progeny of 28 regenerated plants obtained by self-pollination. Based on an average seed number of 57 per plant, numbers of resistant and sensitive seedlings were statistically analyzed by the X2 assay against a 3:1 segregation. The Fl-populations fall into 4 groups (Table 2). Plants of group 1 and 2 inherit the resistance marker according to the expected Mendelian segregation of a dominant character in the case of multiple, unlinked copies of the transgene (group 1) and single or multiple, but tightly linked copies (group 2, example shown in Fig. 4) respectively. Other plants had less than expected (group 3) or no resistant progeny (group 4). This is, however, not correlated with reduced seed setting or lower germination frequency. Seven seed populations derived from progeny of a group 2 plant segregated either 3:1 or 4:0, confirming that the introduced gene can be stably transmitted further to the F2 generation.
DISCUSSION Stable transformation of Arabidopsis thaliana ecotype ZOrich by PEG mediated direct gene transfer to protoplasts, followed by regeneration of fertile plants, has been confirmed by the expression of the resistant phenotype in regenerants, by transgene integration into
high molecular weight DNA and by genetic transmission of the marker gene to subsequent generations. The rate of transformation is as high as described for ecotype Columbia, using a similar protocol (Damm et al. 1989). This is in contrast to the poor response of other genotypes described in that report. The transformation procedure originally developed for tobacco protoplasts (Negrutiu et al., 1987) can therefore be regarded as a starting point for further application in different strains of Arabidopsis thaliana as soon as a protoplast regeneration system is available. Some parameters affecting transformation in other systems (Shillito et al. 1985; Negrutiu et al. 1985) appear to have a similar influence in the Arabidopsis system: the addition of carrier DNA, linearization of the plasmid DNA and use of a pH-stabilized PEG solution are recommended for maximal transformation frequency, whereas the amount of transforming plasmid DNA is not correlated with ATF. Nevertheless, there is a high variation of transformation efficiency (ATF 1.2 x 10.4 +/- 0.6 x 104) in independent experiments even under standardized conditions, suggesting that the source and the treatment of the pretoplasts rather than experimental conditions during the transformation procedure itself are the limiting factor. The influence of other parameters like the magnesium ion concentration may not be clearly visible as long as the overall plating efficiency cannot be increased. The selection system used to screen for transformants is highly reliable. Every analyzed clone surviving the described selective conditions proved to contain integrated hpt-homologous DNA, with a copy number range similar to that found in Nicotiana (Paszkowski et al. 1989). The lack of resistant seedlings in the progeny of some plants was therefore surprising and provoked DNA analysis of their FI plants grown under non-selective conditions. It was found that the hptgene is transmitted but not expressed in the next generation. Although the ratio of plants with a non-Mendelian inheritance of the resistant phenotype is remarkably high in our experiments, similar aberrant segregation ratios were found in other Arabidopsis transformation experiments (Damm et al. 1989; Feldmann et al. 1989; Valvekens et al. 1989). Further investigation of the hpt gene inactivation is in progress.
574
Fig. 3: Hygromycin resistant microcalli embedded in alginate beads. Squares = 1 mm 2
Fig. 4: Fl-seedlings of an untransformed plant (left) and a plant transformed with pGL2 (right) on medium containing 25 mg/I hygromycin B.
Fig. 5: Fertile transgenic plant
Table 2: Segregation of hygromycin resistance in the F1 genera-
tion 3roup
Acknowledgements
REFERENCES
An G, Watson BD, Chiang CC (1986) Plant Physiol.81:301-305 Datum B, Willmitzer L (1988) Mol.Gen.Genet.213:15-20 Datum B, Schmidt R, Willmitzer L (1989) Mol.Gen.Genet.217:6-12 Feldmann KA, Marks MD (1987) MoI.Gen.Genet. 208:1-9 Feldmann KA, Marks MD, Christianson ML, Quatrano RS (1989) Science 243:1351-1354 Gritz L, Davies J (1983) Gene 25:179-188 Karesch H, Bilang R, Potrykus I Plant Cell Rep., this issue Lloyd AM, Barnason AR, Rogers SG, Byrne MC, Fraley RT, Horsch RB (1986) Science 234:464-466 Meyerowitz EM (1989) Cell 56:263-269
Segregation (resistant: sensitive)
Germination No. of F1frequency populations (%)
1
83-100
>3 :1
29- 97
7
2
73- 81
3 :1
38-100
7
3
17- 47
