k. 1992 Oxford University Press
Nucleic Acids Research, Vol. 20, No. 15 3977-3982
Gene rescue in plants by direct gene transfer of total genomic DNA into protoplasts Patrick Gallois*, Keith Lindsey+, Renee Malone§, Martin Kreisf and Michael G.K.Jonesi AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Biochemistry and Physiology Department, Harpenden, Herts AL5 2JQ, UK Received April 30, 1992; Revised and Accepted July 16, 1992
ABSTRACT To study the possibility of gene rescue in plants by direct gene transfer we chose the Arabidopsis mutant GH5O as a source of donor DNA. GH50 is tolerant of chlorsulfuron, a herbicide of the sulfonylurea class. Tobacco protoplasts were cotransfected with genomic DNA and the plasmid pHP23 which confers kanamycin resistance. A high frequency of cointegration of the plasmid and the genomic DNA was expected, which would allow the tagging of the plant selectable trait with the plasmid DNA. After transfection by electroporation the protoplasts were cultivated on regeneration medium supplemented with either chlorsulfuron or kanamycin as a selective agent. Selection on kanamycin yielded resistant calluses at an absolute transformation frequency (ATF) of 0.8 x lo-3. Selection on chlorsulfuron yielded resistant calluses at an ATF of 4.7 x10-6. When a selection on chlorsulfuron was subsequently applied to the kanamycin resistant calluses, 8% of them showed resistance to this herbicide. Southern analysis carried out on the herbicide resistant transformants detected the presence of the herbicide resistance gene of Arabidopsis into the genome of the transformed tobacco. Segregation analysis showed the presence of the resistance gene and the marker gene in the progeny of the five analysed transformants. 3 transformants showed evidence of genetic linkage between the two genes. In addition we show that using the same technique a kanamycine resistance gene from a transgenic tobacco could be transferred into sugar beet protoplasts at a frequency of 0.17% of the transformants.
INTRODUCTION For some important genes, classical methods of gene isolation via library screening with probes and antibodies are not feasible. To isolate genes encoding traits which are monogenic and
dominant, gene rescue is an attractive possibility. At least two strategies are possible. One is screening for a loss of function due to a gene disruption. The loss of function can be caused by the insertion of a transposon or a T-DNA (1,2). As only a few insertions are expected in a given line this approach necessitates the screening of a large population of individuals bearing insertions at different loci. A second strategy is to screen for a
gain of function by the introduction of genomic DNA from a donor genome into a cultivar or a species lacking the trait of interest. Limiting factors in the use of this strategy with crop plants are i) the large number of transformants needed to generate a good representation of the donor genome (this number is inversely related to the amount of the integrated foreign DNA per transformant) and ii) the efficiency of the transformation method which determines the number of cells to be treated for DNA uptake in order to generate the required number of transformants. Model experiments for this second strategy have been carried out where genomic libraries have been transferred into plants via Agrobacterium with a view to recover a gene of interest (3,4,5,6,7). These studies showed that although specific genes could be identified by screening the transformants such an approach was impractical for crop plants because the large sizes of their genomes would necessitate that a high number of transformants be generated and screened at the whole plant level (5). Gene rescue by direct gene transfer could present a viable alternative to Agrobacterium-mediated transfer. It would be expected that the number of transformants to be screened could be smaller, as multiple copies of foreign DNA are integrated in the genome of e.g. tobacco transformants (8,9) as opposed to only a few with Agrobacterium transformation (4). To investigate such a shotgun transformation of plants, we choose the Arabidopsis mutant GH50 as a source of genomic DNA for the following reasons: - GH50 is tolerant of chlorsulfuron, a herbicide from the sulfonylurea class. The source of resistance is well characterised and is due to a point mutation in the gene encoding acetolactate synthase (ALS) (10). The copy number of the gene conferring herbicide tolerance is 1 copy per haploid genome (11).
* To whom correspondence should be addressed at: Laboratoire de Physiologie et Biologie Moleculaire Vegetale, Universite de Perpignan, Avenue de Villeneuve, 66025 Perpignan, France. Present addresses: +Leicester Biocentre, University of Leicester, Leicester LEI 7RH, UK, §Waksman Institute, Rutgers University, Piscataway, NJ, USA, OBiologie du Developpement des Plantes, Batiment 430, Universite de Paris-sud, F-91400 Orsay Cedex, France and ¶Department of Plant Sciences, Murdoch University, Murdoch, Perth, W. Australia 6150
3978 Nucleic Acids Research, Vol. 20, No. 15 - The small size of the Arabidopsis genome (100 000 kb,(12)) is expected to incur a relatively high frequency of integration of the target gene into the host genome, and so should require the screening of relatively few transformants. In this paper we report the feasibility of identifying a dominant plant gene by cotransferring into a host plant cell the genomic DNA carrying the gene and a plasmid carrying a selectable marker gene. This approach allowed us to recover transgenic plants carrying the dominant gene genetically linked to the selectable marker gene. Our results indicate that the transfer of a dominant gene could be carried out in crop plants with a frequency which is compatible with a selection scheme at the whole plant level. The dominant gene could then be isolated by diverse molecular techniques. This constitute a new strategy for plant gene cloning.
MATERIALS AND METHODS Protoplast isolation Leaf protoplasts of Nicotiana tabacum cv. 'petit Havana' SRI were isolated from sterile shoots cultured on Murashige and Skoog (13) medium (Flow Labs) supplemented with 20 g/l sucrose (MS20). Leaf strips were incubated in K3 (14) medium, 0.4 M sucrose, 1.2% cellulase and 0.8% macerozyme for 16 h at 25 'C. The protoplasts obtained were filtered through a sterilized 100 Am stainless steel sieve and washed three times by resuspension in either 0.4 M sucrose adjusted to 80 mM KCl for electroporation treatment or MaMg (15 mM MgC12, 0.1 M MES, 0.45 M Mannitol, pH 5.6) (15) for PEG treatment. Sugar beet protoplasts were isolated from cell suspension cultures as described previously (16).
Protoplast transformation Electroporation. A laboratory-built capacitor discharge system was used as previously described (16,17). After isolation protoplasts were resuspended at a density of 106/ml in an electroporation medium of 80 mM KCl, 4 mM CaCl2, 2 mM KH2PO4 , pH 7.2, with mannitol added to a final osmotic pressure of 640 mosmol/kg and kept for 2 hours at 8°C before electroporation. The electrical parameters used were: 225 V/cm, 100 AF, 2 pulses. Circular plasmid DNA (30 4g/ml final) and HMW DNA (50 jig/ml final) were added to 0.3 ml aliquots of the protoplasts suspension. After electroporation, the protoplasts were left at room temperature for 45 min to allow membrane resealing and were then resuspended in culture medium. Sugar beet protoplasts were electroporated using the conditions described previously (16). PEG. The protocol is essentially as described by Negrutiu et al. (15). Protoplasts were maintained in MaMg for at least 30 min prior to transformation and then resuspended at a density of 1.6 x 106/ml. Circular plasmid DNA (30 4g/ml final) and HMW DNA (50 jig/ml final) were added to 0.3 ml aliquots of the protoplasts suspension and mixed, followed by 0.3 ml of 40% w/v PEG (3350) dissolved in 0.4M Mannitol, 0. IM Ca(NO3)2, and the suspensions were incubated for 25-30 min at room temperature with occasional shaking. 10 ml of W5 solution (154 mM NaCl, 125mM CaC12, 5 mM KCl, 5 mM glucose, pH 6.0) was gradually added over a 20 min period, followed by centrifugation for 10 min at 600 rpm. Protoplasts were then resuspended in culture medium.
Protoplast culture and selection Culture media and procedures were according to Shillito et al. (9) except for the regeneration medium which was according to Installe et al (18). After transfection, protoplasts for stable transformation studies were embedded in medium solidified with 0.6% w/v SeaPlaque (FMC) agarose 45 min after the transformation treatment, cultured 2 d in the dark and then at 25°C with 16 h daylengh. After 10 d culture, the embedded protoplasts were floated on liquid medium supplemented with 100 mg/l kanamycin or different concentrations of chlorsulfuron as selective agent. Chlorsulfuron was a gift of Dr. P. Stougaard, Danisco A/S, Copenhagen, DK. DNA The genomic DNA used for transformation was extracted from mature leaves according to Dellaporta et al (19) and further purified on a CsCl gradient (20). The expected size of the DNA fragments obtained by this method was 50 to 100 Kb. The mutant line GH50 of Arabidopsis thaliana was a gift of Dr. R.Wallsgrove, Rothamsted Experimental Station, Harpenden, UK. and came originally from Dr. C.Somerville, Michigan State University, USA. Carrier DNA was sheared salmon sperm DNA (Sigma). The plasmid used for transformation was pHP23 which consists of a fusion between the 19S and 35S CaMV promoters, a NPT II coding region and a 35S CaMV terminator in pUC 19 (21). The plasmid pSP32 containing the wild type ALS gene from Arabidopsis thaliana was gift of Dr. P.Stougaard, Danisco A/S,Copenhagen, DK. Plasmid DNA was prepared by an alkaline lysis method and quantified according to standard molecular biology procedures
(20). Southern blot analysis Genomic DNA from control plant or transgenic plant was extracted from leaves according to Dellaporta et al (19). Southern blot analysis was carried out on Hybond N (Amersham) according to the manufacturer's instructions. DNA was radiolabelled by oligolabelling to a specific activity of > 1 x 109 dpm/,ug (22).
Agrobacterium-mediated transformation SRI tobacco plants were transformed with A. tumefaciens strain LBA4404 (23) and the binary vector Bin 19 (24) according to a previously described protocol (25).
Segregation analysis Transgenic plants were self pollinated and the mature seeds collected. The seeds were surface sterilised by soaking for 10 min in 10% Domestos (Lever Ltd) and rinsed 5 times with sterile distilled water and allowed to dry under a laminar flow bench. Sterile seeds were sown on 1 % agar plates (MS salts, 20g/l sucrose) complemented with the selective agents. Resistant and sensitive seedlings were scored at 10 days. Chi-square analysis For chi-square analysis (P= 0.05, dF= 1) the ratio obtained for the segregation of chlorsulfuron resistance was used as the expected value and the ratio for the segregation of the double resistance (kan/chlo) as the observed value. An exception was made for plant XI, in that the ratio obtained for the segregation of the kanamycin resistance was used as the expected value, because this ratio was lower than the chlorsulfuron ratio.
Nucleic Acids Research, Vol. 20, No. 15 3979
RESULTS Direct gene transfer of the plasmid pHP23 conferring kanamycin resistance Electrical and PEG-mediated DNA uptake both allow efficient and routine transformation of tobacco protoplasts. In preliminary experiments, electroporation of protoplasts in the presence of 30 MLg/ml of plasmid and 50 .tg/ml of carrier DNA gave an absolute transformation frequency (ATF) of approx. 10-3. The ATF is defined as the number of transformants obtained per total number of protoplasts treated. Further experiments showed that HMW genomic DNA can enter the protoplasts through the pores transiently created in the plasmamembrane by the electric pulse (Gallois et al in preparation). The ATF obtained with PEG (10-4) was an order of magnitude lower than that obtained with electroporation using the same DNA concentrations.
Chlorsulfuron kill curve of tobacco leaf protoplasts After isolation, 106 protoplasts were embedded in 0.6% w/v agarose and cultured for 10 days. Protoplast-derived colonies (in agarose) were then floated in liquid culture medium supplemented with chlorsulfuron at concentrations ranging from 0.2ltg/l to 30 ,ug/l. The minimum concentration of chlorsulfuron required to prevent the development of any escape colonies was found to be 2 Mg1l. Direct gene transfer of Arabidopsis genomic DNA using electroporation To study the possibility of gene rescue in plants following direct gene transfer of genomic DNA, tagging of a mutant ALS gene, present in the GH50 Arabidopsis line, was attempted. Protoplasts were cotransfected with genomic DNA and the plasmid pHP23, encoding neomycin phosphotransferase (NPITH), which confers kanamycin resistance to plants when integrated into the host genome. The expectation was a high frequency of cointegration (26) of plasmid molecules and genomic DNA fragments resulting in the tagging of random Arabidopsis genomic fragments by linkage to selectable plasmid sequences. HMW DNA was extracted from the chlorsulfuron resistant Arabidopsis mutant (GH50) and mixed at a concentration of 50 Mg/ml with the plasmid pHP23 (30 Mg/ml). The mix was transfected into tobacco leaf protoplasts using electroporation. Prior to transfection the molar ratio of HMW DNA and plasmid was approx 1 to 6 (the HMW DNA fragments were at least 10 times larger than the plasmid). Table 1. Screening for chlorsulfuron resistant and kanamycin resistant colonies. Number of colonies obtained after 4 weeks selection on 2 Ag/l chlorsulfuron (a,e) or after 3 weeks on 100mg/l kanamycin followed by 4 weeks on 2 Ag/l chlorsulfuron (b,c,d). Transformants were obtained by either an electroporation or PEG-mediated protocol. nd: not determined.
After transfection the protoplasts were embedded in agarose and cultured at 25°c. After 10 days, agarose strips containing colonies (2 to 8 cells) were floated on the selection medium. One of 2 different selection protocols was then applied to different transformation batches and selected colonies were recovered (Tablel). 1) selection on 2 Mg/l chlorsulfuron yielded green calluses at an ATF of 4.7 x 10-6, of which approx. 60% produced shoots on selection medium. 3) Selection on 100 mg/l kanamycin was in the first instance applied for 4 weeks. Kanamycin resistant calluses were recovered at an ATF of 0.8 x 10-3. Resistant calluses (1 mm diameter) were then subjected to a second selection on 2 Ag/l chlorsulfuron. 8% of the calluses exhibited resistance to chlorsulfuron (ATF of 6.7x 10-5, Table 1). Approx. 75% of the calluses showing the double resistance regenerated shoots in subsequent culture on selective medium.
Direct gene transfer of the Arabidopsis genomic DNA using PEG To compare the efficiency of transfection protocols, the shotgun transformation strategy was repeated using PEG to induce DNA uptake.
copy number E.l
5
1
1/2
C
1
2
3
4
56
kb 2.3 9.4 6.6
..
4.3
2.3 2.0-
Ofk :
t
.
Initial
Protoplasts
Kanar colonies
Chior colonies
3 x 106
nd
14
0.15x106
119
10
1 x 106
148
9
nd x 106
100 nd
0 0
Number
Selection
a) Chlor 2g/1l b) 1) Kana 2) Chlor 2Ag1l PEG c) 1) Kana 2) Chlor 2lg/l Wild type DNA d) 1) Kana 2) Chlor 2Ag/1 untransforned e) Chlor 2Ag/1l
Electroporation
Figure 1. Southern blots of transformed tobacco plant DNA. Genomic DNA was digested with BamH 1, separated by electrophoresis, blotted onto a nylon membrane and hybridized with the radiolabelled Arabidopsis wild type ALS coding sequence. Gene copy number reconstructions correspond to 1/2, 1 and 5 copies per haploid genome of a 1.8 kb fragment corresponding to the wild type ALS gene. Lane c contains DNA from an untransformed tobacco plant. Lanes 1 to 6 contain genomic DNA from different transformed plants: I)plant II, 2) plant V,3) plant VIII, 4) plant IX, 5) plant X, 6) plant XI. The hybridizing 800 bp fragment of the Arabidopsis ALS gene is arrowed. (Stringency 2 xSSC)
3980 Nucleic Acids Research, Vol. 20, No. 15
kb 2.3
-..
9,4
... ...
fi.6 P,t
..
2.3
Figure 2. Southern blots of transformed tobacco plant DNA. Genomic DNA was digested with BamHl, separated by electrophoresis, blotted onto a nylon membrane and hybridized with a radiolabelled probe hybridizing to the 5' end of the Arabidopsis ALS gene. Lane c contains DNA from an untransformed tobacco plant. Lanes 1 to 8 contain genomic DNA from different transformed plants: 1) plant I, 2) plant III, 3) plant IV, 4) plant V, 5) plant VIII, 6) plant IX, 7) plant X, 8) plant XI. The hybridizing fragment is arrowed when present in the track.(Stringency 0.2 xSSC)
106 protoplasts were cotransfected with HMW GH50 genomic DNA and pHP23 at the concentrations used for electroporation. 148 calluses were recovered (ATF of 1.5 x 10-4) after screening on 100 mg/l kanamycin (Table 1). 9 calluses (i.e. 6.1 % of the kanamycin resistant calluses) survived the second screening on 2 ug/l chlorsulfuron. In control experiments, protoplasts were transformed with the plasmid pHP23 plus wild type DNA. Kanamycin resistant colonies were recovered. 100 3 week old colonies were cultured on 2 /4g/l chlorsulfuron and no resistant colonies were recovered. 106 untransformed protoplasts were submitted to selection on 2 izg/l chlorsulfuron and no chlorsulfuron resistant colonies were recovered. This shows that although it may, theoretically, be possible to obtain chlorsulfuron resistant colonies by mutation of a tobacco ALS gene during the selection process, the frequency of chlorsulfuron resistance reported here following transfection with GH50 genomic DNA is higher than the natural mutation rate.
Analysis of regenerated plants resistant to chlorsulfuron Chlorsulfuron-resistant regenerants (Table 1) were studied further by investigating a tissue culture response on a range of chlorsulfuron concentrations. Leaf explants from the regenerants were surface sterilized and cultivated on regeneration medium supplemented with chlorsulfuron at a concentration ranging from 0 to 30 Ag/l. After 4 weeks selection, explants from chlorsulfuron resistant plants were found to be able to produce green adventitious shoots on chlorsulfuron at a concentration of up to 10 .tg/l, while explants from control plants bleached, and failed to regenerate on 5 yg/l. Molecular analysis of chlorsulfuron resistant tobacco plants Genomic DNA was extracted from 6 chlorsulfuron resistant plants, to determine the integration pattern of the mutant ALS
gene by Southern analysis. The DNA was digested to completion with BamHI, blotted on a nylon membrane and hybridized with a radiolabelled probe corresponding to the coding sequence of the Arabidopsis ALS wild type gene. The restriction enzyme BamHI generates different restriction patterns between the tobacco and the Arabidopsis ALS gene (27). BamHI digestion of the Arabidopsis ALS gene generates an 800bp internal fragment which is located within the coding region, and a 7.1 kb fragment which contains 6.1 kb of the 5' flanking DNA and 1 kb of the coding sequence. The 800 bp fragment is absent from the tobacco ALS genes. Hybridizing fragments corresponding to the tobacco ALS genes were detected at low stringency in the control plant and the transformants, whereas a 800 bp hybridizing fragment was only present in the 6 transformants and not in the control (Fig. 1). This latter fragment confirms the presence of the Arabidopsis gene, correlating with the chlorsulfuron resistance expressed in the 6 selected tobaccos (Fig. 1). Reconstruction lines indicated that the hybridization signal of the 800 bp fragment corresponded to approx. one or two copies per 2C genome of tobacco. Southern analysis with a probe hybridizing to the 5' end of the coding sequence detected the 7.1 kb fragment in 4 transformants out of 8 tested (Fig.2). This indicates that in these plants the ALS gene integrated as a fragment of at least 7.9 kb (7.1 +0.8) with no detectable rearrangement. 4 transformants had a hybridizing fragment of a size different from 7.1 kb, indicating that the BamHI site 5' of the ALS gene had been deleted either during the insertion process or at an earlier stage eg. degraded during DNA isolation or protoplasts transformation.
Segregation analysis of the progeny of the double resistant plants Transgenic tobacco plants were self pollinated and their seeds collected for genetic analysis. The ratio of kanamycin resistant to kanamycin sensitive seeds was scored by sowing the seeds on 400 mg/l kanamycin. Kanamycin resistant plants grew normally, while kanamycin sensitive plants stopped developing and bleached as soon as the cotyledons expanded. Segregation of the chlorsulfuron resistance was scored on 100 ,ug/l chlorsulfuron. Chlorsulfuron resistant seedlings grew normally except that germination was delayed compared to wild type seeds on non selective medium. Chlorsulfuron sensitive plants were delayed in their germination, root growth was inhibited, cotyledons bleached partially and did not expand. Double resistant seedlings in the presence of kanamycin and chlorsulfuron developed as chlorsulfuron resistant seedlings did in the presence of chlorsulfuron only. All the plants tested showed segregation of kanamycin resistance as a dominant trait (Table 2). Plant XI had a resistance ratio falling below the ratio predicted for the segregation of a single locus (i.e.3:1) and plant IX had a ratio of 3:1 although 25% of the resistant seedlings exibited a variegated pattern of green and white tissues. These segregations could be associated with abnormal expression of the resistance gene in the progeny. The chlorsulfuron resistance trait segregated in the progeny as a dominant trait and most plants had the segregation ratio falling below 3:1 suggesting loss or inactivation of the resistance gene in some of the offspring. Plant IX and X had the two resistance genes segregating independently (Chi-square, p=0,05). However in plants V, XI and XII the segregation ratio of the double
Nucleic Acids Research, Vol. 20, No. 15 3981 Table 2. Analysis of the progeny of plants resistant to chlorsulfuron and kanamycin. Seeds from different transformants were sown on 400mg/i kanamycin, 100ytg/l chlorsulfuron, or 400mg/i kanamycin plus chlorsulfuron 100ltg/l. Resistant and sensitive seedlings were scored. In the case of double resistance I indicates acceptance of the linkage hypothesis of the two genes (Chi-square, p=0.05), nl indicates rejection of the linkage hypothesis (Chi-square, p=0.05). PARENT
V IX X XI XII Control
KANAMYCIN Resistant Sensitive
Ratio
CHLORSULFURON Resistant Sensitive
Ratio
KANA/CHLOR Resistant Sensitive
370 47 136 275 290 0
3 2,9 3,1 2,6 3,05 /
338 106 280 155 190 0
2,08 1,25 2,8 3,4 1,7 /
265 108 203 212 102 0
123 16 44 106 95 100
resistance was consistent with the genetic linkage of the 2 resistance traits (chi-square, p =0.05). In this case it is suggested that the two genes are located on the same chromosome and possibly at the same locus. Further segregation analysis would establish for each plant the genetic distance between the two foreign genes.
Transfer of kanamycin resistance into sugar beet protoplasts by electroporation of tobacco genomic DNA Most species have a genome size 20 to 30 or more times larger than Arabidopsis and the transformation frequency for these species by direct gene transfer is lower than that of tobacco. To extend to other species the results obtained with Arabidopsis DNA and tobacco protoplasts we carried out direct gene transfer of tobacco DNA (2C content= 7.8 pg) into the crop species sugar beet (Beta vulgaris). An electroporation protocol for the stable transformation of sugar beet protoplasts has been reported elsewhere (16). Tobacco plants were transformed with the NPTII gene by Agrobacterium to obtain kanamycin resistant plants. Southern analysis was carried out and one transformant containing approx. 20 copies of the NPITH gene was selected as a source of HMW DNA with a view to rescuing the NPTII gene following shotgun transformation. Due to the low transformation frequency of sugar beet protoplasts, and the large size of the tobacco genome this high copy number transformant was chosen in order to reduce the number of protoplasts to be treated. According to segregation analysis the NPTII gene copies have integrated at 2 different loci. The DNA of the transformant was digested with EcoRI which does not cut in the NPTII gene but cuts inside the T-DNA construct. The effect of this digestion is to separate each NPTII gene copy onto individual DNA fragments. The digested DNA (5014g/ml) was electroporated into sugar beet suspension cell protoplasts. A total of 25 x 106 protoplasts were treated and cultured in agarose in the presence of 100 mg/l kanamycin. 17 kanamycin resistant colonies were recovered (ATF of 1.5 x 10-6). Southern blot analysis confirmed the presence of the kanamycin gene in the sugar beet genome. In parallel experiments under the same transformation conditions, an ATF of 2 x 0-5 was achieved using plasmid DNA carrying the NPTII gene. Therefore, assuming the same efficiency of transformation from a total of 25 x 106 protoplasts, 500 were expected to give rise to colonies having stably integrated some tobacco genomic DNA. With approx. 20 copies of the resistance gene in the genome of the tobacco used for the experiment, it is estimated that 3,4% of the transformants have therefore taken up a copy of the resistance gene.
162 85 99 45 111 100
151 115 175 82 78 100
Ratio
2' O,n 1,2 2,61 1,31 /
DISCUSSION In this paper we demonstrate the feasibility of using a direct gene transfer system of shotgun transformation as a basis for the rescue of selectable genes. As expected, the frequency of the herbicide resistant calluses obtained via direct gene transfer of genomic DNA was lower than the frequency of kanamycin resistance calluses obtained by direct gene transfer of plasmid. Both electroporation and PEG gave herbicide resistant calluses at an ATF 10 times lower than the ATF of kanamycin resistant calluses (e.g. for electroporation: Kanr:0.8 X10-3; chlor:0.6 XIO-4). More herbicide resistant calluses were recovered by selecting for transformants first with kanamycin rather than by selecting direcfly with chlorsulfuron. This probably reflects the fact that transformed colonies can survive better on the herbicide when at a 1 mm size stage than when at an earlier stage of development. The frequency of transfer of the ALS gene is expected to be direcfly linked to the average amount of foreign genomic DNA integrated in every transformant. It has been shown (3) that a population of 3 x a/b transformants are needed to have a 95% chance of obtaining at least one transformant having integrated a given DNA sequence, (with a corresponding to the size of the genome of the donor plant and b being the amount of integrated DNA). For Arabidopsis recent estimates indicate an haploid genome of approx. 100 000 kb (12). The experiment reported here shows that between 1 out of 17 (6%) and 1 out of 13 (8%) of the kanamycine resistant transformants have integrated the herbicide resistance gene. Applying the above formula using n= 13 to 17, on average the amount of DNA integrated (b) is between 17500-23000 kb. Such a large amount of integrated DNA implies that, as in animal system (28, 29), a second round of transformation and selection with DNA from individual primary transformants should be carried out to establish the physical relationship between plasmid and genomic DNA and to facilitate gene isolation. It has been shown that direct transfer of a single human gene via total genomic DNA occurs with a frequency of 1/5000 transformants in fibroblasts (30) or L cells (31). Given the size of the human genome (3 x 106 kb) the frequency obtained with our protocol for the transfer of a single Arabidopsis gene is 10 times higher, suggesting that more DNA is taken up in tobacco protoplasts. A major point differentiating the results obtained with plant or animal systems is that plant cells are totipotent and thus allow whole plant regeneration and genetic analysis. The segregation analysis showed that in some cases there is no genetic linkage of the dominant trait from Arabidopsis and the co-transferred plasmid DNA, it is suggested that the donor DNA may integrate at more than one locus in the host genome.
3982 Nucleic Acids Research, Vol. 20, No. 15 Ligating the donor DNA to the plasmid before transformation could increase the proportion of transformant showing linkage. Southern analysis showed that the foreign DNA can integrate as fragments larger than 8 kb. This suggests that it should be possible to tranfer dominant gene encoded by 8 kb or less. The results obtained with Arabidopsis DNA are corroborated by the ATF obtained with tobacco genomic DNA transfer to sugar beet protoplasts. If the experiment was repeated with one copy of the resistance gene in the donor genomic DNA it would be expected that 0,17% (3.4 / 20) of the transformants would take up the resistance gene. The frequency of transfer in this case (0. 17 %) would be similar to the frequency of transfer of the ALS gene among kanamycin resistant calluses using Arabidopsis DNA (8%) when it is taken in account that the tobacco genome,used as a donor, is 30 times bigger than the Arabidopsis genome (0.17%x30 = 5.1%). Golz et al (32) reported the transfer of a hygromycin resistance gene using total DNA from a transgenic Brassica nigra (hygromycin resistant) to wild type Brassica napus. They suggest that the frequency obtained was equivalent to that when they transformed with plasmid DNA. We show that such a high frequency of gene rescue (nearly 100% of the transformants) cannot be expected in every case. The frequency obtained may be influenced by the nature of the DNA surrounding the gene of interest: particular sequences of genomic DNA have been shown to boost the transformation frequency of plant protoplasts (33). The authors experiment failed to show the transfer of genes coding for esterase isoenzymes from B. nigra to B. napus. In our case we demonstrate that transfer of an endogenous gene is feasible by direct gene transfer of genomic DNA. Shotgun transformation using Agrobacterium transformation has been shown to be technically possible (5,6) but in practical terms the large size of genomes other than that of Arabidopsis may limit the general applicability of the approach, due to the large effort in screening which would be required. Simoens et al (3) calculated that 17 x 106 transformants must be generated to transfer a particular DNA sequence from the tobacco genome into a host plant via Agrobacterium transformation. The study reported here demonstrates that it is possible to use direct gene transfer (electroporation or PEG mediated) of genomic DNA to identify and possibly isolate selectable genes from any genome larger than that of Arabidopsis. From the data we have obtained from experiments involving 2 different selectable genes, it can be estimated that about 2000 to 3000 transformants would be enough to transfer a dominant gene with a tag linked to it from a given species to a cultivated species. This frequency would allow the application of a selection or screening scheme at the whole plant level to detect the transfer of a dominant trait from a donor species to a crop species, even with a low transformation frequency. The dominant gene could be rescued from the transformed materiel by cloning or plasmid rescue, both of which have been successfully used for gene rescue in animal research (28, 29, 34). Alternatively, if the introduced gene is not expressed in the untransformed line, a cDNA library could be constructed from the transformed line and the relevant cDNA isolated by subtraction with cDNAs from an untransformed line (31).
ACKNOWLEDGEMENTS This work was supported by Danisco A/S and AGC. The authors wish to thank Dr.B.Forde, Dr. N.Halford for helpful discussions,
Dr. M.W.Bianchi and Dr. G.A.Hull for critical reading of the
manuscript. REFERENCES 1. Coen E.S., Romero J.M., Doyle S., Elliot R., Murphy G. and Carpenter R. (1990). Cell 63: 1311-1322. 2. Feldmann K.A. (1991). The Plant Journal I - 1: 71 -82. 3. Simoens C., Alliotte Th., Mendel R., Muller A., Schiemann J., van Lijsebettens M., Schell J, van Montagu M. and Inze D. (1986). Nucleic Acids Res. 14: 8073-8090. 4. Prosen D.E. and Simpson R.B. (1987). Bio/technol. 5: 966-971. 5. Klee H.J., Hayford M.B. and Rogers S.G. (1987). Mol. Gen. Genet. 210: 282-287. 6. Olszewski N.E., Martin F.B. and Ausubel F.M. (1988). Nucleic Acids Res. 16-22: 10765-10782. 7. Lazo G.R., Stein P.A. and Ludwig R.A. (1991). Bio/technol. 9: 963-967. 8. Paszkowski J., Shillito R.D., Saul M., Mandak V., Hohn T., Hohn B. and Potrykus I. (1984). EMBO J. 3-12: 2717-2722. 9. Shillito R.D., Saul M., Paszkowski J., Muller G. and Potrykus I. (1985). Bio/technol. 3: 1099-1103. 10. Haughn G.W., Smith J., Mazur B. and Somerville C. (1988). Mol. Gen. Genet. 211: 266-271. 11. Haughn G.W. and Somerville C. (1986). Mol. Gen. Genet. 204: 430-434. 12. ISPMBNews (1990). Plant Mol. Biol. Rep. 8: 156-159. 13. Murashige T. and Skoog F. (1962). Plant Physiol. 15: 473-497. 14. Kao K.N., Constabel F., Michayluk R. and Gamborg O.L. (1974). Planta 120: 215-217. 15. Negrutiu I., Shillito R., Potrykus I., Biasini G. and Sala F. (1987). Plant Mol. Biol. 8: 363-373. 16. Lindsey K. and Jones M.G.K. (1989). Plant Cell Rep. 8: 71-740. 17. Jones H., Ooms G and Jones M.G.K. (1989). Plant Mol. Biol. 13: 503 -511. 18. Installe P., Negrutiu I. and Jacobs M. (1985). J. Plant Physiol. 119: 443 -454. 19. Dellaporta S.L., Wood J. and Hicks J.B. (1983). Plant Mol. Biol. Rep. 1: 19-21. 20. Sambrook J., Fritsch E.F. and Maniatis T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, ppS45. 21. Paszkowski J., Baur M., Bogucki A. and Potrykus I. (1988). EMBO J.7: 4021 -4026. 22. Feinberg A.P., Vogelstein B. (1983). Anal. Biochem. 132: 6-13. 23. Ooms G., Hooykaas P.J.J., van Veen R.J.M., van Beelen P., RegensburgTuink T.J.G. and Shilperoort R.A. (1982). Plasmid 7: 15-29. 24. Bevan M. (1984). Nucleic Acids Res. 12: 8711-8721. 25. Marris C., Gallois P., Copley J. and Kreis M. (1988). Plant Mol. Biol. 10: 359-366. 26. Schocher R.J., Shillito R.D., Saul M.W., Paszkowski J. and Potrykus I. (1986). Bio/technol. 4: 1093-1096. 27. Mazur B.J., Chui C.F. and Smith J.K. (1987). Plant Physiol. 85: 1110-1117. 28. Perucho M., Hanahan D., Lipsich L. and Wigler M. (1980). Nature 285: 207-210. 29. Lowy I., Pellicer A., Jackson J.F., Sim G.K., Silverstein S. and Axel R. (1980). Cell 22: 817-823. 30. Pinney D.F., Pearson-White S.H., Konieczny S.F., Latham K.E. and Emerson C.P. (1988). Cell 53: 781 -793. 31. Littman D.R., Thomas Y., Maddon P.J., Chess L. and Axel R. (1985). Cell 40: 237-246. 32. Gotz C., Kohler F. and Schieder 0. (1990). Plant Mol. Biol. 15: 475 -483. 33. Marchesi M.L., Castiglione S. and Sala F. (1989). Theor. Appl. Genet. 78: 113- 118. 34. Goldfarb M., Shimizu K., Perucho M. and Wigler M. (1982). Nature 296: 404-409.
Nucleic Acids Research, Vol. 20, No. 15 3977-3982
Gene rescue in plants by direct gene transfer of total genomic DNA into protoplasts Patrick Gallois*, Keith Lindsey+, Renee Malone§, Martin Kreisf and Michael G.K.Jonesi AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Biochemistry and Physiology Department, Harpenden, Herts AL5 2JQ, UK Received April 30, 1992; Revised and Accepted July 16, 1992
ABSTRACT To study the possibility of gene rescue in plants by direct gene transfer we chose the Arabidopsis mutant GH5O as a source of donor DNA. GH50 is tolerant of chlorsulfuron, a herbicide of the sulfonylurea class. Tobacco protoplasts were cotransfected with genomic DNA and the plasmid pHP23 which confers kanamycin resistance. A high frequency of cointegration of the plasmid and the genomic DNA was expected, which would allow the tagging of the plant selectable trait with the plasmid DNA. After transfection by electroporation the protoplasts were cultivated on regeneration medium supplemented with either chlorsulfuron or kanamycin as a selective agent. Selection on kanamycin yielded resistant calluses at an absolute transformation frequency (ATF) of 0.8 x lo-3. Selection on chlorsulfuron yielded resistant calluses at an ATF of 4.7 x10-6. When a selection on chlorsulfuron was subsequently applied to the kanamycin resistant calluses, 8% of them showed resistance to this herbicide. Southern analysis carried out on the herbicide resistant transformants detected the presence of the herbicide resistance gene of Arabidopsis into the genome of the transformed tobacco. Segregation analysis showed the presence of the resistance gene and the marker gene in the progeny of the five analysed transformants. 3 transformants showed evidence of genetic linkage between the two genes. In addition we show that using the same technique a kanamycine resistance gene from a transgenic tobacco could be transferred into sugar beet protoplasts at a frequency of 0.17% of the transformants.
INTRODUCTION For some important genes, classical methods of gene isolation via library screening with probes and antibodies are not feasible. To isolate genes encoding traits which are monogenic and
dominant, gene rescue is an attractive possibility. At least two strategies are possible. One is screening for a loss of function due to a gene disruption. The loss of function can be caused by the insertion of a transposon or a T-DNA (1,2). As only a few insertions are expected in a given line this approach necessitates the screening of a large population of individuals bearing insertions at different loci. A second strategy is to screen for a
gain of function by the introduction of genomic DNA from a donor genome into a cultivar or a species lacking the trait of interest. Limiting factors in the use of this strategy with crop plants are i) the large number of transformants needed to generate a good representation of the donor genome (this number is inversely related to the amount of the integrated foreign DNA per transformant) and ii) the efficiency of the transformation method which determines the number of cells to be treated for DNA uptake in order to generate the required number of transformants. Model experiments for this second strategy have been carried out where genomic libraries have been transferred into plants via Agrobacterium with a view to recover a gene of interest (3,4,5,6,7). These studies showed that although specific genes could be identified by screening the transformants such an approach was impractical for crop plants because the large sizes of their genomes would necessitate that a high number of transformants be generated and screened at the whole plant level (5). Gene rescue by direct gene transfer could present a viable alternative to Agrobacterium-mediated transfer. It would be expected that the number of transformants to be screened could be smaller, as multiple copies of foreign DNA are integrated in the genome of e.g. tobacco transformants (8,9) as opposed to only a few with Agrobacterium transformation (4). To investigate such a shotgun transformation of plants, we choose the Arabidopsis mutant GH50 as a source of genomic DNA for the following reasons: - GH50 is tolerant of chlorsulfuron, a herbicide from the sulfonylurea class. The source of resistance is well characterised and is due to a point mutation in the gene encoding acetolactate synthase (ALS) (10). The copy number of the gene conferring herbicide tolerance is 1 copy per haploid genome (11).
* To whom correspondence should be addressed at: Laboratoire de Physiologie et Biologie Moleculaire Vegetale, Universite de Perpignan, Avenue de Villeneuve, 66025 Perpignan, France. Present addresses: +Leicester Biocentre, University of Leicester, Leicester LEI 7RH, UK, §Waksman Institute, Rutgers University, Piscataway, NJ, USA, OBiologie du Developpement des Plantes, Batiment 430, Universite de Paris-sud, F-91400 Orsay Cedex, France and ¶Department of Plant Sciences, Murdoch University, Murdoch, Perth, W. Australia 6150
3978 Nucleic Acids Research, Vol. 20, No. 15 - The small size of the Arabidopsis genome (100 000 kb,(12)) is expected to incur a relatively high frequency of integration of the target gene into the host genome, and so should require the screening of relatively few transformants. In this paper we report the feasibility of identifying a dominant plant gene by cotransferring into a host plant cell the genomic DNA carrying the gene and a plasmid carrying a selectable marker gene. This approach allowed us to recover transgenic plants carrying the dominant gene genetically linked to the selectable marker gene. Our results indicate that the transfer of a dominant gene could be carried out in crop plants with a frequency which is compatible with a selection scheme at the whole plant level. The dominant gene could then be isolated by diverse molecular techniques. This constitute a new strategy for plant gene cloning.
MATERIALS AND METHODS Protoplast isolation Leaf protoplasts of Nicotiana tabacum cv. 'petit Havana' SRI were isolated from sterile shoots cultured on Murashige and Skoog (13) medium (Flow Labs) supplemented with 20 g/l sucrose (MS20). Leaf strips were incubated in K3 (14) medium, 0.4 M sucrose, 1.2% cellulase and 0.8% macerozyme for 16 h at 25 'C. The protoplasts obtained were filtered through a sterilized 100 Am stainless steel sieve and washed three times by resuspension in either 0.4 M sucrose adjusted to 80 mM KCl for electroporation treatment or MaMg (15 mM MgC12, 0.1 M MES, 0.45 M Mannitol, pH 5.6) (15) for PEG treatment. Sugar beet protoplasts were isolated from cell suspension cultures as described previously (16).
Protoplast transformation Electroporation. A laboratory-built capacitor discharge system was used as previously described (16,17). After isolation protoplasts were resuspended at a density of 106/ml in an electroporation medium of 80 mM KCl, 4 mM CaCl2, 2 mM KH2PO4 , pH 7.2, with mannitol added to a final osmotic pressure of 640 mosmol/kg and kept for 2 hours at 8°C before electroporation. The electrical parameters used were: 225 V/cm, 100 AF, 2 pulses. Circular plasmid DNA (30 4g/ml final) and HMW DNA (50 jig/ml final) were added to 0.3 ml aliquots of the protoplasts suspension. After electroporation, the protoplasts were left at room temperature for 45 min to allow membrane resealing and were then resuspended in culture medium. Sugar beet protoplasts were electroporated using the conditions described previously (16). PEG. The protocol is essentially as described by Negrutiu et al. (15). Protoplasts were maintained in MaMg for at least 30 min prior to transformation and then resuspended at a density of 1.6 x 106/ml. Circular plasmid DNA (30 4g/ml final) and HMW DNA (50 jig/ml final) were added to 0.3 ml aliquots of the protoplasts suspension and mixed, followed by 0.3 ml of 40% w/v PEG (3350) dissolved in 0.4M Mannitol, 0. IM Ca(NO3)2, and the suspensions were incubated for 25-30 min at room temperature with occasional shaking. 10 ml of W5 solution (154 mM NaCl, 125mM CaC12, 5 mM KCl, 5 mM glucose, pH 6.0) was gradually added over a 20 min period, followed by centrifugation for 10 min at 600 rpm. Protoplasts were then resuspended in culture medium.
Protoplast culture and selection Culture media and procedures were according to Shillito et al. (9) except for the regeneration medium which was according to Installe et al (18). After transfection, protoplasts for stable transformation studies were embedded in medium solidified with 0.6% w/v SeaPlaque (FMC) agarose 45 min after the transformation treatment, cultured 2 d in the dark and then at 25°C with 16 h daylengh. After 10 d culture, the embedded protoplasts were floated on liquid medium supplemented with 100 mg/l kanamycin or different concentrations of chlorsulfuron as selective agent. Chlorsulfuron was a gift of Dr. P. Stougaard, Danisco A/S, Copenhagen, DK. DNA The genomic DNA used for transformation was extracted from mature leaves according to Dellaporta et al (19) and further purified on a CsCl gradient (20). The expected size of the DNA fragments obtained by this method was 50 to 100 Kb. The mutant line GH50 of Arabidopsis thaliana was a gift of Dr. R.Wallsgrove, Rothamsted Experimental Station, Harpenden, UK. and came originally from Dr. C.Somerville, Michigan State University, USA. Carrier DNA was sheared salmon sperm DNA (Sigma). The plasmid used for transformation was pHP23 which consists of a fusion between the 19S and 35S CaMV promoters, a NPT II coding region and a 35S CaMV terminator in pUC 19 (21). The plasmid pSP32 containing the wild type ALS gene from Arabidopsis thaliana was gift of Dr. P.Stougaard, Danisco A/S,Copenhagen, DK. Plasmid DNA was prepared by an alkaline lysis method and quantified according to standard molecular biology procedures
(20). Southern blot analysis Genomic DNA from control plant or transgenic plant was extracted from leaves according to Dellaporta et al (19). Southern blot analysis was carried out on Hybond N (Amersham) according to the manufacturer's instructions. DNA was radiolabelled by oligolabelling to a specific activity of > 1 x 109 dpm/,ug (22).
Agrobacterium-mediated transformation SRI tobacco plants were transformed with A. tumefaciens strain LBA4404 (23) and the binary vector Bin 19 (24) according to a previously described protocol (25).
Segregation analysis Transgenic plants were self pollinated and the mature seeds collected. The seeds were surface sterilised by soaking for 10 min in 10% Domestos (Lever Ltd) and rinsed 5 times with sterile distilled water and allowed to dry under a laminar flow bench. Sterile seeds were sown on 1 % agar plates (MS salts, 20g/l sucrose) complemented with the selective agents. Resistant and sensitive seedlings were scored at 10 days. Chi-square analysis For chi-square analysis (P= 0.05, dF= 1) the ratio obtained for the segregation of chlorsulfuron resistance was used as the expected value and the ratio for the segregation of the double resistance (kan/chlo) as the observed value. An exception was made for plant XI, in that the ratio obtained for the segregation of the kanamycin resistance was used as the expected value, because this ratio was lower than the chlorsulfuron ratio.
Nucleic Acids Research, Vol. 20, No. 15 3979
RESULTS Direct gene transfer of the plasmid pHP23 conferring kanamycin resistance Electrical and PEG-mediated DNA uptake both allow efficient and routine transformation of tobacco protoplasts. In preliminary experiments, electroporation of protoplasts in the presence of 30 MLg/ml of plasmid and 50 .tg/ml of carrier DNA gave an absolute transformation frequency (ATF) of approx. 10-3. The ATF is defined as the number of transformants obtained per total number of protoplasts treated. Further experiments showed that HMW genomic DNA can enter the protoplasts through the pores transiently created in the plasmamembrane by the electric pulse (Gallois et al in preparation). The ATF obtained with PEG (10-4) was an order of magnitude lower than that obtained with electroporation using the same DNA concentrations.
Chlorsulfuron kill curve of tobacco leaf protoplasts After isolation, 106 protoplasts were embedded in 0.6% w/v agarose and cultured for 10 days. Protoplast-derived colonies (in agarose) were then floated in liquid culture medium supplemented with chlorsulfuron at concentrations ranging from 0.2ltg/l to 30 ,ug/l. The minimum concentration of chlorsulfuron required to prevent the development of any escape colonies was found to be 2 Mg1l. Direct gene transfer of Arabidopsis genomic DNA using electroporation To study the possibility of gene rescue in plants following direct gene transfer of genomic DNA, tagging of a mutant ALS gene, present in the GH50 Arabidopsis line, was attempted. Protoplasts were cotransfected with genomic DNA and the plasmid pHP23, encoding neomycin phosphotransferase (NPITH), which confers kanamycin resistance to plants when integrated into the host genome. The expectation was a high frequency of cointegration (26) of plasmid molecules and genomic DNA fragments resulting in the tagging of random Arabidopsis genomic fragments by linkage to selectable plasmid sequences. HMW DNA was extracted from the chlorsulfuron resistant Arabidopsis mutant (GH50) and mixed at a concentration of 50 Mg/ml with the plasmid pHP23 (30 Mg/ml). The mix was transfected into tobacco leaf protoplasts using electroporation. Prior to transfection the molar ratio of HMW DNA and plasmid was approx 1 to 6 (the HMW DNA fragments were at least 10 times larger than the plasmid). Table 1. Screening for chlorsulfuron resistant and kanamycin resistant colonies. Number of colonies obtained after 4 weeks selection on 2 Ag/l chlorsulfuron (a,e) or after 3 weeks on 100mg/l kanamycin followed by 4 weeks on 2 Ag/l chlorsulfuron (b,c,d). Transformants were obtained by either an electroporation or PEG-mediated protocol. nd: not determined.
After transfection the protoplasts were embedded in agarose and cultured at 25°c. After 10 days, agarose strips containing colonies (2 to 8 cells) were floated on the selection medium. One of 2 different selection protocols was then applied to different transformation batches and selected colonies were recovered (Tablel). 1) selection on 2 Mg/l chlorsulfuron yielded green calluses at an ATF of 4.7 x 10-6, of which approx. 60% produced shoots on selection medium. 3) Selection on 100 mg/l kanamycin was in the first instance applied for 4 weeks. Kanamycin resistant calluses were recovered at an ATF of 0.8 x 10-3. Resistant calluses (1 mm diameter) were then subjected to a second selection on 2 Ag/l chlorsulfuron. 8% of the calluses exhibited resistance to chlorsulfuron (ATF of 6.7x 10-5, Table 1). Approx. 75% of the calluses showing the double resistance regenerated shoots in subsequent culture on selective medium.
Direct gene transfer of the Arabidopsis genomic DNA using PEG To compare the efficiency of transfection protocols, the shotgun transformation strategy was repeated using PEG to induce DNA uptake.
copy number E.l
5
1
1/2
C
1
2
3
4
56
kb 2.3 9.4 6.6
..
4.3
2.3 2.0-
Ofk :
t
.
Initial
Protoplasts
Kanar colonies
Chior colonies
3 x 106
nd
14
0.15x106
119
10
1 x 106
148
9
nd x 106
100 nd
0 0
Number
Selection
a) Chlor 2g/1l b) 1) Kana 2) Chlor 2Ag1l PEG c) 1) Kana 2) Chlor 2lg/l Wild type DNA d) 1) Kana 2) Chlor 2Ag/1 untransforned e) Chlor 2Ag/1l
Electroporation
Figure 1. Southern blots of transformed tobacco plant DNA. Genomic DNA was digested with BamH 1, separated by electrophoresis, blotted onto a nylon membrane and hybridized with the radiolabelled Arabidopsis wild type ALS coding sequence. Gene copy number reconstructions correspond to 1/2, 1 and 5 copies per haploid genome of a 1.8 kb fragment corresponding to the wild type ALS gene. Lane c contains DNA from an untransformed tobacco plant. Lanes 1 to 6 contain genomic DNA from different transformed plants: I)plant II, 2) plant V,3) plant VIII, 4) plant IX, 5) plant X, 6) plant XI. The hybridizing 800 bp fragment of the Arabidopsis ALS gene is arrowed. (Stringency 2 xSSC)
3980 Nucleic Acids Research, Vol. 20, No. 15
kb 2.3
-..
9,4
... ...
fi.6 P,t
..
2.3
Figure 2. Southern blots of transformed tobacco plant DNA. Genomic DNA was digested with BamHl, separated by electrophoresis, blotted onto a nylon membrane and hybridized with a radiolabelled probe hybridizing to the 5' end of the Arabidopsis ALS gene. Lane c contains DNA from an untransformed tobacco plant. Lanes 1 to 8 contain genomic DNA from different transformed plants: 1) plant I, 2) plant III, 3) plant IV, 4) plant V, 5) plant VIII, 6) plant IX, 7) plant X, 8) plant XI. The hybridizing fragment is arrowed when present in the track.(Stringency 0.2 xSSC)
106 protoplasts were cotransfected with HMW GH50 genomic DNA and pHP23 at the concentrations used for electroporation. 148 calluses were recovered (ATF of 1.5 x 10-4) after screening on 100 mg/l kanamycin (Table 1). 9 calluses (i.e. 6.1 % of the kanamycin resistant calluses) survived the second screening on 2 ug/l chlorsulfuron. In control experiments, protoplasts were transformed with the plasmid pHP23 plus wild type DNA. Kanamycin resistant colonies were recovered. 100 3 week old colonies were cultured on 2 /4g/l chlorsulfuron and no resistant colonies were recovered. 106 untransformed protoplasts were submitted to selection on 2 izg/l chlorsulfuron and no chlorsulfuron resistant colonies were recovered. This shows that although it may, theoretically, be possible to obtain chlorsulfuron resistant colonies by mutation of a tobacco ALS gene during the selection process, the frequency of chlorsulfuron resistance reported here following transfection with GH50 genomic DNA is higher than the natural mutation rate.
Analysis of regenerated plants resistant to chlorsulfuron Chlorsulfuron-resistant regenerants (Table 1) were studied further by investigating a tissue culture response on a range of chlorsulfuron concentrations. Leaf explants from the regenerants were surface sterilized and cultivated on regeneration medium supplemented with chlorsulfuron at a concentration ranging from 0 to 30 Ag/l. After 4 weeks selection, explants from chlorsulfuron resistant plants were found to be able to produce green adventitious shoots on chlorsulfuron at a concentration of up to 10 .tg/l, while explants from control plants bleached, and failed to regenerate on 5 yg/l. Molecular analysis of chlorsulfuron resistant tobacco plants Genomic DNA was extracted from 6 chlorsulfuron resistant plants, to determine the integration pattern of the mutant ALS
gene by Southern analysis. The DNA was digested to completion with BamHI, blotted on a nylon membrane and hybridized with a radiolabelled probe corresponding to the coding sequence of the Arabidopsis ALS wild type gene. The restriction enzyme BamHI generates different restriction patterns between the tobacco and the Arabidopsis ALS gene (27). BamHI digestion of the Arabidopsis ALS gene generates an 800bp internal fragment which is located within the coding region, and a 7.1 kb fragment which contains 6.1 kb of the 5' flanking DNA and 1 kb of the coding sequence. The 800 bp fragment is absent from the tobacco ALS genes. Hybridizing fragments corresponding to the tobacco ALS genes were detected at low stringency in the control plant and the transformants, whereas a 800 bp hybridizing fragment was only present in the 6 transformants and not in the control (Fig. 1). This latter fragment confirms the presence of the Arabidopsis gene, correlating with the chlorsulfuron resistance expressed in the 6 selected tobaccos (Fig. 1). Reconstruction lines indicated that the hybridization signal of the 800 bp fragment corresponded to approx. one or two copies per 2C genome of tobacco. Southern analysis with a probe hybridizing to the 5' end of the coding sequence detected the 7.1 kb fragment in 4 transformants out of 8 tested (Fig.2). This indicates that in these plants the ALS gene integrated as a fragment of at least 7.9 kb (7.1 +0.8) with no detectable rearrangement. 4 transformants had a hybridizing fragment of a size different from 7.1 kb, indicating that the BamHI site 5' of the ALS gene had been deleted either during the insertion process or at an earlier stage eg. degraded during DNA isolation or protoplasts transformation.
Segregation analysis of the progeny of the double resistant plants Transgenic tobacco plants were self pollinated and their seeds collected for genetic analysis. The ratio of kanamycin resistant to kanamycin sensitive seeds was scored by sowing the seeds on 400 mg/l kanamycin. Kanamycin resistant plants grew normally, while kanamycin sensitive plants stopped developing and bleached as soon as the cotyledons expanded. Segregation of the chlorsulfuron resistance was scored on 100 ,ug/l chlorsulfuron. Chlorsulfuron resistant seedlings grew normally except that germination was delayed compared to wild type seeds on non selective medium. Chlorsulfuron sensitive plants were delayed in their germination, root growth was inhibited, cotyledons bleached partially and did not expand. Double resistant seedlings in the presence of kanamycin and chlorsulfuron developed as chlorsulfuron resistant seedlings did in the presence of chlorsulfuron only. All the plants tested showed segregation of kanamycin resistance as a dominant trait (Table 2). Plant XI had a resistance ratio falling below the ratio predicted for the segregation of a single locus (i.e.3:1) and plant IX had a ratio of 3:1 although 25% of the resistant seedlings exibited a variegated pattern of green and white tissues. These segregations could be associated with abnormal expression of the resistance gene in the progeny. The chlorsulfuron resistance trait segregated in the progeny as a dominant trait and most plants had the segregation ratio falling below 3:1 suggesting loss or inactivation of the resistance gene in some of the offspring. Plant IX and X had the two resistance genes segregating independently (Chi-square, p=0,05). However in plants V, XI and XII the segregation ratio of the double
Nucleic Acids Research, Vol. 20, No. 15 3981 Table 2. Analysis of the progeny of plants resistant to chlorsulfuron and kanamycin. Seeds from different transformants were sown on 400mg/i kanamycin, 100ytg/l chlorsulfuron, or 400mg/i kanamycin plus chlorsulfuron 100ltg/l. Resistant and sensitive seedlings were scored. In the case of double resistance I indicates acceptance of the linkage hypothesis of the two genes (Chi-square, p=0.05), nl indicates rejection of the linkage hypothesis (Chi-square, p=0.05). PARENT
V IX X XI XII Control
KANAMYCIN Resistant Sensitive
Ratio
CHLORSULFURON Resistant Sensitive
Ratio
KANA/CHLOR Resistant Sensitive
370 47 136 275 290 0
3 2,9 3,1 2,6 3,05 /
338 106 280 155 190 0
2,08 1,25 2,8 3,4 1,7 /
265 108 203 212 102 0
123 16 44 106 95 100
resistance was consistent with the genetic linkage of the 2 resistance traits (chi-square, p =0.05). In this case it is suggested that the two genes are located on the same chromosome and possibly at the same locus. Further segregation analysis would establish for each plant the genetic distance between the two foreign genes.
Transfer of kanamycin resistance into sugar beet protoplasts by electroporation of tobacco genomic DNA Most species have a genome size 20 to 30 or more times larger than Arabidopsis and the transformation frequency for these species by direct gene transfer is lower than that of tobacco. To extend to other species the results obtained with Arabidopsis DNA and tobacco protoplasts we carried out direct gene transfer of tobacco DNA (2C content= 7.8 pg) into the crop species sugar beet (Beta vulgaris). An electroporation protocol for the stable transformation of sugar beet protoplasts has been reported elsewhere (16). Tobacco plants were transformed with the NPTII gene by Agrobacterium to obtain kanamycin resistant plants. Southern analysis was carried out and one transformant containing approx. 20 copies of the NPITH gene was selected as a source of HMW DNA with a view to rescuing the NPTII gene following shotgun transformation. Due to the low transformation frequency of sugar beet protoplasts, and the large size of the tobacco genome this high copy number transformant was chosen in order to reduce the number of protoplasts to be treated. According to segregation analysis the NPTII gene copies have integrated at 2 different loci. The DNA of the transformant was digested with EcoRI which does not cut in the NPTII gene but cuts inside the T-DNA construct. The effect of this digestion is to separate each NPTII gene copy onto individual DNA fragments. The digested DNA (5014g/ml) was electroporated into sugar beet suspension cell protoplasts. A total of 25 x 106 protoplasts were treated and cultured in agarose in the presence of 100 mg/l kanamycin. 17 kanamycin resistant colonies were recovered (ATF of 1.5 x 10-6). Southern blot analysis confirmed the presence of the kanamycin gene in the sugar beet genome. In parallel experiments under the same transformation conditions, an ATF of 2 x 0-5 was achieved using plasmid DNA carrying the NPTII gene. Therefore, assuming the same efficiency of transformation from a total of 25 x 106 protoplasts, 500 were expected to give rise to colonies having stably integrated some tobacco genomic DNA. With approx. 20 copies of the resistance gene in the genome of the tobacco used for the experiment, it is estimated that 3,4% of the transformants have therefore taken up a copy of the resistance gene.
162 85 99 45 111 100
151 115 175 82 78 100
Ratio
2' O,n 1,2 2,61 1,31 /
DISCUSSION In this paper we demonstrate the feasibility of using a direct gene transfer system of shotgun transformation as a basis for the rescue of selectable genes. As expected, the frequency of the herbicide resistant calluses obtained via direct gene transfer of genomic DNA was lower than the frequency of kanamycin resistance calluses obtained by direct gene transfer of plasmid. Both electroporation and PEG gave herbicide resistant calluses at an ATF 10 times lower than the ATF of kanamycin resistant calluses (e.g. for electroporation: Kanr:0.8 X10-3; chlor:0.6 XIO-4). More herbicide resistant calluses were recovered by selecting for transformants first with kanamycin rather than by selecting direcfly with chlorsulfuron. This probably reflects the fact that transformed colonies can survive better on the herbicide when at a 1 mm size stage than when at an earlier stage of development. The frequency of transfer of the ALS gene is expected to be direcfly linked to the average amount of foreign genomic DNA integrated in every transformant. It has been shown (3) that a population of 3 x a/b transformants are needed to have a 95% chance of obtaining at least one transformant having integrated a given DNA sequence, (with a corresponding to the size of the genome of the donor plant and b being the amount of integrated DNA). For Arabidopsis recent estimates indicate an haploid genome of approx. 100 000 kb (12). The experiment reported here shows that between 1 out of 17 (6%) and 1 out of 13 (8%) of the kanamycine resistant transformants have integrated the herbicide resistance gene. Applying the above formula using n= 13 to 17, on average the amount of DNA integrated (b) is between 17500-23000 kb. Such a large amount of integrated DNA implies that, as in animal system (28, 29), a second round of transformation and selection with DNA from individual primary transformants should be carried out to establish the physical relationship between plasmid and genomic DNA and to facilitate gene isolation. It has been shown that direct transfer of a single human gene via total genomic DNA occurs with a frequency of 1/5000 transformants in fibroblasts (30) or L cells (31). Given the size of the human genome (3 x 106 kb) the frequency obtained with our protocol for the transfer of a single Arabidopsis gene is 10 times higher, suggesting that more DNA is taken up in tobacco protoplasts. A major point differentiating the results obtained with plant or animal systems is that plant cells are totipotent and thus allow whole plant regeneration and genetic analysis. The segregation analysis showed that in some cases there is no genetic linkage of the dominant trait from Arabidopsis and the co-transferred plasmid DNA, it is suggested that the donor DNA may integrate at more than one locus in the host genome.
3982 Nucleic Acids Research, Vol. 20, No. 15 Ligating the donor DNA to the plasmid before transformation could increase the proportion of transformant showing linkage. Southern analysis showed that the foreign DNA can integrate as fragments larger than 8 kb. This suggests that it should be possible to tranfer dominant gene encoded by 8 kb or less. The results obtained with Arabidopsis DNA are corroborated by the ATF obtained with tobacco genomic DNA transfer to sugar beet protoplasts. If the experiment was repeated with one copy of the resistance gene in the donor genomic DNA it would be expected that 0,17% (3.4 / 20) of the transformants would take up the resistance gene. The frequency of transfer in this case (0. 17 %) would be similar to the frequency of transfer of the ALS gene among kanamycin resistant calluses using Arabidopsis DNA (8%) when it is taken in account that the tobacco genome,used as a donor, is 30 times bigger than the Arabidopsis genome (0.17%x30 = 5.1%). Golz et al (32) reported the transfer of a hygromycin resistance gene using total DNA from a transgenic Brassica nigra (hygromycin resistant) to wild type Brassica napus. They suggest that the frequency obtained was equivalent to that when they transformed with plasmid DNA. We show that such a high frequency of gene rescue (nearly 100% of the transformants) cannot be expected in every case. The frequency obtained may be influenced by the nature of the DNA surrounding the gene of interest: particular sequences of genomic DNA have been shown to boost the transformation frequency of plant protoplasts (33). The authors experiment failed to show the transfer of genes coding for esterase isoenzymes from B. nigra to B. napus. In our case we demonstrate that transfer of an endogenous gene is feasible by direct gene transfer of genomic DNA. Shotgun transformation using Agrobacterium transformation has been shown to be technically possible (5,6) but in practical terms the large size of genomes other than that of Arabidopsis may limit the general applicability of the approach, due to the large effort in screening which would be required. Simoens et al (3) calculated that 17 x 106 transformants must be generated to transfer a particular DNA sequence from the tobacco genome into a host plant via Agrobacterium transformation. The study reported here demonstrates that it is possible to use direct gene transfer (electroporation or PEG mediated) of genomic DNA to identify and possibly isolate selectable genes from any genome larger than that of Arabidopsis. From the data we have obtained from experiments involving 2 different selectable genes, it can be estimated that about 2000 to 3000 transformants would be enough to transfer a dominant gene with a tag linked to it from a given species to a cultivated species. This frequency would allow the application of a selection or screening scheme at the whole plant level to detect the transfer of a dominant trait from a donor species to a crop species, even with a low transformation frequency. The dominant gene could be rescued from the transformed materiel by cloning or plasmid rescue, both of which have been successfully used for gene rescue in animal research (28, 29, 34). Alternatively, if the introduced gene is not expressed in the untransformed line, a cDNA library could be constructed from the transformed line and the relevant cDNA isolated by subtraction with cDNAs from an untransformed line (31).
ACKNOWLEDGEMENTS This work was supported by Danisco A/S and AGC. The authors wish to thank Dr.B.Forde, Dr. N.Halford for helpful discussions,
Dr. M.W.Bianchi and Dr. G.A.Hull for critical reading of the
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