[19] Direct gene transfer to protoplasts of dicotyledonous and monocotyledonous plants by a number of methods, including electroporation

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Methyl green, which runs slightly slower than arginine, can be used as a visible marker during electrophoresis, and samples of stock solutions of arginine, nopaline, and octopine are used as markers to help the interpretation of the electropherogram. After electrophoresis (about 1 hr) the paper is dried and then stained with phenanthrene quinone reagent (a freshly prepared mix of equal volumes of 0.02% phenanthrene quinone in ethanol and 10% NaOH in 60% ethanol). After staining the paper is dried with cold air. Areas containing guanidine compounds such as arginine, octopine and nopaline can be visualized as yellow spots by irradiation with long-wave ultraviolet (360 nm).

[19] D i r e c t G e n e T r a n s f e r to Protoplasts o f D i c o t y l e d o n o u s a n d M o n o c o t y l e d o n o u s P l a n t s b y a N u m b e r of M e t h o d s , Including Electroporation

By RAYMONDD. SHILLITO and INGO POTRYKUS Introduction Recent progress in cell and molecular biology has made available a great variety of new techniques for modifying the information content of plant cells. Moreover, in the last few years it has become possible to genetically engineer the plant cells by introduction of defined DNA sequences. This has been achieved mainly by exploiting the natural gene transfer system of the pathogen Agrobacterium tumefaciens (for a review, see Ref. I). This is a convenient and efficient technique, but it can be used only with plants which fall inside its host range. This includes most herbaceous dicots, but only a limited number of monocots, 2 and, as yet, not the graminaceous monocots which make up the bulk of our crop species. The introduction of genes by DNA-mediated transformation is a wellestablished procedure for bacterial, fungal, and animal systems and has proved to be a very powerful technique in the analysis of gene function. It has recently been shown that DNA can be introduced into plant protoplasts and integrated into the chromosomal DNA without intervention of G. Gheysen, P. Dhaese, M. Van Montagu, and J. Schell, in "Genetic Flux in Plants" (B. Hohn and E. S. Dennis, eds.), p. 12. Springer-Verlag, Vienna, Austria, 1985. 2 M. De Cleene and J. De Ley, Bot. Rev. 42, (1976).

METHODS IN ENZYMOLOGY, VOL. 153

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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a bacterial intermediary (direct gene transfer). 3-7 It is also possible, using this technique, to transform graminaceous cells, s,9 A number of other techniques have also been developed recently, employing a viral vector,I° liposomes, II bacterial spheroplasts/2 and microinjection 13 to deliver the DNA. Thus a number of routes are available for introduction of defined pieces of DNA into plant cells, from which one can choose, on the basis of the culture system available, a suitable procedure for the particular species or cell type under study. Transformation of plant cells has the added advantage that in many cases the totipotency of the transformed cells allows regeneration of large numbers of whole plants and genetic and molecular analysis of progeny, which is not available in such an easily accessible manner with transformed animal cells. Methods for the direct introduction of genes into plant protoplasts are presented, along with methods for the subsequent selection of transformed colonies, regeneration of the genetically altered fertile plants, and characterization of the introduced DNA by molecular, biological, and genetic techniques. The methods are drawn from two complementary fields: recombinant DNA and plant tissue culture. In the interests of continuity, methods from both these fields will be presented mixed with one another in the chronological order in which they are needed in a transformation experiment. The frequency previously obtained by these methods was low (10 -5 10-4 of recoverable colonies). 5,6 Such low frequencies seriously hampered the application of the technique of direct gene transfer as a general method for introducing genes into plant cells. However, recent advances 3 M. R. Davey, E. C. Cocking, J. Freeman, N. Pearce, and I. Tudor, Plant. Sci. Lett. 18, 3O7 (1980). 4 j. Draper, M. R. Davey, J. P. Freeman, E. C. Cocking, and B. G. Cox, Plant Cell Physiol. 2,3, 451 (1982). 5 F. A. Krens, L. Molendijk, G. J. Wullems, and R. A. Schilperoort, Nature (London) 296, 72 (1982). 6 j. Paszkowski, R. D. Shillito, M. Saul, V. Mandak, T. Hohn, B. Hohn, and I. Potrykus, EMBO J. 3, 2717 (1985). 7 R. Hain, P. Stabel, A. P. Czernilofsky, H.-H. Steinbiss, L. Herrera-Estrella, and J. Schell, Mol. Gen. Genet. 199, 161 (1985). s I. Potrykus, M. Saul, J. Petruska, J. Paszkowski, and R. D. Shillito, Mol. Gen. Genet. 199, 183 (1985). 9 H. Loerz, B. Baker, and J. Schell, Mol. Gen. Genet. 199, 178 (1985). 10 N. Brisson, J. Paszkowski, J. R. Penswick, B. Gronenborn, I. Potrykus, and T. Hohn, Nature (London) 310, 511 (1984). tmA. Deshayes, L. Herrera-Estrella, and M. Caboche, EMBO. J. 4, 2731 (1985). ~2R. Hain, H.-H. Steinbiss, and J. Schell, Plant Cell Rep. 3, 60 (1984). 13 T. Reich, V. N. Iyer, and B. Miki, Proc. Congr. Plant Mol. Biol., 1st, 1985 p. 28 (Abstr.).

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have increased frequencies into the percentage range. 14Protocols for such efficient transformation, and improvements of the original protocols, are given in this publication. Abbreoiations

MES: 2(N-morpholino)ethanesulfonic acid NAA: Naphthaleneacetic acid 2,4-D: 2,4-Dichlorophenoxyacetic acid BAP: 6-Benzylaminopurine SDS: Sodium dodecyl sulfate TE: 10 mM Tris-HCl, 5 mM EDTA, pH 7.5 dsDNA: Double-stranded DNA Media

Bacterial media, as specified in Ref. 15 Plant culture media are shown in Table I Materials E. coli strain DH115: Sources for the plant material are given with each

individual protocol Table-top centrifuge: Universal 2, (Hettich Zentrifugen, Tuttlingen, West Germany) Osmometer: Roebling Micro-Osmometer (Infochroma AG, Zug, Switzerland) Rocking table: Heidolph Reax 3 rocking table (Salvis AG, Reussbuehl, Switzerland) Stainless steel sieves: Saulas and Company (Paris, France) The 10-cm-diameter containers used for the "bead-type" culture are obtained from Semadani AG (Ostermundigen, Switzerland) Petri dishes: These are obtained from a range of suppliers Electroporator: "DIA-LOG" Elektroporator: DIA-LOG GmbH., (Dusseldorf, West Germany) Resistance meter: AVO B183 LCR meter (Thorn-EMI Ltd., Dover, England) SeaPlaque agarose: Marine Colloids, FMC Corporation (Rockland, Maine) ~4R. D. Shillito, M. W. Saul, J. Paszkowski, M. Mueller, and I. Potrykus, Bio/Technology3, 1099 (1985). ~5 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982.

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Cleaned agar: This is prepared by washing with water, acetone, and ethanol in succession16,17 Tween 80: ICI (Runcorn, England) or Merck-Schuchardt (Munich, West Germany) GreenzitR: Ciba-Geigy AG (Basel, Switzerland) Restriction enzymes (ligase, etc.) can be obtained from a number of commercial sources Cellulase "Onozuka" RI0 and macerozyme R10: Yakult Pharmaceutical Industries Company, Ltd. (Nishinomiya, Japan) Driselase: Fluka AG (Chemische Fabrik, Buchs, Switzerland) Hemicellulase: Sigma Chemical Company (St. Louis, MO) Pectinol: Roehm GmbH (Chemische Fabrik, Darmstadt, FRG) Antibiotics: Kanamycin sulfate: Serva, (Heidelberg, FRG); ampicillin: "Penbritin" Beecham SA (Bern, Switzerland); G418: Gibco Polyethylene glycol (PEG): Merck PEG 6000 and PEG 4000 All other organic and inorganic substances are of the highest purity available from the usual commercial sources.

Protocols for the Preparation, Transformation, and Culture of Protoplasts and Regeneration of Plants We describe here protocols which are in everyday use in our laboratory for four plant species. A number of factors are common to these protocols: (1) Centrifugations are carried out at 60 g except where otherwise stated. (2) Washing solutions (osmoticum) in all protocols are buffered with 0.5% (w/v) MES and adjusted to pH with KOH except where otherwise stated. (3) Counting of protoplasts is carried out by placing a drop of a 1 : l0 dilution of the suspension in the wash solution (where protoplasts will sediment) or in 0.17 M calcium chloride in a haemocytometer, counting, and estimating the density in the original suspension.

1. Source of the Hybrid-Selectable Gene Several hybrid marker genes for use in plant cell transformation, using A. tumefaciens-mediated transformation, have been described in the last 16 A. C. Braun and H. N. Wood, Proc. Natl. Acad. Sci. U.S.A. 48, 1776 (1962). 17 R. D. Shillito, J. Paszkowski, and I. Potrykus, Plant Cell Rep. 2, 244 (1983).

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2 years. ~8-ZlThe elements necessary in such a construction can be summarized as follows: 1. Plant gene expression signals, i.e., promoter and terminator regions for an RNA, which are best derived from a constitutively and highly expressed plant or plant viral gene 2. A protein-coding region joined precisely to the above expression signals which when expressed will give an active product which allows easy selection at the plant cell level: e.g., detoxification of antibiotics lethal for plant cells 3. For DNA-mediated transformation, a region on the bacterial vector plasmid which allows recombination into the plant genome without disruption of the selectable gene A plasmid fulfilling the above requirements has been constructed (pABD1). Details of the construction are given elsewhere. 6 The expression signals used are derived from gene VI of the plant dsDNA virus cauliflower mosaic virus (CaMV). 22 The selectable marker gene joined to these sequences is aminoglycoside phosphotransferase type II [APH(Y)II] 23 and the bacterial plasmid containing this construction is pUC8. 24 Before using the construction in direct DNA transformation experiments it was tested for biological activity and for the ability to be selected by introduction into Nicotiana tabacum cells via the A. tumefaciens method. Any suitable construction which satisfies the above criteria can be used for direct gene transfer.

Preparation of the DNA for Protoplast Transformation Purification. The plasmid pABD1 is grown in Escherichia coli strain DH1 in the presence of 50 /zg/ml ampicillin and isolated by a cleared lysate method. 25 After lysis in Triton X-100 containing lytic mix, supercoiled DNA is purified by a single round of CsCl/ethidium bromide gradient centrifugation. Ethidium bromide is removed by repeated extraction 18 M. W. Bevan, R. B. Flavell, and M. D. Chilton, Nature (London) 304, 184 (1983). 19 R. T. Fraley, S. G. Rogers, R. B. Horsch, P. R. Sanders, J. S. Flick, S. P. Adams, M. L. Bittner, L. A. Brand, C. L. Fink, J. S. Fry, G. R. GaUupi, S. B. Goldberg, N. L. Hoffman, and S. C. Woo, Proc. Natl. Acad. Sci. U.S.A. 80, 4803 (1983). 20 L. Herrera-Estrella, M. De Block, E. Messens, J.-P. Hernalstens, M. Van Montagu, and J. Schell, EMBO J. 2, 987 (1983). 2t L. Herrera-Estrella, A. Depicker, M. Van Montagu, and J. Schell, Nature (London) 303, 209 (1983). 2~ H. Guilley, R. G. Dudley, G. Jonard, E. Balazs, and K. Richards, Cell 30, 763 (1982). 23 S. J. Rothstein and W. S. Reznikoff, Cell 23, 191 (1981). z4 j. Messing and J. Vieira, Gene 19, 269 (1982). 25 y . M. Kuperstock and D. Helsinki, Biochem. Biophys. Res. Commun. 54, 1451 (1973).

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with CsCl-saturated 2-propanol solution. The DNA is then precipitated with ethanol (1 vol DNA solution plus 2 vol TE plus 6 vol 96% ethanol) at - 2 0 ° overnight. The precipitate is collected by centrifugation at 5000 g for I0 min, washed in 70% ethanol, dried briefly in an air stream, and redissolved in TE buffer. After spectrophotometric determination of the DNA concentration this is adjusted to I mg/ml. DNA for transformation is linearized by digestion with SmaI or BgllI overnight and precipitated by addition of one-tenth volume of 3 M potassium acetate followed by 3 vol of ethanol. The precipitate is collected by centrifugation as above, washed in 70% ethanol and 100% (v/v) ethanol in succession, dried in an air stream, and redissolved in sterile double-distilled water at 0.4 mg/ml. All manipulations with the DNA after this sterilization step are carded out under aseptic conditions in a laminar flow cabinet. Physical form of the transforming DNA: All of our early transformation experiments, which have already been well analyzed, were carded out with supercoiled plasmid DNA. We can conclude at this time that both linear and supercoiled molecules can be successfully taken up into plant protoplasts and integrated into the plant genome. However, linear molecules are clearly superior in the efficiency of transformation, amounting to a factor of 3-10 depending on the precise conditions used. Carrier DNA: Experiments are generally carried out using high-molecular-weight calf thymus DNA (Sigma) as carrier, as described by Krens et al. 5 for experiments involving transformation of protoplasts with isolated Ti plasmid. Calf thymus DNA is dissolved in water, precipitated in 70% and washed with 70 and 100% (v/v) ethanol, dried, and redissolved at 2 mg/ml in sterile double-distilled water. The carrier DNA is mixed at a ratio of five times the amount of pABD1 DNA (equal volumes of the two DNA solutions as given). Trials with carrier DNA of other types have shown that salmon sperm DNA and N. tabacum DNA give comparable results but also that transformation is possible at reduced efficiency without any carrier DNA.

2. Preparation, Transformation, and Culture of Protoplasts from a Sterile Shoot Culture of N. tabacum, and Regeneration of Plants The example given is for protoplasts from shoot cultures of the widely used genotype ofN. tabacum cv. "Petit Havana," SR1.26 This material is grown as sterile axenic shoot cultures. The protocol for protoplast isolation is modified from that of Nagy and Maliga. 27 The culture method uses complex media based on that of Kao 24 p. Maliga, A. Breznovitz, and L. Marton, Nature (London) New Biol. 244, 29 (1973). 27 j. I. Nagy and P. Maliga, Z. Pflanzenphysiol. 78, 453 (1976).

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and Michayluk 28 (Table I) and the agarose "bead-type" s y s t e m 17 t o obtain high division frequencies and rates of conversion of protoplasts to colonies. Transformation is carried out using one of the four methods given. Colonies are transferred to agar-solidified medium for one subculture and then placed on regeneration medium to promote the formation of shoots. Regenerated shoots are cultured on the original shoot culture medium. Source of Material. Shoot cultures are established from seeds which are sterilized using mercury chloride (see protocol 2) or sodium hypochlorite [5 min, 1.4% (w/v) containing 0.05% (w/v) Tween 80]. The plants arising are serially subcultured every 6 weeks as cuttings on T medium 29 (Table I) solidified with 0.8% (w/v) Difco Bacto agar and grown at 26° with 16 hr of light (1000-2000 lx) per day in a growth chamber. Preparation of Protoplasts. Just fully expanded leaves of 6-week-old shoot cultures are removed under sterile conditions and wetted thoroughly with enzyme solution. The leaves are then cut into 1- to 2-cm squares and floated on enzyme solution [1.2% (w/v) cellulase "Onozuka" R10, 0.4% (w/v) macerozyme R10 in K3A medium with 0.4 M sucrose] in Petri dishes ( - 1 g leaves in 12 ml enzyme solution in a 9-cm-diameter Petri dish). These are sealed and incubated overnight at 26° in the dark. The digest is gently agitated and then left for a further half-hour to complete digestion. The solution is filtered through a 100-/zm stainless steel mesh sieve and washed through with one-half volume of 0.6 M sucrose (MES, pH 5.6), distributed into capped centrifuge tubes and centrifuged for 10 min. The protoplasts collect at the upper surface of the medium. The medium is then removed from under the protoplasts. A simple method of doing this uses a sterilized cannula (A. R. Howell, Ltd., London, England) attached to a 20-ml disposable plastic syringe. This must be done slowly so as to avoid disturbing the layer of protoplasts excessively. Alternatively, the protoplasts can be collected using a pipet (with a medium orifice). The protoplasts are resuspended in K3A medium (Table I) containing 0.4 M sucrose. Washing of the protoplasts is carried out by repeated (3 x) flotation and replacing of the medium in this way. A sample is taken for counting before the last centrifugation, and the protoplasts resuspended for the last time in the appropriate medium for the transformation protocol to be used. 28 K. N. Kao and M. R. Michayluk, Planta 126, 105 (1975). :9 j. p. Nitsch and C. Nitsch, Science 163, 85 (1969).

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Transformation Method 1. " F medium" method: This method is a modification of the original method described by Paszkowski et a/. 6,3° for transformation of protoplasts of N. tabacum, which was in turn a modification of the method of Krens et al. 5 We have added a heat shock step and changed the order of addition of DNA and PEG. After counting, the protoplasts are adjusted to 2 x 106/ml in K3A medium and aliquots of 1 ml are distributed to 10 ml sterile plastic tubes. Heat shock is then administered by immersing the tubes for 5 min in a water bath at 45 °, followed by cooling to room temperature on ice. Then DNA solution is added to the samples (10/zg of pABD1 plus 50/xg of calf thymus DNA in 50/xl sterile distilled water), followed by gentle mixing. Finally, 0.5 ml of PEG solution [40% (w/v) PEG 6000 in F medium (Table I)] is added with shaking. The protoplasts are incubated with DNA and PEG for 30 min at room temperature with occasional gentle mixing. Then five aliquots of 2 ml of F medium are added at intervals of 5 min. We have noted that the pH of F medium drops to 4.3-4.6 after autoclaving. Since this is likely to be harmful to many protoplast systems we recommend adjustment of the pH after autoclaving with K O H to 5.8. Following transformation, the protoplasts are sedimented by centrifugation for 5 min, resuspended in 2 ml of K3A culture medium, and transferred in 1-ml aliquots to 9-cm Petri dishes. To each dish is added 10 ml of a 1 : 1 mixture of K3A and H media (Table I) containing 0.6% (w/v) SeaPlaque agarose, and the protoplasts dispersed by gentle swirling. This protocol gives transformation efficiencies in the range of 10 -4 to 10 -3. Method 2. Electroporation o f protoplastsl4: Electroporation is a process in which cells or protoplasts are treated with high-voltage electric fields for short periods in order to induce the formation of pores across the membrane. 31,32 In this way it is possible to induce uptake of DNA into animal cells or plant protoplasts, leading to transient expression 33 or to stable transformation. 14,32 Samples of protoplasts are pulsed with high-voltage pulses in the chamber of a " D I A - L O G " Elektroporator. This chamber is cylindrical in form with a distance of 1 cm between parallel steel electrodes and has a 3o j. Paszkowski and M. W. Saul, this series, Vol. 118, p. 668. 31 R. Benz, F. Beckers, and U. Zimmermann, J. Membr. Biol. 48, 181 (1979). 32 E. Neumann, M. Schaeffer-Ridder, Y. Wang, and P. H. Hofschneider, EMBO J. 1, 841 (1982). 33 M. Fromm, L. P. Taylor, and V. Walbot, Proc. Natl. Acad. Sci. U.S.A. 82, 5824 (1985).

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pulsed volume of 0.32 ml. 32 The pulse is applied by discharge of a capacitor across the cell. The decay constant of the pulse (time taken to decay to 1/e of the initial voltage) is in the order of 10 tzsec using a capacitor of 10 nF and a chamber resistance of 1 kfL The resistance across the chamber is measured using an alternating current multimeter operating at 1 kHz. The protocol given is for leaf mesophyll protoplasts of N. tabacum. These have an average diameter of 42/xm. The field strength required is inversely proportional to the diameter of the protoplasts being treated, and may vary a little from species to species in the field strength required for a given size of protoplast. In addition, protoplasts originating from suspension cultures generally require a slightly higher field strength than leaf mesophyll protoplasts. Protoplasts are resuspended, following the last flotation step, in 0.4 M mannitol containing 1 g/liter MES (pH 5.6 with KOH), and containing 6 mM magnesium chloride to stabilize the protoplasts, at a population density of 1.6 x 106/ml. An aliquot of 0.37 ml is transferred to the chamber of the electroporator and the resistance measured. In order to bring the resistance of the mannitol solution in which the protoplasts are suspended to the correct value: it is necessary to add ionic salts. Magnesium chloride is used to adjust the resistance to a value of 1-1.1 k~, by adding 1-3% (v/v) of a 0.3 M solution to the protoplast suspension. Heat shock is carried out before distributing the preparation into tubes, by treating the protoplasts for 5 min at 45 °, followed by cooling to room temperature on ice. Aliquots of 0.25 ml of protoplast suspension are placed in 5-ml-capacity polycarbonate tubes and DNA [4/~g pABDI linearized with Sinai and 20/zg of calf thymus DNA (Sigma) in 20/zl water/aliquot] and one-half volume of the PEG solution [24% (w/v) PEG 6000: Merck] added. This PEG is prepared in mannitol with sufficient magnesium chloride added ( - 3 0 mM) to bring the resistance when measured in the chamber of the electroporator into the region of 1.2 kfl. Ten minutes after addition of the DNA and PEG, samples are transferred to the chamber of the electroporator and pulsed three times at 10sec intervals with pulses of an initial field strength of 1.4-1.5 kV/cm. They are then returned to a 6-cm-diameter Petri dish and held at 20° for 10 min before addition of 3 ml of a 1 : 1 mixture of K3A and H media (Table I) containing 0.6% (w/v) SeaPlaque agarose. This is the optimized protocol. It gives the highest frequencies we have as yet obtained--in the r~gion of 1 to 3% for all colonies recoverable without selection. It is not necessary when using this method to cool the protoplasts on ice, due to the low

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heating effects when using such short pulses, in contrast to when using the method of Langridge et al. 34,35

Method 3. Quick method for transformation without electroporation: Protoplasts are treated in an identical manner to that described for method 2 above. However, there is no need to adjust the resistance of the mannitol with magnesium sulfate, and 40% PEG is added to the protoplasts in place of the 24% (v/v) PEG. The protoplasts are transferred to the Petri dishes 10 min after addition of the PEG, and agarose added 10 min later. This protocol gives efficiencies in the range of 10-4 to 10-3. Method 4. Cotransformation: This is carried out using method 2 given above. The gene to be cotransformed into the protoplasts is added instead of the carrier DNA, i.e., at 50/xg/ml (20/zg/sample). This may be linearized before use, as this appears to give a higher rate of cotransformation. In experiments using a zein gene as the nonselected gene, 88% of the kanamycin-resistant colonies recovered contained sequences hybridizing to the zein gene sequences, with 27% containing a full copy of the zein gene clone u s e d . 36

Protoplast Culture, Selection of Transformed Lines, and Regeneration of Plants. Selection in the agarose bead-type culture s y s t e m 17'37 has been found to be superior to selection in other culture systems tested. In this way a nearly constant selection pressure is maintained during the first 4 weeks of culture, thus suppressing any possibility of background colonies arising due to reduction in the selection pressure by the decay of the drug. The dishes containing the protoplasts are sealed with Parafilm and incubated at 24 ° for I day in the dark followed by 6 days in continuous dim light (500 Ix, cool fluorescent Sylvania "daylight" tubes). The agarose containing the protoplasts is then cut into quadrants and cultured in a 1 : I mixture of K3A and H media in the bead-type culture system 17using one container with 30 ml medium for each 6-cm Petri dish from methods 2 and 3, and three containers with 30 ml of medium/10-cm dish from method 1. Kanamycin sulfate (50/.,g/ml) is added to this and to all subsequent media for selection of transformants. Aliquots of the agarose containing the protoplasts can be cultured as a nonselected control in medium lacking kanamycin. After 1 week one-half of the medium is replaced with a 1 : 1 34 W. H. R. Langridge, B. J. Li, and A. A. Szalay, Plant Cell Rep. 4, 355 (1985). 35 W. H. R. Langridge, B. J. Li, and A. A. Szalay, this volume [20]. 36 R. Scocher, R. D. Shillito, M. W. Saul, J. Paszkowski, and I. Potrykus, Bio/Technology 4, 1093 (1985). 37 I. Potrykus and R. D. Shillito, this series, Vol. 118, p. 549.

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mixture of KC3 and J media (Table I), and thereafter one-half the medium is replaced weekly with a 1 : 1 mixture of K3E and J media (Table I). Resistant clones are first seen 3-4 weeks after the start of the experiment, and after a total time of 5-6 weeks (when 2-3 mm in diameter) they are transferred to LS medium 38 (Table I) solidified with 0.8% (w/v) cleaned agar. Regeneration of Plants. After 3-5 weeks, depending on the size of the original colony, these should reach 1 cm in diameter. Each colony is then split into four parts and two placed on fresh LS medium as above, and two on LS medium with 0.2 mg/liter BAP as the only phytohormone (regeneration medium). These latter dishes are incubated in the dark for 1 week and thereafter in the light (3000-5000 Ix). Shoots arising from the callus on regeneration medium are cut off when 1-2 cm long and placed on LS medium as above, but without hormones, where they produce shoots. When the shoots reach 3-5 cm in length they are transferred to T medium and treated as shoot cultures (see above). They can be transferred to soil once they have an established root system: the agar is gently washed away and the plantlets potted up. They require a humid atmosphere for the first week and can then be hardened off and grown under normal greenhouse conditions. Genetic Analysis. When regeneration of fertile plants from tissue culture is possible then the introduced trait can be followed in its transmission to progeny. The earliest opportunity to observe this is by culture of the male gametes via another culture. 39,4° Haploid plantlets developed from microspores can be tested under selective conditions by transfer to kanamycin-containing media (300 mg/liter kanamycin sulfate) when they are at the "seedling" stage. For a single dominant gene one expects approximately 50% of the plants to be resistant due to the segregation of the trait during meiosis. For genetic analysis, plants of N. tabacum are grown in the greenhouse to flowering. They are then selfed and crossed in both directions with wild-type SR1 plants. Seeds are collected from individual capsules, and stored in a dry place at room temperature. Nonsterilized seed is sown on half-strength NN69 medium 29 (Table I) containing 300/.tg/ml kanamycin sulfate and solidified with 0.8% cleaned agar. 41 Seeds germinate at a 38 E. M. Linsmaier and F. Skoog, Physiol. Plant. 18, 100 (1965). 39 N. Sunderland and J. M. Dunwell, in "Plant Cell and Tissue Culture" (H. E. Street, ed.), p. 233. Univ. of California Press, Berkeley, 1977. 40 E. Heberle-Bors, Theor. Appl. Genet. 71, 361 (1986). 41 I. Potrykus, J. Paszkowski, M. Saul, J. Petruska, and R. D. Shillito, Mol. Gen. Genet. 199, 169 (1985).

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high frequency and, after I week, seedlings can be scored for resistance to kanamycin. Green seedlings are resistant, white sensitive. The counts are compared to the expected segregation ratios for one or more independent loci for the resistance character using the chi-square test. A high proportion of the regenerated plants are fertile and pass the introduced genes to their progeny in a normal dominant Mendelian fashion, but a small proportion of the plants are disturbed in their fertility.

3. Preparation, Transformation, and Culture of Leaf Mesophyll Protoplasts from Greenhouse-Grown Plants (Petunia hybrida) The method described for protoplast isolation is based on that of Durand et al. 42and modified in our laboratory for use with the "cyanidin type, ''43 Mitchell, 44 and other petunias. A high division frequency and rate of conversion to colonies of protoplasts from a number of Petunia species is achieved by this method. Source of Material. Plants of P. hybrida and other Petunia species are clonally propagated via cuttings, grown in clay pots in a controlled environment [12 hr light (5000 Ix), 27/20 ° day/night, 50/70% relative humidity], and watered morning and evening with commercial fertilizer [0.01% (v/v) GreenzitR]. Preparation of Protoplasts. Young leaves at approximately two-thirds to three-quarters of their final size from 4- to 6-week-old plants are washed with tap water and sterilized by immersion for 8 min in 0.01% (w/v) HgC12 solution containing 0.05% (w/v) Tween 80, and then washed carefully with five changes of sterile distilled water (each change 5 min). Leaf halves without midribs are wet with osmoticum P1 [0.375 M mannitol, 0.05 M CaCl2,0.2% (w/v) MES, pH 5.7] and arranged in a stack of six on the lid of a 9-cm Petri dish ready for cutting. They are cut diagonally into clean sections 0.5 mm wide, transferred into a small screw-top flask containing 10 ml of enzyme solution [2% (w/v) cellulase "Onozuka" R10, 1% (w/v) hemicellulase, and 1% (w/v) pectinol in 0.3 M mannitol, 0.04 M C a C I 2 , 0 . 2 % ( w / v ) MES, pH 5.7], and vacuum infiltrated until the leaf tissue is translucent. The vacuum infiltration is carried out by placing the screw-top flask containing the leaf pieces and enzyme in a larger chamber (a small desiccator can be used), and applying a vacuum (-700 mm Hg) while gently shaking the material. The air present in the intracellular spaces in the leaf pieces will expand and bubble out. The

42 j. Durand, I. Potrykus, and G. Donn, Z. Pflanzenphysiol. 69, 26 (1973). 43 D. Hess, Planta 59, 567 (1963). 44 A. Z. Mitchell, B.A. thesis. Harvard Univ., Cambridge, Massachusetts.

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pressure is then gently allowed back through a sterile filter. It may be necessary to repeat the infiltration one or two times to completely remove the air from the leaf spaces. The leaf slices are placed in fresh enzyme solution in a Petri dish (0.5 g/10 ml in a 9-cm Petri dish), which is sealed with Parafilm and incubated at 28 ° for - 3 hr. The incubation mixture is checked periodically under the inverted microscope for the release of protoplasts. The time required may vary, especially with greenhouse-grown material. The digest is gently agitated, filtered through a 100-/zm mesh stainless steel sieve, and transferred in 5-ml aliquots into 10- to 15-ml centrifuge tubes. Osmoticum P1 is added (5 ml) to each tube and, after gentle mixing, these are centrifuged for 5 min to sediment the protoplasts. The supernatant is carefully pipetted off, and the sediment is gently shaken to free the protoplasts before resuspension in 10 ml of osmoticum P1. Washing by sedimentation is repeated two times. If necessary, the suspension is overlaid on 0.6 M sucrose to remove debris and the protoplasts collecting at the interface recovered and resuspended in osmoticum P1. A sample is taken and diluted in osmoticum for counting and the protoplasts sedimented once more and resuspended in medium (K0, 45 Table I) at 1 × 106/ml.

Transformation Method 1. "F medium" method: Transformation is carried out as described for N . tabacum protoplasts above (protocol 2; method 1). After addition of the F medium, the protoplasts are resuspended in K0 culture medium at 2 × 105/ml. To 5 ml of this culture medium in a 9-cm-diameter Petri dish is added 5 ml of liquefied K0 medium containing 1.2% (w/v) SeaPlaque agarose. The protoplasts are dispersed by gentle swirling, and the agarose allowed to solidify. The dishes are sealed with Parafilm and cultured as described below. Method 2. Quick method: The protoplasts are suspended following the last purification step in osmoticum PI at 106/ml. Aliquots of 1 ml are placed in 10-ml polycarbonate centrifuge tubes, the protoplasts subjected to a heat shock as described above, and DNA (10/zg pABD1 plus 50/zg calf thymus DNA in 50/zl water) added. After 1 min, 0.5 ml of the PEG solution [40% (w/v) PEG 6000 in osmoticum P1) is added, followed 10 min later by 10 ml osmoticum P1. The protoplasts are collected by centrifugation, and plated in culture medium as described in method 1 above at 1 x 105/ml. 45 H. Koblitz a n d D. Koblitz, Plant Cell Rep. 1, 147 (1982).

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Method 3. Electroporation: The protoplasts are resuspended after purification at a density of 1.2 × 106/ml in 0.4 M mannitol containing 6 mM MgCI2 as for electroporation of N. tabacum, and treated as described for N. tabacum above (protocol 2, method 2). A pulse voltage of 1.2 kV is delivered from a capacitor of 10 nF. They are then embedded, in the same way as used for N. tabacum, in K0 medium solidified with 0.6% (w/v) SeaPlaque agarose at 1 × 105/ml. Culture and Selection of Transformants. Agarose-solidified cultures are incubated for 6 days in the dark at 26°. For plates containing 10 ml of protoplasts, half the protoplast-containing agarose gel is transferred to each of two 10-cm-diameter containers each containing 40 ml of liquid K0 medium containing 0.2 mg/liter, 2,4-D, 0.5 mg/liter BAP, and 2% (v/v) coconut milk. These are incubated at 26° on a gyratory shaker (60 rpm, 1.2 cm throw) in the light (500-1000 Ix). The liquid medium is replaced weekly, each time reducing the glucose concentration in the original medium by one-quarter so as to reduce the osmotic pressure. After 5-6 weeks the colonies are 1-2 mm in size and can be cultured further. Regeneration of Plants. Colonies are transferred directly to the regeneration medium (NT, 46 Table I) containing 16 mg/liter zeatin and solidified with 1.0% (w/v) SeaPlaque agarose (16 colonies/6-cm-diameter Petri dish). These are cultured in the conditions used for the growth of plants. The dark green calli which arise after 2-4 weeks are transferred to the same medium containing only 2 mg/liter zeatin. Those showing shooting morphology are placed on hormone-free medium to allow outgrowth of these and subcultured further as cuttings. In general, three such passages are necessary before shoots showing a "normal" morphology are obtained and can develop a strong enough root system to be transferred to soil in the greenhouse. 4. Preparation, Transformation, and Culture of Protoplasts from a Nonmorphogenic Suspension Culture of the Graminaceous Monocot Species Lolium multiflorum Protoplasts from leaf or other whole plant tissues of grasses do not in general divide in culture although there have been exceptions to this rule. 47 However, there are a number of suspension cultures of graminaceous species available, and these have been used to produce protoplasts T. Nagata and I. Takebe, Planta 99, 12 (1971). 47 I. Potrykus, in "Advances in Protoplast Research" (L. Ferenczy and G. L. Farkas, eds.), p. 243. Hungarian Academy of Sciences, Budapest, 1980.

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which divide. There have been reports of division and colony formation of protoplasts from morphogenic suspension cultures of these species,a8 but these have not yet proved to be repeatable. We describe a protocol developed in our laboratory for the isolation, transformation, and culture of protoplasts from Lolium multiflorum (Italian ryegrass) suspension culture cells. Source o f Material. The cell line was originally established by P. J. Dale, who has also used it for protoplast culture. 49 Suspension cultures are maintained by weekly serial transfer (1:7 dilution) in CC medium 5° (Table I) without mannitol and with 2 mg/liter 2,4-D on a gyratory shaker (110 rpm, 2 cm throw) in low light levels (500 Ix). Preparation of Protoplasts. Cultures are used for protoplasting, 4, 5, or 6 days after subculture. Cells (I0 ml) are sedimented by centrifugation (5 min) and resuspended in the same volume of enzyme solution [4% (w/v) Driselase in 0.38 M mannitol, 8 mM CaC12, MES, pH 5.6]. The solution is poured into a 9-cm Petri dish and this is sealed and placed on a rocking table for 1 hr at 20° before being incubated overnight (15 hr) without agitation at the same temperature. The preparation is then placed on the rocking table for an hour followed by another hour without agitation. The protoplasts are filtered through a 100-/zm mesh stainless steel sieve, an equal volume of 0.2 M CaC12 (MES, pH 5.8) added, and the suspension distributed into two centrifuge tubes. After centrifugation to sediment the protoplasts (10 min) they are taken up in 3 ml osmoticum L1 (0.25 M mannitol, 0.1 M CaCI2, MES, pH 5.8) and overlayered on a 5-ml sucrose cushion (0.6 M sucrose, MES, pH 5.8). Protoplasts collecting at the interface after centrifugation are carefully removed and washed twice with osmoticum L1, counted, and resuspended in CC medium (Table I) at a density of 2 x 106/ml.

Transformation Method 1. "F medium" method: One-milliliter aliquots of the protoplasts, in CC medium, are heat shocked and treated with DNA as described for N. tabacum in protocol 2, method 1 above. Following the addition of the " F " medium, they are sedimented by centrifugation (5 min), and resuspended in 2 ml of CC culture medium. 4s V. Vasil and I. K. Vasil, Theor. App. Genet. 56, 97 (1980). 49 M. G. K. Jones and P. J. Dale, Z. Pflanzenphysiol. 105, 267 (1982). 50 I. Potrykus, C. T. Harms, and H. Loerz, Theor. Appl. Genet. 54, 209 (1979).

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Culture and Selection of Transformants. The protoplasts are cultured in 3.5-cm-diameter Petri dishes (2 ml/dish) at 26° in the dark. Fresh CC medium (I ml) with 0.2 M mannitol is added after 7 days to dilute the culture and reduce the osmotic pressure. Two weeks after the DNA treatment, the cultures are sedimented and the cells taken up in the 3 ml of CC medium with 0.2 M mannitol containing 25 mg/liter G418. After a further 2 weeks these cultures are diluted 5-fold with CC medium without mannitol, and containing G418 as before. After a total of 6 weeks, calli arising from the cultures are transferred to CC medium as used for the suspension cultures solidified with 0.8% (w/v) cleaned agar and grown at 24° in the dark or in the light (2000 Ix). 5. Preparation, Transformation, and Culture of Protoplasts from a Sterile Shoot Culture of N. plumbaginifolia, and Regeneration of Plants Source of Material. This material is grown as sterile axenic shoot cultures. They are grown as described for N. tabacum above except that the medium used is Murashige and Skoog's medium 51 with 10 g/liter sucrose, as described by Negrutiu) 2 Preparation of Protoplasts. For isolation of protoplasts, sterile leaves from shoot cultures are sliced carefully into 2-mm-wide strips using a sharp razor blade and these incubated overnight at 26° in enzyme solution [0.5% (w/v) Driselase in 0.5 M sucrose, 0.005 M CaCI2, pH 5.5, with KOH]. The released protoplasts are filtered through 100- and 66-~m stainless steel sieves, and centrifuged for 5 min. The protoplasts at the surface are collected and washed two times with W5 salt solution (154 mM NaC1, 125 mM CaCI2,5 mM KC1, 5 mM glucose, pH 5.5, with KOH53]. They are resuspended in W5 solution at a final density of 1-1.6 × 106 and used immediately for transformation. Alternatively, they can be stored in the W5 solution for up to 6 hr at 6-8 ° before use. Transformation Method 1. PEG method: Aliquots (1 ml) of protoplasts are placed in centrifuge tubes, and a heat shock applied as described for N. tabacum. A mixture of linearized plasmid DNA (I0/xg/ml) and carder DNA (50/xg/ml calf thymus DNA) is added, followed by 1.0-1.5 ml PEG solution [45% (w/v) PEG 4000, 2% (w/v) Ca(NO3)2 • 4H20, 0.4 M mannitol, pH adjusted 51 T. Murashige and F. Skoog, Physiol. Plant. 15, 473 (1962). 52 I. Negrutiu, Z. Pflanzenphysiol. 1114, 431 (1981). 53 L. Menczel, G. Galiba, F. Nagy, and P. Maliga, Genetics 100, 487 (1982).

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to 9 with KOH repeatedly over a period of 4 hr and autoclaved; stored at - 2 0 °, final pH 7.5-8.5] with gentle shaking to give a final concentration of 22-27% PEG. After incubation with the PEG for 20-30 min, W5 solution is added 3 × in 1-ml aliquots at 2- to 5-min intervals, and the protoplasts centrifuged for 5 min. The pellet is resuspended in 10 ml culture medium (K3 medium with 0.4 M glucose 27) and cultured in a 10-cm-diameter dish. Alternatively, the protoplasts can be embedded in the same culture medium solidified with 0.6% (w/v) SeaPlaque agarose as described above for N. tabacum. Method 2. Electroporation: The protoplasts can be transformed by electroporation as described for N. tabacum above. Following the enzyme incubation, the released protoplasts are cleaned and transformed exactly as described for N. tabacum in protocol 2. Culture and Selection of Transformants. At the 2- to 4-cell stage, the developing cultures are diluted 6- to 8-fold (to -2000 surviving colonies/ ml) in low-hormone medium MDS 54with 25 mg/liter kanamycin sulfate. In the case of agarose-embedded cells, these are suspended at a similar dilution factor as used for liquid medium in liquid MDS medium with (30 ml) or without (10 ml) shaking. The dishes are incubated at 1000-1500 Ix at 26° for 3-5 weeks, when colonies should become visible. Transformation frequencies of 10-4 to 10-3 should be obtained. It has not yet been possible to attain higher frequencies with this species despite many experiments to this end. Calli are transferred to MDS medium solidified with 0.8% (w/v) cleaned agar containing 50 mg/liter kanamycin. After a further 2-4 weeks they can be transferred to RPZ medium55 for regeneration, and shoots arising are cultured as shoot cultures as described for the source material, and transferred to the greenhouse. Evaluation of Results Colonies which are selected as being resistant should be further analyzed for proof of transformation. Although with the system described we have never observed resistant colonies from control cultures with N. tabacum or P. hybrida, variations in the many factors present, particularly when using other protoplast systems, may lead to misinterpretation of apparently resistant colonies as transformants. 54 I. Negrutiu, R. Dirks, and M. Jacobs, Theor. Appl. Genet. 66, 341 (1983). 55 p. Installr, I. Negrutiu, and M. Jacobs, J. Plant Physiol. 119, 443 (1985).

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We feel that the minimal criteria for confirmation of a transformation event should be the following: 1. A phenotypic change to resistance, or growth under other selective conditions, in a selection scheme which is proved to be "clean" 2. The presence for the transforming DNA in the selected lines in form expected for transformed DNA (integrated in the genome or autonomously replicating) 3. Expression of the foreign DNA at the RNA/protein level In addition, if the plant cell tissue culture system being used is capable of regenerating plants, then genetic data should be obtained.

Phenotypic Change The assumption of direct selection on kanamycin (or G418) is that only transformed cell lines will be phenotypically resistant to the drug. Ideally the level of selection should permit recovery of only transformed clones. Therefore resistant clones should only appear after transformation with the correct vector and not in any control treatments. Selection conditions should be adjusted to produce this situation. The resistant phenotype should be rechecked at later stages in culture by comparison with wild type, for instance at the callus level. In the case of N. tabacum, shoots can be regenerated from transformed callus under selective (100 mg/liter kanamycin sulfate) or nonselective conditions. These shoots can be rooted in medium containing 150 mg/liter kanamycin sulfate. Wild-type SR1 shoots regenerated in the absence of kanamycin never form roots, bleach, and die when cultured in the presence of kanamycin. In order to confirm the resistant phenotype of the cells of such regenerated transformed plants or to show the possible loss of the introduced trait during plant development, mesophyll protoplasts from these plants can be isolated and their resistance checked by culture in kanamycin-containing media.

Molecular Analysis of the DNA of Transformed Clones and Regenerated Plants Analysis ofDNA. DNA of transformed cell lines, regenerated plants, and their progeny should be analyzed using standard Southern blot techniques. 56 We will omit the exact procedures used since they are now standard in molecular biology and the choice of restriction enzymes, etc., 56 E. M. Southern, J. Mol. Biol. 98, 503 (1975).

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will depend on the transforming DNA being analyzed. It should, however, be possible to prove unequivocally the presence of the transforming DNA in high-molecular-weight nuclear DNA of transformed lines in an integrated form in the absence of any hybridization to DNA from control untransformed lines. We shall concentrate here only on a method for the efficient isolation of DNA from small amounts of callus or plant tissue. The relatively slow growth rate of plant cell tissue cultures (-1 week doubling time for callus on solid media) means that the adaption of DNA extraction procedures to small amounts of tissue allows a significant shortening in the length of an experiment. We have adapted a method of Thanh Huynh (personal communication, Dept. of Biochemistry, Standford University), which allows the extraction of pure DNA from - 0 . 5 g of tissue: Samples of 0.5 g of callus or leaf tissue are homogenized in a Dounce homogenizer in 3 ml of a buffer containing 15% sucrose, 50 mM EDTA, 0.25 M NaC1, 50 mM Tris-HC1, pH 8.0. Centrifugation of the homogenate for 5 min at I000 g results in a crude nuclear pellet which is resuspended in 2 ml of a buffer containing 15% sucrose, 50 mM EDTA, 50 mM TrisHC1, pH 8.0. SDS is added to a final concentration 0.2% (w/v). Samples are heated for 10 min at 70°. After cooling to room temperature, potassium acetate is added to a final concentration of 0.5 M. After incubation for 1 hr at 0° the precipitate formed is sedimented for 15 min in an Eppendoff centrifuge at 4°. The DNA in the supernatant is precipitated with 2.5 vol of ethanol at room temperature and redissolved in 10 mM Tris-HCl, pH 7.5, 5 mM EDTA. The DNA samples are then run in a cesium chloride/ethidium bromide gradient in the vertical rotor (Beckman VTi 65) for 17 hr at 48,000 rpm. The DNA is removed from the gradient with a widebore hypodermic syringe needle and the ethidium bromide extracted as for plasmid DNA (see above). The DNA obtained is of high molecular weight and susceptible to various restriction enzyme. For Southern analysis 5-10 /zg DNA is electrophoresed in a 1% agarose gel, transferred to a nitrocellulose membrane, and hybridized with nick-translated DNA 57 (5-10 x 108 cprn//zg). Filters are washed with 2x SSC at 65 °, 3 z for 1 hr, and subsequently exposed to X-ray film with intensifying screens for 24-48 hr.

Activity Assay for the Product of the Transforming Gene The assay for activity of the transformed gene will of course depend on the expected product. In the case described here, a method developed s7 W. J. Rigby, M. Dieckmann, C. Rhodes, and P. Berg, J. Mol. Biol. 113, 237 (1977).

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by Reiss et al. 58 allowed us to detect activity of the APH(Y)II gene product in transformed lines. Callus or leaf pieces (100-200 mg) are crushed in an Eppendorf centrifuge tube with 20/xl extraction buffer [100% (v/v) glycerol, 0.1% (w/v) SDS, 5% mercaptoethanol, 0.005% (w/v) bromphenol blue, 0.06 M Tris, pH 6.8]. Extracts are centrifuged for 5 min at 12,000 g. Proteins in 35/.d of the supernatant are separated on a 10% nondenaturing polyacrylamide gel. The gel is incubated with kanamycin and 32p-labeled ATP and then blotted onto Whatman P81 phosphocellulose paper. The paper is washed five times with deionized water, wrapped in plastic film, and exposed to X-ray film with intensifying screens for 24-48 hr. Kanamycin binds to this paper but ATP does not, therefore radioactive bands on the paper reveal bands on the gel with an activity which transfers radiolabeled phosphate from the [32p]ATP to the kanamycin, i.e., aminoglycoside phosphotransferases. Other transformant nonspecific bands are seen at the top of the gel. These are protein phosphorylases, and the bands can be removed from the P81 paper by treatment with protease if required) 9 General Comments

Comparison with Other Gene Transfer Systems A. tumefaciens-mediated gene transfer was, for a while, the only possible way to introduce foreign genes into plants. Although it is a wellestablished method, there has been little attempt until recently to study the fate of genes introduced in this way. There is also increasing evidence that the integration patterns found when using this method can be complex and show rearrangements of the original T-DNA. One of the great drawbacks of the method is the limited host range, which excludes, for instance, the graminaceous monocots. However, this will remain the method of choice for introduction of foreign DNA in most cases. DNA viruses have been suggested as possible gene vectors. To date there is one example of this, in which cauliflower mosaic virus carrying a modified bacterial methotrexate-resistance gene was used to infect a plant. The foreign gene was thus transported into and systemically spread in the plant) °,6° The advantages of this system are the ease of infection, systemic spread within the plant, and multiple copies of the gene being present per cell. The disadvantages are the narrow host range, lack of 58 B. Reiss, R. Sprengel, M. Willi, and H. Schaller, Gene 30, 217 (1984). 59 p. H. Schreier, E. A. Seftor, J. Schell, and H. J. Bonnert, EMBO J. 4, 25 (1985). 6o N. Brisson and T. Hohn, this series, Vol. 118, p. 659.

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transfer to sexual offspring and the limited space available for passenger DNA. Liposome fusion has been shown to be a method for transformation of plant cells.'1 As with direct gene transfer, this requires protoplasts. It will be of interest to see whether the pattern of integration of DNA by this method is different than that seen when using other methods. Microinjection is an efficient means of transforming plant protoplasts.13 However, the number of transformants that one can obtain is limited by the number which can be injected by the operator. The main use of this technique may be that it could be possible eventually to inject cells, thus circumventing the need for protoplasts which is inherent in other direct gene transfer techniques such as described above. Conclusions The procedures described above require a protoplast system which allows regeneration of callus from protoplasts and a suitable selectable marker gene. If these requirements are satisfied the methods for DNA delivery and selection of transformants are flexible. The protocols described here are certainly applicable to other systems and are reproducible in our hands. Acknowledgments The authors thank all the members of our laboratory, particularly M. W. Saul, J. Paszkowski, I. Negrutiu, and S. KrugerLebusfor help in supplyinginformationand protocols for this publication.

[20] U p t a k e o f D N A a n d R N A into Cells M e d i a t e d by Electroporation By W. H. R. LANGRIDGE, B. J. LI, and A. A. SZALAY

Introduction To study the regulation of eukaryotic gene expression, a reliable and efficient method for introduction of altered genes into cells is required. Zimmerman I described an electric shock method (electroporation) for the 1u. Zimmermann,in "Target Drugs" (E. Goldberg,ed.), p. 153. Wiley,New York, 1983. METHODS IN ENZYMOLOGY, VOL. 153

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.