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Plant Physiol. (1979) 63, 683-686 0032-0889/79/63/0683/04/$00.50/0 Binding of ColEl-kan Plasmid DNA by Tobacco Protoplasts NONEXPRESSION OF PLASMID GENE Received for publication September 13, 1978 and in revised form December 6, 1978 LOWELL D. OWENS United States Department of Agriculture, Science and Education Administration, Agricultural Research, Plant Physiology Institute, Beltsville, Maryland 20705 ColEI-kan plasmid DNA. ColEI-kan plasmid (pML2) is a 9megadalton, nonconjugative hybrid of ColEI and pSC105 plasmids and bears a gene for detoxifying kanamycin (5). ABSTRACT Protoplasts prepared from cultured tobacco cells were treated with ColEl-kan plasmid DNA, a hybrid of ColEl and pSC105 plasmids bearing a gene for kanamycin resistance. The conditions employed permitted the uptake or irreversible binding of 2.9% of the added DNA in acid-insoluble form. Upon commencement of division, the treated cells were plated in agar medium containing kanamycin and differentiating hormones. Plantlets or shoots obtained as presumptive transformants were further tested on kanamycin medium by subculturing small leaf pieces. No evidence was obtained for expression of the kanamycin resistance gene of ColEl-kan in tobacco tissue. MATERIALS AND METHODS Protoplast Isolation and Culture. Protoplasts of Nicotiana tabacum L. "Bright Yellow" were isolated from cell suspension cultures mainly by the methods of Uchimiya and Murashige (14, 15) but with the following changes. One g (fresh weight) of cells was incubated for 2.5 h in 40 ml of enzyme solution (pH 5.7) containing 9.25 mm CaCl2, 0.44 mm Ca(H2PO4)2, 0.85% Cellulysin (Calbiochem),2 0.5% Driselase (Kyowa Hakko), 1% Rhozyme HP150 (Rohm and Haas), and 0.7 M mannitol. The Rhozyme used had been desalted on Sephadex G-25 and lyophilized. The cell suspensions were gently agitated manually at about 15-min intervals. The protoplast wash solution was 0.7 M mannitol, 8.5 mi The demonstration by Aoki and Takebe (1) in 1969 that purified CaCl2, and 0.5 mm Ca(H2PO4)2 (filter-sterilized separately and RNA from TMV' could infect protoplasts of tobacco suggested added after autoclaving) (pH 5.8). Protoplasts were counted in a hemocytometer using the excluthe possibility that protoplasts of higher plants might be genetision of Evan's blue dye (2 ,lI/chamber of 0.1% in 0.7 M mannitol) cally altered by uptake of foreign nucleic acids. Ohyama et al. (11) provided the first evidence that protoplasts could take up DNA, to indicate viability. Washed protoplasts were treated with DNA and a number of investigators subsequently have sought to deter- (see below) and cultured at a density of 105/ml in protoplast mine the physical and biological fate of exogenous DNA within culture medium (14) with 0.045 M glucose and 0.6 M mannitol. protoplasts (3, 6, 8-10, 13). Although methods developed for After 4 days of incubation, fresh medium was added (0.5 of infecting protoplasts with viral RNA have proven highly efficient, original volume), and at 8 days the cells were embedded in a 1nearly 100%o infection rates with production of over 106 viral mm-thick layer of agar medium (15) in plastic Rodac plates (65 particles per protoplast within 48 h (12), parallel results have not x 55 mm) (Falcon). Microcalli were counted by inverting the been achieved with either viral or nonviral DNA. Uchimiya and plate on the stage of a microscope. Screen for Kanamycin Resistance after Treatment with DNA. Murashige (15) regenerated some 500 tobacco plants from protoplasts that had been treated with DNA isolated from plants Thirteen days after embedding in agar, the microcalli were transresistant to TMV, but none of the regenerants proved resistant to ferred en masse in slabs of agar (1.7 x 2.4 cm) to the surface of the virus. Likewise, a preliminary report by Carlson (2) recounted shoot-inducing agar (25 ml in plastic Petri dishes, 95 x 15 mm) an unsuccessful attempt to transform protoplasts from an auxo- medium (14) with tyrosine deleted, 5 ,UM 2-isopentenyl adenine trophic haploid tobacco plant with DNA isolated from a wild type (Sigma) substituted for kinetic, IAA lowered to I t,M, and 10 tIM kanamycin sulfate (Sigma) added. Each dish contained six slabs tobacco plant. with a total of about 1,500 microcalli. In this initial screen for An inherent problem of using eukaryotic chromosomal DNA for transformation experiments is its high mol wt (equivalent to kanamycin resistance, most microcalli turned brown and grew about 6 x 1012 daltons of DNA in diploid tobacco) and the very slowly or not at all. Calli which turned green and differencorrespondingly low concentration of DNA specifying a particular tiated buds or small shoots (2-3 mm) were transferred to fresh gene. Given the DNA uptake rates commonly obtained with medium containing 10 AM kanamycin. Microcalli which tumed protoplasts (15), the probability of a cell taking up one copy of a green but exhibited no shoot differentiation at the 1-mm-diameter stage were transferred to shoot-induced medium (14) having 9.3 gene occurring once in the genome is about 1 in 200 cells. Cloning genes in small plasmid vectors provides a way of ylM kinetin and 11.4 uM IAA but with tyrosine deleted and 10 tIM obtaining DNA that is highly concentrated in a particular gene. kanamysin sulfate added. In parallel experiments without kanaThe probability of obtaining transformation with such molecules mycin, all microcalli turned green and grew very rapidly on the first medium, and some shoot regeneration occurred. When unmay therefore be enhanced (7). In this report we describe an attempt to detect expression of the 2 Mention of a trademark, proprietary product, or vendor does not kanamycin resistance gene in tobacco protoplasts treated with constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other 'Abbreviation: TMV: tobacco mosaic virus. products or vendors that may also be suitable. 683 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 1979 American Society of Plant Biologists. All rights reserved. 684 Plant Physiol. Vol. 63, 1979 OWENS differentiated calli 1 mm in diameter were then transferred to the second medium, over 90o regenerated shoots. Upon reaching a height of 1 to 2 cm, the shoots were transferred to rooting medium (15) with IAA omitted. Plantlets surviving the first kanamycin resistance screen were tested further by excising 10 replicate leaf pieces (2 x 3 mm) and placing them on the first shoot-inducing medium described above containing 0 to 16 yIM kanamycin. Each plastic Petri dish (90 x 24 mm) contained five leaf pieces and 25 ml of medium. The dishes were incubated in continuous light (500 lux) at 27 C for 30 days at which time the tissue, consisting almost entirely of differentiated shoot growth, was harvested, dried, and weighed. Plasmid Isolation. The two strains of bacteria used, JC41 1 thypro-IColE I and C600/ColE I-kan, were derivatives of Escherichia coli K12 (5) and were provided by Dr. Donald Helinski. Stock cultures were maintained on L-broth agar (5). Covalently closed circular plasmid DNA was prepared by growing the bacteria in 500 ml of a modified M9 medium with thymine (2 ,ig/ml) added, where required, as described (5). When the cells reached a density of about 5 x 108/ml, chloramphenicol (300 ,ug/ml) was added, and cells were harvested 16 h later. To prepare labeled plasmid, 2 mCi of [methyl-3H]thymidine (New England Nuclear) were added I h after the chloramphenicol addition. A cleared lysate was prepared and centrifuged for 40 h at 36,000 rpm at 20 C in a dye-CsCl gradient in a Beckman type 65 rotor. The lower DNA band was removed, extracted of dye, and concentrated by ethanol precipitation as described (5). The DNA was stored at -20 C in SSC (0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) containing 5 mM EDTA. Agarose slab gel electrophoresis of purified DNA was performed as described (5). Radioactivity in gel slices was determined by melting the agarose at 100 C in 0.3 ml of water and dissolving in 10 ml of Aquasol-2 liquid scintillation cocktail (New England Nuclear). The various plasmid preparations were shown by agarose gel electrophoresis to be predominantly (75% or more) the covalently closed circular form. Plasmid DNA Uptake. Plasmid DNA was diluted in 0.5 ml of 0.1 x SSC, filter-sterilized (0.2-,um pore), and added to a sterile polystyrene tissue culture tube (16 x 125 mm) containing protoplasts suspended in 1.5 ml of the protoplast culture medium described above except with 0.8 M glucose as the osmoticum and with 0.66 [Lg/ml poly-L-ornithine (mol wt 122,000, Sigma) added. After incubation for indicated times on a tube roller (1 rpm) at 25 C, 13 ml of protoplast wash solution was added, and the suspension was centrifuged (100g, 3 min). The pelleted protoplasts were treated with DNase by additions of 0.9 ml of 0.7 M mannitol containing 66 mt KH2PO4 (pH 5.8) and 10 mM MgCl2; 15 ,lI (10 units) of EcoRI endonuclease (Miles Laboratories, Inc.); and 0.1 ml DNase I (Sigma, bovine pancreas, 1 mg/ml in 30 mm MgCl2). The resuspended protoplasts were incubated at 37 C 10 min. Protoplasts were washed three times by centrifugation, lysed by addition of 30 tdl of 10 x SSC and 30 ,ul of 10%1o sodium lauryl sulfate to the pellet, and heated at 65 C for 10 min. Aseptic conditions were maintained to this point. Acid-insoluble radioactivity was determined by addition of 0.5 mg of BSA followed by precipitation with an equal volume of cold 10%7o trichloroacetic acid. After 20 min on ice, the sample was centrifuged at 10,000 rpm for 10 min, washed with cold 5% trichloroacetic acid, and centrifuged. The pellet was dissolved in 0.4 ml of 0.1 x SSC and counted in 5 ml of Aquasol-2. Where protoplasts were being tested for expression of kanamycin resistance, the DNase treatment was omitted, and the washed (3 X) protoplasts were cultured as described above.3 3Biohazard containment. The work involving the recombinant plasmid ColE 1 -kan was performed under P2 laboratory conditions as specified by the NIH Guidelines for Recombinant DNA Research. All plants regenerated from protoplasts treated with ColEl-kan DNA were cultured in vitro and, along with bacteria and DNA preparations, destroyed by autoclaving before disposal. RESULTS Binding of 3H-labeled DNA. The effect of pH on binding of 3Hlabeled ColEl-kan DNA by tobacco protoplasts was examined. The amount of radioactivity associated with the protoplasts following DNase treatment, ie. the "irreversibly bound" fraction, was about five times greater at pH 5.7 than at pH 7.3. Likewise, the portion of irreversibly bound radioactivity that was polymeric (trichloroacetic acid-insoluble) was about 20 times greater at the lower pH. The kinetics of [3H]ColEl-kan DNA binding by protoplasts at pH 5.7 is presented in Figure 1. The initial binding was very rapid, constituting 70%o of the input DNA by 12 min. The accumulation of irreversibly bound polymeric material was much slower, amounting to about 3% of input DNA in 2 h. Test for Expression of Kanamycin Resistance. A summary of two experiments in which tobacco protoplasts were treated with ColE1 or ColEl-kan DNA, cultured to the microcalli stage and then screened for kanamycin resistance, is given in Table I. Seven hundred-thousand microcalli were screened en masse on a shoot-differentiating medium containing kanamycin at a conI I I I I I --I I -I I I so U0 : . Total bound 4 600- r- 4coR la z 't 8 - D-0 =.C 'o0'r04 ZD 26 0l..~ Irreversibly bound, TCA-insoluble ._ ' 1- 20.0 O T 2 I I ,a 4 6 8 ,I 1 10 12 Hours I I 14 16 I 18 -9 a 20 Kinetics of [3H]ColEl-kan DNA bindi.:- by tobacco protoFIG. plasts. Protoplasts (6 x 106) were suspended in 2 ml of protoplasts culture medium (pH 5.7) containing 3.75 ytg [3H]ColEl DNA (74,000 cpm/,ug) and 0.5 .tg/ml poly-L-ornithine and incubated on a tube roller (I rpm) at 25 C. Aliquots removed at 0.2, 2, and 20 h contained 1.4-, 1.1-, and 0.2 x 106 viable protoplasts, respectively, and were processed as described under "Materials and Methods." "Total bound" radioactivity represents total cpm associated with protoplasts prior to treatment with DNase, and "irreversibly bound" that remaining associated after DNAase treatment. I. Table I, Summary of two experiments testing for expression of the ColEl-kan gene for kanamycin resistance in tobacco cells DNA Treatment ColEl (Control) ColEl-kan 8.2 x 10 7.3 x 10 326,000 32 272,000 52 22 47 No. protoplasts treated No. microcalli screened on kanamycin medium No. plantlets obtained No. plantlets tested further1 for kanamycin resistance 1Plantlets exhibiting gros-s morphological abnormalities or severe stunting were excluded from further testing. Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 1979 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 63, 1979 NONEXPRESSION OF ColEl-kan IN TOBACCO centration (10 ytM) previously determined to exceed by 10%o that required to prevent the growth, greening, and differentiation of microcalli. From this initial screen, 32 plantlets or shoots were obtained from cells receiving the control treatment, ColEl DNA, and 52 from cells treated with ColEl-kan DNA (Table I). Further testing of the presumptive transformants for resistance to kanamycin gave the results presented in Figure 2. The plantlet leaf pieces derived from the DNA-treated protoplasts exhibited varying degrees of tolerance to 16 jM kanamycin relative to their performance on medium lacking kanamycin. The distribution of tolerance among plantlets regenerated from protoplasts treated with ColE 1-kan DNA does not appear to differ significantly from that exhibited by plantlets from protoplasts treated with ColEl DNA. DISCUSSION The "uptake" of [3H]ColEl-kan DNA by tobacco protoplasts was characterized by an initial rapid binding of DNA to the cell membrane where it remained largely accessible to DNase. This reversible binding was followed by a slower, irreversible binding in which the protoplast-associated radioactivity was no longer accessible to exogenous DNase. The irreversible binding or uptake was approximately linear over a 20-h period and amounted to about 3% (7.5 x 10-14g DNA/protoplast) of input DNA at 2.5 h, the incubation time used in experiments where expression of the 7 70 '-1 C U- ze 0 0- e10 0 0 co 510 C 0 co 4'0 co W w 4 *0 'a 3 00~~~~~~ 0 O0. 0 0 685 plasmid-borne gene was tested. These results are quantitatively similar to those obtained by Uchimiya and Murashige (15) using Nicotiana glutinosa ?rotoplasts and [3H]DNA isolated from N. glutinosa (5.08 x 10- 4g DNA/protoplast). However, the numbers of genome equivalents represented by these two similar amounts of DNA are vastly different. In our experiment it represented about 5,000 genome equivalents per protoplast; in the latter experiments, it represented about 0.005 per protoplast. Despite the large number of genome equivalents irreversibly bound by the protoplasts in our experiments, none of the 272,000 microcalli regenerated from protoplasts treated with ColEl-kan DNA gave rise to plantlets that exhibited a greater range of tolerance to kanamycin than did plantlets from protoplasts treated with ColEl DNA. We conclude that the range of kanamycin tolerance exhibited by plantlets from protoplasts receiving ColE 1kan DNA is a manifestation of the natural variance existing in the cell population and not of expression of the gene specifying kanamycin resistance carried by ColEl-kan plasmid. This failure to obtain expression of the plasmid-borne gene in tobacco could be due to any of several reasons. First, the transformation frequency simply may be lower than 2.7 x 10-'. Second, the screening procedure employed required that the kanamycin resistance gene be maintained within the plant cell for many generations. Maintenance via autonomous replication of the plasmid would require that the plasmid be taken up physically intact by the protoplasts. This may not have occurred. The polymeric radioactivity recovered from the protoplasts may have represented DNA that was partially degraded by nucleases. Complete integrity of the plasmid DNA would not be required for maintenance of the kanamycin resistance gene if that DNA segment was integrated into the plant cell genome. Third, molecular barriers at the transcription or translation levels may exist which greatly reduce or prevent expression of cloned prokaryotic (bacterial) genes in eukaryotic cells (4). These might include transcription barriers involving promoter and termination specificities or translation barriers concerning specificities of ribosome binding to mRNA, initiation factors, and termination signals (4). From our results, we conclude that although it may be necessary to have many gene copies taken up by a protoplast, it is not alone a sufficient condition for causing genetic transformation. Careful attention needs to be paid to the design of the transformation molecule. Although it may be permissible to employ a prokaryotic gene vehicle for cloning purposes, it is probably necessary that some portion of the DNA molecule possess segments homologous to one of the DNA species of the recipient plant cell. Acknowledgments-We thankf Dr. Donald Helinski for the two plasmids and for helpful suggestions regarding their isolation. The capable technical assistance of Jean Bellows is gratefully 2 10 acknowledged. 0~ J~~~~~~~~ 1 %J 0-24 25-49 LITERATURE CITED 50- 74 75- 100 Growth of tissue (% of growth on on 16pM kanamycin medium lacking kanamycin) FIG. 2. Response to kanamycin of plantlets regenerated from protoplasts treated with ColEl or ColEl-kan DNA and which survived the initial screen for transformants. Ten replicate leaf pieces (2 x 3 mm) were placed on a differentiating agar medium containing 0 or 16 AEM kanamycin, and the callus and shoot tissue developing therefrom were harvested 30 days later. Bars represent the proportion (per cent) of plantlets tested from each treatment which had tissue dry weights within the range indicated (expressed as per cent of dry weight obtained on medium lacking kanamycin). Average dry weight of the latter was 46 mg. Data are means of 10 replicates. 1. AoKi S, I TAKEBE 1969 Infection of tobacco mesophyli protoplasts by tobacco mosaic virus ribonucleic acid. Virology 39: 439-448 2. CARLSON PS 1972 Attempts to detect DNA mediated transformation in a higher plant. Genetics (Soc Am) 71 Suppl 3 (2), S9 3. COCKING EC 1977 Uptake of foreign genetic material by plant protoplasts. Int Rev Cytol 48: 323-343 4. HELINSKI D 1977 Plasmids as vectors for cloning. In A Hollaender, RH Burris, PR Day, RWF Hardy, DR Helinski, MR Lamborg, L Owens, RC Valentine, eds, Genetic Engineering for Nitrogen Fixation. Plenum Publishing Corp, New York, pp 19-49 5. HERsHFiELD V, HW BoYER, C YANOFSKY, MA LovEn, DR HELINSKI 1974 Plasmid ColEI as a molecular vehicle for cloning and amplification of DNA. Proc Nat Acad Sci USA 71: 3455-3459 6. HFm D 1976 Uptake and expression of foreign genetic material in plant protoplasts. In JF Peberdy, AH Rose, HJ Rogers, EC Cocking, eds, Microbial and Plant Protoplasts. Academic Press, New York, pp 125-144 7. HINNEN A, JB HicKs, GR FINK 1978 Transformation of yeast. Proc Nat Acad Sci USA 75: 1929-1933 8. HUGHEs BG, FG WHTE, MA SmITH 1977 Fate of a bacterial plasmid DNA during uptake by barley protoplasts. FEBS Lett 79: 80-84 9. LiEBKE B, D HESS 1977 Uptake of bacterial DNA into isolated mesophyll protoplasts of Petunia hybrida. Biochem Physiol Pflanz 171: 493-501 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 1979 American Society of Plant Biologists. All rights reserved. 686 OWENS 10. LURQUIN PF, CL KADO 1977 Escherichia coli plasmid pBR313 insertion into plant protoplasts and into their nuclei. Mol Gen Genet 154: 113-121 I 1. OHYAMA K, OL GAMBORG, RA MILLER 1972 Uptake of exogenous DNA by plant protoplasts. Can J Bot 50: 2077-2080 12. SARKAR S. MD UPADHYA, G MELCHERS 1974 A highly efficient method of inoculation of tobacco mesophylJ protoplasts with ribonucleic acid of tobacco mosaic virus. Mol Gen Genet 135: 1-9 Plant Physiol. Vol. 63, 1979 13. SUZUKI M, I TAKEBE 1976 Uptake of a single stranded bacteriophage DNA by isolated tobacco protoplasts. Z Pflanzenphysiol 78: 421-433 14. UCHIMIYA H, T MURASHIGE 1974 Evaluation of parameters in the isolation of viable protoplasts from cultured tobacco cells. Plant Physiol 54: 936-944 15. UCHIMIYA H. T MURASHIGE 1977 Quantitative analysis of the fate of exogenous DNA in Nicotiana protoplasts. Plant Physiol 59: 301-308 Downloaded from on August 11, 2017 - Published by www.plantphysiol.org Copyright © 1979 American Society of Plant Biologists. All rights reserved.