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Plant Physiol. (1975) 55, 689-694

Isolation and Regeneration of Tobacco Mesophyll CellProtoplasts under Low Osmotic Conditions1

Received for publication August 27, 1974 and in revised form December 3, 1974

JAMES F. SHEPARD AND ROGER E. TOTTENDepartment of Plant Pathology, Montana State University, Bozeman, Montana 59715

ABSTRACT

A method is described for the isolation of large numbersof tobacco (Nicotiana tabacum L. cv. Xanthi-nc) mesophyllcell protoplasts under relatively low external osmotic con-ditions. The procedure utilized 0.2 M sucrose as the primaryosmoticum and a mixture of 0.5% macerozyme, 4% cellulase,and 2% polyvinylpyrrolidone, pH 5.4. The viability of re-sultant protoplasts was confirmed through regeneration offertile plants. Plating and regeneration studies revealed,however, that qualitative and quantitative modifications inplating and differentiation media were necessary for proto-plasts prepared in this manner. Over-all, the procedure wasfound to be a simplified alternative to those previously de-scribed for tobacco protoplast regeneration. In addition, thesystem should permit studies related to the influence of differ-ing osmoticum levels on a variety of cell functions.

Protoplasts have been isolated from tobacco mesophyll cellsby a variety of enzymatic techniques (5, 16) and have also beeninduced to regenerate normal plants (4, 10, 11). In each ofthese studies, protoplasts were prepared and subsequentlyplated for regeneration studies in the presence of a con-centrated osmoticum. It would seem desirable, nonetheless,to have available a similar technique in which protoplasts, atleast at the outset, are exposed to lesser degrees of plasmolysis.Plasmolysis is known to influence a variety of cell functions (9)and a more direct determination of additional effects should bepossible if protoplasts could be maintained within a broadrange of osmoticum concentrations.

In this paper, we report a highly efficient and consistent tech-nique for the isolation and regeneration of tobacco mesophyllcell protoplasts in solutions containing relatively low con-centrations of a primary osmoticum. During this investiga-tion, it was found that a considerable number of modificationsover those previously reported (10) were required for regenera-tion of large numbers of plants. Some of these modificationsare presumed to result from the means by which protoplastswere isolated.

MATERIALS AND METHODS

Plant Growth Conditions. The primary experimental plantspecies used for protoplast isolation was Nicotiana tabacum

'This work was supported by National Science FoundationGrant GB-26415 A2, and the study is Montana Agricultural Ex-periment Station Journal Series Paper No. 554.

L. cv. Xanthi-nc. Plants were maintained in environmentallycontrolled growth rooms in 12-inch diameter pots under thefollowing regime: 1500 ft-c of cool white fluorescent light withan 8-hr dark period, relative humidity of 70 to 75%, anda constant 23 C temperature. The soil used was a heavy loamcontaining peat moss (1/3 by vol) and plants were watered witha solution containing 1 g/l of a soluble 20-20-20 fertilizer(Peters, Inc., Allentown, Pa.). Care was taken to prevent thesoil from being continually saturated.

Protoplast Isolation. Tobacco leaves were collected soonafter they had reached 25- to 26-cm length provided that plantswere less than 0.762 m tall and had not begun to flower. Theleaves were surface-sterilized for 15 min in 0.525% sodiumhypochlorite (10% Clorox), rinsed in sterile H20 with freemoisture, then removed in a Microvoid II-C sterile air cham-ber. Leaves were finally placed in sterile paper towels inthe refrigerator overnight. They were then rinsed in 70%ethanol for 3 min, removed, and dried in a sterile air chamber.All ensuing steps were conducted aseptically. Usually, thelower epidermis was removed, but leaves could also beshredded into narrow strips (about 1 mm) to facilitate enzymepenetration. Approximately 2 g of leaf tissue were placed in a250 ml evacuation flask containing 100 ml of the followingfilter sterilized solution: 0.5% macerozyme (Yakult Bio-chemicals Ltd.), 4% cellulase (Onozuka SS, Yakult Bio-chemicals), 2% PVP (mol wt 10,000, Sigma Chemical Co.),and 0.2 M sucrose, pH 5.4. The enzyme preparation at theoutset always contained significant amounts of insolublematerial. This was solubilized by initially increasing the pHof the enzyme mixture to 9.5 with 0.1 N KOH and then after1 min readjusting it back to 5.4 with 0.1 N HCl. Tissue wasvacuum-infiltrated and then incubated at room temperature(about 21 C) on a Model G-76 Gyrorotary shaker (NewBrunswick) at a maximum of 40 cycles/ min. Normally,enzymatic digestion was continued for 4 to 6 hr. Leaf piecescontinued to float on the surface of the enzyme solutionthroughout the digestion period. Tissues that failed to do so,such as those from improperly grown plants, did not producesufficient numbers of viable protoplasts.

Protoplasts were collected by passing the suspensionthrough a small fluted funnel containing a four-layer pad ofcheesecloth into 16.5 cm graduated Babcock milk test bottles(Kimble No. 1000). Bottles were centrifuged for 5 min at 400g in an International model HN-S centrifuge during which timeintact protoplasts floated to the top of the solution (Fig. 1).Protoplasts were withdrawn with a Pasteur pipette and placedin Babcock bottles containing a rinse solution of 0.25 to 0.4 Msucrose (depending upon the experiment) and 'i/o concentra-tion of medium I (Table I). Following centrifugation, theapproximate total number of protoplasts was estimated fromthe depth of the protoplast column(s) in terms of numericaldivisions on each bottle. It was determined through repeated

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SHEPARD AND TOTTEN

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FIG. 1. Protoplasts columns in Babcock bottles following centrifugation. An approximate total of 20.8 X 106 protoplasts were present withinthe two bottles.

FIG. 2. Protoplasts derived colonies after dilution plating and further growth. X 0.9.FIG. 3. Early differentiation of protoplast derived colony on medium III containing 0.3 M sucrose. X 5.FIG. 4. Elongate form of cell growth in 10o concentration medium I. X 150.FIG. 5. Synchronous colony formation in medium II. Photograph taken 10 days after protoplasts were plated. X 600.FIG. 6. Genetic variegation in a plant regenerated from a protoplast. Protoplasts were originally isolated from the mesophyll cells of Xanthi-nc

tobacco infected with potato virus X.

690 Plant Physiol. Vol. 55, 1975

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TOBACCO PROTOPLASTS

cell counts with a hemacytometer that approximately 2 X 106protoplasts were present per numerical division (viz. 1-8)when 25 to 26 cm leaves were used as starting tissue. Proto-plasts were then collected and placed in a known volume ofrinse medium.

Plating and Regeneration. Several culture media were com-pared in protoplast regeneration experiments. Protoplasts atvariable densities were plated (10) in 100-mm plastic culturedishes containing 10 ml of differing concentrations of mediumI or of full strength medium II (Table I). In each instance,the volume of the protoplast suspension added was allowed forin calculating final medium concentrations and cell densities.Each embedment also contained 0.4 to 0.5% washed ionagarNo. 2 and 0.4 M sucrose. Sources of ionagar varied in theirgelling capacity, and the minimum concentration needed toproduce a firm layer was determined for each batch. Afterplating, Petri dishes were sealed with parafilm, incubated under400 lux of white fluorescent light for 72 hr (8) at roomtemperature (21 C), and finally held at 700 lux. The percentageof cells which had given rise to colonies (i.e., plating efficiency)was calculated from three replications of each treatment after3 weeks of culture.

In regeneration experiments, dilution plating of colonies

Table I. Compositiont of Culture Media

Constituent Medium I1 Medium 112 Medium III3

mg,lCa(N03)2.4H20 180NH4NO3 825 82.5 825KNO3 950 167 950CaCl2*2H20 220 22 222MgSO4-7H20 1223 447.3 1223Na2SO4 180KH2PO4 680 68 680NaH2POc4 H20 14.9KCI 58.5Na2 EDTA 37.3 3.7 37.3FeS04*7H20 27.8 2.8 27.8Fe2(S04)3 2.3KI 0.83 0.76 0.83H3B03 6.2 2.0 6.2MnSO4.4H20 4.1MnCI2*4H20 19.8 2.0 19.8ZnSO4*7H20 9.2 2.3 9.2Na2MoO4.2H20 0.25 0.03 0.25CuSO4-5H20 0.025 0.003 0.025CoS04-7H20 0.03 0.003 0.03

meso-Inositol 100 39.7 100Thiamine-HCI 1 0.19 1Glycine 2.7Niacin 0.45PyridoxineHCll 0.1I-Naphthaleneacetic acid 3 1.2Indole-3-acetic acid 46-Benzylaminopurine 1 0.4 1Kinetin 2.56Sucrose Variable Variable 0.05-0.3 MpH 5.6 5.6 5.6

1 Modified from Nagata and Takebe (10).2 Modified from White's medium (12) and from Nagata and

Takebe (10) (9:1, respectively, for most constituents).3IAA and kinetin concentrations from the B3 medium of Sa-

cristan and Melchers (14).

was performed approximately 4 weeks after protoplasts wereplated. In initial experiments, 5 ml of medium I containing0.05 M sucrose was flooded over the surface of the agar layer,followed by 3 additional aliquots at 2-day intervals. However,flooding of the agar surface slowed or terminated colonygrowth, which did not resume until dilution plating. Greaterconsistency was achieved by culturing protoplasts in 0.236 1glass-prescription bottles. A thin agar layer was formed by add-ing 5 ml of the protoplast-agar medium mixture and thenplacing the bottle in a horizontal position. When subsequent2-ml liquid media additions were made (beginning 2 weeksafter culture), bottles were slightly inclined to prevent liquidfrom covering the agar surface. Samples of the same proto-plast-agar medium mixture were also placed in small Petridishes to permit direct observation with a microscope. Dilu-tion plating was performed from bottles after approximately4 weeks of culture. The soft layer was disrupted with a glassrod, and colonies were separated by gentle repipetting. Colonieswere diluted 10-fold in 100-mm Petri dishes by mixing 1 ml ofthe colony suspension with 9 ml of medium I containing 0.05M sucrose and 0.7% agar. Dishes were sealed and incubatedin clear polystyrene boxes at 24 to 26 C under 1500 luxfluorescent light. When most colonies exceeded 1 mm indiameter and had turned green (Fig. 2), they were transferredwith a fine pointed scalpel to Petri dishes containing mediumIII-the hormones of which were similar to the B3 medium ofSacristan and Melchers (14). Colonies grew rapidly anddifferentiated shoots and leaves when placed on this medium.When colonies exceeded 5 mm in diameter and had initiated

primordial shoots, they were transferred with 23-cm Thorek-Brown tissue forceps (Scanlan, St. Paul, Minn.) into CorningNo. 681 Pyrex casserole dishes. Each dish contained 150 ml of0.5 concentration medium I, 0.025 M sucrose, 0.6% ionagar,and no hormones. As a precaution against bacterial contamina-tion, 5 ml/l of gentamicin solution (10 mg/ml, ScheringCorporation) was also added to the medium. Bowls were sealedwith parafilm and incubated under 700 ft-c white fluorescentlight. In this system, differentiation proceeded rapidly with theformation of a single or a limited number of dominant shoots.When shoots were well developed, root formation commenced.Rooted plants were removed from bowls, transplanted intosolid peat growing blocks (Kys-Kubes, Keyes Fiber Co.), andplaced in trays in an environmental chamber at greater than85% relative humidity. When root systems were fully devel-oped and shoot growth had resumed, plants were transplantedinto soil.

RESULTS

Plant Growth Conditions. The plant growth conditions de-scribed herein were arrived at sequentially and in toto werefound to be a critical factor in protoplast isolation. When plantswere grown under normal greenhouse conditions, protoplastyields were low and at best inconsistent. The fertilizer analysisand source was also important. Pronounced deviations in plantgrowth conditions frequently lead to no viable protoplasts atall.

Effects of Osmotic Pressure. Protoplasts from Xanthi-nctobacco leaves were obtained more rapidly and in higher yieldswhen 0.2 M sucrose was present in the enzyme solution thanwhen other concentrations up to 0.6 M were used. PVP, whichis known to complex certain polyphenolic compounds (1), con-sistently increased protoplast yields but also contributed to theosmotic pressure of the isolation medium. When PVP wasomitted, surviving protoplasts could be rinsed and embedded inmedia containing 0.25 or 0.3 M rather than 0.4 M sucrose.

Plant Physiol. Vol. 55, 1975 691



SHEPARD AND TOTTEN

These osmoticum levels are significantly lower than those pre-viously reported as optimal for tobacco mesophyll protoplastisolation (5, 16), and probably related to this is the fact thatprotoplasts were noticeably larger in size and responded differ-ently to cultural conditions. For example, protoplasts incubatedin any of several liquid media displayed a high affinity for oneanother when left undisturbed. Sheets of aggregated protoplaststhat could not be readily dissociated were formed at the sur-face of the culture vessels following an overnight incubation.Necrosis of protoplasts began soon after large clumps or sheetshad formed and was extensive after a 48-hr incubation. Whencell to cell contact was largely prevented through the inclusionof 0.04 g of ionagar/ 100 ml of the incubation medium, com-parable cell necrosis was not observed.

Protoplasts in 0.3 or 0.4M sucrose could be further plasmo-lyzed by exposure to 0.6M sucrose. When this was done, theyimmediately assumed a convoluted and shrunken appearance.After a few hours, however, protoplasts adjusted to the mediumand were again spherical in conformation. Their average di-ameter was significantly decreased, however, and they ap-peared far more compact. Also, protoplasts incubated in mediacontaining 0.6M sucrose displayed a reduced propensity to ad-here to one another.

In initial studies, the osmotic pressure of Medium I whenused for dilution plating and of Medium III for colony trans-fers seemed to influence colony survival and differentiation.Colonies that had received no supplemental media additionsbetween the plating of protoplasts and subsequent dilutionplating were subject to apparent osmotic shock in transfers tomedia containing less than 0.3 M sucrose. Colonies becamebrown in color in a few hours and growth ceased. Similar ef-fects were observed when colonies up to 3 to 4 mm in diameterwere transferred from dilution plates to medium III containing0.05 M sucrose and even those that did survive frequently grewas undifferentiated callus until greater than 5 to 10 mm indiameter. When 0.3 M sucrose was present in Medium III,however, survival and differentiation rates were greatly im-proved. Further experimentation revealed, however, that whenprescription bottles were employed and colonies were repeat-edly supplemented with medium I containing 0.05 M sucrose,there was little evidence of apparent osmotic shock during eitherdilution plating or subsequent colony transfer to media with0.05 M sucrose. Indeed, by this system, colonies differentiatedshoots (Fig. 3) as rapidly if not more so on medium III con-taining 0.05 M instead of 0.3 M sucrose. This suggested thatmedium exhaustion may be at least partially responsible for thereliance in prior experiments of colony survival on higher lev-els of sucrose.

Culture Medium and Plating Efficiency. The basic mediumof Nagata and Takebe (10) was unsatisfactory for platingprotoplasts prepared by our protocol. At densities lower than1 x 10' protoplasts/ml, the medium was toxic and all cellsdied within 4 days (Table II). Above 1 to 1.5 x 10' proto-plasts/ml, however, protoplasts did survive and a moderatepercentage of cells divided and formed colonies. Reductionsin the strength of the medium to one-half and one-third finalconcentrations permitted colony formation at progressivelylower protoplast densities, but plating efficiencies were nothigh (Table II). Additional experiments were also conductedin which the concentration of organic, inorganic, and hormoneconstituents were independently varied. No benefit was ob-served by reducing the level of any one of the classes of com-pounds. One-tenth concentration of medium I permittedgrowth in the form of elongate cells (Fig. 4) for 4 to 6 weeksbut little or no colony formation. The most effective platingembedment was medium It (essentially, with a few alterations.

Table IL.' Influence of Culture Mediuim oni Platinig Efficiency andCell Survival

Results were determined 3 weeks after plating. Data are aver-age of three replications and all were from the same experiment.

Protoplast Density'PlatingAMedium V

5000/ml 10,000/mli 20,000, ml

Per cent colony formation

A.>3 Medium I 21 51 49>3MediumI 15 39 41Medium 1 0 15 37Medium 11 55 71 74

B.Per cent division of living cells2

S3 Medium 1 43 83 59>3 Medium 1 58 57 70Medium I 0 36 71Medium II 84 81 87

C.Per cent of total as dead cells

3 Medium I 53 39 37' Medium 1 72 36 42Medium I 100 56 57Medium II 22 23 21

Approximate protoplast densities within ± 10(.2 Cells alive after 3 weeks that had divided one or more times.

Cell death was denoted by the assumption of an over-all browncoloration under phase microscopy, the absence of cyclosis, anda loss of the ordered appearance of cell contents.

a combination of 90% modified White's medium [ref. 12, page113] and 10% medium I). Cell division began 3 to 5 days afterprotoplasts were plated and appeared relatively synchronous(Fig. 5). Plating efficiencies commonly ranged between 60 and80% at cell densities between 6 x 103 and 2 X 104/ml and inone experiment reached 94% 3 weeks after protoplasts wereplated at 7200/ml. In addition, far fewer cells expired in thismedium than in the others tested (Table II). Between 2 X 10;and 4 x 103 protoplasts/ml, most protoplasts survived butoften grew in an elongate manner and per cent of colony for-mation was highly variable. Medium IL did not, however, pro-vide for the sustained growth of colonies. In initial studies,colonies were removed from medium II after approximately 4weeks and were dilution plated in medium I. However, moreconsistent results were obtained by adding small aliquots ofliquid medium I containing 0.05 M sucrose to prescription bot-tle systems after colony formation was well underway (about 2weeks after culture). This not only provided for sustainedcolony growth and development, but also slowly reduced theosmotic pressure of the culture medium. Dilution plating wasthen performed in medium I after 4 to 5 weeks of culture.

Plant Characteristics. In agreement with prior studies (10),over 90% of the plants regenerated from Xanthi-nc mesophyllcell protoplasts (more than 2000) appeared morphologicallynormal. Randomly selected plants were also found to be fertileand to set seed. Most of these plants, however, were regen-erated from protoplasts initially plated in l/3 concentration ofmedium I. The results with plants regenerated from protoplastsoriginally cultured in medium LI appear comparable but are

692 Plant Physiol. Vol. 55, 1975



TOBACCO PROTOPLASTS

nonetheless preliminary, because only a few hundred havebeen evaluated.Some plants (more then 500) were regenerated from proto-

plasts isolated from leaves systemically infected with potatovirus X. In these, an interesting anomaly in the form of geneticvariegation appeared (Fig. 6) although there is no suggestionas yet that the phenomenon was virus-induced. The plant wasself-fertile and set viable seed. An analysis of the heritabilityof the mutant character is presently in progress.

DISCUSSION

Results of the present study demonstrate that tobacco meso-phyll protoplasts may readily be isolated en masse under rela-tively low osmotic conditions if plants are properly grown.The importance of plant growth conditions on protoplast iso-lation has heretofore received mention but insufficient empha-sis. In but few examples (6) has it been shown that successfulprotoplast regeneration was dependent upon pretreatment ofplants and tissue. These findings may be even more significantwith other plant species from which protoplasts are not aseasily obtained or regenerated. The average osmotic pressureof mesophyll cells at the time of protoplast isolation appearsto be an important consideration in this context, and it is welldocumented that median leaf cell osmotic pressures may bemarkedly influenced by environmental conditions. One ap-proach toward reducing median osmotic pressures, for ex-ample, is to subject plants or leaves to lengthy dark periods.This has been employed successfully by Constable et al. (6) inthe isolation pea leaf protoplasts. Since there are pronouncedintraleaf cell to cell differences in osmotic pressure (2), itshould be of benefit, for enhancing protoplast yields, to at-tempt to reduce the degree of variation in osmotic pressure aswell as median pressures. The results of the present study sug-gest that this is possible in tobacco through a manipulation andsubsequent standardization of plant growth conditions, andwith appropriate modification should apply to other plants aswell.

Protoplasts could be induced to divide and regenerate plantswith high efficiency over a broad range of osmotic conditionsprovided that reductions in osmotic pressure were not made atthe outset. Protoplasts could also be plasmolyzed by exposureto increased concentrations of sucrose (up to 0.6 M). The latterfeature may facilitate uptake studies such as those with bac-teria described by Davey and Cocking (7) but under more con-trolled conditions.The phenomenon of the dependence of cell division and

colony formation on cell density (10) was confirmed in thisstudy, although the minimum cell density for division wasvariable with the nature and concentration of the culture me-dium. This dependence, in combination with the finding thatirradiated moribund cells may be used to "support" divisionof nonirradiated ones at low density (13), raises a questionrelative to suggested (10) protoplast cooperativity in platingexperiments. It would seem to be difficult to assess at this timewhether cell cooperativity is in the form of growth substancesynthesis (10), simply medium "detoxification," or a combina-tion of the two. Either way, the ability of cells to convert enzy-matically or to utilize a number of compounds in the medium(starting with sucrose) may well be influenced by their density.If so, the isolation of a single or group of cell-synthesizedcompounds responsible for growth in such media would bea very difficult task. At any rate, the cell density phenomenoncreates definite problems in protoplast regeneration. Whenprotoplasts were plated at greater than 3 to 4 X 104/ml andformed colonies with high efficiency, supplemental media ad-

ditions, or dilution plating, or both, were required for con-tinued rapid growth. If this was not done, growth becamelimited and eventually ceased. The problem was somewhatless acute when 1/3 or l2 concentration medium I was usedfor plating. This, however, resulted partially from the severeeffects of these media on cell survival, which in turn effectivelyreduced the number of colonies produced. But cell selectionwith regard to different media is not always a desirable phe-nomenon and frequently it is preferable to maintain as higha plating efficiency as possible in a standard medium. Othertechniques such as X- (13) or UV-irradiation of a layer of nursecells over which are layered dilute experimental cells mayempirically assist in minimizing some of the difficulties, butmore work is needed in this area.

Results of the present study indicate that the medium neces-sary to provide maximal plating efficiencies of mesophyll pro-toplasts may be significantly different from that required forlater colony growth and differentiation. Protoplasts isolatedby our procedures required initial exposure to a relativelydeficient plating medium (in quantitative terms) comparedwith those isolated by the procedure of Nagata and Takebe(10). It is uncertain to what extent osmotic pressure is in-volved in this finding, although it should definitely be a con-sideration. Other factors may also be of importance, includ-ing plant growth conditions and the presence of spongymesophyll and vascular parenchyma cells in our protoplastpreparations. Spongy mesophyll cells were eliminated in thesequential enzyme isolation method (10) and the presence ofvascular parenchyma protoplasts was not mentioned. Vascu-lar parenchyma cells did not divide in our systems and if theywere excluded in plating efficiency determinations, the valueswould have been somewhat higher.The addition of supplemental media could be conveniently

accomplished when protoplasts were cultured in prescriptionbottles. If, in this manner, developing colonies were not al-lowed to seriously deplete their ambient nutrient supply, sub-sequent dilution plating and colony transfer were more con-sistently successful than when medium supplements were notprovided. Whereas it should not be unexpected that cells underphysiological stress may react unpredictably when exposed to afresh medium, the influence of sucrose concentration on thisunpredictability is of interest. It is possible that the presumedosmotic shock observed when stressed colonies were trans-ferred from media containing 0.4 to 0.05 M sucrose did not, infact, result from osmotic blow out. Rather, cellular levels ofuptake for certain inorganic and/or organic compounds maysuddenly have become unreasonably high, and cells becamepoisoned.The relative ease and rapidity with which single isolated

tobacco protoplasts may be induced to regenerate plants offersa promising system for the study of a variety of biologicalphenomena (15). We presently are extending these techniquesto virus-infected plant tissue with the basic aim of assessingthe potential of somatic cell selection as a means for securingvirus disease resistance in plants. Simple selection from haploid(3) and even diploid cell populations, may well provide amost useful means for plant varietal improvement when plat-ing techniques are devised to select effectively for desirablegenotypes.

Ack7iouwledgme0ts-The authors gratefully acknowledge the able technicalassistance of 'Mr. Gary A. Secor, Miss J. E. Roddy, 'Miss V. Proue, and MissC. Barry.

LITERATURE CITED

1. BADRAN-. A. MI. AN-D D. E. JON-ES. 1965. Preparation of enzymes from planttissues containing high molecular weight polyphenols (abstr.). Planlt Plhysiol.(Suppl.) 40: lxx.

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694 SHEPARD A

2. BENN-ET-CLARK, T. A., A. D. GREENWOOD, AND J. W. BARKER. 1936. Waterrelations and osmotic pressures of plant cells. New Phytol. 35: 277-291.

3. CARLSON, P. S. 1973. 'Methionine sulfoximine-resistant mutants of tobacco.Science 180: 1366-1368.

4. CARLSON, P. S., H. H. S-MITH, AND R. D. DEARING. 1972. Parasexual inter-specific plant hybridization. Proc. Nat. Acad. Sci. U. S. A. 69: 2292-2294.

5. COCKtING, E. C. 1972. Plant cell protoplasts-isolation and development.Annu. Rev. Plant Physiol. 23: 29-50.

6. CONSTABLE, F., J. WV. KIRKPATRICK, AND 0. L. GAMBORG. 1973. Callusformlation from mesophyll protoplasts of Pisum sativum. Can. J. Bot. 51:2105-2106.

7. DAVEY, M. R. AND E. C. COCKING. 1972. Uptake of bacteria by isolatedligher plant protoplasts. Nature 239: 445-456.

8. EXNZMANN-BECKER, G. 1973. Plating efficiency of protoplasts of tobacco indifferent light conditions. Z. Naturforsch. 28c: 470-471.

9. GREENWAY, H. 1970. Effects of slowly permeating osmotica on metabolismof -acuolated and nonv-acuolated tissues. Plant Physiol. 46: 254-258.

ND TOTTEN Plant Physiol. Vol. 55, 1975

10. NAGATA, T. AND I. TAKEBE. 1971. Plating of isolated tobacco mesophyllprotoplasts on agar medium. Planta 99: 12-20.

11. OHYAMA, K. AND J. P. NITSCH. 1972. Flowering haploid plants obtained fromprotoplasts of tobacco leaves. Plant Cell Physiol. 13: 229-236.

12. PAUL, J. 1970. Cell and Tissue Culture, Ed. 4. Williams & Wilkins Co.,Baltimore.

13. RAVEH, D., E. HUBERMAN, AND E. GALUN. 1973. In vitro culture of tobaccoprotoplasts: use of feeder cells to support division of cells plated at lowdensities. In Vitro 9: 216-222.

14. SACRISTXN, M. D. AN-D G. 'MELCHERS. 1969. The caryological analysis ofplants regenerated frons tumorous and other callus cultures of tobacco. M1ol.Gen. Genet. 105: 317-333.

15. SMITH, H. H. 1974. 'Model systems for somatic cell plant genetics. Bio-science 24: 269-276.

16. TAKEBE, I., Y. OTSUKI, AN-D S. AoKI. 1968. Isolation of tobacco mesophyllcells in intact and active state. Plant Cell Physiol. 9: 115-124.

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