Earle, and John G. Torrey. The Biological ... tere(l cell suspenlsions in agar me(liumii has been ... grown on a simple, completely defined agar medium. (table I) ...
520
PLANT PHYSIOLOGY
15. SOLONION, A. K. 1960. Measuremenlts of equivalent pore radius in cell membranies. In: NIembrane Transport and Metabolism. A. Kleinzeller and A. Katyk, eds. Aca(lemic Press, New York, 1) 94-99. 16. TANADA. T. 1962. Localization anid mechanism of calcium stimulationi of rubidium absorption in the mung bean root. Am. J. Botany 49: 1068-72. 17. VIETS, F. G. 1944. CalciumI1 aInd other polyvaleint
accelerators of ioIn accumllulatioIl by excise(I Planit Physiol. 19: 466-80. 18. \NVAISEF, Y. 1962. Effect of calciumii upoIn uptake of monovalenit ioIns by excised barley roots. Physiol. Plantarum 1 5: 709-24. 19. \NVHITTEIIBU RY G., N. SUGI.O, ANI) A. K. Soi.ormox. 1960. Effect of anitidiuretic hormonie and( calcium on the equivalenit pore ra(lius of kidniey slices from ANcctnirns.v. Nature 187: 699-701. ions as
barley
roots.
Colony Formation by Isolated Convolvulus Cells Plated Elizabeth
D.
Earle, and John G.
on
Defined Media
2,2
Torrey
The Biological Laboratories, Harvard University, Cambridge, Massachusetts In recenit years the cultiv-ationi of smiiall cilumps of vegetative cells and( ev sxingle cells fromli higher plants lhas been achieved in several laboratories. Mluir, Hildebrandt ancd Riker (14, 15) established the first lplant tissue culture clonies (callus tissues of single cell origini) by placing single cells filter paper over nurse callus cultures. T'orrev (22) was able to demonistrate division of sinigle isolated pea root callus cells set arouni(l a callus piece oIn y-eastextract medium. Joines et al. (11) showed that single tobacco cells could clivide and survive for ]ong l)erio(ls in a microchamber containing liquidl en
on
mlledium gYrown
in which calltus tissue had previously been (coinditioned miiediuim ).
Suspension cultur-es of plant cells in agitate(d liquid milediuiil Isee (20) for review are useful for large-scale quantitative exxperiments w\-ith cell populations, btut the fate of individual cells cainnot be followed in suchi ctiltures. A. plating techniique dexeloped by Bergmanini 1() miade it possilble 1)oth to hand(lle malny cells at a timile a(lI obtain siligle cell clones.
In
1
experimenit, Bergmiianuii found that 40 %
of plate(l IPhasco/hs cells dividled at least once, and 13 % fornmed clones of more thani 32 cells. The effectiveness of Bergmanin's technique of plating filtere(l cell suspenlsions in agar me(liumii has been confirnimed usilig cells of Co;ii'ok'lthis arz,cosis (24), Haplophappuis gracilis and carrot (3, 8), and tobacco (
).
TI'he mie(lia tsied in exl)erilments with isolate(l cell., liave alm,,ost always been complex i-athier thaii (defined, containing coconitit milk or xyeast extract or having beeni cond(litionied by the presence of large numibers of cells. Isolated sinigle cells have shown quite exactinog nuitritional requiremilenits. Kato ani( Takeuchi (12) foutid that 1 carrot cell alonie dividede onllv on coniditionecl yeast-extract mie(liumil while manly single cells together on the samile plate cotil(ldivide on unconditioned yeast-extract miediumil. In a recenit paper, Blakelyr anid StewN-ard (3) reporte(l that free carrot cells divided mltuchi more freqtientlv wvhen the cells were plate(l in conditionie(d coconiuit mililk media ancd wheii carrot root explants were l)lace(l the miieditim niear the cells. D)efined media. have usually proved inadequate for large stipporting (ivision in inocula several hiiindred plate(l cells. l'ergmann (1) Torrev anld( IReiniert (24) reported that cells plate(d oni media lackinig cocolinut iilk or east extract were capable of a few divisiolis but nlot of coitillue(1 g,rowth. fin their experinments nio atteml)ts \were made to replace the comilplex components of the miledia with vitamins, hormones, or aminio acids; however, Gibbs aind lDougall (9) also failed to obtaimi clones froml tobacco cells plated oni (lefille(d medliumi which sulpported growth of tobacco callus. The onily report of sustaine(d (livisioin of simiall ntimbers of single plant cells on1 a definiedI me(liti is that of Reinert (17). WNThen friable crown-gall callus fromii l'itis ziinfera was spreadl agar miie(licolitainiilig only- salts, gltcose, and thiamini, 40 % on
as
as
and
a
on
tim
Received November 3, 1964., This study is part of a Ph.D thesis submitted to Harvard University. It was supported by a National Science Foundation Predoctoral Fellowshil) to E. D. Earle and by Research Grant RG-08145 from the Nationial Institutes of Health, Public Health Service. Present address: Department of Biologv, Princeton University, Princetonl, New Jersev. 2
to 70 % of the single cells preseint divide(l. Soime eventually lproduced clones. Larger cell mlZasses were also present oni the agar in this experimenit. It seemils significanit that success was achieved wx ith crown-gall cells, wlhicil have greater synithetic ca.l)acities and less elaborate niutritionial requiremienits thain nlormial cells (5).
EARLE AND TORREY-COLON-Y FORMATION BY ISOLATED CONVOLVULUS CELLS
Development of a defined medium to support division of normal isolated plant cells would help specify their distinctive requirements and would also increase the usefulness of the plating technique for plant physiologists. This report presents the results of a successful attempt to devise such a defined medium for Convolvidths cells.
Materials and Methods Preparationt of Cell Suspensions. Friable callus from cultured roots of Convolvuilus arvensis L. was grown on a simple, completely defined agar medium (table I) similar to the one developed by Shigemura (18) for pea root callus. With the exception of the micronutrients and FeCl3, which were not tested. all components of the medium had been show n to promote tissue growth (7). Suspensions were pro(luced by inoculating Erlenmeyer flasks of liquid medium with callus and agitating them on a horizontal rotary shaker at 80 rpm. [See (13) for details of shaker construction.] Dissociation of the callus into single cells and small clumps was so rapid that satisfactory suspensions were available within a few minutes after inoculation. Flasks usually were agitated not more than a few hours to reduce possible alteration of the defined medium by substances released from cells and to prevent selecTable I. Defined Media for Growth of Convolvulus Calluts and Plated Cells The pH of the media was adjusted to 5.5 after autoclaving. Com,ponent Macronutrient salts Micronutrient salts* * FeCl * 6H.,O (fresh stock) Thiamin * HCI Mllyo-inositol Adenine sulfate 2,4-D Kinetin 3
Reduced nitrogen
mg/liter 4 X Basal level* (Callus) Basal level ( Plated cells) 2.5 0.1
100.0 40.4 (10-4 M) 0.22 (10-6 M) None (Callus)
0.22 (10-6 M) (Cells) Amino acid mixture***
(Callus) Sucrose Bacto-agar *
**
*
146.1 L-glutamifle (Cells) 40,000 7000 (Callus) 6000 (Cells)
Basal level of macronutrients (mg/liter): 242 7H,O; 85 Ca(NO.), * 4H.,O; 42 MgSO4 KNO.,; 61 KCl; 20 KH.,PO,. Micronutrients (mg/liter) :1.5 H3BO3; 1.5 ZnSO4 * 7H.,O; 4.5 MnSO * H.,O; 0.25 Na,MoO4 * 5H.,O. * 2H.,O; 0.04 CuSO Amino acid mixture (mg/liter): 210.7 L-arginine * HCl; 133.1 L-aspartic acid; 132.1 L-asparagine; 147.1 L-glutamic acid; 75.1 glycine; 60.0 urea. A stock solution containing these compounds was adjusted to pH 5.5, sterilized through a Millipore filter, and added to coolinig autoclaved medium.
521
tion of the cells least likely to divide. (Since most cells did not separate immediately after division, single cells present after a few days were probably unhealthy in some way.) For experiments testing the effects of various nutrients on division of plated cells, tissue was shaken in liquid lacking the substances under consideration.
Inoculationl and Incuibationt of Plates. Large tissue clumps were separated from cells and small cell clusters by pouring the suspension through a successive series of stainless steel mesh filters with square openings about 400 IL, 250 u, and 200 IL on a side. Aliquots of stuspension (generally 2 ml) were pipetted on 25 ml of 0.6 % agar medium in 10-cm petri dishes. After inoculation, plates were sealed with nmasking tape or Parafilm "M" (American Can Coim1panyv) to prevent desiccation. Plates were incubated at 240 in darkness, since relatively low intensity (200 ft-c) of white light was found to severely inhibit cell division. Cell Coutnlts and Assavs. The number and distribution of cell units in each ml of filtered suspension were determined by examining 3 to 5 0.1 ml aliquots in a nannoplankton counting cell (A. H. Thomas Company) at 10OX magnification. A tinit was defined as a cell or group of cells potentially capable of dividing to form a cell colony. A healthy single cell or a cluster of several attached cells (at least 1 of which appeared viable) was each counted as 1 unit. Cells with torn walls, damaged nuclei or coagulated cytoplasm were excluded from counts. At a magnification of lOOX cytoplasmic streaming was not visible and so could not be used as a criterion for viability, but at higher magnifications cells counted as living showed definite streaming. At the timre of inoculation no macroscopic cell clusters were present on the plates. Plates were examined periodically for formation of visible clusters. The term colony was used to describe a macroscopic group of cells derived from a plated unit. The more specific term clone was reserved for colonies known to have originated from a single cell. Because plates were assayed only for visible colonies, divisions that did not lead to colonies were undetected; therefore, the number of units dividing at least once was undoubtedly higher than indicated by simple colony counts. Four replicate plates were prepared for each condition in an experiment. Plating efficiency (PE) was calculated by dividing the average number of colonies per plate by the number of units in the inoculum used. Experiments were repeated at least twice. Techniiques for Obtaining Single Cell Clones. For experiments in which specific cells were followed, cells were immobilized within agar medium, rather than plated onto the surface of solidified agar. One-half ml of filtered cell suspension was either mixed with 3 to 5 ml of cooling agar medium and dispensed into 6-cm petri plates or pipetted onto a thin layer of soft agar medium and then covered with more agar medium.
52PLANT I'11 YS1OLOGY 522 Arbitrarv stubdlivisionis of the l)lates were miia(le by inking rows of smiiall circles or attaching aln aluminumii foil (lisc with rows of punched holes onto the bottom surface. Fach sealed l)late was examlinied vith ani inverted imicroscol)e and(I the positions of 20 to 50 single cells anid small clusters withini the subdivisions wvere recor(le(l. Every 2 to 3 (lays these cells were checked microscopically for the occurrence of (livisionis. Once cololnies ha(l reached 1 mm in (liameter, thev xx-ere easily iso1ate(d and transferred to fresh imediumii. \Vheni several colonies were close together, transfer operationis were carried out tunider a (lissecting microscope to eliminiate possible confusioni of clonies with colonies dlerivedl fromn more thanl 1 cell.
Results The number of tunits in a susl)ension of Couzvolcells varied xvith the ratio of calltus inioctulu to volumie of liqui(d imiedIiumiii. Addition of about 1 g freslh weight of callus to 100 mnl of liutid l)ro(luced stuspenisionis containinig about 500()() uits m1l after filtratioll. About 50 % of the unlits were silngle cells, ()1 % ha(l fewer thani 6 cells, and(I 98 %, fewer tllani 11 cells (table II). h'le lonlgest (limelnsionis of the cells ranged fromn 6(0 y to 470 y with the majority ,i/ois
somiietimes dlevelol)e(l. 'I'lTe long- timle lag between ilnoculationi anid the appearance of the small colonlies suggeste(l that the cells wvere iniduce(d to diviide bv somiie actioni of the large coloniies oni the me(litinmi. For this reasoni the seconid crop of coloniies was n1ot inciiudedI in estimates of plating efficiencies. Diision11 On Ycast-Ex.tract iledinin. Inlitial experiments wvere (lone usinlg a complex med(lium con1tainii1g the basal level of salts (table 1), 2.5 mg/liter FeCl,, 0.22 mg, liter ( 10- 1m ) 2. 4-dichlorophenoxyacetic acid (2, 4-1)), 1.0 g/liter Difco powdere(l yeast extract, an(l 40 g/liter sucrose, all autoclave(d togetlher. Platinig efficiency oni this mie(ldium raniged fromn low values of 2 % in earl- experiments ul) to at leas' 35 %. Higher values were somnetimes noted, but accurate counts could n1ot be imadle because the colouties were so close together. ()mission of various comlponents fromn the me(litlll showed that only yeast extract was ani absolute requlremiienit, in its absence, nio coloniies (levelop)ed. A s the concenitrationi of veast extract was raised froml 0 to 2.( g/liter, colony formation iincrease(l. Additioni of 4.1) anid () g liter re(Iuce(l FE ( fig 1). PE(9)|
'
I
0 0.5 1.0
2.0
I
f
I
Table II. Distribnittion of Uniit Si-cs in Filtered Convolvulus Slnspclsiolts
Cells/unit
%/, of Ulnits* 1 53 (43-59) 2 23 (18-28) 3-5 15 (8-19) 6-10 7 (4-9) 11-20 2 (1-4) >20
