Plant Physiol. (1978) 62, 482-485

Intercellular Correlations: Relation between DNA Synthesis andCell Division in Early Stages of in Vitro Bud Neoformation

Received for publication August 30, 1977 and in revised form May 15, 1978

HASSAN CHLYAHLaboratoire de Physiologie Vegetale, Faculte des Sciences, B.P. 1014, Rabat, Morocco

ABSTRACT

As well as showing the existence, during the first stages of in vitro budneoformatlon, of cell populations in a tissue composed of a single ceU layer(stem epidernns of Torenia fournieri Lind), some new physiological char-acteristics of mitosis are defied. Most of the cells which divide duringorganogenesis synthesize their DNA between 20 and 48 hours of culture.On an epidermal strip (10 x 2.5 milimeters composed of about 5,500 cells)20% of the original cells enter the S-phase. The first dIisin takes placeat the 20-hour stage after the entry into the S-phase of a cell population ofabout 25 cells. Ablost none of the cells of this population divide. Thegreatest percentage of divisions occurs in cells which syntbesize DNA nearthe 48-hour stage. The relation

Number of labeled cells at any given stage between 20 and 48 hoursNumber of divided cells after 5 days

has a value of about 25 at the beginning of cell division (20 bours) and fallsto a value of about 1.4 for cells which synthesize DNA near the 48-hourstage. A hypothesis of the existence of a mitotic stimulant in the epidermisis put forward; this stimulant, at first weak, increases progressively.

Corrections (multiple stimulatory or inhibitory interactions be-tween organs, tissues, or cells controlling morphogenetic processes)in any plant are very difficult to study because of their diversityin nature and their complexity. As well as the classic types ofcorrelation, several authors (5, 8, 17, 20) have demonstrated thepresence of some new ones. In order to understand the basis ofthese correlations, one method would be to simplify the materialstudied, using single organs, single tissues, and cells instead of theentire plant. Stem segments of Toreniafournieri cultured in vitroon a simple medium containing sugar but no growth substancesare capable of de novo organogenesis (6). By progressively simpli-fying the material and cultivating different stem tissues separatelyor together, we have demonstrated the existence of intertissuecorrelations in relation to organogenesis (8).

In order to study these correlations even more it seemed nec-essary to work on the cellular level; in 1966, Lang (12) discussedthe problem of "intercellular regulation in plants." In defined invitro conditions, certain epidermal cells of T fournieri stem seg-ments embark on cell division ultimately leading to bud neofor-mation. The correlations which these particular cells undergowould be different from those of other epidermal cells. In aprevious study, it was shown that cell division centers, observed atthe 5-day stage, are not formed at random on the stem epidermisbut in relation to the position of underlying vascular tissue (9). Aninterpretation of this phenomenon, based on hormone movementfrom this vascular tissue, was put forward. This hormone move-ment would be one of the correlative factors unequally influencingepidermal cells. For the same material, it has also been shown that

among the epidermal cells which divide and go on to form celldivision centers, not all form bud meristems (7, 24). A certaincompetition among these centers for nutrient or hormonal factorswould mean further changes in intercellular correlations in theepidermal layer.

It should be possible to define different cell populations amongepidermal cells depending on their position and the differentcorrelations to which they are submitted. The present study hasbeen undertaken to characterize these cell populations at a veryearly stage of organogenesis according to the time of DNA syn-thesis and that of entry into division. Several authors (3, 10, 11,14-16) have analyzed cell populations in stem or root meristemsbut, perhaps for lack of simple techniques, never in single differ-entiated cell layers capable of organogenesis.

MATERIALS AND METHODS

Stem segments of T fournieri Lind, 10 mm long, were culturedaseptically on a simple medium previously described (6), to which[3HJthymidine was added at 0.025 mCi ml-'. The wide face of thestem (which was rectangular in section) was applied to the radio-active medium; only the epidermal cells of this side were studiedin the present article.

After a continuous treatment on this medium for a length oftime (Ti) of 3, 6, 9, 12, 15, 18, 20, 22, 24, 26, 29, 32, 36, 42 or 48hr at 27 C with a 16-hr daily light period, the segments were eithertransferred to an identical medium but without [3Hjthymidine orunderwent immediate fixation. In the latter case, the epidermis(10 x 2.5 mm) which was in contact with the radioactive mediumwas peeled from the segment with no adjacent parenchyma tissueand fixed in Brachet fixative (13) for 1 hr. It was then rinsed for3 hr in tap water and spread and dried on a slide previously coatedwith a 0.5% (w/v) gelatin solution. The slides were then coveredwith Kodak ARIO stripping film for autoradiography in a darkroom where they were left for 7 days. They were then developedusing photographic products before microscopic observation.The explants transferred to a nonradioactive medium were first

rinsed (30 min) in an unlabeled thymidine solution, then placedwith the same epidermal face in contact with the new mediumand in the same environmental conditions as described earlier.the length of culture on the nonradioactive medium (T2) waschosen so that the entire culture period (T, + T2) equaled 5 days(120 hr). At the 5-day stage, it has already been shown that celldivisions ceased on the epidermis, except in the zones of meristemformation (7).For each treatment, 10 stem segments were used and the exper-

iments were repeated three times. In Tables I and II, the meanvalues are followed by the standard deviation from the mean.

RESULTS

Description and Distribution of Epidermal CeRs Whkh HaveIncorporated I'HIThymidine. After 48 hr of continuous culture on

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CELL POPULATIONS IN BUD NEOFORMATION

the radioactive medium, a large number of epidermal cells hadsynthesized DNA. Among the labeled cells, some showed morelabeling than others (Fig. 1). During the first stages (3 and 9 hr ofculture), labeled nuclei were very rare (Fig. 2); after 15 to 26 hr,they became very numerous (Fig. 3). Examination of a largenumber of samples seemed to show that labeled cells had nospecial relation to stomatal cells. Basal hair cells were not labeled.Although often in small groups, the global distribution in labeledzones appeared to be random. The labeled cells could be contig-uous or separated by unlabeled cells. Some large zones of epider-mis persisted with no labeled cells; this was observed particularlyin the median zone all along the epidermal strip, where few or nolabeled cells were seen.For epidermal strips 2.5 mm wide, the width of the median zone

with no labeled cells varied from 0.4 to 1 mm along the epidermalstrip, depending on the particular sample. At a later stage (5 days),a similar disposition of cell division centers on either side of aninactive median zone had been shown previously and was inter-preted as being related to hormone movement from subadjacentvascular tissue (9). It was not surprising that the cells which

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-_ *..w4*nmwin1g.. w-S~ ~ L#'~ 0 0-------1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...:.......FIG. I. One epideral .cell nucleus (L) showing more labeling than

another neighboring cell eI)

_..i~ ~~~~~~~~~~~~....

FIG. 1. One epidermal cell nucleus (L) showing more labeling thananother neighboring cell (I).

S

ay20U rc'M.--

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FIG. 2. Labeled cells (arrows) are very rare during early stages (3-9hr).

s --e-A-' 40

17 LjjR-- l:. ;*.!.-!' r'' 9!.

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r; ..... |~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ .....

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FIG. 3. Large number of labeled nuclei at 26-hr stage.

Table I. Number of labeled cells as a function ofthe length of continuous culture on the

radioactive medium (Tl)T1

Length of culture on Labeled cellsradioactive medium

Hr No.

3 16 6+49 5+412 12 t 815 17 +818 18 +820 48 + 2122 74 ± 3724 85 t 3126 75 3129 188 +9732 311 +13236 394t 16642 506 +20548 910 +256

entered the S-phase of the cell cycle (synthesized DNA) occupiedthe same positions as the cell division centers.Number of Cells Having Incorporated IjHlThymidine after Cul-

ture on Radioactive Medium. Synthesis of DNA by the originalepidermal cells began between 3 and 6 hr culture (Table I) andgrew progressively. Since cell divisions were never observed beforelabeling occurred, it seemed that the cells which synthesized DNAwere in the G1 period at the time of excision from the entire plant.However, they were not all in the same stage of GI since they didnot enter the S-phase simultaneously.

This variation in the duration ofGI depended on the individualreaction of cells to extracellular factors which retarded or accel-erated entry into the S-phase (19), and was due to the physiologicalstate of the cell at the time of excision and culture.At the 48-hr stage (Table I), the mean total number of cells

which synthesized DNA was 910. This number was slightly greaterthan the number of cells which divided on the epidermal surfaceafter 5 or 6 days of culture and even if this duration was prolonged(unpublished results). This would mean that all, or almost all, ofthe cells which divided during bud neoformation synthesized theirDNA between 0 and 48 hr of culture. This number (910) repre-sented about 20%/o of the total 5,000 to 5,500 cells on the epidermalstrip.The results show that the epidermal cells enter the S-phase

gradually and that the greatest number of cells synthesize DNAat the 48-hr stage.

Entry into Division between 0 and 48 Hr. Whereas stainingtechniques such as aceto-carmin and the Brachet (14) do not

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483Plant Physiol. Vol. 62, 1978

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Plant Physiol. Vol. 62, 1978

clearly show the first cellular divisions on the epidermis, this iseasily accomplished using autoradiographic techniques: newlydivided nuclei are relatively small and close together (Fig. 4). Thefirst division was observed after 20-hr culture. At this time, amean two cells over the entire epidermal strip had entered mitosis.The number of divided cells increased progressively. We haveestablished the following relation (R1):

No. of labeled cellsNo. of divided cells

for any given stage. The relation, at the 20-hr stage (time of thefirst cell divisions) had a mean value of about 25 (Fig. 5). Thismeans that about 25 cells were in G2 before the first divisionbegan. This relation (RI) decreased after the 20-hr stage andoscillated around a mean value of 10 until the 48-hr stage (Fig. 5).At the 48-hr stage, the daughter epidermal cells began to divide

and were difficult to distinguish from original epidermal cells.Since this study concerned the fate of these latter cells, theevolution of R1 was followed to the 5-day stage, at which time celldivision in the still undivided epidermal cells become very rare.

Observation of Division In Epmernal Cells Up to the 5-DayStage. When the original labeled cells divide more than once, thelabeling fell to an unobservable level. Any cells which remainedlabeled after transfer to the nonradioactive medium, and whichwere not obviously divided, must have been original undividedcells. The number of such labeled cells at the 5-day stage aftereach treatment is shown in Table II.Comparing the total number of labeled cells after continuous

culture on the radioactive medium (Table I) and the number ofcells which remained labeled at the 5-day stage (Table II), it isevident that cells which synthesize DNA before 20 hr do notdivide. This conclusion is based on the fact that their numbers

.tr Z *

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FIG. 4. Divided nuclei after labeling are visible, during cytokinesis.

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U 25

20

0

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104)

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.0EC

I0 0

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20 22 24 26 29 32 36 42 48h o u r s

FIG. 5. Relation between number of labeled cells and number ofdivided cells at any given stage between 20 and 48 hr on an epidermalstrip 10 x 2.5 mm.

Table II. Number of cells which remain labeled at the5 day stage after transfer to the non

radioactive medium

T T2Length of culture Length of culture Labeled cells

on on after 5 daysradioactive medium non-radioactive medium (T9 + T2)

Hr No

3 117 16 114 19 111 6± 512 108 13 t 815 105 16 ± 818 102 18 t 820 100 46 1522 98 66 3124 96 76 t 3426 94 58 2629 91 136 6632 88 200 9736 84 138 8142 78 182 ± 10548 72 270 + 137

remained nearly constant after 5 days of culture. This comparisonalso shows that the number of cells which synthesize DNA forperiods over 20 hr is always higher than the number of cells whichremain labeled after 5 days. The difference gives the number ofdivided cells on an epidermal strip between the end of the labelingperiod and 5 days of culture; this number increases progressivelywith the length of the labeling period. We have established therelation R2 which follows (Fig. 6):

No. of labeled cells at any given stage between 20 and 48 hrR2 == No. of divided cells after 5 days

The cells which synthesized before 20 hr are not included sincewe have established above that these cells do not divide (TablesI and II).The relation R2 varied from a mean value of about 25 for cells

which synthesized at the 20-hr stage (which confirms the relationpresented in Fig. 5) to a mean value of 1.4 for cells whichsynthesized at the 48-hr stage. Between these two stages, therelation decreased rapidly from 20 to 26 hr of culture and pro-gressively up to 36 hr, thereafter, the relation diminished verylittle.The results show the existence in the epidermis of a cell division

stimulation which increases to reach a maximum for cells whichsynthesize DNA between 36 and 48 hr.

484 CHLYAH

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Plant Physiol. Vol. 62, 1978 CELL POPULATIONS IN BUD NEOFORMATION 485

0V 300

25 |

Z20-E

15-

.010 .

0

05

0 20 22 24 26 29 32 36 42 48h o u r s

FIG. 6. Relation between number of labeled cells at any given stagebetween 20 and 48 hr and number of divided cells at 5-day stage.

DISCUSSION

In this study, it was shown that the cells of the epidermal layerundergo some remarkable caryological activities during the first48 hr of in vitro culture. This activity leads a certain number oforiginal epidermal cells to enter the S-phase of the mitotic cycle.Thus, at the 48-hr stage, most original cells (900- 1,000) which willdivide during bud neoformation have synthesized their DNA.This number is slightly higher than the total number of cells whichdivide over the epidermal surface at the 5-day stage.Two overlapping phases can be distinguished during the first 5

days of the organogenetic process: a phase of nuclear activity from0 to 48 hr and a phase of cell division from 20 to 120 hr.The results have led us to class the epidermal cells into two

populations:1. One cell population never enters the S-phase during the

period studied. This population represents about 80% of the 5,000to 5,500 cells which make up the epidermal strip. For the rootmeristem (2), a similar population represents 55% of cells in somespecies.

2. A second cell population is composed of the remaining 20%of epidermal cells which do enter the S-phase. This populationcan be divided into two subpopulations: (a) cells which synthesizeDNA between 0 and 20 hr; this group is composed of fewer than20 cells out of each 1,000 which synthesize DNA; these cells donot divide; (b) cells which synthesize DNA between 20 and 48 hr;it is mainly the cells which synthesize DNA close to the 48-hrstage which go on to divide, and which would be involved ineventual organogenesis.

This last point could be interpreted as follows: at the beginningof culture, a physiological equilibrium exists in the epidermiswhich is characterized by the state of nondivision. This equili-brium is progressively disrupted, bringing about the initiation ofcell division which is gradually accentuated. This rupture inequilibrium can only be attained after the entry into S-phase of acertain small number of cells: those which synthesize up to the 20-hr stage.The fact that the relation R2 (defined earlier) goes from 25 at

the time of the first cell division to 1.4 in the 5-day stage supportsthis hypothesis. At first, a relatively large number of cells must be

in the S-phase in order that the first cell enter division. From thenon, the equilibrium being broken, this number decreases.

Several authors who have studied nuclear evolution during themitotic cycle have considered the role of hormonal equilibria (1,15, 21-23). The equilibrium mentioned above could be, in thislight, an equilibrium between inhibitors and stimulators of orga-nogenesis. The passage from the nondividing to the dividing statewould take place when the stimulators out-balance the inhibitorsas has already been shown for the diverse correlations controllingapical dominance (4) and flowering. The displacement of thishormonal equilibrium could explain other physiological phenom-ena: the cessation of cell division outside of actively dividing zoneson the epidermis of T. fournieri (7) and the acceleration of celldivision during floral initiation (18).

Acknowledgment The author wishes to thank A. Chlyah for correction of the English style.

LITERATURE CITED

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2. BFNDADIS MC. W-S CHATTI 1976 Modalites de synthese de I'ADN nucleaire dans lesmeristemes radiculaires de diverses especes d'agrumes: etudes autohistoradiographique etcytophotometrique. CR Acad Sci Paris 282: 1101-1104

3. BODSON M 1975 Variation in the rate of cell division in the apical meristem of Sinapis albaduring transition to flowering. Ann Bot 39: 547-554

4. CHAMPAGNAT P 1965 Physiologie de la croissance et de l'inhibition des bourgeons. Dominanceapicale et phenomenes analogues. In Handbuch der Pflanzenphysiologie, Vol XV (1). Berlin.New York, pp 1106-1164

5. CHAMPAGNAT P 1974 Introduction a l'etude des correlations. Rev Cytol Biol Veg 37: 175-2086. CHLYAH H 1973 Neoformation dirigee a partir de fragments d'organes de Toreniafournieri

(Lind) cultives in vitro. Biol Plant 15: 80-877. CHLYAH H 1974 Formation and propagation of cell division centers in the epidermal layer of

internodal segments of Toreniafournieri (Lind) grown in vitro. Simultaneous surface obser-vations of all the epidermal cells. Can J Bot 52: 867-872

8. CHI.YAH H 1974 Inter-tissue correlations in organ fragments: organogenetic capacity of tissues- excised from stem segments of Toreniafournieri (Lind) cultured separately in vitro. Plant

Physiol 54: 341-3849. CHI.YAH H. M TRAN THANH VAN, Y DFMARI.Y 1975 Distribution pattern of cell division

centers on the epidermis of stem segments of Toreniafournieri during de novo bud formation.Plant Physiol 56: 28-33

10. CORSON GE 1969 Cell division studies of the shoot apex of Datura stramonium during transitionto flowering. Am J Bot 56: 1127-1134

11. DAVIDSON D 1972 Morphogenesis of root primordia. In MW Miller. CC Kuehnert, eds, TheDynamics of Meristem Cell Population. Plenum Press, New York, pp 165-185

12. LANG. A 1966 Intercellular regulation in plants. In M Locke, ed, Major Problems in Develop-mental Biology. Academic Press, New York, pp 251-278

13. LISON L 1960 Histochimie et cytochimie animale. Principes et methodes. Gauthier Villars,Paris

14. LYNDON RF 1970 Rates of cell division in the shoot apical meristem of Pisum. Ann Bot 34:1-17

15. MAcLFOD RD, D DAVISON 1966 Changes in mitotic indices in roots of Viciafaba L. II. longterm effect of colchicine and IAA. New Phytol 65: 532-546

16. MAcLEOD RD, SM McLACHI.AN 1974 The development of a quiescent center in lateral rootsof Viciafaba/ L. Ann Bot 38: 535-544

17. MIGINIAC E 1974 Flowering and correlations between organs in Scrofularia arguta Sol. In RLBieleski, AR Ferguson, MM Cresswell. eds, Mechanism of Regulation of Plant Growth.Bulletin 12. The Royal Society of New Zealand. Wellington, pp 539-545

18. MILLER MB, RF LYNDON 1975 The cell cycle in vegetative and floral shoot meristems measuredby a double labelling technique. Planta 126: 37-43

19. MULLER GC 1971 Biochemical perspectives of the G I and S intervals in the replication cycleof animal cells: a study in the control of cell growth in the cell cycle and cancer. Renato BasRega, Vol 1. Department of Pathology. Temple University, Philadelphia, Pa. Marcel Dekker,New York

20. NOZERAN R. L BANCILHON. P NEvII.I.F 1971 Intervention of internal correlations in themorphogenesis of higher plants. In Advances in Morphogenesis. Academic Press, New York,pp 166

21. STERN 1969 Control of the cell cycle. Excerpta Med Int Cong Ser 204: 53-5922. TORREY JG 1966 The initiation of organized development in plants. Adv Morphogen 5: 39-9123. TORREY JG, ED FosiCET 1970 Cell division in relation to cytodifferentiation in cultured pea

root segments. Am J Bot 57: 1072-108024. TRAN THANH VAN M. H CHI.YAH, A CHI.YAH 1974 Regulation of organogenesis in thin layers

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