Plant Physiol. (1974) 54, 341-348

Inter-Tissue Correlations in Organ Fragments

ORGANOGENETIC CAPACITY OF TISSUES EXCISED FROM STEM SEGMENTS OF TORENIA FOURNIERILIND CULTURED SEPARATELY IN VITRO

Received for publication December 12, 1973 and in revised form April 18, 1974

HASSANE CHLYAH1Laboratoire du Phytotron, Centre National de la Recherche Scientifique, 91190-Gif-sur- Yvette, France

ABSTRACT

In order to study the effect of inter-tissue correlations on theorganogenetic capacities of various tissues of stem segments ofTorenia fournieri Lind, different types of explants were excisedand grown separately: epidermis, subepidermal parenchyma,epidermis plus subepidermal parenchyma but devoid of vascu-lar tissue and stem segments devoid of epidermis.The epidermis, normally capable of bud formation in the

context of a stem segment, dies if grown in culture alone;however, it can form buds after callus formation if, after exci-sion, it is replaced on the original stem segment. Subepidermalparenchyma, normally mitotically inactive in the stem segment,forms roots when grown alone and buds and roots when grownwith the epidermis. A stem segment devoid of epidermis formsonly roots. The histological study showed that, in explants com-posed of epidermal and a few subepidermal parenchyma layers,buds can arise exclusively from the epidermis or from bothepidermal and subepidermal cells, whereas roots are exclusivelyformed by parenchyma tissue.The break in correlations among tissues by the culture of each

tissue separately seems to "liberate" masked capacities in cer-tain tissues and to repress the expression of capacities in others.

Whereas correlations among different organs (e.g. buds,bracts, leaves, cotyledons, and roots) of an entire plant havefrequently been studied (1-3, 11, 18, 19), correlations withina single organ or among tissues of an organ fragment have re-ceived little attention.

In the histological study of neoformation from stem seg-ments of Torenia fournieri in which the intertissue correlationsremain intact, we showed (5) the epidermal origin of bud meri-stems and the endogenous perivascular origin of root meri-stems. The parenchyma tissue situated between the epidermisand vascular tissue shows no organogenetic activity in the con-text of the stem segment (5).

It was thus interesting to see whether the epidermis of astem segment, cultured alone, would conserve its budding ca-pacity and to seek the culture conditions which would favor or-ganogenetic expression in the subepidermal parenchyma. Withthese goals in mind, we cultured the following explants: epider-mis alone, subepidermal parenchyma alone, epidermis plus

1 Present address: Institut Agronomique Hassan II, B. P. 704,Rabat-Agdal, Morocco.

subepidermal parenchyma but without vascular tissue, and thestem segment without epidermis.

MATERIALS AND METHODS

The different explants used were excised with a microscalpelfrom stem segments disinfected in a 5% calcium hypochloritesolution and washed three times in distilled water. The stemsegments were taken from the youngest elongated internode ofplants grown at 27 C in a 16 hr-daily light period. The culturemedium was composed of macro- and microelements of Mura-shige and Skoog's (12) revised medium for tobacco to whichwere added thiamine (0.1 mg/ 1), myoinositol (100 mg/l), andsucrose (30 g/1). IAA and kinetin were added in variable con-centrations specified for each experiment. The cytological tech-niques employed have been described in a previous paper (5).

RESULTS

PHYSIOLOGICAL STUDY

Epidermis Cultured Alone. The epidermal layer (single) (Fig.la), when grown on a medium containing IAA and kinetin at1 Mm, dies after 48 hr (neutral red test). The epidermis seemsincapable of showing its bud-forming capacity when isolatedfrom its physiological context. We will see below that the ex-cised epidermis can retain its budding capacity under certainconditions.

Subepidermal Parenchyma Cultured Alone. From a stemsegment with the epidermis stripped off, it is possible to excisean explant composed only of subepidermal parenchyma; thisexplant can form roots when a concentration of 100 Mm IAAis added to the medium, but never buds. Thus, the subepider-mal parenchyma which shows no organogenetic capacity in thecontext of the stem segment is capable of root formation whenisolated.

Epidermis and One or Several Subepidermal ParenchymaLayers without Vascular Tissue. In an effort to re-establish thebudding capacity of the epidermal layer, other explants weregrown in which certain areas of epidermis were attached to nounderlying cells or to one or several layers of subadjacent par-enchymal cells, the parenchyma being in direct contact withthe medium (Fig. 1, d, e, and f). These explants were capable ofproliferation even when only one layer of parenchyma waspresent. However, the areas of epidermis with no subjacentparenchyma (Fig. 1 d) did not proliferate in spite of neighboringparenchyma tissue.

(a) Bud Formation. Explants composed of epidermis andone to seven parenchyma layers in a medium containing IAAand kinetin at 1 uM first form a callus from the subepidermal

341

Plant Physiol. Vol. 54, 1974

d

b e

0FIG. 1. Different types of explant excision employed in epi-

dermis culture. The epidermis is excised from the stem segment andcultured alone (a) or after being replaced directly (c) or indirectly(b) on the original segment. Other explants are excised so that theepidermis stays in contact with 0 (d), 1 (e), or several (f) layersof subepidermal parenchyma.

layers in contact with the medium. This proliferation makes theexplant, which was flat at first, roll up with the epidermis in-side (Fig. 2). After 2 to 3 weeks (depending on the number ofparenchyma layers accompanying the epidermis), the first budprimordia (b) are observed under a binocular microscope (Fig.3) and develop into buds (b) (Figs. 4 and 5) which can flowerunder certain conditions. Thus, a thin explant composed of theepidermis and one to seven subjacent layers with no vasculartissue present is capable of bud formation (Table I).

(b) Root Form?1ation. A stem segment cultured in vitro pro-duces roots which originate in endogenous perivascular tissue(5). However, this formation, which seems to depend on thepresence of neighboring vascular tissue can occur, like budformation, in the absence of this tissue. By adjusting the auxinlevel of the culture medium to 100 tM in the absence of ki-netin, superficial zones of subepidermal parenchyma show theirroot-forming potentiality (Table I). Root neoformation is neverobserved is these zones in the entire stem segment.

(c) Directed Organogenesis. By varying the relative quanti-ties of IAA cnd kinetin in the medium used, neoformation inthese thin explants containing no vascular tissue can be directedtowards the formation of buds only, roots only, or both budsand roots (Table I). Thus, only buds are formed when IAA andkinetin are at the 1 fLM concentration (Table I); no root forma-tion is observed. With IAA alone at 100 tM, only roots are

formed (Fig. 6, Table I). With combinations of varied concen-trations of IAA and kinetin, bud and root formation in differ-ing amounts are obtained (Fig. 7). These results confirm therole of auxin and kinetin in bud and root formation as first de-scribed by Skoog and Miller (14).

Stem Segment Without Epidermis. The stem segment with-out epidermis forms a callus from superficial parenchyma tis-sue and roots from endogenous tissue (Fig. 8). Budding does notoccur even when a high concentration of kinetin at 100 ItM ispresent. However, if the epidermis is incompletely excised,buds appear in these areas.

Epidermis Excised from the Stem Segment and Replaced onthe Same Segment. With the intention of keeping alive the ex-

cised epidermal strip and to demonstrate further its budding po-

tentialities, already shown in the context of the stem segment,different methods of culture were tried. The epidermis was de-tached from the stem segment then replaced immediately, ei-ther directly or after intercalating a thin agar layer or a mil-lipore paper (Fig. 1, b and c). The explant composed in this

manner was then placed on the medium with the epidermis ei-ther on top or against the medium.Bud neoformation, after callus formation, was observed after

5 weeks of culture but only in explants in which the epidermis,applied against the culture medium, was replaced directly onthe stem segment or separated only by a thin agar layer. Thebuds were formed exclusively from the epidermis which wasseparated from the stem segment by a friable callus; Figure 9shows a callus formed from an epidermal strip. The epidermalstrips separated from the stem segment by a thin paper didnot survive; those which were not applied against the culturemedium dried up and died, whether in direct or indirect con-tact with the stem segment.The bud-forming capacity of the epidermis excised and re-

placed on the same segment can thus be re-established, but itseems that direct or indirect contact with the subjacent paren-chyma tissue is indispensable for the expression of this capacity.The principal physiological results obtained are outlined in Ta-be II.

CELLULAR ANALYSIS

The physiological study showed that depending on the IAA-kinetin equilibrium in the medium, explants composed of epi-dermal and a few subepidermal cell layers were capable offorming buds only or roots only. A cellular study of these twotypes of de novo organogenesis was carried out in order to de-termine the participation of epidermal and subepidermal tissue.At the moment of excision of the explants, no meristematicareas were observed (Fig. 10).Buds Exclusively of Epidermal Origin. On the 3rd day of

culture, while the parenchyma cells begin to divide, the epider-mal cells show meristematic characteristics, i.e. large nucleus,and cytoplasmic and nuclear staining with pyronin. On the 4thday, some epidermal cells show an anticlinal division andboth daughter cells have a periclinal division (Fig. 11, pd).

After 5 days, the cytological processes already observed areaccentuated. A growing number of periclinal divisions are ob-served in the epidermis and in places four or five layers of flat-tened ce ls are formed (Fig. 12). From 6 to 8 days, these activa-tion zones undergo intense cell division (anticlinal. periclinal,and ob'ique) giving rise to the formation of a group of smallmeristematic cells which form a dome at the epidermal surface(Fig. 13). These groups of meristematic cells become visibleunder the binocular microscope as meristems after 15 days ofculture (Fig. 14). Two meristems are sometimes observed sideby side (Fig. 15).

Meristematic Zones of Subepidermal Origin. Whereas budformation exclusively of epidermal origin occurs most fre-quently, in some cases the subjacent parenchyma seems in-volved in the origin of meristem formation. Figure 16 shows agroup of meristematic cells with a high nucleoplasmic ratioformed from division of one subepidermal parenchyma cell.The evolution of these meristematic cells is illustrated in Fig-ures 17 and 18. Many of these cellular groups do not seem todevelop into buds (Fig. 19).Buds of Epidermal and Subepidermal Origin. The formation

of these buds follows the same general pattern as that of budsexclusively of epidermal origin except that the cell divisionstake place in subepidermal parenchyma cells as well as epider-mal cells (Fig. 20). After several mitoses, groups of small meri-stematic cells form, which eventuallv become structured as budmeristems (Fig. 21).

Tracheid Differentiation during Bud Formation. On the 3rdday of culture, the first signs of cytoplasmic activation are ob-

342 CHLYAH

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plants.343

Plant Physiol. Vol. 54, 1974

Table I. Nunber of Buds anid Roots Obtain2ed per Explanit fromThini SLiperficial Explanits

Explants contained no vascular tissue. Organogenesis dependedon the respective IAA and kinetin concentrations in the medium(mean values established for 24 explants).

Basal 'Medium +Buds Roots

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1 jM 1 7 ± 3 010 5 + 2 8±i 3

100 3 ± 1 13 + 5

0 10 2 ± 1 10 4100 0 15 6

0 0 0 0

served in the parenchyma cells: the nuclei increase in volumeand stainability (Fig. 22, arrow). Periclinal and oblique divi-sions are observed in certain places in the subepidermal paren-chyma (Fig. 23). These divisions are more intense the 4th day,giving rise to rows of thin cells (Fig. 23, arrows). After about 6days and thereafter, numerous cells formed by this neocam-bial zone differentiate into tracheids (Fig. 24, arrows).

Scar Tissue Formed during Bud Formation. The cells in thezone wounded during excision of the explant which are placedon the culture medium divide actively after 3 days of cultureand produce layers of flat cells which eventually constitute scartissue (Fig. 21). Thus, explants devoid initially of vascular tis-sue grown on a medium which stimulates bud formation onlyshow, after 2 weeks of culture, three distinct zones (Fig. 21): asuperficial zone composed of cells originally in the epidermisor subepidermal layer from which bud meristems (m) arise, amedian zone occupied by parenchyma and many newly differ-entiated vascular elements (v), and an external zone of scartissue (sz).

Formation of Root Meristems. Numerous divisions are ob-served after 3 days of culture in the basal parenchyma layerplaced against the medium (Fig. 25). After 5 days, these divi-sions extend to the next few layers all along the fragment onthe wounded zone; several new cell layers are thus formed. Di-vision, first anticlinal, then periclinal, produce small groups ofmeristematic cells sometimes almost contiguous, later formingthe root meristems which, at 7 days, already have a wellformed root cap (Fig. 26). Numerous roots (10-15) are oftenobserved on the same explant (Figs. 27 and 28).

DISCUSSION AND CONCLUSION

After regulating organogenesis in stem segments (4-6), wewere able to control organogenesis more precisely and to a finerdegree from tissues excised separately from organ segments.(a) Bud neoformation can be obtained from an epidermal stripdetached from the stem segment as long as it is replaced di-rectly on the same segment or separated from it by a thin agarlayer. (b) Proliferation of the parenchymatous cell layers, nor-mally mitotically inactive in the context of the stem segment,occurs when these cells are grown without vascular tissue. (c)From simple homogeneous explants, composed of epidermisand one to seven layers of subjacent parenchyma, all the organsproduced de novo in stem segments can be obtained.

Apart from single cell cultures, we do not know of anotherexample in the literature of proliferation and organogenesisfrom culture of one tissue composed of a single cell layer asdifferentiated and as homogeneous as the stem epidermis ofTorenia fournieri. However, in our experimental conditions,the epidermis only demonstrates its budding capacities if it isin direct or indirect contact with the original stem segment orif it is accompanied by at least one layer of subjacent paren-chyma cells. Without this contact, the epidermal cells die.

Whereas the epidermis, normally capable of bud formation,does not proliferate if grown alone, the opposite is observed insubepidermal parenchyma tissue. Normally mitotically inactivein a stem segment, when isolated from epidermis and vasculartissue, it is capable of cell division and root organogenesis.We have shown that a small number of subepidermal layers

covered by the epidermis and separated from vascular tissuehave budding and rooting capacities. Similar results are ob-served in analogous explants in Nautilocalyx (15) and Nicotianatabacum (16, 17). However, in Verbascum (7) explants devoidof vascular tissue do not proliferate.

It seems likely that the proliferation shown by certain tissuesof the same organ fragment is closely regulated by inhibitorand stimulatory correlations; the parenchyma tissue of a stemsegment only expresses its organogenetic capacities in the ab-sence of vascular tissue. On the other hand, this same paren-chyma cannot form buds in the absence of the epidermis. Thesubepidermal parenchyma seems then to be subject to an inhi-bition from the vascular tissue and a stimulation from the epi-dermis.

This type of antagonistic correlation existing among tissuesof an organ fragment has been more often shown in entireplants. A recent example is that of Scrofularia arguta where theapical bud and the roots have two antagonistic types of corre-lations, respectively inhibitory and stimulatory of vegetativegrowth in cotyledonary buds (11). Also, the inhibitory effect ofa whole plant on the organogenetic capacity of its undetachedorgans (leaves of Begonia and Nautilocalyx, stem and leaf ofTorenia, floral shoot of tobacco, etc.) is similar to that exercisedby one tissue on the organogenetic expression of a neighboringtissue in the same organ fragment.An organ detached from the plant and the explant excised

from an organ fragment, once freed from the inhibitory corre-lations of their original context, acquire a new physiologicalequilibrium which allows them to express their latent capaci-ties. However, for tissue explants, a defined hormcnal --r.nortis often required for the organogenetic expression of those i11i-tially masked potentialities such as in Nautilocalyx (15), car-rot (9), pine tissue (10), Verbascumn (7), and the classic exampleof tobacco pith tissue (8, 13).

Table II. Organls Obtainted cle NovoThe nature of organs obtained de ntovo depended upon the tis-

sues composing the explant grown in culture.

Tissues Composing Explant Grown in Culture Types of Organs FormeddeN1

Epidermis aloneSubepidermal parenchymaEpidermis with one to seven parenchyma

layers but with no vascular tissueStem segment without epidermisExcised epidermis replaced on stem seg-ment

RootsBuds or roots or both

RootsCallus then buds

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345

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Plant Physiol. Vol. 54, 1974

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CORRELATIONS IN ORGAN FRAGMENTS

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FIGS. 22, 23, 25-28: Represent longitudinal sections. Fig. 24 represents a cross section. Fig. 22: beginning of proliferation in the parenchyma.Arrows indicate densely stained nuclei. Fig. 23: formation of flattened cells arranged in rows in the parenchyma (arrows), periclinal divisions (pd);Fig. 24: tracheids (tr) differentiated from cells of the cambial type, parenchyma (p); Fig. 25: beginning of division in the parenchyma layer (p)

in contact with the culture medium, epidermis (e); Fig. 26: root differentiation (r) from parenchyma cells, root cap (c); Figs. 27 and 28: several

roots (r) formed in one explant, epidermis (e).

Acknowledgments-Thanks are due to Dr. Tran Tlianh Van for suggesting the

subject of this work and for her continual guidance. The assistance of Dr. Averil

Chlyah in correcting the English style is gratefully acknowledged.

LITERATURE CITED

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bourgeons: Dominance apicale et phenonmenes analogues. In: W. Ruhland,ed., Encyclopedia of Plant Physiology, Vol. XV/1. Springer-Verlag, Berlin.

pp. 1106-1164.3. CHAMPAGNAT, P. 1965. Carences et inhibition correlative. In: Travaux de

biologie v6g6tale dedies au Professeur Plantefol. Masson & Cie, Paris. pp.

5-19.

4. CHLYAH, H. 1973. N6oformation dirieae Li partir de fragments d'organes de

Torenia fournieri Lind cultiv6s in vitro. Biol. Plant. 15: 80-87.

U.

jo-

* - _

',~ l_--.

IP..

347Plant Physiol. Vol. 54, 1974

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Plant Physiol. Vol. 54, 1974

5. CHLYAH, H. 1974. Etude histologique de la neoformation de meristemescaulinaires et radicularies a partir de segments d'entre-noeuds de Toreniafournieri Lind cultiv6s in vitro. Can. J. Bot. 52: 473-476.

6. CHLYAH, H. 1974. Forrmation and propagation of cell division centers in theepidermal layer of internodal segments of Torenia fouirntieri Lind grown

in vitro. Simultaneous surface observation of all the epidermal cells. Can.J. Bot. 52: 867-872.

7. CARUSO, J. L. 1971. Bud formation in excised stem segments of VerbascumThapsus. Amer. J. Bot. 58: 429-431.

S. JABLONSKI, J. R. AND F. SKOOG. 1954. Cell enlargement and cell division inexcised tobacco pith tissue. Physiol. Plant. 7: 16-24.

9. KATO, H. 1968. The general observations of the adventive embryogenesis inthe microculture of carrot tissue. Sci. papers of the College of Gen. Educ.Univ. of Tokyo. 18: 191-197.

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11. MIGINIAC, E. AND N. LACOMBE. 1973. Influence de ph6nom&nes d'antagonismeentre organes et entre regulateurs sur le d6veloppement floral de bourgeonscotyledonaires chez le Scrofularia arguta Sol. Can. J. Bot. 51: 465-473.

12. MURASHIGE, T. AND F. SKOOG. 1962. Revised medium for rapid growtlh andbioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497.

13. NITSCH, J. P. AND A. LANCE-NOUGAREDE. 1967. L'action conjuguke (les auixineset des cytokinines sur les cellules de mnoelle de Tabac: 6tude physiologiqueet microscopie electronique. Btull. Soc. Franc. Physiol. 13: 81-118.

14. SKOOG, F. AND C. 0. MILLER. 1957. Chemical regulation of gr-owth and organformation in plant tissues culttured in vitro. Symp. Soc. Exp. Biol. 11:118-131.

15. TRAN THANH VAN, MI. AND A. DRIRA. 1970. Definition of a simple experimentalsystem of directed organogenesis de novo: organ neoformation from epi-clermal tissues of Nautilocalyx lynchei. In: Les cultures (le tissus de plantes.Colloques internationaux du CNRS. 193: 169-176.

16. TRAN THANH VAN, M. 1973. In vitro control of de novo flower, btud, root, andcallus differentiation from excised epidermal tissues. Nature 246: 44-45.

17. TRANX THANH VAN, M. 1973. Direct flower neoformation from small explantsof epidermal tissue of Nicotiana tabacum. Planta 115: 87-92.

18. WARDLAW, C. WI. 1965. Organization and evolution in plants. Longmans, Green,London.

19. WARDLAW, C. W. 1968. MIorphogenesis in plants. A contemporary study.Methuen, London.

348 CHLYAH