Development 108, 191-201 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

191

The control of root, vegetative shoot and flower morphogenesis in tobacco

thin cell-layer explants (TCLs)

DEBRA MOHNEN1, STEFAN EBERHARD, VICTORIA MARFA, NANCY DOUBRAVA, PATRICK

TOUBART*, DAVID J. GOLLIN, TERESA A. GRUBER, WALID NURIf, PETER ALBERSHEIM and

ALAN DARVILL

Complex Carbohydrate Research Center and Departments of Biochemistry and Botany, The University of Georgia, 220 Riverbend Road,Athens, Georgia 30602, USA

•Current address: Dipartimento di Biologia Vegetale, Universita di Roma "La Sapienza", Piazzale A. Moro, 00100 Rome, Italyt Current address: 5509 Halpine Place, Apt 201, Rockville, MD 20851, USA1 To whome correspondence should be sent

Summary

Thin cell-layer explants (TCLs) have been proposed asfavorable tissues for the study of root, vegetative shootand flower formation. We tested the effects of pH, lightquality, light quantity, and IB A and kinetin concen-trations on the morphogenesis of TCLs cultured indi-vidually on a liquid medium. Alterations of the amountsof exogenously supplied IBA and kinetin were sufficientto induce the formation of roots, vegetative shoots andflowers on TCLs cultured on otherwise identical media.The type and number of organs formed were sensitive tothe intensity of light (55, 75, 100 and 120 /lEinsteinsm~2sec~') under which TCLs were grown. Evidencewas obtained that the effects of light on TCL morpho-genesis were associated with photochemical degradationof IBA in the medium. Evaluation of the organogenesisthat occurred in TCLs cultured on a medium containinga range of IBA and kinetin concentrations showed thatthe number and type of organs formed, and overall

growth, were dependent upon the initial concentrationsof auxin and cytokinin. We have developed the TCLculture system into a sensitive and reproducible bioassayfor the study of morphogenesis. The advantages of usingthe TCL morphogenesis bioassay for the identificationand study of molecules (e.g. cell wall oligosaccharides)that may regulate morphogenesis are discussed.

Abbreviations: BSTFA, N,O-ftw(trimethylsilyl)trifluoroacetamide; GC, gas chromatographic; IBA, indole-3-butyric acid; IPA, indole-3-propionic acid; 1-NAA, 1-naphthaleneacetic acid; TCL, thin cell-layer explant; TMS,trimethylsilyl; /iEm~2s~', ^Einsteinsm sec"1.

Key words: auxin, cytokinin, IBA, kinetin, morphogenesis,Nicotiana tabacum, oligosaccharin, organogenesis, TCL,thin cell layer, tobacco.

Introduction

Morphogenesis, the sum of processes that gives form toan organism, includes organization of cells into tissues,tissues into organs, and organs into the entire organism.In plants, morphogenesis is a continual process. Agrowing axis that consists of two centers of division, theroot and shoot meristems, is established in the embryo.The entire plant body, including roots, leaves, stemsand flowers, is formed from these meristematic centers.Plant development, however, is notably plastic(Trewavas and Jennings, 1986). Differentiated cells canbecome meristematic during normal development or inresponse to wounding. The totipotency of plant cells isdemonstrated by the ability of in vitro cultured plantorgans, tissues, cells and protoplasts to develop meris-

tematic centers of cells that can develop into roots,vegetative shoots, flowers or entire plants (Skoog andMiller, 1957; Takebe et al. 1971; Tran Thanh Van et al.1974).

Progress has been made in identifying factors thatregulate plant morphogenesis. These factors includeenvironmental stimuli, such as light and temperature,and molecules endogenous to plants, such as thephytohormones cytokinin and auxin (Davies, 1987).Recently, other molecules of plant origin, oligosacchar-ins (oligosaccharides with regulatory properties), havebeen shown to be capable of regulating morphogenesis(Gollin et al. 1984; York et al. 1984; Tran Thanh Van etal. 1985; McDougall and Fry, 1988).

Tran Thanh Van and colleagues (1985), in collabor-ation with our laboratory, presented evidence that the

192 D. Mohnen and others

addition of fragments from plant cell walls to tobaccothin cell-layer explants (TCLs) cultured on variousliquid media caused TCLs to form vegetative shootsrather than flowers or callus, and flowers rather thanvegetative shoots. These observations stimulated us toattempt to use TCLs to study the role of plant cell wallfragments in plant morphogenesis. Unfortunately, inspite of extensive efforts to perform the in vitro TCLbioassay as described (Tran Thanh Van et al. 1985), wewere unable to obtain the expected TCL organogenesis.

TCLs, which have only three to four cell types(epidermis, chlorenchyma, collenchyma and paren-chyma) and initially a total of four to ten cell layers,have been proposed as a favorable tissue to use for thestudy of organogenesis (Tran Thanh Van, 1973; TranThanh Van et al. 1974). The TCLs exhibit organogen-esis of roots, vegetative shoots and flowers within ca.three weeks of culture. Furthermore, the organogenesisoccurs without intervening callus formation. Thus,TCLs allow the study of changes that occur when tissuesare directly induced to undergo organogenesis, asopposed to when intermediate callus formation is firstrequired.

TCLs have been used successfully to study variousaspects of flower formation (Tran Thanh Van et al.1974; Cousson and Tran Thanh Van, 1981; Bridgen andVeilleux, 1985; Altamura etal. 1986; Wilms and Sassen,1987; Heylen and Vendrig, 1988; Kaur-Sawhney et al.1988; Smulders et al. 1988ft; Rajeevan and Lang, 1987;Meeks-Wagner et al. 1989). However, there is noconsensus in the literature as to a standard set ofconditions required to induce TCLs to produce roots orvegetative shoots or flowers. Most researchers haveconcentrated on a subset of morphogenesis programs,such as flower formation or flower and vegetative shootformation. Also, some researchers have used the pedi-cel as the TCL source (Wilms and Sassen, 1987;Smulders et al. 1988a), while others have used the basalinternodes of primary floral branches (Tran Thanh Vanetal. 1974; Heylen and Vendrig, 1988; Kaur-Sawhney etal. 1988; Meeks-Wagner etal. 1989). In addition, TCLshave been incubated on an agar-solidified medium bysome laboratories (Tran Thanh Van etal. 1974; Bridgenand Veilleux, 1985; Wilms and Sassen, 1987; Heylenand Vendrig, 1988; Kaur-Sawhney etal. 1988; Smulderset al. 1988a) and on a liquid medium in others (TranThanh Van et al. 1985; Rajeevan and Lang, 1987;Meeks-Wagner et al. 1989).

We decided to determine which factors had thegreatest effects on TCL morphogenesis and to developthe TCL morphogenesis system into a reproduciblebioassay. Such a bioassay would be useful to studyfactors (e.g. oligosaccharins) that regulate root, veg-etative shoot and flower organogenesis. We were moti-vated by the possibility of obtaining further evidencethat oligosaccharides from the cell wall regulate TCLmorphogenesis, since this would have far-reachingimplications about the function of plant cell wall poly-saccharides in plant development (Albersheim et al.1983). Our study was limited to TCLs taken from basalinternodes of primary floral branches since these

explants have a greater potential than TCLs taken frompedicels, to form vegetative shoots as well as flowersand roots (Altamura et al. 1986; Rajeevan et al. 1987;Tiburcio et al. 1988). Also, TCLs were incubated on aliquid medium rather than on an agar-solidified me-dium, in order to eliminate the possibility of testmolecules binding to the agar matrix. Explants culturedon agar-solidified media also appear to take up mol-ecules (e.g. hormones) more slowly from the mediathan explants cultured on liquid media (Sdnchez-Bravoetal. 1988; Wilms, 1989).

In this paper, we systematically study the effects ofpH, indole-3-butyric acid (IBA), kinetin and light onTCL morphogenesis, and report the development of areproducible TCL bioassay for studies of plant morpho-genesis.

Materials and methods

Plant materialNicotiana tabacum L. cv Samsun plants were grown from seed(gift of K. Tran Thanh Van) in a greenhouse at 23-31 °C.Supplemental lighting was provided by high pressure sodiumlights in order to maintain a 14 h day length. Black shade cloth(51 % shade, Chicopee, Gainsville, GA) was placed over thegreenhouse from April through September to reduce seasonalvariation of solar radiation. Plants were grown in a peat-litesoil mix (Fafard No. 3, Conrad Fafard, Springfield, MA) in20 cm clay pots and fertilized twice each week with Peters20-20-20 fertilizer (containing 473 ppm nitrogen). Sinceambient ozone levels were too high for growth of healthytobacco plants in the greenhouse from late spring to earlyautumn, air was passed through carbon filters (RSE, NewBaltimore, MI) that removed approximately 80% of thenaturally occurring ozone from the air.

Thin cell-layer bioassayThe TCL morphogenesis bioassay used was a modification ofthe assay developed by Tran Thanh Van et al. (1974, 1985).The second and third primary floral branches of a typicaltobacco inflorescence, illustrated in Fig. 1, were harvestedwhen approximately 30 % of the flowers had produced greenfruits. Floral branches were cut into 7-8 cm sections and thesections soaked 2min in 0.5% Tween 20, surface-sterilized8min in 10% commercial bleach (Clorox™ containing5.25% NaOCl), and rinsed three times (total of lOmin) insterile deionized water. Strips of tissue, approximately lmmwide and composed of 1 layer of epidermal cells, 2-3 layers ofchlorenchyma and 3-6 layers of collenchyma and paren-chyma, were cut from the floral branch tissue. TCLs approxi-mately 10 mm long were cut from the tissue strips and floatedindividually on 2 ml of a Linsmaier and Skoog (1965) mediumcontaining 167 mM-glucose (basal medium) and the indicatedamounts of indolebutyric acid and kinetin (Sigma, St. Louis,MO). The media were filter-sterilized using 0.2 tm filtrationunits (Nalgene Labware, Rochester, NY). Unless otherwiseindicated, the pH of each medium was adjusted to 5.8 bytitration with KOH.

TCLs were placed individually in 7 ml wells of 12-well cellculture cluster dishes (Gibco™, Grand Island, NY), and thedishes were sealed with two layers of parafilm (American CanCo., Greenwich, CT). The TCLs were incubated at 24°Cunder continuous cool white F40T120 (natural F40T120 orGro-Lux™ F40GRO where indicated) fluorescent lamps

Control of TCL morphogenesis 193

(Sylvania, Danvers, MA) at 55±5 jtEinsteinsm 2sec '(liEm~2s~l), unless otherwise indicated. After 23-25 days ofculture, the morphogenesis of TCLs was visually scored usinga dissecting microscope. Three types of organs (roots, flowersand vegetative shoots), the general size and the degree ofpolar enlargement of the TCLs (Eberhard et al. 1989) wererecorded. One organ was defined as the root(s), vegetativeshoot(s), or flower(s) that grew from a single initial shoot orroot meristem, or the meristem itself. Thus, an individualflower formed directly on the TCL surface would be recordedas one flower, as would an inflorescence having five flowers.Likewise, a primary root with three lateral roots would berecorded as one root. Vegetative meristems were dis-tinguished from flower meristems according to Waterkeyn etal. (1965). The data were analyzed using an R:Base for DOSdatabase (Microrim™, Redmond, WA).

Quantitation of IB A in the mediumThe basal medium containing 6/iM-IBA and 0.5 jiM-kinetinwas incubated in cluster dishes under cool white fluorescentlight at 55 or 120/iEm~2s~'. Dishes covered with aluminumfoil served as dark controls. IBA concentration was deter-mined by gas chromatographic (GC) analysis of duplicatesamples collected from each treatment after days 0, 2,4,6,12,and 24 of incubation. IBA was extracted from the medium bypartitioning with ethyl acetate (HPLC grade). An internalstandard of 1 ng indole-3-propionic acid (1PA) was added toeach sample after the extraction. Indoles were converted totrimethylsilyl (TMS) derivatives by heating them at 65°C for20min with N,O,-i)«(trimethylsilyl) trifluoroacetamide

Fig. 1. Typical morphology of Nicotiana tabacum L. cvSamsun. T: terminal floral branches. 1-4: consecutiveprimary floral branches. Arrows denote floral branches usedin the TCL bioassay. Total number of leaves varies from16-29. Total number of flower branches with matureflowers varies from 3-6.

(BSTFA). The TMS derivatives were analyzed on a HewlettPackard 5880A series gas chromatograph using a DB-1 30-meter (0.25 mm i.d.) fused silica capillary column (0.25 fanfilm thickness). The GC oven was programmed from 90°C to240°C at ^"Cmin"1. Injections were made in the splitlessmode at 250°C. Quantitative determinations were derivedfrom peak areas using a standard curve for IBA.

Results

Three types of TCL organogenesis can be obtained onmedia of the same pHThe experimental design for the TCL bioassay waschanged from incubation of 20 TCLs in 100x15 mmPetri dishes (Tran Thanh Van et al. 1985) to incubationof a single TCL per well in 12-well cluster dishes. Thischange was made because of the possibility that chemi-cal signaling between TCLs in the same dish mightaffect organogenesis. TCLs in the same Petri dishoccasionally exhibited greater uniformity in morpho-genesis than TCLs in replicate Petri dishes (data notshown). Incubating TCLs individually also reducedTCL loss due to microbial contamination and reducedthe number of TCLs needed for statistically significantresults, since each TCL served as a replicate rather thana subsample of a single replicate containing 20 TCLs.

Tran Thanh Van and colleagues (1985) induceddifferent types of TCL organogenesis by altering the pHand the IBA and kinetin concentrations of the culturemedium. We attempted to simplify the system bydetermining whether root, vegetative shoot and flowerorganogenesis could be induced in media adjusted tothe same pH. Basal medium supplemented with 0.5 JIM-kinetin and 0.5, 5.0, or 15/iM-IBA at pH3.8, 4.5, 5.0,5.8, or 6.15 was used for these experiments, sincepublished results (Cousson and Tran Thanh Van, 1981;Tran Thanh Van et al. 1985) and our preliminary dataprovided evidence that the three types of organogenesiscould be induced using these conditions. Culture ofTCLs in 55^iEm~2s~1 light on a medium containing0.5 iM-IBA and 0.5^M-kinetin resulted in a mixture offlowers and vegetative shoots, regardless of the pH ofthe medium, as shown in Fig. 2A. Very few organs wereformed when TCLs were incubated on media contain-ing 5.0/iM-IBA, except at pH6.15 where flowers wereproduced (Fig. 2B). Roots were formed at each pHtested on a medium containing 15fiM-IBA and 0.5 IXM-kinetin (Fig. 2C). A slightly greater number of flowersand roots were formed on TCLs cultured on media thathad been adjusted to a pH between 5 and 5.8. SimilarpH effects were obtained in duplicate experiments withTCLs incubated in 95/iEm~2s~1 light.

These results demonstrated that, with the exceptionof the formation of flowers at pH6.15 on mediumcontaining 5/XM-IBA, variation of pH within the rangeoriginally used (Tran Thanh Van et al. 1985) causedonly small changes in the number of organs formed onTCLs and had no deciding influence on the type oforgan formed. Our results demonstrated that alteringthe pH of the medium is not required to obtain thethree types of TCL organogenesis. Therefore, the TCL

194 D. Mohnen and others

ooa.mmat

XI

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4 -

3-

2-

1 -

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2-

1 -

A. IBA(0.5»iM) FVSR

B.

C. IBA (15MM)

3.0 4.0 5.0

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6.0 7.0

Fig. 2. Effect of pH on organogenesis of TCLs Culturedunder 55 /dE m~2s~1 of cool white light. Mean numbers(±S.E.) of flowers (F), vegetative shoots (VS), and roots(R) formed per TCL after 23-25 days in culture. Datarepresent two experiments with a minimum of 24 replicatesper experiment. TCLs were cultured on basal mediumcontaining 0.5^M-kinetin and (A) 0.5/iM-EBA, (B) 5/iM-IBA, or(C)

bioassay in cluster dishes was simplified in subsequentstudies by adjusting each medium to pH5.8, a pHwithin the range commonly used for plant tissue culture(George et al. 1987).

Effect of light quality on TCL organogenesisTCLs have traditionally been cultured under cool whitefluorescent lights supplemented at intervals with incan-descent lights as in Cousson and Tran Thanh Van(1983). We were concerned that the variable positioningof incandescent lights would produce unequal lightquality for the TCLs. Therefore, we tested whetherfluorescent light alone could be used to obtain root,vegetative shoot and flower organogenesis. TCLs werecultured on basal medium containing various concen-trations of IBA and kinetin and incubated at50/iEm~2s~1 under one of three types of Sylvaniafluorescent lights: cool white, Gro-Lux™ or natural.All types of organogenesis, as shown in Table 1, wereinduced using fluorescent light without incandescentlight as a supplement. Subsequent experiments wereperformed using cool white fluorescent lights.

Changes in light intensity alter TCL organogenesisLight intensity has been shown to affect the organogen-esis of TCLs (Tran Thanh Van, 1980). We have testedthe effect of light intensity (55, 75, 95 and115^iEm~2s~1) on the organogenesis of TCLs culturedin Petri dishes (20 TCLs per dish). The number offlowers decreased as the light intensity was increasedwhen TCLs were incubated on a medium containing0.5JIM-IBA and 0.5 ,uM-kinetin at pH3.8 (Fig. 3A).TCLs formed roots at the lowest light intensity(55^Em~2s~1), vegetative shoots at the middle lightintensities (75 and 95 /xEm~2s~1) and flowers at the twohighest light intensities (95 and 115^Em~2s~1) wheng )the TCLs were incubated on a medium containingIBA and 0.5/xM-kinetin at pH6.15 (Fig. 3B). Fewerroots formed on TCLs at the higher light intensitieswhen the TCLs were cultured on a medium containing7/iM-IBA and 0.2/iM-kinetin at pH5.8 (Fig. 3C).

In vitro bioassays, such as the TCL assay, are mostuseful for studies of organogenesis and other aspects of

Table 1. Effect of light quality on organogenesis of TCLs cultured on three mediaIBA/JM

0.50.50.5

5.05.05.0

151515

•Cool white (CW), groluxt Mean numbers of flowers

Kin

0.50.50.5

0.50.50.5

0.50.50.5

Light*quality

CWGLNat

CWGLNat

CWGLNat

(GL), or natural (Nat)(F), vegetative shoots

N

826024

846024

836024

fluorescent lights.(VS), or roots (R)

F±S.E.M.t

2.7±0.33.1±0.42.8±0.5

0.3±0.100

000

from N TCLs±SE.

VS±S.E.M.

1.2±0.2l.l±0.20.3±0.1

0.1±000

000

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3.9±0.46.0±0.66.3±0.6

morphogenesis if factors that cause irreproducibilityhave been identified and controlled. Therefore, wedetermined how sensitive TCL organogenesis was tolight quantity in our modified system of incubatingTCLs at pH5.8 in cluster dishes. TCLs were incubatedunder 55, 75, 95 and 120 (±5) f iE i r rV 1 light on aflower-inducing medium, a transition medium (TCLscultured on transition medium form few or no organs),a root-inducing medium, and a vegetative shoot-in-ducing medium. Increasing the light intensity caused adecrease in the number of flowers when TCLs were

80 -

55 75 95 115

Ught (uElnstelns m~2 sec"1)

Fig. 3. Effect of cool white light quantity on organogenesisof TCLs in Petri dishes. Mean numbers of flowers (F),vegetative shoots (VS), and roots (R) formed per Petri dishof 20 TCLs after 23-25 days in culture. Data represent oneexperiment with 7 or 8 replicate Petri dishes. Standard errorbars less than 0.5 mm in length are not shown. TCLs wereincubated on basal medium containing (A) 0.5/iM-IBA and0.5/iM-kinetin, pH3.8; (B) 3^M-IBA and 0.5 j*M-kinetin,pH6.15; and (C) 7/iM-IBA and 0.2/iM-kinetin, pH5.8.

Control of TCL morphogenesis 195

cultured on the flower-inducing medium (Fig. 4A). Thiseffect was similar to that observed in the Petri dishexperiment (compare Fig. 4A to Fig. 3A). Moreflowers were produced under the highest than under thelowest light intensity when TCLs were incubated ontransition medium (Fig. 4B). Conversely, fewer roots

3 -

55 75 95 120

Light O.Eln»tain» m'2 «oc"1)

Fig. 4. Effect of cool white light quantity on organogenesisof TCLs in cluster dishes. Mean number of flowers (F),vegetative shoots (VS), and roots (R) formed per TCL after23-25 days in culture. Data represent two experiments with10-48 replicates per experiment. Standard error bars lessthan 0.5 mm in length are not shown. TCLs were culturedon basal medium, pH5.8, containing (A) 0 . 5 /M- IBA and0.5 /M-kinetin, (B) 4 / M - I B A and 0.5/ZM-kinetin, (C) 15 JJM-

IBA and 0.5/iM-kinetin, and (D) 1/iM-IBA and 6//M-kinetin.

196 D. Mohnen and others

formed on TCLs incubated on root-inducing mediumunder the highest than under the lowest light intensity(Fig. 4C). Light intensity did not have a significanteffect on vegetative shoot formation when TCLs wereincubated on vegetative shoot-inducing medium(Fig. 4D). TCLs were incubated under 55 (±5)^Em~2s~1 light for subsequent studies in order tosimplify the TCL bioassay.

Organogenesis in thin cell-layer explants can bedetermined by the concentrations of IBA and kinetinin the mediumTCLs were cultured on media containing a range ofIBA and kinetin concentrations in order to characterizethe quality, quantity, and reproducibility of TCL mor-phogenesis using our modified culture conditions. ATCL 'organogenesis map' of organ formation in re-sponse to various IBA and kinetin concentrations isillustrated in Fig. 5. The IBA and kinetin concen-trations in the media determined whether TCLs formedroots, vegetative shoots or flowers when pH and lightintensity were held constant. Roots formed when TCLswere cultured on media with relatively high (5-20 fm)IBA and low (0.1-0.75/ZM) kinetin concentrations.Vegetative shoots formed when TCLs were cultured onmedia with high (2-10 ^M) kinetin and a range of IBAconcentrations. Flowers formed when TCLs were cul-tured on media with relatively low (0.1-2JIM) IBA andlow (0.2-2 fJM) kinetin concentrations. Media with IBAand kinetin concentrations falling between those con-centrations that resulted in the formation on TCLs ofroots, vegetative shoots or flowers are referred to astransition media. TCLs cultured on transition mediaformed few or no vegetative shoots, flowers or roots.The mean numbers of organs produced on TCLscultured on the various media tested (Fig. 5) are givenin Fig. 6.

IBA and kinetin can induce asymmetric organogenesisand tissue enlargement of TCLsIncubation of TCLs on media containing a range ofinitial IBA and kinetin concentrations caused hormone-dependent differences in the polarity of organogenesisand tissue enlargement of TCLs. Polar enlargement andpolar organ formation, if it occurs, is always at the basalend (the end that had been nearest the stem) of theTCL. The orientation of the TCLs after 24 days ofculture was recognized by marking the basal or apicalend of the TCL with a diagonal cut at the time theexplant was removed from the floral branch, or,alternatively, by maintaining the TCL's orientationduring the course of the experiment by growing theTCLs on paper wicks kept in contact with the medium.

The following general conclusions can be made basedon observations of TCLs cultured on media with a rangeof IBA and kinetin concentrations. (1) Pronouncedpolar enlargement occurs on TCLs cultured on mediacontaining < 5 ^ M IBA and <5/ZM kinetin, while uni-form enlargement occurs when the concentration ofeither IBA or kinetin in the media exceeds 5JIM. (2)Organs form either predominantly on the basal end of

the TCL or over the entire TCL, depending upon theorgan type and the IBA and kinetin concentrations inthe media. TCLs cultured on root-inducing mediumusually form roots with a nonpolar distribution, excepton root-inducing medium with relatively low levels (e.g.4JIM) of IBA (see Fig. 5). Vegetative shoots form in anonpolar fashion, except on vegetative shoot-inducingmedia containing <3^M-kinetin. Flowers form in apolar manner, but this polarity is reduced on media with>l;UM-kinetin. (3) The polar distribution of organformation is not always accompanied by polar tissueenlargement. For example, roots can form in a polarmanner on TCLs that show little or no polar tissueenlargement (4^M-IBA and O.l^M-kinetin), andflowers can form in a nonpolar fashion on TCLs thathave well-defined polar tissue enlargement (1 /iM-IBAand ljiM-kinetin). Thus, IBA and kinetin are able toregulate the polarity of tissue enlargement and organo-genesis.

The effect of light intensity on TCL organogenesis:correlation with the light-induced degradation of amedium componentThe sensitivity of TCL organogenesis to light couldhave resulted from a direct physiological response ofTCLs to light or, alternatively, as a secondary responseto light-induced degradation of a medium component.The latter possibility was tested by comparing theorganogenesis of TCLs cultured on fresh media to theorganogenesis of TCLs cultured on media that had beenpreincubated either in the dark or in light. A flower-inducing medium (FM) and two transition media (TM1and TM2) were preincubated for six days at 55 and120fiEm~ s"1 in unwrapped (light-pretreated) and alu-minum foil-wrapped (dark-pretreated) cluster dishes.After preincubation of the media, TCLs were placed onpretreated or fresh media and cultured under 55 orUOfjEm^s'1 light.

The types of organogenesis differed when TCLs werecultured on fresh versus light-pretreated media, asshown in Tables 2 and 3. For example, TCLs incubatedon fresh FM medium or on dark-pretreated FM me-dium produced flowers. However, no flowers formedon TCLs cultured on the light-pretreated FM medium.Alternatively, TCLs formed roots when incubated onfresh or dark-pretreated TM2 medium, while TCLscultured on light-pretreated TM2 medium formedflowers rather than roots.

These results demonstrated that the changes in TCLorganogenesis that occurred when TCLs were incu-bated at increasingly higher light intensities (see Figs. 3and 4) could be mimicked by preincubation of themedia in light. This similarity strongly suggested thatthe light-induced changes in TCL organogenesis weredue to degradation of a medium component(s). Com-parison of the organogenesis of TCLs incubated under55 versus 120^Em~2s~1 light (Fig. 4) with the types oforganogenesis obtained by culturing TCLs on mediawith a variety of IBA and kinetin concentrations at55 fiEm"2s"' (see Fig. 5) showed that a decrease in theamount of IBA in each medium tested could cause the

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ed.

Control of TCL morphogenesis 197

KIN000000.10.10.10.10.10.10.10.10.10.10.10.10.10.10.10.10.10.10.10.10.150.150.150.20.20.20.20.20.20.20.20.20.20.20.20.20.20.20.20.250.250.250.250.250.250.250.30.30.30.30.30.30.30.30.30.30.30.30.30.30.30.30.30.40.40.40.50.50.50.50.50.50.50.50.50.50.50.50.50.50.50.50.50.50.50.50.5

T4

0.10.51.0

10.000.10.20.30.40.50.61.02.03.04.03.06.07.08.010.013.020.025.040.06.07.0B.O0.10.20.30.40.50.61.02.04.05.06.07.08.010.015.020.05.06.07.08.0

10.020.023.00.10.20.30.40.50.61.02.03.04.05.06.07.08.010.015.020.02.03.04.000.10.20.30.40.50.61.01.52.03.04.03.06.07.08.010.013.020.023.040.0

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000000000.30.10.20.10.10.20000000000000000.20.31.21.32.21.40.70000000000000000.10.31.22.33.23.42.71.30.3000000001.00001.01.42.63.94.33.33.73.42.01.00.30.300000000

f00000000.30.10.10.10.10.10000000000000000.10.20.30.40.40.30.20000000000000000.10.10.50.30.40.30.40.30.2000000000.50000.30.40.50.50.10.40.30.30.30.300.100000000

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f0000000000000.10.70.40.70.60.90.70.60.20.91.40.90.80.80.6000000000.41.00.60.30.71.30.40.90.40.60.90.50.91.41.5000000000.10.30.70.50.70 81.20.81.000.20.500000000000.10.10.20.20.30.60.70.10.4O.B1.7

KIN

0^750.750.750.750.750.750.750.750.750.750.750.730.750.750.750.750.750.80.80.80.90.90.91.01.01.01.01.01.01.01.01 01.01.01.01.01.01.01.01.01.01.02.02.02.02.02.02.02.02.02.02.02.03.03.03 03.03.03.03.03.03.03.03.04 04.04.04.04.05.05.05.05.05.05.08.0B.OB.O6.06.06.06.07.07.08.08.09.09.010.010.010.0

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A16141623162221743346463824IB242323152215153215132915161612B1676162313312129161626231627151114631644813B13191513165416381620162035348913493313813912124396

8149141222222727221727269

I4.66.07.66.85.85.84.42.20.8

2.21.10.80.30.10000.12.92.00.54.40.50.504.66.65.35.46.66.34.71.110.10.60.30.10.100001.65.33.63.44.32.10.70000.12 12.52.61.41.30.60.300.30.10.31.0000.40.20.4000.3001.21.21.80.10.10.10000000000

0.60.61.00.80.60.70.60.20.50.30.30.20.10000.10.60.50.30.90.20.200.60.60.50.90 40.70.50.40.30.10.30.40.10.100000.40.91.10.90.50.70.30000.10.50.70.70.50.30.30.100.20.10.30.3000.20.10.1000.3000.50.20.10.100.10000000000

VS.1.40.91.00.91.11.40.20.10.10.40.20.30.100.30000.60.400.90.10.501.82.12.81.73.01.62.30.61.21.90.3O.B0.50.40.10.10.102.64.48.310.27.58.36.34.62.64 40.64.56.99.310.110.86.38.64.32.92.02.412.57.84.84.11 910.910.64.84.03.43.001.2

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0 30.30.20.30.30.20.100.10.10.10.100.10000.30.100.30 10.200.40 30.70 50.50.40.30.20.40.50.10.40.20.20.10.10.100.6O.B1.41.10.61.10.81.10.71.20 30.80.91.31.10.61.20.80.80.70.50.50.91.01.31.30.50.7O.B0.81.31.01.520.50.90.41.50.60.80.91.51.41.50.91.31.11.21.20.6

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00000000000.20.20.20.20.50 71.21 100000000000000000000.10.20 20.10.31.30000000000000000000000000000000000.1000000000000000

Fig. 6. Effects of IBA and kinetin concentrations on the mean number of organs produced on TCLs at pH5.8. Meannumbers (±S.E.) of flowers (F), vegetative shoots (VS), and roots (R) from N TCLs after 23-25 days in culture. Datarepresent a minimum of two experiments. The TCLs were incubated on the basal medium supplemented with the indicatedconcentrations of kinetin (KIN) and IBA.

198 D. Mohnen and others

observed light-induced changes in organogenesis. It hasbeen shown that indoleacetic acid is unstable in light(Yamakawa et al. 1979). Therefore, we determinedwhether incubation of a medium in 55 and120 jiEirT2 s~' light caused degradation of IBA in themedium.

Effect of light irradiation on stability of IBA in themediumThe stability of IBA in the medium was determined bymeasuring the amount of IBA remaining in the basalmedium containing 6fiM-IBA and 0.5^M-kinetin afterthe medium was placed in cluster dishes and maintainedfor various lengths of time under low or high lightintensity. Medium in cluster dishes wrapped in alumi-num foil served as a dark control. The time-dependentdecrease in the concentration of IBA in mediumexposed to 55 and 120 p>Em~2 s"1 cool white fluorescentlight is shown in Fig. 7. The IBA concentration in themedium was reduced by 50 % and 88 % after 6 days ofincubation under 55 and 120/iEm~2s-1 light, respect-ively. The concentration of IBA was reduced by greaterthan 98% in the medium incubated at either lightintensity for 24 days. However, the IBA concentrationof the medium in cluster dishes covered with aluminumfoil decreased by only 9-12% after 24 days of incu-bation. The decreased amount of IBA remaining in the

medium incubated under 55 and 120 Em 2s 1 lightcorrelates with the changes in organogenesis that oc-curred when TCLs were cultured on light-pretreatedmedia (Tables 2 and 3). Thus, light-induced degra-dation of IBA appears to be largely or entirely respon-sible for the observed light-induced changes in TCLorganogenesis.

Discussion

We have evaluated the effects of pH, light quality, lightquantity, and initial IBA and kinetin concentrations onorganogenesis of TCLs cultured on liquid media. Con-trary to previous reports (Cousson and Tran ThanhVan, (1981; Tran Thanh Van et al. 1985), it was notnecessary to alter the pH in order to obtain root,vegetative shoot and flower formation in TCLs culturedon liquid media. All of the types of organogenesis couldbe obtained by varying only two factors: the initialexogenous auxin (IBA) and cytokinin (kinetin) concen-trations. The intensity of light illuminating the TCLsduring culture had a dramatic effect on TCL organo-genesis. We have provided evidence that the observedeffect of light on TCL morphogenesis results from alight-induced degradation of IBA; however, the photo-destruction of endogenous IAA (Jarvis and Shaheed,1987; Fang and Butts, 1957), light activation of the

-2,-)Table 2. Effect of preincubation of media in light on organogenesis of TCLs cultured at 55 \xEm sMedium*

FM

FM

FM

FM

TM1

TM1

TM1

TM1

TM2

TM2

TM2

TM2

Expt #t

12121212

12121212

12121212

Treatment:):

Control

Dark

55

120

Control

Dark

55

120

Control

Dark

55

120

F±S.E.M.§

3.5±0.56.2±0.92.6±0.64.7±0.8

0000

0.3±0.2000

3.5±0.84.8±1.1

00

0000

1.2±0.54.3±1.30.7±0.5

0

VS±S.E.M.

O.ltO.l0.8±0.50.5±0.21.0±0.5

0000

0000

0.1±0.10.2±0.2

00

0000

0.1±0.11.3±0.9

00

•Basal medium supplemented with 0.5/un-IBA and 0.5/iM-kinetin (FM), 4/iM-lBA and 0.5/iM-kinetin (TM1), <kinetin (TM2).

tTCLs were cultured for 19 days (expt 1) or 24 days (expt 2).$ Fresh medium (Control), medium pretreated for 6 days in foil-wrapped cluster dishes placed under 55/iEm~2

pretreated for 6 days in 55/tE~2s~' light (55), or medium pretreated for 6 days in 120/JE~2S~' light (120).§Mean numbers±s.E.M. of flowers (F), vegetative shoots (VS), and roots (R). Data represent a minimum of 6

R±S.E.M.

00000000

00

0.1±0.100000

2.3±0.90

1.8±0.71.7±0.8

0000

or 6/iM-IBA and 0.5 /.IM-

S'' light (Dark), medium

replicates per experiment.

Control of TCL morphogenesis 199

-2 r-iTable 3. Effect of preincubation of media in light on organogenesis of TCLs cultured at 120 fiEm s

Medium* Expt # t Treatment^ F±S.E.M.§ VS±S.E.M. R±S.E.M.

FM

FM

FM

FM

TM1

TM1

TM1

TM1

TM2

TM2

TM2

TM2

Footnotes: see Table 2.

12121212

12121212

12121212

Control

Dark

55

120

Control

Dark

55

120

Control

Dark

55

120

2.4±0.81.8±0.63.0±0.72.5±0.5

0000

1.4±0.70

0.9±0.40.3±0.31.6±0.33.3±0.70.5±0.4

0

1.6±0.80

0.3±0.20

3.9±0.43.8±1.42.3±0.9

0

0.110.11.010.60.110.10.510.2

0000

0.110.10.310.2

00.710.7

00.210.2

00

0.110.10.210.20.110.10.510.5

00.410.2

00

00000000

00

0.310.300000

0.810.31.210.81.010.60.510.5

0000

8 12 16

Days of Incubation

20

Fig. 7. Effect of cool white light quantity on stability ofFBA in the medium. Mean concentration ( ± S . E . ) of IBAremaining in the medium after exposure of the mediumcontaining 6/an-IBA and 0.5^M-kinetin to 55 (55L) or 120(120L) /*Em~2sec~' of cool white fluorescent light for theindicated length of time. The media not exposed to lightwere also tested (55D and 120D). Data represent duplicatesamples from one (55/iEm~2s and 12 day point from120/iEm"2s"1) or two (120/iEm~2s~') experiments.

IAA-oxidase system (Galston et al. 1953), or modifi-cation of the TCL response to auxin (Klein, 1967) mayalso be involved.

We have shown that the cytokinin and auxin concen-trations in the medium can determine the degree of

asymmetric growth of TCLs as well as the type andnumber of organs formed. Asymmetric growth is typi-fied by greater tissue enlargement and/or organogen-esis at the basal end of TCLs cultured under low(<5 ^M) auxin and cytokinin concentrations. Hormone-dependent asymmetric flower and asymmetric callusformation on TCLs cultured on solid media has pre-viously been reported (Van den Ende et al. 1984;Smulders et al. 1988a). Tissue enlargement at the basalend of TCLs cultured on media containing relativelylow levels of auxin is consistent with a hypothesis thatbasipetal movement of auxin is involved in establishingthe polarity (Goldsmith, 1977; Sachs, 1984). Smuldersand colleagues have shown that the polar transport ofthe auxin analog 1-naphthaleneacetic acid (1-NAA)leads to the formation of flowers on the basal end ofTCLs cultured on solid media (1988a), and that TCLscultured on a solid medium do not secrete 1-NAA intothe medium (19886). If TCLs cultured on a liquidmedium also do not secrete IBA into the medium, a netbasipetal transport of IBA would lead to an accumu-lation of IBA in the basal end of TCLs. Since auxin inthe presence of cytokinin is known to induce celldivision and enlargement (Davies, 1987), our data areconsistent with an accumulation of auxin at the basalend of the TCL being the cause of polar tissue enlarge-ment. A similar conclusion was reached by Niedergang-Kamien and Skoog (1956) using the internal tissues oftobacco stems. They reported that polar callus andpolar organ formation on the basal end of tobacco stemsegments and stem cylinders (consisting of cambium,

200 D. Mohnen and others

xylem, internal phloem, and pith) was dependent on anauxin gradient.

The results of our experiments complement andextend previous work by Skoog and Miller (1957), TranThanh Van et al. (1974), and others which establishedthat the concentrations of auxin and cytokinin in mediacan determine the type of organogenesis that occurs incallus and tissue cultures. It was by working with arelatively simple system under controlled conditionsthat Skoog and Miller (1957) recognized that cytokininand auxin can regulate organogenesis. We have modi-fied the tobacco thin cell-layer bioassay in order to usethe system to study plant morphogenesis. The TCLbioassay, as described here, meets Skoog and Miller's(1957) criteria for a useful system to study the manyfactors that must interact in a growth response ascomplex and variable as organogenesis. The TCLs arean example of a tissue "capable of 'multiple growthresponses' and grown under as rigidly standardizedconditions as reasonably could be attained" (Skoog andMiller, 1957).

The significance of the results reported here is thatthey (i) give the methodology to use the same tissueexplants effectively for studies of root, vegetative shootand flower morphogenesis, and growth in general; (ii)set the foundation, both conceptually and experimen-tally, for subsequent studies of molecules that regulateTCL morphogenesis (Eberhard et al. 1989), and (iii)establish that changes in the initial exogenous concen-trations of cytokinin and auxin are sufficient to induce,in a predictable fashion, different types of organogen-esis.

TCLs can be grown on the same medium, at the samestarting pH, in the same light, with the same amount ofglucose and be induced to form different types andnumbers of organs by altering only the concentrationsof auxin and cytokinin. This study, in comparison toprevious studies, most clearly demonstrates the effectsof exogenous hormone concentrations on flower, veg-etative shoot and root organogenesis. This point is nottrivial as a controversy exists as to whether the decidingfactors in plant development are the concentrations ofhormones or the sensitivity of plant cells to the hor-mones (Guern, 1987). Although the results describedhere show that alterations in the concentrations ofexogenous auxin and cytokinin are sufficient to initiate,in 'identical' TCLs, a series of events which culminate inthe formation of roots, vegetative shoots and flowers,they do not rule out that changes in sensitivity to thehormones occurs during the organogenesis process.TCL morphogenesis is an attractive system to test thehypothesis that the plant hormones regulate the sensi-tivity of plant tissues to hormones, for example, bytesting whether hormones regulate the number ofhormone receptors (Guern, 1987).

We have developed a TCL IBA-kinetin 'organogen-esis map' to characterize the effect of various para-meters on TCL organogenesis. The 'organogenesismap' enables us to predict the effects that changes inauxin and cytokinin concentrations will have on TCLorganogenesis. TCLs cultured on transition media were

particularly useful in deciphering the effects of light onorganogenesis, since relatively small, light-inducedchanges in the concentration of a hormone resulted inan easily observed change in organogenesis. Thus,TCLs cultured on transition media are a particularlysensitive source of tissue to use in studying the bio-chemical changes associated with morphogenesis. Wehave recently confirmed that the TCL bioassay de-scribed here can be used to study and identify molecules(i.e. oligosaccharins) that regulate morphogenesis. TheTCL bioassay was used to show that cell wall fragmentsreleased from sycamore suspension cells could, depen-ding upon the concentrations of auxin and cytokinin inthe medium, inhibit root formation, cause roots to formin a polar manner, and induce polar tissue enlargementand flower formation (Eberhard et al. 1989). TCLmorphogenesis can be used to dissect, into specificdevelopmental and molecular components, the eventsthat occur during organ formation.

We thank Kiem Tran Thanh Van for introducing us to thethin cell-layer system, William S. York for writing the pie-chart (Fig. 5) plotting program, Ray F. Severson (USDA-ARS, Athens, GA) for information on the GC analysis ofIBA, Carol Hahn for preparation of the figures, Andrew Tulland Donna Jarnagin for growing the tobacco plants, andmembers of the CCRC for helpful comments and criticism.This work was supported by NIH postdoctoral fellowship no.F32 GM11857-O2 (to D.M.), DOE grant no. DE-FGO9-85ER13425, and in part by Department of Energygrant no. DE-FG09-87ER13810 as part of the USD A/DOE/NSF Plant Science Centers Program.

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{Accepted 18 October 1989)