identification of multiple stages in the conversion of...

Development 112, 891-898 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

891

Identification of multiple stages in the conversion of maize meristems from

vegetative to floral development

ERIN E. IRISH* and TIMOTHY M. NELSON

Department of Biology, Yale University, New Haven, CT 06511-7444, USA

'Current address: Department of Botany, University of Iowa, Iowa City, IA 52242, USA

Summary

Vegetative growth in most lines of maize is terminated ata predictable stage in development by the conversion ofthe shoot apical meristem into an inflorescence, thetassel. The conversion from vegetative to floral develop-ment is under developmental control, the basis of whichis obscure. We have assayed the developmental potentialof the shoot apical meristem in order to identify the stageat which it is determined to form a tassel. We show,using shoot apex culture, that meristems are notdetermined to form a tassel until after all vegetative

nodes have been initiated. We also show that floraldetermination is a separate, later event in the develop-ment of a maize inflorescence. Intermediate stages canbe distinguished in which the meristem is determinateand has the phyllotaxis of a tassel when cultured butdevelops vegetative shoots from buds that normally giverise to sets of florets.

Key words: determination, flowering, meristem,determinate growth, maize.

Introduction

The shoots of maize (Zea mays) become determinate,or limited in growth, by the differentiation of the shootapical meristem into the terminal inflorescence, thetassel. During vegetative growth, the meristem initiatesa predictable number of leaf primordia. Once allvegetative nodes have been initiated, the meristemchanges its pattern of development and begins toinitiate primordia that will ultimately give rise toflorets, in which such specialized processes as sexdetermination, meiosis and microsporogenesis takeplace (Bonnett, 1948; Kiesselbach, 1949; Sass, 1976).The meristem is ultimately consumed in the process ofinitiating primordia, so that the extent of growth of theshoot becomes limited.

Little is known about the signal(s) responsible for thedramatic changes in the activity of the shoot apicalmeristem and its derivatives. Most lines of maize areclassified as quantitative short day plants (short dayscan induce premature flowering); thus, the conversionfrom vegetative development to flowering can beinfluenced by environmental conditions (Hanway andRitchie, 1985; Heslop-Harrison, 1961). However, innormal field conditions in the continental US, which arelong days, the meristem initiates a predictable numberof vegetative nodes, then converts to tassel develop-ment (Russell and Stuber, 1983). Thus, most maizeplants flower in response to a developmental cue.

Approaches to understanding the developmental

regulation of flowering have included assays fordetermination. Determination is operationally definedas a stable developmental state in which an organ willdevelop in isolation or in a new location (environment)in the same pattern as it would have had it been left insitu (Gehring, 1972; McDaniel, 1984). Determination ofthe shoot apical meristem to develop into a flower orinflorescence was shown to occur during vegetativegrowth, well before the onset of floral development, intwo dicots: sunflower (Habermann and Sekulow, 1972)and tobacco (Singer and McDaniel, 1986). Like maize,sunflower and tobacco have an unbranched growthhabit and a terminal inflorescence. Sunflower wasshown by grafting experiments to be determined toform a terminal inflorescence (at approximately nodenumber 16) while still at the seedling stage (nodenumber 2-3), approximately 14 nodes in advance ofmorphological differentiation (Habermann and Seku-low, 1972). McDaniel and coworkers have demon-strated by decapitating, grafting and rooting of terminalbuds (Singer and McDaniel, 1986) that tobacco isdetermined to form a terminal inflorescence when it hasfour nodes remaining to be initiated before differen-tiation of the apical meristem into the terminal flower(approximately node 30).

We previously showed that meristems of maizeseedlings that had initiated 7-9 nodes are not yetdetermined to form the tassel that appears approxi-mately 10 nodes later (Irish and Nelson, 1988).Determination was assayed by shoot apex culture, in

892 E. E. Irish and T. M. Nelson

which the apical meristem along with 1-2 youngest leafprimordia were allowed to continue development afterremoval from the rest of the plant body. Here weidentify the distinct stage at which the meristem isLimited in the number of nodes it can form. In contrastto sunflower and tobacco, maize meristems are notcommitted to form a terminal inflorescence until afterall vegetative nodes have been laid down. We also showthat complete tassel determination occurs in a step-wisefashion: first the conversion to an axis with limitedgrowth and a new phyllotaxis, followed by the floraldetermination of the buds that it has initiated.

Materials and methods

All plant material used was the maize inbred line W23(source: Scott Poethig, University of Pennsylvania).

Meristem isolation, culture and plant growth conditionswere as described previously (Irish and Nelson, 1988), usingmedium without added hormones for initial culturing and rootdevelopment. Isolated shoot apices, consisting of the apicalmeristem plus 1-2 leaf primordia, were grown initially in 2 mlof medium (Polowick and Greyson, 1982) in wells of tissueculture plates. Liquid culture (25 ml in 125 ml flasks), with theaddition of 1 janolar kinetin, was used for some immaturetassels longer than 7 mm. Meristems were measured bycomparison with an ocular reticle in the dissecting microscopejust prior to culture. Height was measured from the upperedge of the newest leaf primordium attachment point(estimated when growth of the primordium obscured thatpoint) to the apex of the meristem.

The assay for floral determination (see Results) wascomplicated by the requirement of slightly different media forvigorous vegetative or floral development. Solid mediumwithout added hormones promoted vegetative growth (resultsnot shown), while culture in shaking liquid medium contain-ing kinetin favored floral development, as had been shownpreviously (Polowick and Greyson, 1982). Although eithermedium promoted both vegetative and floral patterns ofdevelopment, we chose to assay for floral determination in thetwo different conditions in order to optimize the response ofthe tissue. In this way the earliest stages, at which floraldevelopment occurred in only one or two primordia, could bescored with confidence. Duplicate sets of various sizedenlarged meristems/immature tassels were grown in bothmedia. Floral development was scored after one week ofculture. Although each medium favored one pattern ofgrowth (vegetative or floral), it was possible to classify thestructures that grew from the cultured meristems/immaturetassels as either floral or vegetative in both growingconditions. The differences seen in the two media were theresults of vigor and extent of growth, not pattern. As similarresults were obtained in the two media, the data have beenpooled (Fig. 3).

As previously described, explants maintain morphologicalorganization and continue to initiate normal organs withoutapparent reorganization or formation of callus. Once theexplants had attained at least lcm in length, they weretransferred to culture tubes containing approximately 15 ml ofthe same medium. Cultures in both plates and tubes weregrown in a plant growth chamber (20°C), which provided30/iEinsteinsm~2 of illumination from fluorescent bulbs for aperiod of 16 h per day. Plantlets with shoots at least 3 cm longand well-developed adventitious roots were transferred to soiland grown in the greenhouse. The data presented here are

from several sets of experiments done throughout the year,during which greenhouse conditions were variable: thedaytime temperatures were approximately 24 °C in the winterand as high as 40°C in the summer. Daylength in thegreenhouse was 16 h±2 h, and light intensity in the winter wasmaintained by supplemental fluorescent and sodium vaporlights. Despite variable greenhouse conditions, the number ofvegetative nodes initiated before flowering in W23 plants wasrelatively constant, as determined from counting the vegetat-ive nodes of control plants that were simply grown from seedto flowering in soil-filled pots.

The different stages of meristems were obtained by plantinga large number of seeds at one time in the greenhouse andsampling every 2-3 days. As controls, some plants from theinitial population were allowed to complete flowering, atwhich time the number of nodes per plant was determined.When cultured plantlets were transferred to soil, a second setof controls was planted. As the average number of nodesformed during each experiment varied by less than 0.5 nodes(data not shown), all of these have been pooled.

Plants from vegetative meristems (see Results) developedessentially as if they were seed-derived: leaves were initiatedin a distichous pattern, internodes above the first fiveelongated, and the plants terminated with a tassel. There weresome abnormalities: the plants were slightly smaller thanseed-derived control plants, and occasionally there werechanges in phyllotaxis.

The initial culturing stage, from isolation until the plantletswere large enough to be transferred to culture tubes, wasapproximately one month long. The second stage, duringwhich the plantlets grew adventitious roots and a vigorousshoot, lasted another month. Plantlets transferred to soil inthe greenhouse initiated tassels within two months. Survivalrate was approximately 75% (not shown).

Scanning electron microscopy, done as previously de-scribed (Irish and Nelson, 1988), was used for morphologicalcomparisons of the shoot apices at different stages (heights)

Results

In order to identify the stage at which the shoot apicalmeristem of maize is committed to cease vegetativegrowth and begin to form an inflorescence, thedevelopmental potential of isolated meristems atvarious stages was assayed by shoot apex culture. Inshoot apex culture, vegetative meristems continue theirfunction of initiating leaves separated by internodes(Irish and Nelson, 1988). Meristems that had initiatedfrom 11 to 16 nodes were excised, along with one or twomost recently initiated leaf primordia, and placed inculture. The plants resulting from the continued activityof the cultured meristems were tranferred to soil andgrown to flowering in the greenhouse. The number ofnodes formed after culture by meristems isolated andcultured at the various stages is shown in Table 1. Thisexperiment identified two classes of meristems. Thefirst group, which had initiated between 11 and 16 nodesbefore isolation, initiated a full complement of nodes(approximately 16) after isolation. The second group,which had initiated 15 or 16 nodes, did not form anynew vegetative nodes after culturing, but insteaddifferentiated into a tassel-like structure. There were nomeristems that when cultured initiated less than a fullcomplement of vegetative nodes, then developed a

Vegetative to floral conversion in maize 893

Table 1. Developmental potential of meristemsisolated at different stages of development (number of

nodes initiated)

Nodes initiatedat culture

Control

111213141516

1516

Total nodeson plant atflowering(mean)

15.8

16.115.31416.315.916.0

00

Numberscored

257

743998

48116

Standarddeviation

0.65

1.11.03.02.73.30.9

0.00.0

The first group represent meristems that were capable ofcontinuing vegetative development: these all reformed a wholeplant before differentiating into a tassel. The second grouprepresent meristems isolated after they had lost the ability toinitiate vegetative nodes. Control plants were not cultured.

Table 2. Comparison of number of nodes initiatedbefore flowering in control (noncultured) plants and

number of nodes initiated by meristems thatdifferentiated into tassel-like structures when cultured

Control meristemsthat flowered

Cultured meristemsthat differentiated

Node number

1-141516

02865

02970

tassel-like structure. Table 2 shows a comparison of thepercentage of plants that flowered at a given node to thepercentage of meristems that had initiated the samenumber of nodes differentiating into a tassel-likestructure when cultured. The similarities indicate thatthe meristems that differentiated into a tassel-likestructure were most likely meristems that had com-pleted the vegetative phase of development. Thus, theshoot apical meristem of W23 maize is not determinedto form an inflorescence until vegetative development iscomplete, and meristems isolated before vegetativedevelopment is completed are 'reset' to the beginningof the vegetative phase of development.

It was possible to isolate meristems that were at thesame stage but exhibited completely different behaviorsin culture. These meristems had completed vegetativedevelopment but may or may not have been determinedto form a tassel. Meristems of that stage were examinedfor morphological indications of developmental phase.Fig. 1 shows scanning electron micrographs of a seriesof freshly dissected meristems spanning the develop-mental stage at which meristems are determined toform an inflorescence. The morphological transition ofthe vegetative meristem to an immature tassel showedno abrupt changes that could be associated with thedetermination of the tassel. During vegetative growth,

the height of the meristem increased from 0.12 to0.16 mm tall during the period from germination to theinitiation of the last leaf (approximately one month,data not shown). Once all vegetative nodes wereinitiated, there was a more rapid increase in height andgirth of the tassel primordium, which produced budsacropetally in a spiral pattern (Fig. 1). Buds weredetectable first as surface irregularities on the otherwisesmooth meristem. Basal buds will give rise to tasselbranches; buds on the branches and above the brancheson the main axis will give rise to spikelet pairs.

The tassel-like structures formed by meristems thatdifferentiated instead of reiterating vegetative develop-ment in culture were not normal tassels. As in normaltassels, the structures were determinate and exhibitedspiral phyllotaxis. Unlike normal tassels, each bud onthe differentiating meristem developed into a vegetat-ive shoot instead of a tassel branch or spikelet bearingflorets. In most cases these shoots showed very limitedgrowth, consisting of approximately 4-6 leaflets notgreater than lcm long, with no internode elongation.However, some of the cultured meristems of this stageexhibited more vigorous growth, in which a fraction ofthe buds developed into shoots showing substantialvegetative development, including the initiation ofadventitious roots (Fig. 3A). Such structures are typicalof proliferous inflorescences (Battey and Lyndon,1990), in which vegetative shoots develop in place offlowers on an inflorescence axis. This pattern ofdifferentiation identifies an intermediate stage in theprocess of converting the vegetative maize meristeminto a tassel with normal florets.

The development of proliferous tassels from meri-stems cultured at the earliest stages of tassel develop-ment indicated that although the tranisition to determi-nate growth had occurred, floral determination had notoccurred at those stages. Shoot apex culture was used toidentify the later stage at which floral determinationoccurs and to define more precisely intermediate stagesof tassel development. Meristems of various stageswere isolated and cultured from plants that had acomplete set of vegetative nodes (16-17 nodes). Eachdevelopmental stage corresponded to a characteristicmeristem height. Fig. 2 shows the developmentalpotential of the various stages of meristems assayedfrom several sets of experiments. Meristems less than0.30 mm high were always vegetative and developed fullsets of nodes, then flowered, after isolation and culture.Meristems between 0.70 mm and 7.0mm always devel-oped into proliferous tassels. There was some overlapbetween these two classes, as some meristems as tall as0.60 mm were able to initiate full sets of vegetativenodes, while some meristems 0.40 mm tall developedinto proliferous tassels. In addition, there was anintermediate size class that developed in culture into asmall (approximately 5 mm) green cylinder with nolateral outgrowths that most closely resembled a thorn.Determination to form an inflorescence (between0.4 mm and 0.7 mm) was associated with stages betweenthose shown in Fig. 1C.-E. Stages such as those shownin Fig. IB have undergone morphological changes


Fig. 1. Scanning electron micrographs of freshly excised and fixed vegetative and prefloral meristems. (A) 0.16 mm,(B) 0.28mm, (C) 0.40mm, (D) 0.55 mm, (E) 0.66mm, (F) 0.89mm, (G) 1.08mm, (H) 1.4mm, (I) 2mm. If left on intactplants, the basal buds would give rise to branches; all other buds to spikelet pair primordia and ultimately four florets.Bar=100 microns. Bar in A applies to A-F; bar in G applies to G-I. See text and Fig. 2 for description of stages, b,branch primordium; 1, leaf primordium; m, meristem; sp, spikelet pair primordium.


days8 11 1621

floraltassel

glumes only/sterile tassel

proliferoustassel

thorn-like -

vegetativemeristem

1 10meristem length in mm

100

Fig. 2. The developmental fate of different stages (heights) of meristems excised and cultured after a complete set ofvegetative nodes had been initiated, as determined by the average number of nodes produced before flowering on controlplants at the time of the experiment. Each point represents a single meristem; these are the pooled results of several setsof experiments. See text for descriptions of the various classes. Days represent the average amount of time elapsed fromthe beginning of tassel development to successive stages (0 days represents the end of the period in which the averagemeristem could start over in vegetative development). Scanning electron micrographs of meristems from 0.16mm to2.0mm, spanning the first three stages, are shown in Fig. 1.

towards tassel differentiation into a tassel yet are notdetermined to form a tassel when assayed in culture.

Meristems between 7 and 10 mm developed determi-nate shoots with approximately four leaf-Like organs onan elongated axis (Fig. 3B); the determinate shootswere interpreted as sterile florets with only floral bractsand no floral organs. Meristems greater than 10 mm tallwere able to develop florets containing stamens withnormal morphology (Fig. 3C). In all cases the culturedimmature tassels showed a developmental gradient: themost recently initiated primordia (at the tip) did notdevelop to the same extent as. did more matureprimordia (at the base). Meristems were scoredaccording to the most mature stage present. Tasselscultured after reaching more than 10 mm tall showed agreater percentage of normal florets.

Discussion

We have assayed the developmental potential of theshoot apical meristem in culture during its switch fromvegetative growth to determinate, floral development.We have established that the meristem does not becomecommitted to form a tassel until after it has initiated allvegetative nodes (Tables 1 and 2). We have usedculture experiments to identify sequential steps inflowering and have found that floral determinationoccurs late in tassel development (Fig. 2), after all floralorgans have been initiated. During the period betweentassel determination and floral determination, the tasselprimordium makes no abrupt morphological changes,

but instead gradually increases in height and girth whileinitiating buds acropetally in a spiral pattern (Fig. 1).

The stages that culturing experiments have identifiedare summarized in Fig. 4. The different stages representtransitions which cannot occur in the isolatedmeristem/immature tassel, but require input fromother portions of the plant. These transitions are: (1)indeterminate to determinate growth of the meristem,(2) initiation of buds on meristem, (3) indeterminate todeterminate growth of shoots from the buds, (4) sterileto fertile shoots (florets). Previous experiments onintact maize plants, in which daylength and tempera-ture were varied, identified points at which environ-mental cues could affect the development of the tassel(Helsop-Harrison, 1961). These points overlap with,but are not identical to, those identified in this study.

This study showed that maize meristems are deter-mined to form a terminal inflorescence at a later stagethan was seen in the dicots sunflower and tobacco.Because the experiments with the dicots analyzed thedevelopmental behavior of explants with several moreleaves "and associated stem tissue than was present inthe experiments presented here, it is not possible todirectly compare those results with these obtained inmaize. Experiments addressing this problem are under-way; however, because our experiments used littlemore than the shoot apical meristem itself, it is likelythat results obtained are a more accurate reflection ofthe ability of the meristem to undergo the varioustransitions in the switch from vegetative to floraldevelopment.

Maize plants show a gradient of juvenile to adultnodes, in which characters such as leaf shape, waxy


Fig. 3. Structures that developed from cultured immature tassels at various stages. (A) 0.70 mm immature tassel culturedon solid medium; proliferous tassel. The immature tassel showed limited elongation in culture and is not visible in thisfigure. Several spikelet pair primordia have developed into shoots that developed adventitious roots. These shoots have anindeterminate pattern of growth. (0.9x magnification). (B) 8.5 mm tassel cultured in liquid medium. The most matureflorets are sterile, with only leaflike organs. These shoots are vegetative and determinate. (2x magnification). (C) normalflorets from a 30mm tassel cultured in liquid medium. Only the most mature florets have well-formed stamens. Lessmature florets have only leaf-like organs, ( l . lx magnification). Arrow indicates most mature region.

vegetativemeristem"

elongated^meristem

.bud _ ^initiation

branchinitiation

spikelet pair_initiation

spikeletinitiation

glumeinitiation

floretinitiation

flora) organ' initiation

J Tvegetative thorn proliferous tassel sterile tassel fertile tassel

Fig. 4. A schematic summary of the stable developmental phases of the transition from indeterminate, vegetativedevelopment to determinate, floral development. The upper portion represents the morphologically identifiable stages intassel development, in which the vegetative meristem becomes elongated and initiates buds. The buds may develop intobranches bearing spikelet pairs or directly to spikelet pairs. Spikelet pair primordia give rise to spikelets, which initiateglumes and florets. Each floret initiates a lemma, palea, and floral organs. The lower portion represents the type ofdevelopment observed when meristems of those stages are cultured.

versus hairy leaf epidermis, presence of adventitiousroots, and the developmental fate of lateral buds varyfrom the base to the tip of the plant (Poethig, 1988).Nodes initiated at the top of the maize plant do notnormally initiate adventitious roots: these are usually

limited to the first 4-6 nodes and may be consideredjuvenile traits. Nevertheless, when the uppermostvegetative nodes from plants that had initiated up to allof the normal complement of nodes were put in culture,they were able to initiate normal adventitious roots.


Similarly, the basal leaves on explants from late nodeshad shapes and epidermal characters similar to seedlingleaves, not adult leaves, as had been found in plantsderived from seedling-stage meristems (Irish andNelson, 1988). The one or two nodes left attached to themeristem when cultured developed in a pattern mostsimilar to the basal one or two nodes of a seedling.Thus, even though nodes high on the plant normallyhave adult characteristics, the nodes are not limited toan adult pattern early in their development. Thedifferentiation of organs into adult types depends oninteractions with previously formed body parts (Irishand Nelson, 1988). The juvenile versus adult charactersof the maize plant are not a reflection of the ageing ofthe meristem.

Transition-stage meristems (0.32-0.67 mm) devel-oped, in culture, into determinate cylinders with nolateral outgrowths (Fig. 2). This suggests that the tasselis limited in growth in advance of the initiation of anylateral buds. Determinate growth of shoots is notalways associated with flowering; while the majority ofshoots terminate by the differentiation of the apicalmeristem into a flower or inflorescence, some becomedeterminate by the formation of a thorn, such as inGleditsia (Steeves and Sussex, 1989). Thus, it is notunexpected that limitation of growth potential andfloral determination are separable events. However,the cylindrical meristems were isolated during a periodof rapid growth. The extremely limited development ofmeristems of this stage in culture may simply be theresult of suboptimal medium and/or a wound responsethat interferes with the normal pattern of development.

In the maize tassel, florets are initiated and developsequentially, such that each tassel has a gradient ofdevelopmental stages (Fig. 2). Excised and culturedtassels at least 10 mm long showed at least one floretthat developed normally. Florets in less maturepositions on these cultured tassels were not able todevelop florally even though the floral signal must havebeen present. Thus, primordia are required to becomecompetent to flower in order to respond to the floralsignal.

Vegetative or proliferous inflorescences have beenobserved in a variety of plants (Battey and Lyndon,1990). Galinat and Naylor (1951) induced proliferoustassels on maize plants homozygous for the indetermi-nate mutation, which confers a short day requirementfor flowering, by giving the plants an insufficientnumber of short days to complete floral development.Proliferous tassels have also been observed in strains ofmaize from Mexico when grown farther north (Collins,1909); these presumably are short day strains. Thedescriptions of these proliferous tassels fit the vegetat-ive tassels that we obtained when early-staged tassels(between 0.5 and 5 mm) were isolated from the plant.This phenomenon is not limited to maize: chrysanthe-mum, also a short-day plant, will develop vegetativeinflorescences when raised under short day conditionsand then switched to long days after receptacleformation (Schwabe, 1959). These examples demon-strate that the flowering signal is required in these

species for a prolonged period or for separate eventsduring floral development. The cases of proliferousinflorescences described previously were obtained indaylength-dependent plants. We obtained proliferoustassels with the maize inbred line W23, which is day-neutral. Presumably, isolation of the developing inflor-escences of a day-neutral plant has the same effect asremoval of the floral induction signal during flowerdevelopment in daylength-sensitive plants. Thus, therequirement for prolonged presence of the floweringsignal(s) is not limited to plants that flower in responseto daylength but is probably typical of flowering inmany species.

If perturbed early in development, flowers fromdifferent species show a range of ability to proceed withnormal development. Tobacco flowers excised from theplant at the stage at which only the sepals had beeninitiated can continue to initiate the normal sequence ofpetals, stamens and pistils and grow to maturity insterile culture (Hicks and Sussex, 1970). Similar resultswere obtained with the short day plant Chenopodium,which produced small but normal inflorescences inculture from shoot apices that had been excised six daysafter floral induction (Wetmore et al. 1959). Thus, floraldetermination occurs early in these plants and havingoccurred, the completion of organogenesis requires nomore from the plant than simple nutrients. In contrast,Impatiens balsamina, a short day plant, when shiftedfrom short day to long day and then back to short day,will develop organs intermediate between leaf and petalas the plant reverts to flowering (Battey and Lyndon,1988). In this plant, cells in the developing organsrespond continuously to environmental cues as theydifferentiate: organ identity is not stably determined atinitiation. Cultured maize tassels follow the pattern of/. balsamina. The earliest staged tassel on which budswere able to develop into fertile florets in culture was10 mm high. However, all floral organs have beeninitiated in florets of tassels less than 10 mm high (datanot shown). Thus, maize floral development is notautonomous until floral organs are partially developed.A similarly late stage of floral determination wasobserved in chrysanthemum (Schwabe, 1959).

Short day growing conditions have been shown toaccelerate the transition from vegetative to tasseldevelopment in maize (Heslop-Harrison, 1961). Theexperiments presented here assayed the developmentalpotential of meristems of various stages from plantsgrown in long days. We predict that meristems fromplants grown in continuous short day conditions wouldshow determination at an earlier stage. It may bepossible to identify a critical period in which day lengthcontrols the timing of tassel determination by combin-ing day length shifts with meristem culture.

The molecular nature of the flowering signal, which isrequired for a prolonged developmental period toinitiate and complete reproductive development, re-mains obscure. The signal to convert the meristem intoa tassel comes from other parts of the plant. Thisappears to be a node-counting mechanism in which themeristem responds to the gradual accumulation of some


factors(s) as the plant develops beneath it, or to a morerapid appearance of a flowering factor in the meristemafter the required number of nodes have accumulated.There are no morphological differences between plantsderived from meristems that had initiated 14-16 nodesand those that had produced 7-9 nodes (Irish andNelson, 1988) before culturing. This suggests that if aflowering signal acts by accumulating in the meristemduring development, it is not stable when the meristemis isolated and cultured.

We thank Ian Sussex, Scott Poethig, and Gene Szymkowiakfor critically reading this manuscript. E. E. I. was supportedby grants from Pioneer Hi-Bred International, Inc. and theMcKnight Foundation.

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(Accepted 4 April 1991)

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