Identification of multiple stages in the conversion of maize meristems from vegetative to floral development

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 at a predictable stage in development by the conversion of the shoot apical meristem into an inflorescence, the tassel. The conversion from vegetative to floral development is under developmental control, the basis of which is obscure. We have assayed the developmental potential of the shoot apical meristem in order to identify the stage at which it is determined to form a tassel. We show, using shoot apex culture, that meristems are not determined to form a tassel until after all vegetative nodes have been initiated. We also show that floral determination is a separate, later event in the development of a maize inflorescence. Intermediate stages can be distinguished in which the meristem is determinate and has the phyllotaxis of a tassel when cultured but develops vegetative shoots from buds that normally give rise 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 shoot apical meristem into the terminal inflorescence, the tassel. During vegetative growth, the meristem initiates a predictable number of leaf primordia. Once all vegetative nodes have been initiated, the meristem changes its pattern of development and begins to initiate primordia that will ultimately give rise to florets, in which such specialized processes as sex determination, meiosis and microsporogenesis take place (Bonnett, 1948; Kiesselbach, 1949; Sass, 1976). The meristem is ultimately consumed in the process of initiating primordia, so that the extent of growth of the shoot becomes limited. Little is known about the signal(s) responsible for the dramatic changes in the activity of the shoot apical meristem and its derivatives. Most lines of maize are classified as quantitative short day plants (short days can induce premature flowering); thus, the conversion from vegetative development to flowering can be influenced by environmental conditions (Hanway and Ritchie, 1985; Heslop-Harrison, 1961). However, in normalfieldconditions in the continental US, which are long days, the meristem initiates a predictable number of vegetative nodes, then converts to tassel development (Russell and Stuber, 1983). Thus, most maize plants flower in response to a developmental cue. Approaches to understanding the developmental regulation of flowering have included assays for determination. Determination is operationally defined as a stable developmental state in which an organ will develop in isolation or in a new location (environment) in the same pattern as it would have had it been left in situ (Gehring, 1972; McDaniel, 1984). Determination of the shoot apical meristem to develop into a flower or inflorescence was shown to occur during vegetative growth, well before the onset of floral development, in two dicots: sunflower (Habermann and Sekulow, 1972) and tobacco (Singer and McDaniel, 1986). Like maize, sunflower and tobacco have an unbranched growth habit and a terminal inflorescence. Sunflower was shown by grafting experiments to be determined to form a terminal inflorescence (at approximately node number 16) while still at the seedling stage (node number 2-3), approximately 14 nodes in advance of morphological differentiation (Habermann and Sekulow, 1972). McDaniel and coworkers have demonstrated by decapitating, grafting and rooting of terminal buds (Singer and McDaniel, 1986) that tobacco is determined to form a terminal inflorescence when it has four nodes remaining to be initiated before differentiation of the apical meristem into the terminal flower (approximately node 30). We previously showed that meristems of maize seedlings that had initiated 7-9 nodes are not yet determined to form the tassel that appears approximately 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 leaf primordia were allowed to continue development after removal from the rest of the plant body. Here we identify the distinct stage at which the meristem is Limited in the number of nodes it can form. In contrast to sunflower and tobacco, maize meristems are not committed to form a terminal inflorescence until after all vegetative nodes have been laid down. We also show that complete tassel determination occurs in a step-wise fashion: first the conversion to an axis with limited growth and a new phyllotaxis, followed by the floral determination 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 conditions were as described previously (Irish and Nelson, 1988), using medium without added hormones for initial culturing and root development. Isolated shoot apices, consisting of the apical meristem plus 1-2 leaf primordia, were grown initially in 2 ml of medium (Polowick and Greyson, 1982) in wells of tissue culture plates. Liquid culture (25 ml in 125 ml flasks), with the addition of 1 janolar kinetin, was used for some immature tassels longer than 7 mm. Meristems were measured by comparison with an ocular reticle in the dissecting microscope just prior to culture. Height was measured from the upper edge of the newest leaf primordium attachment point (estimated when growth of the primordium obscured that point) to the apex of the meristem. The assay for floral determination (see Results) was complicated by the requirement of slightly different media for vigorous vegetative or floral development. Solid medium without added hormones promoted vegetative growth (results not shown), while culture in shaking liquid medium containing kinetin favored floral development, as had been shown previously (Polowick and Greyson, 1982). Although either medium promoted both vegetative and floral patterns of development, we chose to assay forfloraldetermination in the two different conditions in order to optimize the response of the tissue. In this way the earliest stages, at which floral development occurred in only one or two primordia, could be scored with confidence. Duplicate sets of various sized enlarged meristems/immature tassels were grown in both media. Floral development was scored after one week of culture. Although each medium favored one pattern of growth (vegetative or floral), it was possible to classify the structures that grew from the cultured meristems/immature tassels as either floral or vegetative in both growing conditions. The differences seen in the two media were the results of vigor and extent of growth, not pattern. As similar results were obtained in the two media, the data have been pooled (Fig. 3). As previously described, explants maintain morphological organization and continue to initiate normal organs without apparent reorganization or formation of callus. Once the explants had attained at least lcm in length, they were transferred to culture tubes containing approximately 15 ml of the same medium. Cultures in both plates and tubes were grown in a plant growth chamber (20 C), which provided 30/iEinsteinsm~ 2 of illumination from fluorescent bulbs for a period of 16 h per day. Plantlets with shoots at least 3 cm long and well-developed adventitious roots were transferred to soil and grown in the greenhouse. The data presented here are from several sets of experiments done throughout the year, during which greenhouse conditions were variable: the daytime temperatures were approximately 24 C in the winter and as high as 40 C in the summer. Daylength in the greenhouse was 16 h±2 h, and light intensity in the winter was maintained by supplemental fluorescent and sodium vapor lights. Despite variable greenhouse conditions, the number of vegetative nodes initiated before flowering in W23 plants was relatively constant, as determined from counting the vegetative nodes of control plants that were simply grown from seed to flowering in soil-filled pots. The different stages of meristems were obtained by planting a large number of seeds at one time in the greenhouse and sampling every 2-3 days. As controls, some plants from the initial population were allowed to complete flowering, at which time the number of nodes per plant was determined. When cultured plantlets were transferred to soil, a second set of controls was planted. As the average number of nodes formed 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) developed essentially as if they were seed-derived: leaves were initiated in a distichous pattern, internodes above the first five elongated, and the plants terminated with a tassel. There were some abnormalities: the plants were slightly smaller than seed-derived control plants, and occasionally there were changes in phyllotaxis. The initial culturing stage, from isolation until the plantlets were large enough to be transferred to culture tubes, was approximately one month long. The second stage, during which the plantlets grew adventitious roots and a vigorous shoot, lasted another month. Plantlets transferred to soil in the greenhouse initiated tassels within two months. Survival rate was approximately 75% (not shown). Scanning electron microscopy, done as previously described (Irish and Nelson, 1988), was used for morphological comparisons of the shoot apices at different stages (heights) Results In order to identify the stage at which the shoot apical meristem of maize is committed to cease vegetative growth and begin to form an inflorescence, the developmental potential of isolated meristems at various stages was assayed by shoot apex culture. In shoot apex culture, vegetative meristems continue their function of initiating leaves separated by internodes (Irish and Nelson, 1988). Meristems that had initiated from 11 to 16 nodes were excised, along with one or two most recently initiated leaf primordia, and placed in culture. The plants resulting from the continued activity of the cultured meristems were tranferred to soil and grown to flowering in the greenhouse. The number of nodes formed after culture by meristems isolated and cultured at the various stages is shown in Table 1. This experiment identified two classes of meristems. The first group, which had initiated between 11 and 16 nodes before isolation, initiated a full complement of nodes (approximately 16) after isolation. The second group, which had initiated 15 or 16 nodes, did not form any new vegetative nodes after culturing, but instead differentiated into a tassel-like structure. There were no meristems that when cultured initiated less than a full complement of vegetative nodes, then developed a

Vegetative to floral conversion in maize 893 Table 1. Developmental potential of meristems isolated at different stages of development (number of nodes initiated) Nodes initiated at culture Control 11 12 13 14 15 16 15 16 Total nodes on plant at flowering (mean) 15.8 16.1 15.3 14 16.3 15.9 16.0 0 0 Number scored 257 7 4 3 9 9 8 48 116 Standard deviation The first group represent meristems that were capable of continuing vegetative development: these all reformed a whole plant before differentiating into a tassel. The second group represent meristems isolated after they had lost the ability to initiate vegetative nodes. Control plants were not cultured. 0.65 1.1 1.0 3.0 2.7 3.3 0.9 0.0 0.0 Table 2. Comparison of number of nodes initiated before flowering in control (noncultured) plants and number of nodes initiated by meristems that differentiated into tassel-like structures when cultured Node number 1-14 15 16 Control meristems that flowered 0 28 65 Cultured meristems that differentiated tassel-like structure. Table 2 shows a comparison of the percentage of plants that flowered at a given node to the percentage of meristems that had initiated the same number of nodes differentiating into a tassel-like structure when cultured. The similarities indicate that the meristems that differentiated into a tassel-like structure were most likely meristems that had completed the vegetative phase of development. Thus, the shoot apical meristem of W23 maize is not determined to form an inflorescence until vegetative development is complete, and meristems isolated before vegetative development is completed are 'reset' to the beginning of the vegetative phase of development. It was possible to isolate meristems that were at the same stage but exhibited completely different behaviors in culture. These meristems had completed vegetative development but may or may not have been determined to form a tassel. Meristems of that stage were examined for morphological indications of developmental phase. Fig. 1 shows scanning electron micrographs of a series of freshly dissected meristems spanning the developmental stage at which meristems are determined to form an inflorescence. The morphological transition of the vegetative meristem to an immature tassel showed no abrupt changes that could be associated with the determination of the tassel. During vegetative growth, 0 29 70 the height of the meristem increased from 0.12 to 0.16 mm tall during the period from germination to the initiation of the last leaf (approximately one month, data not shown). Once all vegetative nodes were initiated, there was a more rapid increase in height and girth of the tassel primordium, which produced buds acropetally in a spiral pattern (Fig. 1). Buds were detectable first as surface irregularities on the otherwise smooth meristem. Basal buds will give rise to tassel branches; buds on the branches and above the branches on the main axis will give rise to spikelet pairs. The tassel-like structures formed by meristems that differentiated instead of reiterating vegetative development in culture were not normal tassels. As in normal tassels, the structures were determinate and exhibited spiral phyllotaxis. Unlike normal tassels, each bud on the differentiating meristem developed into a vegetative shoot instead of a tassel branch or spikelet bearing florets. In most cases these shoots showed very limited growth, consisting of approximately 4-6 leaflets not greater than lcm long, with no internode elongation. However, some of the cultured meristems of this stage exhibited more vigorous growth, in which a fraction of the buds developed into shoots showing substantial vegetative development, including the initiation of adventitious roots (Fig. 3A). Such structures are typical of proliferous inflorescences (Battey and Lyndon, 1990), in which vegetative shoots develop in place of flowers on an inflorescence axis. This pattern of differentiation identifies an intermediate stage in the process of converting the vegetative maize meristem into a tassel with normal florets. The development of proliferous tassels from meristems cultured at the earliest stages of tassel development indicated that although the tranisition to determinate growth had occurred, floral determination had not occurred at those stages. Shoot apex culture was used to identify the later stage at which floral determination occurs and to define more precisely intermediate stages of tassel development. Meristems of various stages were isolated and cultured from plants that had a complete set of vegetative nodes (16-17 nodes). Each developmental stage corresponded to a characteristic meristem height. Fig. 2 shows the developmental potential of the various stages of meristems assayed from several sets of experiments. Meristems less than 0.30 mm high were always vegetative and developed full sets of nodes, then flowered, after isolation and culture. Meristems between 0.70 mm and 7.0mm always developed into proliferous tassels. There was some overlap between these two classes, as some meristems as tall as 0.60 mm were able to initiate full sets of vegetative nodes, while some meristems 0.40 mm tall developed into proliferous tassels. In addition, there was an intermediate size class that developed in culture into a small (approximately 5 mm) green cylinder with no lateral outgrowths that most closely resembled a thorn. Determination to form an inflorescence (between 0.4 mm and 0.7 mm) was associated with stages between those shown in Fig. 1C.-E. Stages such as those shown in Fig. IB have undergone morphological changes

894 E. E. Irish and T. M. Nelson 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 intact plants, 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.

floral tassel glumes only/ sterile tassel Vegetative to floral conversion in maize 895 days 8 11 1621 proliferous tassel thorn-like - vegetative meristem 1 10 meristem length in mm 100 Fig. 2. The developmental fate of different stages (heights) of meristems excised and cultured after a complete set of vegetative nodes had been initiated, as determined by the average number of nodes produced before flowering on control plants at the time of the experiment. Each point represents a single meristem; these are the pooled results of several sets of experiments. See text for descriptions of the various classes. Days represent the average amount of time elapsed from the beginning of tassel development to successive stages (0 days represents the end of the period in which the average meristem could start over in vegetative development). Scanning electron micrographs of meristems from 0.16mm to 2.0mm, spanning the first three stages, are shown in Fig. 1. towards tassel differentiation into a tassel yet are not determined to form a tassel when assayed in culture. Meristems between 7 and 10 mm developed determinate shoots with approximately four leaf-like organs on an elongated axis (Fig. 3B); the determinate shoots were interpreted as sterile florets with only floral bracts and no floral organs. Meristems greater than 10 mm tall were able to develop florets containing stamens with normal morphology (Fig. 3C). In all cases the cultured immature tassels showed a developmental gradient: the most recently initiated primordia (at the tip) did not develop to the same extent as. did more mature primordia (at the base). Meristems were scored according to the most mature stage present. Tassels cultured after reaching more than 10 mm tall showed a greater percentage of normal florets. Discussion We have assayed the developmental potential of the shoot apical meristem in culture during its switch from vegetative growth to determinate, floral development. We have established that the meristem does not become committed to form a tassel until after it has initiated all vegetative nodes (Tables 1 and 2). We have used culture experiments to identify sequential steps in flowering and have found that floral determination occurs late in tassel development (Fig. 2), after all floral organs have been initiated. During the period between tassel determination and floral determination, the tassel primordium makes no abrupt morphological changes, but instead gradually increases in height and girth while initiating buds acropetally in a spiral pattern (Fig. 1). The stages that culturing experiments have identified are summarized in Fig. 4. The different stages represent transitions which cannot occur in the isolated meristem/immature tassel, but require input from other portions of the plant. These transitions are: (1) indeterminate to determinate growth of the meristem, (2) initiation of buds on meristem, (3) indeterminate to determinate growth of shoots from the buds, (4) sterile to fertile shoots (florets). Previous experiments on intact maize plants, in which daylength and temperature were varied, identified points at which environmental 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 determined to form a terminal inflorescence at a later stage than was seen in the dicots sunflower and tobacco. Because the experiments with the dicots analyzed the developmental behavior of explants with several more leaves "and associated stem tissue than was present in the experiments presented here, it is not possible to directly compare those results with these obtained in maize. Experiments addressing this problem are underway; however, because our experiments used little more than the shoot apical meristem itself, it is likely that results obtained are a more accurate reflection of the ability of the meristem to undergo the various transitions in the switch from vegetative to floral development. Maize plants show a gradient of juvenile to adult nodes, in which characters such as leaf shape, waxy

896 E. E. Irish and T. M. Nelson Fig. 3. Structures that developed from cultured immature tassels at various stages. (A) 0.70 mm immature tassel cultured on solid medium; proliferous tassel. The immature tassel showed limited elongation in culture and is not visible in this figure. Several spikelet pair primordia have developed into shoots that developed adventitious roots. These shoots have an indeterminate pattern of growth. (0.9x magnification). (B) 8.5 mm tassel cultured in liquid medium. The most mature florets are sterile, with only leaflike organs. These shoots are vegetative and determinate. (2x magnification). (C) normal florets from a 30mm tassel cultured in liquid medium. Only the most mature florets have well-formed stamens. Less mature florets have only leaf-like organs, ( l. l x magnification). Arrow indicates most mature region. branch initiation vegetative meristem" elongated^.bud _ ^ spikelet pair_ meristem initiation initiation J T vegetative thorn proliferous tassel glume initiation spikelet initiation floret initiation sterile tassel flora) organ ' initiation fertile tassel Fig. 4. A schematic summary of the stable developmental phases of the transition from indeterminate, vegetative development to determinate, floral development. The upper portion represents the morphologically identifiable stages in tassel development, in which the vegetative meristem becomes elongated and initiates buds. The buds may develop into branches bearing spikelet pairs or directly to spikelet pairs. Spikelet pair primordia give rise to spikelets, which initiate glumes and florets. Each floret initiates a lemma, palea, and floral organs. The lower portion represents the type of development observed when meristems of those stages are cultured. versus hairy leaf epidermis, presence of adventitious roots, and the developmental fate of lateral buds vary from the base to the tip of the plant (Poethig, 1988). Nodes initiated at the top of the maize plant do not normally initiate adventitious roots: these are usually limited to the first 4-6 nodes and may be considered juvenile traits. Nevertheless, when the uppermost vegetative nodes from plants that had initiated up to all of the normal complement of nodes were put in culture, they were able to initiate normal adventitious roots.

Vegetative to floral conversion in maize 897 Similarly, the basal leaves on explants from late nodes had shapes and epidermal characters similar to seedling leaves, not adult leaves, as had been found in plants derived from seedling-stage meristems (Irish and Nelson, 1988). The one or two nodes left attached to the meristem when cultured developed in a pattern most similar to the basal one or two nodes of a seedling. Thus, even though nodes high on the plant normally have adult characteristics, the nodes are not limited to an adult pattern early in their development. The differentiation of organs into adult types depends on interactions with previously formed body parts (Irish and Nelson, 1988). The juvenile versus adult characters of the maize plant are not a reflection of the ageing of the meristem. Transition-stage meristems (0.32-0.67 mm) developed, in culture, into determinate cylinders with no lateral outgrowths (Fig. 2). This suggests that the tassel is limited in growth in advance of the initiation of any lateral buds. Determinate growth of shoots is not always associated with flowering; while the majority of shoots terminate by the differentiation of the apical meristem into a flower or inflorescence, some become determinate by the formation of a thorn, such as in Gleditsia (Steeves and Sussex, 1989). Thus, it is not unexpected that limitation of growth potential and floral determination are separable events. However, the cylindrical meristems were isolated during a period of rapid growth. The extremely limited development of meristems of this stage in culture may simply be the result of suboptimal medium and/or a wound response that interferes with the normal pattern of development. In the maize tassel, florets are initiated and develop sequentially, such that each tassel has a gradient of developmental stages (Fig. 2). Excised and cultured tassels at least 10 mm long showed at least one floret that developed normally. Florets in less mature positions on these cultured tassels were not able to develop florally even though the floral signal must have been present. Thus, primordia are required to become competent to flower in order to respond to the floral signal. Vegetative or proliferous inflorescences have been observed in a variety of plants (Battey and Lyndon, 1990). Galinat and Naylor (1951) induced proliferous tassels on maize plants homozygous for the indeterminate mutation, which confers a short day requirement for flowering, by giving the plants an insufficient number of short days to complete floral development. Proliferous tassels have also been observed in strains of maize from Mexico when grown farther north (Collins, 1909); these presumably are short day strains. The descriptions of these proliferous tassels fit the vegetative 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: chrysanthemum, also a short-day plant, will develop vegetative inflorescences when raised under short day conditions and then switched to long days after receptacle formation (Schwabe, 1959). These examples demonstrate that the flowering signal is required in these species for a prolonged period or for separate events during floral development. The cases of proliferous inflorescences described previously were obtained in daylength-dependent plants. We obtained proliferous tassels with the maize inbred line W23, which is dayneutral. Presumably, isolation of the developing inflorescences of a day-neutral plant has the same effect as removal of the floral induction signal during flower development in daylength-sensitive plants. Thus, the requirement for prolonged presence of the flowering signal(s) is not limited to plants that flower in response to daylength but is probably typical of flowering in many species. If perturbed early in development, flowers from different species show a range of ability to proceed with normal development. Tobaccoflowersexcised from the plant at the stage at which only the sepals had been initiated can continue to initiate the normal sequence of petals, stamens and pistils and grow to maturity in sterile culture (Hicks and Sussex, 1970). Similar results were obtained with the short day plant Chenopodium, which produced small but normal inflorescences in culture from shoot apices that had been excised six days after floral induction (Wetmore et al. 1959). Thus, floral determination occurs early in these plants and having occurred, the completion of organogenesis requires no more from the plant than simple nutrients. In contrast, Impatiens balsamina, a short day plant, when shifted from short day to long day and then back to short day, will develop organs intermediate between leaf and petal as the plant reverts to flowering (Battey and Lyndon, 1988). In this plant, cells in the developing organs respond continuously to environmental cues as they differentiate: organ identity is not stably determined at initiation. Cultured maize tassels follow the pattern of /. balsamina. The earliest staged tassel on which buds were able to develop into fertile florets in culture was 10 mm high. However, all floral organs have been initiated in florets of tassels less than 10 mm high (data not shown). Thus, maize floral development is not autonomous until floral organs are partially developed. A similarly late stage of floral determination was observed in chrysanthemum (Schwabe, 1959). Short day growing conditions have been shown to accelerate the transition from vegetative to tassel development in maize (Heslop-Harrison, 1961). The experiments presented here assayed the developmental potential of meristems of various stages from plants grown in long days. We predict that meristems from plants grown in continuous short day conditions would show determination at an earlier stage. It may be possible to identify a critical period in which day length controls the timing of tassel determination by combining day length shifts with meristem culture. The molecular nature of thefloweringsignal, which is required for a prolonged developmental period to initiate and complete reproductive development, remains obscure. The signal to convert the meristem into a tassel comes from other parts of the plant. This appears to be a node-counting mechanism in which the meristem responds to the gradual accumulation of some

898 E. E. Irish and T. M. Nelson factors(s) as the plant develops beneath it, or to a more rapid appearance of a flowering factor in the meristem after the required number of nodes have accumulated. There are no morphological differences between plants derived from meristems that had initiated 14-16 nodes and those that had produced 7-9 nodes (Irish and Nelson, 1988) before culturing. This suggests that if a flowering signal acts by accumulating in the meristem during development, it is not stable when the meristem is isolated and cultured. We thank Ian Sussex, Scott Poethig, and Gene Szymkowiak for critically reading this manuscript. E. E. I. was supported by grants from Pioneer Hi-Bred International, Inc. and the McKnight Foundation. References BATTEY, N. H. AND LYNDON, R. F. (1988). Determination and differentiation of leaf and petal primordia in Impatiens balsamina. Ann Bot. 61, 9-16. BATTEY, N. H. AND LYNDON, R. F. (1990). Reversion of flowering. Bot. Rev. 56, 162-189. BONNETT, O. T. (1948). Ear and tassel development in maize. Ann. Missouri Bot. Card. 35, 269-287. COLLINS, G. N. (1909). Apogamy in the maize plant. U. S. Nat. Mus. Contrib. National Herb. 12, 453-455. GALINAT, W. C. AND NAYLOR, A. W. (1951). Relation of photoperiod to inflorescence proliferation in Zea mays L. Am. J. Bot. 38, 38-47. GEHRJNG, W. (1972). The stability of the determined state in cultures of imaginal disks in Drosophila. In Results and Problems in Cell Differentiation (H. Ursprung and R. Nothinger, ed.), Vol. 5, pp. 35-58, Springer, Berlin. HABERMANN, H. M. AND SEKULOW, D. B. (1972). Development and ageing in Helianthus annuus L. Effects of the biological milieu of the apical meristem on patterns of development. Growth 36, 339-349. HANWAY, J. J. AND RITCHIE, S. W.(1985). Zea mays. In CRC Handbook of Flowering 4, 525-541. HESLOP-HARRISON, J. (1961). The experimental control of sexuality and inflorescence structure in Zea mays L. Proc. Linn. Soc. (London) 172, 108-123. HICKS, G. S. AND SUSSEX, I. M. (1970). Development in vitro of excised flower primordia of Nicotiana tabacum. Can. J. Bot. 48, 133-139. IRISH, E. E. AND NELSON, T. M. (1988). Development of maize plants from cultured shoot apices. Planta 175, 9-12. KIESSELBACH, T. A. (1949). The structure and reproduction of corn. Res. Bull. 161, Agric. Exp. Sta., University of Nebraska College of Agric. Reprinted 1980, U. Nebraska Press. MCDANIEL, C. N. (1984). Competence, determination, and induction in plant development. In Pattern Formation' A Primer in Developmental Biology (G. Malacinski, ed.), pp. 393-412. Macmillan, New York. POETHIG, R. S. (1988). Heterochronic mutations affecting shoot development in maize. Genetics 119, 959-973. POLOWICK, P. L. AND GREYSON, R. I. (1982). Anther development, meiosis and pollen formation in Zea tassels cultured in defined liquid medium. PI. Sci. Lett. 26, 139-145. RUSSELL, W. K. AND STUBER, C. W. (1983). Effects of photoperiod and temperatures on the duration of vegetative growth in maize. Crop Sci. 23, 847-850. SASS, J. E. (1976). Morphology. In Corn and Corn Improvement (G. F. Sprague, ed.). Amer. Soc. AgTonomy, Madison, pp. 89-110. SCHWABE, W. W. (1959). Some effects of environment and hormone treatment on reproductive morphogenesis in the Chrysanthemum. J. Linn. Soc. London (Bot.) 56, 254-261. SINGER, S. R. AND MCDANIEL, C. N. (1986). Floral determination in the terminal and axillary buds of Nicotiana tabacum L. Devi Biol. 118, 587-592. STEEVES, T. A. AND SUSSEX, I. M. (1989). Patterns in Plant Development Second Edition, Cambridge University Press, pp. 176-202. WETMORE, R. H., GIFFORD, E. M., JR AND GREEN, M. C. (1959). Development of vegetative and floral buds. In Photoperiodism and Related Phenomena in Plants and Animals (R. B. Wilson, ed.). Am. Assn. Adv. Sci. Publ. 55, 255-273. (Accepted 4 April 1991)