DEVELOPMENTAL

BIOLOGY

47, 376-383 (1975)

Morphogenesis III. Meristem

in Selaginella

Determination ZACHARY

and Cell Differentiation

S. WOCHOK’ AND

I.

M.

SUSSEX

Biology Department, Yale University, New Haven, Connecticut 06520 Accepted July 7,1975 stem. At Selaginella willdenovii Baker is a prostrate vascular cryptogam with a dorsiventral each major branching of the stem tip a dorsal and a ventral angle meristem are formed. The ventral meristem becomes determined as a root and the dorsal meristem as a shoot. Indoleacetic acid (IA& is transported basipetally in the stem and has been found to be the regulatory agent for meristem determination both in vitro and in viuo. Growth measurements of intact plants indicated that the sequence of development for each stem unit is frond expansion, internodal elongation, ventral meristem growth as a root, and dorsal meristem growth as a shoot. The principal experimental findings of this study are as follows. Triiodobenzoic acid (TIBA), an inhibitor of auxin transport alters the normal pattern of development in intact plants, causing ventral meristems to develop as shoots and dorsal meristems to develop precociously. Dorsal meristems grown in sterile culture on an auxin-free medium develop as shoots, but in the presence of IAA develop as roots. Meristems transferred after excision from auxin-free to plus-auxin medium on successive days showed an increasing tendency to develop as shoots, with more than 50% doing so by day 5. The mitotic index is low at the time of excision of the meristem, rises to a peak on day 5 and then declines.

are subject to developmental regulation (Cusick, 1953, 1954). This developmental At opposite poles of the plant embryo are pattern eliminates the necessity for worktwo meristems, each with a different develing on the initial determination of meriopmental potential. One is determined as stems in the embryo and facilitates experia shoot-producing organ and the other as a mental manipulation of the material. root. The mechanism of meristem determiThe usual pattern of angle meristem nation remains obscure, but in recent growth in the intact plant ofS. willdenovii years tissue culture techniques have been is for a shoot to develop from each dorsal used to study some of the biochemical asangle meristem and a root from each venpects of this question (Murashige, 1964; tral angle meristem. The determination of Pillai and Hildebrandt, 1969; Skoog and organ type arising from either meristem Miller, 1957; Wochok, 1973). has been shown to be under the control of Selaginella willdenovii is especially useexternally applied auxin (Webster, 1969; ful for studies of meristem determination Williams, 1937). From these experiments and of the degree of stability of the determined state. The reason for this is that at it has been implied that the level of endogeeach major branching of the stem, called nous auxin controls meristem determinahere the branch junction, this plant has tion in the intact plant. However, no detailed study of meristem determination angle meristems that remain morphologihas been made to establish this point. cally undetermined for some time and that There is now evidence for specificity of basipetal auxin transport in S. willdenovii 1 This study was supported by NSF Grant No. GB 37945 to z. s. w. which indicates that: (i) auxin is transINTRODUCTION

376 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

WOCHOK

AND ,%JSSEX

Morphogenesis

in Selaginella.

III

377

ported more rapidly in vascular than corti- were made every third day for a 30-day cal tissues, and (ii) twice as much auxin is period. The youngest frond visible behind transported in the dorsal than ventral tis- the stem tip at the initial observation was sues in branch junctions where roots and designated FI. The branch junction formed shoots are determined (Wochok and Sus- by the bifurcation of this frond and the sex, 1973). The objectives of this study main axis was designated as I, and the were to establish the developmental pat- root developing from the ventral meristem tern of growth in S. willdenovii and to of the first branch junction was designated reconcile the regulatory action of auxin in RI. Internode I was identified as that recontrolling the determination of an angle gion of the main axis acropetal to branch junction I. Each new root, frond and intermeristem as root or shoot. node that developed during the period of MATERIALS AND METHODS observation was numbered in succession II, III. . . n, as shown in Fig. 1. Plant material. Selaginella willdenovii Triiodobenzoic acid. Transport of radioBaker plants grown under greenhouse conactively labeled indoleacetic acid (IAA) ditions were used in these experiments. This species is a dorsiventral scrambling has been shown to be basipetal in the stem of Selaginella willdenovii (Wochok and plant that typically grows horizontally. The stem tip branches terminally, and at Sussex, 1973). In order to observe the efeach bifurcation a dorsal and a ventral fects of IAA transport inhibition in the angle meristem occur. The former typi- intact plant triiodobenzoic acid (TIBA) was cally develops into a shoot and the latter used. TIBA was made up to 0.1 mM in into a root in the intact plant. hydrous lanolin and applied around the Growth measurements. Fifteen young stem behind the tip and around the basal plants were selected at random for the pur- portion of the youngest frond. Sterile cultures. Dorsal meristems were pose of obtaining growth data for roots, internodes and fronds. Measurements excised aseptically from the first or second

FIG. 1. Selaginella willdenovii Baker. noted. The numbering system used in numerals). In the system used the lowest growth. The highest number represents

The apical tip (A), root CR), frond (F), and branch junction (BJ) are studying growth of greenhouse grown plants is shown (Roman number represents the first growth measurement and most mature the most recent growth and measurements.

378

DEVELOPMENTALBIOLOGY

branch junctions which had been surface sterilized in a 0.5% sodium hypochlorite solution for 10 min and rinsed three times with sterile distilled water. The first or second branch junctions were selected since their meristems were undeveloped. The triangular-shaped wedge of tissues measured on the average 1.5-2.0 mm per side and about 1.0-1.5 mm thick. Excised meristems were transferred singly to 25 x 120-mm culture tubes containing 20 ml of solidified medium which were covered with squares of Parafilm sterilized with ethanol. The basal medium was Knop’s and consisted of 250 mg/liter MGSO,*7H,O, 60 mg/liter KN03, 250 mg/liter Ca(NO&. 4Hz0, 10 ml/liter White’s vitamins, and 20 g/liter sucrose in a 0.8% agar substrate. Two media were used: Knop’s without any additives and Knop’s + filter-sterilized 5 $V IAA. Cultured meristems were kept in a Hotpack growth chamber maintained at 23°C and an 18-hr photoperiod. Isolated meristems were maintained on Knop’s for 1,3,5 and 7 days before transfer to Knop’s + IAA. Meristems maintained throughout the culture period on Knop’s or Knop’s + IAA served as controls. At least three meristems were used for each treatment with seven replicates. Histology. Meristems maintained on Knop’s for 0, 1, 3, 5, and 7 days were fixed in ethanol:acetic acid (3:1), sectioned at 5 pm and stained according to the Feulgen procedure. Mitotic counts (number of cells in prophase through telophase) were made in serial sections of meristems. At least three meristems for each treatment were counted.

VOLUME 47, 1975

used by Cusick (1953). A single root grows from the ventral meristem and is usually first observed in the third branch junction behind the stem tip. The branch junction at which the dorsal meristem was observed to begin developing as a shoot varied from the sixth to ninth position. Thus, the ventral root usually develops in advance of the dorsal shoot by several weeks. Pattern of growth. Growth measurements of 15 greenhouse-grown plants were taken every third day. Three parameters were used: root length, internode length and frond length at successively initiated branch junctions. Data obtained from one plant representative of the group are presented graphically in Figs. 2-4. Internode elongation proceeds uniformly with an average interval of 7.2 days (SE ? 0.36) between initiation of elongation in successive internodes (Fig. 2). The frond is the first organ to develop at a given branch junction, a new frond expanding on the average every 6.2 days (SE * 0.33) (Fig. 3). Frond development was found to precede internode elongation. Roots develop from the ventral angle meristem 11 days after frond growth from the same branch junction (Fig. 4). Roots develop on the average every 6.8 days (SE 2 0.42). The shoot develops from the dorsal

RESULTS

Morphology. Branching of the shoot in S. wilkienovii is terminal, unequal, and in

one plane, resulting in a flattened pseudomonopodial branching system (Fig. 1). At each branch junction are found two opposing meristems: one dorsal and one ventral. This terminology is in accord with that

20

10

30

DAYS

FIG. 2. Internode growth of S. willdenovii. Measurements of internode elongation were made every third day. Numbers represent successive internodes, with I being the oldest and IV the youngest.

WOCHOK

AND

Morphogenesis

SUSSEX

1

It

. ” .

. ”

. .

.‘”

. .

. .

n”

. . .

. L.-

. ~__

20

10

30

DAYS

FIG. 3. Frond growth in S. willclenovii. Measurements of frond length were made every 3 days. Numerals represent fronds growing from a specified branch junction. Frond II was the first measured and V the last. For reference, frond II subtends internode II.

.

. . .

.



A

111

in Selaginella.

III

379

TIBA treatment. To test the hypothesis that the pattern of organ formation observed in Selaginella may be under hormonal control, 0.1 n&f TIBA was applied in a lanolin paste around the stem behind the shoot tip of intact plants. In some cases epinasty occurred and the meristems immediately behind the treated area remained dormant throughout the period of observation. All of the remaining plants responded to TIBA treatment by the nearsimultaneous development of both dorsal and ventral meristems as leaf-bearing shoots (Fig. 5). Protuberances from both meristems were usually first observed 15 20 days after treatment. It was also noted that the TIBA was less effective if applied to the second internode, in which case half of the ventral meristems produced shoots and half roots. This indicates that the regulatory controls influencing root development are exercised at an early stage of development. This hypothesis was tested in another experiment by applying a smaller amount of TIBA paste to the subapical stem region and to the base of the lateral developing frond. In this experi-

FIG. 4. Root growth in S. wilklenouii. Root growth was measured every third day. Root I is oldest and IV is youngest proceeding in the acropetal direction. For reference, compare root III with frond III.

meristem of this same branch junction 4060 days after root development is first observed. The above values are based on 40 measurements from 15 test plants. In summary, the sequence of developmental events at any given branch junction of S. willderwvii is: frond development, internode elongation, ventral root development, and dorsal shoot development.

FIG. 5. Effect of triiodobenzoic acid (TIBA) on organogenesis. Dorsal and ventral meristems developed as shoots Cl) as the results of TIBA application immediately below the shoot apex (arrow). The frond has developed normally (F), but the growth of the main axis (A) is reduced. Shoots were first observed I4 days after TIBA application. Photographed 2 months after the TIBA treatment.

380

DEVELOPMENTAL BIOWGY

ment all of the meristems immediately basal to the treated tissue formed dorsal and ventral shoots. The precocious development of the dorsal meristem as a result of TIBA treatment offers strong support for the hypothesis of auxin regulation of organogenesis in this plant. Meristem culture. Since dorsal meristems remain in a developmentally arrested state for a longer period than ventral meristems, they were chosen for culture studies. Isolated meristems cultured aseptically on solidified Knop’s medium developed almost exclusively as shoots, as shown in Fig. 6. Meristems cultured on medium supplemented with 5 a IAA developed exclusively as roots. The results support the contention of Williams (1937) and Webster (1969) that auxin influences organogenesis in Selaginella. They also suggested to us that dorsal meristem cells may be in an undetermined state during their developmentally arrested period in the intact plant. If this is the case then there must be some time following the resumption of growth when the meristem becomes determined for shoot development. The in vitro method lends itself to such a study because of the ease of manipulation and the precise control over the chemical environment in which meristems are cultured. As isolated meristem grown in culture develops into an identifiable shoot within

VOLUME 47, 1975

14 days as opposed to the 40-60 days required by comparable meristems on the intact plant. If there is a specific time at which determination occurs in uitro, it was hypothesized that it should be prior to 14 days when the meristem has developed a recognizable shoot. To test the hypothesis, meristems were cultured for 1, 3, 5, and 7 days on Knop’s medium and then transferred to the same medium supplemented with 5 a IAA. If irreversible determination occurred prior to transfer to the IAA medium it was expected that the meristems would develop as shoots. The results presented graphically in Fig. 7 support the hypothesis. The data indicate that meristems have not been irreversibly determined as shoots after 1 day of culture on Knop’s medium prior to transfer to IAA medium. After 3 days on Knop’s medium there is a significant increase in shoot development. Forty-five percent of meristems have been determined for shoot development by this time. More than half of 100

100 IL

l

4

I

I

80

I 4D : -605

t 4 60 F 8 oc

1

FIG. 6. Shoot development from a dorsal angle meristem cultured on Knop’s medium. Photographed after 5 week’s growth. x 10.

‘/

2

3

4

5

6

7

FIG. 7. Organogenesis in excised dorsal meristems transferred from basal to IAA medium. Dorsal meristems were excised and cultured on Knop’s medium for 1, 3, 5 and 7 days preceding transfer to medium containing 5 @f IAA. the percentage of roots and shoots developed is indicated. A total of 266 meristems were cultured with an average of 39 meristams for each treatment. Data were based on six experiments. Final determinations were made after 5 weeks.

WOCHOK

AND SUSSEX

381

Morphogenesis in Selaginella. III

the meristems developed as shoots after 5 days on Knop’s medium and nearly 80% did so after 7 days. After 9 days or more of culture in Knop’s prior to transfer, 95% of the meristems developed as shoots. The critical period at which the majority of meristems in culture become determined for shoot development appears to be day 5. Cell division in the meristem. The results of in vitro experiments indicated that in the majority of meristems cultured determination as a shoot occurred at day 5. Our results support the contention that auxin is the regulatory factor controlling meristem determination, the meristem developing as a shoot in its absence and as a root in its presence. Meristems are most receptive to the influence of externally applied auxin prior to day 5, and it was apparent that some further clues regarding the regulation of developmental potential in meristems might be observed at the cell level. The question asked in the following study was: Can changes that might be associated with determination be correlated with mitotic indices in cells of meristems cultured on Knop’s medium over a 7-day period? The total number of cells and mitotic figures in sectioned meristems were counted. Meristematic cells were easily recognized by virtue of their small size and relative cytoplasmic density as compared to the larger, highly vacuolated cells immediately surrounding them. “Mitotic index” refers to the average percentage of mitotic nuclei observed in a meristem. The results in Table 1 show about 0.9% mitoses in a meristem fixed immediately after excision, called 0 day. After 1 day in culture the mitotic index increased to 3% and was maintained at 3% in 3-day meristems. Another increase to 5.6% occurred at 5 days which represents an entrainment of shoot determination. The mitotic index decreased to 1.6% by day 7. At this time the majority of meristems are determined as shoots hence it is doubtful whether the decreased mitotic activity represents a sig-

TABLE THE

MIT(YTIC

EXPRESSED

INDEX

1

IN DORSAL

AS PERCENTAGE

MERISTEMS

OF MITOSES

AND 7 DAYS AETER 0, 1,3,5 IN CULTURES

Days on Knop’s

Number of nuclei

Number of mitoses

Mitotic index

0 1 3 5 7

10,044 11,209 12,641 13,877 17,535

92 342 374 779 273

0.9 3.0 3.0 5.6 1.6

n Data represent counts of at least four meristems from two experiments.

nificant ess.

event in the determination

proc-

DISCUSSION

The principal points to emerge from this investigation are the following: (i) TIBA, an inhibitor of auxin transport, upsets the normal pattern of meristem determination and growth, causing the ventral angle meristem to be determined and develop as a shoot, and the dorsal meristem to develop several weeks earlier than it would in normal development. (ii) Dorsal meristems excised and grown in sterile culture on an auxin-free medium develop as shoots, but in the presence of auxin they develop as roots. (iii) Transfer of cultured dorsal meristems from an auxin-free to an auxin-contaming medium at successive times after excision from the plant indicates that determination occurs in the majority of meristems by day 5. (iv) The mitotic index is low at the time of excision of the meristem, rises to a peak at day 5 and then declines. The significance of these results can now be discussed in the light of previous work on meristem determination. Auxins are known to influence the determination of meristems in Selaginella. The angle meristem of S. martensii grows as a root on the intact plant. When stem segments were removed from the plant 93% of the meri-

382

DEVELOPMENTAL BIOLOGY

stems developed as shoots, but if an auxin paste was applied to the cut ends of the segment, the meristem grew as a root (Williams, 1937). Indolebutyric acid applied to cut ends of excised branch junctions of S. marten&i induced the formation of roots from meristems that would have been expected to develop as shoots on the intact plant (Webster, 1969). We have previously shown that auxin is transported basipetally in the main stem axis of S. willdenovii (Wochok and Sussex, 1973). Based on the results of auxin transport studies the following hypothesis was advanced: If basipetal auxin movement was inhibited, a change in the developmental pattern of both dorsal and ventral meristems would occur. To test this hypothesis, TIBA, an inhibitor of auxin transport, was applied subjacent to the terminal shoot tip meristem of the intact plants. Winter (1967) has shown that TIBA increases immobilization of auxin so that diffusion as well as metabolic transport might be reduced. The experimental results supported the hypothesis in that dorsal and ventral meristems below the site of TIBA application developed as shoots in 15-20 days. This represented a change in the developmental pattern normally found in the ventral meristem and also a temporal change in development of the dorsal meristem which normally exhibits shoot development only after 8 weeks. The proposal for determination of dorsal meristems on the intact plant of S. willdenovii has support on the basis of in vitro experiments. Dorsal meristems under constant exposure to IAA will form roots in culture after about 3 weeks, while those on auxin-free medium develop as shoots after 2 weeks. Shoot development in vitro is significantly accelerated temporally in comparison to normal development time (6-8 weeks). The results from culture experiments compare favorably with the rapid shoot development observed with TIBA treatments. The results of experiments in which mer-

VOLUME 47, 1975

istems were transferred from control medium to IAA medium supported the contention that dorsal meristems underwent a change in developmental potential. After l-3 days in culture most dorsal meristems were not determined as shoots and could still be influenced by exogenous auxin. After 5 days the majority of meristems had been determined as shoots and could not be redetermined by auxin. These results suggest that IAA has a direct influence on meristem determination in vitro. At the time of excision the meristems must be in an undetermined state and they become determined in the culture environment. This interpretation is supported by experiments (Wochok, unpublished) in which dorsal meristems excised from branch junctions 1, 2, and 3 were cultured on an auxin supplemented medium and developed as roots. Thus meristems are undetermined in at least the first three branch junctions, and can be influenced by exogenous IAA to form roots in vitro. Excised frond tips consisting of the terminal meristem and a few leaf primorida of S. willdenovii cultured on Knop’s + IAA (Sussex, unpublished) develop normally and in no case was the meristem redetermined as a root. Relationship to other developmental systems. The present study with Selaginella

bears some resemblance to a study of axillary root development in the cress plant (Ballade, 1970). The axillary radicular plate of cress, which usually develops a root, is similar to a meristem as a site of organogenesis . Application of kinetin to isolated cuttings from which roots were growing inhibited growth and then induced the formation of a bud at the root tip. Once roots grew to a certain length they became irreversibly determined and did not respond to the kinetin treatment. This is similar to S. willdenovii where auxin can elicit a redirection of development only early in the culture period. Holtzer has proposed that differentiation of presumptive myoblasts follows a

WOCHOK AND SUSSEX

Morphogenesis

“quantal” cell division which is unlike preceding “proliferative” cell divisions in that the cell now exhibits an altered genetic expression (Holtzer et al. 1972). Other investigators have proposed that unequal cell division is a required precedent for certain kinds of differentiation in plant cells (Stebbins, 1965; Torrey, 1967). In order to determine whether phenomena of the above types might precede determination of Selaginella meristems we investigated the relationship between the time of cell division, and meristem determination. Since we showed that meristem cells were already dividing at the time of excision, determination in excised meristems does not precede the initiation of division. It would be of interest to determine the role of nucleic acid and protein synthesis in this system. Quatrano (1968) attempted to determine whether RNA synthesis or protein synthesis were required for rhizoid formation of the first cell division in Fucus. He found that the irreversible determination of a specific region of the cytoplasm was controlled at the level of translation rather than transcription. These levels of developmental regulation in Selaginella will be pursued in future studies. The authors acknowledge the preliminary experiments of William Packard done with I.M.S. The technical assistance of Ms. Janice Innes is most appreciated. REFERENCES BALLADE, P. (1970). Precisions nouvelles genese apical des racines axillaires Planta (Berlin) 92, 138-142.

sur la caulodu cresson.

in Selaginella.

III

383

CUSICK, F. (1953). Morphogenesis in Selaginella wilkdenouii Baker. I. Preliminary morphological analysis. Ann. Bot. (London) 17, 369-383. CUSICK, F. (1954). Morphogenesis in Selaginella willdenouii Baker. II. Angle-meristems and angle shoots. Ann. Bot. (London) 28, 171-181. EBERT, J. and SUSSEX, I. M. (1970). “Interacting Systems in Development.” Holt, Rinehart and Winston, New York. HOLTZER, H., SANGER, J. W., ISHIKAWA, H., and STRAHS, K. (1972). Selected Topics in Skeletal Myogenesis. Cold Spring Harbor Symp. Quant. Biol. 37.549-566. MURASHIGE, T. (1964). Analysis of the inhibition of organ formation in tobacco tissue culture by gibberellin. Physiol. Plant. 17, 636-643. PILLAI, S. K., and HILDEBRANDT, A. C. (1969). Morphogenetic changes of Geranium callus in suspension cultures. Phytomorphology 19, 154-158. QUATRANO, R. (1968). Rhizoid formation in Fucus zygotes: Dependence on protein and ribonucleic acid synthesis. Science 162.468-470. SKOOC, F., and MILLER, C. 0. (1957). Chemical regulation of growth and organ formation in plant tissues cultured in uitro. Symp. Sot. Exp. Biol. 11, 118-131. STEBBINS, G. L. (1965). Some relationships between mitotic rhythm, nucleic acid synthesis, and morphogenesis in higher plants. Brookhaven Symp. Biol. 18, 204-221. TORREY, J. G. (1967). “Development in Flowering Plants,” pp. 104-107. Macmillan, New York. WEBSTER, T. R. (1969). An investigation of angle meristem development in excised stem segments ofSelaginella martensii. Can. J. Bot. 47, 717-722. WILLIAMS, S. (1937). Correlation of phenomena and hormones in Selaginella, Nature (London). 139, 966. WINTER, A. (1967). The promotion of the immobilization of auxin in Arena coleoptiles by triiodobenzoic acid. Physiol. Plant. 20, 330-336. WOCHOK, Z. S., and SUSSEX, I. M. (1973). Morphogenesis in Selaginella: Auxin transport in the stem. Plant Physiol. 5 1, 646-650. WOCHOK, Z. S. (1973). DNA synthesis in cultured tissues of wild carrot. Biol. Plant. 15, 107-111.

Morphogenesis in Selaginella. III. Meristem determination and cell differentiation.

DEVELOPMENTAL BIOLOGY 47, 376-383 (1975) Morphogenesis III. Meristem in Selaginella Determination ZACHARY and Cell Differentiation S. WOCHOK’ A...
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