Developmental potentialities of leaf primordia of Osmrrnda cinnamomea. 11. Further studies on the influence of determined leaf

primordia on undetermined leaf primordial

CHARLES CARROLL KUEHNERT Department of Bacteriology and Botany, Syracuse University, Syracuse, New York

Received May 21, 1968

KUEHNERT, C. C.. 1969. Developmental potentialities of leaf primordia of Osmunda cinnamornea. 11. Further studies on the influence of determined leaf primordia on undetermined leaf primordia. Can. J. Botany, 47: 59-63.

In the fern Osmunda cinrzamomea, leaf primordia may be excised and grown in sterile culture before or after their irreversible determination as leaves. It has been demonstrated that P3 primordia (third most recently formed primordia) are not irreversibly determined as leaves at the time of excision for they exhibit a tendency to develop as whole shoots (approximately 67.0% of the time) whether grown singly, or as pairs grown in physiological contact. Results from the present investigation support the hypothesis that a morphogenetic factor(s) is found in older primorida which is transmitted to younger primordia to influence the latter to develop as leaves rather than shoots, for P3's grown in physiological contact (as pairs) with Plo, P12, or PI3 primordia are expressed as leaves at a level approximately twofold or greater than P3's grown singly (controls). Results from the present investigation do not support the hypothesis that increased bulk of tissue, and therefore increased capacity for nutrient productivity by older and larger primordia, is responsible for imposition of leafness on undetermined P3 primordia, for P14 pri- mordia grown in physiological contact with P3 primordia (as pairs) are observed to increase the per- centage of P3's expressing leafness at a level only slightly greater than P3's grown singly, and slightly less than when P3's are grown in physiological contact with other P3's as pairs. The slight enhancement in leafness exhibited by undetermined P3's grown as pairs with other P3's, or with P14's has been shown to be non-significant at the 5% level (P < 0.05) by :he standard t test and Kramer's (1956) modification of Duncan's (1955) new multiple range test.

Introduction and (or) isolation from the shoot apical meri-

~h~ two unique characteristics of the typical stem which is instrumental in determining a

leaf, dorsiventrality and determinate growth, are primordium as a leaf.

largely determined during the very early stages Steeves (1961a) originally demonstrated that

of growth at the shoot apical meristem. The excised, cultured undetermined leaf primordia

difference between the shoot and leaf expression- the fern osnzLmcja lainnanzotnea L.

potential of undetermined leaf primordia has as leaves, Or as whole plants. The per-

been the point of departure for investiga- 'entage of shoots formed is highest in the Young-

tions dealing with the problem of the establish- est leaf primordia and decreases in progressively

ment of the leaf pattern. older primordia. The 10th and all older pri-

The investigations of Sussex (1955) on S01atzut12 mOrdia as leaves. Kuehnert and fuberosum L., and those of Wardlaw, and Ward- demonstrated that law and Cutter on D,.vopte,.is Druce bisected primordia of fourth-youngest to ninth-

(1949-1963) have suggested the hypothesis that youngest leaves of '. cit2namomea had the the active site of leaf determination resides in the "'pacity as leaves, Or

shoot apex and that control of expression of the plants (a prilnordia

leafness characteristic in developing primordia (Steeves 1961a)). This variable response of

is mediated by the prevascular tissue of the primordia or fragments of primordia

shoot apical meristem. Other investigators suggested that one could influence the leafness

(steeves 1961a, 1961b, 1962; Kuehnert and or shootness expression of undetermined pri-

Steeves 1962; Kuehnert 1967) have suggested mordia by growing them in varying degrees of

that the fate of an undetermined leaf primordium coiltact with older primordia, primordia which

might depend on its content of a specific leaf- invariably develop into leaves. This would test

forming substance(s) at the time of its cxcision the hypothesis that a morphogenetic factor, or factors. is found in older ~rimordia which

lWork performed under Contract No. AT(30-1)-3597, diffuses from the older to the iounger primordia Atomic Energy Commission, ~ i ~ i ~ i ~ ~ of ~ i ~ l ~ ~ ~ and influencing the latter to develop as leaves rather Medicine, Biology Branch. than shoots. Again, using the fern Osmunda

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60 CANADIAN JOURNAL OF BOTANY. VOL. 47, 1969

cinnamonzen, it has been demonstrated (Kueh- nert 1967) that P3 primordia" are not irreversibly determined as leaves for when excised Pq primordia are cultured they develop as sfzoots at a level not less than 66.0% of the time. Furthermore, excised, cultured P3 primordia grown as paired units, i.e., P3's growing in physiological contact with other P i s (point of contact between primordia in all cases of pairing being the respective leaf excision scars) exhibit a tendency to express as shoots at nearly the same level as unpaired P3 primordia, and P3 primordia grown in physiological contact as pairs with Plo or P12 primordia exhibit an approximately twofild increase in their capacity to express as leaves over controls. In view of the fact that paired P3 primordia produce slightly more leaves than do P3's grown separately, and further, contact with a Plo or P12 greatly increases the tendency to leafness, the question may be raised whether the suggested transmitted influence is something non-specific rather than a definite leaf-forming substance, perhaps something of a nutritive nature present in all primordia, and perhaps all apical tissues. Experiments have been designed to test this hypothesis, and the results are reported here.

Materials and Methods The organism used was Os~nunda cinnamornea L. and

the methods for sterile culture of excised fern leaves essentially those previously reported by Sussex and Steeves (1953). Sterilization of the whole bud consisted of a surface treatment of the buds in a 7y0 (by weight) solution of "Percloron" (a commercial product contain- ing 70% calcium hypochlorite manufactured by the Pennsalt Chemicals Corporation, Philadelphia, Pennsyl- vania). P1.4'~~ P13's. PlZ1s, P~o 's (14th-youngest, 13th- youngest, 12th-youngest, and 10th-youngest primordia), which invariably produce leaves when cultured asepti- cally, and P3 primordia (third-youngest primordia), which tend to develop as shoots rather than leaves (Steeves 1961a; Kuehnert 1967), were grown together in sterile culture. The paired leaves were placed in such a way that their adaxial faces, at the point of the respective excision scars, were in physiological contact, and these paired leaf primordia were placed in 1-oz square tablet bottles on a nutrient medium which contained a modified Knudson's solution of mineral salts (Steeves et al. 1955), 2% sucrose, and 0.8yo agar (Fig. 1). P3 primordia as

*A P3 primordium is the third rnost recently formed primordium, and therefore is the third-yortngest pri- mordium in a sibling series; however, in a previous pub- lication, P3, incorrectly, was referred to as the third- oldest primordium in a sibling series (cf. Kuehnert 1967).

FIG. 1. P3 growing in physiological contact with a P12. A = apical cell; V = vasc~ilar and prevascular tissue.

FIG. 2. Donor leaves to undetermined P3 primordia grown in physiological contact (as pairs).

pairs, as well as unpaired P3, Plo, Pl2, P13, and Pl4 pri- mordia, as controls, were cultured in a similar manner. The primordia were maintained in a Percival model E-57 Environator controlled-environment chamber for 10 weeks at a temperature of 24" + 1 "C and an illumina- tion of 75-100 ft-c supplied by a combination of cool white fluorescent tubes and incandescent bulbs. The photoperiod was maintained as an alternating 12-h light : 12-h dark period.

Results The developmental potentialities of undeter-

mined third-youngest leaf primordia of Osi?zundo cinnntnornea grown under the influence of un- determined P3 primordia and determined Plo and Pi2-P14 primordia are summarized in Tables I and TI; see also Fig. 2. Survival rntes of all

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KUEHNERT: DEVELOPMENTAL POTENTIALITIES OF LEAF PRIMORDIA 61

cultured primordia and the results obtained are characteristic of three additional experiments performed. With regard to control primordia, the data show results similar to those reported in an earlier publication (Kuehnert 1967). P3 pri- mordia are not irreversibly determined as leaves. When cultured as paired units, P3's develop as shoots 67.0% of the time (Table I). Similarly, cultured P3 primordia grown as pairs, i.e., P3's growing in physiological contact with other P3's (point of contact in all cases of pairing being the respective leaf excision scars) exhibit a tendency to develop as shoots at approximately the same level as unpaired P3's (Table 11). At no time were Plo, and P12, PI3, or P14 primordia observed to develop as shoots. By contrast, Plo and P12 pri- mordia grown in physiological contact with P3 primordia caused the latter to be expressed as leaves at a level almost twofold greater than un- paired P3 primordia, and if compared with P3's growing as paired units, the level of increase is approximately the same. Further, as can be seen from the data in Table 11, P13 primordia showed an enhancement of the phenomenon. In sharp contrast, initial experiments with P14 prilnordia showed that P14's infl~lence P3 primordia to be expressed as leaves at a level approximately equal

TABLE I Fates of individual excised leaf primordia successfully

cultured for 10 weeks

Primordium Leaves Shoots Leaves, %

TABLE 11 Fates of excised P3 primordia cultured in physiological contact (as pairs) with P3, Plo, P12, P13, or P14 primordia

for 10 weeks

Morphogenetic response

Pairing No. No. conditions leaves shoots Leaves, %

P3 with P3 16 26 38.0 (controls)

P3 with Plo 20 11 65.0 P3 with Plz 24 13 65.0 Pq with PI? 13 5 72.0 P; with P;: 8 15 35.0

to that of P3's, and though, from the data pre- sented, P14's appear to enhance the leafness ex- pression of P3's when P3's and Pl4's are grown in physiological contact, the level of enhancement appears always to be approximately equal only to that of P3's grown either singly, or P3's grow- ing as pairs (Tables I and 11). These are totally unexpected results, but ones which have been borne out in subsequent experiments.

Discussion That simply increasing size or bulk of tissue is

not the deciding factor influencing undetermined P3 primordia of the cinnamon fern to develop as leaves rather than shoots under the influence of older primordia is borne out by the results from the present investigation. P3 primordia growing in physiological contact with other P3 primordia do exhibit a tendency to develop as leaves at a percentage level slightly greater than that ob- tained when P3's are grown singly on a simple, defined medium, increasing from 33.0% to 38.0% (compare Tables I and I!). A similar per- centage increase in leafness was reported earlier (Kuehnert 1967). Furthermore, with increasing bulk of donor leaf primordial tissue, the effect is considerably enhanced. When P3's are grown in physiological contact with Ploys, P12)s, or P13'~, the level of leaf expression in P3's rises to 72.0y0 (see Table 11). However, the trend of increasing bulk, and therefore increasing capacity for nu- trient productivity, as expressed by PI4's, does not maintain a concomitant increase in percentage of P3's being expressed as leaves. Quite to the contrary, P3's growing in physiological contact with P14's are expressed as leaves at a level ap- proximately only equal to that of P3's grown sin- gly, or aspairs. The slight enhancement in leafness exhibited by undetermined P3's grown as pairs with other P3's, or with P14)s has been shown to be statistically non-significant at the 5% level (P < 0.05) using both the standard t test and Kramer's (1956) modification of Duncan's (1955) new multiple range test. Thus, the results re- ported here would seem to argue strongly in favor of morphogenetic substances being pro- duced by certain of the older primordia, sub- stances which are transmitted from older deter- mined primordia to younger undetermined primordia influencing the latter to be expressed as leaves rather than shoots.

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62 CANADIAN JOURNAL O F BOTANY. VOL. 47, 1969

There is much previous work implicating the shoot apical meristem in leaf determination (Wardlaw 1949a, 1949b, 1949~; 1955a, 1955b; Wardlaw and Cutter 1954, 1956; Cutter 1954, 1956, 1957 Sussex 1955; Snow 1951; Richards 1948). From these extensive investigations of leaf development, both in ferns and in angiosperms, a hypothesis has been put forward which sug- gests that the ultimate and active site of control for leaf expression resides primarily, if not solely, within the shoot apical meristem, and that the control factors for this expression are mediated through the prevascular tissues of the shoot apex (see Cutter 1965, for citations). Results from the present investigation would suggest, at the least, that determination of a primordium as a leaf may be under the influence of more than one control center. Such a control center is associated with certain of the older primordia of a sibling series, for undermined P3 primordia of Osmunda cinnamomea are influenced to develop as leaves rather than shoots when grown under the influ- ence of Plo, Plz, or P13 primordia.

Though the factor(s) emanating from these certain older primordia controlling expression of an undetermined primordium asa leaf rather than a shoot is as yet unidentified, it would appear that the system herein described offers exciting possibilities for identification of such a morpho- geneticsubstance(s).This would seem so especial- ly in light of the fact that expression of the sub- stance's, or substances', influence to determine a primordium as a leaf, at least in cinnamon fern leaf primordia, appears to be tripped much as a lever is tripped, at a point in time before a P3 becomes determined as a P3 leaf primordium, but no longer can be brought into play by the time a primordium has "aged" to the point of a p14.

Results from these experiments, and those previously reported (Kuehnert 1967) do not deny the fact that, in the adult shoot, similar control may be exerted by the shoot apical meristem. Further, it is self-evident that the first leaf pro- duced by an embryo, or an adventitious bud is established without the influence from other leaves. The question being raised here, however, is not whether certain of the older prjmordia of a sibling series are the sole source of control exerted on younger leaf primordia to cause them to be expressed as leaves. Rather, it is the pre- eminent role of the center of the meristem which

is being questioned in this regard. The results from this investigation, as well as other evidence accumulating, strongly suggest that, in the cin- namon fern, control of morphogenetic expres- sion of a letgf'primordium only as a leaf can be exerted through older primordia of a sibling series, and that control from the older primordia is restricted to only certain of these primordia.

Acknowledgments The author expresses his appreciation to Dr.

James R. Florini for his helpful and stimulating discussions, and to Mr. Thomas H. Haight for his technical assistance.

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KUEHNERT, C. C. and STEEVES, T. A. 1962. Capacity of fragments of leaf prirnordia to produce whole leaves. Nature, 196: 187-189.

RICHARDS, F. J. 1948. The geometry of phyllotaxis and its origin. Symp. Soc. Exptl. Biol. 2: 217-245.

SNOW, M. 1951. Experiments on spirodistichous shoot apices. I. Phil. Trans. Roy. Soc. London, Ser. B, 235: 131-162.

STEEVES, T. A. 1961a. The development of leaves in sterile nutrient culture. In Recent advances in botany. Vol. I. University of Toronto Press. pp. 823-827.

1961b. The developmental potentialities of excised leaf prirnordia in sterile culture. Phytomorphology, 11: 34fi-359. - - . - . . . . . .

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KUEHNERT: DEVELOPMENTAL POTENTIALITIES OF LEAF PRIMORDIA 63

WARDLAW, C. W. 1949a. Experimental and analytical - 1955b. Experimental investigation of leaf for- studies of pteridophytes. XIV. Leaf formation and mation, symmetry and orientation in ferns. Nature, phyllotaxis in Dryopteris aristnta Druce. Ann. Botany 175: 115-117. (N.S.) 13: 163-198. WARDLAW, C. W. and CUT~ER, E. G. 1954. Effect of

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