DEVELOPMENTAL POTENTIALITIES OF LEAF PRIMORDIA OF OSMUNDA CINNAMOMEA

THE INFLUENCE OF DETERMINED LEAF PRIMORDIA ON UNDETERMINED LEAF PRIMORDIA1

CHARLES CARROLL KUEHNERT Department of Bacteriology and Botany, Syraczise University, Syraczise, hTew York

Received May 23, 1967

I n the fern Osnzunda cinnamomea, leaf primorida may be excised and grown in sterile culture before or subsequent to their irreversible determination as leaves. I t has been demonstrated tha t P g prilnordia (third-oldest primordia) are not irreversibly determined as leaves a t the time of excision for they eshibit a tendency to develop as shoots rather than leaves when cult~lred aseptically. When grown singly they become shoots 75.0% of the time; when grown in physiological contact as paired units they exhibit a tendency to become shoots a t approxi- mately the same level. The hypothesis that the active site of leaf determination is through the direct or indirect control of the shoot apical meristem is questioned and a n alternative hypothesis is proposed which suggests that a morphogenetic factor is formed in older primordia which will diffuse to younger prirnordia to influence younger prirnordia to develop as leaves rather than shoots. The latter hypothesis is supported by experiments in which P3 prirnordia grown in physio- logical contact (as pairs) with P I" or P I? prirnordia produce more than twice as many leaves as unpaired or paired P 3 primordia.

Introduction The phenomena concerned \\.it11 the establishment of the leaf pattern have

recently received considerable attention. There are two major characteristics of tlle typical leaf nrhich appear during its morphogenesis and which stand in sharp contrast to the organization of the whole shoot: dorsiventrality, and determinate or restricted growth. Both inicrosurgical and sterile culture techniques have been used to study the phenomena. Surgical isolation of pre- suinptive leaf positions, or even young emergent leaf primordia may lead to the forination of centric leaves (Sussex 1955) or even indeterminate buds (Wardla\\- 1949; Cutter 1956). Steeves (1961~) has shown that any of tlle nine youngest leaf prinlordia of the fern Osmunda cinnamomea L., after excision and explanta- tioil to a sterile nutrient medium of simple con~position, may develop as leaves, or as nrhole plants. The percentage of slloots formed is highest in the youngest and decreases in progressively older priillordia with the 10th and older primordia aln~ays becoming leaves. These investigations suggest that tlle mech- anism of leaf determination, once begun, is a process arhich sets in lnotion a sequence of morphogenetic events \vliich is largely self-controlled within the developing primordium.

Cutter (1956), einploying microsurgical methods, concluded that, in Dryop- tevis, determination bf a priinordiuin as a leaf approximately coincides with the establishment, in tlle primordial apex, of a distinct lenticular cell. Steeves (1961~) was not able to relate determination to the fornlation of a tetrahedral apical cell in 0. cinnamomea. Leaves with and without an apical cell developed into shoots. I<uehnert and Steeves (1962) attempted to deterinine \\il~ether a particular configuration of cells in the intact, undetermined primordium could

'Work performed under Contract No. AT(30-1)-3597, Atornic Energy Commission, Division of Biology and Medicine, Biology Branch.

Canadian Journal of Botany. Volume 45 (196i)

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2110 CANADIAN JOURNAL O F BOTANY. VOL. 45, 1967

be correlated with its capacity to develop as a leaf. These investigators grw- the halves of sagittallj, split, undetermined primordia of 0. cinlzamomca in sterile culture. Such fragments were observed to have the capacity to develop as ivhole leaves. This led these authors to conclude that no particular configura- tion of cells conferred determination as a leaf upon the primordium.

I t has been repeatedly suggested (Steeves 1961b, 1962 ; Kuelinert and Steeves 1962) that the fate of a primordium might depend on its content of a specific leaf-forming substance a t tlie time of excision. The variable response of the excised, cultured first nine leaf prinlordia of 0. cinnamornea suggested that it would be I\-orth~vhile to attempt to influence the leaf-ness or shoot-ness expression of undetermined prirnordia by gro~ving them in varying degrees of contact ~vith older priniordia, priinordia nrhich invariably develop into leaves. This \~oiild test the hypothesis that a morpliogenetic factor is formed in older priiiiordia \\-hich \\-ill diffuse from older primordia to younger primordia to cause younger prilllordia to develop as leaves rather than shoots.

Materials and Methods Tlle organism used was Osmunda cinlzamomea L. and the methods are essenti-

ally those previously reported for sterile culture of excised fern leaves (Sussex and Steeves 1953). Sterilization of the ~vhole bud consisted of a surface treat- ~nei l t of tlie buds in a 7% (by ~veight) solution of "Percloron" (a commercial product containing 70% calcium liypocl~lorite manufactured by tlie Pennsalt Chemicals Corporation, Philadelphia, Pennsylvania). Ill2 priinordia (12th- oldest primordia) or 1'10 priinordia (10th-oldest primordia), which invariably produce leaves ~vhen cultured aseptically, and P3 priinordia (3rd-oldest primordia), 11.11ich tend to develop as shoots rather than leaves (see Steeves 1961a), \\-ere grown together in sterile culture. Tlie paired leaves \\ere placed so that their adaxial surfaces, a t the point of the respective leaf scars, were in contact, and these paired leaves \\-ere placed in 1-oz square tablet bottles on a nutrient medium ~\rhich contained a modified Knudson's solution of ininera1 salts (Steeves ct al. 1955), 2% sucrose, and 0.8% agar. P3 priiilordia as pairs, as \\-ell as unpaired PI?, 1'10, and Pg primordia, as controls, were cultured in a similar manner. primordia were illaintained in a Percival controlled- environinent chamber for 10 weelts a t a temperature of 24" f 1 "C and an illumii~atioi~ of 75-100 ft-c supplied by a coinbination of cool white fluorescent tubes and incandescent bulbs. Tlie photoperiod was maintained as an alter- nating 12-11 light : 12-11 dark period.

Results Tlle developliiental potentialities of undetermined leaf primordia of Osmztlzda

cinnan~omea gro\\-n under the influence of determined leaf primordia are suin- marized in Tables I-IV. In control primordia, the data sho~v that P3 priniordia (third-oldest primordia) are not irreversibly deterinined as leaves for, when excised, cultured P3 primordia are grown singly, they develop as shoots 75.0% of the time (Table I). Similarly, excised, cultured Pa priinordia grown as paired units, i.e. P3's growing in pllysiological contact with other P3's (point of contact in all cases of pairing being the respective leaf scars) exhibit a tendency to develop as shoots a t approximately the same level as unpaired P3 primordia (Table 11).

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KUEHNERT: DEVELOPMENTAL POTESTIALITIES O F LEAF PRIMORDIA 21 11

TABLE I Fates of individual excised leaf prilnordia successfully

cultured for 10 weeks

Primordium Shoots Leaves Shoots (%)

TABLE I 1 Fates of nine excised P 3 primordia cultured in physiological

contact with nine add~tional P3 primordia for 10 meelcs

Morphogenetic response Number Leaf or shoot (%)

P3 pri~nordia Leaf Shoot

Totals

TABLE I11

Fates of 31 P 3 prinlordia c u l t ~ ~ r e d in physiological contact with 31 Plo primordia for 10 weeks

-

Morphogenetic response N u n ~ t ~ e r Leaf or shoot ( C j , )

P a primordia Leaf Shoot

Totals

I t is apparent from the data in all tables that unequal numbers of prinlordia are being compared. The unequal numbers result from problems of contamina- tion. Survival rates of all excised primordia and the results obtained are typical of three additional ex~eriments ~erforined.

At no time did Plo or Plz primordia, whether paired with P3's or unpaired, develop as shoots. By contrast, P3 prirnordia grown in physiological contact with Plo prirnordia produce more tlian twice as many leaves as control P3 prii-nordia grown as single units; and, tlie percentage difference is essentially tlie same ivhen P3 prirnordia grown in pllysiological contact \\;it11 Plo priniordia are compared \\it11 control P3's in ~vhich P3 priniordia were grou7n (as pairs) \\;it11 other Pa primordia (Tables 1-111). I t is apparent from the data dlat excised P3 priinordia exhibit a greater tendency to develop as leaves under tlie influerlce of Plo primordia; and further, this tendency is enhanced \vIien 1'3 primordia are grown in pliysiological contact with Plz priinordia. Table IV slio\vs that tlie morphogenetic response of P3 prirnordia cultured, in pl~ysiological contact, \\.it11 P1? primordia results in leaf-ness in 75.0y0 of tlie P3 primordia scored for leaf-ness or shoot-ness. This represents a better tlian t\vofold increase in leaf- ness over paired P3 prinlordia and a threefold increase in leaf-ness over ~~npaired P3 primordia.

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CAN.-\DI=\N JOURNAL OF BOTASY. VOL. 45, 1967

TABLE IV Fates of 12 P3 pri~nordia cultured in physiological conLact

with 12 P12 primordia for 10 weeks -

i\/Iorphogenetic response Number Leaf or shoot (Lj;;)

P3 primordia Leaf Shoot

Totals

Discussion Llany recent investigations have been concerned with factors which will

favor either the leaf expression or the shoot expression in developing leaf primordia nhich are not irreversibly determined as leaves (Miardlaw 1949c, 1955a,b; Cutter 1954, 1956; Wardlaw and Cutter 1954, 1956; Sussex 1955; Steeves 1 9 6 1 ~ ; Kuelinert and Steeves 1962). Frorn extensive investigations of leaf development in the fern Dryopteris aristata Druce, in I\-hich the technique of microsurgical isolation was utilized, a hypothesis emerged \vhich suggested that the ultimate and active site of control for leaf morphogenesis resided in the shoot apical rneristein and that factors controlling leaf development \\,ere mediated through the prevascular tissue of the shoot apex (\\Tardla\\- and Cutter 1956).

Results from tlie present investigation are not in agreement \vith this hypo- thesis for they clearly demonstrate that primordia determined as leaves will influence undetermined primordia, \vhich still retain a potentiality for shoot development, to develop as leaves rather than shoots. Tlie data strongly indicate that a primordium a t the 1'3 stage of development is not irreversibly determined as a leaf since excised, cultured P3 priinordia of Osmzrnda cinna- morlzea gro\vn separately or under the influence of other P3 primordia exhibited a tendency to develop as shoots a t a level twice that of P3 priinordia gronrn under tlie influence of older determined leaf primordia, \I-hich influence primordia to develop as leaves. Thus it appears that an alternative liypotliesis is \\ orlliy of consideration, i.e. that determination of the primordium as a leaf occurs progressively, and under the influence of as yet unidentified factors einanating from older leaves \\rliich niay influence younger priinordia to develop as leaves rather than shoots during that period of time in \\ihich a priinordiuin is capable of more than one rnorphogenetic expression. Subsequent development of the primordium as a leaf most likely depends upon factors produced within the developing primordium as suggested froin the work of 11. and R. Snow (1933, 1947, 1948), Ball (1948), Wardlaw (1949a,b), Steeves (1961b), and Kuehnert and Steeves (1962). 111 further support of this, Cutter and Wardlaw recently (1963) suggested that the leaf apex has an important function in controlIing developruent \vitliin the leaf and Cutter (1965) further suggested that the leaf apex, rather than the apical cell \vithin the primordial apex, is probably the vital controlling center to the extent that it may have a role in leaf morphogenesis equivalent to that \\-hich the shoot apical cell group exercises control \vithin the shoot apical meristenl of ferns.

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K C E H S l < R T : DEVELOPh.llCSTAL POTBNTI.\LITll<S OF LEA17 PKIMORDIA 2113

Acknowledgments The author expresses his sincere appreciation to 1 I iss Judith I-Ioffman and

;\ lrs. Catlly h/Ial~le for their technical assistance. Me also expresses his apprecia- tion to Dr. Herbert B. Tepper for reviexving the manuscript.

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