foliar ontogeny in podocarpus macrophyllus, with speical reference to transfusion tissue

Foliar Ontogeny in Podocarpus macrophyllus, with Speical Reference to Transfusion TissueAuthor(s): Mildred M. GriffithSource: American Journal of Botany, Vol. 44, No. 8 (Oct., 1957), pp. 705-715Published by: Botanical Society of AmericaStable URL: http://www.jstor.org/stable/2438637 .

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October, 1957] GRIFFITH-FOLIAR ONTOGENY IN PODOCARPUS 705

secretion is believed to be a phenomenon allied to the formation designated earlier by the writer as 44pectic warts." Anatomical differences occur between species; inflorescence bracts are alike throughout the genus. Wilkesia is differentiated from Argyroxiphium in its "normal" leaf structure

and in its nodal anatomy. Ecological, phylogenetic, and taxonomic implications of foliar anatomy are indicated.

THE CLAREMONT GRADUATE SCHOOL,

RANCHO SANTA ANA BOTANIC GARDEN,

CLAREMONT, CALIFORNIA

LITERATURE CITED

CARLQIJIST, S. 1955. Maui, Kauai, and five silverswords, Pacific Discovery 8 (3): 4-9.

-. 1956. On the occurrence of intercellular pectic warts in Compositae. Amer. Jour. Bot. 43: 425-429.

1957. The genus Fitchia (Compositae). Univ. California Publ. Bot. 29: 1-144.

DEGENER, 0. 1946. Flora Hawaiiensis (as a unit; parts published variously). Privately printed.

ESAU, KATHERINE. 1945. Vascularization in the vegetative shoots of Helianthus and Sambucus. Amer. Jour. Bot. 32: 18-29.

FOSTER, A. 1936. Leaf differentiation in angiosperms. Bot. Rev. 2: 349-372.

HABERLANDT, G. 1914. Physiological plant anatomy (trans. by Montagu Drummond). Macmillan & Co. London.

HILLEBRAND, W. 1888. Flora of the Hawaiian Islands. Privately published.

HOFFMANN, 0. 1890. Compositae. In ENGLER & PRANTL, Die natiirlichen Pflanzenfamilien 4 (5): 87-391.

JOHANSEN, D. 1940. Plant microtechnique. McGraw Hill. New York.

KECK, D. 1936. The Hawaiian silverswords. Occ. Pap. Bishop Mus. 11 (19): 1-38.

MANGIN, L. 1893. Recherches sur les composes pectiques. Jour. Bot. (Paris) 7: 37-41, 121-131., 325-343.

FOLIAR ONTOGENY IN PODOCARPUS MACROPHYLLUS, WITH SPECIAL REFERENCE TO TRANSFUSION TISSUE'

Mildred M. Griffith

THE EARLY ontogeny of leaves in gymnosperms has been described in three genera of the Taxo- diaceae (Cross, 1940, 1941, 1942), in Zamia (John- son, 1943) and in Pseudotsuga (Sterling, 1946). Foliar development in Dacrydium taxoides has been studied by Lee (1952), and Sacher (1955) described cataphyll ontogeny in Pinus lambertiana. Additional information on the ontogeny of leaves in this group of plants is necessary before broad comparisons within the group as well as with angiospermous plants can be made. Since the development of transfusion tissue and accessory transfusion tissue has received very limited attention, special emphasis was given to these tissues in this study.

Transfusion tissue generally is associated with the vascular bundle in the leaves of gymnosperms (Esau, 1953)., This tissue always includes transfusion tracheids, but in many genera, parenchyma also is present. Transfusion tissue was first described by Frank (1864) and received attention in the anatomical studies of leaves of gymnosperms in the late nineteenth and early twentieth centuries. These accounts were concerned with the arrangement of the tissue, its cellular components, and its phylogenetic and physiological significance. The literature is adequately reviewed by Worsdell

1Received for publication May 13, 1957. The author expresses appreciation to Professor Ralph H.

Wetmore for the use of the facilities at the Biological Laboratories, Harvard University, and to Professor Adri- ance S. Foster for reading the manuscript and offering helpful suggestions.

(1897) and Van Abbema (1934). Huber (1947) described the arrangement of cells in the transfusion tissue of Pinus, and Lederer (1955) ex- tended this type of study to include other genera.

Accessory transfusion tissue (fig. 1) occurs in the laminae of the leaves of a number of species of Podocarpus (Buchholz and Gray, 1948). This tissue consists of lignified elements and bordering parenchyma, both of which are elongated at right angles to the vein. Precise descriptions of this tissue are not available and no developmental studies have been reported.

MATERIALS AND METHODS. Shoot apices, young leaves and old leaves were obtained from shrubs of Podocarpus macrophyllus (Thunb.) D. Don., grow- ing on the campus of the University of Florida in Gainesville. Collections were made bimonthly during the winter months and weekly from March through July. Craf III was used as a killing and fixing agent. Materials were dehydrated and em- bedded in Tissuemat according to the schedule described by Ball (1941). Serial longisections of twenty-five apices were prepared at 10,u. Young leaves were sectioned at 10,u and older ones at 12-18,u. Tannic acid-iron chloride-safranin (Fos- ter, 1934) were used in staining the material. In addition, leaves were macerated in chromic and nitric acid for the study of isolated cells. Leaves, cleared in sodium hydroxide solution and stained with safranin, were useful in determining the dis- tribution of tissues.

ANATOMY OF MATURE LEAVES. Leaves of P.



706 AMERICAN JOURNAL OF BOTANY [Vol. 44

at tr x p

Lrc Fig. 1. Diagram of cross section of leaf of Podocarpus

macrophyllus. at, accessory transfusion tissue; P, phloem; rc, resin canal; tr, transfusion tissue; x, xylem.

macrophyllus are 5-7 cm. long, 0.5-0.8 cm. wide, and have a tapering base and a short petiole. They are arranged spirally on the stem. The general arrangement of tissues in the blade is shown diagram- matically in fig. 1. The epidermal cells are heavily cutinized, and the stomata are arranged in rows and limited to the abaxial surface. The sunken guard cells are overtopped by subsidiary cells. An interrupted layer of hypodermal fibers occurs on the adaxial side of the leaf. These fibers are present only at the margins and beneath the vein in the abaxial part of the leaf.

No endodermis can be recognized. The single collateral vascular bundle has the elements of the xylem and phloem arranged in radial rows, and in both of these tissues parenchymatous cells occur in continuous files. Three resin canals are located abaxial to the phloem. The lateral canals are larger than the median one.

Transfusion tissue occurs in a wing-like strand on either side of the vascular bundle. This tissue consists of tracheids and has its greatest development in the distal two-thirds of the leaf where in addition to the laterally situated transfusion tracheids, an occasional element is present adaxial to the xylem. In the middle of the leaf (fig. 2) transfusion tissue occurs only at the sides of the vein. The number of cells making up this tissue is pro- gressively smaller in the basal part of the blade and petiole. Some of these elements are in contact with the tracheids of the xylem. Thin-walled, elongated living cells occur at the sides of the phloem of the bundle. They have a marked radial alignment and appear to remain small and relatively undifferentiated in the mature leaves of P. macrophyllus. Strasburger (1891) described these in certain conifers as albuminous cells and termed them "transi- tional cells." Lederer (1955) included these cells as part of the transfusion tissue.

The transfusion tracheids vary in size and shape. Although many are approximately isodiametric

(fig. 2, 4), others are about as long as tracheids of the xylem. The longest transfusion tracheids are usually adjacent to the xylem with their long axes parallel to the vein. The majority of cross walls are transverse, and the cells are frequently arranged in longitudinal rows.

The structural pattern of the secondary wall (fig. 4, 5) of the transfusion tracheid is variable, and may be scalariform, scalariforin-reticulate or have pits with circular borders. Occasionally, a cell may have a combination of one of the first two types with bordered pits. There seems to be no precise relationship between the location of the transfusion tracheid in the strand of tissue and its wall type. In mature leaves of P. macrophyllus most of these cells appear to contain nuclei and highly vacuolate cytoplasm.

The accessory transfusion tissue, consisting of tracheids and parenchyma, is situated between the adaxial palisade and the abaxial spongy mesophyll, and extends from near the edge of the transfusion tracheids to within a few cells of the margin (fig. 2, 3) of the leaf. Most of the cells of the accessory transfusion tissue are conspicuously elongated at right angles to the vein, but there are all gradations between these and a few that are approximately isodiametric (fig. 13-16). Some of the elongated elements are 0.4 mm. long, and the isodiametric ones have an average diameter of 0.1 mm. Some have a distinctly wavy outline; others have forked or knob-like tips (fig. 14). The tracheids have lignified secondary walls and prom,inent bordered pits. The apertures of the pits vary from circular to oval, and the borders are oval or asymmetrical. Pits are not distributed uniformly over the wall, but occur in groups on the cross walls or at places along the side walls where two cells overlap. Large intercellular spaces occur between the cells of the accessory transfusion tissue, and in paradermal section, the arrangement suggests a network. In mature leaves the tracheids of this tissue appear to be devoid of contents. Parenchyma cells, elongated at right angles to the vein, occur largely as an adaxial and abaxial border to the accessory transfusion tracheids, and because of their arrangement are included as components of the accessory transfusion tissue. These living cells contain chloroplasts. Parenchyma cells, varying in shape from isodiametric to somewhat elongated, separate the tracheids of the transfusion tissue from those of the accessory transfusion tissue (fig. 2, 4).

The lignified elements of the accessory transfusion tissue have been termed tracheids (De Bary, 1884; Worsdell, 1897) and sclereids (Buchholz and Gray, 1948). Lee (1952) referred to similarly located cells in Dacrydium as osteosclereids. In

Fig. 2-5.-Fig. 2. Transverse section of midrib region. X 100.-Fig. 3. Transverse section of margin of lamina. X100.-Fig. 4. Transverse section of leaf near base of the blade, showing part of vein and transfusion tissue. X632. -Fig. 5. Longisection of blade at location of transfusion tissue. X 137. a, albuminous cells; at, accessory transfusion tissue; p, phloem; rc, resin canal; tr, transfusion tracheids; x, xylem.



October, 19571 GRIFFITH-FOLIAR ONTOGENY IN PODOCARPUS 707

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Fig. 6-8 -Fig. 6. Longisection of apical meristem. Note insertion of periclinal wall in surface layer. X 380.- Fig. 7. Longisection of foliar pri'mordium 80 i. long. X 632.-Fig. 8. Transverse section of marginal part of primor. diuim 297 mm lnong sahowing, elongatio-n of grrondir meristem eplls toward midldle. part of lamina. 6329-. ati, accessory

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October, 1957] GRIFFITH FOLIAR ONTOGENY IN PODOCARPUS 709

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tials; tr, transfusion tracheid; x, xylenm.

spite of the fact that descriptions of accessory trans- ftusion tissue and its cellular components are highly variable, the term, accessory transfusion tissue, has been used since it was first proposed by W'orsdell (1897). The literature describing accessory transfusion tissue in the Podocarpaceae has been reviewed by Orr (1944). The presence of this tissue in some species of I'odocarpus has been used by Bluchholz and Gray (1948) in a taxonomic revision of the genus.

SHOOT APEX AND EARLY FOLIAR ONTOGENY.-The apical meristem of P. macrophyllus (fig. 6) has the form of a rounded cone. Since new walls are frequently inserted in a perielinal plane in the surface layer, a tunica corpus organization is not present. The zonal pattern includes apical initials, central

mother cells, the leripheral zone and the rib meristem. The cytological features of these zones are most p)ronounced during the period of cataphyll and leaf production. In Gainesville, Florida, buds open through the period from March to November, but the major part of the new growth appears in March and April. Seven to eight cataphylls sur- round the embryonic leaves of the winter bud.

Foliar initiation involves radial elongation and predominately periclinal divisions in the two outer rows of the subsurface cells of the peripheral zone. Divisions in the surface layer are limited to the anticlinal plane. Continued divisions result in the production of a finger-like protuberance (fig. 7). Elongated procambial cells are well differentiated at this stage, and all observations indicated a con-




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tinuous and acropetal development of the procambium. Subapically, a group of particularly active cells divides both periclinally and anticlinally. Derivatives of these cells divide largely at right angles to the length of the primordium, and the resultant rows of cells constitute a conspicuous feature that is retained until the organ is approximately 750V, long (as measured along the adaxial surface). By the time the primordium is 1500,u long, the cells of the apical region are enlarged and highly vacuolate, and cell divisions are restricted to the basal two-thirds of the leaf. Thus along the length of such a primordium different stages of maturation are encountered.

Increase in thickness begins when the primordium is approximately 100 % long and is due to divisions and enlargement of cells throughout the organ. The development of the lamina is initiated when the primordium is about 150,u long by the meristematic activity of marginal and submarginal cells. Divisions in the marginal initials are limited to the antclinal plane, but those in the submarginal initials and their derivatives are variable.

FOLIAR DIFFERENTIATION AND MATURATION.-The primary meristematic tissues are differentiated early in the development of the leaf. The surface layer is distinct from the time of foliar initiation and constitutes a protoderm. Maturation of epidermal cells was not followed.

The first phloem element is present at the base of the primordium when it is approximately 300V, long and by the time the foliar organ is 400, long, sieve cells occur throughout the length of the procambial strand. At this stage a single tracheid is usually present in the basal part of the primordium.

Initials of the transfusion tracheids can be recognized when the primordium is approximately 50OV, long. These initials are arranged in a compact group on each side of the developing vascular strand. Their relatively dense, deeply-staining cytoplasm contrasts sharply with the surrounding cells of the lamina. The nuclei are large and numbers of mitotic figures are present in the tissue. In size, the cells have much smaller diameters than those of the adjacent ground meristem, but are larger than those of the procambium. These initials are recognizable earliest near the apex of the foliar primordium. Protophloem has been differentiated in this part of the developing leaf, but no mature xylem elements are present.

As the primordium elongates the contrast between the enlarged and vacuolated cells of the mesophyll and the deeply staining initials of the transfusion tissue becomes more pronounced. In the middle

part of a leaf 1.5 mm. long (fig. 9) these initials are a conspicuous feature. Cellular alignment indicates that some walls have been inserted in a longitudinal plane. However, paradermal sections of leaves 3 mm. long (fig. 11) and 10 mm. (fig. 12) show that new walls are inserted predominately in a transverse plane. The resulting growth pattern is that of a rib meristem. Cells in the immediate vicinity of the procambium are frequently elongate, but those most distant from the procambium are approximately isodiametric or enlarged at right angles to the length of the primordium.

The earliest mature transfusion tracheids occur in the terminal part of the primordium when it is approximately 2 mm. long (fig. 20). Differentia- tion proceeds basipetally, and by the time the foliar organ is 10 mm. long (fig. 21) numbers of transfusion tracheids are found throughout the distal half of the developing primordium. These early maturing cells have scalariform walls, and even after the development of the secondary walls the nuclei stain deeply. Usually the first mature transfusion tracheids (fig. 12) are separated by several cells from the tracheids of the xylem, but their exact position does not show any uniformity. In shape, the earliest maturing transfusion tracheids are isodiametric or elongated in a direction parallel to the length of the primordium (fig. 20, 21). In contrast, many of the later differentiated transfusion tracheids are elongated approximately at right angles to the vein (fig. 22).

In the terminal part of the leaf, the initials of the transfusion tissue appear to have matured by the time elongation is completed. However, in the middle and basal parts of the leaf undifferentiated cells occur among the lignified transfusion tracheids when the leaf is full length (fig. 22). Since parenchyma is not interspersed with the tracheids in the transfusion tissue of P. macrophyllus, it can be assumed that the maturation of a considerable number of the lignified cells occurs after elongation has ceased. Most of the immature cells are in the vicinity of the xylem.

Initials of the accessory transfusion tissue can be identified in the middle part of the primordium when it is about 2 mm. long. These initials (fig. 8) are located in the lamina about equidistant between the upper and lower protoderm, and are elongated at right angles to the vascular bundle. At this early stage, other cells of the ground meristem are small, and mitoses in the submarginal initials and their recent derivatives contribute to the extension of the blade. Tannins occur in many cells of the lamina, but are absent in the initials of the accessory trans-

Fig. 11-16.-Fig. 11. Longisection through midrib region of a primordium 3 mm. long, showing procambium and adjacent transfusion tissue initials.-Fig. 12. Longisection through midrib region of a primordium 10 mm. long. Note differentiated transfusion tracheids.-Fig. 13-16. Cells of accessory transfusion tissue isolated by maceration. Note points of contact and intercellular spaces between adjacent accessory transfusion tracheids in fig. 16. ati, accessory transfusion tissue initials; pc, procambiuim; ti, transfusion tissue initials; x, xylem. Drawn with the aid of a camera lucida.



712 AMERICAN JOURNAL OF BOTANY [o.4

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Fi. 722 ig 1.Trnvesescto trog mdlepatofflir rmodum7 m ln, hoig ntil of rasfsin isuean acesoy rasfsin isue Fg. 8.Trnsere ecio o mdde ar o flir rioriu 2.2cm lng Fg.19.Trnsere ecio ofmidl prtoffoiarprmodim .1cm log.Fi. 0.Ti o cleared foliar primordium 2.5 mm. long. Fig. 21. Tip of cleared primordium 9 mm. long. Fig. 22. Part of vein and~~~~~. .... . .... ............. ~; ;. 'R!R-M.i-i i0I.E adjacent transfusion tracheids in cleared primordium 4 cm. long. All X90.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-1, il 5 '




fusion tissue. The longer initials of this tissue are near the vein and those close to the margins are small and approximately isodiametric (fig. 8, 17, 18). All of the initials have vacuolate cytoplasm and a nucleus that stains deeply (fig. 8). The phases in the maturation of these initials include elongation, the formation of prominent intercellular spaces and the development of the lignified secondary wall.

Until a primordium is about 2 cm. long only elongation is involved in the differentiation of the initials of the accessory transfusion tissue. The early development appears to be relatively uniform, and the tissue retains its compactness. Although detailed studies of the wall and the protoplast were not made, at an early stage, the wall appears to be thin, the nuclei are oval in outline, and the cytoplasm is very vacuolate. Prominent intercellular spaces are first noticed when the primordium is 2-3 cm. long (fig. 18). They appear to result from the separation of lateral walls at restricted areas. With the increase in intercellular spaces, the cells become more irregular in shape. The lacunose structure of the tissue increases with growth in length. When the, leaf is approximately full length, the initials of the accessory transfusion tissue form a loose network. By the time the leaf is about 5 cm. long (fig. 19) the walls of some initials are slightly thickened. In a few instances, thin areas conform- ing to the arrangement and shape of pits in mature cells were observed. Complete maturation of the wall and the loss of cell contents in tracheids occur after the primordium has reached its full length. As stated earlier, parenchyma cells are located between the tracheids of the transfusion tissue and those of the accessory transfusion tissue. It was not possible in the present study to determine the origin of these cells.

DIsCUSSION.-Foliar initiation, early elongation of the primordium, axial thickening and marginal growth in Podocarpus macrophyllus agree with the descriptions of these phases of development in leaves of the Taxodiaceae (Cross, 1940, 1941, 1942). In all of the investigated conifers, apical growth is of short duration and meristematic activity at the base of the primordium accounts for the major part of foliar elongation.

Although transfusion tissue is of universal occurrence in the leaves of gymnosperms, the spatial arrangement and the kinds of cells included in it vary in the different genera (Bernard, 1904; Wors- dell, 1897). In contrast with the leaves of Pinus, where transfusion tissue surrounds the vein, and with leaves of certain species of Araucaria, where it is located largely adaxial to the xylem, most of the transfusion tissue of P. macrophyllus occurs in wing-like arrangements on either side of the vascular bundle. In the transfusion tissue of Pinus, tracheids and parenchyma are arranged in an inter- lacing pattern (Huber, 1947), but in P. macrophyllus the only cells that might be designated as

-transfusion parenchyma are located at either side of the phloem. Lederer (1955) has pointed out that "the classical accounts of transfusion tissue tend to ignore transfusion parenchyma cells and to empha- size only the anatomical peculiarities of transfusion tracheids." Lederer also suggests that there may have been a strong evolutionary tendency toward the "loosening up" of primitive compact tracheidal transfusion tissue by increasing amounts of parenchyma, like the progressive development of rays and wood parenchyma in xylem itself. In this con- nection it is perhaps noteworthy that transfusion tracheids in mature leaves of P. macrophyllus contain distinct nuclei and vacuolate cytoplasm. Van Abbema (1934) observed living contents in the transfusion tracheids of a number of gymnosperm leaves.

Most of the early descriptions of transfusion tissue (Bernard, 1904; Worsdell, 1897) were concerned with whether it should be considered modi- fied parenchyma of the mesophyll or part of the vascular system. Some adherents of the latter view (Bernard, 1904) believed it to be a remnant of ancestral centripetal xylem. Although the present study offers no evidence on the phylogeny of the tissue, its ontogeny affords certain comparisons. In P. macropihyllus, the development of transfusion tissue resembles vascular tissue in the early differentiation of its initials and in the early maturation of some elements. However, in the rib meristem type of growth and in the general basipetal direction in which maturation proceeds, it is comparable to the parenchyma of the mesophyll.

Accessory transfusion tissue is characteristic of a number of species of Podocarpus. The designa- tion of these lignified cells as sclereids (Buchholz and Gray, 1948) appears to have been made on the basis of their variation in shape and the inter- pretation of the walls as having simple pits. Al- though the shape of these cells in the leaf of P. macrophyllus is somewhat variable, the pits have prominent borders typical of elements of the xylem. De Bary's (1884) reference to them as tracheids appears to be justified in the species considered in the present study.

There is conisiderable variation in different genera in the relative proportion of parenchyma and tracheids in the accessory transfusion tissue. In a comparison of this tissue in Cycas and in Podo- carpus neriifolia, Lederer (1955) emphasizes the relatively large amount of parenchyma in both. However, according to this author, a distinct inter- lacing pattern between the tracheids and parenchyma occurs only in Cycas. In P. macrophyllus, transversely oriented, elongated parenchyma cells are frequently in contact with the tracheids of the accessory transfusion tissue. These living cells border the accessory transfusion tracheids and separate them from the palisade and spongy mesophyll. Lee (1952) described the elongated, thick-walled cells of the accessory transfusion tissue of Dacrydium




taxoides as forming a loose network with a relatively large proportion of parenchyma.

In view of the fact that there has been a tendency to link the cells of the transfusion tissue with those of the accessory transfusion tissue, a comparison of them in P. macrophyllus seems pertinent. On the basis of wall structure, the lignified cells of both tissues are classified as tracheids. The walls of the transfusion tracheids may be scalariform, reticulate or bordered-pitted, but only the latter occurs in accessory transfusion tracheids. The dis- tribution of pits, their shape and the appearance of the borders is in contrast in the cells of the two tissues. In the bordered-pitted transfusion tracheids, the pits have circular apertures and circular borders, and are distributed uniformly in the wall. In the accessory transfusion tracheids, the pits have apertures that vary from elongate to circular, and the borders are oval or asymmetrical. Furthermore, these pits occur in groups in the walls of the accessory transfusion tracheids. The tracheids of the transfusion tissue are compactly arranged, but conspicuous intercellular spaces occur among those of the accessory transfusion tissue. Transfusion tracheids retain their cell contents in the mature leaf, but the lignified cells of the accessory transfusion tissue are devoid of protoplasm. The contrast in the shape of the tracheids of the two tissues is marked. A relatively large proportion of those in the transfusion tissue are parenchymatous, but accessory transfusion tracheids are characteristically prosenchymatous.

The ontogeny of these two tissues affords some contrasts. Initials of the transfusion tracheids are discernible when the primordium is about 500 pt long, but the initials of the accessory transfusion tissue are not morphologically defined until the leaf is about 2 mm. in length. The first transfusion tracheids mature when the foliar organ is approximately 2 mm. long, and additional ones differen- tiate during elongation. None of the accessory transfusion tracheids are completely matured until after the leaf has reached its full length.

SUMMARY

The mature leaf of Podocarpus macrophyllus (Thunb.) D. Don., is approximately 6 cm. long and 0.6 cm. wide, and has a tapering base and a short petiole. The epidermal cells are heavily cutinized, and stomata occur in rows on the abaxial surface. Hypodermal fibers form an interrupted layer adaxially and occur in the midrib region on the abaxial side of the leaf. The mesophyll is differentiated into adaxial palisade and abaxial spongy parenchyma. Three resin canals are located abaxial to the phloem. The single vein is flanked on each side by wing-like arrangements of transfusion tracheids. These cells are parenchymatous in shape and have lignified secondary walls. Nuclei and cytoplasm are present. The accessory transfusion tissue occurs between the palisade aind

spongy mesophyll and extends from the vicinity of the transfusion tissue nearly to the margin. The tracheids of this tissue have lignified walls with bordered pits and are devoid of protoplasmic contents.. A tunica corpus organization is not present in the apical meristem. Foliar initiation involves periclinal divisions in the subsurface cells and anticlinal divisions in the surface cells. Apical and subapical initials contribute to early foliar elongation. Further growth in length is attributed to a basal meristem. Marginal and submarginal initials account for laminal extension. Procambium and protophloem are acropetal in development. Initials of the transfusion tissue consist of small, highly mitotic cells and are present on either side of the vascular strand when the primordium is 500,u long. The first transfusion tracheids occur at the distal part of the primordium when it is approximately 2 mm. long, and the course of further maturation is largely basipetal. Elongated initials of the accessory transfusion tracheids are recognized when the primordium is 2 mm. long. Their maturation involves enlargement, the development of lignified walls and the loss of cell contents, and is not completed until the leaf has attained its full length.

DEPARTMENT OF BOTANY, UNIVERSITY OF FLORIDA,

GAINESVILLE, FLORIDA

LITERATURE CITED

BALL, E. 1941. Microtechnique for the shoot apex. Amer. Jour. Bot. 28: 233-243.

BERNARD, C. 1904. Le bois centripete dans feuilles de conife'res. Beih. Bot. Centralbl. 17: 241-310.

BUCHHOLZ, J. T., AND NETTA E. GRAY. 1948. A taxonomic revision of Podocarpus. I. The sections of the genus and their divisions with reference to leaf anatomy. Jour. Arnold Arboretum 29: 49-63.

CROss, G. L. 1940. Development of the foliage leaves of Taxodium distichum. Amer. Jour. Bot. 27: 471-482. . 1941. Some histogenetic features of the shoot of Cryptomeria japonica. Amer. Jour. Bot. 28: 573-582.

-. 1942. Structure of the apical meristem and development of foliage leaves of Cunninghamia lanceolata. Amer. Jour. Bot. 29: 288-301.

DE BARY, A. 1884. Comparative anatomy of the vegetative organs of the phanerogams and ferns. Clarendon Press. Oxford.

ESAU, K. 1953. Plant anatomy. John Wiley and Sons. New York.

FOSTER, A. S. 1934. The use of tannic acid and iron chloride in staining cell walls in meristematic tissues. Stain Tech. 9: 91-92.

FRANK, A. B. 1864. Beitrage zur kenntnis der Gefass- biindel. Bot. Zeit. 22: 167-169.

HUBER, B. 1947. Zur Mikrotopographie der Saftstrome im Transfusionsgewebe der Koniferennadel. Planta 35: 331-351.

JOHNSON, M. A. 1943. Foliar development in Zamia. Amer. Jour. Bot. 30: 366-378.

LEDERER, B. 1955. Vergleichende Untersuchungen fiber Transfusionsgewebe einigen rezenter Gymnospermen. Vergleich-Anat. Untersuchungen aus den Forst- botanischen Institut Miinchen. 1-42.



October, 1957] MCCORQUODALE AND DUNCAN-PLANT GROWTH INHIBITIONS 715

LEE, C. L. 1952. The anatomy and ontogeny of the leaf of Dacrydium taxoides. Amer. Jour. Bot. 39: 393- 398.

ORR, M. Y. 1944. The leaf anatomy of Podocarpus. Trans. Proc. Bot. Soc. Edinburgh 34: 1-54.

SACHER, J. A. 1955. Cataphyll ontogeny in Pinus lambertiana. Amer. Jour. Bot. 42: 82-91.

STERLING, C. 1946. Organization of the shoot of Pseudot- suga taxifolia (Lamb.) Britt. I. Structure of the shoot apex. Amer. Jour. Bot. 33: 742-750.

STRASBURGER, E. 1891. fiber den Bau und die Verricht- ungen der Leitungsbahnen in den Pflanzen. Histo- logische Beitrage. Band 3.

VAN ABBEMA, T. 1934. Das Transfusionsgewebe in den Blattern der Cyadinae, Ginkgoinae und Coniferae. Rec. Trav. Bot. Ceerland. 31: 309-390.

WORSDELL, W. C. 1897. On "transfusion tissue": its origin and function in the leaves of gymnospermous plants. Linn. Soc. London Trans., Bot. Ser. II. 5: 301-319.

PLANT GROWTH INHIBITIONS BY CERTAIN IMIDAZOLE COMPOUNDS AND THEIR PREVENTIONS WITH METAL IONS

D. J. McCorquodale and R. E. Duncan'

A USEFUL APPROACH to the elucidation of the biochemical mechanisms controlling cell division and cell enlargement has been the use of chemicals which modify the mitotic cycle and/or the course of cell enlargement. For most favourable results, the chemicals chosen for such studies should have easily identifiable biochemical activities. Com- pounds believed to act as antimetabolites (e.g., structural analogs) should be especially suitable. Certain of the imidazole compounds have been reported to possess such antimetabolic properties; benzimidazole (BZI) is a reported antagonist of adenine (Woolley, 1944) and imidazole has been observed to act as an anti-histamine (Morris and Dragstedt, 1945). A study of the inhibitory properties of a number of imidazole compounds was therefore initiated with the hope that some of the biochemical processes underlying cell division and elongation would be revealed if the precise manner in which the compounds were interfering with cell division and elongation could be determined.

MATERIALS AND METHODS.-The primary roots of seedlings obtained by germinating seeds of the broad bean, Vicia faba, were used as experimental material. The seeds (batches 9C-9F) were obtained from the L. L. Olds Seed Co., Madison, Wis. The method of seed germination and conditions for growth of the roots have been previously described (Setterfield and Duncan, 1955). Essentially, the seeds were soaked in water, stripped of their seed coats, and germinated in well-watered vermiculite. After 4-5 days the seedlings were removed and selected as to healthy appearance, thickness, and length of roots (8-12 cm.). Treatments were car- ried out by immersing the roots of the seedlings in aqueous solutions of the chemicals under study in 800-ml. beakers. Eight seedlings were supported in

'Received for publication March 4, 1957. Contribution from the Program in Cytology, Department

of Botany, University of Wisconsin, Madison, supported in part by grants to the late Dr. C. Leonard Huskins from the American Cancer Society, Inc., The Rockefeller Founda- tion, and the Research Commnittee of the Graduate School with funds supplied by the Wisconsin Alumni Research Foundation.

each beaker by passing their roots through holes in plastic lids resting on top of the beakers. Treat- ment solutions were aerated during all experiments with C02-free air. At the beginning of all treatments the epicotyls were removed from all seedlings.

Two quantitative measurements served as an assay for the inhibitory properties of the compounds studied-rate of root elongation and frequency of mitotic stages in the root meristem.

Root-length increase was determined by measur- ing, before and after each time period, the distance between the root apex and an India-ink mark placed arbitrarily beyond the region of elongation (usually 6-7 cm. from the root tip) at the beginning of each experiment. Figures denoting length increase at 24 hr. (or 12 hr. in histamine-treated roots) are arithmetic means of values from 8 roots while figures at 48 hr. (or 24 hr. in histamine-treated roots) are the means of 4 values.

Mitotic frequency was determined by the cell- counting method described by Setterfield et al. (1954). Free-cell suspensions, obtained by macer- ating pectinase-treated meristems previously fixed in 3:1 alcohol-acetic acid and stained after the Feulgen technique, were concentrated by sedimenta- tion and resuspended in a buffered "Karo" syrup solution. One drop of this suspension was then placed on a microscope slide and the cells scored as to mitotic stage with the aid of a microscope (by taking high power transects). Usually, the first 2 mm. from each root used for length increase determinations was macerated individually and a random sample of 1000 cells tallied. All mitotic frequency values reported are arithmetic means of four separate determinations.

Since, in these experiments, there was no signifi- cant shift in the relative proportions of the actual division stages (i.e., prophase, metaphase, ana- phase, and telophase), the (lata are reported as percent nuclei (or cells) in mitosis or "mitotic frequency."

The sources of the chemicals used in this investi- gation were as follows: BZI and imidazole from



foliar ontogeny in podocarpus macrophyllus, with speical reference to transfusion tissue

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