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Premise of the study New growth in the spring requires resource mobilization in the vascular system at a time when xylem and phloem function are often reduced in seasonally cold climates. As a result, the timing of leaf out and/or flowering could depend on when the vascular system resumes normal function in the spring. This study investigated whether flowering time is influenced by vascular phenology in plants that flower precociously before they have leaves. Methods Flower, leaf, and vascular phenology were monitored in pairs of precocious and non-precocious congeners. Differences in resource allocation were quantified by measuring bud dry mass and water content throughout the year, floral hydration was modelled, and a girdling treatment completed on branches in the field. Key results Precocious flowering species invested more in floral buds the year before flowering than did their non-precocious congeners, thus mobilizing less water in the spring, which allowed flowering before new vessel maturation. Conclusions A shift in the timing of resource allocation in precocious flowering plants allowed them to flower before the production of mature vessels and minimized the significance of seasonal changes in vascular function to their flowering phenology. The low investment required to complete floral development in the spring when the plant vascular system is often compromised could explain why flowers can emerge before leaf out.

Premise of the study Dimensions and spatial distribution of vessels are critically important features of woody stems, allowing for adaptation to different environments through their effects on hydraulic efficiency and vulnerability to embolism. Although our understanding of vessel development is poor, basipetal transport of auxin through the cambial zone may play an important role. Methods Stems of Populus tremula ×alba were treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) in a longitudinal strip along the length of the lower stem. Vessel lumen diameter, circularity, and length; xylem growth; tension wood area; and hydraulic conductivity before and after a high pressure flush were determined on both NPA-treated and control plants. Key results NPA-treated stems formed aberrant vessels that were short, small in diameter, highly clustered, and angular in cross section, whereas xylem formed on the untreated side of the stem contained typical vessels that were similar to those of controls. NPA-treated stems had reduced specific conductivity relative to controls, but this difference was eliminated by the high-pressure flush. The control treatment (lanolin + dimethyl sulfoxide) reduced xylem growth and increased tension wood formation, but never produced the aberrant vessel patterning seen in NPA-treated stems. Conclusions These results are consistent with a model of vessel development in which basipetal polar auxin transport through the xylem-side cambial derivatives is required for proper expansion and patterning of vessels and demonstrate that reduced auxin transport can produce stems with altered stem hydraulic properties.

UNLABELLED • PREMISE OF THE STUDY The presence of gelatinous (tension) fibers (GFs) in the roots of two extant cycadales (Cycas and Zamia) in a recent publication raises interesting issues of GF distribution in seed plants. An immediate question that arises from this discovery is whether GFs occur consistently in the radicle of all extant cycad genera and therefore might have a similar role in root contraction. We present results of a survey of nursery-grown material of all 10 genera.• METHODS We sequentially sectioned seedling root material and used simple staining and histochemical methods to follow anatomical changes along the radicle of all 10 genera.• KEY RESULTS We found GFs in nine genera; Stangeria appears to be the only genus without them. In all genera, there is a wide variation in the number of GFs and also variation in the development of thickened, fleshy roots. "Tertiary expansion" is a useful term to describe late cell division and enlargement of both primary and secondary parenchyma, the latter produced by the vascular cambium. Certain other histological features can be diagnostically useful at the generic level.• CONCLUSIONS The functional interpretation of GFs as being wholly responsible for apparent tissue contraction is now somewhat compromised, especially as distortion of tracheary elements by changes in dimensions of parenchyma cells can falsely suggest root contraction when it may not occur. These preliminary results point the way to a more precise investigation of study material grown in more uniform environments using advanced technological methods.

Premise of the study Secondary xylem (wood) produced by a vascular cambium supports increased plant size and underpins the most successful model of arborescence among tracheophytes. Woody plants established the extensive forest ecosystems that dramatically changed the Earth's biosphere. Secondary growth evolved in several lineages in the Devonian, but only two occurrences have been reported previously from the Early Devonian. The evolutionary history and phylogeny of wood production are poorly understood, and Early Devonian plants are key to illuminating them. Methods A fossil plant preserved anatomically by cellular permineralization in the Lower Devonian (Emsian, ca. 400-395 million years old) Battery Point Formation of Gaspe Bay (Quebec, Canada) is described using the cellulose acetate peel technique. Key results The plant, Franhueberia gerriennei Hoffman et Tomescu gen. et sp. nov., is a basal euphyllophyte with a centrarch protostele and metaxylem tracheids with circular and oval to scalariform bordered multiaperturate pits (P-type tracheids). The outer layers of xylem, consisting of larger-diameter P-type tracheids, exhibit the features diagnostic of secondary xylem: radial files of tracheids, multiplicative divisions, and a combination of axial and radial components. Conclusions Franhueberia is one of the three oldest euphyllophytes exhibiting secondary growth documented in the Early Devonian. Within the euphyllophyte clade, these plants represent basal lineages that predate the evolution of stem-leaf-root organography and indicate that underlying mechanisms for secondary growth became part of the euphyllophyte developmental toolkit very early in the clade's evolution.

Premise of the study Primary vascular tissues of angiosperm and gymnosperm roots have significant anatomical differences. In gymnosperms, lack of protophloem sieve elements indicates a lengthy parenchymatous pathway for nutrient transport to the root apical meristem (RAM). Because F-actin is an essential component of transport in parenchyma cells, the distribution of F-actin was determined and compared among roots of several angiosperm and gymnosperm species. Methods Roots were chemically fixed and sectioned by hand to enable rapid production of many sections for labeling F-actin with phalloidin. Key results In angiosperm and gymnosperm root tips, relative intensity of F-actin labeling was highest in primary vascular tissues. Parenchyma cells in and around protophloem tended to have more F-actin while cells in cortical and protoxylem tissues tended to have less. In gymnosperms, phloem parenchyma was intensely labeled for several millimeters distal to the root apical meristem (RAM), and the F-actin is mostly composed of bundles that lie parallel to the root longitudinal axis. This orientation differed from the multidirectional arrangement of F-actin filaments in cortical cells. In angiosperms, intense F-actin labeling of pericycle and phloem parenchyma cells occurred around the first mature sieve elements. Conclusions F-actin is concentrated in the vascular cylinder, commonly in primary phloem parenchyma. In gymnosperms, the absence of sieve elements suggests that cytoplasmic streaming has a role in some aspect of phloem transport or unloading. In angiosperms, the region of intense F-actin labeling in the phloem parenchyma is limited to the extreme terminal portion of primary phloem where unloading of the earliest mature sieve elements occurs.

The Middle Triassic flora of the Fremouw Formation in the central Transantarctic Mountains consists of conifers, cycads, ferns, pteridosperms, and sphenophytes. Stems with an unusual anatomy have been discovered within silicified peat from the same locality. The diameters of the stems range from 1.4 to 1.7 cm; the longest specimen is approximately 12 cm. In transverse section the vascular system consists of segments that occur as single traces or are connected in the center and anastomose at varying levels within the stem. Each segment contains a bifacial vascular cambium. Secondary tissues of each segment surround a central area of parenchyma and small tracheids presumed to represent primary xylem. Surrounding the stem is a periderm. Traces are produced near the periphery of the axis and consist of radially arranged secondary xylem and a thick periderm. The absence of leaves and reproductive organs leads to uncertain phylogenetic relationships. We are unaware of any Triassic plants with this type of vascular tissue organization, and those plants with a similar type of arrangement occur only in the Devonian and Carboniferous. Possible phylogenetic affinities with the Cladoxylales and Lycophyta are examined, but the anatomical differences, along with stratigraphic age, preclude formal assignment to any known taxon at this time. Therefore, we have assigned it to a new taxon: Hapsidoxylon terpsichorum gen. et sp. nov.

We report the longitudinal, biomechanical, and anatomical trends observed for tissue samples drawn from the parallel aligned, prismatic woody vascular bundles running the length of a Pachycereus pringlei plant measuring 5.22 m in height. The main vertical stem of this plant was cut into five segments (labeled A through E in the acropetal direction) measuring ;1.02 m in length. Four of the 14 vascular bundles in each segment were surgically removed to obtain 20 vascular bundle segments that were tested in bending to determine their stiffness measured in the radial ER and tangential ET direction. We also determined the lignin content of representative samples of wood. A nonlinear trend in stiffness was observed: ER and ET were highest in segments B or C (1.67 GN/m 2 and 1.09 GN/m2, respectively), lower in segment A (ER 5 1.18 GN/m 2 and E T 5 0.35 GN/m 2), and lowest in segment E (E R 5 0.03 GN/m 2 and ET 5 0.20 GN/m2). Similar longitudinal trends were seen for axial tissue volume fraction and fiber wall thickness, which achieved their highest values in segment B (69.8% and 6.59 mm, respectively). Wood stiffness also correlated significantly with cell wall lignin content: with respect to segment B (which had the highest lignin content, and was thus used as the standard reference for percent lignin content), lignin content, was 15, 60, 85, and 43% in segments E, D, C, and A, respectively. Fiber cell length, which increased toward the base of the stem and toward the vascular cambium in the most proximal vascular bundle segment, did not correlate with ER or ET. Basic engineering principles were used to calculate stem stresses resulting from self-loading and any wind-induced bending moment (produced by drag forces). Calculations indicated that the less stiff wood produced in segment A eliminates a rapid and potentially dangerous increase in stresses that would otherwise occur in segments B or C. The less stiff wood in segment A also reduces the probability of shear failure at the cellular interface between the wood and surrounding tissues in this portion of the stem. We conclude that P. pringlei wood stiffness is dependent on the volume fraction and lignification of axial tissues, less so on fiber wall thickness, and that wood development in this species is adaptively responsive to self-loading and differentially applied external mechanical forces

The reorganization of vascular cylinders of pea (Pisum sativum, cv. Alaska) primary roots following the formation of vascular cavities was examined by light and electron microscopy. Cavities usually began forming ;20 mm from the root tip and were continuous to ;90 mm from the tips in roots 150 mm long, where they began filling with specialized parenchyma cells (SP cells). SP cells were usually produced by enlargement of parenchymous cells of the primary xylem at cavity margins. Depending on the extent and shape of the cavity, they were also sometimes produced by primary phloem parenchyma and early derivatives of the vascular cambium. Enlargement and some divisions of SP cells continued until a cavity was completely filled by them. SP cells proceeded through a series of cytoplasmic changes as they developed. First the cytoplasmic layer became thicker and more electron dense than surrounding cells. As SP cells enlarged there was an increase in vesicular traffic and the cytoplasm became less electron dense. Ultimately the cytoplasm thinned further, organelles degenerated, and the tonoplast sometimes broke down. SP cells did not form secondary walls. X-ray microanalysis revealed that SP cells accumulated potassium and rubidium to the same degree as cortical and xylem parenchyma cells and to a greater degree than immature secondary and late-maturing tracheary elements.

The developmental anatomy of root buds and root sprouts was examined in the clonal tree Sassafras albidum. Root samples from 13 clones that varied widely in age and vigor were sectioned and two types of buds were found, "additional" buds and "reparative" buds. Additional buds form during the early growth of uninjured roots and they perennate by growing outwards in concert with the vascular cambium such that bud traces are produced in the secondary xylem. Reparative buds form de novo in response to senescence, injuries, or other types of disturbance. Reparative buds were found on the roots of seven of the clones, whereas additional buds were found on the roots of all 13 clones. The reparative buds had originated in the proliferated pericycle, where they were subtended by sphaeroblasts, or spherical nodules of wood. Few of the reparative buds were vascularized and none were connected with the vasculature of their parent roots. In contrast, most of the additional buds were vascularized, and the leaf traces of several of the additional buds appeared to be contiguous with the conducting xylem of their parent roots. To determine whether both bud types were functional, 82 field-collected root sprouts and 44 incubation-induced sprouts were sectioned at the root-sprout junction and examined for evidence relating to their mode of origin. None of the sprouts were subtended by sphaeroblasts, but 98% were subtended by bud traces, which indicated that they had originated from additional buds. Although reparative buds were more common than additional buds on some of the root samples, they appear to be dysfunctional at sprouting. Additional buds, on the other hand, are able to sprout both as a normal part of clonal spread and from root cuttings.

Developmental and physiological studies of roots are frequently limited to a post-germination stage. In Arabidopsis, a developmental change in the root meristem architecture during plant ontogenesis has not previously been studied and is addressed presently. Arabidopsis thaliana have closed root apical organization, in which all cell file lineages connect directly to one of three distinct initial tiers. The root meristem organization is dynamic and changes as the root ages from 1 to 4 wk post-germination. During the ontogeny of the root, the number of cells within the root apical meristem (RAM) increases and then decreases due to changes in the number of cortical layers and number of cell files within a central cylinder. The architecture of the initial tiers also changes as the root meristem ages. Included in the RAM's ontogeny is a pattern associated with the periclinal divisions that give rise to the middle cortex and endodermis; the three-dimensional arrangement of periclinally dividing derivative cells resembles one gyre of a helix. Four- or 5-wk-old roots exhibit a disorganized array of vacuolated initial cells that are a manifestation of the determinate nature of the meristem. Vascular cambium is formed via coordinated divisions of vascular parenchyma and pericycle cells. The phellogen is the last meristem to complete its development, and it is derived from pericycle cells that delineate the outer boundary of the root.

Nucleolar involvement in the regulation of the activity-rest-quiescence cycle of the vascular cambium was assessed by determining the seasonal variation in number, diameter, and volume of nucleoli in fusiform cells of Abies balsamea (L.) Mill. The cells were isolated from 1- and 19-yr-old cambia and stained with either silver nitrate or Feulgen + naphthol yellow-S. The ability of fusiform cells to incorporate [5-3H]-uridine into nuclei and nucleoli was also determined. In the 1-yr-old cambium, the activity of the nucleoli, as evidenced by their diameter, total volume per cell, and intensity of staining with silver nitrate, exhibited two maxima during the year-a large one during cambial reactivation in April-May and a small one during the rest-quiescence transition in October. Incorporation of radiolabeled uridine at 20 C was low at the end of the active period and increased during the rest-quiescence transition, suggesting that the quiescent, but not the resting, cambium can rapidly resume nucleolar activity when the temperature is permissive. The number of nucleoli per cell varied between two and eight, and was higher during the dormant than the active period. The increase in number took place during the autumnal activity-rest-quiescence transition, when cambial cells were arrested in the G, phase of the cell cycle. Similar seasonal changes in nucleolar morphology were observed in the 19-yr-old cambium. Nucleolar diameter and total nucleolar volume were larger in the 19-yr-old cambium than in the 1-yr-old cambium, whereas nucleolar number was lower. The results suggest that repression of rRNA genes underlies the development of rest when the cambium will not produce new cells.

Meristematic activity in Stigmaria ficoides is interpreted from a combination of historical information and newly developed data on stelar diameter, wood production, and morphology of the apex. Many specimens of Stigmaria preserved as mold/casts display little or no recognizable change in the width of the stelar groove between successive forks. Thickness of the xylem cylinder in permineralized axes of similar diameters also is relatively uniform. Over a distance of 1.5 m along one axis the mean number of tracheids per radial row diminishes from 56 to 46 toward the apex. While this is a statistically significant decrease, in biological terms it is consistent with the reduction in primary growth that one would expect in a zone of apoxogenesis. Coupled with our knowledge of the morphology of the apex, these data allow us to interpret radially aligned tracheids in the steles of most stigmarian axes as having been produced largely from the action of a primary thickening meristem, rather than from a secondary vascular cambium as commonly believed.

Observations of the vascular tissue of Cycas shoots have provided supporting evidence that the first vascular cambium as well as subsequent successive cambia are simultaneously active. The establishment of the second cambium occurs during the seedling stage, and differentiates mainly within the cortical cells. However, cambial activity also occurs within phloem parenchyma cells of the first vascular cylinder. Tracheids in the first and the successive vascular cylinders are generally of the same length; however, there is a trend toward increasing length within the successive cylinders, possibly because the successive cambia are long-lived.

Xylem and phloem tissue samples were collected from various-aged Great Basin bristlecone pine (Pinus longaeva D. K. Bailey) stems in southern Utah and southeastern California to determine whether the vascular cambia of older trees produce fewer xylem rays, shorter-lived xylem and phloem ray cells, fewer phloem sieve cells, and a thinner phloem. Increment cores were examined to determine whether 'aged' cambia produced narrower tracheids that might reduce water translocation. Sapwood thickness was measured and sapwood growth layers were counted on these cores. Regression and Classification and Regression Tree (CART) analyses of sample data found no age-related changes in cambial products. Phloem and xylem production appeared normal at all ages, with no evidence of cambial malfunction. AS A TREE ages, its vascular cambium continues to form xylem and phloem elements. Xylem cells formed by young and mature cambia have been compared anatomically (Dinwoodie

The branched anastomosed laticifer system in the primary body of Cichorium intybus L. originates in embryos from files of laticiferous members at the boundary between phloic procambium and ground meristem. Upon seed germination, laticiferous members develop perforations in the end walls which become entirely resorbed. Perforations also develop in the longitudinal walls of contiguous laticiferous members and from lateral connections between developing laticifer branches. Additional laticiferous members originate as procambium differentiation proceeds, and their differentiation follows a continuous acropetal sequence in leaf primordia of the plumule. In roots, laticifers closely associated with sieve tubes in the secondary phloem originate from derivatives of fusiform initials in the vascular cambium. These laticifers develop wall perforations and in a mature condition resemble laticifers in the primary body. As the girth of the root increases, laticifers toward the periphery, unlike associated sieve tubes, resist crushing and obliteration. Laticifers vary in width from about 4 to 22 ,um; the widest ones occur in involucral bracts and the narrowest ones in florets. There was no evidence that intrusive growth occurs during development of the laticifer system, although such growth may occur during development of occasional branches which extend through ground tissue independent of phloem and terminate in contact with the epidermis. Presence of amorphous callose deposits is related to aging of laticifers and mechanical injury.

In the mitotically active vascular cambium of conifers and broadleaved trees there is tension across the radial middle lamellae. This tension seems to be important for intrusive growth of fusiform cells. In the vascular cambium of Fraxinus and Robinia there is tension across the tangential middle lamellae also, which makes possible the increase in diameter of differentiating vessel members.

The primary xylem connection between the diarch parent root and the diarch lateral root was derived from the pericycle and stelar parenchyma. Early in lateral root development stelar parenchyma that was positioned between the parent xylem and the primordium divided transversely. These transverse divisions produced a plate of cells, most of which subsequently differentiated into vessel element connectors. After emergence of the lateral root, xylem maturation began in the stelar vessel element connectors and maturation proceeded acropetally into the lateral root. Protoxylem of the lateral root was connected to the metaxylem of the parent root via stelar vessel element connectors. The circular phloem connection was pericyclic in origin. Axial phloem connections which vascularized the lateral root were established with sieve tube elements of both parent phloem poles. Maturation of the phloem connection occurred prior to lateral root emergence. Transaxial phloem, positioned in arches above and below the lateral root vascular cylinder, was derived from the pericycle; and each arch consisted of three to four sieve tube elements. No transfer cells were found in the transaxial phloem. BOTH THE DEVELOPMENT and the structure of a root vascular connection between the primary and secondary root were first described in detail for soybean (Byrne, Pesacreta and Fox, 1977a). We reported three major developmental and structural features of the soybean vascular connection. First, the primary xylem connection was derived from stelar parenchyma rather than the pericycle (Esau, 1965). Second, primary xylem maturation began within the parent root and proceeded acropetally into the lateral root. Third, the phloem connection was circular and consisted of two parts developmentally: the axial connection which was derived from the pericycle, and the transaxial connection which was derived from stelar parenchyma. The major purpose of this investigation was to compare the vascular connection of the diarch tomato root with that of the tetrarch soybean root. From previous experience and the literature (Hayward, 1938; Byrne et al., 1975; Aung, Bryan and Byrne, 1975), we knew that lateral roots of tomato originate between a xylem and phloem pole. Also, unlike soybean, outer stelar parenchyma is absent be1 Received for publication 29 October 1980; revision accepted 3 April 1981. Part of this paper represents research done for a B.S. Honors degree in Biology. 3 Present address: 21567 South Park Dr., Fairview Park, OH 44126. tween the protoxylem and pericycle in the tomato root. Although there are at least three modern reports concerning the development of lateral roots from diarch parent roots, various aspects of lateral root vascularization have not been adequately studied (Esau, 1965, Byrne et al., 1977a). In addition, if transaxial phloem existed in the tomato root, we hoped transmission electron microscopy (TEM) would shed some light on the proposed functional relationship (Byrne et al., 1977a) between the transaxial phloem and the vascular cambium. MATERIALS AND METHODs-Seedlings of Lycopersicon esculentum Mill. cv Fireball were grown in Seed-Pack Growth Pouches (Scientific Products, Evanston, Illinois; Haas and Carothers, 1975). The seedlings were maintained on 0.5% Hoagland's Solution in the greenhouse. Histological methods reported previously (Byrne et al., 1977a, b) were used to investigate the root material taken from 10to 14-day-old seedlings. Material for TEM was fixed in cold, buffered (pH 7.2), 3% glutaraldehyde. Specimens were trimmed to size while submerged in the fixative. Specimens were post-fixed in 2% OSO4 for 1 hr, dehydrated in a graded series of ethanol and block stained with 0.5% uranyl acetate. The tissue was embedded in Spurr's Low Viscosity Resin and sectioned on a Sorvall M1 ultramicrotome. Sections were post-stained

Mature needles and elongating current year's needles of Pinus strobus growing in Massachusetts and P. brutia growing in Israel were collected monthly or bimonthly for seasonal analysis of leaf cambial activity. Mature needles produced secondary phloem but no xylem, and, regardless of the season, had a cambial zone from 2 to 3 cell layers wide. In the current year's needles maturation was basipetal and the procambium differentiated into primary xylem, primary phloem, and the phloem-producing vascular cambium before needle maturity. Oneand 2-year-old needles of Pinus strobus produced slightly over 4 cell layers of phloem between April 15 and September 1 of 1983, with a peak production rate of about 2 cell layers per month in May and early June. One-year-old needles of P. brutia produced about 6 phloem cell layers in 1983, with phloem being produced throughout the year except in midsummer. This was contrasted by fall and winter dormancy in needles of P. strobus. THERE HAVE BEEN many seasonal time-course studies on cambial activity in stems of plants (Esau, 1965; Philipson, Ward and Butterfield, 1971; Fahn, 1982). These include studies of xylem and phloem production in Pinus strobus (Wilson, 1966; Alfieri and Evert, 1968). Most studies involve temperate plants, but plants from Mediterranean (e.g., Fahn, 1962; Liphschitz, Lev-Yadun and Waisel, 1981; Liphschitz et al., 1984; Liphschitz, Lev-Yadun and Waisel, 1985; Liphschitz and Lev-Yadun, 1986) and tropical regions (e.g., Paliwal and Prasad, 1970; Amboni, 1974; Dave and Rao, 1982; Fahn, 1982) have also been examined in this way. Cambial activity in evergreen leaves has received little attention, mostly because it has not been widely appreciated that evergreen leaves commonly undergo secondary growth (Ewers, 1982a, b). Our previous approach to studying secondary growth in leaves was to compare leaves of different known ages (Ewers, 1 982a, b). For example, we examined 1 to 33year-old needles of Pinus longaeva and found that the needle vascular cambium remained active throughout the life of the needle. In each of twelve taxa of conifers that we have examined, including P. strobus and P. brutia, the I Received for publication 17 July 1986; revision accepted 11 November 1986. This work was initiated while Ewers was a Cabot PostDoctoral Fellow and Aloni was Bullard Fellow at Harvard Forest in Petersham, Massachusetts. We thank Ms. Monica Mattmuller for technical assistance and Drs. N. Liphschitz, R. Schmid, and P. Baas for reviewing the manuscript. mature needles produce secondary phloem each year but no secondary xylem (Ewers, 1982a, b; Ewers and Aloni, 1984, 1985). The secondary phloem arises from a unidirectional phloem-producing needle vascular cambium (Ewers, 1982a). In the present study we examined the current year's needles, 1-year-old, and 2-year-old needles of Pinus strobus and P. brutia many times throughout the year for a more detailed seasonal analysis of vascular differentiation. The objectives were to determine the time of onset of secondary phloem production and the time of peak phloem production in developing as well as mature needles. We also examined the width of the needle vascular cambium throughout the year. The two species were chosen to represent two very different climatic regions. P. strobus is from temperate Massachusetts and P. brutia is from Mediterranean Israel. Taxonomically, these two species are from different subgenera. P. brutia is a diploxylon pine whereas P. strobus is a haploxylon species (Mirov, 1967). Coniferous leaves grow by means of basal meristem; the tip matures while the needle base is still elongating (Doak, 1935; Mirov, 1967; Ewers, 1982a, b). To follow vascular development in elongating current year needles, we compared over time the vascular tissue at specific distances from the needle tip (Fig. 6). We define secondary growth as growth (i.e., cell production) after elongation of the plant part has been completed. This is consistent with the classical definitions strongly advocated by Esau (1943, 1965). Due to the basipetal maturation

Experimental results show that predominant cell arrest in G2 and the presence of endoreduplicated cells are coincident with presence of secondary vascular tissues while preponderant cell arrest in GI and absence of polyploid cells are coincident with an absence of secondary vascular tissues in mature root tissues of intact and cultured roots of Raphanus sativus L. In mature tissues of intact seedling roots, most cells arrest in G2, and both polyploid cells and secondary vascular tissues are present. If excised roots are grown on simple medium, most mature cells arrest in G 1, none undergo endoreduplication, and only primary vascular tissues are present. When bases of these excised roots are later placed in a medium with auxin, cytokinin, and myo-inositol that produces secondary vascular tissues in vitro, preponderant cell arrest occurred in G2 with some polyploid cells. The general relationship of predominant period of cell arrest, presence of polyploid cells, and presence of secondary vascular tissues in mature roots among plants of various taxa is surveyed. ROOT MORPHOGENESIS and maturation at the root meristem have presented researchers with a proliferative cell population in which cells differentiate to produce widely divergent cell types both structurally and functionally. Patterns of cell differentiation are observed in transverse tissue sections at various distances from the proliferative cell population (Williams, 1947; Heimsch, 1951; Popham, 1955; Jensen and Kavaljian, 1958). Many cell types in roots retain a potential to proliferate some distance from the meristem and to produce new tissues. For example, pericycle cells proliferate to produce secondary roots, vascular cambium cells proliferate to produce secondary vascular tissues; adventitious shoots may develop from root tissues. These examples illustrate the potential of specific root cell types to proliferate in response to intracellular chemical and/or physical influences. Specific targets for initiation of cell cycle activity remain obscure, but evidence is available to suggest that cycle controls for continued proliferation are located in GI and G2 [See Prescott (1976) for review]. Loomis and Torrey (1964) demonstrated that particular chemicals in appropriate concentrations could stimulate cell proliferation in the vas1 Received for publication 1 April 1977; revision accepted 10 June 1977. Research supported in part by Grant #1-ROl-GM23232-01 from the National Institutes of General Medical Sciences. The authors gratefully acknowledge the use of a computerized microspectrophotometer at Brookhaven National Laboratory by Dr. J. Van't Hof, and the excellent technical assistance of Sophia Almeida. cular cambium of excised roots of radish to produce secondary vascular tissues in vitro. This developmental system has been described in great detail (Torrey and Loomis, 1967a, b; Radin and Loomis, 1969; Webster and Radin, 1972). The applied constituents initiate cell proliferation in the vascular cambium, specifically, and the latter subsequently produces both secondary xylem and phloem in a manner similar to intact roots. In another experimental technique, Van't Hof, Hoppin, and Yagi (1973) and Evans and Van't Hof (1974) demonstrated that cells in intact roots of Vicia faba and Pisum sativum normally arrest in G2 (with a 4C amount of DNA/nucleus), but when roots are excised and grown in aseptic culture, cells arrest predominantly in GI. Moreover, excised roots of V. faba and P. sativum produce only primary vascular tissues in vitro (Torrey, 1963; personal observation), while intact roots produce secondary vascular tissues. The experiments described in this paper integrate elements of the cell cycle with proliferation of the vascular cambium and subsequent morphogenesis in excised roots of R. sativus. Two questions are asked. First, is there a correlation of predominant cell arrest in G1 and G2 and presence of polyploid cells with stimulation of the vascular cambium in excised roots of R. sativus? Second, is the nuclear DNA content of mature root cells prescribed in meristems of experimentally manipulated roots of R. sativus? A general relationship between predominant period of cell arrest, presence of polyploid cells, and pres

Seedlings of Atriplex hortensis were studied to ascertain; 1) in which organ the primary thickening meristem (PTM) first differentiates; 2) the direction of differentiation of the PTM, and 3) the pattern of differentiation of conjunctive tissue. The PTM initially differentiates in pericycle of the primary root base 11 days after emergence of the primary root. It then differentiates in the transition region of the hypocotyl, mostly in cells of pericycle between pairs of vascular bundles. In the upper hypocotyl, PTM differentiates by day 20 in the inner layer qf cortical parenchyma. In the epicotyl, PTM apparently differentiates in the inner layer of cortex, by day 24. Desmogic xylem differentiates from radial files of internal conjunctive tissue cells and desmogic phloem differentiates opposite desmogic xylem strands from newly formed cells of external conjunctive tissue. No interfascicular cambium differentiates in the root, hypocotyl, or epicotvl. ATRIPLEX HORTENSIS L., a member of the Chenopodiaceae, is an example of a dicotyledonous plant that grows in diameter principally by a primary thickening meristem (PTM), instead of a vascular cambium. These two lateral meristems differ in that: 1) PTM differentiates outside of primary phloem whereas vascular cambium differentiates centripetally of primary phloem; 2) PTM produces parenchymatous cells mainly to the inside, internal conjunctive tissue, and fewer cells to the outside, external conjunctive tissue (Stevenson and Popham

LIKE THE other vegetative organs of the peanu (Arachis hypogaea L.), the stem has been describe( only in a cursory manner by earlier authors an( largely in terms of its external features. Its histo logical features have not been adequately describe( and in particular there is no account of the anatom ical changes which occur in the stem during matur ation of the plant, nor any morphological evalu ation of the tissues of the stem in relation to the other vegetative organs of the plant. This papei presents a study of the form and structure of thstem of Arachis hyp,ogaea and, together with twc preceding papers (Yarbrough, 1949, 1957), com pletes an account of seedling development in ithi species. The materials used and methods followed here were identical with those described in the latte paper (1957). FORM OF THE STEM.-The stem of Arachis hac been described by Waldron (1919) and Reed (1924) as presenting no features unusual amono herbaceous dicotyledons. It is green and pubesceni with simple hairs having two basal cells. Very young internodes (i.e., those within the last 4-> expanded leaves) of the main axis show some angu larity in transverse section (fig. 4) while youn& internodes of lateral branches are, in contrast usually flattened elliptical in form. In both cate gories lower internodes, where the pith begins ta break down, may be angular in appearance. Stil' older internodes in which the vascular cambium has formed abundant secondary tissue are usually circular in cross-section (fig. 5). The orientatior of stems of Arachis has been described (Gregory et al., 1951) and is different in erect and prostrate varieties. The main axis, however, in all varietieJ is erect. ANATOMY OF THE STEM.-The epidermis of the stem is composed of small cells (fig. 2, 3) with a layer of cutin somewhat thicker than that found or most herbs. Stomates and crystal-bearing cells, de scribed earlier for the leaf (Yarbrough, 1957), are also found in this tissue. The hypodermis consist, of collenchyma, largely of the simple, "angular' type. Adjacent to the stomates the collenchyma i9 only a single layer in thickness while in interstomatal areas it may consist of 3-4 cell layers. The cortex consists of two main types of tissue, (1) the specializ,ed chlorenchyma which is concentrated beneath and lateral to the stomates but may be con. tinuous in branching lines of cells between these points, and (2) the inner, larger, thin-walled paren.

A B S T R A C T Excised cultured roots of Raphanus sativus L. cv. White Icicle elongate and produce a few lateral roots but do not increase in diameter. Lateral expansion is effected when both an auxin (indoleacetic acid or naphthaleneacetic acid) and a cytokinin (benzyladenine) are applied at the cut end of the root. The growth regulator effects are apparent first in the pericycle and subsequently in the procambium. Both of these groups of cells divide, producing large numbers of secondary derivatives. The increase in number of cells is reflected in an increase in root diameter. When cultured roots are treated with auxin only, a limited number of pericycle cells divide and lateral roots develop. When roots are treated with cytokinin only, all pericycle cells divide and a multiseriate zone of pericycle-derived cells develops. The procambium is not markedly affected by application of a single growth regulator. The distinct and separable responses of pericycle cells to different regulators suggest that the pericycle can be characterized in a functional, as well as a topographic, sense. STUDIES OF THE factors which control the growth and development of roots are comparatively few compared with those of shoots. In intact plants, hormones synthesized in the shoot and transported to the root may be important in controlling patterns of root growth. By isolating and culturing roots, one can evaluate the effects of applied growth regulators on root development. The radish root is particularly well suited for this type of study since it grows well in culture and readily responds to applied growth regulators. Torrey and Loomis (1967a) induced secondary thickening in cultured radish roots by adding an auxin and a cytokinin to the culture medium and using a "polar feeding" technique (Loomis and Torrey, 1964). Anatomical examination of their treated roots indicates that the increased diameter is attributable to activity of the vascular cambium, which Torrey and Loomis noted, and also to proliferation of the pericycle. Pericycle development has not been assessed, although it contributes to the normal secondary thickening in intact radish roots (Hayward, 1938). This study emphasizes the early changes in the pericycle of cultured radish roots following the application of growth regulators, and the comparative time and extent of development of the pericycle and the cambium. The effects of growth regulator treatment on root elongation are also reported.

The peculiar secondary growth in Doxantha unguis-cati provides several developmental problems concerning cambial activity. One of the most interesting of these problems is the presence of both unidirectional and bidirectional arcs of cambium within the same stem. This investigation reports the ontogenetic development of these two kinds of cambial arcs. The first cambial divisions are observed in the fascicular regions of the 11th to 16th internodes from the shoot tip. This event is initiated after internode elongation is completed. In the initial stages, secondary tissues have a cylindrical configuration, but subsequently four grooves become apparent. These grooves signify the first evidence of unidirectional cambial activity. The four unidirectional arcs occur near the four major vascular strands to which all of the leaf traces connect. As secondary growth continues, the bidirectional and unidirectional arcs of cambium become separated and radial fissues can be seen between the furrows of phloem and the lobes of secondary xylem. Additional furrows originate either as sets of four between the original set of four or as single furrows to either or both sides of an existing furrow. All furrows are bordered by multiseriate rays. The initials of the bidirectional and unidirectional cambial arcs are non-stratified and are similar in size and appearance. The phloem produced within the furrow differs in several respects from that produced by the bidirectional arcs. The two types of cambial activity and the precise locations of the unidirectional cambial arcs in the stem (i.e. near the four major strands) suggests that transported products from the leaves are involved in the control of unidirectional cambial activity. THIS REPORT is the second in a series on the anomalous cambial activity in the cross vine (Doxantha unguis-cati) (L.) Rehder. At least four arcs of a normally functioning vascular cambium cease or reduce the production of xylem while continuing the production of phloem (Hovelacque, 1888; Schenck, 1893, 1895; Pfeiffer, 1924, 1926). Thus, the secondary xylem becomes lobed by deep furrows of phloem. The first report of this series described the ontogeny of the vascular system up to the first cambial activity. The present work describes secondary growth with emphasis on the initiation of unidirectional cambial activity. MATERIALS AND METHODS-Clonal material3 of Doxantha un9uis-cati (L.) Rehder (= Bignonia unguis-cati L.) was used for this study. All plants ' Received for publication 3 August 1970. Based on a dissertation submitted to the Graduate School, University of Massachusetts, in partial fulfillment for the degree of Doctor of Philosophy. The author ,vould like to acknowledge the advice and assistance of Drs. Otto L. Stein, Seymour Shapiro, Brayton Wilson, and John R. Rowley. 2 Present address: Department of Biological Sciences University of Lethbridge, Alberta, Canada. I Clonal material from Dobbins' stock, number 311. were grown in the greenhouse under coinstant fluorescent illumination. Under these conditions a uniform growth rate of the shoots was obtained. Specimens for microscopical examination were prepared either for paraffin sectioning, plastic sectioning (Araldite-Epon), or fresh sectioning on the sliding microtome. All staining was accomplished with toluidine blue 0 or hematoxylinsafranin. Measurements of cell dimensions were made with an ocular micrometer or directly from photomicrographs. Terminology-A "bidirectional" arc of cambium in this study is defined as that portion of the cambial cylinder that produces as much or more xylem than phloem. A "unidirectional" arc of cambium is defined as that portion of the cambial cylinder that produces little or no xylem and extensive amounts of phloem. The arms of phloem produced by the unidirectional arcs of cambium appear to radiate from the core of secondary xylem to the outer cylinder of secondary phloem. These arms will be called "furrows" (from "Furche," Pfeiffer, 1926). RESULTs-Mature primary axis-The primary axis, prior to the first cambial divisions, has a [The JOURNAL for August (58: 595-696) was issued 13 August 19711 AMERICAN JOURNAL OF BOTANY, Vol. 58: No. 8, 1971

BROWN, CLAUD L. (U. Georgia, Athens), and KARL SAX. The influence of pressure on the differentiation of secondary tissues. Amer. Jour. Bot. 49(7): 683-691. Illus. 1962.-When longitudinal bark strips of Populus trichocarpa and Pinus strobus are separated from the bole wood during early spring and maintained in a humid environment while attached to the parent tree, the cambial zone along the inner surface of the segments rapidly proliferates producing an extensive parenchymatous callus. Subsequently, a new phellogen and cambium differentiate and extend tangentially around the outer periphery of the callus pad, this resulting in the formation of a new stem-like structure. Whenever bark strips are separated from the bole wood by a layer of polyethylene plastic film and held firmly against the tree by externally applied pressure, the cambium continues to function normally, setting aside derivatives which differentiate and mature into normal, elongate xylary and phloic elements. The importance of mutual pressures and spatial relationships in controlling patterns of differentiation in secondary tissues is clearly demonstrated. THE MANY complex phenomena associated with the differentiation and maturation of cells and tissues in higher plants constitute a most fascinating and fertile area of experimental research. It is not surprising that much current research in morphoand histogenesis centers around the general area of apical meristems because, ultimately, it is here that complete control is exercised over the entire sequence of developmental events in the higher plant. It is rather odd, however, that so few workers have recognized lateral meristems as material par excellence for various experimental approaches to other problems of histogenesis. Perhaps this is because many view the vascular cambia of woody species only as a meristematic zone, where orderly cell divisions produce a more or less stereotyped pattern of xylem and phloem inherently peculiar to a given species. Obviously the biological mechanisms controlling cellular differentiation and maturation at this level are no less dynamic than those occurring elsewhere in the intact plant. Although a large body of literature is available on the physiology of cambial activity per se, little is actually known about specific mechanisms controlling the pattern of histological development in secondary tissues. Some of the most pertinent, yet indirect, work in this general area is that concerned with the healing of tissues around wounds and more specifically *to the insitu regeneration of tissues from the xylem side of the cambium on plants subjected to various surgical treatments. Of these, the work of Sharples and Gunnery (1933) on the formation of callus and regeneration of secondary tissues in several I Received for publication May 10, 1961. woody species is of special interest, as are also the more recent anatomical observations of Soe (1959) on the ancient practice of scoring trees to increase fruit or seed production. The present study describes the observed pattern of tissue differentiation in bark segments in which the vascular cambium is: (1) released from apparent pressure and normal spatial relationships; or (2) maintained under different amounts of externally applied pressure. MATERIALS AND METHODS-Forty-five vigorous, 5-year-old seedling trees of northern black cottonwood (Populus trichocarpa Torr. and Gray) were selected for treatment from among numerous seed sources in an experimental plantation established by the Cabot Foundation of Harvard University near Weston, Massachusetts. In addition, 35 white pine (Pinus strobus L.) seedlings ranging from 3 to 5 years of age and growing in adjacent blocks were selected for experimental use. The experimental treatments were initiated in early May during the period of active shoot elongation when the cambium of individual trees was exceedingly active. The surgical techniques used were simple. Two parallel, longitudinal incisions, approximately 8.0 cm in length and 1.5 cm apart, were made with a sharp grafting knife through the bark into the xylem at breast height. The basal portion of the bark strip was severed transversely and carefully lifted outward from the bole as shown in Fig. 1. The bark strips were left attached at the acropetal end to provide nourishment, and, upon separation from the bole, were immediately encased in a small, thin-gauge, polyethylene bag to prevent desiccation. Thus the strips were maintained in a humid environment and in an atmosphere which permitted gas AMERICAN JOURNAL OF BOTANY, Vol. 49, No. 7, 1962 [The Journal for July (49: 547-682) was issued July 18, 1962]

A comparative study was made of the effectiveness of various horinoine anid metabolite mixtures in inducing vascular cambium initiation and secondary vascular tissue formation in isolated first-transfer oots of the radish, Raphanus sativus L. 'White Icicle,' when provided to the cut basal end of the root grown in sterile culture. An auxin, such as indoleacetic acid (IAA) at 10-5 M, a cytokinin, such as 6-benzylamino purine at 5 X 10M, a cyclitol, such as myo-inositol at 5 X 10-1 M and sucrose at 8% were all required for maximum response. Requirements for auxin and cytokinin were absolute; in their absence no cambium was formed. The addition of cyclitol, while not an absolute requirement for cambium initiation, increased the magnitude of the response markedly. Alternative auxins such as a-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid were equally as effective as IAA. Alternative effective cytokinins included 6-furfurylaminlopurine, 6-phenylaminopurine and 6-(y,--dimethylallylaminio)purine. Alternative cyclitols equivalent to myo-inositol were seyllitol and pinitol. Other related cyclitols tested were much less effective or totally inactive. ISOLATED EXCISED ROOTS growIn in sterile nutrient culture usually do Inot form a vascular cambium or secondary vascular tissues. Only under unusual culture conditionis (Dormer and Street, 1948; Seeliger, 1956; Torrev, 1951) are secondary vascular tissues observed. Using a technique devised by Raggio and Raggio (1956) for culturing excised roots by providing organic nutrients via medium iintroduced into the cut basal enid of the root, Torrey (1963) was able to induce vascular cambium and limited secondary vascular tissue formation in excised roots of Pisum sativum L. Loomis and Torrey (1964) demonstrated with the same techniques that subcultured isolated I Received for publication 21 November 1966. This investigation was supported in part by research grant (GM-08145) and a special fellowship (no. 1F3GM2024-01) to RSL from the National Institutes of Health, U. S. Public Health Service. The technical assistance of Sharon Hosley and Pauline Borsari is gratefully acknowledged. roots of the radish, Raphanus sativus L., could be induced to form cambium and secondary vascular tissues bv introducinig appropriate metabolites and hormone mixtures through the cut basal end. They showed that, in additon to sugar in the form of sucrose, the excised radish roots must be provided an auxiin such as indoleacetic acid and a cytokinin, such as 6-beiizylaminopurine, both at appropriate low coincenitrations, in order to induce vascular camabium iniitiation. The addition of myo-iiiositol increased the response markedly. A further study has been made to determine the optimum coniditions in vitro for cambial initiation and for extenided vascular cambium activity in the excised radish root. A broad range of concentrations of the essenitial components, alone and in combinationi, was tested on first-transfer radish roots in. culture, and the ailatomical responses were determined in fixed and sectioned material. In the studies presenited below a comparisoin is made of ainatomiical responses observed October, 1967] TORREYl AND LOOM1-VASCULAR TISSUE FORMATION 1099 at the end of a period of approximately 4 weeks of treatment of roots subjected to a wide range of experimental conditions. Conclusions concerning the optimum concentrations of the chemical constituents required for cambial initiation and maintained activity are presented. Detailed considerations of the ontogenetic sequence in cambial initiation and secondary vascular tissue formation are presented elsewhere (Torrey and Loomis, in press). MATERIALS AND METHODS Ro0ots of radish, Raphanus sativus L. 'White Icicle,' were used in the anatomical studies described here. Mlethods of culturing and treating the excised roots have already been described in detail (Loomis and Torrey, 1964) and may be summarized briefly. Ten-mm root tips from 3-day-old seedlings germinated aseptically in sterile distilled water in the dark at 23 C were excised and cultured in a modified Bonner nutrient agar medium. After 4 days, when the isolated root tips were about 50 mm in length, 15-mm first-transfer tips were excised with a sharp scalpel and transferred to fresh nutrient medium in 11-cm petri dishes. The basal 5-mm portion of each root wtas inserted into a separate agar medium contained in a 12 X 35 mm glass vial. Ussually two roots were cultured in each 11-cm dish. The roots N-ere grown in the dark at 23 C for additional periods up to about 5 weeks. After various periods of culture, whole roots were removed, fixed in formalin-acetic acid-alcohol, dehydrated through an ethyl-butyl alcohol series, embedded in Tissuemat, and sectioned at 10 p. Sections were stained for anatomical study using Heidenhaiin's iron haematoxylin and safranin. From the studies reported earlier (Loomis and Torrey, 1964), it was know-n that aniatomical differences, especially with respect to the initiation of the vascular cambium and the development of secondary vascular tissuies, were dependent upon the preseince of hormones and hormone-like materials in the vial medium. Auxins, cytokinins, and cyclitols, as well as the concentration of sugar provided, appeared especially important. A large number of different combinations of nutrients both in the vial and in the plate medium had been tested for physiological response. Macroscopic observation of roots in culture usually showed whether secondary thickening of the roots had occurred, but it was necessary to make careful anatomical investigations in order to assess the extent and nature of the response to the various treatments. In the present study anatomical analyses were made of roots taken from approximately 64 different treatments. In the results described below, emphasis has been placed on the following aspects: the effects of auxin concentration including omission, auxin type; cytokinin concentration including omission, cytokinin type; cyclitol omission and type of cyclitol. A comparative study was made of structures formed in response to optimum concentration of these factors and to their absence from the vial medium. In making such an anatomical study large numbers of roots must be sampled and some variation in response is inevitable. Some arbitrary selection must be made of roots to be studied in this way. Usually each treatment involved 8-16 roots, but fewer than half of the roots wiere fixed and even fewer were selected for sectioning. Among those sectioned were the roots showing the maximum response as measured by root diameter. Despite this selection, the number of different reatments was sufficiently great so that a wide range of responses could be studied. The anatomical responses to similar treatments were remarkably consistent. Photographs of sections are selected as typical of the particular treatment and as showing good physiological response. In most cases any of a number of roots sectioned could have been used as illustrations. In the presence of optimum concentratioins of substances stimulatory to cambial activitv, the anatomical response was dramatic and a large root diameter resulted; in the absence of any one critical component, the response was markedly reduced or absent. IPhotographic comparison among roots from various treatments was made difficult, since useful magnifications varied markedly. In the accompanying photographs some compromise has been made in choice of magnification so that, where possible, cell detail can be seen. Large-diameter oots are reproduced to show overall response rather than cell detail. Some attention must be paid by the reader to the magnification of the photographs in comparing responses. In most instanices, the thickeening was macroscopically complete wN-ithin 14-21 days, but to be sure that all effects were observed the experiments were sometimes continued for longer periods. Some of the cambial derivatives continued to differentiate after cambial activity ceased, but the anatomical patterns in young (14-21 days) and old (28-35 days) roots were generally quite similar. The principal difference was that a cambial region of small, rapidly dividing cells was not as clearly evident in the old roots. The reasons for cessation of cambial activity constitute an important problem (Loomis and Torrey, 1964) w-hich is being investigated further. RESULTS As has been described briefly by Loomis and Torrey (1964), isolated 'White Icicle' radish roots in culture show maximum secondary vascular-tissue formation under the following cultural conditions. Plate medium: M\odified Bonner medium containing 2 % sucrose and vitamins (Bonner and Devirian, 1939). 1100 AMERICAN JOURNAL OF BOTANY [Vol. 54

MILLER, ROBERT H. (U. Nevada, Reno.) Morphology of Humulus luppulus. 11. Secondary growth in the root and seedling vascularization. Amer. Jour. Bot. 46(4): 269-277. Illus. 1959.In the primary state the roots of Humulus lupulus L. have a diarch xylem plate with 2 strands of primary phloem lying on either side of the primary xylem. Secondary histogenesis is described for the primary root. Fibrous and fleshy storage roots are developed by the hop plant and their respective developmental and anatomical structures are described. Lateral roots are initiated in the pericycle opposite the protoxylem poles. The architecture of these secondary roots is similar to that of the primary root. The seedling develops a fleshy storage organ through secondary growth of the primary root and the hypocotyl. The hypocotyl eventually resembles a fleshy taproot throughout most of its extent. The vascular cambium differentiates large amounts of parenchymatous tissues. A relatively smaller amount of tracheary tissue is formed. The secondary phloem comprises a high percentage of phloem parenchyma and ray cells containing numerous large starch grains, and constitutes the larger portion of the fleshy storage root. Numerous thickwalled lignified fibers occur throughout the secondary vascular tissues. Resin and tannin cells are abundantly distributed. A phellogen is differentiated from the pericycle and develops a persistent periderm on the outer surface of the fleshy storage organ. A relatively short transition region occurs in the upper part of the hypocotyl. The transition takes place from a radially alternate arrangement of the vascular tissues in the root to a collateral arrangement in the cotyledons. THE ONTOGENETIC DEVELOPMENT of the primary tissues in the root of Humulus lupdulus L. was described in detail in a previous paper (Miller, 1958). In the present paper the development of the secondary tissues of the primary root, lateral root formation, and seedling vascularization will be described. As was pointed out in the earlier paper, the literature on the developmental anatomy of hops is limited. Aside from the earlier investigations of Lermer and Holzner (1896) on hop anatomy in general and the more recent and unpublished work of Runner (1950) on hop roots, there have been no developmental studies of the vegetative organs of this economically important plant. MATERIALS AND METHODS. The materials and methods were those presented in the earlier paper. However, mention should again be made of the clearing technique which facilitated the rapid determination of the arrangement of the vascular tissues in the "transition region." Entire young seedlings ('Early Fuggles' and 'Late Clusters' varieties) were aspirated in a 5.25% solution of sodium hypochlorite and then again aspirated in water. They were dehydrated in an ethyl alcohol series, to which safranin alone was added at the 70%7 level, and finally stored in xylene. SECONDARY HISTOGENESIS.-As described in the previous paper, the vascular cambium originates from parenchymatous cells situated between the diarch primary xylem and primary phloem. The first cells of the cambial region are located as isolated strips on each side of the primary xylem plate, 1 Received for publication Novemlber 10, 1958. outwardly bordered by the primary phloem. As secondary xylem is cut off toward the inside of the cambium and secondary phloem toward the outside, the cambium takes on the appearance of 2 "arcs," more or less bulging toward the outside of the vascular cylinder. With further development the cambial region assumes an undulated appearance in its encirclement of the vascular cylinder. With still further development, the cambium becomes a more or less cylindrical layer of tissue around the circumference of the differentiating secondary xylem, except for interruptions at the protoxylem poles. In the meantime, the pericycle immediately abutting the protoxylem poles undergoes periclinal divisions resulting in the differentiation of cambial cells in this region also. With the formation of these additional cells, the "gaps" in the vascular cambium are filled and the cambium completely encircles the xylem. The vascular camibium of the root of Huimulus lZp'iuus is composed of both ray and fusiform initials. Those pericyclic cells abutting the protoxylem give rise to multiseriate ray initials. The developing rays eventually radiate from the protoxylem poles outwardly through the secondary vascular tissues. These are the first multiseriate rays in the secondary xylem. Ray initials are also produced by the cambium between the xylem poles; however, these are at first uniseriate in nature. Runner (1950) reported that the vascular cambium was observed to be of an "anomolous type" with the cambium "-multiseriate" between the vascular rays. "Sieve tubes and companion cells are differentiated in the center of this region., while

WILSON, BRAYTON F. (U. California, Berkeley). Increase in cell wall surface area during enlargement of cambial derivatives in Abies concolor. Amer. Jour. Bot. 50(1): 95-102. Illus. 1963.Dimensions of fusiform cells (tracheids and sieve cells) and ray cells were measured from samples of the 1960 xylem and phloem increment of 5 trees felled at monthly intervals from April through July, 1960. Calculations using these measurements gave the magnitude, direction and rate of increase in cell wall surface area during enlargement. Although 14 times more tracheids than sieve cells were produced, both cell types enlarged mostly in a radial direction (up to 400%) at the same rate (20-33 X 103Au2 wall surface area/day) to the same final size. Fusiform cambial cells doubled their wall area between successive pericinal divisions. Calculations showed that / of this increase was in the radial walls of the daughter cells at a rate comparable to that in enlarging tracheids and sieve cells; the other % was from cell plate formation at an estimated rate of 187327 X 103/A2/day. Enlargement of derivatives in the radial direction largely determined the amount of increase in wall area. Besides radial enlargement, tracheids also elongated (up to 13%) and phloem cells enlarged tangentially (sieve cells up to 36%; pholem ray cells up to 60%). The relationships of enlarging tracheids and xylem ray cells are discussed, and it is suggested that slippage may occur between the developing walls. CELL WALL formation is an important part of the process of cell enlargement. The aim of this investigation was to determine the amount, orientation and rate of increase in cell wall surface area, and the relationships between adjacent cells during enlargement of cambial derivatives. As a specific example of the general process of cell enlargement, the enlargement of cambial derivatives has several characteristic features. The vascular cambium, in contrast to apical meristems, produces cells in 2 directions which differentiate respectively into secondary xylem and phloem. Also, both the cambium and its derivative tissues are composed of 2 cell systems, the fusiform cells oriented parallel to the stem axis and the ray cells oriented perpendicular to the stem axis. Thus, there is an opportunity to contrast wall formation in cells of the same origin which are enlarging in different environments and are separated spatially, and also to contrast the enlargement of 2 cell types which are already differentiated within the cambium. Abies concolor is well suited for such a study because, as in many conifers, there are few cell types in the secondary xylem and phloem. Ray and fusiform cells in the cambium produce ray and fusiform derivatives: fusiform tracheids and ray cells in the xylem; fusiform sieve cells and ray cells in the phloem. The only additional cambial derivatives are parenchyma in the 'Received for publication June 19, 1962. This study represents a portion of a thesis submitted in partial satisfaction of the requirements for the Ph.D. at the University of California, Berkeley. 2 Present address: Cabot Foundation, Harvard Forest, Petersham, Massachusetts. I wish to thank Dr. W. A. Jensen for his guidance throughout this study. phloem. The cambial cells and their derivatives are large and relatively easy to measure, fusiform cells up to 4 mm long and ray cells up to 1 mm long. In addition, derivatives at a given distance from the cambium are at a comparable stage of differentiation so that averaged dimensions are meaningful. Interest in dimensions of conifer cambial derivatives has centered on tracheids, which are the major component of the wood. Bailey (1920) noted that enlargement of tracheids was due mainly to radial expansion rather than elongation. However, because of interest in paper-making and cambial development, the literature has emphasized final tracheid length and long-term changes in the length of fusiform cambial cells, rather than dimensional changes during cell enlargement (Dinwoodie, 1961). While these studies are important to the understanding of changes in the cambium, they yield little information about the process of enlargement and increase in wall surface area of cambial derivatives. As an approach to the study of the actual process of enlargement, measurements were made of cell wall surface dimensions of fusiform and ray cells at various stages during enlargement. These measurements were used to show the magnitude, direction, and rate of the increase in wall area. Comparisons were made of the data from 5 trees to show the differences in enlargement which result in the formation of annual rings in the xylem and phloem. MATERIALS AND METHODS-Five white fir trees (Abies concolor Lindl. & Gord.), selected for uniformity, were felled at approximately 1-month intervals from April through July, 1960 (Table

ROBERTS, LORIN W. (U. Idaho, Moscow.) Protein-bound sulfhydryl groups in Coleus wound meristems. Amer. Jour. Bot. 47(2): 110-114. Illus. 1960.-A histochemical examination was conducted of the developmient of the Item wound-meristem of Coleus blumei Benth. with 2,2'-dihydroxy-6,6'-dinaphthyl disulfide (DDD) and 1(4-chloromereciriphenylazo) -naphthol-2 (Mercury Orange) to test for protein-bound sulfhydryl groups. Specificity for the histochemical reactions was indicated by complete blockinfgf of staining in sections pretreated with 0.001 M iodine in npropanol and 0.1 M aqueous solutions of iodoacetamide. Both histochemical methods yielded comparable results in protein-SH localization (zone of cell division and differentiating xylem elemments) and staining intensity. The zone of cell division did not exhibit staining at the time of initial cell division (3 days). However, the cytoplasm of the dividing cells of the wound-meristem demonstrated protein-SH 4-5 days following wounding. A high concentration of protein-SH was observed in the cytoplasm of the dividing cells 7. days after wounding. Redifferentiating parenchyma cells, destined to become wound-xylem elements, first exhibited wall staining at the time of protoplast contraction. The subsequent walled xylem stage exhibited strong staining in the reticulated striations of the secondary w alls. THE FREQUENT literature references to the possible role of sulfhydryl in cell division are too wellknown to reiterate in detail (Barron, 1951). Although determinations have been made on proteinbound SH during cell division, there has been little experimental evidence to demonstrate significant changes of this chemical group during division. Mazia (1954) reported evidence that protein-SH may have a structural function by the oxidative formation of the disulfide linkage in the mitotic spindle. However, he could find no significant difference in protein-SH staining between the cytoplasm of nondividing cells and cells at various stages of mitosis using the DDD histochemical procedure of Barrnett and Seligman (J952). Similarly, amperometrically determined protein-SH indicated no significant chanre in concentration during mitosis of microspores of Lilium longiflorum var. 'Croft' (Stern, 1956). Previous results from my laboratory, employing 2,2'-dihydroxy-6,6'dinaphthyl disulfide (DDD method of Barrnett and Seligman, 1952), 1(4-chloromercuriphenylazo) naphthol-2 (Mercury Orange method of Bennett, 1951), and 4-iodoacetamido-naphthol-1 (IAN method of Roberts, 1956), have demonstrated a high concentration of protein-SH in apical meristems, embryonic tissues, and the vascular cambium of higher plants (Roberts and Lucchese, 1955; Roberts, 1956). These results suggested that changes in protein-SH during cell division in plant tissues might be approached by investigating a stem wound-meristem. By employing this approach, changes in protein-SH could be determined prior 1Received for publication June 7, 1959. This investigation was supported by a research grant (G-5708) from the National Science Foundation. The writer wishes to express his appreciation for the services of Miss M. S. Dempsey, Department of Botany, University of Michigan, for her technical assistance. to, during, and following cell division. Followirng the act of wounding, one could predict the histological location of the dedifferentiating parenchyma cells that would form the zone of cell division. Also the time required for the initiation of cell division could be determined with reasonable accuracy. The Coleus stem wound-meristem was chosen because it has been extensively investigated (Surrey, 1955) and the plant is easily propagated. This paper is devoted to a brief description of the changes in protein-SH content of the Coleus stem tissues during the development of the wound-meristem. MATERIALS AND METHODS. A single clone of Coleus blumei Benth.2 was vegetatively propagated in the University of Idaho greenhouse by taking repeated stem cuttings from the original plant. Approximately 100 plants were employed in the experiments described in this report. Although a considerable number of these plants were sacrificed for specificity tests and for developing suitable histochemical staining procedures, stem wound-meristems from approximately 60 plants were sectioned. Slides prepared from each stem wound-meristem were stained by both the Mercury Orange and DDD methods, respectively. Supplementarv lighting was required during the winter months to facilitate growth. The plants were maintained under reasonably uniform environmental conditions throughout the experimental period. For a given experiment, plants that had approximately the same stem diameter, internode lencth, and leaf area were selected fronm the same age group. These criteria applied to the second internode and second leaf pair of the test plants. Although Erickson and Miche2The experimental plant was taxonomnically identified by Prof. W. H. Snyder, Department of Horticulture, University of Idaho.

LIANAS IIAVE LONG BEEN a favorite source of mtiaterial for the study of anomalous stem structure. In addition to the usual vascular structure, the principal anomalies generally recognized, and some of the plants in which they occur are: (1) inverted medullary bundles (Wisteria); (2) cortical vascular bundles (Serjania, Paullinia, and Thinouia); (3) secondary tissues produced by concentric cylinders or arcs of accessory vascular cambium (Bauhinia); (4) secondary tissues distorted by the growth of isolated parenchyma (Bauhinia) and (5) vascular patterns resulting from the production of unequal amounts of xylem and phloem or from the restriction of cambial activity to certain parts of the original vascular cylinder (Bauhinia, Malpighiaceae, Bignoniaceae, and certain members of the Sapindaceae). The accounts of liana structure in the textbooks of DeBary (1884), Pfeiffer (1926), Eames and MacDaniels (1947), and Esau (1953) are drawn largely from the investigations of Nageli (1864), Radlkofer (1875, 1876, 1877, 1886), and Schenck (1893). These early studies were almost exclusively confined to mature structure, and little attention was devoted to the origin and development of the anomalies, or to tracing the vascular path through a complete phyllotactic spiral. The present report contains a description of the vascular system of Serjan a subdentata Juss. which has cortical bundles as described in type 2 above (fig. 3), and of S. mexicana Willd. in which the stem has unequal amounts of xylem and phloem arranged in a definite pattern as in type 5 (fig. 7). The path of the vascular system proved to be so complicated in both species that it was decided to reserve the study of the. origin and development of the vascular system for a second paper. MATERIALS AND METHODS.-Material of Serjania subdentata Juss. was obtained from a single plant which had grown into the tree tops of a small stand of native forest in the Atkins Garden, Cuba. This collection was made during the rainy season in August, 1951. Old stems and young branches of Serjania mexicana Willd. were collected from the U.S.D.A. Plant Introduction Garden, Coconut Grove, Florida, in September and December, 1954. All material was fixed in formalin-acetic acid

A B S T R A C T Circular patches of bark were surgically isolated on the sides of sugar maple (Acer saccharum Marsh.) trees at breast height at various times during the dormant and growing seasons. Subsequently, samples of wood and attached bark were taken from isolated and control sites to determine the effects of isolation of the bark on cambial activity and xylem and phloem development. In control sites cambial activity and xylem and phloem development occurred normally. Isolation of bark during the dormant season (in November, February, or March) prevented initiation of cambial activity and xylem and phloem development in isolated areas of half of the trees. Varying degrees of cambial activity (periclinal divisions) occurred in the remaining isolated areas, but normal cambial activity and xylem and phloem development were prevented. Isolation of bark after initiation of cambial activity and phloem differentiation, but prior to initiation of xylem differentiation, resulted in the formation of very narrow xylem and phloem increments with atypically short vessel members and sieve-tube members, respectively. The xylem increments consisted primarily of parenchyma cells. Isolation of bark after initiation of xylem differentiation resulted in curtailment of secondary wall formation in the last-formed part of many increments. The last-formed vessel members of all these xylem increments were atypically short. Similarly, the last formed sieve-tube members of corresponding phloem increments were atypically short. The atypically short cells in the xylem and phloem of isolated areas reflected the effect of isolation on the cambial region, viz., the subdivision of all fusiform cells into strands of cells. Ultimately, the strands of short fusiform cells lapsed into maturity, leaving only strands of parenchymatous elements between xylem and phloem. NORMAL CAMBIAL ACTIVITY in woody plants requires a continuous supply during the growing season of phloem-translocated products from the shoots (Kozlowski, 1971). Thus, severing the phloem so as to prevent transport of carbohydrates and hormonal growth regulators to the lower stem greatly modifies production and maturation of xylem and phloem cells below the wound. The effects of phloem blockage on cambial growth vary with species and particularly with the time of treatment (Wareing, 1951; Reines, 1959; Schneider, 1954; Wilcox, 1962; Noel, 1970). When the phloem of the diffuse-porous species, Populus tremuloides, was severed during the dormant season, initiation of seasonal cambial actvity and phloem differentiation below the point of blockage were not prevented, but subsequent cambial activity was abnormal and xylem differentiation was largely prevented (Evert and Kozlowski

Developmental changes in the vascular cambium of Polygonum lapathtifolium were determined primarily by an analysis of the secondary xylem. The cambium and xylem consist of fascicular and interfascicular regions in this herbaceous dicotyledon. Near the pith vessels are restricted to the fascicular regions of the xylem. During secondary growth vessels are formed in some radial files in the interfascicular regions. Anticlinal divisions are of two types, oblique and lateral. In interfascicular files consisting of fibers only, about two-thirds of the anticlinal divisions are oblique. The oblique partition averages 31% of the length of the dividing initials. In interfascicular files consisting of vessel elements and fibers, there are almost equal numbers of oblique and lateral divisions. The oblique partition averages 37% of the length of the dividing initials in these files. Lateral divisions account for approximately three-fifths of the anticlinal divisions in the fascicular regions, consisting of vessel elements and fibers. The partitions formed in oblique anticlinal divisions average 64% of the length of the dividing cells in the fascicular regions. The frequency of anticlinal division is much higher in files consisting of vessel elements and fibers than in those consisting of fibers only. There is no loss of fusiform initials, except by ray formation. Ray initiation occurs by simple subdivision of fusiform initials. The findings are discussed in relation to the developmental changes in the vascular cambium in plants of different habits. THE MOST intensive studies on the developmental changes in the vascular cambium associated with the increase in circumference of this meristem have been made on conifers (Bannan, 1950, 1957, 1960b; Whalley, 1950; Hejnowicz, 1961) and on some species of arborescent dicotyledons (Evert, 1961; Cheadle and Esau, 1964; Cumbie, 1967). Except for an investigation of a species of Hibiscus (Cumbie, 1963), there have been no intensive studies on the developmental changes in the vascular cambium in the herbaceous dicotyledons. This study describes the developmental changes in the vascular cambium of another herbaceous species, Polygonurn lapathifolium L. (Polygonaceae). In this species the fibrous elements in the secondary xylem, in addition to vessel elements and axial parenchyma strands, do not elongate to any appreciable extent during differentiation. Thus the cells in the secondary xylem replicate the basic cell pattern in the vascular cambium, and it was possible to follow the developmental changes in serial tangential sections of the xylem and cambial region. MVATERIALS AND METHODS-Developmental changes in the vascular cambium of P. lapathifolium were studied in three plants collected near Columbia, 1\/Io. Each plant for which a voucher specimen was prepared was approximately 1 m tall and 1 cm in diam near the base. At this level the radial thickness of the xylem ' Received for publication 19 April 1968. was 2.4-2.7 mm. This probably represents all or most of a season's growth because the plants were collected in late August and September. The secondary xylem in P. lapathifolium consists of fascicular and interfascicular regions. From serial tangential sections, 12 A in thickness, of the xylem and cambial region, developmental changes were followed in 163 radial files in the interfascicular regions and 40 radial files in the fascicular regions. In addition, the origin of 70 rays was studied. OBSERVATIONS-The primary vascular system in P. lapathifoliurn is in the form of discrete, widely spaced strands. The continuous vascular cambium and secondary xylem which develops later, therefore, consist of fascicular and interfascicular regions. Near the pith, vessels in the xylem are restricted to the fascicular regions (Fig. 1), but later vessels are produced in some radial files in the interfascicular regions. The fibrous elements in the xylem are thinwalled, living cells which occur in regular, radial rows (Fig. 1). Near the pith, the end walls of the fibers are transverse to abruptly tapered in tangential view (Fig. 2). A short distance out, however, the end walls are more tapered (Fig. 3). Axial xylem parenchyma is scanty, paratracheal and consists of strands of 2-7 cells. There are no rays in the first-formed secondary xylem (Fig. 2), but after the formation of 2.5 mm of xylem there are about 6.0 rays per mm2 of xylem (Fig. 3). All of the rays are uniseriate

Developmental changes in the vascular cambium of Leitneria floridana, a slhrub, were determined primarily by an analysis of the secondary xylem. During the productioni of the first growth ring of secondary xylem, 37% of the anticlinal divisions in, the fusiform iniitials were lateral, the remaining were oblique. The oblique partitioll averaged 1/2 of the lelngth of the dividing initials during this period of growth. Following their origin in antielilial division, daughter cells elongated at a rapid rate until they were about as long as the mean for all cells, and then most cells elongated at a slow rate. Almost all initials survived during the formation of the inner secondary xylem (growth rings 1-10), and few niew rays were formed from fusiform initials. During the production of the outer secondary xylem (growth rings 22-26), lateral divisions accounted for less thani 5% of all antielinal divisionis. The oblique partition averaged only 1/4 of the length of the dividing cells durinig this period, although the mean length of dividing initials was relatively constant throughout seconidary growth. About 20% of the initials studied during the deposition of the outer seconidary xylem disappeared from the cambium, and many others were transformed inito ray iniitials. The filndings are discussed in relation to the developmental changes in the vascular cambium in plants of different habits. THE MOST intensive studies on the developmental changes in the vascular cambium associated with the increase in circumference of this ineristem have been made on conifers (Whalley

The vascular tissues of the leafless form of the "lanceolate" mutant in tomato resemble those of the normal hypocotyl in the lower portions. A-divergence in development occurs in the middle region of the mutant hypocotyl: two xylary strands split acropetally into five to seven units, while in the normal hypocotyl four main vascular bundles are established. The three to four week old mutant hypocotyl shows a marked proliferation of cells in its central cylinder when it is grown on a simple nutrient medium; proliferation also occurs in older specimens grown in soil. This condition is averted if the mutant is grafted beneath an actively growing normal shoot tip. Instead of having numerous, random cell divisions in the central cylinder, the grafted mutant shows large pith parenchyma cells and an active vascular cambium. These features characterize the normal hypocotyl and they have not been observed in the intact leafless mutant. The observed behavior of intact and grafted mutant hypocotyls can be interpreted in terms of the normal shoot tip acting both as physiological sink, with respect to cell-division stimuli produced mainly in the root system, and physiological source, with respect to cambial activating hormones.

The hybrid of Gossypium hirsutun X G. gossypioides develops normally until 10-12 nodes have been produced at which time neoplasms form in the cortex, phloem, and pith of the lower portion of the shoot axis. Neoplasms are characterized by irregular whorls of unusually large cells the central portions of which contain apparently cytolysed cells and a reddish-brown substance that probably represents the products of cytolysis. Neoplastic invasion is followed by inactivation of the vascular cambium, disorganization of the phloem and cortex, and tylosing and plugging of many xylem vessels. Subsequent growth of the shoot, which continues at a very slow rate for up to several years, involves the proliferation of parenchymatous tissue which becomes secondarily vascularized. HYBRID NECROSIS occurs in the interspecific progenies of several species of Gossypium, being

A B S T R A C T Anomalous secondary thickening occurs in the main axis of Bougainvillea spectabilis as a result of a primary thickening meristem which differentiates in pericycle. The primary thickening meristem first appears in the base of the primary root about 6 days after germination and differentiates acropetally as the root elongates. It begins differentiating from the base of the hypocotyl toward the shoot apex about 33 days after germination. The primary thickening meristem is first observable at the base of the first internode about 60 days after germination. It then becomes a cylinder in the main axis of the seedling. No stelar cambial cylinder forms in the primary root, hypocotyl, or stem because vascular cambium differentiation occurs neither in the pericycle opposite xylem points in the primary root nor in interfascicular parenchyma in the hypocotyl or stem. The primary vascular system of the stem appears anomalous because an inner and an outer ring of vascular bundles differentiate in the stele. Bundles of the inner ring anastomose in internodes, whereas those of the outer ring do not. Desmogen strands each of which is composed of phloem, xylem with both tracheids and vessels, and a desmogic cambium, differentiate from prodesmogen strands in conjunctive tissue. The parenchymatous cells surrounding desmogen strands then differentiate into elongated simple-pitted fibers and thick-walled fusiform cells that are about the same length as the primary thickening meristem initials.

A B S T R A C T Circular patches of bark were surgically isolated on the sides of trembling aspen (Populus tremuloides Michx.) trees at breast height at various times during the dormant and growing seasons. Subsequently, samples of wood and attached bark were taken from isolated anld control sites to determine the effects of isolation of the bark on cambial activity and xylem and phloem development. In control trees cambial activity and xylem and phloem development occurred normally. Isolation of bark during the dormant season (in November, February, or March) did not prevent initiation of cambial activity and of phloem differentiation in spring but continued normal cambial activity and phloem developmented were prevent. Xylem differentiation was essentially prevented by isolation of tissues during the dormant season. The ultimate effect of isolation of the bark on the cambium, either during the dormant season or during the growing season, was subdivision of all fusiform cambial cells into strands of parenchymatous elements; the ultimate effect on the newly formed phloem was early death of the sieve elements. The most conspicuous effect of isolation of the bark after xylem differentiation had beguni was the curtailment of secondary wall formation. Shortening of cells of the cambial region was reflected in the length of the vessel members which differentiated from such cells. These results indicate that normal cambial activity and xylem and phloem development require a supply of currently translocated regulatory substances from the shoots.