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Made available in DSpace on 2022-04-28T19:27:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 This study aimed to analyze the leaf anatomy of Manihot caerulescens, with emphasis on the ontogeny and secretion composition of the laticifers. Leaf samples were submitted the usual techniques in plant anatomy and histochemical tests to identify the main classes of secondary metabolites. The analyzes were performed using light and scanning electron microscopy. The leaves have a single layered epidermis coated by a uniform cuticle. The epidermal cells are elongated and papillose on the leaf abaxial surface of leaves. The mesophyll is isobilateral and the vascular bundle of the midrib is collateral. The laticifers are originated from ground meristem and procambium, and are of articulated anastomosing type. Besides the laticifers, two types of idioblasts were found: the secretory idioblasts, with a dense content, and the calcium oxalate druses idioblasts. Histochemical tests revealed different classes of chemicals for both secretory idioblasts and laticifers. In idioblasts, a positive reaction was detected for the general phenolic compounds, proteins, total polysaccharides and tannins. The laticifers reacted positively to total lipids and acids, essences, total polysaccharides and rubber. The individuals analyzed here are from caatinga and their leaf anatomical structure showed to be different from that described for the same species growing in the cerrado. Programa de Pós-Graduação em Ciências Biológicas (Biologia Vegetal) Universidade Estadual Paulista “Júlio de Mesquita Filho” Instituto de Biociências Departamento de Botânica, Av 24A, 1515 Programa de Pós-Graduação em Recursos Genéticos Vegetais Universidade Federal do Recôncavo da Bahia Centro de Ciências Agrárias Ambientais e Biológicas Universidade Federal do Recôncavo da Bahia Centro de Ciências Agrárias Ambientais e Biológicas Programa de Pós-Graduação em Ciências Biológicas (Biologia Vegetal) Universidade Estadual Paulista “Júlio de Mesquita Filho” Instituto de Biociências Departamento de Botânica, Av 24A, 1515

At present, the lysosome pathway (LP) and proteasome pathway (PP) are known as major clearance systems in eukaryotic cells. The laticifer, a secretory tissue, degrades some cytoplasm during development. In this study, we investigated the distribution of LP and PP in non-articulated laticifers of Euphorbia helioscopia L. Electron microscopy revealed that, plastids, mitochondria and some cyotsol were degraded in the late development laticifers, where there were numerous vesicles originated from dicytosomes. Accordingly, some key proteins in LP and PP were detected in E. helioscopia latex using isobaric tags for relative and absolute quantitation (iTRAQ) proteomics. Further immunohistochemistry analysis revealed that the clathrin heavy chain (CHC) belonging to LP and the ubiquitin-mediated proteasome degradation increases gradually as the laticifer develops. Immuno-electron microscopy revealed that the cysteine protease, CHC and AP-2 complex subunit beta-1 belonging to LP were mainly distributed in vesicles deriving from dicytosomes, which we called lysosome-like vesicles. Ubiquitin was widely distributed in the cytosol, and proteasome activity was significantly reduced when various concentrations of the inhibitor MG132 were added to the latex total protein. We hypothesize that LP and PP are distributed in E. helioscopia laticifers; and it was speculated that LP and PP might be involved in the degradation of organelles and some cytoplasmic matrix in E. helioscopia laticifers.

SWEET proteins play an indispensable role as a sugar efflux transporter in plant development and stress responses. The SWEET genes have previously been characterized in several plants. Here, we present a comprehensive analysis of this gene family in the rubber tree, Hevea brasiliensis. There are 36 members of the SWEET gene family in this species, making it one of the largest families in plant genomes sequenced so far. Structure and phylogeny analyses of these genes in Hevea and in other species demonstrated broad evolutionary conservation. RNA-seq analyses revealed that SWEET2, 16, and 17 might represent the main evolutionary direction of SWEET genes in plants. Our results in Hevea suggested the involvement of HbSWEET1a, 2e, 2f, and 3b in phloem loading, HbSWEET10a and 16b in laticifer sugar transport, and HbSWEET9a in nectary-specific sugar transport. Parallel studies of RNA-seq analyses extended to three other plant species (Manihot esculenta, Populus trichocarpa, and Arabidopsis thaliana) produced findings which implicated MeSWEET10a, 3a, and 15b in M. esculenta storage root development, and the involvement of PtSWEET16b and PtSWEET16d in P. trichocarpa xylem development. RT-qPCR results further revealed that HbSWEET10a, 16b, and 1a play important roles in phloem sugar transport. The results from this study provide a foundation not only for further investigation into the functionality of the SWEET gene family in Hevea, especially in its sugar transport for latex production, but also for related studies of this gene family in the plant kingdom.

The extraction of high-purity proteins from the washing solution (WS) of rubber particles (also termed latex-producing organelles) from laticifer cells in rubber tree for proteomic analysis is challenging due to the low concentration of proteins in the WS. Recent studies have revealed that proteins in the WS might play crucial roles in natural rubber biosynthesis. To further examine the involvement of these proteins in natural rubber biosynthesis, we designed an efficiency method to extract high-purity WS proteins. We improved our current borax and phenol-based method by adding reextraction steps with phenol (REP) to improve the yield from low protein concentration samples. With this new method, we extracted WS proteins that were suitable for proteomics. Indeed, compared to the original borax and phenol-based method, the REP method improved both the quality and quantity of isolated proteins. By repeatedly extracting from low protein concentration solutions using the same small amount of phenol, the REP method yielded enough protein of sufficiently high-quality from starting samples containing less than 0.02 mg of proteins per milliliter. This method was successfully applied to extract the rubber particle proteins from the WS of natural rubber latex samples. The REP-extracted WS proteins were resolved by 2DE, and 28 proteins were positively identified by MS. This method has the potential to become widely used for the extraction of proteins from low protein concentration solutions for proteomic analysis.

The plant secondary metabolites glucosinolates (GSL) have important functions in plant resistance to herbivores and pathogens. We identified all major GSL that accumulated in S-cells in Arabidopsis by MALDI-TOF MS, and estimated by LC-MS that the total GSL concentration in these cells is >130 mM. The precise locations of the S-cells outside phloem bundles in rosette and cauline leaves and in flower stalks were visualised using sulphur mapping by cryo-SEM/energy-dispersive X-ray analysis. S-cells contain up to 40% of the total sulphur in flower stalk tissues. S-cells in emerging flower stalks and developing leaf tissues show typical signs of programmed cell death (PCD) or apoptosis, such as chromatin condensation in the nucleus and blebbing of the membranes. TUNEL staining for DNA double-strand breaks confirmed the occurrence of PCD in S-cells in post-meristematic tissues in the flower stalk as well as in the leaf. Our results indicate that S-cells in post-meristematic tissues show an extreme degree of metabolic specialisation in addition to PCD. Accumulation and maintenance of a high concentration of GSL in these cells are accompanied by degradation of a number of cell organelles. The substantial changes in cell composition during S-cell differentiation indicate the importance of this particular GSL-based phloem defence system. The specific anatomy of the S-cells and the ability to accumulate specialised secondary metabolites is similar to that of the non-articulated laticifer cells in latex plants, suggesting a common evolutionary origin.

Efficient sucrose loading in rubber-producing cells (laticifer cells) is essential for retaining rubber productivity in Hevea brasiliensis, but the molecular mechanisms underlying the regulation of this process remain unknown. Here, we functionally characterized a putative Hevea SUT member, HbSUT3, mainly in samples from regularly exploited trees. When expressed in yeast, HbSUT3 encodes a functional sucrose transporter that exhibits high sucrose affinity with a K(m) value of 1.24 mm at pH 4.0, and possesses features typical of sucrose/H(+) symporters. In planta, when compared to the expression of other Hevea SUT genes, HbSUT3 was found to be the predominant member expressed in the rubber-containing cytoplasm (latex) of laticifers. The comparison of HbSUT3 expression among twelve Hevea tissues demonstrates a relatively tissue-specific pattern, i.e. expression primarily in the latex and in female flowers. HbSUT3 expression is induced by the latex stimulator Ethrel (an ethylene generator), and relates to its yield-stimulating effect. Tapping (the act of rubber harvesting) markedly increased the expression of HbSUT3, whereas wounding alone had little effect. Moreover, the expression of HbSUT3 was found to be positively correlated with latex yield. Taken together, our results provide evidence favouring the involvement of HbSUT3 in sucrose loading into laticifers and in rubber productivity.

This study found that the latex capacity (mg latex mg −1 dry weight capsule) of opium poppy capsules is fixed early in capsule development. Latex capacity, which represents the proportion of the capsule wall allocated to laticifers (specialised cells for latex storage), had peaked in the capsule at 1 week after flowering. In contrast, the morphine content of capsules continued to increase with capsule development until commercial harvest. Morphine content was correlated with capsule mass and total latex mass, but there was no correlation between latex capacity and morphine yield. The most important morphological characteristic in terms of morphine end yield (commercial harvest stage) was capsule mass. The findings of this study demonstrate that although latex yield per plant is a highly heritable morphological characteristic, it may have limited potential for use in a breeding strategy aimed at increasing the morphine yield from capsules.

The plant kingdom has elaborated several conducting systems. Three are primarily for mass transport: the aerenchyma (for gas exchange in submerged parts), the phloem (for exchange of nutrients within the plant), and the xylem (largely for transport of water from soil to transpiring leaves). Two others are believed to be primarily defensive and to store under pressure aversive contents which they exude when punctured: the laticifer and the secretory duct. This review provides for the latter two systems the highlights of what is known about their general physiology and ecophysiology but not their metabolism and their molecular biology. It is argued that, given the importance of laticifers and secretory ducts to plant defense against insect herbivory, these structures are under-investigated and deserve more intensive study.

Extraction of banana juice using the traditional physical method is limited to Mbidde banana cultivars. Most other banana cultivars (including Matooke cultivars) do not produce juice by this method. A study of the microstructure and tannin content of banana fruit (Mbidde and Matooke cultivars) was carried out in order to understand the underlying factors in the juice extraction process and develop a working hypothesis to explain the mechanism of juice separation. Microscopic examination of pulp sections of Mbidde bananas revealed that their microstructure contained many more intensely stained and larger laticifer cells than pulp tissues of Matooke cultivars. On ripening, the intensity of staining of laticifer cells in Mbidde fruit appeared to increase, while there was limited or no staining in fruit of Matooke cultivars. There was also higher tannin content in Mbidde than in Matooke bananas at both unripe and ripe stages. The high tannin concentration corresponding to the unique presence of high numbers of laticifer cells in fruit of Mbidde cultivars suggests that the ability of the bananas to release juice is linked to tannins. During juice extraction the physical working of the banana pulp ruptures the laticifer cells, resulting in the release of tannins that interact with celluloses, hemicelluloses, proteins and, most importantly, pectins to form insoluble complexes, thus facilitating juice release.

The opium poppy (Papaver somniferum) belongs to the group of latex-containing plants. Latex is the milky-like fluid within laticifer cells. In this study, poppy latex was analyzed with respect to ultrastructure, alkaloid, and protein content. The main goal of this project was the examination of the proteins by two-dimensional gel electrophoresis. In a proteomics approach, we investigated two main fractions of the latex, namely the cytosolic serum and the sedimented fraction containing the alkaloid-accumulating vesicles. Of the serum, representing the protein-rich part of the latex, 75 spots were analyzed by internal peptide microsequencing, followed by a database searching. For 69 proteins a function could be assigned due to homology to known proteins, whereas six spots could not be identified. Furthermore, codeinone reductase, a representative of the specific enzyme system in morphine biosynthesis, could be detected within the cytosolic serum fraction. In the vesicle-containing pellet, 23 protein spots were analyzed. An attempt was also made to separate the vesicle pellet by density centrifugation, followed by investigation of the alkaloid content, ultrastructure, and protein pattern. This study describes the first database of soluble proteins present in the latex of P. somniferum

Himatanthus drasticus (Apocynaceae), a laticifer plant, is popularly known as janaguba. This latex has been widely used in traditional folk medicine to treat diseases such cancer and ulcer. This st...

The purpose of our work was to investigate the functioning of K + channels in protoplasts of laticifers of Hevea brasiliensis Muell. Arg., anastomosed into a network devoid of large central vacuoles, after tapping stress. Physiological functions such as proton pump activity and uptake of sucrose (a rubber precursor) were maintained, when the voltage-clamp method was used in vivo to record the whole-cell K + current during the stress response. A time-dependent inward current was induced in 50 mM KCl and rapidly inactivated (about 100 ms). The activation potential of this inward K + channel was not closely dependent on E K . This would be coherent with the 'valve model' of Schroeder and Fang (1991, Proc. Natl. Acad. Sci. USA 88 :11583-11587) involving the activation of a H + -pump accounting for the K + uptake observed in laticiferous cells under stress. The activation half-time of outward currents was clearly voltage dependent : from about 350 to 60 ms for 125 and 155 mV, respectively. Time-dependent outward current sensitivity to 5 mM BaCl 2 or CaCl 2 or to 5 μM Erythrosin B showed that the K + channels could be Ca 2+ -dependent. Because of the positive values of the activation potential of the outward current, the possibility opens that an action potential exists, these cells being specialized for stress response.

A comparative and phylogenetic significance of colleters of 19 members of the family Apocynaceae are studied on the basis of both morphological and anatomical features. Colleters are present on the adaxial side of petiole, bract, bracteole, calyx and corolla. In many members colleters are intermingled with petiolar hairs. Structurally a colleter consists of a central core of parenchyma cells surrounded by radially elongated epithelial cells. Besides the normal structure, unicellular epithelial hair, sub-epidermal cells, vasculature and laticifer are also observed in the colleter tissue of various genera. It is considered that the colleters are multicellular secretory structures protecting the meristem. Auf der Basis morphologischer und anatomischer Eigenschaften wurde die Bedeutung von Kollateren zum Vergleich und in der Phylogenie von 19 Gattungen der Familie Apocynaceae untersucht. Kollateren sind auf der adaxialen Seite der Blattstiele, der Brakteen, der Brakteolen, dem Kelch und der Krone vorhanden. Bei etlichen Gattungen sind die Kollateren mit den Blattstiel-Haaren vermischt. Strukturell besteht ein Kollater aus dem zentralen Kern der Parenchymzellen, umgeben von radial gestreckten Epithelzellen. Neben, der normalen Struktur wurden im Kollateren-Gewebe verschiedener Gattungen einzellige Epithelhaare, subepidermale Zellen. Gefase und Laticifer beobachtet. Die Kollateren werden als mehrzellige Sekretionsstrukturen. die das Meristem schutzen, gedeutet.

Non-inactivating outward rectifying K+ channel currents have been identified in a variety of plant cell types and species. The present study of laticifer protoplasts from Hevea brasiliensis, cells which are specialized for stress response, has revealed, through a switch-clamp method, an outward rectifying current displaying rapid inactivation. The inactivation depended on the external K+ concentration and on the voltage. This current inactivation appeared clearly different from all those previously described in plant cells and it shared homology with current kinetics of animal Shaker family channels. These results, given the recent cloning of plant K+ channel β-subunits, shed new light on possible plant K+ channel regulation.

Latex is a frequently milky plant exudate, often containing rubber, found in specialized cells called laticifers. Latex from Hevea brasiliensis is commercially important as the source of natural rubber. Keywords: latex; laticifer; rubber; polyisoprenoid

Latex isolated from laticifer cells of Euphorbia lathyris maintained its ability to synthesize triterpenols (and their esters) from acetate. When the latex was centrifugated at 5 000 g for 15 min. this biosynthetic activity could be subdivided into two separate fractions; the acetate to β-hydroxymethyl glutaryl-coenzyme A activity remained in the supernatant, while the β-hydroxymethyl glutaryl-coenzyme A to triterpenol activity was pelleted. Further purification of the pellet by isopycnic centrifugation on Percoll gradients yielded at least three particles: latex particles, starch grains, and a single membrane-bound organelle. Electron micrographs were made of all of these latex particles. The single membrane-bound organelle was only observed in the region of the density gradient that exhibited the ability to incorporate mevalonic acid into the triterpenoids. In addition, the enzyme β-hydroxymethyl glutaryl-coenzyme A reductase (EC 1.1.1.34) was found in the 5000 g pellet, while β-hydroxymethyl glutaryl-coenzyme A lyase (EC 4.1.3.4) remained in the supernatant.

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.

Laticifer starch accumulation was compared to laticifer growth for developing leaves of Euphorbia pulcherrima Willd. (poinsettia). Measurements of the laticifer-specific triterpenol, cycloartenol, in latex and in whole leaf extracts were used to calculate the total latex volume in leaves of different developmental stages. Latex volume and starch concentration in the latex were used to determine total laticifer starch and to compare laticifer growth and starch synthesis. Young leaves contained the highest latex and laticifer starch contents on dry wt and leaf area bases. In older expanding leaves, laticifer growth produced an increase in total latex volume accompanied by an increase in total laticifer starch. Laticifer growth and starch accumulation stopped upon cessation of leaf expansion. Starch concentration was similar in latex from all leaves, but differed between plant organs. Thus, laticifer starch accumulation correlated with laticifer growth, but mobilization of the starch out of the laticifer was not observed in old or senescent leaves. This is evidence that laticifer starch grains function within the laticifer independently of degradation or export to other cell types. STARCH GRAINS in latex of Euphorbia differ in their metabolic processes and biophysical-biochemical properties from grains of other plant cells (Biesboer and Mahlberg, 1978; Spilatro and Mahlberg, 1985). The period of laticifer starch grain accumulation in relation to growth and development of the laticifer is unclear. If starch grain formation only occurs in the laticifer tip, then the total starch content should be established prior to the expansion phase of laticifer and organ growth, as for a leaf. In this

A technique for the partial purification of a-amylases from latex of Euphorhbia heterophylla, E. marginata, and E. tirucalli is described. The enzymes were found to be similar to other higher plant amylases using the criteria of molecular weight, pH characteristics, kinetics, number of isozymes, and blue value-reducing value patterns. Carbohydrases other than a-amylases were not detectable in latex. The amylases were employed to examine their capacity to digest latex starch grains which are common components of the laticiferous cell in this genus. Laticifer starch grains are not susceptible to in vitro amylolysis. Removal of the starch grain membrane with Triton X-100, damaging the grain, or treating the grains with a-amylases from diverse biological sources had little effect upon hydrolysis. Grains incubated with pullulanase followed by a-amylase caused a slight but significant increase in hydrolysis of raw laticifer starch grains. These studies indicate that the nonarticulated laticifer in Euphorbia is a cul de sacc for certain primary and secondary metabolic products and that the indigestible and morphologically complex starch grains in the latex have evolved to function in a secondary role within the laticifer. MANY SPECIALIZED PLANT products have been identified in the latex exudate of the nonarticulated laticifer in the genus Euiphor-biai. These products include rubber, alkanes, glycerides (Nielsen et al., 1977), C28-C30 triterpenes and their esters (Ponsinet and Ourisson, 1968), various polyfunctional polycyclic diterpenes and cryptic irritants, the fatty acid esters of which are cocarcinogenic (Hecker, 1975), and starch (Mahlberg, 1975). Although much attention has been directed toward isolation and identification of these ergastic substances, very little is known about the enzymatic or structural proteins present in latex. In a previous paper we studied the accumulation and degradation of starch in Euphorbia, and it was determined that laticifer starch grains form a metabolically inaccessible polysaccaride pool in the specialized cytoplasm of the laticifer (Biesboer and Mahlberg, 1978). We noted that a-amylases, which can effect a rapid fragmentation of the entire starch molecule by cleaving a-D-(1 -> 4) linkages, also occurred in substantial quantities in latex. The purpose of the present study is to: 1) de' Received for publication 24 March 1980; revision accepted 11 June 1980. 2 Present address: Dept. of Botany, 220 Biological Sciences Center, University of Minnesota, St. Paul, Minnesota 55108. The authors thank D. McCracken, Illinois State University, for his suggestions during the course of the experimental work. This study was supported by NSF grant DEB-78-02045. scribe a general technique for the partial purification of active enzymes from latex; 2) clarify what carbohydrases besides a-amylase (1 ,4-glucan-4-glucanohydrolase, EC 3.2.1.1.) are present in latex; 3) compare and partially characterize a-amylases from latex of several selected species of Euphorbia, and 4) determine if latex starch grains are susceptible to a-amylase digestion. MATERIALS AND METHODS-Sources of latex-Latex was taken from greenhouse or garden grown populations of Euphorbia heterophylla L., E. marginata Pursh., and E. tirucalli L., by scarification of stem or petiolar regions of the plants. Exuded latex was collected directly into graduated vials containing cold 0.1 M citrate-phosphate buffer (pH 5.9) and 0.2% calcium acetate (buffer; latex, 1:1) and maintained at 2 C. Purification procedure-The cold latexbuffer suspension was centrifuged at 30,000 x g for 1 hr or slightly longer until three distinct fractions appeared in the centrifuge tube. The hard, upper rubber layer and milky-white lower layer were carefully removed and discarded. The remaining clear latex serum fraction was retained, and cold acetone (-5 C) was added to it (2:1, v/v) to precipitate the proteins. The resulting precipitate, separated from the remaining serum by centrifugation, was air dried and subsequently dissolved in cold 0.2% calcium acetate in 0.1 M citrate-phosphate buffer

The ultrastructure of developing and mature nonarticulated laticifers in Asclepias syriaca L. (the common milkweed) was studied by conventional fixation and staining techniques and by osmium impregnation techniques. The mature laticifer protoplast in A. syriaca possesses a large central vacuole with an intact vacuolar membrane. Formation of this vacuole apparently results from dilation and subsequent enlargement of endoplasmic reticulum and possibly in part by fusion of smaller vacuoles and limited cellular-lytic autophagy. Widespread digestion or autophagy of cytoplasm within vacuoles is not evident. Nuclei, mitochondria, dictyosomes, and small vesicles are the most prominent components distributed in the peripheral cytoplasm. Plastids appear to degenerate as the laticifer matures. The specialized cellular component, latex, which is the vacuolar content of the laticifer, is interpreted to be produced in the cytoplasm and subsequently incorporated into the large central vacuole. Rubber globules, the most prominent latex component, are surrounded by a membrane that does not have a trilaminate structure. Globules are associated with an electron-dense fibrillar component in the vacuole.

Anatomical examination of 37 species of Jatropha (Euphorbiaceae) has revealed the occurrence of two distinct types of laticifers-articulated, and nonarticulated-in addition to idioblasts that are irregularly shaped individual cells. With the notable exception of J. augustii Pax & K. Hoffm. (section Peltatae (Pax) Dehgan & Webster), these idioblasts occur in the two most advanced sections of the subgenus Curcas (Adans.) Pax, namely Loureira (Cav.) Muell. Arg. ex Pax and Mozinna (Ortega) Pax. The presence of two laticifer types and "idioblastic laticifers" in the genus, in association with the morphological reduction series found with evolutionary advancement are, therefore, significant in the delimitation of sections and subsections. Further evidence is presented by the occurrence of 'chambered crystalliferous cells" in subgenus Curcas, but not in subgenus Jatropha (=Adenorhopium Griseb.). Reexamination of laticifers in other genera of the Euphorbiaceae is suggested as a possible means of alleviating the long-standing taxonomic dilemma of this large and morphologically diverse

Plastids were observed in all stages of laticifer differentiation in Papaver somniferum L. Plastids in laticifer initials were present as proplastids that later developed electron-dense inclusions, but never possessed the thylakoids or starch grains that characterize chloroplasts in other cells. Electrondense inclusions in laticifer plastids were membrane-bound and appeared to arise from the accumulation of material within an invagination of the inner plastid membrane. Cytochemical studies of these plastid inclusions indicated that their matrix was not composed of crystalline protein, a-amylose, amylopectin or polysaccharide. The results suggest that the electron-dense, membrane-bound inclusions in laticifer plastids may be composed of lipoprotein. PREVIOUS ULTRASTRUCTURAL STUDIES of laticifers in Papaver somniferum L. (Nessler, 1976; Thureson-Klein, 1970) and its latex have re

The development and structure of the laticifers in several species of the section Subhydrochylus of the genus Mammillaria (Cactaceae) were examined. These laticifers were found to be similar to those of the section Mammillaria in that both types develop from the complete lysis of several rows of parenchyma cells, and both types consist of long, branching, tubular lumens which are lined by epithelia. The laticifers of the section Subhydrochylus differ from those of the section Mammillaria in that those of the former are more irregular in shape, lumen development, and epithelium form. Also, the Subhydrochylus laticifers occur only as a single ring in the outermost cortex and tubercle bases, whereas those of section Mammillaria can be found in pith, medullary rays, cortex and throughout the tubercles. Because the structure of the laticifers in the section Mammillaria is much more regular and orderly, it is postulated that they are the derived type and that the laticifers of the section Subhydrochylus more closely resemble the ancestral condition. Two species, M. elegans and M. tegelbergiana, were found to be intermediate in nature, having characteristics of both types of laticifer systems. Solisia pectinata was found to have laticifers similar to those in section Subhydro

The micromorphology of starch grains in the nonarticulated laticifer was examined in 200 taxa of succulent and nonsucculent Euphorbia. Rod, spindle, osteoid, discoid and intermediate shapes were found in latex. These unusual starch grain shapes are assumed derivatives from more rounded grains present in other angiospermous cells. Unbranched, rod–shaped grains were present in latex of annual and perennial herbaceous taxa. Spindle, osteoid, and discoid shapes, often branched, occurred in xerophytic, succulent taxa. Leafy taxa in several subgenera possessed rod–shaped grains. Taxa of the primitive subgenus, Esula, possessed the shortest rod–shaped grains derived from rounded grains common in parenchyma. Length of rod–shaped grains increased through herbaceous subgenera and culminated in Poinsettia where grain length may be extremely great. Dwarf or shrubby succulents with thickened roots have osteoid grains. Shrubby or arborescent coralline and cactiform species, specialized taxa, possessed large branched grains. Grain morphology of Euphorbia was compared with that from latex of other genera of the Euphorbiaceae. These in–depth analyses of laticifer plastids demonstrates the applicability of starch grain morphology for interpreting the progressive evolution of the nonarticulated laticifer as a cell type within the genus Euphorbia.

Comparative anatomical studies of the mature stems of two species each of Trichipteris and Cyathea (Cyatheaceae) are described. The outermost boundary of the stem is typically a twolayered hypodermis. Mucilage-sac cells are randomly distributed in all parenchymatous areas of the stem and form articulated laticifer systems. Localized areas of sclerenchyma tissue occur in the cortex of both T. microphylla and C. suprastrigosa. All species studied possess medullary bundles, whereas cortical bundles are found only in T. trichiata. Accessory bundles occasionally are associated with indentations in the internal stelar sheath of T. trichiata. The stelar pattern in each genus is a dictyostele and consists of individual meristeles. Distinctive cubical cells typically occur wherever sclerenchymatous fibers and parenchyma cells abut one another. Tangential cells occur within the primary phloem of each meristele, and occasionally within the larger accessory bundles. The primary xylem of the adventitious roots is typically diarch, although triarch and tetrarch xylem may occur. Leaf traces and petiole strands are similar anatomically to the accessory bundles. Based upon this study Trichipteris and Cyathea show striking anatomical similarities, and appear to be closely-related taxa. ALTHOUGH MORPHOLOGICAL similarities exist, Trichipteris (90 species) and Cyathea (110 species) represent separate evolutionary lines. Tryon (1970) revised the classification of the family Cyatheaceae and recognized three basic evolutionary lines among the squamate genera. The genera Trichipteris and Cyathea (together with Cnemidaria) possess structurally marginate petiole scales without apical setae and constitute one evolutionary line. Three groups of species indicate a close relationship between these two genera, based upon lamina architecture, venation, petiole and lamina length, and cytology (Tryon, 1970). Characters of paraphyses and details of petiole scales also indicate evolutionary affinity between these two taxa. The exindusiate Trichipteris gave rise to the indusiate Cyathea through the independent development of an indusium.

Laticifers of Papaver bracteatum Lindl., population Arya II, seedlings were examined by electron microscopy. Laticifers were first differentiated in procambium of the radicle associated with phloem about 72 hr after seeds were sown. Proliferation of membrane-bound vesicles of apparent endoplasmic reticulum origin distinguished laticifers from adjacent cells. Vesicles developed electron-dense caps from the internal condensation of small particles. Laticifer initials possessed the usual complement of organelles that became obscured in mature cells by the large, closely packed vesicles. Plastids contained an electron-dense, membrane-bound inclusion, but never developed lamellae or starch grains. Articulation and anastomoses between laticifer elements resulted from gradual removal of wall materials by both cells on opposite sides of the common walls at a perforation site. Differentiation of the laticifer initials and the micromorphology of the protoplast of P. bracteatum is similar to that reported for P. somniferum. THE ARTICULATED character of laticifers in the Papaveraceae was recognized in the classical literature. Members of this family show a wide range in development of this cell which may be of importance for determining the pattern of alkaloid accumulation frequently associated with this cell and for interpreting the phylogenetic significance of laticifers in the family (Esau, 1965). The laticifer system of Papaver bracteatum Lindl. was recognized early as being of the articulated, anastomosing type (Trecul, 1865), whereas this system in Chelidonium is nonanastomosing in character (Schacht, 1851). Sanguinaria is reported to develop perforations between laticifer members in the stem, but not in the leaf (Holm, 1908). Glaucium and Eschscholzia possess articulated laticifers that lack perforations between laticifer members (Leger, 1894

Development of alkaloidal vesicles in laticifers of opium poppy, Papaver somniferum L., was investigated at the ultrastructural level. Laticifer initials possessed abundant endoplasmic reticulum throughout their dense cytoplasm. During differentiation the endoplasmic reticulum organized into long, folded sheets that were parallel to the longitudinal walls along the periphery of the cell. Vesicles appeared to be derived from dilation of endoplasmic reticulum. This relationship was confirmed through cytochemical data obtained with zinc iodide-osmium tetroxide and osmium tetroxide impregnation. Alkaloidal vesicles had electron-dense regions or caps that occurred early in laticifer differentiation, but these caps became less conspicuous in mature cells. Caps appeared to be derived from small particles which condensed along the inner surface of the vesicle membrane and subsequently accumulated at one or two positions along the membrane of the vesicle. LATICIFERS in opium poppy, Papaver somniferum L., can be distinguished from other cell types at the ultrastructural level by the large, irregularly shaped vesicles that fill the mature cell. Origin of these vesicles from endoplasmic reticulum was first suggested by Sarkany et al. (1964) in an abstract unaccompanied by micrographs. In a more detailed report, Thureson-Klein (1970) described abundant, elongated profiles of endoplasmic reticulum in undifferentiated poppy laticifers that appeared to give rise to vesicles through local distension. The bottom fraction of ultracentrifuged latex from P. somniferum has been shown to consist of vesicles that contained alkaloids within their vacuolar plasm (Fairbairn, Hakim, and El Kheir, 1974). Dickenson and Fairbairn (1975) investigated the ultrastructure of these "alkaloidal vesicles" in both ultracentrifuged latex and intact plants. In the present investigation the ontogeny of alkaloidal vesicles in laticifers of Papaver somniferum is described and cytochemical evidence is presented for their origin from endoplasmic reticulum. MATERIALS AND METHODS-Plants of Papaver somniferum L. cv. "Neuga" were grown from seed in the greenhouse. Seeds were also germinated in glass jars on cheese cloth discs supported by filter paper cylinders to obtain straight radicles that were uncontaminated with soil particles. 'Received for publication 9 September 1976; revision accepted 2 February 1977. This research was supported by a grant from the United Nations, UN 444-75, and the Indiana University Foundation. D. E. A. Registration No. PM0084537. Tissue processing for general electron microscopy-For general electron microscopy, tissues from various portions of plant axes and seedlings were fixed for 8-12 h at room temperature in one of the following fixatives: 1) 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2; 2) 1% glutaraldehyde, 3 % paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 (Karnovsky, 1965); or 3) 4% glutaraldehyde, 0.7 % picric acid in 0.1 M sodium cacodylate buffer, pH 7.0 (Turner, 1970). After several rinses with 0.05 M sodium cacodylate, pH 7.0, tissues were post-fixed by immersion in 1 % osmium tetroxide in 0.05 M sodium cacodylate, pH 7.0, for 2 h, rinsed with glassdistilled water and prestained in 1 % aqueous uranyl acetate at 4 C for 6-12 h. Tissues were then rinsed with glass-distilled water, dehydrated through an ethanol-acetone series and embedded in Spurr's (1969) low-viscosity resin. Some tissues were fixed in 1 % osmium tetroxide in 0.1 M phosphate buffer, pH 7.2, for 2 h and dehydrated and embedded as described above. Tissue processing for cytochemical electron microscopy-Procedures described above were modified for cytochemical electron microscopic investigations as follows: 1) Zinc iodide-osmium tetroxide (ZIO) impregnation. Tissues were fixed in the dark for 24 h in a zinc iodide-osmium tetroxide solution as described by Niebauer et al. (1969). ZIO fixed tissues were rinsed with glass-distilled water, prestained with uranyl acetate, dehydrated and embedded as above. A modification of this procedure consisted of treating tissues with Turner (1970) fixative for 8-16 h before ZIO impregnation.

Although the laticifers of several species of Mammillaria can technically be classified as being of the articulated type, they differ significantly from all other reported articulated laticifers. They are derived from cells which differentiate only in older tissues, never in meristematic or young regions. The development involves the complete lysis of masses of cells, not just the perforation or resorption of the end walls in a single file of cells. At maturity, the laticifer lumen is lined with a one-to-several layered epithelium which may be quite thick. The laticifers increase in diameter with age, apparently by the lysis of the inner epithelial cells. Laticifers occur in the pith, cortex and tubercles of the vegetative body but were not observed in the roots, flower parts or in seedlings up to eight months old. Seven species were studied, all of which have 'milky sap," and the laticifers of each were virtually identical to the laticifers of the others. IT HAS LONG been known that laticifers, or at least cells which produce a "milky sap," occur in certain members of the Cactaceae (Lauterbach, 1889). There has been no detailed study of them, although they have been mentioned by Lauterbach (1889), Craig (1945), Buxbaum (1950), and Boke (1960). While examining the apical meristems of certain species of Mammillaria for another study (1978), I noticed that the anatomy of the laticifers in these plants appeared to be quite different from either of the usual types. An investigation of seven species has shown that the laticifer system in MAammillaria is highly unusual, and although it can be classified as articulated, to my knowledge it is not like any other reported articulated laticifer. MATERIALS AND METHODS-The genus Mammillaria is a large group which contains ca. 320 species (Britton and Rose, 1923; Craig, 1945). They are divided into three groups: 1) those which have "milky sap," 2) those which have "semi-milky sap," and 3) those which have "watery sap." For this study, seven species were selected which all have milky sap. This represents a sample of ca. 6% of the 113 species in this group. The material was fixed in either purified glutaraldehyde (prepared as described previously: Mauseth, 1976) or Randolph's modified Navashin solution (Johansen, 1940). After fixation, the ' Received for publication 2 August 1977; revision accepted 29 November 1977. Support for this work was provided in part by Grant PCM 76-21492 from the National Science Foundation and by Grant 14-7499-0225-URI from The University Research Institute of The University of Texas. material was handled with normal paraffin methods (Johansen, 1940), then stained with Heidenhain's haematoxylin and Orange G (Mauseth, 1976). To avoid tearing the delicate walls which are present at some stages of development, many samples were embedded in Epon plastic, microtomed at I pm on glass knives, and stained with Richardson's stain (Mauseth, 1977). RESULTs-Distribution of laticifers in the plant body-Laticifers occur in both the pith and the cortex of the vegetative shoot, but they have not been found in, or associated with, either the primary or the secondary vascular tissues. The predominant orientation of the laticifers is vertical, but lateral branches which extend through the medullary rays connect the pith laticifers with those of the cortex. The laticifers do not extend to the vicinity of the shoot apical meristem, but terminate, at least in Mammillaria magnimamma, 5,000 ,um below the youngest leaf primordium. The leaves of Mammillaria are extremely reduced and are soon disrupted by tubercle growth (a tubercle is a highly modified leaf base: Buxbaum, 1950; Fig. 1, 2); thus, laticifers do not occur in leaves. Likewise, the roots and flowers (fruits were not available) do not contain laticifers. Seeds and seedlings up to eight months old of Mammillaria compressa, M. brandegeei, and M. geminispina were examined; none had yet developed laticifers. The tubercles are richly supplied with laticifers that connect with those of the cortex (Fig. 4, 5). Because laticifers do not extend into the apical regions of the plant, tubercle primordia and young tubercles are without laticifers. In all cases examined, tracheary elements and spine

The morphology of plastid starch grains from several succulent and nonsucculent species of Euphorbia was examined in parenchyma and in non-articulated laticifiers. Several classes of grains were identified: small oblong-round, rod, somewhat osteoid, osteoid, lobed osteoid, discoid, and round grains. Parenchyma possessed only small oblong-round grains, whereas grains of different morphology were present in laticifers. Different species of this genus can be characterized by the morphology of the large, or mature, starch grains present in the laticifer. The rod-shaped grain, which was somewhat wider at the midregion than at the ends, was present in several nonsucculent forms, E. terracina, E. pulcherrima and E. heterophylla. The somewhat osteoid grain was represented by the succulent species E. viguieri, E. milii, and E. nzauritanica, where the mature grains developed somewhat enlarged ends. Grains with much enlarged ends were represented in the succulent species of E. abyssinica, E. pseudocactus, and E. tirucalli. Alteration of the pattern of starch deposition in which several lobes were formed at the ends of the grain has given rise to a lobed osteoid class (E. inconstantia). Lobes also may be formed with greater frequency along the midregion of the grain in this than in other species. Euphorbia lactea had the most complex grain in which lobing was frequent at the ends as well as along the midregion, resulting in a large discoid grain. Subclasses in which grains differed significantly in length between species were evident in all classes containing several species. The average length of grains in any subclass was similar for subclasses between the classes. The study suggests that the elongated grain of the laticifer was derived from the round or oblong grain present in the more primitive parenchymatous cell. Progressive changes in the pattern of starch deposition have given rise to osteoid and discoid grains of increasing morphological complexity which is interpreted to represent trends in laticifer evolution between different species of Euphorbia as reflected by subtle changes for starch deposition within the plastids of this cell. PLASTIDS DERIVED from the latex of two different species of Euphorbia were shown in a previous report to possess starch grains characteristic for each species (Mahlberg, 1973). The morphology of these starch grains also was observed to differ from those present in other cells within the plant. Although the peculiar shape of these starch grains had been reported in the literature for a few species (Demiriz, 1969; Gaucher, 1902; Molisch, 1901), no investigations have utilized the morphology of the starch grain to interpret the phylogeny of laticifers within this genus. Since these differences for starch grain morphology between the laticifer and other cells in a species, and between laticifers in different species, reflect a control mechanism associated with either the plastid or nuclear genome, it can be hypothesized that the observed morphological changes also reflect phylogenetic changes within the plastid which in turn may reflect subtle changes in the evolution 1 Received for publication 14 June 1974. This research was supported by the Indiana University Foundation, in part, and the Indiana Academy of Scienceof the laticifer between species in this genus. This report describes the morphology of starch grains from eleven species of Euphorbia and supports an interpretation that different species possess a specific grain morphology. The present data are incorporated into a working hypothesis for interpreting the phylogeny of the laticifer in the genus Euphorbia as reflected in the morphology of its starch grains. MATERIALS AND METHODS-The species utilized in this study were maintained under similar conditions in a greenhouse and were adult flowerproducing plants. They included: Euphorbia abyssinica Gmel. (iu 1), E. heterophylla L. (iu 44), E. inconstantia Dyer (iu 46), E. lactea Haw. (iu 48), E. mauritanica L. (iu 58), E. pseudocactus Berger (iu 83), E. pulcherrima Willd. (iu 87), E. milii Desmoul. (iu 62), E. terracina L. (iu 99), E. tirucalli L. (iu 10]) and E. viguieri Denis (iu 106). The iu numbers refer to herbarium vouchers or greenhouse specimens. Latex was collected from the shoot as reported previously (Mahlberg, 1973). However, a '/2-1 hr rather than 12-hr fixation period was adequate.

A B S T RA CT A pectinase with a pH optimum of 5.2 is present in the latex of the common milkweed, Asclepias syriaca L. The enzyme was partially purified from the serum fraction of fresh latex by dialysis and ammonium sulfate fractionation. Enzyme activity was detected by a viscometry assay and by the dinitrosalicylic acid assay for reducing sugars. Pectin and polygalacturonic acid could serve as substrates for the enzyme. Pectolytic activity in latex presents a basis for describing the development of the non-articulated branched laticifer system. Enzyme activity may facilitate intrusive tip growth of the laticifer among other cells by solubilizing pectic substances of the middle lamella and also may be important for loosening wall material of the laticifer itself to facilitate extension growth. THE INTRUSIVE GROWTH and extensive elongation

Thick mats of cellular remains from Eocene brown coal deposits of the Geiseltal near Halle, DDR, were determined to be fossil nonarticulated laticifers. Nuclear magnetic resonance analyses of intact strands showed they consisted of cis-1,4-configuration rubber representing the polymerized isoprenoid contents of individual laticifers. Only remains of laticifers are present; other cells are absent as a result of biodegradation. The long laticifers, often with a surrounding cell wall, retained a tubular shape during their preservation. The isoprenoid content, which filled the entire lumen, possessed a cribriform structural character. The interstices within the rubber represent areas of former protoplasm of the cell. Various configurations in the protoplasm molded by the rubber during the initial phase of fossilization appear as negative images of former nuclei, organelles, and possibly membrane surfaces. The laticifer axes possess branches of several configurations comparable in morphology to those in branched, nonarticulated laticifers in extant plants. Acetone extracts of the rubber contents analyzed by gas-liquid chromatography identified the presence of several hydrocarbons which form a characteristic profile for the laticifer. It is suggested that the distinctive cellular micromorphology, rubber configuration, and hydrocarbon profile of these laticifers can be employed as markers in comparative studies with extant plants to identify the generic or species origin of these laticifers. FOSSIL remains from Eocene brown coal beds were reported in the classical literature as Milchsaftgefasse, or latex vessels (Hartig, 1848). Available information indicates that these cellular remains are limited in distribution to the Geiseltal, near Halle in central Europe, where they occur along with deposits of diverse flora and fauna (Krumbeigel, Ruffle and Haubold,1983). Gothan (1924,1927) supported the interpretation that these remains represented fossilized laticifers. The density and stiScharacteristics of these hairlike masses and their dark color prompted Jurasky (1928,1930) to refer to them inappropriately as Affenhaar, a term which remains in the literature. Kindscher (1924) interpreted the material to be comparable to rubber. Early studies of its composition indicated that the material decomposed upon ozonization to aldehydes and also that it could be vulcanized (Gothan,1927; Jurasky,l928). The origin of these materials has been controversial even from these few reports, and authors have conjecturally associated them with s Received 23 November 1983; revision accepted 3 April 1984. Research performed at the Colorado State University Regional NMR Center was supported by National Science Foundation Grant CHE 78-18581. the purported remains ofthe Apocynaceae, Euphorbiaceae, Moraceae, and Sapotaceae in the Geiseltal deposits (Hartig, 1848; Kirchheimer, 1935). More recently, Gottwald (1976), in a comparative examination of the fossil lignitecontaining laticifers with extant woods, reported the wood anatomy to be similar to the genus Couma (Apocynaceae) and assigned the remains to the form genus, Coumoxylon har

The leaf primoidia of Lactutca sativa 'Meikoningen' develop from a subapical initial in the second layer of the tunica on the side of a flat shoot apex. Subsequent growth of the subsurface lamina is initiated by submarginal initials which divide anticlinally to produce an adaxial layer and abiseriate abaxial layer, and periclinally to produce a middle layer from which procambium differentiates. The protoderm is derived from the first tunica layer by continuous anticlinal divisions. The activity of the subapical and submarginal initials is completed when the leaf is 0.3 mm in length and 4.0 mm in width, respectively. Continued growth of the leaf to 130-150 mm results from intercalary cell division and enlargement. The mature venation is visibly delineated when the leaf is 25-30 mm in length. Laticifer and phloem cells are initiated by the same mother cells in the procambium. The former become non-septate laticifers by resorption of cross walls. They mature concurrently with the phloem and before the xylem. THERE ARE numerous reports concerning various aspects of leaf histogenesis in dicotyledonous plants. The work on foliar venation has been reviewed by Foster (1952), and leaf histogenesis and vascular differentiation by Esau (1965a, b). The latex-containing elements of plants are classified as articulated or non-articulated laticifers. The development of the articulated laticifer, which is the type found in Lactuca sativa, has been studied in the embryo, root, stem and apical meristem in several diverse genera (Scott, 1882, 1884, 1886; Sperlich, 1939; Krotkov, 1945). Descriptions of laticifer structure and leaf development in Lactuca sativa are explained by Hayward (1938). This investigation was undertaken to follow the development of the lamina of Lactuca sativa with emphasis on the ontogeny of the laticifers. The results contribute to an explanation of a physiological breakdown in lettuce (tipburn) resulting from abnormal laticifer development and subsequent rupture in rapidly enlarging leaves (Tibbitts, Struckmeyer, and Rao, 1965; Olson, Tibbitts, and Struckmeyer, 1967). MATERIALS AND METHODS-A small butterhead cultivar of lettuce ,'Meikoningen,' was used in this study. Seeds were planted in well-fertilized peat 1 Received for publication 15 April 1969. This work represents a portion of a dissertation in partial fulfillment of the requirements for the Ph.D. degree in the Department of HorticultuLre, University of Wisconsin. Appreciation is expressed to Dr. Ray Evert for his counsel during the course of this investigation. Published with the permission of the Director, Research Programs Division, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706. 2 Present address: Biology Department, Hiram Scott College, Scottsbluff, Nebraska 69361. soil in gallon containers and placed in a growth chamber with a non-barrier type light bank of both fluorescent and incandescent light at approximately 1,800 ft-c for 16 hr daily. The temperature was maintained at 70 F ?i 2 F day and night and the relative humidity above 80%7. Parts of leaves sampled at different stages of development were prepared for study by paraffiin sectioning and by tissue clearing. The samples for sectioning were killed in FPA, dehydrated with n-butyl alcohol, and infiltrated and embedded in Tissuemat. Paradermal and transverse sections were cut 6-10 , thick and stained with safranin and fast green FCF (Sass, 1958). Two clearing techniques were employed which provided excellent, precise methods for viewing the different layers within the leaf. The lactic acid method of Simpson (1929) was rapid and effective. Immersion of the entire leaf in 75 % lactic acid for 2 to 3 days at 54 C was found most effective. These leaves were not stained. Other leaves were killed in FAA, treated with NaOH and chloral hydrate, stained with safranin and mounted in Harleco's synthetic resin (Brady, Wemple, and Lersten, 1965). RESULTS Initiation and development of leaf primordia-Leaf primordia are initiated on the flanks of a broad, flat shoot apex (Fig. 1). In the second tunica layer a cell becomes a subapical initial. The subapical initial divides periclinally, the outer derivative remaining a subapical initial and the inner one giving rise to the middle tier of cells of the primordium (Fig. 2). The first and second tunica layer grow with the developing primordium by frequent anticlinal divisions. Subsequently, cells of the second tunica layer divide periclinally, adding to the width of the pri

The multinueleate condition in the non-articulated laticifers of embryos of Euphorbia marginata arises as a result of mitosis. Successive stages of mitosis in the nuclei of the laticifer appear in the form of a wave. No sequence of mitotic stages has been noticed in the neighboring longitudinal tiers of cells. This difference in the mitotic pattern in the latieifer and other parenchymatous cells of the embryo suggests that the synthesis of factor (s) responsible for triggering mitosis occurs within the laticifer and does not diffuse to the surrounding cells. The mitotic waves originate distally from the meristems, either in the cotyle~donary or hypocotyl portion of the laticifer, and move uni- or bidirectionally along its longitudinal axis. The mitotic stimulus does not start simultaneously in all the laticifers. The variable velocity of the mitotic substance results in aphasic mitotic waves in laticifers of the same embryo. Mitotic aberrations have not been observed in the dividing nuclei of the laticifer. A chromosome estimation made from a polar view of metaphase does not suggest polyploidization in the observed laticifers. A SALIENT characteristic of the non-articulated laticifer is the development of a multinucleated protoplast. The absence of cytokinesis within this cell is an enigma in differentiation, in particular, when one reflects upon- laticifer ontogeny. This cell becomes distinguishable from other cells of the embryo's meristematic mass upon initiation of the cotyledons at which time the incipient laticifer commences to undergo a somewhat nonpolarized pattern of unlimited cell wall extension. The growing tips of the cell ramify in different directions permeating various tissues of the shoot and root axes, a process which continues throughout the growth of the plant. At the inception of the laticifer a sudden alteration in the mitotic process ensues: the meehanism controlling cell plate formation is deactivated. The nucleus of the laticifer initial undergoes division without concomitant wall formation, a phenomenon which persists during the subsequent development of this cell. The few cytological observations which have

MAHLBERG, P. G. (U. Pittsburgh, Pittsburgh, Pa.) Embryogeny and bistogenesis in Nerium oleander. II. Origin and development of the non-articulated laticifer. Amer. Jour. Bot. 48(1): 90-99. Illus. 1961.-Laticifer initials, collectively considered as a laticifer system, are differentiated in the globular embryo from meristematic cells which occupy a position within the potential procambial tissue. A, total of usually 28 initials, in Nerium oleander, arise as an irregular ring of cells directly below the embryonic shoot apex, during initiation of the cotyledonary primordia. No anastomoses occur between laticifer initials. During subsequent development of the embryo, the laticifer initials grow in a bi-directional manner and penetrate into the root, cotyledons and toward the shoot apex. Upon enlargement the initials bifurcate repeatedly, many branches penetrate into the cotyledons, others grow into the cortex of the hypocotyl or penetrate between cells of the procambium. Repeated nuclear divisions within each initial result in the formation of a multinucleated protoplast in this cell type. The tips of laticifers' occupy intercellular spaces during their growth; they do not penetrate into or through adjacent cells. A plexus of laticifer branches is formed within the cotyledonary node of the mature embryo. No new initials are formed during subsequent growth of the plant, rather certain branches from the cotyledonary nodal plexus penetrate into the enlarging shoot system. The nature of their growth habit and branching suggests that the tips of laticifer initials exhibit an intrusive form of growth.

Cellulase activity was localized at the ultrastructural level in the articulated, anastomosing laticifers of Papaver somniferum.. Electron-dense crystalline deposits indicating the presence of cellulase activity were restricted to discrete patches along the laticifer wall in regions of recently formed perforations. This report presents the first direct evidence for the involvement of cellulases in the cell wall perforation process in articulated laticifers. LATICIFER TYPES are defined on the basis of their mode of origin and development within plants. Two general types have been recognized -articulated and nonarticulated (Hartig, 1862). Articulated laticifers are of compound origin being formed from a series of initials in which adjacent walls may undergo partial or complete perforation. Nonarticulated laticifers, in contrast, originate from single cells that greatly elongate and often become coenocytic through karyokinesis without cytokinesis. Cell wall perforation in the articulated, anastomosing laticifers of the opium poppy, Papaver somniferum L., has been described as a gradual process resulting from the thinning of walls on both sides of the middle lamella at a perforation site (Nessler and Mahlberg, 1977). The progressive bilateral thinning of laticifer