Part-I Development, Histochemistry and Ultrastructure of Gum-resin Ducts in Commiphora mukul Engl

8/4/2019 Part-I Development, Histochemistry and Ultrastructure of Gum-resin Ducts in Commiphora mukul Engl.

1/8

Ann. Bot. 41, 999-1004, 1977

Development, Histochemistry and Ultrastructure ofGum-resin Ducts in Commiphora mukul Engl.

R. c. SETIA, M. v. PARTHASARATHY* and J. J. SHAHDepartment of Botany, Sardar Patel University, Vallabh Vidyanagar, 388120, India, and *Division of; Biological Sciences, Section of Genetics, Development andPhysiology, Cornell University,

I Ithaca, N. Y.I4853, U.sA.Received: 13September 1976

ABSTRACTGum-resin ducts are present in the primary and secondary phloem of Commiphora mukul Engl. The impor-tant gum-resin, known commercially as 'guggul' , is secreted and collected in ducts which develop schizo-genously. The duct init ials have dense cytoplasm, large nuclei, increased cytoplasmic RNA and proteins.The lumen of newly-formed ducts widens accompanied by anticlinal divisions and subsequent tangentialelongation of epithelial cells. Histochemical tests reveal that the epithelial cells have apparently largeamounts of proteins, cytoplasmic RNA, and DNA in the nucleus. Lipid globules are also present in thesecells. Epithelial cell walls in contact with the duct are thin and. of a loose fibrillar mesh. Microtubules,randomly oriented in the epithelia l cells are always parallel and adjacent to the wall. The cytoplasm isrich in ribosomes, endoplasmic reticulum, mitochondria, plastids and vacuoles containing osmiophilicsubstances. At the peripheral region of the duct, electron-transparent bodies containing densely-stainedmaterial are present close to the tangential wall.

INTRODUCTIONThere have been several recent studies on the structure of secretory ducts in plants (Amel-unxen, 1964,1965; Wooding and Northcote, 1965; Ame1unxenand Arbeiter, 1967, 1969;Ame1unxen and Gronau, 1969; Werker and Fahn, 1968; Schnepf, 1969a, b, Fahn andEvert, 1974; Fahn and Benayoun, 1976) but none on the development, histochemistry"andultrastructure of gum-resin ducts in tropical trees. A number of trees of the familyBurseraceae are known to yield gum-resin in ducts which occur both in the bark and wood.Commiphoramukul, an important member of this family, is a source ofIndian Bdellium-a gum-resin which is commercially known as 'guggul'. 'Guggul' is largely used as incense,as a fixative in perfumery and has a wide range of usefulness in medicines (Anonymous,1972).The term gum-resin indicates that it consists of two components, the gum and theresin. The gum consists of the polysaccharide material, and the resin that can be separatedfrom the gum by solvent extraction methods is completely soluble in most of the organicsolvents, castor oil, drying oils and turpentine (Anonymous, 1950).The purpose of the present investigation was to study the mode of development of gum-resin ducts on the basis of broad histological and histochemical changes; the ultrastruc-ture ofmature gum-resin ducts and the possible site of origin of gum-resin in the epithelialcells of these ducts.

MA n!RIALS AND METHODSThe young stemand the bark ofCommiphora mukul were collected from Mahi river ravinesin Gujerat State, India. For light microscopic studies the plant material was fixed in FAA(Sass, 1958),dehydrated using a TBA seriesand embedded in Embeddol (Harleco, U.S:A.).Serial sections, transverse and longitudinal, ten microns thick, were cut on a rotary micro-


2/8

1000 Setia, Parthasarathy and Shahtome. Staining techniques used for these sections and sections from the fresh materialwere: Safranin and fast green (Jensen, 1962),and tannic acid-ferric chloride and safranin-fast green combination (Foster, 1934)for general histology, Azure B for DNA and RNA,periodic and Schiff's (PAS) reaction for insoluble polysaccharides, ruthenium red forpectic material, Sudan black B for lipids, I2KI for starch and I2KI and H2S04 for cellulose(Jensen, 1962). For general proteins mercuric bromphenol blue (Mazia, Brewer andAlfert, 1953),and for suberin and resin Sudan IV (Werker and Fahn, 1969)were used.For electron microscopy the plant material was fixed in 5 per cent glutaraldehyde andpost-fixed in osmium tetroxide. The tissues were dehydrated in an acetone series andembedded in Epon 812 embedding medium. Sections ofthe material were obtained with aReichert OMU2 ultramicrotome, stained with uranyl acetate and lead citrate, viewed andphotographed with a Philips EM300 or EM201 electronmicroscope. One micron thicksections were stained with toluidine blue 0 at pH 68(O'Brien, Feder and McCully, 1964).

RESULTSIn transverse sections of young Commiphora mukul stem, round to oval gum-resin ductsare seen distributed irregularly in the phloem (Plate 1A).They are oriented parallel to thelongitudinal axis and anastomose in the tangential planes. In the bark, the irregularlyarranged ducts form a network. Each duct islined by a singlelayer of tangentially-flattenedcells (Plate IB). As the resin is soluble in most of the organic solvents, it presumablydissolves out during processing of the plant material for microtomy, but the sites of itspresence appear as empty round spaces in the lumen of the duct (Plate 1B).The poly-saccharide material, i.e. the gum component of the gum-resin, appears densely stained(Plate 1B).Sections from fresh plant material, when stained witha1coholic solution ofSudan IV, show that the resin droplets are stained red in the lumen of the duct (Plate Ie).With PAS reaction a part of the substance in the duct and bordering cells is stained red(Plate 1D).With ruthenium red some of the substance in the epithelial cells is stained red.Thus the accumulated material in the duct and the cells surrounding it consists of resindroplets and polysaccharide material.

DEVELOPMENT OF GUM-RESIN DUCTIn the young stem, gum-resin ducts develop from the procambium in the phloem region,while in the stem showing secondary growth they develop in the cambial zone towards thephloem. The duct initials are early recognized by their dense stainability, and large nuclei(Plate IE). Specific staining with mercuric bromphenol blue and Azure B indicates anapparent increase in the amount of proteins (Plate IF) and cytoplasmic RNA (Plate 2A).The region where the walls of contiguous duct initials meet is the first region to show theduct initiation. As a result of breakdown of the middle lamella (indicated by rutheniumred staining) at this site an intercellular space is formed (Plate 2B).The intercellular spacefurther expands and the cells lining it become epithelial cells (Plate 2e, D). Thus the modeof development of a gum-resin duct is schizogenous.The widening of a newly-formed duct occurs probably due to the pressure exerted onthe epithelial cells by the gum-resin which gets accumulated in the duct. This may beaccompanied by anticlinal divisions of the epithelial cells and their subsequent tangentialelongation (Plate 2E).Due mainly to the pressure of the duct contents the epithelial cellsappear compressed and narrow (Plate 1B).The epithelial cellsmay sometimes break downand the cell contents are released into the duct (Plate 2F). Sometimes the epithelial cellsappear separated from one another along their radial walls (Plate 1B,arrow head). Inboth cases, underlying cells lining the duct behave as epithelial cells (Plate 2G).


3/8

Gum-resinDucts in Commiphora mukal Engl. 1001The epithelial cells show an apparently increased amount of proteins which appeargranular and uniformly distributed when stained with mercuric bromphenol (Plate 2H).

Specific staining with Azure B indicates dense staining for DNA in the nucleus as comparedto. that in the surrounding cells (Plate 2r). The nucleus appears large. Dense staining forcytoplasmic RNA is also indicated. Lipid bodies of various sizes are also present in theepithelial cells (Plate 2J). The walls of the epithelial cells facing the duct show dense stainingfor polysaccharide material (Plate ID). When tested for cellulose the thin walls of the epi-thelial cells facing the duct appear very lightly stained (Plate 2K).

ULTRASTRUCTURE OF EPITHELIAL CELLSEpithelial cells that line the gum-resin duct are more electron-dense than the cells adjacentto them, presumably due to the large number of organelles and osmiophilic droplets pre-sent in the former cells. The cell wall in contact with the duct is thinner than the other walls(Plate 3). The microfibrillar material of the thin wall appears as a loose fibrillar mesh(Plates 4-6). Presumably such a wall-structure facilitates an easy transfer of material intothe duct. Microtubules are frequently present adjacent to the tangential wall of theepithelial cell that is in contact with the duct (Plates 4, 6, 8). The microtubules appear to berandomly oriented but always parallel to the wall. Plasmodesmatal connections are presentfrequently in the radial walls and only occasionally in the outer tangential wall. The cellwall lining the duct does not have any plasmodesmata.

The plasmalemma is wavy at places and typically triple-layered. Paramural bodies arefrequently present along the tangential wall that lines the duct (Plates 3, 4,5, 7, 9).The nucleus of the epithelial cell appeared similar in structure to those in other nucleatecells. It is oval to amoeboid in shape with a double-membraned envelope that has pores(Plates 3 , 7 ). The cytoplasm is rich in ribosomes, ER, mitochondria, plastids and vacuolescontaining osmiophilic substances. The ER is mostly rough but with ribosomes that arein patches rather than in continuous array (Plates 3, 4, 5, 6, 8). Mitochondria often haveosmiophilic inclusions (Plates 4, 5, 7). Plastids do not have well-differentiated internalmembranes nor do they contain starch granules. They do, however, contain materialcomparable to lipid droplets (Plate 8). A most striking structural feature is the associationof ER with plastids. Most plastids appear to be partly or completely ensheathed by ER(Plates 5, 8). Although dictyosomes are not as abundant as mitochondria or rough ER,they are nevertheless frequently present (Plates 4, 5,9). Thedictyosome vesicles are usuallyelectron-dense. Large numbers oflipid droplets are present in the cytoplasm (Plates 3, 7,9).Vacuoles of various shapes and sizes that contain osmiophilic droplets are typicallypresent in the epithelial cell. It is possible that these vacuoles are in fact dilated cisternae ofER.

GUM-RESIN DUCTFine, granular and filamentous material fills the entire duct along with large oval tospherical bodies that vary in electron density (Plate 9). Osmiophilic droplets comparablein size and opacity to those in the epithelial cell-vacuoles are also frequently present in theduct (Plate 9). At the peripheral region of the duct, electron-transparent bodies that con-tain densely-stained material are frequently seen close to the walls of the epithelial cellsthat line the duct (Plates 4, 5, 6, 9). Their similar orientation with respect to the contourof the cell wall surface lining the duct is also significant. One gets the impression that thismaterial is being secreted into the duct by the epithelial cells. .


4/8

1002 Setia, Parthasarathy and ShahDISCUSSION

Our observations suggest that the cavity of the gum-resin duct develops schizogenously.It is similar to that found in Rhus glabra (Fahn and Evert, 1974)and Pinus halepensis(Werker and Fahn, 1969; Fahn and Benayoun, 1976).As both gum and resin are produced simultaneously in the ducts of Commiphora mukul,it is obvious that two systems are operating in the epithelial cells for their synthesis.Commonly there is either gum formation as in Sterculia urens (Shah and Setia, 1977) orresin asinPinus. Unlike Sterculia urens inwhich the gum isformed as aresult'ofbreakdownof epithelial cells, particularly the cell wall constituents (Shah and Setia, 1977),the histo-logical evidence of gum formation in Commiphora mukul remains obscure. From thenature ofthe cellwall ofthe epithelial cellsfacing the duct in Commiphora mukul it appearsthat the polysaccharide material of the gum may be continuously derived from it (the cellwall). The resin component of the gum-resin in Commiphora mukul first appears in theepithelial cells as tiny droplets which are then secreted into the duct. The process of thissecretion, observed in the fresh material (unpublished), is similar to that inPinus halepensisreported by Zamski and Fahn (1972). The site of resin synthesis still remains undeter-mined.The manner by which lipophilic and other material passes through the plasmalemmaand the cellwallinto the gum-resin duct isnot yet clear. The frequent presence of paramuralbodies between the plasmalemma and the cellwall lining the duct and absence ofplasmo-desmata suggest that the material passes across the cell wall region through vesicles thatoriginate from dictyosomes or endoplasmic reticulum. Thematerial could then conceivablydiffuse into the duct through the relatively porous cell walL Fahn and Benayoun (1976)suggest that resin droplets in Pinus halepensis are secreted in the duct by their becomingsurrounded by plasmalemma invaginations.The significance of the endoplasmic reticulum that ensheaths plastids has not beenresolved. Wooding and Northcote (1965) have reported similar sheathing endoplasmicreticulum in the resin ducts ofPinus pinea.The role of microtubules found adjacent to the thin tangential wall of the epithelialcells can be at present only speculated. Their presence could indicate that new wallmaterialis being constantly added while the outer wall layers gradually become part ofthe contentsof the duct (Fahn and Evert, 1974),or they could serve as a form of mechanical supportfor the thin and apparently fragile cellwall.

ACKNOWLEDGEMENTSWe are thankful to Dr. B. P. Deshpande for her suggestions during the preparation of themanuscript. One of us (R.C.S.) thanks the University Grants Commission, India, for aResearch Fellowship.

LITERATURE CITEDAMELUNXEN,F., 1964. Elektronmikroskopische Untersuchungen an den Drusenhaaren von Mentha

piperita L. Planta Med. 12, 121-9.--1965. Elektronmikroskopische Untersuchungen an den Drusenhaaren von Mentha piperita L. Ibid.13,457-73.-- and ARBEITER,H., 1967. Untersuchungen an den Spritzdrusen von Dictamnus albus L. Z. Pflanzen-physiol, 58,49---69.-- -- 1969. Untersuchungen an den Drusenhaaren von Cleome spinosa L. Ibid. 61, 73-80.-- and GRONAU,G., 1969. Elektronmikroskopische Ungersuchungen an den Olzellen von Acornuscalamus L. Ibid. 60, 156-68. .ANONYMOUS,972. Indian Forest Utilization, Vol. 2, p. 733. Forest Research Institute (F.R.I.) Press,DehdrDun, India.


5/8

Gum-resin Ducts in Commiphora mukul Engl. 1003ANONYMOUS,950. The Wealth of India, Vol. 5.C.S.I.R., New Delhi, India.FAHN,A. and EVERT,R. F., 1974. Ultrastructure of the secretory ducts ofRhusglabraL. Am.J. Bot. 61,1-14.-- and BENAYOUN,., 1976. Ultrastructure of resin ducts in Pinus halepensis development, possible sitesofresin synthesis, and modeofits elimination from the protoplast. Ann. Bot. 40, 857-63.FOSTER,A. S., 1934. The use of tannic acid and iron chloride for staining cell walls in meristematic tissue.Stain Tech. 9, 91-2.JENSEN,W. A., 1962. Botanical Histochemistry. W. H. Freeman, London.MAZIA,D., BREWER,P. A. and ALFERT,M., 1953. The cytochemical staining and measurement of proteinwith mercuric bromphenol blue. Biol. Bull. 104,57-67.O'BRIEN,T. P., FEDER,N. and MCCULLY,M. E., 1964. Polychromatic staining of plant cell walls by tolui-dine blue O. Protoplasma 59,367-73.SASS,J. E., 1958.Botanical Microtechnique. Iowa State College Press, Ames, Iowa.SCHNEPF,E., 1969a. Uber den Feinbau von Oldrusen III. Die Olgange von Solidago canadensis und dieExkretschlauchen von Arctium lappa. Protoplasma 67, 205-12.--1969b. Uber den Feinbau von Oldrusen IV. Die Olgange von UmbeIliferen: Heracleum spondylinumind Dorema ammoniacum. Ibid. 67, 375-90.SHAH,J. J. and SETIA,R. C., 1977. Histological and histochemical changes during the development of gumcanals in Sterculia urens Roxb. Phytomorphology (in press).WERKER,E. and FAHN,A., 1968. Site of resin synthesis in cells ofPinus halepensis Mill. Nature, Lond. 218,388-9.-- -- 1969. Resin ducts of Pinus halepensis Mill.- Their structure, development and pattern ofarrange-ment. Bot. J. Lin. Soc. 62, 379-411.WOODING,F. B. P. and NORTHCOTE,D. H., 1965. The fine structure of the mature resin canal cells ofPinusptnea. J. Ultrastruct. Res. 13, 233-44.ZAMSKI,E. and FAHN,A., 1972. Observations on resin secretion from isolated portions of resin ducts ofPinus halepensis Mill. Israel J. Bot. 21, 35-8.

EXPLANATIONS OF PLATESABBREVIATIONS

~ytoplasmic protrusion into the vacuole containing osmiophilic materialD-dictyosomeDU-ductEC-epithelial cellER-endoplasmic reticulumM-mitochondrionMT-microtubuleN-nucleusP-plastidPB-paramural bodyPM-plasmalemmaR-Resin dropletVO-vacuole with osmiophilic material

PLATE 1A. Transverse section of the stem. Irregularly distributed gum-resin ducts (DU) are present in the phloemx50.B. A gum-resin duct (DU) with tangentially-flattened epithelial cells (arrows). Arrow heads indicateseparation of epithelial cells along their radial walls. x315.c. Transverse section of the duct (DU) from fresh plant material showing resin droplets (R) stained withSudan IV. x450.D. A gum-resin duct (DU) stained with PAS reaction. Polysaccharide material in the duct lumen is not asintensely stained as the polysaccharide material of the inner tangential wall of the epithelial cells(arrows). x275.E. The duct initials showing larger nuclei (arrows). x550.F. The duct initials (arrows) showing apparently increased amounts of proteins (stained with mercuricbromphenol blue). x275.


6/8

1 0 0 4 Setia, Parthasarathy and ShahPLATE 2

A. The duct init ials showing an apparently increased amount ofcytoplasmic RNA (stained with Azure B).xSSO.B. Formation of an intercellular space (arrow) during duct initiation. xS50.c. Arrow indicates the expansion of intercellular space. xS50.D. Further expansion of the intercellular space isseen along with formation ofepithelial cells (EC). x550.E. Transverse section of part of a mature duct. Arrow indicates anticlinal division in the epithelial cell .x31S.F. Breakdown of an epithelial cell (arrow) and release of its contents into the duct. x315.G. After the breakdown of the epithelial cell (F), the outlying cellbehaves as an epithelial cell (arrow). x315.H. Surface view of epithelial cells as seen in longitudinal section. Note the large amount of proteins whichappear granular and uniformly distributed in the epithelial cells (stained with mercuric bromphenolblue.) x315. .I. Surface view of epithelial cells as in H. Note dense staining of DNA in the large nuclei. (Stained withAzure B). x315.1. Surface view of epithelial cells. Note dark lipid droplets of varying sizes (stained with Sudan Black B).x5S0.K. Transverse section of a part of a duct, treated with I2KI and H2S04 for cellulose. The inner tangentialwall of epithelial cells is very Iightlystained (arrows). xSSO.

PLATE 3Electron micrograph showing transverse section of a duct epithelial cell and a small portion of the duct(DU). Note that the tangential cell wall (large arrow) lining the duct is thin as compared to the outer walls.Vacuoles with osmiophilic material (YO) are abundant, as are lipid droplets (L). Rough endoplasmicreticulum (small hollow arrows) and free ribosomes are present in abundance. Several paramural bodiesare seen (short solid arrows) between the plasmalemma and the thin tangential cell wall. x13 000.

PLATE 4A portion of an epithelial cell and the duct with the tangential wall (CW) separating them. Note the para-mural body (PB), microtubule and other organelles. The duct (DU) has vesicular material that con-tains (polysaccharides?) electron dense substances similar to that also seen in the wall (unlabelled arrows).x32900.

PLATE SSame as Plate 4. Note the sheathing ER (unlabelled arrow) and the plastid (P) in the epithelial cell. Unlabelled arrows in the duct indicate material (that could be interpreted as polysaccharide) that has ap-parently been secreted by the epithelial cell . x43 000.

PLATE 6A magnified view of the tangential wall of the epithelial cell illustrated in Plate 4. Note that the cell wall(CW) is composed of a loose mesh of fibrous material. x91 400.

PLATE 7An epithelial cell containing lipid droplets (L) and most of the organelles. Unlabelled arrow indicatesmicrotubule. x21 000.

PLATE 8A portion ofthe epithelial celland the duct. Note the endoplasmic reticulum (ER) that ensheathes the plastid(p). Lipid droplets (0) seem to be present in the plastids. Note also some membrane material (MM), anda microtubule (MT) adjacent to the cell wall (CW). Unlabelled arrows indicate paramural bodies. x42 000.

PLATE 9An oblique transverse section showing a portion of the epithelial cell and the duct. Note the large vacuole(YO) containing osmiophilic material in the epithelial cell, and the lipid droplets (L). A dictyosome (D) isalso present between the vacuole and the cell wall (large arrow). Small unlabelled arrows indicate material(presumably polysaccharides) that has apparently passed into the duct from the epithelial cell. The largeoval to spherical bodies in the duct vary in electron transparency. Some of the bodies contain dark materialcomparable to the osmiophil ic bodies in the vacuole of the epithelial cell. x14 000.


7/8

SETIA ET AL.-Gum-resin Ducts in Commiphora mukul Engl.

PLATE 1

Ann. Bot. 41, 999-1004, 1977 (facing p. 1004)


8/8

SETIA ET AL.-Gum-resin Ducts inCommiphora mukul Engl.

Ann. Bot. 41, 999-1004, 1977 PLATE 2

Documents

Part-I Development, Histochemistry and Ultrastructure of Gum-resin Ducts in Commiphora mukul Engl