Developmental and Histochemical studies on Gum Ducts in Terminalia crenulata ROTH

8/6/2019 Developmental and Histochemical studies on Gum Ducts in Terminalia crenulata ROTH

1/10

Flora (1981) 171: 86-94

Developmental and Histochemical Studies on Gum Ducts in Terminaliacrenulata ROTH

R. C. SETTADepartment of Botany, Punjab Agricultural University, Ludhiana, India

SummaryIn Terminalia crenulata the gum ducts in the young stem and leaf develop lysi-schizogenously.

The cells lining the duct show the presence of gum substance and lipid bodies. The cell walls facingthe duct lumen show fan shaped radiating fibrillar structures during disorganization, and contri-bute to gum formation.

IntroductionNot much information in details is available on the process of gum formation in

plants except for few recent studies by SHAH & SETIA (1976a, b) and SETIA & SHAH(1979). They reported histological and histochemical changes in the gum producingstructures in some plants. In the present investigation an effort has been made tostudy the development and histochemistry of gum ducts in Terminalia crenulatawhich is a member of the family Combretaceae and is a source of "Terminalia" gum.Though this gum is comparatively of less economic value, yet it finds its usefulnessas an incense in the preparation of cosmetics and as an item of food for santals (WATT1972). True gums are water soluble or water dispersible polysaccharides. Thesegums in the plants may originate due to physiological or pathological disturbancesor injury or a normal metabolic activity but are formed as a result of breakdownof the walls and cell contents (SETIA & SHAH 1979).And the process of gum formationis generally known as "gummosis".

Materials and MethodsThe young sterns and leaves of T'erniinalia crenulata were collected from Dangs Forest, Gujarat

State. The plant material was fixed on the spot in Carnoy's fluid and FAA (SASS1958) and aspira-ted. Fixed material was dehydrated using TBA series and embedded in Embeddol (Harleco,U.S.A.). Serial sections, transverse and longitudinal, 10microns thick were cut on a rotary micro-tome. Staining techniques used for these sections and sections from the fresh material were:Safranin and fast green for general histology, Azure B for DNA and RNA, I2KI for starch, I2KIand H2S04 for cellulose, Sudan black B for lipids, ruthenium red and hydroxylamine ferric chloridefor pectins, periodic acid and Schiff's (PAS) reaction for insoluble polysaccharides (.JENSEN1962). General proteins were localized using mercuric bromophenol blue (MAZIA,BREWER&ALFERT1953). For localizing gum in the plant tissue three stains were used - PAS reaction, rutheniumred (.JENSEN1962) and toluidine blue 0 (O'BRIEN, FEDER & MCCULLY1964). Photomicrographs


2/10

Developmental and Histochemical Studies 87were taken on Carl Zeiss photomicroscopc with planopochromatic objectives using Kodak panato-nic-X 402 film with yellow and blue filters.

ObservationsThe transverse section of a young stem showing secondary growth has a single

layer of epidermis (Fig. IA). The cortex consists of 4-5 layers of parenchyma cells.The phloem and xylem form a continuous cylinder. Internal phloem is present atthe periphery of the parenchymatous pith. Four gum ducts are present in associationwith the internal phloem and pith. Internal phloem completely surrounds two op-posite ducts (Fig. IA, C1C1)while the remaining two opposite ducts are surroundedpartly by phloem and partly by pith cells (Fig. lA, C2C2). Fig. IB shows two ducts,one surrounded by phloem only (CI)and the other by phloem and pith (C~)(paren-chyma cells of pith are apparently larger than those of phloem). Occasionally sievetubes occur close to the ducts (Fig. I C, D).In transection these ducts appear oval to round (Fig. IB) and in longisection they

are parallel to the axis of the stem (Fig. I E). The diameter of the duct is almost uni-form throughout its length.In petiole the solitary crescent shaped vascular strand is dorsally flattened with

outwardly directed ends. Four to six round to oval ducts are present in the vascularstrand. In the midvein 4-6 gum ducts are present in the vascular bundles. In thelamina one duct is present above each vascular bundle.

Development of the gum duct in the young stemIn young stem, the gum ducts develop in the peripheral region of the pith just

close to the procambium (Fig.2A, arrow). A group of 6-8 densely stained cellsform the gum duct initials (Fig. 2B). Their nuclei are large and showdense stainabilityfor DNA (Fig. 2C). There is no change in the staining intensity for RNA (both nuclearand cytoplasmic) in these cells. Starch grains are absent. A small intercellular spaceis formed where a fewgum duct initials are contiguous (Fig. 2D). This space is formedas a result of breakdown of middle lamella substance at this point. Slowly the inter-cellular space enlarges and the walls of the cells lining the space appear flattened(Fig. 2E). Thus the initiation of the duct lumen is schizogenous. The cells lining theduct lumen show degeneration and the degraded cell contents become indistinct(Fig. 2F). The material in the cavity shows positive test with ruthenium red (Fig. 2F)and PAS reaction (Fig. 2G). As more cells lining the duct lumen degenerate moregum is formed and the duct lumen widens (Fig. 2H). Thus the mode of wideningof the duct is lysigenous. The gum duct follows schizolysigenous mode of develop-ment.

Gum ductBesides ruthenium red (Fig. 3A) and PAS reaction (Fig. 3B) (two histochemical

tests used for localization of gum in cells and duct lumen), toluidine blue 0 (a dif-ferential histological stain) also stains this gum red to violet (Fig. 3C). The cells


3/10

Fig. 1. A-E. 'I'erminalia crenulaia, A. Transverse section of young stem. Note four ducts in thepith region (011:0202)' B. Two ducts enlarged. Note 01 surrounded by phloem only and 02surrounded partly by phloem and partly by pith cells. 0, D. Note the sieve tubes lining the duet.E. Note two ducts running parallel, as seen in longitudinal section.


4/10

Developmental and Histochemical Studies 89

Fig. 2. A-H. 'I'errninalia crenulata. A. Arrow indicates 20 group of densely stained cells, the ductinitials. B. The duct initials. C. The nuclei of duct initials show dense stainability for DNA (stainedwith Azure B). D. Arrow indicates the formation of a space. E. Enlargement of the space. F.The degraded product gives positive test with ruthenium red. G. Note the darkly stained gumwith PAS reaction. H. A wide gum duct.


5/10

90 R. C. SETIA

lining the lumen of a duct lack any distinct pattern of their form and arrangementand their broken walls project in the duct lumen. The presence of gum in thesecells is indicated by ruthenium red (Fig. 3D), PAS reaction (Fig. 3F) and toluidineblue 0 (Fig. 3E). An important event is the appearance of lipid bodies in these cells.These cells show negative test for general proteins. Nucleus does not show any changein DNA and RNA contents. The starch grain size and number appear similar tothose in the surrounding cells.

Formation of gum in the ductAs more cells lining the lumen disintegrate, additional gum is formed. The de.

gradation of the cells involves certain histological and histochemical changes. Withruthenium red, the disintegrating wall facing the lumen of the duct shows the forma-tion of fan shaped radiating fibrillar structures (Fig. 31). Itprojects into the ductlumen. The fibrillar structures are not stained with hydroxylamine ferric chlorideindicating the absence of pectins of high esterification (JENSEN 1962). Thus, thefibrillar mass may be the gum substance containing pectic material of low esterifica-tion. Sometimes remnants of walls are seen in the duct lumen and show the similarfibrillar masses (Fig.4A). The middle lamella of these cells appears darkly stainedand swollen (Fig. 4A, B arrows) probably undergoing degradation. Sometimes thecell wall facing the duct separates and is seen in the duct lumen. Itshows degradation(Fig. 4C, D). When stained with PAS reaction, the degrading material of the wallfacing the lumen of duct is dark red while the gum stains red (Fig. 4E). The outerwall of the cell also shows degradation (Fig. 4E, arrow). When tested for cellulose,the disintegrating wall appears light blue (Fig. 4F), thus indicating some changein the cellulosic component of wall.The cell wall facing the duct lumen appears swollen (Fig. 4G, arrow head). The

small bubble like structures observed in the densely stained cell lumen are lipidbodies (Fig. 4G, H). The outer wall of this cell also appears swollen (Fig. 4G, arrow).The components of these walls appear degrading as they show positive test for thegum (Fig. 41). During degradation of the cell wall, first the cellulosic material isaffected followed by the pectic material of the middle lamella. Ultimately the cellcomponents degrade to form a homogenous mass which shows positive test for gum.During the disorganization of the cell walls and the cell contents, the starch grains

are present in the duct lumen alongwith the gum. Similarly lipid bodies are alsopresent.The nucleus in the cells, lining the duct lumen may degenerate. Sometimes it

persists in the duct lumen before its disorganization. The breakdown of the nuclearenvelop is followed by the disintegration of the nuclear material.


6/10

90 R. C. SETIA

lining the lumen of a duct lack any distinct pattern of their form and arrangementand their broken walls project in the duct lumen. The presence of gum in thesecells is indicated by ruthenium red (Fig. 3D), PAS reaction (Fig. 3F) and toluidineblue 0 (Fig. 3E). An important event is the appearance of lipid bodies in these cells.These cells show negative test for general proteins. Nucleus does not show any changein DNA and RNA contents. The starch grain size and number appear similar tothose in the surrounding cells.

Formation of gum in the ductAs more cells lining the lumen disintegrate, additional gum is formed. The de-

gradation of the cells involves certain histological and histochemical changes. Withruthenium red, the disintegrating wall facing the lumen of the duct shows the forma-tion of fan shaped radiating fibrillar structures (Fig. 31). Itprojects into the ductlumen. The fibrillar structures are not stained with hydroxylamine ferric chlorideindicating the absence of pectins of high esterification (JENSEN 1962). Thus, thefibrillar mass may be the gum substance containing pectic material of low esterifica-tion. Sometimes remnants of walls are seen in the duct lumen and show the similarfibrillar masses (Fig. 4A). The middle lamella of these cells appears darkly stainedand swollen (Fig. 4A, B arrows) probably undergoing degradation. Sometimes thecell wall facing the duct separates and is seen in the duct lumen. Itshows degradation(Fig. 4C, D). When stained with PAS reaction, the degrading material of the wallfacing the lumen of duct is dark red while the gum stains red (Fig. 4E). The outerwall of the cell also shows degradation (Fig. 4E, arrow). When tested for cellulose,the disintegrating wall appears light blue (Fig. 4F), thus indicating some changein the cellulosic component of wall.The cell wall facing the duct lumen appears swollen (Fig. 4G, arrow head). The

small bubble like structures observed in the densely stained cell lumen are lipidbodies (Fig. 4G, H). The outer wall of this cell also appears swollen (Fig. 4G, arrow).The components of these walls appear degrading as they show positive test for thegum (Fig. 41). During degradation of the cell wall, first the cellulosic material isaffected followed by the pectic material of the middle lamella. Ultimately the cellcomponents degrade to form a homogenous mass which shows positive test for gum.During the disorganization of the cell walls and the cell contents, the starch grains

are present in the duct lumen alongwith the gum. Similarly lipid bodies are alsopresent.The nucleus in the cells, lining the duct lumen may degenerate. Sometimes it

persists in the duct lumen before its disorganization. The breakdown of the nuclearenvelop is followed by the disintegration of the nuclear material.


7/10


Fig. 3. A-I. 'I'erminalio. crenulata, A. Arrows indicate gum (stained with ruthenium red) in theduct. B. Note gum in the duct stained with PAS reaction. C. Note gum in the duct stained withtoluidine blue O.D- F. Note the cells lining the cavity of the duct. They are densely stained withruthenium red, toluidine blue 0 and PAS reaction respectively. G. Note lipid bodies (stained withSudan black B) in the cells lining the duct (arrow). H. Note the presence of starch grains in thecells lining the duct (treated with I2KI). J. Note fan-shaped radiating fibrillar structure projectinginto duct (stained with ruthenium red).


8/10

92 R. C. SETIA

Fig. 4. A - 1. 'I'ermimalia creriulata, A. Note the fibrillar masses in the duct. Arrow indicatesdegrading middle lamella. B. Enlarged view of A. Arrow indicatos degrading middle lamella.e, D. Note cell walls showing degradation in the duct. E. The cell wall facing the duct showsdegradation (stained with PAS reaction). The outer tangential wall of the cell also shows degrada-tion (arrow). F. Note, at arrow, the lightly stained cell wall facing the duct (treated with 12KI-H2S04 for cellulose). G. Arrow head and arrow indicate degrading walls (stained with toluidineblue 0). H, 1. Further degradation of wall (stained with toluidine blue 0).


9/10


DiscussionAs in Sterculia urens (SHAH& SETIA1976a), the gum ducts in Terminalia crenulata

form the essential structures of the plant body. The sites of their development arepredetermined and appear to be genetically controlled. And the process of gum for-mation in the duct appears to be a feature of normal plant metabolism. As reportedby STEWART1966) the secretion of gum in Acacia, Albizia and Melia may representthe removal of waste metabolic products from the centre of production to an areawhere they become non-toxic. But the physiological role of the gum in plants as8, whole is undetermined so far. The physical property of the gum particles to absorbmoisture might reflect its one of the functions as water retainer in the plant body.The appearance of gum substance and lipid bodies in the cells lining the duct

shows that these cells are at different metabolic level as compared to the surroundingcells. Unlike Sterculia urens (SHAH& SETIA1976a) the starch grains, DNA and RNAin the cells lining duct lumen do not show any change.Itappears that the major part of the gum is derived from the cell walls surrounding

the gum duct. Sugars such as glucose, galactose, arabinose and galacturonic acidform SOl'n6 of the monomers of plant cell wall polysaccharides (CLOWES& JUNIPER1968; STRANGE1972). The "Terminalia" gum contains D-galactose, L-arabinose,L-rhamnose, D-galacturonic acid in different proportions (ANDERSON& BELL1974).Possibly certain enzymes produced in the plant itself in the gum duct zone bringabout the degradation of the complex cell wall polysaccharides. An investigation ofthe aspect of study should further add to the understanding of gummosis in plants.

AcknowledgementI am thankful to Professor J. J. SHAHfor guidance and encouragement and to Professor C. P.

1VIALIKor encouragement.

ReferencesANDRRRON, D. M. W., & BELL, P. C. (1974): The composition and properties of gum exudatesfrom Terminalio sericea and T. su/perba, Phytochemistry 13: 1871-1874.

CLOWES,F. A. L., & JUNIPER, B. E. (1968): Plant cells. Blackwell, Oxford.JENSEN, ''Y. A. (1962): Botanical Histochemistry. London.MAZIA,D., BREWER, P. A., & ALFERT, M. (1953): The cytochemical staining and measurementof protein with mercuric bromophenol blue. Biol. Bull. 104: 57- 67.O'BRIEN, '1'. P., FEDER, M., & MCCULLY,M. E. (1964): Polychromatic staining of plant wallsby Toluidine blue O . Protoplasma 59: 367 -373.

SASS,J. E. (1958): Botanical Microtechnique. Iowa State College Press, Ames.BETIA,R. C., & SHAH,J. J. (1979): Histological, histochemical and ultrastructural aspects of gumand gum-resin producing structures in plants. In: Ann. Rev. Plant Sciences (ed, C. P. MAI,IK)1: 315-330.

SHAH,J. J., & SETIA, R. C. (1976a): Histological and histochemical changes during the develop-ment of gum canals in Sterculia urens, Phytomorphology 26: 151-158.


10/10

94 R. O. SETIA, Developmental and Histochemical Studies

(1976b): Histological and histochemical studies in gum formation in Pterocar-pus marsupiumRoxn. Indian Forester 102: 161-167.

STEWART,M. O. (1966): Excretion and heartwood formation in living trees. Science 153: 1068-1074.

STRANGE,R. N. (1972): Plants under attack. Sci. Prog. 60: 365-385.WATT,G. (1972): A dictinonary of economic products of India, Oosmo Publications, Delhi.Received August 8, 1980.

Author's address: Dr. R. O. SETIA, Department of Botany, Punjab Agricultural University,Ludhiana, India.

Verantwortlich fur die Redaktion: Prof. Dr. H. Meusel, 4020 Halle (Saale). Verlag: VEB GustavFischer Verlag, 6900 Jena, Villengang 2, Telefon 27332. Satz und Druck: Druckerei ..Magnus Poser",6900 Jena. - Ver6ffentlicht unter der Lizenznurnrner 1658 des Presseamtes beirn Vorsltzenden desMinisterrates der Deutschen Dernokratischen Republik. Aile Rechte beirn Verlag. Nachdruck (auchauszugsweise) nur mit Genehmigung des Verlages und des Verfassers sowie mit Angabe der Quellegestattet. Printed in the German Democratic Republic, Artikel-Nr, (EDV) 57415

Developmental and Histochemical studies on Gum Ducts in Terminalia crenulata ROTH

Documents

Transcript of Developmental and Histochemical studies on Gum Ducts in Terminalia crenulata ROTH