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Structure and macromolecular composition of the seed coat of Musaceae

Graven, P.; de Koster, C.G.; Boon, J.J.; Bouman, F. DOI 10.1006/anbo.1996.0013 Publication date 1996

Published in Annals of Botany

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Citation for published version (APA): Graven, P., de Koster, C. G., Boon, J. J., & Bouman, F. (1996). Structure and macromolecular composition of the seed coat of Musaceae. Annals of Botany, 77, 105-122. https://doi.org/10.1006/anbo.1996.0013

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UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:04 Oct 2021 Annals of Botany 77: 105–122, 1996

Structure and Macromolecular Composition of the Seed Coat of the Musaceae

PETER GRAVEN*†, CHRIS G. DE KOSTER†, JAAP J. BOON† and FERRY BOUMAN* * Hugo de Vries-Laboratory, UniŠersity of Amsterdam, Kruislaan 318, 1098 SM Amsterdam and † FOM Institute of Atomic and Molecular Physics, Unit for Mass Spectrometry of Macromolecular Systems, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands

Received: 20 June 1995 Accepted: 30 August 1995

Mature seed coats of representatives of all three genera of Musaceae were analysed for macromolecular composition with various mass spectrometric techniques and compared with scanning electron microscopy and light microscopy in combination with histochemical techniques. Mass spectrometric techniques are more sensitive and more specific in identifying macromolecular compounds than histochemical methods. The macromolecular ‘fingerprint’ of the seed coats of Musaceae showed unique components of aromatic phenols. The seed coat structure of all three genera is homogeneous within the Musaceae. It is characteristic at the family level and most complex within the Zingiberales. Very remarkable are the separation of the outer cell walls from the exotestal layer, exposing a secondary surface with silica crystals, and the relatively thick mesotesta which protects the seed, e.g. against the biting forces and passage through the digestive tracts of dispersing agents. Germination takes place with an operculum and is facilitated by a predetermined rupture layer in the micropylar collar. The musaceaous seed presents a good example of the solution of conflicting demands of protection and germination. # 1996 Annals of Botany Company

Key words: Musaceae, Musa, Ensete, Musella, seed coat, pyrolysis (gas chromatography) mass spectrometry, histochemistry, anatomy, macromolecules, silica, lignin, cellulose, vegetable polyphenols, operculum, germination.

to which the bananas belong, there are three genera: Musa INTRODUCTION L., with about 35 species, Ensete Bruce., with three species Hardly anything is known about the chemistry of seed and a more obscure genus Musella (Fr.) C. Y. Wu with one coats. The biochemical and histochemical literature on species. The acceptance of the Musella as a distinct genus is seeds is almost fully focused on the composition of storage only provisional (Manchester and Kress, 1993). A number material in embryo and endosperm. The functional in- of genera formerly mostly included in the Musaceae are terpretation of the seed coat is an almost unexplored field of nowadays placed in the separated families Strelitziaceae, research, because of the lack of reliable data on the presence Heliconiaceae and Lowiaceae (Nakai, 1941; Tomlinson, and distribution of macromolecular components. Currently 1962; Cronquist, 1981; Dahlgren and Rasmussen, 1983). it is possible to describe the anatomy in more detail with Each taxon possesses a comparable range of anatomical microscopical and histochemical techniques and to study variation which overlaps to a minimum with other groups the macromolecular composition of the testal layers of the within the Zingiberales (Tomlinson, 1962). The family is mature seed with mass spectrometrical techniques. tropical-subtropical Asian (Musa, Musella) and African In source pyrolysis mass spectrometry (PyMS) is a relative (Ensete), but Musa-cultivars are nowadays cultivated simple method to obtain information from plant tissue at troughout the tropical regions of the world. the molecular level (Boon, 1989). The PyMS analysis Most edible cultivated clones are seedless whereas other provides a chemical ‘fingerprint’ of the seed coat. Chemical plants, wild or cultivated, produce one to many seeds. identification of individual pyrolysis products is performed Breeding programs are hindered by the low seed set, as well by using pyrolysis gas chromatography (PyGC) combined as by slow and non-uniform germination (Shepherd, 1954; with mass spectrometry (PyGCMS). Stotzky, Cox and Goose, 1962). The ovary of Musa is three The order Zingiberales is a well-defined and coherent locular, contains numerous ovules, and develops into a taxon. Within this order there are questions about the longish berry. The fruit of Musa contains a higher number relation of seed coat structure and chemistry to germination. of seeds than that of Ensete. The seeds rarely attain 1 cm in The Musaceae represents the most basal lineage in the order diameter, whereas the seeds of Ensete are mostly larger, up (Manchester and Kress, 1993). This basal position of the to 1n8 cm, although there may be some overlap in size. The Musaceae, its special type of germination and its well- shape of the seeds varies greatly, is often irregularly and differentiated seed coat forms an interesting subject to study sharply angular, either rarely globose or smooth. The the relation of structure, macromolecular composition and variability in shape of the pale or dark brown to black seeds functions of the testal layers, and moreover to produce a is due to compression between neighbouring seeds new data set relevant for macrosystematic research. (Simmonds, 1960). The embryological literature has been In most recent classifications the family of the Musaceae, summarized by Davis (1966), that on seed anatomy by 0305-7364\96\020105j18 $12.00\0 # 1996 Annals of Botany Company 106 GraŠen et al.—Composition of Musaceaous Seed Coats Netolitzky (1926) and Takhtajan (1985). In earlier studies with a hexane\dichloromethane mixture (1:1 v\v), after 3 h McGahan (1961a) described the anatomy of the seed of it was centrifuged, the pellet was suspended in deionized Musa balbisiana and Bouharmont (1963) the development water, dried and extracted with an ethanol\acetone mixture of the ovule to mature seed in Musa acuminata. (1:1 v\v) for another 3 h, and finally washed several times The ovule is anatropous, bitegmic, and crassinucellar, in deionized water. The homogenized and extracted seed with a multi-layered outer integument and a thin inner coat material was suspended in a few drops of water. integument. The micropylar part of the seed coat develops into an operculum or seed lid. This operculum is surrounded Transmethylation by a characteristic micropylar collar. During germination the seed lid is displaced by the elongating radical-hypocotyl For methylation of phenolic hydroxyl and carboxyl axis (McGahan, 1961b). The mature seed contains mainly groups, a small droplet of 2n5% (w\v) tetramethyl- endosperm, which is copious, starchy and mealy, with large, ammonium (TMAH) was added to the wet sample on the eccentric, compound starch grains, and some perisperm wire of the direct insertion probe prior to PyMS and (Cronquist, 1981). The endosperm is initially nuclear, PyGCMS experiments. becoming cellular later, and extends during its development at the expense of the perisperm. The embryo remains Silylation relatively small, is situated under the operculum and is partly enclosed by the micropylar collar. In the ovular stage An off-line pyrolysate was prepared by placing a few a trichomatous aril develops out of the funicle surrounding drops of an aqueous suspension of extracted seed coat on a the seed coat (Netolitzky, 1926; Friedrich and Strauch, wire with a Curie point of 510 mC. The wire was dried under 1975). According to Friedrich and Strauch (1975) the aril rotation in vacuum. After drying the wire was placed in a becomes reduced and only some small vestiges can be found glass liner and the glass liner was flushed for a minute with near the funicle on the mature seed. argon, directly followed by pyrolysis at atmospheric Seeds of Ensete can be distinguished from those of Musa pressure. The procedure was repeated several times with a by a rimmed hilar depression that is nearly as wide as the new wire in the same glass liner and the final condensate seed itself. Seeds of Musella resemble those of Musa, being that formed on the inner wall of the glass liner was collected. distinguished from Ensete by the lack of a wide hilar Silylation of the off-line pyrolysate was performed by depression and rim (Manchester and Kress, 1993). Most adding 70 µl of BSTFA\TMSCL [N.O.bis(TMS) species of Musa have a rough verrucate seed coat, whereas trifluoroamide\trimethylchlorosilane] in 235 µl pyridine to the seed coat of Musella and Ensete is smooth. the sample in the dark. After 24 h the pyridine was In this study data from scanning electron microscopy evaporated by flushing with nitrogen and the reaction (SEM), light microscopy (LM), histochemistry and mass product was resuspended in dichloromethane and analysed spectrometry are compared, to acquire insight into the with GCMS. functions and chemical composition of specialized testal layers. Acid transesterification

MATERIALS AND METHODS For hydrolysis a few milligrams of the extracted seed coat were placed in a tube with a screw top and 12  sulphuric Plant material acid (approx. 2 ml) was added. The tube was flushed with Mature seeds of Musa acuminata Colla, Musa balbisiana nitrogen before sealing and the sample was incubated at Colla, Musa mannii H. Wendland, Musa paradisiaca L., room temperature for 3 h. The acid was diluted to 1  and Musa rosacea Jacquin, Musa textilis Ne! e, Musa Šelutina H. the sample was further hydrolysed at 100 mC overnight. The Wendland & Drude, Musa sp., Ensete Šentricosum sample was then centrifuged and the residue was hydrolysed (Welwitsch) Cheesman, Ensete glaucum (Roxb.) Cheesman, again with 0n25  sulphuric acid. The tube was flushed with and Musella lasiocarpa (Fr.) C. Y. Wu used in this study nitrogen and incubated at 100 mC overnight. The solid were collected via botanical gardens all over the world. The residue was separated from the liquid hydrolysate by seeds were stored dry at room temperature in closed plastic centrifugation and washed several times with deionized containers. water and dried overnight over phosphorus pentoxide in a vacuum dessicator at 50 mC (Pastorava, Oudemans and Boon, 1993). Germination Ethyl esters were prepared by using approx. 1 mg of the Seeds of Musa Šelutina and Musa rosacea were soaked in extracted sample in a plastic tube with a screw top with a water for 24 h, and sown in petri dishes on wet filter paper 20 µlH#SO%\ethanol solution (1n25 µlH#SO% in 1 ml and in soil under greenhouse conditions. ethanol). The tube was sealed with a Teflon tape and incubated at 100 mC for 4 h. Sample preparation for mass spectrometry Direct temperature resolŠed or pyrolysis mass spectrometry Samples of seed coats of Musaceae were collected and homogenized in a few drops of water in a small glass mortar A small drop of a suspension of the unextracted or with a glass pestle. The homogenized seed coat was extracted homogenized and extracted seed coat was placed on a GraŠen et al.—Composition of Musaceaous Seed Coats 107

F. 1. A, Mature seed of Musa balbisiana (i10). B, Musa sp. showing, partly removed, the thin brittle remains of the aril (a), the outer epidermis (oe) of the outer integument and crystalline silica bodies (si). Irregular radial divisions arise at the outer layer of the mesotesta (arrowhead), which gives the seed a rough appearance in some species (i90). C–E, Musa balbisiana; The mesotesta (me) has developed into 20–25 layers of sclerotic cells. C, The cells of the four to five uppermost layers are more thickened and less elongated (i140). D, Backscatter image showing the silica bodies as a thin layer on top of the mesotesta (i135). E, Sclerotic cells with pits at the lower part of the mesotesta (i1010). F, The inner epidermis of the testa develops into an endotesta (en) consisting of U-shaped thickened cells. The tegmen (te) consists of two layers of tangentially elongated cells. A thick cuticle bounds the inner surface of the tegmen and has a tegmic and nucellar origin (Musa balbisiana)(i610). G, In the mature seeds the perisperm (pe) is completely compressed and only the cell walls remain (Musa mannii)(i1380). H, The tegmic cells are filled with a spongy like structure (Musa balbisiana)(i2690). 108 GraŠen et al.—Composition of Musaceaous Seed Coats

F. 2. For legend see facing page. GraŠen et al.—Composition of Musaceaous Seed Coats 109 Pt\Rh (9:1) wire of the insertion probe for in-source gold\palladium and examined with an ISI-DS130 Dual pyrolysis mass spectrometry (PyMS) on a JEOL DX-303 Stage electron microscope (9 kV) fitted with an ISI- double focusing mass spectrometer. The wire was inserted Robinson Detector RBSE 130R (19 kV) for backscatter into the evacuated ion source, after drying the sample. The imaging. ion source temperature was 180 mC. The wire was resistively Mature seeds of Musa balbisiana, Musa paradisiaca, " heated at 16 mCs− to a final temperature of 800 mC. Musa textilis and Musa Šelutina were examined for the Compounds, formed upon desorptionor pyrolysis, were presence and localization of silica in the seed coat. The seeds ionised by 16 eV electron impact (EI) or ammonia chemical- were sectioned transversely, fixed on aluminium stubs ionization (CI) PyMS. The scan cycle time was 1 s and the cemented with carbon cement and air dried under low selected massrange was m\z 20–1000. relative humidity at room temperature. The specimens were carbon coated and examined in a Cambridge Stereoscan 150 (at 20 kV) fitted with a backscatter detector (KE Develop- Curie-point pyrolysis gas chromatography mass ments, Cambridge, UK) and a LINK energy-dispersive X- spectrometry ray analyser [Department of Molecular Cell Biology (EMSA) of the University of Utrecht, The Netherlands]. Curie-point pyrolysis gas chromatography mass spec- The presence of silica was detected with the help of the trometry under EI conditions was conducted on a Finnigan diagrams of the X-ray spectra. The specific peak (keV, 1 74) INCOS 50 quadrupole mass spectrometer connected to a n for silica in the spectrum was isolated and the spectrum HP 5890 series II gas chromatograph. About 5 µgofa pulses were simultaneous with the scanning of the sample suspension of a sample was applied to a ferromagnetic wire carried back so that an element distribution image of silica with a Curie-point temperature of 610 mC. The pyrolysis could be created (X-ray mapping). products were flushed towards a 25 m CPSILS-5 CB fused silica capillary column (i.d. 0n32 mm; film thickness 1n2 µm) " using He as carrier gas (flow 30 cm s− ). The GC oven was Light microscopy and histochemistry kept at 30 mC during pyrolysis and was subsequently " programmed to 320 mC at a rate of 6 mC min− . The Hand-cut sections were stained for vegetable polyphenols compounds were ionised at 70 eV electron impact energy. (‘tannins’) with ferric chloride, vanillin\HCl and the nitroso The scan cycle time was 0n5 s with a selected mass range of reaction, for lipids with Magdala red, Nile blue sulphate m\z 20–500. method, for cuticle (neutral fats and fatty acids) with Sudan IV, for ‘cutin’ with azure B method and auramine O method, for suberin with phosphate buffer method and On column injection gas chromatography mass cyanin method, for lignin with sodium hypochlorite\HCl, spectrometry phloroglucinol\HCl and lignin pink, for callose with soda method, Corallin and aniline blue , for pectin with ruthenium On column injection gas chromatography mass spec- red, and for cellulose with chlor-zinc-iodide (Gahan, 1984; trometry (GCMS) was performed using a HRGC MEGA 2 Krisnamurthy, 1988). Sections for staining with ruthenium series FISONS instruments gas chromatograph which was red and for the cyanin\potassium method were also connected to a Jeol DX-303 double focusing mass spec- pretreated with Eau de Javelle (potassium hypochlorite). trometer. The column used was a 25 m SIMDIST (i.d. Samples for semi-thin sectioning were fixed in FAA or 0n32 mm, film thickness 0n15 µm). Helium was used as −" Craf III, or were softened in 10% ammonia (1 h at 50 mC) carrier gas (flow 30 cm s ). The GC oven was kept at 30 mC " or a mix of glycerol and Aerosol OT in water (Schmid and and subsequently heated to 375 mC at a rate of 6 mC min− . Turner, 1977; Setoguchi, Tobe and Ohba, 1992), dehydrated Compounds were ionized at 70 eV electron impact energy, in a n-butyl alcohol series and embedded in glycerol at a source temperature of 180 mC. The scan cycle time was methylacrylate polymer (Feder and O’Brien, 1968). 0n8 s over a mass range of m\z 20–500.

RESULTS Scanning electron microscopy Mature seed coat anatomy and histochemistry Seeds were sectioned transversely and fixed in FAA (formalin, ethanol, acetic acid), dehydrated in acetone, In mature seed coats the cells are difficult to interpret with incubated in tetramethylsilane and air dried (Dey et al., light microscopy due to the extreme hardness of the tissue. 1989). The seeds were coated with a thin layer of This has hampered the observations on the mature seed

F. 2. A, Each exotestal cell contains a multi-angular crystalline silica body (Musa sp.) (i500). B–C, The exotesta easily releases and while separating, the tips of the silica bodies break off and stick to the membrane (Musa sp.) (B, i220; C, i665). D, The silica bodies are situated against the inner periclinal wall of the epidermal cells (Musa sp.) (i270). E, The tip of the silica bodies breaks off after the release of the outer periclinal wall and the radial walls (Musa sp.) (i1010). F, Backscatter image of the silica bodies of Musa balbisiana (i605). G, Backscatter image of the seed coat of Musa balbisiana with partly released epidermis and remains of the aril (i75). H–I, Backscatter image and bright dot mapping on silica in the seed coat of Musa paradisiaca (H, I i165). J, Backscatter image of released epidermal wall of Musa balbisiana and silica bodies which are situated on the inner periclinal wall (i300). K–L, Backscatter and bright dot mapping view on the inner side of the separated epidermal wall, showing the broken tips of the silica bodies and the silica encrusted walls and membrane of the exotesta of Musa balbisiana (K, L i630). 110 GraŠen et al.—Composition of Musaceaous Seed Coats

hi mi

op se ab

mc em ii

oi end

nu

pt

cd

F. 3. Schematic drawing of a longitudinal section of the Musa seed coat (i30). ab, abscission layer; cd, chalazal disk; em, embryo; end, endosperm; hi, hilum; ii, inner integument; mc, micropylar collar; mi, micropyle; nu, nucellar tissue or perisperm; oi, outer integument; op, operculum; pt, parenchymatic tissue between chalaza and nucellus; se, silicified exotesta. coats of Musaceae. Softening of the seeds facilitates body remains attached to the seed coat after release of the sectioning of the material, but it releases some of the epidermal wall layer (Fig. 2A, D–F). components in the seed coat (e.g. silica). Most stains used The middle layers of the outer integument have developed were negative or aspecific. into 20–25 layers (180–350 µm) of sclerotic cells (Fig. The seed coat anatomy of all three genera in Musaceae 1C–E). These vary greatly in shape (length up to 300 µm; was practically homogeneous therefore the structure of the width about 10 µm), size and orientation. The secondary Musa sp. only is described here. cell wall contains phenolic compounds in the S2 layer, During the development of the seed the aril jellifies and which gave a very weak response with the nitroso reaction dries out. In the mature seed (Fig. 1A) the remains of the and the vanillin\HCl test. These compounds increase in aril adhere closely to the seed coat (Fig. 1B). The aril relative amount from the inner to the outer side of the surrounds the ripe seed partly as a thin brittle layer and, if mesotesta. The phenolic compounds are more concentrated not released, swells up during germination. in the four to five uppermost cell layers of which the cell The cells of the outer epidermis of the outer integument walls are more thickened and the cells are less elongated. are radially stretched and vary in length (65–190 µm). The The histochemical tests on lignin were mostly negative, only epidermis is covered with a thin cuticle which stains in very thin sections a light pink colouration could be seen positively with sudan IV, azure B and auramine O. This in the S1 layer of the secondary wall, using lignin pink. indicates ‘cutin’ and fatty acids in the cuticle. Each In some species locally irregular periclinal divisions lead epidermal cell contains a multi angular crystalline silica to protrusions on the outer layer of the mesotesta, which are body, which is situated against the inner periclinal wall (Figs the cause of a rough surface on the seed of most Musa 1D, 2A, G–J). The walls of the epidermal cells remain species (Fig. 1A–B). The innermost layer of the outer relatively thin and are encrusted with a net-like silica integument develops into an endotesta consisting of U- structure. The outer periclinal and radial walls easily shaped thickened cells (Fig. 1F). Testa and tegmen are not separate from the seed coat (Fig. 2G, J). During the release, separated by a distinct cuticular layer. The tegmen consists the tips of the silica bodies may break off and stick to the of two layers of longitudinally elongated cells (Fig. 1F–G). membrane (Fig. 2B–C, J–L). The main part of the silica The inner wall of the outer layer and the inner and outer

F. 4. A, An abscission layer (ab) has developed through the outer integument and delimitates the operculum (op) (Musa balbisiana)(i40) B, The tegmen and nucellus (nu) rupture just beneath the abscission layer (Musa sp.) (i100). C–D, The operculum consists of three distinct layers. The raphal zone is followed by a parenchymatic tannin zone and a sclerenchymatic zone (Musa sp.) (C i60, D i30). E, The silicified vascular bundle ends in the chalazal disk (cd) consisting of thin walled cells (Musa paradisiaca)(i55). F, The powdery endosperm (end) forms a thick layer along the inner side of the seed (Musa balbisiana)(i705). GraŠen et al.—Composition of Musaceaous Seed Coats 111

F. 4. For legend see facing page. 112 GraŠen et al.—Composition of Musaceaous Seed Coats T 1. List of a number of compound classes and the layer of the inner integument and partly has a nucellar pyrolysis mass spectrometric and PyGCMS techniques by origin (Fig. 1F–G). which they can detected In the mature seed the perisperm is completely compressed and only the cell walls remain (Fig. 1G). The powdery PyMS PyGCMS GCMS endosperm (Fig. 4F) of the mature seed forms a thick layer along the inner side of the seed and leaves a cavity in the NH$ TMAH TMAH middle of the seed (Fig. 3). EI CI EI EI EI EI At the micropylar side a typical micropylar collar is Polysaccharide (x) x (x) formed by a local ingrowth of the outer integument into the Protein* x x x inner integument and the nucellus (Figs 3 and 4A). An Lignin x (x) x abscission layer continuous with the micropylar collar has Vegetable xx developed through the outer integument and envelops the polyphenol (tannin) operculum (Fig. 4A). The operculum (width 1–1n5 mm) is Cutin x (x) x x† mainly formed by the exostomal part at the outer integument Phenolic acids (x) (x) x x and hilar\raphal tissue (Fig. 4D). The operculum consists Lipids x (x) (x) (x) x of three distinct zones (Fig. 4B–D). The raphal top zone is followed by a parenchymatic ‘tannin’ zone and a scleren- * Poor response. chymatic zone. The hilum and micropyle are situated at the † After acid transesterification. top of the operculum (Fig. 3). The silicified vascular bundle is widely branched through the top zone of the operculum, walls of the innermost layer are thickened. The tegmic cells running through the raphal mesotesta and ending in the are filled with a spongy-like structure of unknown com- chalazal disk (Figs 3 and 4E). During germination the position (Fig. 1H). A thick cuticle, which strongly stains operculum is easily pushed out by the embryonic root, due with Sudan IV and Nile blue, is grafted to the inner surface to the abscission layer. Removing the operculum does not of the tegmen. This cuticle partly originates from the inner result in faster or higher percentage of germination. The

F. 5. Pyrolysis (EI) mass spectrum of the non-extracted seed coat of Musa Šelutina showing hydroxymethoxybenzyl cation (m\z 137) as major fragment ion. #, guaiacyl lignin; $, syringyl lignin; >, polysaccharides; j, lipids; P: Musaceae characteristics; f, ferulic acid; pc, p-coumaric acid; t, vegetable polyphenol markers. GraŠen et al.—Composition of Musaceaous Seed Coats 113

× 3

576 100

602

550 618 704 620 691 572 592 732

632 648 662 676

0

500550 600650 700 750

× 3 Relative abundance 100

882

857

830 798 911

0

750 800850 900 950 1000 m/z

F. 6. Lipid fraction of pyrolysis (EI) mass spectrum of the total seed coat of Musa Šelutina. j, di- and triglycerides; ', waxes.

tegmen and nucellus rupture also just beneath the abscission consisting of thin-walled cells (Fig. 4E). In the developing layer in the outer integument (Fig. 4B–C). The epidermal seed these cells are filled with small reddish brown vacuoles cells at the nucellar apex are radially elongated and fill the which fuse later on to a single vacuole. According to White bottom of the micropylar channel (Fig. 3). (1928), McGahan (1961a) and Grootjen (1981, unpubl. res.) The inner and outer integument are discontinuous at the those cells are filled with a gelatinous substance. The chalazal side. At this place a central disk has developed contents become brittle in the mature seed. On top of the 114 GraŠen et al.—Composition of Musaceaous Seed Coats

TIC 338, 356, 374 which are prominent for the Musaceae seed coats. The lignin pyrolysis products and the prominent peaks for the Musaceae are not extractable and are part of 180 the polymer system of the Musaceae seed coat, as shown in G Fig. 8. The presence of these compounds is also confirmed by PyGCMS as shown in Fig. 9 and Table 2. 210 S The spectra of the extracted and non-extracted seed coat show also pyrolysis products indicating the presence of 576 polysaccharides as shown by m\z 43, 57, 60, 73, 126 and 144 Dg for hexose sugars and m\z 58, 85 and 114 for pentose sugars (Pouwels, Eijkel and Boon, 1989). The presence of poly- 676 Wx saccharides is confirmed by ammonia chemical ionization. Figure 10 shows the pyrolysis ammonia chemical ionization 10 20 30 4050 60 70 mass spectrum of the extracted seed coat of Musa. This Scans spectrum contains mass peaks for ammoniated hexose sugar F. 7. Total ion current and mass chromatograms of the total seed monomers (m\z 134, 144, 162, 180, 222) which are prominent coat of Musa Šelutina. The ions of the mass chromatogram comprise for cellulose and ammoniated pentose sugar monomers markers of several compound classes as guaiacyl lignin (G), syringyl (m\z 132, 150) and dimers (m\z 264, 282), which are lignin (S), diglycerides (Dg) and waxes (Wx). prominent for hemi-cellulose (Pouwels and Boon, 1990). The abundance of dimeric and trimeric sugars is low due to chalazal disk, between the insertion of the inner integument, the presence of anorganic matter as indicated by the relative a parenchymatic tissue is situated. high m\z 134 (Scheijen and Boon, 1989). The ammonia CI data also confirm the presence of guaiacyl (m\z 163) and syringyl (m\z 193) lignin (Hage, Mulder and Boon, 1993; Macromolecules Hage, Weeding and Boon, 1995). No single technique exists to identify all macromolecular In the mass spectrum (Figs 5, 8) of the extracted and non- components at once. The evidence for the presence of the extracted seed coat samples mass peaks are observed for macromolecular components has to come from a com- phenolic compounds (m\z 94, 110, 124), which amongst bination of separation and ionisation methods. Table 1 lists others can indicate the presence of vegetable polyphenols. a number of component classes which can be recognized These compounds are also components of the polymeric with direct mass spectrometric and PyGCMS techniques. system and appear at high temperatures in the mass By addition of TMAH as a reagent it is possible to directly chromatogram as shown in Fig. 7. The presence of these transesterify ester bound components which facilitates their compounds is confirmed with Py (EI) GCMS. detection by mass spectrometry. Monocotyledonous plants usually have relatively high Figure 5 shows the overall spectrum of the non-extracted m\z 120 and 150 for p-coumaric acid and ferulic acids either seed coat of Musa. This spectrum contains markers of a present ester bound to the polysaccharide fraction or ether volatile fraction and a non-extractable part of the polymer bound to the true lignin structure (Boon, 1989). Also the system. All the three genera of the Musaceae gave the same extracted seed coat of the monocotyledonous Musaceae characteristic chemical ‘fingerprint’ of their seed coat. A contains relative high amounts of these phenolic acids. The lipid fraction of fatty acids (m\z 256, C"':!; 262, C"):#[M- Py-EI-MS (Fig. 8) shows peaks indicating ester bound (m\z H#O]; 264, C"):"[M-H#O]; 280, C"):#; 282, C"):"; 284, C"):!), 120) and ether bound (m\z 164) p-coumaric acid, and ester and as shown in Fig. 6, diglyceridic fragments of triglycerides bound (m\z 150) and ether bound (m\z 194) ferulic acid. (e.g. m\z 550, C"',"' [M-H#O]; 576, C"',") [M-H#O]; 602, Also the ammonia CI spectrum indicates the presence of p- C"),") [M-H#O]), triglycerides (e.g. m\z 830, C"',"',") [M- coumaric acid and ferulic acid as indicated by the H#O]; 857, C"',"),") [M-H#O]; 882, C"),"),") [M-H#O]) and pseudomolecular ions m\z 121 (vinylphenol) and m\z 151 wax esters (m\z 592jn.28), are thermally desorbed in an (vinylguaiacol). The PyGCMS of the TMAH treated early stage of the temperature ramp (Fig. 7), whereas the samples of the seed coat confirm the presence of these biopolymeric materials are pyrolysed at higher compounds as shown in Fig. 11. In this figure the mass temperatures. The presence of C"' and C") hydroxy fatty chromatograms of the masses m\z 192 and m\z 222 indicate acids is confirmed with Py-TMAH-EI-GCMS and Py- p-coumaric acid and ferulic acid, respectively (Mulder, TMAH-(ammonia)CI-GMS (not shown), indicating the Hage and Boon, 1992). presence of cutin (Boon, unpubl. res.). The Musaceae seed coat contains a number of charac- Evidence for lignin in the mass spectrum of the non- teristic compounds. The Py-EI-MS shows a peak at m\z extracted seed coat sample of Musaceae can be seen as the 194, which is still present after extraction. This peak is monomeric pyrolysis products at m\z 124, 138, 150, 152, identified with Py-EI-GCMS as a caustic secondary metab- 164, 166, 178, 180 and pyrolysis products of dimers at m\z olite zingirone. However, the most remarkable peaks are 272, 328, 340, 358 of guiacyl lignin. A low abundance of aromatic compounds which all have the fragment ion m\z syringyl lignin, indicated by m\z 154, 167, 168, 180, 182, 137 (hydroxymethoxybenzyl cation). Additional mass 194, 196, 208 and 210, is present in the mass spectrum. spectrometric techniques did not provide sufficient in- Furthermore the spectrum yields peaks at m\z 137, 200, formation to identify these compounds. GraŠen et al.—Composition of Musaceaous Seed Coats 115

× 2

46 100

pc 120

pc 61 137 164

85 58 151 180 114 73 98 t 126 200

t

0

406080 100 120 140160 180 200

× 2 Relative abundance 100

338 244 356 374 224 272 256 302

0

220240 260 280300 320 340 360 380 m/z F. 8. Pyrolysis (EI) mass spectrum of the seed coat of Musa Šelutina after extraction with hexane\dichloromethane and acetone\ethanol. #, guaiacyl lignin; $, syringyl lignin; >, polysaccharides; T, lipids; P, Musaceae characteristics; f, ferulic acid; pc, p-coumaric acid; t, vegetable polyphenol markers.

fleshy fruits and are predominantly zoochorously dispersed. DISCUSSION It seems that the aril has lost its function during evolution The main structure of the seed coats of the Musaceae taxa and probably the fruit has taken over the nutritive function that have been examined are similar. As mentioned before, for the dispersal of the seeds by animals (Friedrich and vestiges of the aril are still present on the mature seed, Strauch, 1975). although it is easily released, especially when the aril and Very characteristic in Musaceae is the separation of the exotesta cells are dried out and compressed. Musaceae have outer cell wall layer from the silicified exotesta. The silica on 116 GraŠen et al.—Composition of Musaceaous Seed Coats

31 100

29 34 46 54

27

50 23 Relative intensity

30 32 36 9 26 53 1 28 50 35 40 4345 47 55 56 33 38 8 18 39 71 7 21 13 14 37 62 19 41 51 24 48 57 74 4 12 42 52 76 78 2 5 11 15 22 25 44 49 61 69 6 17 20 58 64 70 72 68 73 79 3 10 16 5960 63 65 67 66 0 1000 2000 3000 4000 scan number 10:58 21:55 32:52 43:49 retention time F. 9. Total ion chromatogram of a pyrolysis (EI) gaschromatograph mass spectrometric run of a solvent extracted seed coat of Musa Šelutina. the exposed surface of the mature seed coat of the Musaceae combination with outer layers. The tightly packed, thick may function as a mechanical barrier against environmental walled cells of the mesotesta are probably impermeable for forces, pathogens, chewing insects, other predators, and water by the presence of water repellent substances, such as passing alimentary tracts of dispersers. In combination with lignin, deposited in the polymeric network of the cell walls. vegetable polyphenols, which are located more to the inner Also the unknown phenolic compounds may contribute to side of the seed coat, in the mesotesta and in the tegmen, it a tight polymeric network. The vegetable polyphenolic can be a sufficient barrier against fungi. In other mono- substance in the tegmen probably hampers the diffusion of cotyledons with silicified seed coats more often the silica is oxygen through the seed coat (Guan, 1991). In addition, the located in the endotesta. In Commelinaceae the siliciferous other possible openings at the micropylar side and the endotestal layer splits along its radial walls as a result of chalazal side (chalazal disk) of the seed coat are blocked. which the exo- and mesotestal layers of the seed become The germination of Musa is variable and relatively separated. Netolitzky (1926) mentioned the occurrence of difficult under artificial conditions. Stotzky et al. (1962) siliciferous endotestas in Commelinaceae, Zingiberaceae scarified the lateral side of Musa balbisiana seeds, which and of a siliciferous exotesta in Musaceae, and suggested a raised the germination percentage up to 80% and shortened protection of embryo and endosperm as one of the main the time to germinate from 3–6 weeks to 6–10 d. Probably functions. water entry and gas exchange are made possible by to the The silica layer may help to prevent the seed coat from interruption of the silica layer, cuticle, and mesotesta. oppression and shrinking during germination. The role of The hard seed coat of Musa leads to conflicting demands silica in the cell wall seems to be analogous to that of lignin of protection and germination. Hard seed coats offer an as a compression resisting element, i.e. more rigid re- efficient protection during maturation, dispersal and dor- placement for the water between the microfibrils and other mancy; however, it hampers germination, because the carbohydrate components of the wall of non-lignified cells embryo needs strong forces to rupture the seed coat. (Raven, 1983). Furthermore, the silica can also restrict Germination lids with predetermined rupture layers fa- water loss by hydration of the silica gel (Kaufman et al., cilitate germination. In Musaceae germination takes place 1981; Yoshida, Ohnishi and Kitagishi, 1962). by squeezing out the operculum as a result of the elongating The thick cuticle at the innermost side of the seed coat radical-hypocotyl axis, without rupturing the seed coat may function as a last barrier to water diffusion in (McGahan, 1961b). Removal of the operculum of the GraŠen et al.—Composition of Musaceaous Seed Coats 117 T 2. PyGCMS data on Musa velutina seed coat. Identified and unknown peaks from a chromatogram after extraction. From eŠery peak the molecular ion is giŠen (M+d);PS,Polysaccharide; L, Lignin; Lp, Lipid; M, aromatic phenols of unknown structure with fragment ion m\z 137

Peak + no. Compound Origin Scan M d Remarks

1 carbon dioxide 120 44 2 propanedialdehyde PS 162 72 3 2,3-butanedione PS 226 86 4 2-butanone PS 235 72 5 2-methylfuran PS 252 82 6 3-methylfuran PS 262 82 7 acetic acid PS 291 60 8 2-butanol cis\trans PS 302 70 9 hydroxypropane PS 321 74 10 2,5-dimethylfuran PS 409 96 11 but-3-enal-2-one PS 491 84 12 3-hydroxypropanal PS 500 74 13 unknown 526 92 m\z 65, 77, 92 14 pyruvic acid PS 531 102 15 (2H)-furan-3-one PS 553 84 16 3-furaldehyde PS 591 96 17 4,2-pentadienal PS 628 82 18 2-furaldehyde PS 637 96 19 1-(acetyloxy) propane-2-one PS 718 116 20 (5H)-furan-2-one PS 789 84 21 2,3-dihydro-5-methylfuran-2-one PS 872 98 22 5-methyl-2-furaldehyde PS 958 110 23 phenol 1038 94 24 2-hydroxy-3-methyl-2-cyclopentene-1-one PS 1128 112 25 4-methyl-phenol L 1212 108 26 2-methyl-phenol (o-cresol) L 1263 108 27 guaiacol L 1280 124 28 2-ethylphenol 1469 122 29 4-methylguaiacol L 1527 138 30 1,2 dihydroxybenzene 1550 110 31 4-vinylphenol L 1578 120 pc 32 guaiacylethane (l4-ethylguaiacol) L 1711 152 33 methyl-1,2-dihydroxybenzene 1740 124 34 4-vinylguaiacol L 1781 150 f 35 syringol L 1837 154 36 guaiacolaldehyde L 1923 152 37 4-methylsyringol L 2031 168 38 4-(trans-2-propenyl)guaiacol L 2047 164 39 4-butene guaiacol L 2062 178 40 4-acetyl guaiacol L 2094 166 41 methyl 2-isopropylbenzoate 2101 178 42 levoglucosan PS 2080– 162 2175 43 methyl α-methyl cinnamate 2172 176 44 unknown lignin component L 2229 178 45 4-vinylsyringol L 2246 180 46 methyl β-methyl cinnamate 2294 176 47 2-butanone, 4-(4 hydroxy-3 methoxy 2383 194 phenyl; zingirone 48 syringylaldehyde L 2391 182 49 4-(prop-cis-2-enyl-syringol L 2397 194 50 4-(prop-trans-2-enyl-) syringol L 2482 194 51 4-acetyl syringol L 2518 196 4-(prop-2-enol) guaiacol L 2518 178 52 unknown * 2534 202 m\z 137, 159, 169, 187, 202 53 unknown 2653 192 m\z 89, 117, 145, 177, 192 54 unknown 2679 200 naphthalene skeleton, m\z 128, 139, 157, 185, 200 55 unknown * 2688 202 m\z 137, 159, 169, 187, 202 56 unknown * 2720 202 m\z 137, 159, 169, 187, 202 57 phtalaat artefact 2750 (149) 118 GraŠen et al.—Composition of Musaceaous Seed Coats T 2. (cont.)

Peak + no. Compound Origin Scan M d Remarks

58 unknown * 2773 220 m\z 137, 169, 187, 202, 220 59 unknown * 2796 220 m\z 137, 151, 162, 202, 220 60 phtalaat artefact 2888 (149) 61 C"':! fatty acid Lp 2920 256 62 unknown 3028 228 m\z 77, 105, 123, 151, 185, 228 63 C"):# fatty acid Lp 3155 280 64 unknown * 3161 264 m\z 137, 151, 216, 264 65 C"):" fatty acid Lp 3167 282 66 C"):! fatty acid Lp 3200 284 67 unknown * 3552 288 m\z 137, 149, 225, 241, 257, 273, 288 68 phtalaat artefact 3665 (149) 69 3-methoxy-4,4h-hydroxy stilbene L 3752 272 70 unknown * 3893 338 m\z 137, 141, 169, 183, 201, 214, 338 71 unknown * 3902 336 m\z 137, 152, 169, 181, 197, 211, 275, 303, 321, 336 72 unknown * 3945 338 m\z 137, 152, 169, 181, 197, 211, 275, 303, 321, 336 73 unknown * 3955 338 m\z 137, 141, 169, 183, 201, 214, 310, 338 74 unknown * 4026 350 m\z 137, 141, 169, 195, 200, 310, 338, 350 75 unknown * 4062 342 m\z 137, 151, 163, 281, 342 76 unknown * 4111 358 m\z 137, 151, 163, 179, 190, 222, 340, 358 77 unknown * 4137 334 m\z 137, 151, 163, 175, 188, 202, 231, 270, 274, 281, 303, 317, 334 78 unknown * 4150 340 m\z 137, 151, 203, 340 79 unknown * 4333 340 m\z 137, 151, 163, 175, 190, 203, 340

Musaceae seeds permitted water to reach the embryo, but described the seed coat as not lignified. The PyMS fingerprint did not result in a higher percentage of germination. This of Musaceae indicated the presence of guaiacyl and syringyl indicates that water entering the seed is unable to hydrate lignin components. This observation was confirmed with Py the embryo. So Musaceae also have an embryo imposed (EI) GCMS. dormancy. Harris and Hartley (1976) stated that cell walls containing The mass spectrometric ‘fingerprint’ of the macro- ferulic acid bound to polysaccharides give a negative molecular composition of Musaceae shows some unique phloroglucinol\HCl test for lignin. The macromolecular and characteristic mass peaks. Although no structure can be data of Musaceae show that the seed coat indeed contains assigned, these mass peaks seem to be specific for Musaceae, ferulic acid, but is also lignified. A combination of several and will be very useful as an additional data set for components in the cell wall is probably the reason why most macrosystematic research of Angiosperm families (Graven, stains are negative. unpubl. res.). Further studies are in progress to obtain more Monocotyledonous plants usually have relatively high information about the chemical structure of these unknown m\z 120 and 150 from p-coumaric and ferulic acids, either Musaceae components. present ester bound to the polysaccharide fractions or ether With the phloroglucinol\HCl test or other tests no lignin bound in the true lignin structure (Boon, 1989). Ferulic acid components could be detected, although the test with lignin bound to the primary wall has been recorded in nearly half pink gave the indication of the presence of lignin in the S1 of the monocotyledon families and in about 10 families of layer of the secondary wall of the mesotesta. These findings the dicotyledons only (Harris and Hartley, 1980). are supported by the results of McGahan (1961a), who also Many studies have shown that phenolic acids are able to obtained negative results for lignin when routine tests for affect germination, but usually via the germination rate lignin were used. Also Stover and Simmonds (1987) (time needed for germination) rather than via the total seed GraŠen et al.—Composition of Musaceaous Seed Coats 119

180 100

134 pc 151 150 pc 121 162 193 132 144 98 224 222

0

6080 100 120 140160 180 200 220

× 5 Relative abundance 100

242 339

356 391 279

264 282 300 376

0

240 260280300 320 340 360 380 400 m/z F. 10. Pyrolysis (ammonia CI) mass spectrum of the extracted seed coat of Musa Šelutina. #, Guaiacyl lignin; $, syringyl lignin; >, polysaccharides; P, Musaceae characteristics; 4, phenolic compounds; f, ferulic acid; pc, p-coumaric acid. germination (Kuiters, 1990). Seeds of Musa Šelutina and attachement of staining material. If a lipid is attached to Musa rosacea germinated relatively easily in 3–8 weeks. other macromolecular components or even not attached but Within the Musa seed coat the phenolic acids seem to be covered by other macromolecules, the result of a staining genuine structural components of the cell walls, not affecting will be negative. The presence of the insoluble cutin polymer germination. in the cuticle is not specifically detectable with histochemical The use of histochemical methods for the detection of methods. The PyMS and PyGCMS data still contained C"' lipids shows the limitation of this approach (Gahan, 1984). and C") fatty acids after extraction of lipids, which can There is no specific functional group in lipids for indicate the presence of insoluble polymeric cutin. The 120 GraŠen et al.—Composition of Musaceaous Seed Coats

TIC

192 3 OCH 3 C O C C OCH

222 3 3 OCH OCH 3 C O C C OCH

1000 2000 3000 4000 Scans F. 11. Py (EI) GCMS partial mass chromatogram of the TMAH treated samples of the extracted seed coat of Musa Šelutina for m\z 192 (p-coumaric acid) and m\z 222 (ferulic acid). evidence for the presence of cutin hydroxy fatty acids was al., 1993). The identification of zingirone in the Musa seed given by using methylated samples of the extracted seed coat confirms the relationship of the Musaceae with coat using Py(EI)GCMS and Py(NH$-CI)GCMS. Zingiberaceae. Zingirone was first described in the rhizome Waxes are also associated with the polymeric material in of Zingiber officinale. The presence of products as zingirone the cuticle to provide an effective diffusion barrier in the seed coat of Musa is another defence line against (Kolattukudy, Espelie and Soliday, 1981). This may explain specific chewing insects and other predators. the relatively high contribution of wax esters in the mass The thermal stability of condensed vegetable polyphenols spectrum of the Musa seed coat. The cuticle between tegmen is high. The occurrence of the components 1,2- and the remains of the nucellus of the Musaceae have dihydroxybenzene (m\z 110) and methyl-1,2- several functions as a last barrier against water loss, ion dihydroxybenzene (m\z 124) indicates the presence of diffusion and several pathogens. proanthocyanidins. Lignins have been reported to contain The mass spectrometric techniques are sensitive and only negligible amounts of catechol (Galletti and Reeves, specific methods in identifying macromolecular compounds. 1992). And so it is reasonable to use catechol as a diagnostic It is not always possible to locate the different compounds fragment for condensed vegetable polyphenols (Galletti and in the differentiated tissues of the seed coat, unless these can Reeves, 1992). Vegetable polyphenols are extremely complex be separated from each other and analysed separately. The compounds. They may be highly specific in at least some isolation of the different seed coat sections by hand is still and probably most of their possible interactions, via an the limiting factor. Histochemical methods are mainly enormous potential for stereo specificity (Zucker, 1983). useful in localising certain compounds. However, it has Within the seed vegetable polyphenols have several mixed been shown that especially in mature seed coats the stains functions, involving a defence mechanism against living are not always succesful in attaching a specific compound. plant enemies, a delay in decomposition when the seed is a Many pyrolysis products were identified using PyGCMS, component of the seed bank, and influencing dormancy in confirming the general findings of the PyMS spectra. seeds. Caustic products are widely distributed within the family The real cause of dormancy of Cornus seeds is due to a Zingiberaceae. Members of the family Zingiberaceae have high level of vegetable polyphenols in the seed coat attracted continuous phytochemical interest due to their restricting the embryonal development (Guan, 1990). considerable importance as natural spices or as medicinal Proanthocyanidins are present in most families of the plants (Pandji et al., 1993). In literature a lot of rhizomes monocotyledons. According to Dahlgren, Clifford and Yeo and seeds are described as tasting caustic (Hegnauer, (1985) cells with vegetable polyphenols of the Musaceae 1962–1990). Taxa of Zingiberaceae have been studied for may contain proanthocyanidins. Ellis, Foo and Porter insecticidal activity (Grainge and Ahmed, 1988; Pandji et (1983) have described the occurrence of proanthocyanidins GraŠen et al.—Composition of Musaceaous Seed Coats 121 in the fruit skin of Musa sapientum. Within the line of this Feder N, O’Brien TP. 1968. Plant microtechnique: some principles and study no specific information on vegetable polyphenolic new methods. American Journal of Botany 55: 123–142. structures has been found. Friedrich WC, Strauch F. 1975. Der Arillus der Gattung Musa. Botaniska Notiser 128: 339–349. Many monocotyledons have anatomically relatively Gahan PB. 1984. Plant histochemistry and cytochemistry. London: simple seed coats. Endotestal seeds as found in Dioscoreales, Academic Press. Zingiberales, Commelinales and others may be original for Galletti GC, Reeves JB. 1992. Pyrolysis\gas chromatography\ion- monocotyledons. The seed coat structure of the Musaceae is trapdetection of polyphenols (vegetable tannins): preliminary characteristic at the family level and the most complex one results. Organic Mass Spectrometry 27: 226–230. Grainge M, Ahmed S. 1988. Handbook of plants with pest-control within the Zingiberales. Very remarkable are the micropylar properties. New York: John Wiley. collar, the relative thick mesotesta, the unique macro- Guan KL. 1990. Studies on dormancy of Cornus officinalis seed. molecular ‘fingerprint’ with the typical Musaceae phenolic International Conference on Seed Science and Technology, 12–15 compounds of still unknown composition, and the breaking March 1990, Hangzhou, China, 52. of the cell walls in the exotestal layer resulting in a surface Guan KL. 1991. Studies on complex dormancy of Cornus officinalis with silica crystals. More often the endotesta is silicified, as seed. Chinese Journal of Botany 3: 145–150. Hage ERE van der, Mulder MM, Boon JJ. 1993. Structural charac- in Commelinaceae, Strelitziaceae, Rapateceae, and terization of lignin polymers by temperature-resolved in-source Marantaceae. In Commelinaceae also a silicified surface pyrolysis-mass spectrometry and Curie-point pyrolysis-gas remains due to a breaking of the endotesta. The Zingiberales chromatography\mass spectrometry. Journal of Analytical and is one of the most indisputable natural suprafamilial groups Applied Pyrolysis 25: 149–183. (Dahlgren et al., 1985). The seed coat structure of the Hage ERE van der, Weeding TL, Boon JJ. 1995. Ammonia chemical ionization mass spectrometry of substituted phenylpropanoids Musaceae is characteristic and the most complex one of the and phenylalkyl phenyl ethers. Journal of Mass Spectrometry 30: Zingiberales, whereas that of the Cannaceae is considered as 541–548. the most derived one within this order. The rather complex Harris PJ, Hartley RD. 1976. Detection of bound ferulic acid in cell seed coat is characteristic for this family and seems to be walls of the Graminae by ultraviolet fluorescence microscopy. unique if compared to other families of the Zingiberales and Nature 259: 508–510. Harris PJ, Hartley RD. 1980. Phenolic constituents of the cell walls to other monocotyledons. monocotyledons. Biochemical Systematics Ecology 8: 153–160. Hegnauer R. 1962–1990. Chemotaxonomy der Pflanzen Vol. 1–9. Basel: Birkha$ user Verlag. ACKNOWLEDGEMENTS Kaufman PB, Dayanandan P, Takeoka Y, Bigelow WC, Jones JD, Her The authors wish to thank J. B. M. Pureveen, N. Devente, R. 1981. Silica in shoots of higher plants. In: Simpson TL , Volcani BE, eds. Silicon and siliceous structures in biological systems. J. Dahmen, F. D. Boesewinkel and M. M. Mulder for Berlin: Springer Verlag, 409–449. technical assistance. Special thanks are for B. Ursem of the Kolattukudy PE, Espelie KE, Soliday CL. 1981. Hydrophobic layers Hortus Botanicus in Amsterdam for obtaining most of the attached to cell walls, cutin, suberin and associated waxes. seed material and W. J. Kress for sending seeds of the genus Encyclopaedia of Plant Physiology, New Series 13B: 225–254. Musella. Krisnamurthy KV. 1988. Methods in plant histochemistry. Chetput, The investigations were supported by the Life Science Madras: S. Viswanathan (Printers & Publishers) Private Limited. Kuiters AT. 1990. Role of the phenolic substances from decomposing Foundation (SLW), which is subsidised by the Netherlands forest litter in plant soil interactions. Acta Botanica Neerlandica Organisation for Scientific Research (NWO). 39: 329–348. Manchester SR, Kress WJ. 1993. 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