Development and morphology of secretory trichomes of Calceolaria volckmanni (Scrophulariaceae

Nordic Journal of Botany

Development and morphology of secretory trichomes of Calceolaria volckmanni (Scrophulariaceae)

Gianni Sacchetti, Alessandro Bruni, Giuseppe Dall’Olio, Marcello Nicoletti, Antonella Di Fabio and Ferruccio Poli

Sacchetti, G., Bruni, A,, Dall’Olio, G., Nicoletti, M., Di Fabio, A. & Poli, F. 1997. Development and morphology of secretory trichomes of Calceolaria volckmanni (Scrophulariaceae). - Nord. J. Bot. 16: 505-51 3. Copenhagen. ISSN 0107-055X.

The development and morphology of secretory trichomes of Calceolaria volckmanni was examined with light and electron microscopy. The formation and development of the glandular trichomes began with the outgrowth of a single epidermal cell which progressively increased in height and evolved into a pear-shaped cell. Subsequent divisions generated a mature trichome formed by a basal cell, a stalk “endodermal“ cell and an 8-celled glandular head. Histochemical tests revealed the lipophilic nature of the secretion, the presence of terpenes and flavonoids, and displayed a particular cutinization of the walls of the stalk cell. The observed ultrastructural features of the lipophilic glandular hairs suggested the function of plastids in the secretory process.

G. Sacchetti, A. Bruni, and G. Dall’Olio, Department of Biology, Section of Botany, University of Ferrara. C.so Porta Mare 2, 1-44100 Ferrara, Italy. - M. Nicoletti and A . Di Fabio, Department .f Plunt Biology, “La Sapienza” University, P le Aldo Moro 5, 1-00185 Roma, Italy. - I? Poli, Institute of Botany and Botanical Garden, Univer.sitv of Cagliari, Viale Frci lgnazio 13, 1-09123 Cagliari, Italy.

Introduction

Calceolaria is a genus of the Scrophulariaceae, mostly composed by medium sized herbs, widespread in tropi- cal and temperate South America. Ten per cent of Cal- ceolaria spp. are endemic to Chile (Navas 1979) and some are known and appreciated in floriculture for the characteristic shape of their corolla with a peculiar slip- per sized lower lip. Besides their ornamental use, the aerial parts of many Calceolaria spp. are employed in Chilean folk medicine as stomach tonic, sweetener, an- tibacterial and cicatrizant agents (Navas 1979; Muiioz et al. 1981).

Regarding Scrophulariaceae, Schnepf ( 1969) described the production of a fatty oil by glandular hairs in flowers of a cultivated Calceolaria, while Vogel (1971) reported a floral secretion of insect attracting fatty-oil in

plants belonging to the genera Calceolaria, Angelonia and Dacia.

Recently, it was reported that the aerial parts of some Calceolaria spp. contain phenylpropanoid glycosides and small amounts of flavonoids and terpenic compounds (Nicoletti et al. 1988; Wollenweber et al. 1989; Chamy et al. 1989, 1990; Garbarino et al. 1992; Di Fa- bio et al. 1995). Other phytochemical investigations revealed that terpenic compound distribution in Calceo- laria is complex. Some species, i.e. C. adscendens, C. glandulosa, C. foliosa, C. hypericina and C. latifolia, produce diterpenes peculiar to the genus, while in other species, as C. volckmanni, C. crassifolia and C. scabi- osafolia, diterpenes were not found (Garbarino et al. 1992).

This study, carried out by conventional, fluorescence and electron microscopy (SEM and TEM), examines the

Accepted 12- 12- 1995 0 NORDIC JOURNAL OF BOTANY

Nord. J . Bot. lh(51 1996 505

development, ultrastructure and secretion of leaf trichomes of the endemic Chilean Calceolaria volckmanni. The aim was to evaluate possible correlations between the phytochemical data and the morphology of the secretory trichomes in which secondary compounds are synthesized and stored.

Materials and methods

Seeds of Calceolaria volckmanni from Chilean wild plants were germinated in the dark on moistened paper. Then, seedlings were cultivated in small pots with com- mon soil in a phytotrone (HERMUS, VEPHQ 5/1350) with a photoperiod of 16 h light (10 000 lux), 20 f 1°C and with 80 f 10 % of humidity.

Conventional and fluorescence microscopy

Small pieces of vegetative apices, young and mature leaves were fixed and embedded following previously reported procedures (Bruni & Modonesi 1983). For the histological investigations, embedded sections were processed with the following methods: a) toluidine blue 0 (TBO) as general staining (Feder & O’Brien 1968); b) periodic acid-Schiff reagent (PAS) (Jensen 1962) and periodic acid-fluorescent Schiff reagent (F-PAS) for polysaccharides (Bruni & Vannini 1973).

Fresh sections of leaves at different stages of development were made using razor blades, and were stained as follows: a) Sudan Black B for lipids (Jensen 1962); b) NADI reagent for terpenes (David & Carde 1964); c) ferrous thiocianate and concentrated H,SO, for sesquiterpenes (Cappelletti et al. 1986); d) SbCI, for terpenes containing steroids (Hardman & Sofowora 1972); e) Al- C1, for flavonoids and lead basic acetate for flavones (Guerin et al. 1971); f) silver nitrate for calcium oxalate crystals (Franceschi & Homer 1980).

For all the above-cited histochemical methods, con- trol reactions were made following the suggestions of the respective authors. A Zeiss Axiophot microscope, with UV exciting illumination (UV-H 365:BP 365/12, FT 395, LP 397 excitation filter), was used for fluorescence and conventional investigations.

Scanning and transmission electron microscopy

For SEM, pieces of leaves (approximately 2 mm long), at different stages of development, were fixed and processed as reported in Bruni et al. (1987). The samples were viewed with a Stereoscan 360 Cambridge microscope (Electronic Microscopy Center, University of Fer- rara).

For TEM, the same kinds of samples were fixed and processed as cited in Poli et al. (1989). Some thin sections were processed with ThiCry reaction for polysaccharides (ThiCry 1967). A Zeiss electron microscope EM 109 N was used for the observations.

To test the occurrence of calcium having crystalline structure in the vacuoles, X-ray microanalysis was employed (Franceschi & Homer 1980). 100 nm thin sections of the same specimens used for TEM were laid down on carbon supports for SEM. The X-ray microanalysis for calcium (Ka=3,693 KeV and Kb=4,014 KeV) was performed with a Stereoscan 360 cambridge microscope equipped with a Link Analytical W. D. S . spectrometer (Electronic Microscopy Center, University of Ferrara) and focusing the beam on the vacuoles.

Mature leaves of Calceolaria volckmanni were coated with a sticky material both on the adaxial surface and, scantily, on the abaxial one. This substance embedded

~ ~~

Figs 1-9. Calceolaria vo1ckmanni.- Figs 1 and 2 (a, b). Fresh sections of a mature leaf at conventional and fluorescence microscope respectively. The secreted material embeds the glandular trichomes (arrows; Figs I ; 2a) and fluoresces at UV light (asterisks; Fig. 2a). Fig. 2b. After dipping in chloroform, only the fluorescent heads of the trichomes (arrows) remained visible. Bars = 50 pm (x 400; x 180; x 180). - Fig. 3. SEM micrograph of the adaxial surface of the mature leaf. Note non-secreting trichomes (arrows), glandular hairs (arrowheads) and hydathodes (H). Bar = 100 pm (X 120). - Fig. 4. Detail of Fig. 3 showing a mature glandular trichome formed by a stalk and a globe-shaped multicellular head. Bar = I0 pm (x 1600). - Figs 5-7. Longitudinal resin sections of glandular trichomes at different stages of development at fluorescence microscope. F-PAS staining. - Fig. 5. Pre-secretory stage. Outgrowth of a single epidermal cell which becomes pear-shaped. Bar = 25 pm (x 600). - Fig. 6. Pre-secretory stage. The first anticlinal division generates a plasma rich upper cell and a vacuolated lower cell. Bar = 25 pm (x 850). - Fig. 7. Two-celled and three-celled glandular trichomes at the pre-secretory stage and a mature glandular hair at the secretory stage. Bar = 25 pm (x 550). Fig. 7a. Transverse section of a mature glandular hair showing an eight-celled secretory head. Bar = 25 pm (x 400). - Fig. 8. Longitudinal fresh section of a glandular trichome at the beginning of the secretory stage stained with Sudan Black B observed at conventional microscope. Note the positive reaction to the histochemical staining of the secretory products (arrow) filling up the subcuticular space. Bar = 25 pm (x 780). - Fig 9. Longitudinal fresh section at conventional microscope showing a mature trichome with a tight dome built up by the secreted material (asterisk) deposited between the cell walls (arrowheads) and the cuticular sheath (arrows). Bar = 25 pm (x 520).

Nord. J Boi. 16(5) I996 506

Tab. 1 . Autofluorescence and different reactivity of the glandular trichomes at the secretory stage (- negative; +- weakly positive; ++ positive). Note the positive reaction of the secretion, of the stalk cell and of the head cells to Sudan Black B and NADI reagent, specific for lipids and terpenes respectively. The stainings of sesquiterpenes with Fe(SCN), and concentrated H,SO,, and of steroidic terpenes with SbCI, were negative. AICI, and lead basic acetate, both specific for flavonoids, weakly stained the head cells and the secretory products. The specific reaction for calcium oxalate with silver nitrate gave a positive response in the head cells.

Staining Target Cells of the head Cell of the stalk Secretion compounds

Autofluorescence 350-380 nm +- ( yellow-orange)

++ (red)

++ (yellow-orange)

Sudan Black B lipids ++ +- ++ NADI reagent terpenes

(blue-violet) ++

(pink) ++

(blue-violet) ++

Fe( SCN), sesquiterpenes

Concentrated H,SO, sesqui terpenes

SbCI, steroidic terpenes

AICI, flavenoids +- +- Lead basic acetate tlavenoids +- +- Silver nitrate calcium oxalate ++

numerous glandular hairs responsible for the secretion (Fig. 1). The secreted material fluoresced bright yellow under UV light (Fig. 2a). A quick immersion of fresh leaf samples in a non-polar solvent (chloroform) re- moved all the fluorescent oleoresin and only the fluorescent heads of the trichomes remained visible (Fig. 2b).

SEM investigations showed that the glandular trichomes were numerous and uniformly distributed on the adaxial surface (Fig. 3). On the abaxial surface, there were fewer trichomes, mainly localized along the major veins. In the leaf margin, there were mostly non-secreting trichomes and some hydathodes at the apex of the leaf teeth. When mature, adaxial and abaxial glandular trichomes had a stalk and a globular multicellular head

(Fig. 4). Trichome development had three distinct stages: pre-secretory. secretory and post-secretory.

At the pre-secretory stage, the outgrowth of a single epidermal cell progressively increased in height and evolved to a pear-shaped cell. The nucleus of this cell was centrally located at the beginning, then migrated to- ward the apex. The lower part of the cell was occupied by a large vacuole (Fig. 5 ) . Thereafter, the first pericli- nal division generated a plasma rich upper cell and a vacuolated lower cell (Fig. 6) . The lower one remained an epidermal cell, while the upper one underwent two successive penclinal divisions forming a 3-celled hair comprised of a basal cell, a stalk cell and a mother cell of the head (Fig. 7). The lower cells were small and

Figs 10-16. Culceoluriu volckmonni - Figs 10-12. Longitudinal resin sections of glandular trichomes at different stages of development observed at conventional microscope. PAS-staining. - Fig. 10. During the pre-secretory stage, the lateral walls of the stalk cell were PAS-positive (arrows). Bar = 25 pm (x 750). - Fig. 11. Glandular hairs at the secretory stage showing the lateral walls of the stalk cell (arrow) not stained by PAS reaction. Bar = 25prn (x 500). - Fig. 12. At the post-secretory stage, the trichomes display a collapsed glandular head. The lateral walls of the stalk cell are still PAS-negative (arrows). Bar = 25 prn (x 570). - Figs 13-14. TEM micrographs of a glandular hair at the pre-secretory stage. - Fig. 13. Note a dense cytoplasm with large nuclei and nucleoli (NU) and vacuoles with osmiophilic droplets (D). Bar = 5 prn (x 4000). - Fig. 14. Detail of Fig. 13 showing numerous profiles of RER (R), Golgi bodies ( G ) , mitochondria (M) and plastids (P). Bar = 1 pm (x 16200). - Figs 15-16. TEM micrographs of a glandular hair at the secretory stage. - Fig. 15. The trichome presents an eight-celled head (H), a stalk cell ( S ) and a coin-like basal cell (B). Arrows = photosynthetic plastids. Bar = 10 pm (x 2000). - Fig. 16. Enlargement of the wall between the stalk cell (above) and the basal cell. Note the numerous plasmodesmata (arrowheads) and photosynthetic plastids (P). Bar = I pm (x 18000).

Nord. J . But. 1601 1996 508

Nord. J . Bot. 16(5) 1996 509

X-RAY Live= 1 00s P r e s e t = 1 00s

4 ca I

N ’ 0 A 0s I rn

2 - 2 I -

0.0 2.6 KeV 5.2

Fig. 17. X-ray spectrum obtained from the analysis of the crystalline structures in the vacuoles of the glandular head. The beam of the X-ray microanalysis is focused on the vacuoles of the head cells.

coin-like while the terminal one was large and globe- shaped. By a series of anticlinal divisions, the mother cell of the head formed an 8-celled secretory head (Fig. 7a).

As revealed by the Sudan Black B reaction, secretory lipophilic products slowly filled up the sub-cuticular space. The secretory process started early, when the glandular head consisted of two cells (Fig. 8). At maturity, the trichome contained an 8-celled secretory head beneath a tight dome which slowly swelled as the secreted material was deposited between the cell wall and cuticular sheath (Fig. 9). At this stage, the observations under UV light at 365 nm of fresh samples showed a bright yellow-orange fluorescence of the trichome head and a red fluorescence of the cytoplasm of the stalk cell.

Tab. I presents the histochemical reactions performed. Sudan Black B stained the secretions, the head cells and partially stained the stalk cell indicating the presence of lipophilic compounds. NADI reagent, spe-

cific for terpenes, stained positively the secretions and partially stained the head cells blue-violet, while the material in the vacuole of the stalk cell was stained pink, which means the presence of resiniferous acids. However, other specific reactions for terpenic compounds [Fe(SCN),, H,SO, and SbCI,] .were negative. The reactions with aluminium chloride and lead citrate employed for checking the presence of flavonoids were weakly positive. The histochemical technique for evalu- ating the presence of calcium oxalate crystals showed a positive staining in the secretory cells of the head.

During trichome ontogenesis, the composition of the lateral walls of the stalk cell changed. In the pre-secretory stage, these walls were PAS-positive indicating the presence of polysaccharidic compounds (Fig. 10). while, when the trichome had a secretory head composed by at least two cells and evolved to a mature glandular structure, the lateral walls of the stalk cell became PAS-negative (Fig. 11). In the post-secretory stage, the leaf trichomes had a collapsed glandular head devoid of secretory products (Fig. 12)

Ultrastructurally, in the pre-secretory stage all the cells of the glandular hair had a dense cytoplasm, containing large nuclei with nucleoli and vacuoles with osmiophilic droplets (Fig. 13). In addition, the cells had numerous profiles of RER, Golgi bodies, a few mitochondria and plastids (Fig. 14). At the secretory stage, the coin-like basal cell displayed a large vacuole and few plastids with few thylakoids. The stalk cell revealed a large vacuole filled with osmiophilic material and a dense cytoplasm (Fig. 15). Photosynthetic plastids were also observed (Figs 15 and 16). In the cross wall between the stalk cell and the basal one, numerous plasmodesmata were noted (Fig. 16). The secreting cells of the head showed an electrondense cytoplasm, rich in ri- bosomes, dictyosomes and RER, as observed in the stalk cell. The vacuoles presented electron translucent crystalline structures. Focusing the beam of the X-ray microanalysis on the vacuoles appreciable amounts of calcium were pointed out (Figs 17 and 18). The plastids showed a particular stroma full of a tubular network that became more electrondense when the secretory trichome reached maturity (Fig. 18).

At first, the secretory products synthesized in the glandular head were mainly extruded into the spaces

~~

Figs 18-22. Calceolaria volkrnanni. - Figs 18 and 19. TEM micrographs of a glandular hair at secretory stage.-- Fig. 18. The cells of the head shows an electrondense cytoplasm with RER (R), Golgi bodies (G), electrondense plastids (P) with tubular networks and crystalline structures (C) in the vacuoles. Bar = 5 pm (x 4600). - Fig. 19. The secretory products are extruded within the spaces (asterisks) between the walls of two cells of the head. Bar = 1 pm (x 20000). - Figs 20-21. TEM micrographs after ThiCry reaction. - Fig. 20. Detail of the lateral wall of the stalk of a glandular hair at the pre-secretory stage. The reaction stained the polysaccharidic network of the walls (asterisks) while the thin cuticle (arrows) remains unreactive. Bar = 1 pm (x 2oooO). - Fig. 21. Detail of the lateral walls of the stalk cell of a glandular hair at the secretory stage. Note the weakly positive polysaccharidic network (asterisks), the thick cutinized wall and the cuticle (arrows). Bar = 1 pm (x 18000). - Fig. 22. TEM micrograph of a glandular head at the post-secretory stage. Note the degradation of the cellular constituents and the progressive disconnection of the walls of the head cells which trended away one from another (arrows = cuticle). Bar = 5 pm (x 5600).

510 N o d J . But. Ih(S) 1YY6

Nord. J . Bot. 16(.0 IY9h 5 1 1

formed between the cell walls. Later, these spaces increased as the cells moved away from each other (Fig. 19). Finally, the secretion, filling up both the spaces and the subcuticular area, induced the glandular head of the trichome to become spherical in shape.

The histochemical staining for TEM confirmed the peculiar property of the lateral walls of the stalk cell observed through the optical microscope. At the pre-secretory stage the lateral walls displayed clear polysaccharidic structures and an unreactive thin cuticle (Fig. 20). while at the secretory stage there was a thick cutinized wall with a weak polysaccharidic network (Fig. 21).

In the post-secretory stage, trichomes underwent a progressive degradation of their cellular constituents and the walls of the head cells were progressively dis- connected. The cuticle was constantly present and displayed no particular ruptures or structures as pores (Fig. 22).

The results obtained by histochemical reactions indi- cate that the secretions in C. volckrnanni trichomes are lipophilic, as revealed by Sudan Black B. The test with aluminium chloride and lead citrate and the bright yellow-orange fluorescence obtained under UV light evi- denced that the glandular trichomes secrete flavonoids, supporting previous phytochemical data of some Cul- ceolaria spp. (Wollenweber et al. 1989). As for terpenes, the general staining suggested by David & Carde (1964) was positive, in accordance with Di Fabio (per- sonal communication) who also found triterpenes and flavones in the secretion of C. volckmanni. The negative response to Fe(SCN), and H,SO, established the ab- sence of sesquiterpenes in the secretion of the glandular trichomes. In the same way, the negative result with Sb- CI, excluded the occurrence of steroidic terpenes. Crys- tals of calcium oxalate in the vacuoles of head cells were demonstrated by silver nitrate and X-ray microanalysis. This kind of crystal has been frequently described in glandular hairs with lipophilic secretion (Werker & Fahn 1981; Ascensko & Pais 1987).

The ultrastructural characteristics of glandular hairs occurring in flowers of a cultivated Calceolaria were described by Schnepf (1969), who found that the oil was stored in small subcuticular spaces and released outside by the rupture of the cuticle. In addition, in several kinds of external glands secreting lipophilic substances, oleoresin frequently accumulates in the subcuticular space and is discharged in the same way (Ascensko & Pais 1987).

In conclusion, the glandular trichomes of leaf surfac- es of C. volckmanni present a secretion with both flavonoid and terpene compounds, thus supporting the phytochemical results previously reported (Wollenweber et al. 1989).

Discussion

The secretory peltate trichomes of Calceolaria volckmanni exhibit an ontogeny and morphology similar to those previously described in Geraniaceae, Solanaceae and Cannabaceae (Fahn 1988).

The fully developed trichome is composed of a basal cell, which supplies nourishment to the head, a stalk cell and a glandular head typically made up of eight cells responsible for the secretory process.

As the trichomes develop, the histochemical tests ap- plied showed that the side walls of the stalk cell at first mainly consist of polysaccharides only. Afterwards, when the head begins its glandular activity a cutinization process starts. Thus, the cells acquire endodermal like features that probably prevent the backflow of secreted compounds. Furthermore, the cutinized cell walls are peculiar of oil secreting trichomes (Fahn 1988).

Of particular relevance are the morphological differ- ences observed about the plastids in the head cells and the photosynthetic ones in the stalk cell. Similar features in the lipophilic secreting structures were reported by several authors for trichomes of several plants (Cheniclet & Carde 1985; Ascensio & Pais 1987; Fahn 1988). The presence of plastids in the glandular head with an electrondense matrix and tubular networks, completely or partly surrounded by endoplasmic reticu- lum, in parallel with the different stages of the glandular trichome development, confirms the involvement of these organelles to the lipophilic secretion (Figueiredo & Pais 1994). In fact, it is known that in the secretion of lipophilic substances various cell organelles are in- volved, particularly the plastids (Fahn 1988). It was also demonstrated that plastids are important sites of isoprenoid and lipid biosynthesis (Kleinig 1989).

Acknowledgements - Investigations supported by grants from Consiglio Nazionale delle Ricerche (CNR), Minis- tero dell’universita e della Ricerca Scientifica (MURST) of Italy and INTERREG.

References Ascensio, L. & Pais, M. S . S . 1987. Glandular trichomes of

Artemisia campestris ssp. maritima: ontogeny and histochemistry of the secretory product. - Bot. Gaz. 148: 221- 227.

Bruni, A. & Vannini, 0. L. 1973. Possibiliti di impiego dell’arancio di acridina e dell’acriflavina nella ricerca isto- chimica dei polisaccaridi insolubili della cellula vegetale. - Giorn. Bot. Ital. 107: 201-206.

- & Modonesi, P. 1983. Development, oil storage and dehis- cence of peltate trichomes in Thymus vulgaris (Lamiace- ae). - Nord. J. Bot. 3: 245-25 1.

- , Tosi, B. & Modonesi, P. 1987. Morphology and secretion

512

of glandular trichomes in Tamus communis. - Nord. J. Bot.

Cappelletti, E. M., Caniato, R. & Appendino, G. 1986. Locali- zation of cytotoxic hydroperoxyeudesmanolides in Artemi- siu umbelliformis. - Biochem. Syst. Ecol. 14: 183-190.

Chamy. M. C., Piovano. M., Garbarino, J. A., Gambaro, V. & Miranda, C. 1989. Foliosate, a bis-diterpene and 9-epi-ent- 7.15-isopimaradiene derivatives from Calceolaria foliosa. - Phytochemistry 29: 57 1-574.

- , Piovano, M., Garbarino, J. A,, Miranda, C. & Gambaro, V. 1990. Diterpenes from Calcenlariu lepida. - Phyto- chemistry 29: 2943-2946.

Cheniclet, C. & Carde, J. P. 1985. Presence of leucoplasts in secretory cells and of monoterpenes in the essential oil: a correlative study. -‘Israel J. Bot. 34: 219-238.

David, R. & Carde, J. P. 1964. Coloration difftrentiele des in- clusions lipidiques et terpeniquks des pseudophilles du Pin maritime. - C.R. Acad. Sc. Paris SCr. D 258: 1338- 1340.

Di Fabio, A., Bruni, A,, Poli, F., Garbarino, J. A., Chamy, M. C., Piovano, M. & Nicoletti, M. 1995. The distribution of phenylpropanoid glycosides in Chilean Calceolaria spp. - Biochem. Syst. Ecol. 23: 179-182.

Fahn. A. 1988. Secretory Tissues in Plants. - New Phytol.

Feder, N. & O’Brien, T. P. 1968. Plant Microtechnique: some principles and new methods. - Am. J. Bot. 55: 123-142.

Figueiredo, A. C. & Pais, M. S. S. 1994. Ultrastructural as- pects of the glandular cells from the secretory trichomes and from the cell suspension cultures of Achilleu millefolium ssp. millefolium. - Ann. Bot. 74: 179-190.

Franceschi, V. R. & Horner, H. T. Jr. 1980. Calcium oxalate crystals in plants. - Bot. Rev. 46: 361-427.

Garbarino, J. A,, Chamy, M. C. & Piovano, M. 1992. Diter- penos de Schrophulariaceae. - In: Orlando MuAoz M. (ed.), Quimica de la Flora de Chile. Universidad de Chile, Departamento tecnico de Investigacion, Santiago de Chile,

Guerin, H. P., Delaveau, P. G. & Paris, R. R. 1971. Localisa- tions Histochimiques. 11: procCdts simples de localisation

7: 79-84.

108: 229-257.

pp. 95-1 15.

de pigments flavoniques. Application ti quelques phantro- games. - Bull. SOC. Bot. Fr. 1 18: 29-36.

Hardman, R. & Sofowora, E. A. 1972. Antimony trichloride as a test reagent for steroids, especially diosgenin and yamogenin, in plant tissues. - Stain Techno]. 47: 205-208.

Jensen, W. A. 1962. Botanical histochemistry. - Freeman, San Francisco.

Kleinig, H. 1989. The role of plastids in isoprenoid biosynthesis. - Annu. Rev. Plant Physiol. Plant Mol. Biol. 40: 39- 59.

Muiioz, M., Barrera, E. & Meza, I. 1981. El us0 medicinal y limenticio de plantas nativas y naturalizadas en Chile, pub- lication ocasional no. 23. - Museo Nacional de Historia Natural, Santiago de Chile.

Navas, L. E. 1979. Flora de la Cuenca de Santiago de Chile (Scrophulariaceae), Tomo 111. - Ediciones de la Universi- dad de Chile, Santiago de Chile.

Nicoletti, M., Galeffi, C., Multari, G., Garbarino, J. A. & GambdrO, V. 1988. Polar constituents of Calceolaria as- cendens. - Planta Med. 54: 347-348.

Poli, F., Bonora, A., Vannini, G. L., Bruni, A. & Fasulo, M. P. 1989. Callus formation, cell suspension culture and plant regeneration in Ranunculus serbicus Vis. - J. Plant Physi-

Schnepf, E. 1969. Uber den Feinbau von Oldrusen. 11.: Die Driisenhaare in Calceolaria - Bluten. - Protoplasma 67:

ThiCry, J. P. 1967. Mise en Cvidence des Polysaccarides sur coupes fines en microscopie Clectronique. - J. Microsc. (Paris) 6: 987-1018.

Vogel, S. 1971. Oldproduzierende Blumen, die durch olsam- melnde Bienen bestaubt werden. - Naturwissenschaften 58: 58.

Werker, E. & Fahn, A. 198 1. Secretory hairs of hula viscosa L. (Ait.) - Development, ultrastructure and secretion. - Bot. Gaz. 142: 461-476.

Wollenweber, E., Mann, K. & Roitman, J. N. 1989. Flavonoid aglycones excreted by three Calceolaria species. - Phyto- chemistry 28: 2213-2214.

01. 135: 637-639.

195-203.

Nurd. J. Bot. 1615) 1YY6 513

Development and morphology of secretory trichomes of Calceolaria volckmanni (Scrophulariaceae

Documents

Transcript of Development and morphology of secretory trichomes of Calceolaria volckmanni (Scrophulariaceae