Cyanogenic glycosides in leaves and callus cultures of Schlechterina mitostemmatoides

Leaf material and callus cultures of Schlechterina mitostemma toides (Passifioraceae), an endangered species from northern KwaZulu-Natal, was screened for cyanogenic glycosides. Both leaf and callus material tested positive. Extracts from both sources were further investigated by thin-layer chromatography. This indi cated that the callus culture produced the same cyanogenic com pound as the intact plant.

graphy -To whom correspondence should be addressed.  Tydskr.Ptanlk. 1995,61(5): 274-275 SchleClerina milostemmacoides Harms (Passifioraceae) is a creeper growing in the coastal forests of northern Zululand and Mozambiq ue. The Zulu name for the species is Mhlalanyosi and the plan! is used for cleansing steam baths . It is reported to be a favourite food plant for elephants. As S. milOst emmatoides is now scarce in the wild, efforts to micropropagate the plant by in vitro culture were undertaken. h is well known that many members of the Passifloraceae con tain cyanogenic glycosides (Russell & Reay 1971;Siegler 1977. Adsersen er al. 1993 but S. milO· slemma/aides has never been investigated for cyanogenesis. Leaf and callus material were therefore screened in this study for cyano gen ic compou nds.
Callus cu lture of S. milaslemmalOides was initiated from juvenile leaves which had been surface sterilized for 10 min in 3.5% NaOCl. The explants were placed on Murashige and Skoog med ium supplemented with 3% sucrose, 100 mg I-I m yo-in ositol, I mg I'] NAA and I mg I'] BA, at a pH af 5.8, far the induction af callu s. This medium was also used for maintenance of the callus and the cultures were kept in the light at 26°C.
Two tes ts that detect cyanide release from cyanogenic glycosides were used to screen leaf and callus material for the presence of these compounds. Fresh leaf (50 mg) and callus material (500 mg) were transferred to 25-ml Erlenmeyer fl asks . In order to break down cyanogenic glycosides , and thereby release cyanide, 100 ~I of 0.1 mg I-I J3-glucosidase or 1 ml ch loroform was added to the flasks. Chloroform breaks the cell walls, releasing cyanogenic glycosidcs, which come into contact with any gJycosidases stored in other cells. Amygdalin (0 .01 mg and 0.1 mg) in aq ueous soluti on was used as a standard . Controls contained either I ml chloroform or 100 !J.1 of the J3-glucosidase solution. Stri ps of sodium picrate paper (Whatman no. 1 filter paper d ipped in a sol ution of 5 g sodium carbonate, 0.5 g picriC acid and 100 ml water, and dried) were hung in the closed flasks . After 30 min , the strips in the fl as ks containing the amygdalin standard as well as leaf and callus material turned reddish-brown, indicating the presence of cyanide, whereas the con trols remained unchanged. A second test was performed similarly. using different lcst strips. These strips were prepared by dipping Whatman no . 1 fil· lef paper frrstly into a solution of 1 mg mI' ) guaia gum in ethanol.

CALLUS ST
drying, and then into 0.1 mg mi-' Cu50 4 • Strips in flasks contain· iog the amygdalin standard, leaf and callus material turned prus· sian blue in col OUT, indicating the presence of cyanide . The guaia gum control strips did not change colour.
To investigate the chromatographic propert ies of the cyanogenic glycoside(s) present in the leaf and callu s material. leaves and callus were extracted for cyanogenic gJycosides. Callus (2 g) or fresh leaf material (150 mg) were cut into small pieces and boiled in 80% ethanol for 5 min. The extracts were cooled, ftl~ {ered and the filtrates dried in vacuo. The residues were redis~ solved in eluent and app lied to Merck Silica 60 F254 TLC pl ates. Only one-third of the leaf extract was applied, whereas all of the callus extract was used. Amygdalin (5 ~g) was used as standard. The TLC plates were developed (one ascent) using ethyl acetate:acetone:chlorofonn:methanol: water (40:30: 12: 1 0:8) (Brimer el at. 1983). To detect the cyanogenic comJXJunds, the TLC plates were sandwiched by a modified method from Tantisew ie el al. (1969). The plate was sprayed with 0. 1 mg mi" D-glucosidase. A piece of synthetic net fabric and a sheet of sodium picrate paper was immediately layered on the TLC plate, followed by a glass plate. The edges were taped and the sandwich placed in an ove n at 35°C. After 90 min . a distinct reddish-brow n band was visible on the sodium picrate paper in both the leaf and callus culture lane, as well as a reddish~brown spot for the slandard, amygdalin (Figure 1). The cyanide bands in both samples occurred at Rr 0.8 . They did not co~chromatograph with any of the bands detected un de r UV light at either 254 nm or 366 nm . This indicates that the callus culture probabl y produced the same cyanogenic compound(s) as the intact plant.
There is only one other known report on the production of cya~ nogenic compounds in cell cultures (Hosel el ai. 1985), where these compounds of an unknown stru cture were produced and accumulated in osmotically stressed cell cultu res of Eschscholtzia californica. However. they were not identical to the cyano~ gen ic glycosides of the intact plant and were only produced when the cultures were under stress. Islock el ai. (1990) established a cell culture of Phaseolus lunatus from highly cyanogenic seed~ lings. The callus cells were free of cyanogenic glycosides, bu t the callus maintained enzyme activity of several of the enzymes involved with cyanogenic glycosides. Our study appears to be the flrst report where cyanogenic glycoside(s) similar to those produced in the intact plant were apparently synthesized in a callus culture. Keywords: Plant~derived smoke, seed germination, smoke source, Them eda triandra.
-To whom correspondence should be addressed.
Smoke derived from the burning of natural mixtures of plant species stimulates the germination of seed from a wide range of plant species common to flre~dependent floral communities (de Lange & Boucher 1990;Baxter el al. 1993;Brown 1993.). Seeds of T lriandra exposed to smoke derived from burning a mixture of species do not lose the enhanced effect of such exposure during storage. This provides an effective means of pre-treatment for seeds which may prove difficult to germinate (Baxter & van Staden 1994). In addition, smoke has been shown to stimulate germination of soil-stored seed (de Lange & Boucher 1990). Neither the mechani sm by which smoke acts to stim ulate seed germina~ lion, nor the active cornponent(s) in plant-derived smoke are