Plant Cell Rep (2002) 21:125129 DOI 10.

1007/s00299-002-0497-1

CELL BIOLOGY AND MORPHOGENESIS

G. Bringmann T. Noll H. Rischer

In vitro germination and establishment of tissue cultures of Bulbine caulescens and of two Kniphofia species (Asphodelaceae)

Received: 15 March 2002 / Revised: 11 June 2002 / Accepted: 11 June 2002 / Published online: 24 July 2002 Springer-Verlag 2002

Abstract A protocol for the in vitro germination and the establishment of tissue cultures of Bulbine caulescens Linn., Kniphofia pumila Kunth, and K. uvaria Hook has been developed. The members of the family Asphodelaceae contain interesting substances with antiplasmodial activity against several strains of Plasmodium falciparum. With respect to species of Bulbine, this is the first time that in vitro germination has been achieved. For seeds of K. pumila and K. uvaria, a facilitated germination was attained by an optimized pretreatment of the seeds. After in vitro germination, root explants of Kniphofia and bulb explants of B. caulescens were successfully transformed into callus cultures. From these, liquid cultures of the two species of Kniphofia were easily produced. Studies on shoot multiplication of Kniphofia in vitro were also performed. Keywords Bulbine caulescens Kniphofia pumila Kniphofia uvaria In vitro germination Abbreviations BAP: 6-Benzylaminopurine 2,4-D: 2,4-Dichlorophenoxyacetic acid GA3: Gibberellic acid NAA: -Naphthaleneacetic acid PAR: Photosynthetically active radiation TDZ: Thidiazuron

Introduction
Plants of the genera Kniphofia Moench, Bulbine Wolf, and Bulbinella Kunth (Asphodelaceae) are known to be rich in axially chiral phenylanthraquinones (Thomson 1997). These are pharmaceutically interesting constituents of which knipholone and especially knipholone anthrone show considerable activity against the asexual erythrocytic stages
Communicated by W. Barz G. Bringmann () T. Noll H. Rischer Institute of Organic Chemistry, University of Wrzburg, Am Hubland, 97074 Wrzburg, Germany e-mail: bringman@chemie.uni-wuerzburg.de Tel.: +49-931-8885323, Fax: +49-931-8884755

of two strains of Plasmodium falciparum in vitro (Bringmann et al. 1999). For this reason, highly efficient total syntheses have been developed to build up such molecules chemically (Bringmann and Menche 2001). Recent studies on secondary metabolites in the genus Bulbine (Qhotsokoane-Lusunzi and Karuso 2001; Bringmann et al. 2002) and also classical reports on the isolation of knipholone (Dagne and Steglich 1984) have stimulated investigations on the biosynthetic origin of these bioactive natural products. Plant cell culture technologies are notable tools for studying plant secondary metabolism (Bourgoud et al. 2001). A precondition for our biosynthetic investigations, therefore, was first to be able to raise the plants in vitro. There are, however, as yet no reports on the in vitro cultivation of any species of the genus Bulbine, while only a few such reports exist for several species of Kniphofia. Nayak and Sen (1989) induced organogenesis in K. nelsonii and K. uvaria using half-strength MS (Murashige and Skoog l962) basal medium as germination medium. McAlister and van Staden (1996) worked on the in vitro propagation of K. pauciflora for conservation purposes. For Bulbine bulbosa R. Br. Borzi, a species indigenous to the natural grasslands of southeastern Australia, germination is strongly temperature-dependent (Willis and Groves 1991). Since there are similarities in germination responses within savannah plants in many parts of the world for example, in South African fynbos and Californian chaparral (Keely and Bond 1997) it seemed possible to transfer the finding of temperature dependency in B. bulbosa to the cultivation of B. caulescens from South Africa. In order for us to raise the three abovementioned species for biosynthetic purposes in vitro, existing knowledge about ecological germination cues (Willis and Groves 1991) proved to be very useful.

Materials and methods

Plant material Seeds of Kniphofia pumila Kunth were obtained from B&T World Seeds, Paguignan, Olonzac, France. The seeds of K. uvaria Hook

126 were collected from a plant in our laboratory, which had been purchased at Maingarten, Hanau (Germany), and seeds of Bulbine caulescens Linn. were obtained from the Botanical Garden of the University of Wrzburg. In vitro culture Seeds were sown on half-strength MS basal medium containing half-strength MS mineral salts and full-strength MS organics. Gelrite (Carl Roth, Karlsruhe, Germany) was used for gelling. The medium was supplemented with 30 g/l sucrose and adjusted to pH 5.80.1. Sterilization was achieved by autoclaving for 30 min at 120 kPa and 120C. Some seeds were initially subjected to stratification in the darkness at 7C for 3 days, while the remaining seeds were grown directly at 2223C. The photoperiod was 14/10 h (day/night) with light supplied by a combination of Osram L58W/77 Fluora and Osram L58W/11 Lumilux (daylight) fluorescent lamps at an intensity of 2,000 lux. Routinely, 50 ml of medium was dispensed into 100-ml Erlenmeyer flasks; the flasks were then closed with cellulose plugs (Neolab Migge, Heidelberg, Germany) and the plugs covered with aluminum foil. All cultures were generally transferred to fresh medium every month. Aseptic seedlings of K. pumila The seeds of K. pumila were divided up into three batches, each batch consisting of four seeds. The seeds of batch A were scarified with emery paper (Lux K100 100-510, Emil Lux, Wermelskirchen, Germany) before sterilization to remove the testa. The other seeds were not treated this way. Batch B was stratified and batch C was not pretreated. The seeds of Kniphofia were sterilized for 10 min in 50 ml of a 1:1 mixture of sterile water and saturated aqueous sodium hypochlorite solution that contained two drops of the detergent Triton X 100 (Carl Roth) per 50 ml. The seeds were then washed thoroughly twice, 10 min each time, in sterile water and aseptically transferred to the medium. Aseptic seedlings of K. uvaria Each of the ten seeds was pretreated with emery paper as mentioned above. The sterilization procedure, the medium, and the cultivation parameters were identical, but these seeds were not subject to stratification. Aseptic seedlings of B. caulescens These seeds were treated differently: some were scarified with emery paper (Lux K100 100-510, Emil Lux) to remove the testa; alternatively, seeds were put for 10 s into the flame of a Bunsen burner to perforate the testa. Sterilization was carried out as mentioned above. One portion of seeds was placed for 24 h in sterile water after sterilization; a second portion was put in fresh solution of GA3 (1 mg/ml) for 12 h; a third portion was treated with an aqueous extract of plant ashes overnight. The remaining seeds were laid into an aqueous extract that had been produced by combusting dried parts of the savannah plants Euryops pectinata (Asteraceae), Aloe pectinata (Asphodelaceae), and Bulbine caulescens (Asphodelaceae) and passing the smoke through water. The residue of this burning process was the crude material for the extract of ashes mentioned before. Five grains were placed into a 100-ml Erlenmeyer flask and treated with a combination of the various possibilities mentioned above. One portion of the seeds was maintained under the same light and temperature regime as K. pumila; the other portion of the plant material was grown under alternating temperature conditions: 12 h at 2224C and 2,000 lux (fluorescent light at 51 mol m2 s1 PAR) and overnight at 7C in the dark. Explant production and the establishment of aseptic cultures Roots of 3-week-old K. pumila and K. uvaria seedlings were cut into 1-cm pieces. These explants were transferred to petri dishes containing solid one-fifth MS medium with full-strength organics supplemented with 0.5 mg/l BAP, 0.5 mg/l NAA, and 0.5 mg/l 2,4-D and grown under low light conditions (700 lux) at 2223C and a 14/10-h (day/night) photoperiod. In the case of B. caulescens, the tiny bulbs were cut into halves and transferred to the same medium in petri dishes under the same temperature and light conditions as described for the species of Kniphofia. Establishment of in vitro liquid cultures of K. pumila and K. uvaria Calli of both Kniphofia species were cut into smaller pieces using a sterile scalpel. The small fragments were placed into 500-ml flasks containing 100 ml liquid one-fifth strength MS medium with full-strength organics supplemented with 0.5 mg/l BAP, 0.5 mg/l NAA, and 0.5 mg/l 2,4-D. The flasks were placed on a shaker at 84 rpm under low light conditions (700 lux). Multiplication of K. pumila and K. uvaria in vitro Plants of these two species were transferred to two kinds of medium: medium A, which contained 0.01 mg/l NAA and 2.0 mg/l BAP, and medium B, which contained 0.01 mg/l NAA and 0.01 mg/l TDZ. Observations were carried out for up to 6 weeks.

Results and discussion

Aseptic seedlings of K. pumila and K. uvaria Based on good experience with pretreating seeds of tropical plants by scarification with emery paper to remove the testa (Bringmann et al. 2000), we treated a few seeds of K. pumila in this way. This technique, also known as mechanical scarification, is often used in the horticulture of tropical plants; for example, in the cultivation of Indian medical plants (Kasera et al. 2000). Chemical scarification with acid also helps break the hard seed coat dormancy, for example in Pistacia mutica (Caloggero and Parera 2000). Following mechanical scarification, the seeds were sown on half-strength MS medium, as shown by Nayak and Sen (1989). Mechanical scarification of the seeds with emery paper had quite a remarkable effect on germination, as shown in Fig. 1. Within a few days the first seedlings of K. pumila arose from seeds that had been pretreated with emery paper (batch A), while the other seedlings (batches B and C) did not appear until 4 weeks later in the aseptic Erlenmeyer flasks. Since the medium was always the same, this acceleration of germination has to be attributed to the special treatment given to the seeds of batch A. In order to further test the efficiency and the scope of this method within this genus, we chose K. uvaria from South Africa. This plant is of horticultural importance and therefore easily available in market gardens. Since there is an existing protocol for the aseptic germination of K. uvaria (Nayak and Sen 1989), it was possible to

127

Fig. 1 Time and rate of germination of seeds of Kniphofia pumila in vitro (batch sizes: four seeds each)

proper in vitro germination of the genus Kniphofia. Various studies have been carried out on the specific primary barrier to the diffusion of oxygen, water, and solutes into the seeds. Brits et al. (1999) assumed that in the genus Leucospermum (Proteaceae). the exotesta acts as the primary barrier to oxygen diffusion to the embryo. In Emmenanthe penduliflora (Hydrophyllaceae) from Australia, Egerton (1998) showed that the seed coat is responsible for the proximal regulation of dormancy and that a waxy sub-testa cuticle is the primary barrier to the diffusion of water and small-diameter solutes. However, a mechanical scarification affects both exotesta and subtesta and therefore alters the permeability to oxygen, water, and small-diameter solutes. Germination of the two species K. pumila and K. uvaria was achieved rapidly under aseptic conditions following mechanical scarification of the seeds with emery paper before sowing them aseptically on half-strength MS medium. Both species exhibited a healthy and uncomplicated growth on this solid medium, especially K. pumila. These findings could probably be correlated to other Kniphofia species or, with a few modifications, even to other genera of the monocotyledon family of the Asphodelaceae. Aseptic seedlings of B. caulescens Bulbine caulescens grows in the savannahs of different parts of South Africa. Due to its remarkable similarities in germination response to species of South African fynbos and Californian chaparral (Keely and Bond 1997) and because of the lack of information about the South African species, B. caulescens, we adapted findings about savannah plants in general and those of Australia in particular. There are a few reports on the Australian species B. bulbosa (G.Br.) Haw (Willis and Groves 1991) that show that the germination of this species is strongly temperature-dependent. Morgan and Lunt (1994), however, noted the failure of some Australian Liliaceae to germinate even in their natural environment. Many Australian native species demonstrate low germination responses using conventional nursery propagation methods (Tieu et al. 1999). To investigate the role of germination cues in breaking seed-dormancy mechanisms in B. caulescens, we tested the influences of various techniques. Smoke released from burning vegetation contains chemical signals that trigger germination of species from different parts of the world. It stimulates seed germination in wildflower species from fire-dependent plant communities in South Africa and Australia (Brown and van Staden 1998). Smoke is used in horticulture to stimulate seed germination and can improve the germination of such crops as lettuce and celery. Aqueous smoke-water, which is commercially available, is also active in this respect. The unidentified active constituents are volatile, thermostable, water-soluble, and long-lasting in aqueous solution and in the soil (van Staden et al. 2000). Additional fire-related cues like heat-shock and an aqueous extract of plant ash

Fig. 2 Time and rate of in vitro germination of K. uvaria (number of seeds tested: ten)

compare and correlate the findings of germination time with the results of our studies. Following the pretreatment, the sterilization of the seeds and the sowing on half-strength MS medium was carried out as described above. Again, scarification with sand paper had an accelerating effect on aseptic germination in Kniphofia (Fig. 2). Nayak and Sen (1989) found that seeds of both K. uvaria and K. nelsonii pretreated with HgCl2 as a means of sterilization require a germination time of about 1520 days under aseptic conditions. In their experiments, probably both the seed quality and the HgCl2 treatment influenced the time needed for germination. Our studies have shown that seeds of K. uvaria germinate as early as 310 days following a pretreatment with emery paper, which is an impressive improvement in the time required for germination for this species. Following scarification, a germination rate of almost 100% was obtained. We can therefore conclude that a better perforation of the protecting testa seems to be necessary for

128 Table 1 Scheme of treatments and germination response of the seeds of Bulbine caulescens Pretreatment Emery paper Emery paper Emery paper Emery paper Emery paper None None Bunsen burner Additional treatment None None Sterile water Aqueous smoke extract Aqueous ash extract GA3 Aqueous smoke extract None Temperature regime Regular Altered Regular Regular Altered Regular Altered Regular Germination rate [%] 0 (0 of 10 seeds) 40 (4 of 10 seeds) 0 (0 of 10 seeds) 0 (0 of 10 seeds) 40 (4 of 10 seeds) 0 (0 of 10 seeds) 40 (4 of 10 seeds) 0 (0 of 10 seeds)

Fig. 3 a Development of callus at roots of K. pumila. b Tiny bulb of Bulbine caulescens with formation of callus

were tested, although Baldwin et al. (1994) had shown that ash of burnt wood does not contain potent cues that stimulate germination. The other techniques that we used are more conventional with respect to the pretreatment of seeds; for a scheme of all the treatments and results, see Table 1. We found that the main factor affecting germination was the alternation in the temperature regime. A difference of about 17C between day and night temperatures seems to be necessary for germination in B. caulescens. Similar differences in diurnally alternating temperatures have been found to improve germination in Solanaceae from Ethiopia (Teketay 1998). All other treatments in addition to this temperature regime are only co-initiating factors to the germination; for example, the treatment with the aqueous extract of smoke of burning savannah plants. Except for the seeds treated with the aqueous extract of smoke, the pretreatment with emery paper again seemed to have at least a certain positive effect on germination. One fraction of seeds treated with smoke-water and altered temperatures even germinated without mechanical scarification. Egerton (1998) showed that smoke treatment produces an intense chemical scarification at the seed surface and alters the permeability of the subtesta cuticle. Therefore, the treatment with smoke-water might also be considered as a kind of scarification and, in addition to the temperature shift, a germination cue in this experiment. The results of the ecological studies made by Willis and Groves (1991) and by Morgan (1998) were very useful in the context of finding the adequate temperature regime. The problem of strongly temperature-dependent germination does not only exist in some of the Australian species of Bulbine but also in B. caulescens from South Africa. For a successful in vitro propagation

of plants knowledge about their ecological background is an important precondition. The seedlings of B. caulescens grow very slowly under aseptic conditions. In our study, this species exhibited a faster growth when the aerial parts and the roots were cut from time to time. It was also more effective to subcultivate the seedlings on fullstrength MS medium after germination. Callus induction of Bulbine and the Kniphofia species Callus tissues of the two Kniphofia species formed spontaneously (Fig. 3a) on cut margins of aseptically cultured roots on solid one-fifth strength MS medium with full strength organics and supplemented with 0.5 mg/l of BAP, NAA, and 2,4-D, respectively. The calli of Kniphofia showed a yellowish color that was derived from the production of anthraquinones. Callus formation was induced on cut bulb margins from sterile plants of B. caulescens using the same medium formulation (Fig. 3b). This callus was pure white. Establishment of liquid cultures of K. pumila and K. uvaria Cut parts of the aseptic calli were subcultured on the same medium without the gelling compound. These callus suspensions (aggregates about 0.8 cm in diameter) exhibited a growth similar to that of the cultures on solid medium as above, and showed the formation of short roots. They also produced yellowish compounds, which were detected in the medium. The color originates from various anthraquinones, of which chrysophanol was

129

identified by thin layer chromatography and by coelution on high-performance liquid chromatography. Formation of shoots of K. pumila and K. uvaria Without added phytohormones, spontaneous multiplication in Kniphofia was observed only a few times. For a directed induction of multiplication, we used two kinds of medium (media A and B; see above). The induction of shoots was enhanced within a short time by medium A. Up to three shoots per plant were formed after 3 weeks. On the other hand, root production was reduced under these conditions. With medium B, shoot production took more time and led to very thick roots approximately 3 cm in length. Following division, the plants were transferred to medium without hormones, where they showed normal growth. Conclusion The objectives of the study reported here were the in vitro germination and the establishment of tissue cultures of three Asphodelaceae species within the framework of investigations on the biogenesis of knipholone and other bioactive substances produced by these plants in which stable isotope-labeled precursors are used. In view of the permanent need of new agents against malaria, further studies on the antiplasmodial constituents of the genera Bulbine, Bulbinella and Kniphofia supported by in vitro cultures seem promising.
Acknowledgements We are grateful to F. Thiele and A. Kreiner (Wrzburg Botanical Garden) for Bulbine caulescens seeds and for competent technical advice and to Prof. M. Mller (Chair of Pharmacognosy, University of Wrzburg) for useful discussions. This work was supported by the Fonds der Chemischen Industrie.

References
Baldwin IT, Stazak KL, Davidson R (1994) Up in smoke. I. Smoke-derived germination cues for postfire annual, Nicotiana attenuata Torr. Ex. Watson. J Chem Ecol 20:23452371 Bourgaud F, Gravot A, Milesi S, Gontier E (2001) Production of plant secondary metabolites: a historical perspective. Plant Sci 161:839851 Bringmann G, Menche D (2001) First, atropo-enantioselective total synthesis of the axially chiral phenylanthraquinone natural products knipholone and 6-O-methylknipholone. Angew Chem Int Ed Engl 40:16871690

Bringmann G, Menche D, Merhatibeb B, Abegaz BM, Kaminsky R (1999) Antiplasmodial activity of knipholone and related natural phenylanthraquinones. Planta Med 65:757758 Bringmann G, Rischer H, Wohlfarth M, Schlauer J (2000) Biosynthesis of antidesmone in cell cultures of Antidesma membranaceum (Euphorbiaceae): an unexpected class of glycine-derived alkaloids. J Am Chem Soc 122:99059910 Bringmann G, Menche D, Brun R, Msuta T, Abegaz B (2002) Bulbine-knipholone, a new axially chiral phenylanthraquinone from Bulbine abyssinica (Asphodelaceae): isolation, structural elucidation, synthesis, and antiplasmodial activity. Eur J Org Chem 6:11071111 Brits GJ, Calitz FJ, Brown NCA (1999) Heat desiccation as seed scarifying agent in Leucospermum ssp. (Proteaceae) and its effect on the testa, viability and germination. Seed Sci Technol 27:163176 Brown NAC, van Staden J (1998) Plant-derived smoke: an effective seed pre-soaking treatment for wildflower species and with potential for horticultural and vegetable crops. Seed Sci Technol 26:669673 Caloggero S, Parera CA (2000) Improved germination and emergence of Pistacia mutica by presowing chemical scarification. Seed Sci Technol 28:253260 Dagne E, Steglich W (1984) Knipholone: a unique anthraquinone derivate from Kniphofia foliosa. Phytochemistry 23:17291731 Egerton WLM (1998) A smoke-induced alteration of the sub-testa cuticle in seeds of the post-fire recruiter, Emmenanthe penduliflora benth. (Hydrophyllaceae). J Exp Biol 49:13171327 Kasera PK, Shukla JK, Prakash J, Saharan P, Chawan DD (2000) Biology, conservation and mediculture of important medicinal plants from Indian Thar desert. J Med Aromatic Plant Sci 22 23:432443 Keeley JE, Bond WJ (1997) Convergent seed germination in South African fynbos and Californian chaparral. Plant Ecol 133:153167 McAlister BG, van Staden J (1996) In vitro propagation of Kniphofia pauciflora Bak. for conservation purposes. S Afr J Bot 62:219221 Morgan JW (1998) Comparative germination responses of 28 temperate grassland species. Aust J Bot 46:209219 Morgan JW, Lunt ID (1994) Germination characteristics of eight common grassland and woodland forbs. Victorian Nat 111: 110117 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473497 Nayak S, Sen S (1989) Differential requirement for organogenesis in two species of Kniphofia. Indian J Exp Biol 28:171173 Qhotsokoane-Lusunzi MA, Karuso P (2001) Secondary metabolites from Basotho medicinal plants. J Nat Prod 64:13681372 Teketay D (1998) Environmental factors that control the germination of five Solanum species from Ethiopia. Trop Ecol 39:7987 Thomson RH (1997) Naturally occurring quinones, VI. Chapman & Hall, London Tieu A, Dixon KA, Sivasithamparam K, Plummer JA, Sieler IM (1999) Germination of four species of native Western Australian plants using plant-derived smoke. Aust J Bot 47:207219 Van Staden J, Brown NAC, Jager AK, Johnson TA (2000) Smoke as a germination cue. Plant-Species-Biol 15:167178 Willis AJ, Groves RH (1991) Temperature and light effects on the germination of seven native forbs. Aust J Bot 39:219228