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Abstract A protocol for the in vitro germination and theestablishment of tissue cultures of Bulbine caulescensLinn., Kniphofia pumila Kunth, and K. uvaria Hook has

been developed. The members of the family Asphodelac-eae contain interesting substances with antiplasmodial ac-tivity against several strains of Plasmodium falciparum.With respect to species ofBulbine, this is the first timethat in vitro germination has been achieved. For seeds ofK. pumila and K. uvaria, a facilitated germination was at-tained by an optimized pretreatment of the seeds. After invitro germination, root explants ofKniphofia and bulb ex-plants ofB. caulescens were successfully transformed intocallus cultures. From these, liquid cultures of the two spe-cies ofKniphofia were easily produced. Studies on shootmultiplication ofKniphofia 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, andBulbinella Kunth (Asphodelaceae) are known to be rich inaxially chiral phenylanthraquinones (Thomson 1997).These are pharmaceutically interesting constituents ofwhich knipholone and especially knipholone anthrone showconsiderable activity against the asexual erythrocytic stages

of two strains of Plasmodium falciparum in vitro (Bring-mann et al. 1999). For this reason, highly efficient total syn-theses have been developed to build up such molecules

chemically (Bringmann and Menche 2001). Recent studieson secondary metabolites in the genus Bulbine (Qhotsoko-ane-Lusunzi and Karuso 2001; Bringmann et al. 2002) andalso classical reports on the isolation of knipholone (Dagneand Steglich 1984) have stimulated investigations on thebiosynthetic origin of these bioactive natural products.

Plant cell culture technologies are notable tools forstudying 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 culti-vation of any species of the genus Bulbine, while only afew such reports exist for several species of Kniphofia.Nayak and Sen (1989) induced organogenesis in K. nel-sonii and K. uvaria using half-strength MS (Murashigeand Skoog l962) basal medium as germination medium.McAlister and van Staden (1996) worked on the in vitropropagation of K. pauciflora for conservation purposes.ForBulbine bulbosa R. Br. Borzi, a species indigenous tothe natural grasslands of southeastern Australia, germina-tion is strongly temperature-dependent (Willis andGroves 1991). Since there are similarities in germinationresponses within savannah plants in many parts of theworld for example, in South African fynbos and Cali-fornian chaparral (Keely and Bond 1997) it seemedpossible to transfer the finding of temperature dependen-cy inB. bulbosa to the cultivation ofB. caulescens fromSouth Africa. In order for us to raise the three above-mentioned 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 ofKniphofia pumila Kunth were obtained from B&T WorldSeeds, Paguignan, Olonzac, France. The seeds of K. uvaria Hook

Communicated by W. Barz

G. Bringmann () T. Noll H. RischerInstitute of Organic Chemistry, University of Wrzburg,Am Hubland, 97074 Wrzburg, Germanye-mail: bringman@chemie.uni-wuerzburg.deTel.: +49-931-8885323, Fax: +49-931-8884755

Plant Cell Rep (2002) 21:125129DOI 10.1007/s00299-002-0497-1

CELL BIOLOGY AND MORPHOGENESIS

G. Bringmann T. Noll H. Rischer

In vitro germination and establishment of tissue cultures

ofBulbine caulescensand of two Kniphofiaspecies (Asphodelaceae)

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

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were collected from a plant in our laboratory, which had been pur-chased at Maingarten, Hanau (Germany), and seeds ofBulbinecaulescens Linn. were obtained from the Botanical Garden of theUniversity of Wrzburg.

In vitro culture

Seeds were sown on half-strength MS basal medium containinghalf-strength MS mineral salts and full-strength MS organics. Gel-

rite (Carl Roth, Karlsruhe, Germany) was used for gelling. Themedium was supplemented with 30 g/l sucrose and adjusted topH 5.80.1. Sterilization was achieved by autoclaving for 30 minat 120 kPa and 120C. Some seeds were initially subjected tostratification in the darkness at 7C for 3 days, while the remain-ing seeds were grown directly at 2223C. The photoperiod was14/10 h (day/night) with light supplied by a combination of OsramL58W/77 Fluora and Osram L58W/11 Lumilux (daylight) fluores-cent lamps at an intensity of 2,000 lux. Routinely, 50 ml of medi-um was dispensed into 100-ml Erlenmeyer flasks; the flasks werethen closed with cellulose plugs (Neolab Migge, Heidelberg, Ger-many) and the plugs covered with aluminum foil. All cultureswere generally transferred to fresh medium every month.

Aseptic seedlings ofK. pumila

The seeds ofK. pumila were divided up into three batches, eachbatch consisting of four seeds. The seeds of batch A were scarifiedwith emery paper (Lux K100 100-510, Emil Lux, Wermelskir-chen, Germany) before sterilization to remove the testa. The otherseeds were not treated this way. Batch B was stratified andbatch C was not pretreated. The seeds ofKniphofia were sterilizedfor 10 min in 50 ml of a 1:1 mixture of sterile water and saturatedaqueous sodium hypochlorite solution that contained two drops ofthe detergent Triton X 100 (Carl Roth) per 50 ml. The seeds werethen washed thoroughly twice, 10 min each time, in sterile waterand aseptically transferred to the medium.

Aseptic seedlings ofK. uvaria

Each of the ten seeds was pretreated with emery paper as men-tioned above. The sterilization procedure, the medium, and thecultivation parameters were identical, but these seeds were notsubject to stratification.

Aseptic seedlings ofB. caulescens

These seeds were treated differently: some were scarified withemery paper (Lux K100 100-510, Emil Lux) to remove the testa;alternatively, seeds were put for 10 s into the flame of a Bunsenburner to perforate the testa. Sterilization was carried out as men-tioned above. One portion of seeds was placed for 24 h in sterilewater after sterilization; a second portion was put in fresh solutionof GA3 (1 mg/ml) for 12 h; a third portion was treated with anaqueous extract of plant ashes overnight. The remaining seedswere laid into an aqueous extract that had been produced bycombusting dried parts of the savannah plants Euryops pectinata(Asteraceae), Aloe pectinata (Asphodelaceae), and Bulbine caul-escens (Asphodelaceae) and passing the smoke through water. Theresidue of this burning process was the crude material for the ex-tract of ashes mentioned before.

Five grains were placed into a 100-ml Erlenmeyer flask andtreated with a combination of the various possibilities mentionedabove. One portion of the seeds was maintained under the samelight and temperature regime as K. pumila; the other portion of theplant material was grown under alternating temperature condi-tions: 12 h at 2224C and 2,000 lux (fluorescent light at51 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 cutinto 1-cm pieces. These explants were transferred to petri dishescontaining solid one-fifth MS medium with full-strength organicssupplemented with 0.5 mg/l BAP, 0.5 mg/l NAA, and 0.5 mg/l2,4-D and grown under low light conditions (700 lux) at 2223Cand a 14/10-h (day/night) photoperiod.

In the case ofB. caulescens , the tiny bulbs were cut into halvesand transferred to the same medium in petri dishes under the same

temperature and light conditions as described for the species ofKniphofia.

Establishment of in vitro liquid cultures ofK. pumilaand K. uvaria

Calli of both Kniphofia species were cut into smaller pieces usinga sterile scalpel. The small fragments were placed into 500-mlflasks containing 100 ml liquid one-fifth strength MS mediumwith 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 ashaker at 84 rpm under low light conditions (700 lux).

Multiplication ofK. pumila and K. uvaria in vitro

Plants of these two species were transferred to two kinds of medi-um: medium A, which contained 0.01 mg/l NAA and 2.0 mg/lBAP, and medium B, which contained 0.01 mg/l NAA and0.01 mg/l TDZ. Observations were carried out for up to 6 weeks.

Results and discussion

Aseptic seedlings ofK. pumila and K. uvaria

Based on good experience with pretreating seeds of trop-ical plants by scarification with emery paper to removethe testa (Bringmann et al. 2000), we treated a few seeds

ofK. pumila

in this way. This technique, also known asmechanical scarification, is often used in the horticultureof tropical plants; for example, in the cultivation of Indi-an medical plants (Kasera et al. 2000). Chemical scarifi-cation with acid also helps break the hard seed coat dor-mancy, for example in Pistacia mutica (Caloggero andParera 2000).

Following mechanical scarification, the seeds weresown on half-strength MS medium, as shown by Nayakand Sen (1989). Mechanical scarification of the seedswith emery paper had quite a remarkable effect on ger-mination, as shown in Fig. 1.

Within a few days the first seedlings of K. pumilaarose from seeds that had been pretreated with emery pa-per (batch A), while the other seedlings (batches B andC) did not appear until 4 weeks later in the aseptic Erlen-meyer flasks. Since the medium was always the same,this acceleration of germination has to be attributed tothe special treatment given to the seeds of batch A. In or-der to further test the efficiency and the scope of thismethod within this genus, we chose K. uvaria fromSouth Africa. This plant is of horticultural importanceand therefore easily available in market gardens. Sincethere is an existing protocol for the aseptic germinationof K. uvaria (Nayak and Sen 1989), it was possible to

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compare and correlate the findings of germination timewith the results of our studies. Following the pretreat-ment, the sterilization of the seeds and the sowing onhalf-strength MS medium was carried out as describedabove. Again, scarification with sand paper had an accel-erating effect on aseptic germination in Kniphofia(Fig. 2).

Nayak and Sen (1989) found that seeds of both K. uv-aria and K. nelsonii pretreated with HgCl

2as a means

of sterilization require a germination time of about1520 days under aseptic conditions. In their experi-ments, probably both the seed quality and the HgCl2treatment influenced the time needed for germination.Our studies have shown that seeds of K. uvaria germi-nate as early as 310 days following a pretreatment withemery paper, which is an impressive improvement in thetime required for germination for this species. Followingscarification, a germination rate of almost 100% was ob-tained. We can therefore conclude that a better perfora-tion of the protecting testa seems to be necessary for

proper in vitro germination of the genus Kniphofia. Vari-ous studies have been carried out on the specific primarybarrier to the diffusion of oxygen, water, and solutes intothe seeds. Brits et al. (1999) assumed that in the genus

Leucospermum (Proteaceae). the exotesta acts as the pri-mary barrier to oxygen diffusion to the embryo. In Em-menanthe penduliflora (Hydrophyllaceae) from Austra-lia, Egerton (1998) showed that the seed coat is responsi-

ble for the proximal regulation of dormancy and that awaxy sub-testa cuticle is the primary barrier to the diffu-sion of water and small-diameter solutes. However, amechanical scarification affects both exotesta and sub-testa and therefore alters the permeability to oxygen, wa-ter, and small-diameter solutes.

Germination of the two species K. pumila and K. uv-aria was achieved rapidly under aseptic conditions fol-lowing mechanical scarification of the seeds with emerypaper before sowing them aseptically on half-strengthMS medium. Both species exhibited a healthy and un-complicated 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 theAsphodelaceae.

Aseptic seedlings ofB. caulescens

Bulbine caulescens grows in the savannahs of differentparts of South Africa. Due to its remarkable similaritiesin germination response to species of South Africanfynbos and Californian chaparral (Keely and Bond 1997)and because of the lack of information about the SouthAfrican species,B. caulescens, we adapted findings about

savannah plants in general and those of Australia in par-ticular. There are a few reports on the Australian speciesB. bulbosa (G.Br.) Haw (Willis and Groves 1991) thatshow that the germination of this species is strongly tem-perature-dependent. Morgan and Lunt (1994), however,noted the failure of some Australian Liliaceae to germi-nate even in their natural environment. Many Australiannative species demonstrate low germination responses us-ing conventional nursery propagation methods (Tieu et al.1999). To investigate the role of germination cues inbreaking seed-dormancy mechanisms in B. caulescens,we tested the influences of various techniques. Smoke re-leased from burning vegetation contains chemical signalsthat trigger germination of species from different parts ofthe world. It stimulates seed germination in wildflowerspecies from fire-dependent plant communities in SouthAfrica and Australia (Brown and van Staden 1998).Smoke is used in horticulture to stimulate seed germina-tion and can improve the germination of such crops aslettuce and celery. Aqueous smoke-water, which is com-mercially available, is also active in this respect. The un-identified active constituents are volatile, thermostable,water-soluble, and long-lasting in aqueous solution and inthe soil (van Staden et al. 2000). Additional fire-relatedcues like heat-shock and an aqueous extract of plant ash

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Fig. 1 Time and rate of germination of seeds ofKniphofia pumilain vitro (batch sizes: four seeds each)

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

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were tested, although Baldwin et al. (1994) had shownthat ash of burnt wood does not contain potent cues thatstimulate germination. The other techniques that we usedare more conventional with respect to the pretreatment ofseeds; for a scheme of all the treatments and results, seeTable 1.

We found that the main factor affecting germinationwas the alternation in the temperature regime. A differ-ence of about 17C between day and night temperatures

seems to be necessary for germination in B. caulescens.Similar differences in diurnally alternating temperatureshave been found to improve germination in Solanaceaefrom Ethiopia (Teketay 1998). All other treatments in ad-dition to this temperature regime are only co-initiatingfactors to the germination; for example, the treatmentwith the aqueous extract of smoke of burning savannahplants. Except for the seeds treated with the aqueous ex-tract of smoke, the pretreatment with emery paper againseemed to have at least a certain positive effect on germi-nation. One fraction of seeds treated with smoke-waterand altered temperatures even germinated without me-chanical scarification. Egerton (1998) showed that smoketreatment produces an intense chemical scarification atthe seed surface and alters the permeability of the subtes-ta cuticle. Therefore, the treatment with smoke-watermight also be considered as a kind of scarification and, inaddition to the temperature shift, a germination cue in thisexperiment. The results of the ecological studies made byWillis and Groves (1991) and by Morgan (1998) werevery useful in the context of finding the adequate temper-ature regime. The problem of strongly temperature-de-pendent germination does not only exist in some of theAustralian species ofBulbine but also in B. caulescensfrom South Africa. For a successful in vitro propagation

of plants knowledge about their ecological background isan important precondition. The seedlings ofB. caulescensgrow very slowly under aseptic conditions. In our study,this species exhibited a faster growth when the aerialparts and the roots were cut from time to time. It was alsomore effective to subcultivate the seedlings on full-strength MS medium after germination.

Callus induction ofBulbine and the Kniphofia species

Callus tissues of the two Kniphofia species formed spon-taneously (Fig. 3a) on cut margins of aseptically culturedroots on solid one-fifth strength MS medium with fullstrength organics and supplemented with 0.5 mg/l ofBAP, NAA, and 2,4-D, respectively. The calli ofKnipho-

fia showed a yellowish color that was derived from theproduction of anthraquinones. Callus formation was in-duced on cut bulb margins from sterile plants ofB. caul-escens using the same medium formulation (Fig. 3b).This callus was pure white.

Establishment of liquid cultures ofK. pumilaand K. uvaria

Cut parts of the aseptic calli were subcultured on thesame medium without the gelling compound. These cal-lus suspensions (aggregates about 0.8 cm in diameter)exhibited a growth similar to that of the cultures on solidmedium as above, and showed the formation of shortroots. They also produced yellowish compounds, whichwere detected in the medium. The color originates fromvarious anthraquinones, of which chrysophanol was

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Pretreatment Additional treatment Temperature regime Germination rate [%]

Emery paper None Regular 0 (0 of 10 seeds)Emery paper None Altered 40 (4 of 10 seeds)Emery paper Sterile water Regular 0 (0 of 10 seeds)Emery paper Aqueous smoke extract Regular 0 (0 of 10 seeds)Emery paper Aqueous ash extract Altered 40 (4 of 10 seeds)None GA3 Regular 0 (0 of 10 seeds)None Aqueous smoke extract Altered 40 (4 of 10 seeds)

Bunsen burner None Regular 0 (0 of 10 seeds)

Table 1 Scheme of treatmentsand germination response ofthe seeds ofBulbine caulescens

Fig. 3 a Development of callusat roots ofK. pumila. b Tinybulb ofBulbine caulescenswith formation of callus

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identified by thin layer chromatography and by coelutionon high-performance liquid chromatography.

Formation of shoots ofK. pumila and K. uvaria

Without added phytohormones, spontaneous multiplica-tion in Kniphofia was observed only a few times. For a

directed induction of multiplication, we used two kindsof medium (media A and B; see above). The induction ofshoots 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 underthese conditions. With medium B, shoot production tookmore time and led to very thick roots approximately3 cm in length. Following division, the plants were trans-ferred to medium without hormones, where they showednormal growth.

Conclusion

The objectives of the study reported here were the in vit-ro germination and the establishment of tissue culturesof three Asphodelaceae species within the framework ofinvestigations on the biogenesis of knipholone and otherbioactive substances produced by these plants in whichstable isotope-labeled precursors are used. In view of thepermanent need of new agents against malaria, furtherstudies on the antiplasmodial constituents of the genera

Bulbine, Bulbinella and Kniphofia supported by in vitrocultures seem promising.

Acknowledgements We are grateful to F. Thiele and A. Kreiner(Wrzburg Botanical Garden) for Bulbine caulescens seeds andfor competent technical advice and to Prof. M. Mller (Chair ofPharmacognosy, University of Wrzburg) for useful discussions.This work was supported by the Fonds der Chemischen Industrie.

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