Development of in vitro Techniques for the Important Medicinal Plant Veratrum californicum

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Initiation and maintenance of callus cultures
• Plant regeneration
• Extraction and targeted HPLC/ESI/MS of plantlets
• Initiation of cell suspension cultures
• Cryopreservation of the cell suspension lines A, B and C
• Growth optimization of the suspension cell line B
• Statistical analysis
• Results and Discussion
• Acknowledgements
• References

Abstract

Veratrum californicum (Liliaceae) is an important monocotyledonous medicinal plant which is the only source of the anticancer compound cyclopamine. An in vitro culture system for somatic embryogenesis and green plant regeneration of Veratrum californicum was developed. Embryogenic calli were induced from mature embryos on induction medium. Five basal media supplemented with different growth regulators were evaluated for embryogenic callus induction, modified MS medium with 4 mg/L picloram showing the best result for embryogenic callus production. Fine suspension cell lines were established by employing friable embryogenic calli as starting material and AA medium and L2 medium as culture media. The suspension cell lines cultured in AA medium with 4 mg/L NAA appeared to be fresh yellow and fast growing. The suspension cells were cryopreserved successfully and recovered at a high rate. Green plants were regenerated from embryogenic calli maintained on solid medium with 73 % regeneration ability (green plants/100 calli) in 27-month-old culture. The in vitro plantlets contained the steroid alkaloids cyclopamine and veratramine. This in vitro system will form the basis for metabolic engineering of Veratrum cells in the context of biotechnological production of pharmaceutically important secondary metabolites.

Abbreviations

DMSO:dimethyl sulfoxide

fw:fresh weight

NAA:naphthaleneacetic acid

2,4-D:2,4-dichlorophenoxyacetic acid

picloram:4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid

dicamba:3,6-dichloro-2-methoxybenzoic acid

Introduction

The genus Veratrum comprises up to 45 species of perennial herbs distributed throughout the northern temperate to Arctic regions of Europe, Asia and North America. Its exact systematic position is still under debate but modern systems place it within the tribe Melanthieae of Melanthiaceae whereas traditionally the genus belongs to the large family of Liliaceae [1]. Veratrum is phytochemically characterised by the presence of steroid alkaloids which exhibit interesting pharmacological properties already recognised in ancient times.

The teratogenity of Veratrum californicum Durand, a species distributed throughout the mountains of the western USA and often referred to as corn lily, was noticed when a high percentage of sheep grazing these areas gave birth to deformed lambs. Only offspring of ewes which had consumed Veratrum during early pregnancy (on day 14 of gestation) developed the anomalies varying from cyclopia, i. e., extreme craniofacial malformation, to mildly deformed upper jaws [2]. Two steroid alkaloids cyclopamine (11-deoxojervine) and jervine have been identified as the responsible teratogens (Fig. [1]). More recently the molecular mode of action for cyclopamine-induced teratogenesis has been investigated and it has been revealed that the compound selectively blocks Sonic hedgehog signal transduction [3]. This pathway has a central role in a multitude of developmental processes. Interestingly, a number of genes in the Sonic hedgehog signalling network have been associated with certain human tumours. Therefore, cyclopamine and its derivatives are proposed as potential therapeutic agents for the treatment of tumours arising from disruption of components of the hedgehog pathway [4]. Cyclopamine has already shown promise in the treatment of medulloblastoma tumour [5], basal cell carcinoma [6] and small cell lung cancer [7] in vitro and in whole animal systems. Currently the compound is in clinical trials for medulloblastoma patients, phase I studies being completed by the middle of 2006 (www.curis.com).

At present, Veratrum californicum is the only source for cyclopamine because the compound with its complex chemical structure cannot be totally synthesised at an economical price. Isolation of the substance from wild plants is impeded by the low and highly variable quantity, and is therefore very expensive. With metabolic engineering tools recently developed [8], [9] it would be possible to produce these alkaloids and new derivatives in cell cultures [10]. However, an in vitro culture system for Veratrum has so far not been available although this is a prerequisite for such metabolic engineering.

Here we report for the first time an in vitro culture system for Veratrum californicum comprising the initiation of callus, the regeneration of plants, the establishment of suspension cultures and their long-term storage by cryopreservation. In addition, in vitro plantlets are shown to contain steroid alkaloids.

Materials and Methods

Initiation and maintenance of callus cultures

Seeds of Veratrum californicum Durand were peeled, rinsed with 94 % ethanol and surface sterilized with 1 % (v/v) sodium hypochlorite supplemented with a few drops of Tween 20 for 10 minutes, and finally rinsed three times with sterile water. Seeds were allowed to germinate for 2 days at 22 °C in the dark on moist filter paper. Embryos were excised and cultured on MS medium [11] solidified with gelrite (Carl Roth GmbH; Karlsruhe, Germany) (3 % w/v) and supplemented with 1 mg/L kinetin (Sigma; St. Louis, MO, USA) and 1 mg/L NAA (Sigma) in the dark at 22 °C. Produced calli were subcultured to fresh plates at two to three weeks intervals. After four months of culture calli were transferred to L2-medium [12] solidified with gelrite (3 % w/v) and supplemented with 2.5 mg/L 2,4-D and were subsequently subcultured to fresh plates at one month intervals.

Embryogenic callus cultures from isolated V. californicum embryos were induced by the following media: Basal media MS [11], R2M [13], L2 [12], modified MS medium (mMS, consisting of MS basic salts and vitamins, 146 mg/L glutamine, 200 mg/L casein hydrolysate) and AA [14] supplemented with different hormones [picloram (Duchefa; Haarlem, The Netherlands), NAA, 2, 4-D (Sigma), dicamba (Duchefa)] at three levels (2, 4 and 7 mg/L) all solidified with gelrite (3 % w/v). The cultures were kept in the dark at 25 °C and embryogenic calli were subcultured at one month intervals.

Plant regeneration

In order to test the regeneration abilities of embryogenic calli during the subculturing, some of the calli were transferred to solid regeneration medium L2 (without hormones), and cultured in light (light intensity about 30 - 40 µmol mm^-2 s^-1 Osram cool white/Osram fluora, 1 : 1 on Watt basis) at 25 °C. After 4 - 5 weeks green shoots were moved to hormone-free medium R2M in a plastic container (Greiner Bio-one 68/11 mm; Frickenhausen, Germany) for root development. Well developed plantlets were potted into peat soil and transferred to the greenhouse.

Extraction and targeted HPLC/ESI/MS of plantlets

100 mg lyophilised plant material were suspended in 4 mL 5 % NH₄OH and extracted with 20 mL toluene for 10 min in an ultrasonic bath. Following centrifugation (7000 rpm, 10 min) the organic layer was collected and the sample residue was twice re-extracted with another 20 mL toluene. The organic phases were combined and the solvent was evaporated to dryness. For analysis the extract was redissolved in 200 µL of the LC solvent which is described below.

HPLC separation was performed using a Waters HT-Alliance 2795 system and was monitored with a Micromass Quattro Micro triple quadrupole mass spectrometer equipped with an electrospray source. The ion source was operated at a capillary voltage of 4.00 kV and a cone voltage of 60 V. Source and desolvation temperatures were 130 °C and 290 °C, respectively. Desolvation gas flow was 911 L/h and cone gas flow 30 L/h. The scan mode function was applied to record the protonated molecular ions (m/z = 200 - 900). An aliquot of 50 μL of sample was loaded onto a reverse-phase C18 column (Xterra MS C18, 4.6 × 150 mm, 5 μm, Waters; Milford, MA, USA) at 35 °C. The sample was eluted within 30 min using isocratic conditions of acetonitrile and 0.1 % TFA (30 : 70) applying a flow of 1 mL/min and a split of 0.2 mL/min reaching the mass spectrometer. Commercially available alkaloids cyclopamine (TRC; North York, Canada), jervine (TRC), veratramine (Sigma) and solanidine (Sigma) were used as reference compounds.

Initiation of cell suspension cultures

Suspensions were initiated from Veratrum californicum calli grown on solid L2-medium. They were maintained in liquid L2-medium with 2,4-D (2.5 mg/L) and without hormones. Suspensions were subcultured in 10 to 14 days intervals by subculturing 20 mL of the 10-day-old suspension (2 - 3 g fresh weight cells) to 30 mL of fresh medium. Produced suspension lines A, B and C were grown at + 25 °C on a rotary shaker (90 rpm) in the dark.

Cryopreservation of the cell suspension lines A, B and C

The suspension lines A, B and C were cryopreserved by using a Kryo10 device (Planer Biomed; Sunsbury-on-Thames, UK). The suspensions were subcultured 4 to 5 days prior to the start of the cryopreservation experiment. A dehydration procedure as follows was applied: I) Sugar alcohol concentration of the suspension was adjusted to 0.2 M by adding five times small aliquots of 4 M sorbitol during a period of 30 minutes. After sorbitol additions, the suspensions were incubated under normal growth conditions for 24 hours [in the dark, 25 °C, on a rotary shaker (90 rpm)]. II) Sugar alcohol concentration was adjusted to 0.4 M by 4 M sorbitol and incubations were carried out as in step I). The dehydrated suspensions were transferred to ice and DMSO was added to reach 5 % (v/v) concentration. Cell : medium ratio was adjusted to 1 : 2 and suspensions were packed into ampoules. Protectant-treated suspensions were kept on ice for a period of 100 minutes. A freezing protocol as follows was applied: I) A rate of 10 °C/min to reach 0 °C was followed by II) incubation at 0 °C for 20 minutes. The freezing was finalized by using III) a rate of 1 °C/min to reach -40 °C and then IV) samples were immersed in liquid nitrogen. Thawing of cryopreserved samples was carried out by immersing the suspension ampoules straight from liquid nitrogen to a 40 °C water bath for 2 minutes. Cells were transferred to sterile filter paper on solid culture medium originally used for the culture of that particular cell suspension line. Cell division and growth of the cultures were monitored.

Growth optimization of the suspension cell line B

Growth of suspension line B on L2-medium with 2,4-D (2.5 mg/L) was further optimised. About 3 g (fw) of cells were transferred to an Erlenmeyer flask containing 50 mL liquid AA medium (with 2, 4-D and NAA at levels of 2, 4 and 7 mg/L or without hormones), and incubated in the light (light intensity about 30 - 40 µmol mm^-2 s^-1 Osram cool white/Osram fluora, 1 : 1 on a Watt basis) on a rotary shaker (130 rpm) at 25 °C. The volume of the liquid medium was made up to 100 mL after the second subculture. The suspensions were subcultured at three weeks intervals by replacing about half of the old culture with an equal volume of fresh medium.

Statistical analysis

Frequencies of embryos producing embryogenic calli and frequencies of regenerated green and albino plants were recorded. Oxidative browning percentages of embryos were calculated. Completely randomized designs (CRD) were used in the experiments. Each Petri dish with 20 embryos was considered an experimental unit and each treatment contained five replicates. Data analyses with two treatment levels were carried out by t test, and with more than two treatment levels by the ANOVA procedure. Multi-range comparisons were performed by the LSD test.

Results and Discussion

Callus cultures from isolated embryos of Veratrum californicum were successfully initiated. Several aspects (e. g., influence of embryo size, culture medium and growth regulators) were studied in detail in order to clarify their role in the induction of embryogenesis.

For monocotyledonous plant tissue culture, embryo size, indicating the physiological stage of the embryo development, plays an important role in somatic embryogenesis and is critical for the establishment of embryogenic callus. The influence of embryo size of mature seeds on somatic embryogenesis of V. californicum was studied (Table [1]). Embryo size significantly affected embryogenic callus induction (P < 0.001). Bigger embryos (> 2 mm) produced significantly higher embryogenic callus yield than smaller embryos (< 2 mm). It was found that embryogenic callus induction from small embryos was difficult. Most probably small mature embryos had not developed well and had less active cells compared to big embryos. In previous works for immature embryo culture of monocot cereal plants, optimal embryo size was shown to vary from 0.5 - 2 mm [15], [16]. Our results indicate that the influence of embryo size on somatic embryogenesis differs between mature and immature embryo cultures, and therefore requirements for embryo sizes are different. An interaction of embryo size and medium was not found (P > 0.05).

Culture medium, providing both nutrients and an osmotic environment, is an important factor influencing somatic embryogenesis. Composition of basal medium including the carbon source, the source and amount of total nitrogen, vitamins and growth regulators are crucial for embryogenic callus induction. Requirements for medium vary with species, genotype and culture conditions. For embryogenic callus induction of V. californicum (Figs. [2] A, B, and C), culture media and plant growth regulators were compared (Table [2]). Both media (P < 0.001) and plant growth regulators (P < 0.001) significantly influenced embryogenesis. Among the five tested media mMS produced the highest embryogenic callus yield. Addition of picloram resulted in the highest embryogenic callus production. Interactions of media and growth regulators on embryogenesis were not found (P > 0.05). In previous studies on somatic tissue culture of monocot plants, e. g., of Liliaceae [17], [18], MS or modified MS medium were most commonly used as induction media for somatic embryogenesis. Reduction of ammonium and nitrate nitrogen was generally important in embryogenic culture systems [15]. Amino acid supplements have also been used as nitrogen source, and certain amino acids were found to be beneficial in somatic embryogenesis [19]. In the present study addition of amino acids (glutamine and casein hydrolysate) to MS significantly improved the embryogenic callus induction of V. californicum. AA medium, i. e., a medium enriched with glutamine and other amino acids as nitrogen source, was found not to be suitable for embryogenic callus induction (Table [2]).

Exogenous addition of growth regulators to culture medium is usually necessary for embryogenic callus induction. The type and concentration of auxin in induction medium plays an important role in obtaining high efficiency of somatic embryogenesis. Requirements vary with species, genotype and plant growth conditions [20]. Effects of different concentrations of plant growth regulators in media were compared for V. californicum. In Table [3] it is shown that somatic embryogenesis was significantly influenced by the levels of growth regulators (P < 0.001). Concentrations 4 mg/L and 7 mg/L gave significantly higher embryogenic efficiencies than 2 mg/L. Significant differences between concentrations of 4 mg/L and 7 mg/L were not found. Efficiency of auxins on somatic embryogenesis has been investigated previously for monocot cereal crops [15]. The auxin 2,4-D has most often been used in induction medium for embryo culture initiation. However, in embryo culture of barley [15] and rye [16], dicamba was found to be more suitable for somatic embryogenesis induction than 2,4-D. In Lilium longiflorum, picloram has more often been used in somatic embryogenesis than 2,4-D [21], [22]. Our results indicate that picloram is more suitable than 2,4-D, and is the best of the four growth regulators tested (Table [2]). Similar findings have, for example, been reported in tritordeum [23]. We could not find a significant (P > 0.05) difference of embryogenic frequency between picloram and NAA suggesting that NAA would be another choice for embryogenic callus induction of V. californicum.

Oxidative browning has been a major problem associated with plant tissue culture and has limited the application of the culture techniques in many plants [24]. Also a high percentage of V. californicum embryos released phenolic compounds during embryo culture. Culture medium (P < 0.001) and growth regulators (P < 0.001) significantly influenced phenolic exudation. The lowest percentage (18.9 %) of embryos producing phenolic exudates was observed on mMS medium containing picloram (see Table [1]S in the Supporting Information). Browning is considered to be the result of oxidation of phenolic exudates released from plant cells into the surrounding medium and can even cause necrosis of plant cells. Supplementing the media with polyvinylpyrrolidone and activated charcoal did not prevent browning of the Veratrum cultures (data not shown).

Plant regeneration in tissue culture is a complex morphogenic phenomenon in which both extrinsic and intrinsic factors have important roles. For monocot cereal crops, regeneration ability has been shown to be genetically controlled [15]. Green plants of V. californicum were successfully regenerated from embryogenic calli maintained on solid medium (Figs. [2] D and E). Regeneration abilities of the embryogenic calli subcultured on solid medium mMS (with 4 mg/L picloram) were evaluated on L2 medium (without growth regulators). The percentage of green plants regenerated from the embryogenic calli was excellent, i. e., 107 % (green plants/100 calli) after 3 months culture (Fig. [3]). Maintenance of embryogenic capability and regeneration potential has been a critical problem in efficient in vitro culture systems [15]. In the case of V. californicum regeneration capacity also decreased but only to 73 % after 27 months culture (Fig. [3]). Whole green plant regeneration is a crucial requirement for most current methods of plant tissue culture and genetic transformation. Rooted green plants (Fig. 2F) were also successfully weaned in the green house (Fig. [2] G).

A fraction containing free steroid alkaloids was extracted from in vitro plantlets of V. californicum and subjected to liquid chromatography (LC) which was monitored by a mass spectrometer. The reference alkaloids cyclopamine, jervine, veratramine and solanidine were analysed to compare retention times and mass spectra. Veratramine and traces of cyclopamine were detected in the plantlets. The identity of these compounds was confirmed by samples spiked with authentic references (See Fig. [1]S in the Supporting Information). Jervine has been reported from V. californicum earlier [25] and solanidine has been found in several Solanum and Veratrum species. Both veratramine and jervine are biosynthethic products of cyclopamine which is derived from cholesterol [26]. Plant material collected from the wild can contain on average 0.35 g total alkaloids per 100 g dry material depending on the location and growth stage [27]. The targeted LC method for the analysis of steroid alkaloids was chosen because it avoids a derivatisation step which is usually employed for alternative gas chromatography analysis. Low solubility and problems with bad peak shape were circumvented by the selected solvent mixture as described before [25].

Embryogenic suspension cultures are finely dispersed and fast growing. Embryogenic cells aggregate in small groups and are highly cytoplasmic and non-vacuolated. The initiation of suspension cultures from isolated embryos of V. californicum resulted in A, B and C suspension lines grown in L2-medium. All these lines were also able to grow without growth regulators. In order to maintain the viability of the cultures, the cell suspensions were subcultured at the beginning of the stationary phase. According to earlier studies Lilium suspensions have been initiated from embryogenic calli, shoot apices and meristematic nodular cell clumps [21], [27]. Liquid media MS, N6 or derivatives of them with different auxins have been commonly employed as culture media. AA medium, an amino acid-based culture medium [14], has been used as culture medium for the rapid establishment of rice suspension cultures [29]. This finding is in accordance with our observation on V. californicum. AA medium with the optimal concentration level of 4 mg/L NAA or 2,4-D improved the growth of the suspension line B (Fig. [2] H). However, the long-term maintenance of suspensions was better with NAA than with 2,4-D. The growth of suspension cell line B is shown in Fig. [4]. Our result differs from previous work for monocotyledonous plants where the medium suitable for embryogenic callus induction has been also suitable for suspension culture.

The produced suspension lines A, B and C were cryopreserved by using a classical slow-freezing protocol. A summary of the cryopreserved lines is shown in Table [4]. The lines were deposited in the VTT Culture Collection (VTT, Espoo, Finland). VTT Culture Collection codes will be used hereafter to specify the suspension lines. A recovery rate of 83 % for the cryopreserved lines was recorded. For successful cryopreservation, the initiation of the dehydration procedure must be started when the suspensions are in the beginning of the exponential growth phase. The cells in exponential growth phase survive the freezing-thawing procedure better than the larger more vacuolised cells already reaching the stationary phase [30]. This is most probably due to the nature of the cells in the exponential growth phase: dense cytoplasm, small vacuoles and low water content. Osmotic dehydration was needed and 5 % (v/v) DMSO was sufficient as a cryoprotectant. Dehydration has a positive influence on the freezing tolerance [31] since cell damage caused by high intracellular water content is prevented. In addition, the use of DMSO improves the viability and cell recovery as observed earlier [32]. This may be due to the fact that the protection mechanism of DMSO is different when compared to sugar alcohols, since DMSO penetrates the cell membranes. The immediate thawing in a + 40 °C water bath gave the best recovery for the cryopreserved lines. In Fig. [2] I, a cryopreserved cell line is shown after thawing and one month of culture. The cells have been kept under liquid nitrogen so far for one year, and been thawed successfully.

In summary, an in vitro culture system of the monocotyledonous medicinal plant Veratrum californicum was established for the first time. Modified MS medium (mMS) with 4 mg/L picloram was found to be suitable for embryogenic callus induction. In vitro plantlets contained steroid alkaloids but green plant regeneration ability was partly lost for embryogenic calli maintained on solid medium. Suspension cultures were established in L2 medium with and without 2,4-D and their growth was further optimised by using AA medium with 4 mg/L NAA. For long-term storage the cells were cryopreserved and recovered at a high rate. The developed culture system of Veratrum californicum Durand can now be used for metabolic engineering of the plant to efficiently exploit secondary metabolite production.

Acknowledgements

This research was funded by the National Technology Agency of Finland (Tekes), the ”NeoBio” program to K.-M. O.-C. Rui Ma’s post-doctoral work was supported by the grant from the Academy of Finland (to K.-M. O.-C). The authors are indebted to Dr. T. Seppänen-Laakso for analytical support, to Dr. L. Nohynek and P. Oza for tissue culture work and to T. Teikari, J. Rikkinen, A. Heikkinen for their technical assistance. Dr John Londesborough is kindly acknowledged for critically reading the manuscript.

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Kirsi-Marja Oksman-Caldentey

Biotechnology

VTT Technical Research Centre of Finland

P. O. Box 1000

Tietotie 2

FIN-02044 Espoo

Finland

Phone: +358-20-722-4459

Fax: +358-20-722-7071

Email: Kirsi-Marja.Oksman@vtt.fi

References

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  CrossrefPubMedSearch in Google Scholar
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Kirsi-Marja Oksman-Caldentey

Biotechnology

VTT Technical Research Centre of Finland

P. O. Box 1000

Tietotie 2

FIN-02044 Espoo

Finland

Phone: +358-20-722-4459

Fax: +358-20-722-7071

Email: Kirsi-Marja.Oksman@vtt.fi

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