4. Special Part
4.5. Axenic In Vitro Cultures of Corsinia coriandrina
Axenic in vitro cultures of Corsinia coriandrina were established in cooperation with Dr. Klaus von Schwartzenberg at the Biozentrum Klein-Flottbek, University of Hamburg.
Plant in vitro cultures consist of whole plants, plant tissues or cells, which grow under axenic conditions (free from contaminating organisms) on artificial (defined) nutrient media under controlled conditions (Dixon, 1985). Techniques for liverwort in vitro cultures have been reviewed (Bopp & Knoop, 1984; Ohta et al., 1990; Duckett et al., 2004; Hohe & Reski, 2005).
Apart from various biological and molecular biological investigations, liverwort in vitro cultures were previously used for isolation of secondary compounds (Takeda & Katoh, 1981;
Sauerwein & Becker, 1990; Morais & Becker 1991; Ono et al., 1992; Becker, 1990, 1994, 1995; Grammes et al., 1994, 1997; Geis et al., 1999), bioconversion of exogenous substrates (Speicher & Roeser, 2002; Speicher et al., 2003), and application experiments to study precursor-product relationships in terpenoid (Takeda & Katoh, 1983; Warmers & König, 2000; Adam et al., 1998; Nabeta et al., 1998; Tazaki et al., 1999; Hertewich et al., 2001;
Barlow, et al., 2001; Karunagoda et al., 2001; Spiteller et al., 2002; Itoh et al., 2003) or bis(bibenzyl) (Friederich, 1999a, 1999b) biosynthesis.
4.5.1. Collection of Corsinia coriandrina Spores
Axenic in vitro cultures were established from the spores of Corsinia coriandrina, collected by the late W. A. König near St. Bartolomeo, Andalusia, in April 2004. Male antheridia and female archegonia were exclusively located on separate thalli, in agreement with the dioecious, haploid cytotype described from Atlantic areas (Bousselier-Dubayle & Bischler, 1998). In contrast to most other liverworts sporogons of Corsinia coriandrina do not emit the mature spores, which are only released upon decay of the surrounding plant tissue. Infertile spores isolated from sporogons surrounded by a fleshy calyptra did not germinate. About 80 mature spores were obtained from a single sporogon pit located at the decaying thallus end (Plate 7, page 213). Consequently, spores were considered highly contaminated with algae, bacteria and fungi. To eliminate these concomitant organisms, spores were sterilized for 5 min using 1 % calcium hypochlorite (Ca(OCl)2) solution, washed 5 times with sterile water and centrifuged. Sterilized spores were stored in sterile water at 7 °C for up to one month. Spore morphology was observed by scanning electron microscopy (SEM) (Plate 8, page 213) and proved to be identical to literature descriptions of Corsinia coriandrina (Jovet-Ast, 1973).
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4.5.2. Germination of Corsinia Spores and Propagation of Monoclonal Strains
From about sixty spores approximately fourtyfive germinated at 15 °C within 2 – 3 weeks when placed on Knop agar supplemented with the antibiotics ampicilline, amphotecerin B, and nystatine to suppress bacterial and fungal growth. The germ tube stadium was short and plantlets differentiated to form rhizoids and calli (Plate 9, page 214). Within one month twenty-nine calli could be transferred to fresh Knop agar before being overrun by resistant bacteria, fungi or algae. Calli were subcultured every month using Knop agar supplemented with abovementioned antibiotics. After 3 months, antibiotics were finally withdrawn and axenic culture conditions were regularly tested by transfer of plant fragments and media to liquid broth agar (LB) or potato dextrose agar (PDA). Cultures contaminated with bacteria or fungi were discarded. Twenty-one calli grown under axenic conditions were employed for vegetative propagation of the plant material, performed by using excised plant fragments as new inoculums. To ensure the highest possible comparability of the application experiments, three monoclonal strains (CC1 – 3) were raised from 3 selected plantlets of which CC1 exhibited fastest growth and proofed the most useful for vegetative propagation (Plate 10, page 214).
4.5.3. Determination of Ploidy Levels
In addition to the monoclonal Corsinia coriandrina strains CC 1 – 3 derived from the spores of a dioecious haploid specimen, in vitro cultured polyploid C. coriandrina strain Lorbeer/33 was obtained from the Culture Collection of Autotrophic Organisms (CCALA, Trebon, Czech Republic). Originating from G. Lorbeers collection of liverworts, the specified strain was cultivated at CCALA since the 1950‟s, but information on the collection site or date was subsequently lost. Comparison of the relative chromosome number per nuclei using flow-cytometry enabled different ploidy levels to be distinguished (Bousselier-Dubayle & Bischler, 1998). As shown in figure 75 the polyploid Lorbeer/33 strain contained twice the relative amount of DNA per nuclei in comparison to the dioecious haploid CC1 strain from Andalusia.
The occurrence of additional 4-methoxystyrenes like (Z)-corsiandrenin ((Z)-92) (Figure 44, page 48) in polyploid Corsinia strain Lorbeer/33 is in agreement with the proposed allopolyploid origin (derived from a cross of two genetically distinct species) with the haploid cytotype as one of the putative progenitors (Boisselier-Dubayle & Bischler, 1998).
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Figure 75: Relative chromosome counts per nuclei for haploid Corsinia coriandrina strain CC1 and polyploid Corsinia coriandrina strain Lorbeer/33 determined by flow-cytometry.
4.5.4. Optimization of Culture Media and Conditions
For propagation of Corsinia coriandrina chemically defined basal salt media like Knop, Gamborg B5 (Dixon, 1985), and Murashige Skoog (MSK) (Murashige & Skoog, 1962) were investigated. These high salt media were originally developed for in vitro cultures of higher plants, but have been successfully applied for liverwort cultures as concentrated or diluted media (Ohta, 1977; Katoh, 1980; Takeda & Katoh, 1981). Concentrations of macro nutritients including optimized MSKP/5 medium are given in table 4. A 100 µM Fe(III)NaEDTA supplement and trace elements listed in the Experimental Part (7.3.1, page 166) were identical for all media.
[mM] NH4+ K+ Ca2+ Mg2+ Na+ NO3– Cl– PO43– SO42–
Knop – 5.5 4 1 – 8 4 1.5 1
GB 2 25 1 1 1 25 2 1 2
MSK 41 20 3 1.5 – 60 3 1.25 1.5
MSKP/5 8 4.25 0.6 0.3 – 12 0.6 0.5 0.3
Table 4: Concentrations [mM] of macro nutritients in basal salt media for plant in vitro cultures.
Poylploid strain Lorbeer / 33 (N = 3401 nuclei)
Haploid strain CC1 (N = 3712 nuclei)
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To optimize the culture conditions for Corsinia coriandrina, plantlets were cultivated at 15 °C or 25 °C on Knop, GB, or MSK basal salt agar for 2 months using a 16/8 day-night cycle (10 µmol*m 2*s 1). Morphological features like plant differentiation, increase in callus or thallus size, and plant colour were compared in weekly intervals. Overall growth characteristics observed at 15 °C were superior over those at 25 °C, in agreement with the observation that Corsinia exhibits active growth during the moist winter season (Schuster, 1992). Growth of Corsinia on GB and MSK basal media was significantly better than Knop, indicating preferred NH4+
uptake over NO3–
as previously reported for other liverworts like Marchantia polymorpha (Katoh et al., 1980), Reboulia hemisphaerica (Morais & Becker, 1991), and Jungermannia subulata (Ohta et al., 1981). Nevertheless, after one month of vigorous growth plantlets started to decay, indicating the requirements for further optimization. To improve the concentration of macro-nutritients, concentrated and 1/2, 1/5, and 1/10 diluted MSK basal salt agar were compared over a period of ˂ 6 months, indicating the MSK/5 medium to be the most advantageous for constant growth and long durability in comparison with higher concentrated media.
4.5.5. Mixed Photo-Heterotrophic Growth
Plant growth of in vitro cultures using basal salt media is restricted through the photosynthetic capacity of the plants. Optimization of light intensity, composition and regime, as well as increased CO2 levels have been reported to result in increased growth of plant cultures under photoautotrophic conditions (Dixon, 1985). Cultures supplemented with suitable carbon sources to be incorporated into the plants primary metabolism become independent from these limitations, and increased growth is often observed under mixed photo-heterotrophic conditions. In some cases, plant cultures even become independent from the photosynthetic process itself and grow in complete darkness under chemoheterotrophic conditions (Takio et al., 1990). A growth promoting effect of carbohydrates in liverwort cultures has been reported, with D-glucose and D-sucrose (saccharose) being the most effective. In addition to plant growth, carbohydrate supplements also affect secondary metabolism and either higher or lower concentrations of secondary metabolites have been reported (Chopra & Sood 1973, Morais & Becker 1991, Takeda & Katoh, 1981). For mixed photo-heterotrophic growth of Corsinia coriandrina supplements of 25, 50, or 100 mM D-glucose, or 25 and 50 mM sucrose were found to be effective leading to increased growth. Differentiation of thalli was only
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observed with the lowest carbohydrate concentration of 25 mM D-glucose, whereas ≥ 50 mM resulted in dedifferentiation and callus formation (Plate 11, page 215).
4.5.6. Aerated Liquid Submersion Cultures
Overall growth of Corsinia coriandrina on agar media even under optimized mixed photo-heterotrophic conditions was painfully slow, thus, making propagation extremely time-consuming. Consequently, liquid submersion cultures were tested using thallus or callus parts placed in liquid media. Aerated liquid cultures were established in 250 or 500 ml glass vessels by bubbling filter sterilized air into the media via a glass tube. Full strength Knop, GB, and MSK, as well as diluted MSK/x media (dilution factor x = 2, 5, or 10) were tested using a 16/8 h day-night cycle (25 µmol*m 2*s 1). Comparable to agar cultures the best results were obtained with MSK/5 basal salt medium. Because phosphate has often been recognized as a growth limiting factor, especially in diluted media (Katoh, et al., 1980), a phosphate enriched media designated MSKP/5 with doubled phosphate concentrations was also tested and found to be advantageous.
When using MSK media for liquid cultures of Corsinia coriandrina a severe drop from the initial pH 5.7 to about pH 3.9 – 3.7 was observed during the first week, followed by a slow decrease within the next three weeks, thus, indicating preferred and rapid NH4+
/H+ exchange.
With MSK/2 media pH values reached 3.0, and cultures decayed within 3 weeks. With the diluted MSK/5 media pH values ranged at 3.4, and cultures decayed after 6 – 8 weeks. No significant difference between MSK/5 and phosphate enriched MSKP/5 media was observed, indicating that doubled phosphate levels were insufficient to buffer pH values. Comparable acidification upon preferred ammonium uptake is well known in agriculture and has previously been described for in vitro cultures of higher plants like Nicotiana tabacum, Solanceaceae (Behrend & Mateles, 1976) and liverworts (Ohta et al., 1981, 1990). Organic acids of the Citric-Acid cycle have been reported to efficiently buffer pH values during ammonium uptake (Behrend & Mateles, 1976; Katoh et al., 1980; Takami & Takio, 1987;
Ohta et al., 1981, 1990).
Figure 76: Citric acid (169) and fumaric acid (170) supplemented to liquid media.
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Consequently, potassium salts of citric acid (169) or fumaric (170) (Figure 76) at c = 2.5 mM were tested for their ability to buffer pH values during ammonium uptake by Corsinia coriandrina using MSK/5 agar. Fully differentiated monoclonal thalli (CC 1) were cut and transferred to test media adjusted at pH 5.7. Cultures were inspected in weekly intervals and pH values of the media were determined after 1 month. Plantlets cultivated with citrate (169) supplement were of yellowish colour, exhibited significantly less growth in comparison to control, and finally decayed within 3 weeks when the pH value ranged at 6.4, which is close to the pKs3 of citric acid. Culture development on agar media supplemented with 2.5 mM fumarate (170) was comparable to control, and pH values were efficiently buffered exhibiting pH 4.9 versus pH 3.9 for control after 1 month.
Aerated liquid submersion cultures using MSK/5 medium supplemented with 2.5 mM potassium citrate (169) decayed within 3 days, whereas 2.5 – 10 mM potassium fumarate (170) was tolerated without any notable negative effect on plant growth and development.
Culture media were tested in weekly intervals, revealing a potent stabilization of pH values for ˂ 4 weeks. After 8 weeks pH values finally increased to 7.0 – 8.0, indicating ammonium (NH4+) depletion followed by subsequent nitrate (NO3–) uptake. In conclusion, while citrate (169) was found to have an undefined toxic effect on Corsinia coriandrina, 2.5 mM fumarate (170) was shown to efficiently buffer pH values during NH4+/H+ exchange without disturbing plant development or ammonium uptake.
Aerated liquid cultures of monoclonal haploid Corsinia coriandrina strain CC1 were finally established in 500 or 1000 ml glass vessels at 15 °C using MSKP/5 basal salt medium supplemented with 25 mM D-glucose and 2.5 mM potassium fumarate. Under these conditions plantlets failed to differentiate forming callus-like structures (Plate 12, left, page 215) exhibiting a doubling time of approximately 30 days. Nevertheless, when partly differentiated thalli obtained with identical media under temporary immersion conditions with RITA® (Section 4.5.7.) were retransferred to liquid culture conditions, no dedifferentiation was observed (Plate 12, right, page 215). Microscopic investigation of differentiated thalli of in vitro cultured Corsinia coriandrina indicated the presence of large air chambers, rhizoids, ventral scales and oil bodies, comparable to the plant material from natural habitats (Plate 15, page 217). Liquid cultures of undifferentiated calli and differentiated thalli of monoclonal
haploid Corsinia coriandrina strain CC1 (Plate 12, page 215) were maintained for more than 2.5 years, produced almost the same constituents throughout, and were successfully
employed for the incorporation experiments with stable isotope labelled precursors (Sections 4.6., page 84).
82 4.5.7. Temporarily Immersed Cultures using RITA®
Temporary immersion systems (TIS), which combine unlimited ventilation of the plant tissue and intermittent contact between its entire surface and the liquid medium, have recently been described for in vitro cultures of higher plants (Etienne & Berthouly, 2002). Prevention of hyperhydricity and more natural conditions are among the major advantages of the TIS technique, which, however, requires optimization of the culture medium as well as immersion frequency and duration (McAlister et al., 2005; Zhu et al., 2005). Although temporary immersion systems appear highly suitable for reproducing the natural conditions of liverwort habitats, this technique has not been applied to liverwort in vitro cultures before.
Temporary immersed cultures of Corsinia coriandrina strain CC1 were established using the
»Recipient for Automated Temporary Immersion« system (RITA®) shown in Plate 14 (page 216) (Teisson & Alvard, 1995). The plant material is placed in the upper compartment on a strainer, while the liquid medium can be pumped up from the lower compartment by applying pressure, generated by a timer controlled aquarium-pump. Diluted MSKP/2, MSKP/5, and MSKP/10 media were tested. Immersion times were synchronized with the light regime (16/8 h day-night cycle, 25 µmol*m 2*s 1), with immersion times for 3½ or 17½ minutes at 0, 6, 12 and 18 h (1.0 or 5.0 % of total immersion times of liquid cultures). By using optimized diluted MSKP/5 medium supplemented with 25 mM D-glucose and 2.5 mM potassium fumarate and applying immersion times of 3½ minutes every 6 hours a doubling time of approximately 30 days was observed. Furthermore, spontaneous partial differentiation of callus cultures occurred under these conditions (Plate 13, page 216). However, the factors responsible for differentiation could not be identified. Resulting thalli could be transferred to liquid culture conditions using identical media and propagated without dedifferentiation (Plate 12, right, page 215). In addition, TIS cultures of Corsinia coriandrina were used to limit amino acid uptake in order to prevent excessive reversible transamination in application experiments with L-tyrosine (154) (Section 4.6.1.2., page 90).
4.5.8. Chemical Investigation of In Vitro Cultured Corsinia coriandrina
GC and GC-EIMS investigation of in vitro cultured Corsinia coriandrina indicated the presence of those compounds present in plant material from the natural habitat, although quantitative differences were apparent (Figure 77). These results unambiguously established the liverwort origin of Corsinia styrenes and stilbenoids. The predominance of (Z)-coriandrin ((Z)-65) in the in vitro cultured material indicated that (Z)-isomers constitute the initial
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metabolites and are not derived from UV induced (E/Z)-isomerisation of (E)-isomers because UV permeability of glass vessels and the RITA® compartment is low.
Figure 77: TIC chromatograms of diethyl ether / Na2SO4 extracts of Andalusian Corsinia coriandrina and corresponding in vitro cultured plant material from liquid cultures and RITA® cultures.
in vivo material
RITA® in vitro culture
Liquid in vitro culture
10
5 15
18
61
61 62
64 64
(E)-65
(E)-65
(Z)-72
RT [min]
RT [min]
RT [min]
TIC [%]
TIC [%]
66 (Z)-67
74
18
73 C16 acid
66
C16 acid (Z)-65
(Z)-65
(Z)-65
73 (Z)-72
62
(Z)-67
73 50
100
100
50
100
50 TIC [%]
84