Acta Physiol Plant (2011) 33:1547–1551 DOI 10.1007/s11738-010-0660-1
SHORT COMMUNICATION
Development of an optimal culture system for callogenesis of Chrysanthemum indicum protoplasts Tom Eeckhaut • Johan Van Huylenbroeck
Received: 11 June 2010 / Revised: 10 November 2010 / Accepted: 25 November 2010 / Published online: 11 December 2010 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2010
Abstract This study is a comparison of four methods to induce calli formation in a protoplast culture of Chrysanthemum indicum. Culture in liquid medium (17.6 calli/105 protoplasts) was preferable to culture in solid agarose beads, although the efficiency of the latter could be improved by layering them on glass beads (12.5 vs. 0.83 calli/105 protoplasts). Culture of protoplasts on moistened filter paper was unsuccessful. In the liquid media, microcalli and calli were induced efficiently and easily after 6 weeks. These effects may be explained by reduced toxicity due to cell breakdown and improved aeration. Keywords Aeration In vitro Liquid medium Microcalli Regeneration
Introduction Interspecific hybridisation is the main source of innovation in ornamentals. Although sexual crosses have generated interspecific prebreeding material in many genera, problems such as albinism, hybrid vigour and sterility frequently occur (Eeckhaut et al. 2006). Somatic fusions can be an alternative (Waara and Glimelius 1995). The design of an efficient regeneration protocol for various genotypes is indispensable when implementing protoplast fusion breeding programmes in any crop.
Communicated by B. Borkowska. T. Eeckhaut (&) J. Van Huylenbroeck Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit-Applied Genetics and Breeding, Caritasstraat 21, 9090 Melle, Belgium e-mail:
[email protected]
Commercial chrysanthemum, hybridised from Chrysanthemum indicum L. (Asteraceae) (2n = 6x = 54), is the second most economically important floricultural crop worldwide after rose. In the past, breeders have focused on enhancing chrysanthemum’s ornamental value through improving flower colour, size and form, vegetative height, growth form and sensitivity to light quality/quantity (Teixeira Da Silva 2004). A wide range of breeding techniques has been developed for chrysanthemum. Tissue culture has been extensively studied as a breeding tool (Teixeira Da Silva 2003). Somatic fusion has occasionally been successful (Furuta et al. 2004). Both environment and medium are known to influence protoplast division rates (Davey et al. 2005). A number of culture components, technical tools and other factors have been evaluated to improve division rates of Chrysanthemum protoplasts or regeneration of microcalli. Several parameters were found to be significant, such as genotype (Sauvadet et al. 1990), cell density and complex additives such as coconut water or calf serum (Fujii and Shimizu 1990), leaf age and osmoticum type (Endo et al. 1997), rate of medium refreshment (Amagasa and Kameya 1989), conditioned medium (Zhou et al. 2005) and start material. In some genotypes leaf-derived de novo shoots yield more sustained division than regular stock cultures (Lindsay and Ledger 1993). A few similarities among all these protocols can be found, apart from the reduction or even complete removal of ammonium from the regeneration medium, as previously demonstrated by Okamura et al. (1984). A relatively neglected parameter in studies on Chrysanthemum protoplasts has been the screening of alternative culture types. As reviewed by Davey et al. (2005), different culture types affect oxygen availability to protoplasts, which can significantly influence the overall efficiency of the system. For instance, Anthony et al. (1995) succeeded in
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stimulating division of cassava protoplasts by including glass rods in the culture medium, thus improving aeration at the menisci. Our general aim is to induce callus formation from Chrysanthemum indicum protoplasts as a first and necessary step towards complete protoplast regeneration. A more specific objective is to design a culture system that efficiently promotes protoplast division into callus. We have compared several culture systems and determined which was the most efficient one.
Materials and methods Stock cultures of Chrysanthemum indicum ‘Yoko Ono’ were maintained in Meli-jars at 23 ± 2°C under a 16 h photoperiod at 40 lmol m-2 s-1 photosynthetic active radiation, supplied by cool white fluorescent lamps (OSRAM L36 W/31). Shoots were transferred every 6–10 weeks on fresh medium (eight 2 cm explants per jar) (Table 1). Leaves from 6- to 10-week-old stock plants were chopped into small pieces and incubated in 0.4 M mannitol for 1 h. They were subsequently incubated on a shaker in an enzyme solution containing 0.5% cellulase Onozuka R-10 (Duchefa Biochemie BV, The Netherlands), 0.3% macerase R-10 (Duchefa Biochemie BV, The Netherlands) and 0.1% driselase from Basidiomycetes sp (SigmaAldrich, Belgium) dissolved in 0.4 M mannitol (16 h, 10 rpm, dark, 22°C). After incubation, the enzyme/protoplast mixture was sieved through a 100 lm nylon sieve. After centrifugation (100g, 10 min) in an Eppendorf 5810R centrifuge equipped with a swing-out rotor, the pellet was resuspended in washing medium (Table 1). After three centrifugation/washing cycles, the protoplast concentration was counted in a Bu¨rker counting chamber. Protoplasts were diluted to 2 9 105 pp/ml in washing medium.
Four different culture systems were attempted, using the same solid and liquid media throughout except for the presence of a solidifier (Table 1). Light conditions were the same as described above for culture maintenance throughout the experiment. Method 1 used a Petri dish (5 cm diameter) to start the culture by incubating freshly isolated protoplasts during the first week at a concentration of 105 pp/ml in 10 ml liquid medium. Subsequently, protoplasts were collected by centrifugation and divided in four new Petri dishes filled with 10 ml fresh liquid medium at a concentration of 2.5 9 104 pp/ml. After 3 and 5 weeks, the liquid medium was again replaced by centrifuging the protoplasts, but the concentration was kept stable at 2.5 9 104 pp/ml. After 5 weeks, the mannitol concentration was reduced to 0.2 M. Method 2 used four Petri dishes (5 cm diameter) filled with 3 x 100 ll beads. The beads were a 1:1 mixture of solid medium:washing medium with 2.105 pp/ml (at 35–40°C). Subsequently, 4 ml of liquid medium was added. After 5, 12, 19, 26 and 33 days, 1 ml of the liquid medium was removed and substituted with identical medium but without mannitol. Method 3 used four Petri dishes (5 cm diameter) filled with 4 ml of the solid medium. Afterwards, 3 9 100 ll aliquots of the same mixture (1:1 solid medium:washing medium with protoplasts) were pipetted onto the freshly solidified solid medium. Those aliquots were layered around three groups of four sterile glass beads. Two millilitres of the liquid medium was added; 0.5 ml was replaced by the medium without mannitol after the same time intervals as in Method 2. Method 4 used four large Petri dishes (9 cm diameter) lined with sterile filter paper and moistened with 2.5 ml of the liquid medium. The same solid medium:protoplast mixture as in Methods 2 and 3 was placed on the filter
Table 1 Composition of media for stock maintenance and protoplast isolation and culture of Chrysanthemum indicum ‘Yoko Ono’ Stock medium
Washing medium
Liquid and solida medium
Salts
MS
MS without NH4
1/2 MS without NH4
Vitamins
KMb,c
KMc
KMc
Sucrose (g/l)
20
20
20
Mannitol (M)
0
0.4
0.4
Others
2 mg/l glycine, 1 mg/l kinetin, 0.01 mg/l NAAd pH 6.2
KM organic acids, 1 g/l MES; pH 5.6
KM organic acids, 1 g/l MES, 1 mg/l BAP, 2 mg/l NAA, 1 mg/l 2,4-D; pH 5.6
2,4-D 2,4-dichlorophenoxy acetic acid, BAP benzyl amino purine, KM Kao and Michayluk (1975), MES 2-(N-morpholino) ethanesulfonic acid, MS Murashige and Skoog (1962), NAA napthyl acetic acid a
Solidified with 12 g/l low melting point agarose (Duchefa Biochemie BV, The Netherlands)
b
Except retinol and cholecalciferol
c
Vitamins were filter sterilized and added after autoclaving (121°C, 30 min, 500 hPa)
d
Solidified with 6 g/l MC29 agar (Lab M Limited, UK)
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paper (3 beads of 100 ll per Petri dish). At last, an extra volume of 1.5 ml of the liquid medium was added. After the same time intervals as mentioned above, 1 ml of the liquid medium was replaced by liquid medium without mannitol. All Petri dishes were sealed with a parafilm. During 6 weeks, the cultures were microscopically (Leica DMIRB/Leica WILD MZ8) monitored. After that period, the formation of visible microcalli was evaluated. Digital pictures were made with Leica IM500 software. Size determination was done by measuring 15 calli when possible. On the obtained data, one way analysis of variance (ANOVA) was conducted through Statistica 9; significantly different means were separated by the Duncan (P B 0.05) method.
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Results and discussion Method 4 (culture on moist filter paper) yielded no calli (Table 2). All other treatments yielded initial divisions during the first week. Method 2 induced callogenesis, but very inefficiently; in 12 agarose beads, callus was formed only once. The efficiency of this method was significantly improved by layering the agarose beads around small glass beads (as in Method 3) (Fig. 1). However, it was clear that the liquid culture system (Method 1), gave better results than the other culture systems examined (Table 2). As protoplasts can be more easily cultured in relatively high liquid volumes than in small agarose beads, this difference is reinforced when considering callogenesis efficiency per Petri dish. The number of calli was more than tenfold
Table 2 Callus formation in different culture systems of Chrysanthemum indicum ‘Yoko Ono’ protoplasts after 6 weeks Number of calli/100,000 protoplastsy
Number of calli/Petri dishy
Callus size (lm)
Method 1
17.60 ± 0.57 a
44.00 ± 1.41 a
Method 2
0.83 ± 0.83 b
0.25 ± 0.25 c
250z
Method 3
12.50 ± 3.44 a
3.75 ± 1.03 b
247 ± 26
Method 4
0b
0c
676 ± 46
–
Data are means ± SE (n = 4) y
a,b,c significant differences based on Duncan’s multiple range test, P B 0.05
z
Single measurement; standard error could not be defined
Fig. 1 Culture of Chrysanthemum indicum ‘Yoko Ono’ protoplasts: formation of microcolonies (a) and macroscopically visible callus (b); callus formed adjacent to glass beads (Method 3) (c) and in liquid medium (Method 1) (d)
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higher than that obtained through Method 3 (Table 2), the second best method. In turn, Method 3 resulted in a higher number of calli than Method 2 (the similar culture system without glass beads). Calli obtained through Method 1 were larger than those induced by methods 2 and 3 (Table 2), although for every culture system (except Method 4) initial division started in the first week. In additional independent experiments with ‘Yoko Ono’, Methods 1 and 3 showed similar efficiencies, with average yields of 32.5–51.5 and 1.5–5 calli/Petri dish, respectively. Our results can therefore be considered reproducible. A specific advantage of the liquid culture system is the possibility to reduce or increase protoplast concentration during the experiment. In our experiment, the protoplast concentration in Method 1 was reduced from 105/ml to 2.5 9 104/ml after 1 week (after the first divisions had already occurred). We found 105 pp/ml as a required start concentration for sustained division in introductory experiments (data not presented). Nonetheless, in a later stage, the better availability of nutrients and the decreased concentration of toxic waste from dead cells in a less concentrated culture may account for a more rapid callus growth. We propose that more rapid dilution of waste molecules and easier access to nutrients is the main explanation for the high efficiency of Method 1, as a solid agarose matrix can be expected to inhibit the dispersal of both groups. Method 3 is significantly more efficient than Method 2. This is in accordance with results previously obtained by Anthony et al. (1995); the stimulating effects of this culture system modification may be due to the occlusion of small air bubbles near the agarose beads that enable a better gas exchange between the dividing protoplasts and their environment. Indeed, calli formed exclusively adjacent to the glass beads. Finally, Method 4 was conceived as a culture system with a maximal air supply, but this clearly inhibited the presence of other vital nutrients. Liquid culture has been documented as being partly successful in Chrysanthemum protoplast culture (Fujii and Shimizu 1990, Endo et al. 1997) but according to Zhou et al. (2005) embedding in agarose is preferential, as they found plating efficiencies to be positively correlated with increasing agarose content. Our results are opposed to the latter findings; Zhou et al. (2005) probably have used a genotype that tolerates the accumulation of toxic breakdown products of dead cells very well, as they also included a nursing cell suspension in their culture system. Sauvadet et al. (1990) favoured a liquid system, but did so without quantifying or attempting to explain its benefits compared to a semisolid system. Lindsay and Ledger (1993) and Furuta et al. (2004) applied a solid/liquid system exclusively. Our results suggest that their protocols might increase in efficiency by switching to a liquid culture system.
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Liquid culture and culture near glass beads both have a significant positive effect on the development of calli from protoplasts of the test cultivar ‘Yoko Ono’. However, the manipulation of relatively large volumes is more feasible when using liquid cultures. This makes it far more efficient. As protoplast regeneration in Chrysanthemum has been found to be genotype dependent, we will now begin to evaluate the system for several different genotypes. The glass bead system may be preferable for recalcitrant genotypes. We will also study possible long-term effects with regard to callus regeneration in more detail. Acknowledgments The authors wish to thank Ronald Van Den Oord for his technical assistance.
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