Plant Cell, Tissue and Organ Culture 77: 221–230, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 221 Review of Plant Biotechnology and Applied Genetics Micropropagation of Ranunculus asiaticus: a review and perspectives Margherita Beruto1,∗ & Pierre Debergh2 1 Istituto Regionale per la Floricoltura, Via Carducci 12, 18038 Sanremo (IM), Italy; 2 Department of Plant Sciences, Laboratory of Horticulture, Faculty of Agricultural and Applied Biological Sciences, University Gent, Coupure links 653, B-9000 Gent, Belgium (∗ requests for offprints: Fax: +39-184-542111; E-mail: beruto@regflor.it) Received 10 April 2003; accepted in revised form 12 November 2003 Key words: axillary bud stimulation, commercial production, micropropagation, Ranunculus asiaticus L., somatic embryogenesis Abstract Ranunculus asiaticus is an important ornamental species mainly cultivated in the countries surrounding the Mediterranean sea. So far the multiplication of this plant has been mainly carried out by seed and rhizome division; however, these systems present many drawbacks. Tissue culture is an attractive alternative for accelerated propagation of selected and indexed genotypes. In this paper we review the work carried out on in vitro culture of R. asiaticus and present a flow chart for its commercial production, using axillary budding and embryogenesis. Although the price of micropropagated plants is higher compared to traditional material (seedlings and rhizomes from seed populations), it should be considered that tissue cultured plants have a better rhizome yield per plant; moreover, the tissue culture approach allows to offer clonal material of selected lines. Abbreviations: 2,4-D – 2,4-dichlorophenoxyacetic acid; BA – N6 -benzyladenine; IBA – indole-3-butyric acid; MS – Murashige and Skoog medium (1962); NAA – 1-naphthaleneacetic acid Introduction The ornamental species Ranunculus asiaticus L. is quite important in the Mediterranean countries, but also in South Africa, California, Israel and Japan. Meynet (1993) estimated 30–50 ha are used in rhizome production, and 65 ha in cut flower production [France (31%), Italy (46%) and Israel (23%)]. During the last decade it gained in importance; 13 million ranunculus cut flowers were sold in the auctions of the Netherlands in 1994, 22 in 1999 and 34 in 2001 (Statistisch Jaarboek, 2001). Data recorded at the Flower Market of Sanremo, the most important market in Italy, show it is a leader product with a high percentage (about 84%) of dealers involved in its commercialisation. Ranunculus is normally grown from seeds, which yield rhizomes at the end of the first year. These rhizomes are graded and cultivated to produce flowers during the second year. Propagation by division of the tuberous roots is possible, but the annual multiplication rate is only 2–5. Moreover, different diseases caused by viruses (e.g., Cucumber Mosaic Virus (CMV); Tomato Spotted Wilt Virus (TSWV); Tobacco Necrosis Virus (TNV); Tobacco Rattle Virus (TRV) and different potyviruses) and by fungi (e.g., Fusarium oxysporum f.sp. ranunculi, Fusarium tabacinum, Pythium sylvaticum and Rhizoctonia sp.) are major factors requiring consideration in vegetative propagation (Meynet, 1993). To overcome the aforementioned problems, breeders developed seed lines of Ranunculus flowering the first year: sowing in September–October, harvesting flowers in March– April, and rhizomes are discarded at the end of the cultivation cycle (May–June). Although this system enables growers to start from healthy propagation 222 Figure 1. Micropropagation of R. asiaticus L. through axillary bud stimulation; in vitro multiplication (a) and rooting (b). Figure 2. Scheme for the micropropagation of R. asiaticus L. by standard protocol of axillary bud stimulation (Beruto et al., 1989). 223 Figure 3. Micropropagation efficiency of different R. asiaticus accessions multiplied through axillary bud protocol (Beruto et al., 1989). For 27 selected genotypes the following parameters were evaluated: percentage of contaminated (a) or non-developed (b) explants at the end of stage 1; nr of weeks to start the multiplication phase (c); mean multiplication rate recorded after 4 weeks in culture (d); percentage of in vitro rooting (e) and in vivo survival of plantlets (f). material (viruses are not transmitted through seed (Meynet, 1985)) and allows to grow the crop under glass as an annual, it prevents winter production, normally realised from stored rhizomes. Moreover, the breeding programmes for the selected seed lines is quite complex, and homogeneity of the populations is not guaranteed. Therefore tissue culture is an attractive alternative for accelerated propagation of selected and healthy genotypes. In this paper, we describe our research carried out over the past 15 years, enabling to develop appropriate micropropagation protocols of R. asiaticus and to bring clones onto the market. Particularly, two different methods of propagating Ranunculus via tissue culture have been considered: axillary bud stimulation and somatic embryogenesis. We also make reference to available relevant literature. 224 Figure 4. Figure 5. Figure 6. 225 In vitro culture of R. asiaticus L. Axillary shoot culture Axillary bud development has proven to be the most applied and reliable system for true-to-type in vitro propagation in general. Maia et al. (1973) produced virus-indexed ‘Barbaroux’ Ranunculus (a Turban Ranunculus clone) by meristem-tip culture, however, they faced major difficulties due to systematic bacterial contaminations. To overcome this problem Lercari et al. (1985) tried to improve the technique by using seed as starting material, and subsequent field selection of the most promising lines, while the material was still maintained in vitro. This system is still used in some commercial laboratories and is based on in vitro seed germination, their axillary multiplication till a satisfactory number of individuals is obtained, and subsequent rooting before evaluation under in vivo conditions. The most important bottleneck is the enormous amount of labour required in selecting clones with interesting agronomic characteristics; from the 264 in vitro germinated seedlings, 168 clones were micropropagated but only 29 (±17%) were finally selected. Moreover, the micropropagation procedure of the selected lines took 2 years before the required amounts (about 80–100 thousand plantlets per line) for commercial exploitation had been produced. During the subsequent subcultures and the weaning procedure, different problems occurred (physiological disorders, delay in flowering, reduction in flower diameter, lowered productivity, presence of abnormalities and somaclonal variation) (Lercari et al., 1985). ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 4. Effect of gels (a), plant density (b) and fructose (c) on development and multiplication of Ranunculus plantlets micropropagated through axillary bud stimulation. The effect of replacement of sucrose with fructose was evaluated by adding increasing amounts of fructose in the medium, while the same carbohydrate concentration (0.09 M) was maintained. For more details, refer to Beruto et al. (1999, 2001) and Beruto and Portogallo (2000). Figure 5. Three months under in vivo conditions, plantlets micropropagated by axillary budding (right) show a more advanced development and an increased number of shoots with respect to seedlings of the same age (left) (Beruto et al., 1996a). Figure 6. Rhizomes collected at the end of the cultural cycle of micropropagated plantlets. The acclimatisation period and the genotype affect the number of rhizomes that can be harvested from each ex vitro plantlet (Beruto et al., 1996a). Because of the labour intensive character of the aforementioned approach, we developed a reliable micropropagation system by axillary budding, starting from selected adult mother plants (Beruto et al., 1989). To our knowledge, this is the only micropropagation protocol available for commercial propagation of selected plants of R. asiaticus. Apical buds (about 0.5 cm) are cultured on basic MS-medium (Murashige and Skoog, 1962) with a combination of the growth regulators BA, kinetin and NAA (Figures 1a and 2); subsequently the plantlets are easily rooted on the same MS-medium supplemented with IBA (Figures 1b and 2) and acclimatization under in vivo conditions can be successfully accomplished for a large number of genotypes (Figure 2). In Figure 3, presenting the results for 27 selected lines, it is clearly illustrated there are significant differences among lines; the percentage of contaminated or not developing explants scored at the end of stage 1 varied considerably. Further experiments showed that the physiological status of the mother plant and the time of the year the explant was sampled greatly influenced the success rate in stage 1: explants taken 1 month before flower opening were most appropriate for successful establishment. The multiplication stage started 8–20 weeks after initiation. The duration of stage 2, as well as the rate of multiplication, was genotype dependent. However, apart from the genotype, many other factors were of considerable importance for successful micropropagation. Among others, the water status and availability of nutrients (Beruto, 1997; Beruto et al., 2001), the type of gelling agent (Figure 4a; Beruto et al., 1999, 2001), the type of sugar, plant density, frequency of subculture, and ventilation of the tissue culture container (Beruto and Portogallo, 2000). Appropriate plant density was found to be 10–15 shoots/vessel (Figure 4b) [320 ml Meli jars (De Proft et al., 1985) filled with 100 ml of gelled multiplication medium]. Subculturing on fresh medium every 3 weeks resulted in improved fresh and dry weight gain, and it also improved the quality of the shootlets (Beruto and Portogallo, 2000), but the propagation ratio was improved by subculturing every 4-weeks (Beruto, 1997). Full or partial replacement of sucrose by fructose reduced the impact of chlorotic leaves (Figure 4c), improved shoot length, and leaf number when compared to the control (only sucrose in the medium). When considering the aforementioned most appropriate conditions, each shoot produced 1.5–4.5 new shoots every 4 weeks depending on genotype (Figure 3). Under field conditions those plants grew vigorously (Beruto et al., 226 1996a), and 3 months after planting they surpassed seedlings of the same age (Figure 5): increased number of shoots, slightly earlier and homogeneous flowering and, at the end of the culture cycle, they yielded more than one rhizome, although the rhizome production per plant was genotype-dependent (Figure 6). The collected rhizomes sprouted regularly and their flowering was homogeneous (Figure 7). No phenotypic differences were recorded (however, appropriate selection remains advisable for each micropropagated genotype). Continuous subculturing (more than 12–22 months in culture, depending on the genotype) leads to a lowered flowering percentage and some abnormalities (plant vigour, increased percentage of aberrant stems and flowers) in some lines. An integrated production schedule is presented in Figure 8. To guarantee a healthy production scheme, rigorous controls must be integrated to test the phytosanitary status of the mother plants grown as ‘nuclear stock’ in insectproof greenhouses (Figure 8). After micropropagation, plantlets were transferred to soil, and subsequently the production phase could take place (Figure 8). When the process addresses the production of rhizomes, which are subsequently used by the growers, the ex vitro plantlets are grown under controlled conditions to prevent any possible infection and pooled controls are carried out during the whole process (Figure 8). The results of our research are now used for commercial propagation. Somatic embryogenesis Figure 7. Ranunculus asiaticus plantlets micropropagated by axillary budding: homogeneous flowering and no phenotypical variations. Different authors documented that Ranunculus can be regenerated in vitro from different tissue sources (Table 1). Apart from the work presented by Pugliesi et al. (1992) which describes the callus induction and adventitious shoot regeneration from seedling tissues (system not useful for commercial application due to the use of an unknown genotype), Ranunculus regenerations have been reported by using different floral organs (Meynet and Duclos, 1990a, b, c; Beruto and Debergh, 1992; Beruto et al., 1996a, b). In our work, we have developed a system for somatic embryogenesis from thalamus derived calli of R. asiaticus, which can be used for mass clonal propagation, although careful evaluation is required to evaluate true-to-typeness (Beruto and Debergh, 1992). The starting material for this protocol is the thalamus of immature flower buds. The choice of an appropriate developmental stage of the thalamus, and the period of the year in which explants are taken can influence the further development of the cultures. Thalamus portions collected from immature floral buds (±8 mm), 1 month before anthesis and maturity, are suitable primary explants; the pale-yellow immature anthers are removed together with petals and sepals and the thalamus portion above the stamen insertion is used as explant (Beruto, 1997). Calli are initiated and maintained on MS-medium supplemented with different concentrations of 2,4-D and cytokinins (BA or kinetin). Callus formation and subsequent embryo regen- 227 Figure 8. Propagation scheme for R. asiaticus through in vitro techniques. Details are given on the different steps of production and the operations to be carried out in the nursery to ensure that healthy material is obtained. eration are observed after an average culture period of 150 days. The genotype and the inoculated thalamus portion, as well as the medium, can greatly influence the response; in particular, the percentage of regenerating calli, the time after which regeneration can occur, and the number of somatic embryos produced. Callus induction and proliferation depend on the thalamus tissue used (apical, medial and basal); more abundant callus growth was observed by culturing the apical tissues. The percentage of regenerating calli, the time along which regeneration occurs, and the number of somatic embryos obtained strictly depend on the 2,4-D concentration, when this hormone was supplied in the absence of any other growth regulator. In particular, explants on a higher 2,4-D concentration (7.2 µM) gave more embryoids per callus in a shorter time (150 days from the culture beginning) than on lower 2,4-D (3.6 µM or less); but the percentage of regenerating calli is lower (±28% instead of 80%) (Beruto and Debergh, 1992). The absence of growth regulators had a negative effect on thalamusderived callus growth; 2,4-D was an important factor 228 Table 1. Summary of the literature on adventitious regenerations of R. asiaticus cultured in vitro (direct regeneration (D) or through callusing (C)) Explant Medium (µM) Mode of development Response Reference Thalamus MS + BA 2.2 µM + 2,4-D 0.45 µM; MS + BA 2.2 µM + 2,4-D 0.45 µM + NAA 2.68 µM MS (half strength of macroelements) + BA 2.2 µM + 2,4-D 0.45 µM MS + NAA 5.4 µM + KIN 4.6 µM; MS + NAA 5.4 µM + KIN 23.2 µM MS + 2,4-D 7.2 µM; MS + 2,4-D 0.92 µM + BA 4.4 µM D, C Adventitious shoots Meynet and Duclos (1990a) D, C Somatic embryos Meynet and Duclos (1990b) C Adventitious shoots Pugliesi et al. (1992) C Somatic embryos Beruto and Debergh (1992); Beruto et al. (1996a, b) Anther Cotyledon Thalamus to stimulate callus growth and an appropriate balance between this auxin and glutamine (0.3–3.4 mM) was responsible for an improved production of embryogenic calli (Beruto, 1997). However, best results (% of embryogenic calli) were obtained by supplying a low concentration of 2,4-D (<3.6 µM) and a cytokinin. It is proven that BA is an important factor to stimulate callus growth and regeneration in Ranunculus, while kinetin is detrimental (Beruto et al., 1996b). Best callus growth and embryogenesis (100% embryogenic calli with ±55 somatic embryos regenerated from each callus culture (Beruto et al., 1996b)) were observed for calli initiated on MS-medium supplemented with 2,4-D 0.9 µM, BA 4.4 µM and glutamine 0.68 mM (Figure 9). Somatic embryos developed into complete plants when callus was transferred to MS-medium Figure 9. Somatic embryogenesis in R. asiaticus on thalamus derived calli. without hormones; however, a variable percentage (±20%) of abnormal embryos, which did not convert, was observed. Further studies are in progress to avoid or to select against these abnormalities when subculturing. In literature it is well documented that a major limitation of the extensive use of somatic embryogenesis is somaclonal variation. Indeed, also for Ranunculus, Meynet and Duclos (1990c) reported that anther-derived embryos presented somaclonal variation to a large extent, leading to new, stable characteristics, which can be sexually transmitted as nuclear mutations. When plantlets were regenerated from thalamus tissues, true-to-typeness was the rule, but abnormalities cannot be excluded (Beruto et al., 1996a), and virus eradication by this system is not guaranteed (Meynet and Duclos, 1990a). The developmental stage of the in vitro cultured thalamus tissues, the hormonal balance, and the genotype can influence the appearance of abnormalities. The most common deviations concerned were: leaf morphology, plant habit and flowering delay. These aberrations could persist for different cultural cycles as permanent or they were only observed during the first cultural cycle as transitional (plant vigour and high percentage of aberrant leaves) (Beruto et al., 1996a). Further investigations are required to evaluate if somatic embryogenesis is a suitable alternative for mass propagation. When comparing the time schedule and productivity of the studied embryogenic and axillary protocols, it is evident that with the embryogenic pathway the propagation ratio is 3–4 times higher than with axillary bud stimulation. Moreover, a considerable saving of labour and time is achieved by somatic embryogenesis (Figure 10). 229 Figure 10. Scheme for the micropropagation of R. asiaticus L. by standard protocol for somatic embryogenesis (Beruto et al., 1996b). Prospects: economics importance of micropropagation Ranunculus asiaticus is wanted as cut flower, pot or border plant. The area of production is not limited to the countries surrounding the Mediterranean sea; other cooler oceanic climates are also suited. Compared to seedlings, rhizome propagation guarantees earlier and more plentiful flowering. For this reason, appropriate vegetative propagation of selected genotypes can be efficient to introduce performant varieties. Tissue culture techniques are an attractive alternative for rapid clonal multiplication. Although the cost price of in vitro produced plants (±0.45 Euros) is higher than that of traditional propagation units [seedlings (0.14– 0.17 Euros) and rhizomes (0.29–0.34 Euros)], many benefits can be envisaged. Micropropagation enables to supply growers with more performant and healthy genotypes and a better production schedule can be envisaged. Moreover, Ranunculus clones provide growers with a ‘new product’ which can be marketed as a novelty. A similar situation has already been witnessed in the past: nowadays, commercially well known plants (orchids, gerbera, marantha. . .) became commercially important because of micropropagation techniques. Micropropagated Ranunculus can be directly commercialised or used as mother plants for subsequent rhizome propagation under controlled conditions. Our research work allowed commercial com- panies to produce several hundreds of thousands of micropropagated plantlets and the reaction on the market has been very positive. Further prospects of the research will focus on the reduction of the costs of the tissue-cultured plants by both optimising the axillary bud propagation protocol and implementing the studies on somatic embryogenesis for a wider number of cultivars with the aim of distributing the obtained somatic embryos to several private companies. Acknowledgements This work was supported by the Regional Institute for Floriculture of Sanremo, Italy. The authors thank Miss C. Portogallo for her helpful technical assistance. References Beruto M (1997) Agar and gel characteristics with special reference to micropropagation systems of Ranunculus asiaticus L. PhD Thesis, Gent, Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, University Gent, Belgium, pp. 21–44, 125–184 Beruto M & Debergh P (1992) Somatic embryogenesis in Ranunculus asiaticus L. hybr. thalamus cultivated in vitro. Plant Cell Tiss. Org. 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