In vitro propagation of Helleborus species

Emmy Dhooghe; Marie-christine Van Labeke

In vitro propagation of Helleborus species

Plant Cell, Tissue and Organ Culture, 2007

By Emmy Dhooghe and Marie-christine Van Labeke

RESEARCH NOTE In vitro propagation of Helleborus species Emmy Dhooghe Æ Marie-Christine Van Labeke Received: 27 October 2006 / Accepted: 2 August 2007 / Published online: 25 September 2007 Ó Springer Science+Business Media B.V. 2007 Abstract A generalin vitro cloning system was established for four Helleborus species: H. argutifo- lius, H. foetidus, H. niger and H. orientalis. The plant material was introduced in vitro from axillary buds. A Murashige and Skoog (MS)—basedmedium (Murashige and Skoog 1962) was used supplemented with 2% (w/v) sucrose, 2-isopentenyladenine (2-iP) and 6-benzylaminopurine (BA). Multiplication rates depended on the genotype and varied from 1.3 for H. foetidus till 3.8 for H. niger. The ﬁrst results showed that the rooting phase could be done ex vitro. Rooting was induced by a drench for one week in a solution of indole-3-butyric acid (IBA -3 mg l –1 ) and 1-naph- thaleneacetic acid (NAA-1 mg l –1 ) at 5°C. Keywords Argutifolius Axillary bud Foetidus Niger Orientalis Ranunculaceae Abbreviations BA 6-benzylaminopurine IBA Indole-3-butyric acid 2-iP 2-isopentenyladenine MS Murashige and Skoog salts NAA 1-naphtaleneacetic acid NOA Naphthoxyacetic acid PPM TM Preservative for Plant Tissue Culture Media Helleborus is an early spring ﬂowering perennial and memberof the family ofthe Ranunculaceae. The most well knownspeciesare H. niger L. and H. orientalis Lam. (Rice and Strangman 1993). There is an increasinginterestfor these speciesin ornamental horticulture, but Helleborus species have also large potentials as medicinal plants (Bu ¨ssing and Schweizer 1998; Watanabe et al. 2005).To explore these advantages an eﬃcient propagation system fo Helleborusspeciesiscrucial.Unfortunately, until now there isno easy way to multiply Helleborus (Seyring 2002). Methods ofgenerative propagation are limited sincethe degreeof variation in the oﬀspringis oftentoo high. Moreoverthe seeds require several monthsto germinate after sowing (Niimi et al. 2006). Vegetative propagation methods by means of division are time-consuming and cannot always guarantee a successful multiplication rate. In vitro methodscould providea solutionfor this species. However, information of in vitro propagation of Helleborus is scarce and often missing crucial details forsuccess. The choice ofthe explants for initiation of an aseptic culture is important. Starting in vitro propagation with rhizome buds lead to a high degree ofpathological contamination due to their subterranean origin (Seyring 2002). Successful E. Dhooghe (&) M.-C.Van Labeke Department of Plant Production, Genth University, Coupure links 653, 9000 Gent, Belgium e-mail: Emmy.Dhooghe@UGent.be 123 Plant Cell Tiss Organ Cult (2007) 91:175–177 DOI 10.1007/s11240-007-9280-x

in vitro protocols for H. niger were initiated from seedlings (Seyring 2002) or from meristem tip culture (Onesto et al. 2004).In an attempt to increase the eﬃciency of propagation and to reduce the variation in the oﬀspring we searched for an in vitro propa- gation system using axillary buds as initial explants. This system was tested for four Helleborus species: H. argutifolius Viv., H. foetidus L., H. niger L.and H. orientalis Lam. In H. niger and H. orientalis the buds are located at the base of the plant more speciﬁc at the shoot-root transition zone at the soillevel, while the buds of H. foetidus and H. argutifolius can be taken on the elongated stem, because the inter- nodes are stretched. Plant material of the four Helleborus species was cultivated in the greenhouse. To circumvent conta- mination water was only supplied to the pot and no pestswere tolerated. The plantmaterial used was 2 years old for H. niger and H. orientalis, only 1-year- old for H. argutifoliusand H. foetidus. After the outgrowth of 2–3 newly formed leaves, the in vitro initiation could start. The leaves and the roots were cut oﬀ. Since Helleborus is very susceptible for contam- ination,a disinfection procedure of three steps was generated. First the unleaved upper part was washed thoroughly with tap water and subsequently a 15% Haztab TM solution (resulting in an available chlorine content of 0.26%) with Tween1 was used for 5 min. Aftera rinse with sterile distilled water and a ﬁrst furtherdissection oftheplantmateriala second disinfection step was performed using HgCl 2 (0.5%) and Tween1 for3 min.Afterrinsing the material with sterile water the last disinfection step, a drench of 15 min in a 7% Haztab TM solution (resulting in an available chlorine content of 0.12%) with Tween1 was done. Subsequently, the plant material was rinsed three times and then axillary buds were dissected. The species yielded a diﬀerent number of isolated buds per plant (Table 1).Before in vitro introduction the length of the excised buds was measured (Table 1). These axillary buds were inoculated on an adapted MS medium adjusted to a pH of 5.8 and enriched with 500 mg l –1 Cagluconatemonohydrate, 2% sucrose,0.7% agar,3 ml l –1 PPM, 5 mg l –1 2-iP and 0.2 mg l –1 BA. The axillary budswere incu- batedin a climateroom (16 h light/8 hdark, 60 lmol m –2 s –1 ) at 21°C. After 7–14 weeks upon the in vitro initiation the explants were transferred to a multiplication medium (pH 5.8),which was a supplemented MS medium with 500 mg l –1 Ca gluconate monohydrate, 2.5 mg l –1 riboﬂavin, 3% sucrose, 0.8% agar, 1 ml l –1 PPM, 2 mg l –1 2-iP, 0.1 mg l –1 NOA and 5 mg l –1 BA. The choice of the multiplication medium with hormones was based on a preliminary test where the multipli- cation ratefor H. argutifoliusafter6 weekswas superior on a medium with hormones (2 mg l –1 2-iP, 0.1 mg l –1 NOA and 5 mg l –1 BA) (1.7 ± 0.3) com- pared to the same medium withouthormones (1.2 ± 0.1). As a coolclimate enhances leaf forma- tion,the plantlets were then transferred to a climate room (day/night period of 16 h/8 h, 60 lmol m –2 s –1 ) at a temperature of 14–16°C.Due to the severe sterilization procedure, which was essential to obtain an asepticin vitro culture,a high percentage of axillary buds did not survive (Table 2).After 8 weeksthe multiplication rates were counted for the four Helleborus species, which were now vigo- rously growing (Fig. 1). The multiplication rate was signiﬁcantly higher for H. niger than for H. argutifo- lius,H. foetidus and H. orientalis (Table 2). After the in vitro multiplication, the plantlets were adapted to an in vivo environment. This Table 1 Number and length of the buds per species isolated for in vitro inoculation Species Number of buds/plant* (n = 10) Length of dissected bud* (mm) (n = 15) H. argutifolius 7.7 ab 5.1 a H. foetidus 13.4 ab 3.1 a H. niger 5.7 b 3.9 a H. orientalis 16.3 a 5.5 a *Tukey test:meansdenoted by thesameletterwerenot signiﬁcantly diﬀerent (P \ 0.05) Table 2 Survival and multiplicationrate of Helleborus species Species n Survival rate* (%) Multiplication rate* H. argutifolius 37 53.3 a 2.0 b H. foetidus 92 43.3 a 1.3 b H. niger 51 58.7 a 3.8 a H. orientalis 246 32.1 a 2.0 b *Tukey test:meansdenoted by thesameletterwerenot signiﬁcantly diﬀerent (P \ 0.05) 176 Plant Cell Tiss Organ Cult (2007) 91:175–177 123

Plant Cell Tiss Organ Cult (2007) 91:175–177 DOI 10.1007/s11240-007-9280-x RESEARCH NOTE In vitro propagation of Helleborus species Emmy Dhooghe Æ Marie-Christine Van Labeke Received: 27 October 2006 / Accepted: 2 August 2007 / Published online: 25 September 2007 Ó Springer Science+Business Media B.V. 2007 Abstract A general in vitro cloning system was established for four Helleborus species: H. argutifolius, H. foetidus, H. niger and H. orientalis. The plant material was introduced in vitro from axillary buds. A Murashige and Skoog (MS)—based medium (Murashige and Skoog 1962) was used supplemented with 2% (w/v) sucrose, 2-isopentenyladenine (2-iP) and 6-benzylaminopurine (BA). Multiplication rates depended on the genotype and varied from 1.3 for H. foetidus till 3.8 for H. niger. The first results showed that the rooting phase could be done ex vitro. Rooting was induced by a drench for one week in a solution of indole-3-butyric acid (IBA -3 mg l–1) and 1-naphthaleneacetic acid (NAA-1 mg l–1) at 5°C. Keywords Argutifolius Axillary bud Foetidus Niger Orientalis Ranunculaceae Abbreviations BA 6-benzylaminopurine IBA Indole-3-butyric acid 2-iP 2-isopentenyladenine MS Murashige and Skoog salts NAA 1-naphtaleneacetic acid E. Dhooghe (&) M.-C. Van Labeke Department of Plant Production, Genth University, Coupure links 653, 9000 Gent, Belgium e-mail: Emmy.Dhooghe@UGent.be NOA PPMTM Naphthoxyacetic acid Preservative for Plant Tissue Culture Media Helleborus is an early spring flowering perennial and member of the family of the Ranunculaceae. The most well known species are H. niger L. and H. orientalis Lam. (Rice and Strangman 1993). There is an increasing interest for these species in ornamental horticulture, but Helleborus species have also large potentials as medicinal plants (Büssing and Schweizer 1998; Watanabe et al. 2005). To explore these advantages an efficient propagation system for Helleborus species is crucial. Unfortunately, until now there is no easy way to multiply Helleborus (Seyring 2002). Methods of generative propagation are limited since the degree of variation in the offspring is often too high. Moreover the seeds require several months to germinate after sowing (Niimi et al. 2006). Vegetative propagation methods by means of division are time-consuming and cannot always guarantee a successful multiplication rate. In vitro methods could provide a solution for this species. However, information of in vitro propagation of Helleborus is scarce and often missing crucial details for success. The choice of the explants for initiation of an aseptic culture is important. Starting in vitro propagation with rhizome buds lead to a high degree of pathological contamination due to their subterranean origin (Seyring 2002). Successful 123 176 in vitro protocols for H. niger were initiated from seedlings (Seyring 2002) or from meristem tip culture (Onesto et al. 2004). In an attempt to increase the efficiency of propagation and to reduce the variation in the offspring we searched for an in vitro propagation system using axillary buds as initial explants. This system was tested for four Helleborus species: H. argutifolius Viv., H. foetidus L., H. niger L. and H. orientalis Lam. In H. niger and H. orientalis the buds are located at the base of the plant more specific at the shoot-root transition zone at the soil level, while the buds of H. foetidus and H. argutifolius can be taken on the elongated stem, because the internodes are stretched. Plant material of the four Helleborus species was cultivated in the greenhouse. To circumvent contamination water was only supplied to the pot and no pests were tolerated. The plant material used was 2 years old for H. niger and H. orientalis, only 1-yearold for H. argutifolius and H. foetidus. After the outgrowth of 2–3 newly formed leaves, the in vitro initiation could start. The leaves and the roots were cut off. Since Helleborus is very susceptible for contamination, a disinfection procedure of three steps was generated. First the unleaved upper part was washed thoroughly with tap water and subsequently a 15% HaztabTM solution (resulting in an available chlorine content of 0.26%) with Tween1 was used for 5 min. After a rinse with sterile distilled water and a first further dissection of the plant material a second disinfection step was performed using HgCl2 (0.5%) and Tween1 for 3 min. After rinsing the material with sterile water the last disinfection step, a drench of 15 min in a 7% HaztabTM solution (resulting in an available chlorine content of 0.12%) with Tween1 was done. Subsequently, the plant material was rinsed three times and then axillary buds were dissected. The species yielded a different number of isolated buds per plant (Table 1). Before in vitro introduction the length of the excised buds was measured (Table 1). These axillary buds were inoculated on an adapted MS medium adjusted to a pH of 5.8 and enriched with 500 mg l–1 Ca gluconate monohydrate, 2% sucrose, 0.7% agar, 3 ml l–1 PPM, 5 mg l–1 2-iP and 0.2 mg l–1 BA. The axillary buds were incubated in a climate room (16 h light/8 h dark, 60 lmol m–2 s–1) at 21°C. After 7–14 weeks upon the in vitro initiation the explants were transferred to a multiplication medium 123 Plant Cell Tiss Organ Cult (2007) 91:175–177 Table 1 Number and length of the buds per species isolated for in vitro inoculation Species Length of dissected bud* (mm) (n = 15) Number of buds/plant* (n = 10) 7.7 ab H. argutifolius H. foetidus H. niger H. orientalis 5.1 a 13.4 ab 3.1 a 5.7 b 3.9 a 16.3 a 5.5 a *Tukey test: means denoted by the same letter were not significantly different (P \ 0.05) (pH 5.8), which was a supplemented MS medium with 500 mg l–1 Ca gluconate monohydrate, 2.5 mg l–1 riboflavin, 3% sucrose, 0.8% agar, 1 ml l–1 PPM, 2 mg l–1 2-iP, 0.1 mg l–1 NOA and 5 mg l–1 BA. The choice of the multiplication medium with hormones was based on a preliminary test where the multiplication rate for H. argutifolius after 6 weeks was superior on a medium with hormones (2 mg l–1 2-iP, 0.1 mg l–1 NOA and 5 mg l–1 BA) (1.7 ± 0.3) compared to the same medium without hormones (1.2 ± 0.1). As a cool climate enhances leaf formation, the plantlets were then transferred to a climate room (day/night period of 16 h/8 h, 60 lmol m–2 s–1) at a temperature of 14–16°C. Due to the severe sterilization procedure, which was essential to obtain an aseptic in vitro culture, a high percentage of axillary buds did not survive (Table 2). After 8 weeks the multiplication rates were counted for the four Helleborus species, which were now vigorously growing (Fig. 1). The multiplication rate was significantly higher for H. niger than for H. argutifolius, H. foetidus and H. orientalis (Table 2). After the in vitro multiplication, the plantlets were adapted to an in vivo environment. This Table 2 Survival and multiplication rate of Helleborus species Species n Survival rate* (%) Multiplication rate* H. argutifolius 37 53.3 a 2.0 b H. foetidus 92 43.3 a 1.3 b 51 58.7 a 3.8 a 246 32.1 a 2.0 b H. niger H. orientalis *Tukey test: means denoted by the same letter were not significantly different (P \ 0.05) Plant Cell Tiss Organ Cult (2007) 91:175–177 177 Fig. 1 Overview of the Helleborus species in vitro: H. argutifolius (a), H. foetidus (b), H. orientalis (c) and H. niger (d) acclimatization was done simultaneously with a rooting phase. The plantlets were incubated in a solution of IBA (3 mg l–1) and NAA (1 mg l–1) at 5°C to induce rooting. This lower temperature promotes root formation in vivo for this ornamental. One week later, shoots were transplanted in a peatsand substrate in an environment of 100% humidity. To prevent fungal growth a spray with Rovral1 (1 ml l–1) was done the first day after transplanting in vivo and was repeated after 1 week. Planted shoots were incubated for 12 weeks under 100% humidity, whereafter they were transplanted to normal greenhouse conditions. Eight weeks after the start of the acclimatization phase the plants were fertilized with Kristalon1 Blue (NPK + Mg 19:6:30 + 3) (0.5 g l– 1 ). The first preliminary results of the rooting phase showed that acclimatization was possible after a rooting phase ex vitro. In conclusion, we have described a protocol that has proven to be workable for the species H. argutifolius, H. foetidus, H. niger and H. orientalis. As the in vitro introduction of Helleborus has been considered as difficult, this is a promising beginning for developing a mass production system, although the multiplication ratio is not always as high. A further optimization of the protocol can be worked out to result in an efficient multiplication system for this ornamental. Acknowledgements The authors thank T. Versluys for the technical supports and ‘het Wilgenbroek’ (Oostkamp, Belgium) for the supply of high quality plants. References Büssing A, Schweizer K (1998) Effects of a phytopreparation from Helleborus niger on immunocompetent cells in vitro. J Ethnopharmacol 59:139–146 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plantarum 15:473–497 Niimi Y, Han DS, Abe S (2006) Temperatures affecting embryo development and seed germination of Christmas rose (Helleborus niger) after sowing. Sci Hortic 107:292– 296 Onesto JP, Cardin L, Poupet R et al (2004) Healthy in vitro propagation by meristem tip culture of Helleborus niger’ selected clone for cut flower. Paper presented at the 5th IVCHB symposium, Hotel Aranybika, Debrecen, 12–17 September 2004 Rice G, Strangman E (1993) The gardener’s guide to growing Hellebores. David & Charles, Devon Seyring M (2002) In vitro cloning of Helleborus niger. Plant Cell Rep 20:895–900 Watanabe K, Sakagami H, Mimaki Y (2005) Four new steroidal saponins from the rhizomes of Helleborus orientalis. Heterocycles 65(4):775–785 123

This study aimed to develop an efficient method for micrpropagation of true-to-type date palm plants through direct organogenesis. Nodular cultures were obtained from shoot tips on MS medium plus 2 mg/l 2ip and 1 mg/l NAA. Among combinations of 2ip and 2,4-D added to culture medium for direct shoot buds proliferation, 5 mg/l 2ip alone gave the highest organogenesis frequency. For in vitro multiplication, culture medium amended with 5 mg/l 2ip + 2 mg/l Kin gave the maximum shoot bud proliferation and shoot bud length. The response of three (1, 5, and 10 mg/l) concentrations of silver nitrate in the presence of 2ip regarding shoot bud multiplication was examined. Supplementation of culture medium with 5 mg/l silver nitrate was superior to the other concentrations used. For in vitro rooting, NAA (1 mg/l) was the best for in vitro root formation in comparison with IAA or IBA at same concentration. Acclimatization was achieved by transferring the plantlets into pots contained equal volumes of peat moss and vermiculite under high humidity. Tissue cultured plantlets were subjected to assessment of genetic stability using RAPD analyses. The patterns of DNA amplification showed a very high level of genetic similarity between regenerated plants and their mother plant. Introduction Date palm (Phoenix dactylifera L.) tree is one of the important fruit crops cultivated in arid and semi-arid regions. The tremendous advantages of the tree are its resilience, its requirement for limited inputs, its long-term productivity and its multiple purposes attributes (Bircher 1990). Due to the high degree of genetic heterozygosity and dioecious nature of date palm, sexual propagation method cannot be used for propagation the cultivars of interest in a true-to-type manner. It has to be propagated vegetatively by the offshoots which arise from the base of mother plants. This method of propagation is limited since mother plant produces 15 to 20 offshoots during palm's life, depending on the cultivar and this reduces considerably the expansion of the existing palm growers (Zaid, and de Wet 1999). One more disadvantage of this method is the spread of dangerous diseases and pests such as Bayoud disease or Red Palm Weevil which can be transported by contaminated offshoots. To satisfy the increasing demand of healthy date palm plants for national and international markets, it necessary to develop alternative methods of vegetative propagation to produce large numbers of disease-free plants from selected genotypes. The use of tissue culture is the most suitable approach for large-scale plant propagation of such recalcitrant crop. High quality and uniform planting material can be multiplied on a year-round basis under disease-free conditions anywhere irrespective of the season and weather. The date palm micropropagation process, like other large-scale plant propagation processes, carries a number of risks. Off-types, that is, non true-to type and genetically not identical to the mother plant, may be among the resulting plants (Moghaieb et al. 2011). Date palm is micropropagated by two main methods, the first is known as propagation by the somatic embryogenesis and the other is by forming auxiliary buds (organogenesis). Somatic embryogenesis method is characterized by giving in vitro plants in a comparatively, shorter time as well as high propagation ranges. But the most disadvantage of this method is the possibility of mutation and abnormalities occurrence during growing in vitro which appear in the field later on. In this respect, several attempts of date palm regeneration have been done based on somatic embryogenesis (Sharma et al. 1986; Madhuri and Shankar 1998; Bekheet et al. 2001). Conversely, the most advantage of micropropagation via organogenesis is getting in vitro plantlets highly identical in their genetic and vegetative characteristics with the mother plant (Tisserat, 1984; Abo El-Nil 1986; Aaouine 2000). The organogenesis technique consists of four steps: initiation of vegetative buds; bud multiplication; shoot elongation and rooting. The success of this technique is highly dependent on the success of the first step (Abahmane 2011). Hence, direct organogenesis presents an advantage of use of low concentrations of plant growth regulators and consequently callus-phase is avoided.

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In vitro propagation of Helleborus species