Bulletin UASVM Horticulture, 66(1)/2009 Print ISSN 1843-5254; Electronic ISSN 1843-5394 In Vitro Multiplication, Conservation and Ex Vitro Acclimation of Drosera rotundifolia Doina CLAPA 1), Alexandru FIRA 1), Ioan PACURAR 2) 1) Fruit Research Station Cluj, 5 Horticultorilor St., Cluj-Napoca 400457, Romania; www.scdpcluj.ro, doinaclapa@yahoo.com 2) University of Agricultural Sciences and Veterinary Medicine, 3-5 Manastur St., Cluj-Napoca 400372, Romania Abstract. At the Fruit Research Station of Cluj experiments were carried out for the optimization of in vitro proliferation of Drosera rotundifolia as well as for the in vitro conservation, in vitro rooting and ex-vitro acclimation of this species. For proliferation, modified Murashige & Skoog (MS) media containing either 200 ml/l coconut water or 5 mg/l Kinetin proved to be optimal. BAP in the media proved not to be suitable. For in vitro conservation a double layer hormone-free MS medium was used, which ensured the long-term storage and in vitro proliferation of Drosera rosettes. For in vitro rooting, two variants of MS medium were used, one with macroelements diluted to ¼ and the other with macroelements diluted to ½. Both variants proved to be suitable for in vitro rooting and increasing plantlet size; they provided 100 % rooting percentage and there were no significant differences between the two variants regarding the biomass of the resulting plantlets. Ex-vitro acclimation was done most efficiently by hydroculture, in uncovered trays containing water with neutral ph. Keywords: sundew, micropropagation, hydroculture, conservation INTRODUCTION Drosera rotundifolia - round leafed sundew is one of the few species of carnivorous plants that can be found also in Romania especially in acidic peat bogs in the mountains. It is protected by law, most of its areal is part of natural reservations. It has weak roots and the leaves are grouped in a basal rosette. The petiole is long and the lamina is covered with hairs. The hairs, also termed tentacles, secrete sticky substances that gather in the form of some sparkling droplets, from which the name of the plant originates. An insect that sits on a leaf of this plant gets stuck in the sticky secretion and, by trying to free itself it gets stuck to more hairs. The hairs entangle the insect and secrete an abundant sticky juice that suffocates the insect. Then the insect is digested by certain proteolytic enzymes in a few days, so that only the skeleton remains. One plant consumes approximately 50 insects/year. In late spring and early summer the pentamere, pinkish-white or violet flowers appear, forming a scorpioid inflorescence. Recently some Drosera species became important also for pharmaceutical purposes as they contain plumbagin; and for molecular biology, as they contain genes for important enzymes. Plant tissue culture can provide a means for the conservation, and propagation of this species and the ex-vitro acclimated plantlets could be used for repopulating its natural habitats. Plant tissue culture could also provide the means for obtaining large amounts of 34
biomass for plumbagin production by using bioreactors. Research regarding tissue culture of several Drosera species was done by a few researchers, such as: Bekesiova et al., (1999), Cachiţă-Cosma et al.,(2008), Ichiishi et al.,(1999), Jayaram et al.,(2009), Sauerwein et al., (1994), Turcuş, (2009). Having an unusual, interesting shape and beautiful colours, Drosera rotundifolia can also be used as an ornamental. At the Fruit Research Station of Cluj a protocol was optimized for the in vitro propagation and ex-vitro acclimation of Drosera rotundifolia. MATERIALS AND METHODS In our study the experiments were started from Drosera rotundifolia cultures already available in vitro. For the in vitro multiplication experiments Magenta vessels a modified MS medium was used (Table 1, variant 0), to which various growth regulators were added (Table 2). The coconut water used in the experiments resulted from ripe fruit. All the components were added to the media before autoclavation. The media were poured into Magenta GA7 vessels (cca 50 ml/vessel) and autoclaved at 121 C for 20 minutes. For the proliferation experiments, 5 rosettes ca 2 cm in diameter were inoculated into each vessel. For the rooting experiments, 9 rosettes of the same size were inoculated into each vessel. The cultures were kept at cca 300 lux and 16-hour photoperiod, at temperatures of 23-26 C in the growth chamber. Both for in vitro multiplication and rooting the cultures were kept for 2 months. For the rooting experiments, 2 variants of MS medium were used (Table 1, Variants 1 and 2) as well as liquid MS medium with perlite as solid support. In the latter case, cca 10 g of perlite+100 ml MS medium were used/magenta vessel For conservation, liquid MS medium (cca 100 ml/vessel) was poured onto Drosera rotundifolia cultured in 2 Magenta vessels in solid, hormone-free MS medium). Tab. 1 The media used for Drosera maintenance and rooting Component Variant 0 Variant 1 Variant 2 MS macroelements Full concentration Diluted to 1/4 Diluted to la 1/2 MS microelements Full concentration Full concentration Full concentration FeNaEDTA 36.7 mg/l 36.7 mg/l 36.7 mg/l Myo-inositol 100 mg/l 100 mg/l 100 mg/l Vitamin B1 1 mg/l 1 mg/l 1 mg/l Vitamin B6 0.5 mg/l 0.5 mg/l 0.5 mg/l Nicotinic Acid 0.5 mg/l 0.5 mg/l 0.5 mg/l Sugar 30 g/l 30 g/l 30 g/l Plant Agar (Duchefa) 6 g/l 6 g/l 6 g/l PH adjusted to 5.8 35
The experimental variants usedfor proliferation and the resulting multiplication rate Tab. 2 Variant Mediul nutritiv Vessel no. 1 Hormone-free MS 1 169 2 140 No. of rosettes resulted/vessel Rosettes resulted/ vessel 154.5 30.9 Multiplication rate (rosettes resulted/inoculm 2 Hormone-free MS without agar 3 MS+ 100 ml/l coconut water 4 MS+ 200 ml/l coconut water 1 107 2 114 1 219 2 243 1 271 2 209 110.5 22.1 231 46.2 240 48 5 MS+ 2 mg/l kinetin 1 168 168 33.6 6 MS+ 5 mg/l kinetin 1 256 256 51.2 7 MS+ 10 mg/l kinetin 1 154 154 30.8 8 MS+ 0.5 mg/l BAP 1 183 171.5 34.3 2 160 9 MS+3 mg/l BAP+0.2 mg/l kinetin 1 No viable plant For ex-vitro acclimation, several variants were tested: plastic trays and pots containing peat, perlite, peat+perlite in 1:1 volume to volume ratio, peat+soil, perlite+soil. Acclimation in hydroculture was also tested; for this purpose plastic trays and glass jars containing a layer of 2-3 cm of tap water from Cluj-Napoca with the ph=7 were used. The plant material used on the solid substrates consisted in Drosera plantlets cultured on full-strength MS medium (Tab. 1, Variant 0). In hydroculture, plantlets cultured on all 3 variants were tested. RESULTS AND DISCUSSION In the proliferation stage, the highest multiplication rate was provided by variant 4 (MS+ 200 ml/l coconut water), but without great differences from variant 3 (MS+ 100 ml/l coconut water). MS+ 5 mg/l kinetin also provided a high proliferation rate. BAP did not increase multiplication rate very significantly and the resulting rosettes were rather deformed. Hormone-free MS gelled with agar provided a higher proliferation rate and better developed plantlets in comparison with liquid MS (Tab. 2 ) The rooting percentage was of 100 % on full-strength hormone-free MS medium (Table 1, variant 0) as well as the variants tested for rooting (Variants 1 and 2). On Variant 0 the plantlets proliferated and rosette sizes were much smaller than those on Variants 1 and 2, where there was weak proliferation and the plantlets grew larger, with robust leaves covered 36
with well-developed tentacles, each bearing a red droplet of digestive secretion. The green biomass of the Drosera plantlets grown on the rooting media was remarkably high (Fig. 1) and the two rooting variants gave remarkably similar results. The Drosera plantlets also rooted on liquid hormone-free MS medium containing perlite as substrate; rooting percentage was also 100 %, but the plantlets were less robust than in media gelled with agar (Fig. 2). a. b. Fig. 1. Green biomass of Drosera rotundifolia on the rooting media: a) on MS with macroelements diluted to ¼; b) on MS with macroelements diluted to ½; a b c d Fig 2. Drosera in vitro: a) and b) on modified MS (Table 1, Variant 1); c) on modified liquid MS + perlite; d) on full-strength liquid MS For conservation (Fig. 3), liquid MS medium (cca 100 ml/vessel) was poured onto Drosera rotundifolia cultured in Magenta vessels in solid, hormone-free MS medium)for 2 months. After another 9 months in culture the plantlets were still alive, resulting a mass of abundantly proliferated plantlets. The results regarding the number of rosettes harvested from the 2 vessels and plantlet biomass are presented in Table 3. Drosera acclimation (Fig. 4) failed in all the experiments using solid substrate for acclimation, except for the plastic pots containing perlite, where the plantlets were covered with glass jars. In the latter case, the plantlets were acclimated in 3 months, with a survival rate of cca 1/3. 37
Number and biomass of plantlets during conservation Tab. 3. Vessel no. No. of resulting plantlets Total biomass (g) Biomass/plantlet (g) 1 625 65.48 0.1047 2 942 79.88 0.0847 Average 783.5 72.68 0.0947 Fig. 3. Drosera conservation on double layer medium Drosera plantlets cultured on full-strength MS medium could not be acclimated in hydroculture; mortality rate was of 100 %. Acclimation in hydroculture was entirely successful in the case of Drosera plantlets cultured on modified MS media with low amounts of macroelements (Table 1, Variants 1 and 2), where survival rate was of 100 % during a 2-month culture period in all types of vessels used for acclimation (plastic trays, glass jars), the plantlets grew during the acclimation phase, they flowered and slightly proliferated. The leaves became visibly more robust and the number and size of the tentacles containing droplets of digestive secretion increased. Fig. 4. Acclimated Drosera plantlets CONCLUSIONS Drosera rotundifolia is a rare, endangered and interesting species. It is an indicator of soil acidity and moisture. For the in vitro propagation of this species, several media proved to be suitable. Among the variants that we tested, modified MS medium containing coconut water proved to be suitable, but high multiplication rates were obtained on MS medium containing 5 mg/l Kinetin as well as on hormone-free MS gelled with agar. In vitro storage was achieved on double-layer MS medium, on which massive proliferation of the rosettes took place during the storage period. In vitro rooting was successful on all the variants of hormone-free MS medium and ex- 38
vitro acclimation was achieved by using the technique of hydroculture, in the case of plantlets rooted on MS media with reduced amounts of macroelements. REFERENCES 1. Bekesiova, I., J. P. Nap and L. Mlynarova (1999). Isolation of High Quality DNA and RNA from Leaves of the Carnivorous Plant Drosera rotundifolia, Plant Molecular Biology Reporter 17: 269 277. 2. Cachiţă-Cosma, D., V. Turcuş, A. Petruş-Vancea, L. Barbutudoran and C. Crăciun (2008). Drosera Rotundifolia L. Vitroculture Associated with a Saprophyte Fungus, Sudia Universitatis, pp.103-105. 3. Ichiishi S., T. Nagamitsu, Y. Kondo, T. Iwashina, K. Kondo and N. Tagashira (1999). Effects of Macro-components and Sucrose in the Medium on in vitro Red-color Pigmentation in Dionaea muscipula Ellis. and Drosera spathulata Labill., Plant Biotechnology, 16 (3), 235-238. 4. Jayaram, K., and M. N. V. Prasad (2005). Rapidly in vitro multiplied Drosera as reliable source for plumbagin bioprospection, Current Science, Vol. 89, No. 3, pp 407-408. 5. Sauerwein M., S. Schmidt, J. Reichling and M. Wink (1994). Naphtoquinone production in in vitro cultures of Drosera communis St. Hil., BIOForum Extra, Prague, pp. 26-27. 6. Turcuş, V. (2009). Studii Morfo Studii anatomice si citologice efectuate la nivelul vitroplantulelor de Drosera Rotundifolia L., Teza de doctorat. 39