Propagation of Drosera rotundifolia and Drosera capensis in an in vitro Culture System 1* 1 Ileana MICLEA , Marius ZĂHAN 1 *Department of Animal Reproduction,University of Agricultural Sciences and Veterinary Medicine, 3-5, Mănăştur Street, 400372, Cluj-Napoca, Romania. Corresponding author, e-mail: [email protected] Bulletin UASVM Animal Science and Biotechnologies 74(2)/ 2017 Print ISSN 1843-5262; Electronic ISSN 1843-536X DOI:10.15835/buasvmcn-asb: 0018 ABSTRACT Drosera rotundifolia and Drosera capensis in vitro (Droseraceae) are carnivorous plants grown as ornamentals and sources for homeopathic medicine. The aim of this study was to optimize nutrient and growth regulator concentrations for the propagation of these species. Half strength MS medium (1/2MS) was supplemented with kinetin (0.5, 2, 5 mg/l) or 6-benzyladenine (3, 5 mg/l) and plantlets were transferred to 1/2MS with or without cytokinins. After 8 weeks rosette diameter, plant height, number of roots, root length were recorded and plants were cultured in fullD. strength rotundifolia MS, 1/2MS or 1/2MS with 0.5 mg/l α-naphthaleneacetic acid for the same period of time. Afterwards, plant characteristics (number of roots, root length, number of shoots, number of flower stalks) were assessed. For , shoot development and rosette diameter increased significantly in the medium with 0.5 mg/l kinetin and 3 mg/l 6-benzyladenine, whileD. capensis root development decreased. Plant growth regulator free medium was more suitable for root development than medium with α-naphthaleneacetic acid and thus supported the formation of significantly more flower stalks. For , kinetin was detrimental for shoot development, the optimumKeywords: medium auxins, for cytokinins, both shoot Drosera and root rotundifolia, formation Drosera being MS capensis, without in plant vitro growth regulators. INTRODUCTION Drosera et al. are also valued for their medicinal properties The genus (sundews) is a large (Kawiak , 2011) and are widely used in Droseragroup of carnivorous plants comprising more folk medicine. Extracts from these plants are than 200 species (McPherson, 2010). Numerous included in herbal preparations used in the species are distributed worldwide across therapy of respiratory tract infections (Egan and tropical to tundra regions, except for deserts van der Kooy, 2013). Therapeutic properties and Antarctica. The largest number of species is are thought to result from the presence of concentrated in Australiaet al. and New Zealand but etbioactive al compounds which can be used in the Droseraothers can be found only in America, South Africa Droseraproduction of pharmaceutical drugs (Banasiuk or Europe (Rivadavia , 2003). The leaves of ., 2012). The main secondary metabolites in plants have developed sticky traps that plants are 1,4 naphthoquinones, among attract, trap and digest prey using the resulting which the most abundant are plumbagin and nutrients to supplement what they can obtain 7-methyljuglone (ramentaceone). Besides these fromDrosera poor growing substrates (Rice, 2011). sundews synthesize several minor, but unique,et The unique biology and carnivorous habit al.naphthoquinones as wellet al.as a broad spectrumet al. of of spp. have gained theet interestal. of flavonoids such as myricetin, quercetin (Devi horticulturists and made sundewsDrosera economically species , 2016;Drosera Braunberger rotundifolia , 2015; Krolicka , important ornamentals (Barthlott , 2004). 2008). Besides their ornamental traits has a prostrate growth 145 habit with leaves in contact with the soil (Anthony, MS was supplemented with kinetin (0.5 mg/l, 2 1992) and is oneet of al. the most broadly distributed mg/l, 5 mg/l) or 6-benzyladenine (BA)D. rotundifolia (3 mg/l, 5 species, occurring throughout much of the andmg/l), D. with capensis cytokinin concentrations being chosen Holarctic (Wolf , 2006). It is among the few based on other published studies. in species of carnivorous plantset al. that can be found in vitro plantlets (10-15 mm in length, Romania with a habitat made of acidic peat bogs 7-10 leaves) were selected from an established in the mountains (Clapa , 2009). It has been culture and transferred to 1/2 MS without prescribedet al. as a diuretic, a laxative, and treatment plant growth regulators (1/2MS0) or to 1/2MS for aDrosera variety ofcapensis kidney, stomach, and liver problems containing one of the two cytokinins. Plants were (Wolf , 2006). kept for 8 weeks in the growing chamber at 20- is a small, erect perennial 25°C under a 18/6 h light/dark regime provided sundew native to the Cape region of South by cool white fluorescent2 lamps with a light Africa (Pavlovič et al., 2014) andD. capensisits plumbagin leaves intensity of 40 µmol/m /s. Afterwards, rosette production has been reviewed by Egan and van diameter, plant height, number of shoots, numberD. Staphylococcusder Kooy (2013). aureus Extract from et al. rotundifoliaof roots, root length, were measured and number could be effective in fighting antibiotic-resistant of roots and shoots per explant was calculated. strains (Krolicka , grows in horizontalD. capensis rosettes which 2008). can be accurately defined by rosette diameter The unfortunate side effect of the interest while in our experiment formed stems generated by sundews is overharvesting from which were better assessed by measuring plant natural sources. Together with a loss of their height. Plants were placed in fullMS without plant natural habitats this has ledet toal. their protection growth regulators (fullMS0), 1/2MS0 or 1/2MS and inclusion in the European Red List of supplemented with 0.5 mg/l α-naphthaleneacetic Threatened Plants (Kawiak , 2003).in vitro Another acid (α-NAA) for another 8 weeks. At the end of this result has been the surge in research regarding the time the number of roots, and root length, number micropropagation and the use of -grown of shoots and flower stalks were assessed. These plants as alternative sources of biomass (Hook, measurements were used to calculate number of 2001). roots, shoots and flower stalks per explant. Five This experiment aimed to find the most plantlets per vessel were inoculated for each suitable nutrient concentration and to study the treatment and treatments were repeated 5 times. ineffect vitro of several plant growth regulators (kinetin,D. Data were analysed by the analysis of variance rotundifolia6-benzyladenine, and D. α-naphthaleneaceticcapensis. acid) for and differences were estimated using the Tuckey shoot development and rooting of and t tests at p<0.05. These were performed using MATERIALS AND METHODS GraphPad InStat version 3.05 for Windows 95 (GraphPadRESULTS Software, AND DISCUSSIONSSan Diego California, USA). in vitro D. rotundifolia D. rotundifolia and TheD. capensis experiments used plant material from established cultures of In kinetin and BA promoted maintained on half-strength MS significantly larger rosette diameter than medium medium (Murashige and Skoog, 1962) without without plant growth regulators, the best being plant growth regulators. These cultures originated 0.5 mg/l kinetin (Tab. 1.). The same treatment from seeds provided by the Cluj-Napoca Botanical resulted in the largest number of shoots/explant Garden. Unless otherwise stated all reagents were while 3 mg/l BA was second and no shoots were acquired from Sigma-Aldrich, Germany. generated in 1/2MS0. The largest number of The employed media were: full strength MS roots/explant was counted for 0.5 mg/l kinetin (fullMS) and MS with half-strength macronutrients, but their average length was smaller than that half-strength micronutrients and full vitamins of roots grown in medium lacking plant growth (1/2MS). Both types of media were enriched with regulators. None of the other treatments resulted 30 g/l sucrose, solidified with 5 g/l phytagel and in growth and explants died before the end of the pH was adjusted to 5.4-5.5 before autoclaving at 8 weeks. 121°C for 20 min. After sterilization half-strength ThisBulletin suggests UASVM Animalthat Science plants and Biotechnologiesdevelop better 74(2) / 2017 in 146 et al. MICLEA Tab. 1. Drosera rotundifolia and Drosera capensis Effect of cytokinins on growth and developmental characteristics of Drosera rotundifolia roots/explant shoots/explant Rosette Number of Root length Number of Treatment diameter (cm) (cm) 1/2MS0 1.73±0.071a 2.01±0.06a 2.40±0.08a 0.00±0.00 0.5 mg/l kinetin 3.61±0.101b 3.83±2.07a 1.30±0.63a 3.43±0.07a 2 mg/l kinetin 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 5 mg/l kinetin 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 3 mg/l kinetin 3.21±0.043c 0.00±0.00 0.00±0.00 2.45±0.06b Drosera 5 mg/l kinetin 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 capensis roots/explant shoots/explant Plant height Number of Root length Number of Treatment (mm) (mm) 1/2MS0 2.81±0.41a 2.22±0.40a 2.92±0.17a 6.67±0.84a 0.5 mg/l kinetin 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 2 mg/l kinetin 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 5 mg/l kinetin 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 3 mg/l BA 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 5 mg/l BA 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 Note: Values expressed are mean ± standard error of the mean (SEM). Different letters between means within the same column denote significant differences (p < 0.05). medium with reduced cytokinin concentrationet al.