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Evaluation of Seed Germination and Plant Regeneration in Brugmansia Suaveolens – a Trophane Alkaloid Producer Plant

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Evaluation of Seed Germination and Plant Regeneration in Brugmansia Suaveolens – a Trophane Alkaloid Producer Plant Int. J. Med. Arom. Plants, ISSN 2249 – 4340 RESEARCH ARTICLE Vol. 2, No. 3, pp. 396-405, September 2012 Evaluation of seed germination and plant regeneration in Brugmansia suaveolens – a trophane alkaloid producer plant Cleuza A.R. MONTANUCCI1, Fernando FURLAN1, A. Adeline NEIVERTH1, Walkyria NEIVERTH1, Izabel V. ZADINELO2, Raquel M. SERENISKI2, Isaac ROMANI2, Robson F. MISSIO2, Marise F. dos SANTOS2, Eliane C. G.VENDRUSCOLO2*, Márcia M. ECHER1 1Universidade Estadual do Oeste do Paraná-Brazil 2Universidade Federal do Paraná, Campus Palotina-Brazil *Corresponding Author, Tel.: +55 44 32118577 Article History: Received 7th August 2012, Revised 11th September 2012, Accepted 12th September 2012. Abstract: Brugmansia suaveolens is known by its pharmaceutical importance. The aims of this study were to evaluate seed germination under different treatments and the establishment of a plant regeneration protocol. In vitro germination capacity of coated and uncoated seeds under different conditions was evaluated. Calli induction and plant regeneration were conducted using 9 different matches between 2,4-D and KIN dosages. Coated seeds did not germinate and the un- coated seeds germinated in MS medium as well as other treatments. Exposure to sulfuric acid and soaking for 24 hours reduced germination. The plant regeneration protocol was established from mature embryos and 0.5 mg L-1 of 2,4-D and 1.0 mg L-1 KIN was the most suitable dosage for Brugmansia suaveolens. Keywords: Solanaceae; tissue culture; growth regulators; regeneration. Abbreviations: 2, 4-D: 2, 4 - dichlorophenoxy acetic acid; KIN: Kinetin; GA3: Giberellic acid. Introduction and a complex of bases tropine and scopine, ex- Brugmansia suaveolens, a member of hibiting hallucinogenic, antispasmodic, diapho- solanaceae family, native to tropical South retic and diuretic activities (Iranbakhsh et al. America, is an ornamental and medicinal plant, 2007; Pitta-Alvarez et al. 2000). considered toxic, popularly known as angel Considering its pharmaceutical importance, trumpet (Corrêa 1984; Zayed and Wink, 2004). knowledge about germinative behavior and es- Brugmansia suaveolens was formely described tablishment of an efficient protocol for tissue as Datura suaveolens and in 1823, Bercht & culture and plant regeneration is a pre-requisite Presl reclassified this species in Brugmansia for biotechnological processes (Valizadeh and suaveolens. Brugmansia plants are woody trees Valizadeh 2009) and for breeding programs or bushes, with pendulous, not erect, flowers, (Benesi et al. 2010). Among these processes, without spines on their fruit while Datura genetic transformation using the Agrobacterium species are herbaceous bushes with erect (not system for insertion of genes and obtaining of pendulous) flowers, and presenting spines on hairy roots in high and commercial scale is im- their fruit (Smith and Downs, 1966). portant (Kieran 2001; Robins et al. 1991; Sheludko 2010). Species from the genera Brugmansia and Datura produce the atropine and scopolamine Seed is the main way of reproduction for the alkaloids. Both are organic esters formed by the majority of woody species, and its storage and combination of an aromatic acid, tropanic acid growing methods may cause the losing in *Corresponding author: (E-mail) vendruscolo <@> ufpr.br http://www.openaccessscience.com © 2012 Copyright by the Authors, licensee Open Access Science Research Publisher. [email protected] This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC- ND 3.0) License (http://creativecommons.org/licenses/by-nc-nd/3.0) 397 Int. J. Med. Arom. Plants Seed germination and plant regeneration in Brugmansia suaveolens germinative capacity (Amorim et al. 1997). claved and distilled water. The effect of Studies regarding germination are also im- gibberellic acid (GA3) was assessed by adding portant for obtaining in vitro tissue culture 20, 30, 40 and 50 mg L-1 to the MS medium (Verpoorte 2000). (Group IV). The soaking effect was evaluated In vitro procedures and plant regeneration keeping seeds immersed in 50 mL of autoclaved are used to some degree in almost every major and distilled water for 24, 48 and 72 h at room plant species. The success of such biotechnolo- temperature (Group V). gy requires an efficient protocol for plant regen- After the application of the treatments, coat- eration from different explants (Sultana and Bari ed and uncoated seeds were submitted to the Miah 2003). Many studies in the literature as- aseptic treatment in a laminar flow. They were sessed the performance of the explant in regard submersed for 10 min in 70% alcohol, followed to tissue culture and plant regeneration: anthers by 2% sodium hypochlorite with two drops of (Datura stramonium) (Sundar and Jawahar Tween 80 for 20 min. Seeds were subsequently 2010); adventitious stems and leaves (Datura washed three times with distilled and autoclaved metel) (De 2003); stems (Datura innoxia) water. After asepsis, the seeds were dried on (Zayeda et al. 2006); hypocotyls (Datura metel) filter paper and inoculated in jars (600 mL vol- (Muthukumar et al. 2000); buds (Datura ume) containing 50 mL of MS medium. Each insignis) (Dos Santos et al. 1990), mature em- treatment consisted of 4 jars containing 5 seeds bryo (Datura stramonium) (Amiri et al. 2011). each. Seeds remained 7 days in the dark and However, in the literature, there are no studies then were transferred to the culture growth room that have checked the plant regeneration in B. (16 h light /8 h dark) with temperature at 23 ± 2 suaveolens ºC. The germination experimental design was randomized and the accumulated germination The purpose of this study was to carry out the evaluation of seeds germination behavior was measured after 7, 14, 21, 28, 35 and 42 and the establishment of a regeneration protocol days. from mature embryos from Brugmansia suaveolens. In vitro calli induction and plant regeneration Brugmansia suaveolens seeds were collected Materials and methods from the same plant (white biotype) and their In vitro germination coats were mechanically removed. Asepsis was performed as described above. The embryos Twenty two accessions of B. suaveolens were rescued with the help of a tweezers and a were collected on rural and urban areas in scalpel in a stereomicroscope. The MS medium Palotina city, Paraná, Brazil. Coated (Experi- (Murashige and Skoog 1962) was used for in- ment I) and uncoated seeds (Experiment II) duction, maintenance and regeneration with half were submitted to 17 different conditions to the concentration of macro and micronutrients, check germination viability. MS culture medium 30 g L-1 of sucrose, pH 5.8, 8.0 g L-1 of agar and (Murashige and Skoog 1962) was used with half 1.0 g L-1 of activated charcoal with decreasing concentration of macro and micronutrients, 1% concentrations of 2,4-D and KIN, (Table 1). Re- -1 sucrose, activated charcoal at 1.0 g L , pH 5,8 generation phase consisted of absence of growth -1 and 7.0 g L agar without growth regulators. regulators. All medium was adjusted to. Group I treatment consisted of submitting At induction phase, the embryos remained in the seeds to 4 ºC for 24, 48 and 72 h. To verify the dark for 7 days, and, at the maintenance and the effect of high temperatures on the embryo regeneration phases, exposed to 25 ± 2 ºC with a and its germination, B. suaveolens seeds were photoperiod (16 light /8 hours darkness). submitted to 50 ºC in a water bath for 5, 10 and 15 min (Group II). Group III treatment consisted After 30 days of culture at the induction me- to keep seeds under sulfuric acid at 50% for 5, dium, the embryos were evaluated according to 10 and 15 min, followed by 3 rinses of auto- their calli induction competence. They were http://www.openaccessscience.com Montanucci et al. [email protected] 398 Int. J. Med. Arom. Plants Seed germination and plant regeneration in Brugmansia suaveolens subcultured every 7 days and the number and Statistical analysis size of calli were gotten. At maintenance phase th Germination data were submitted to descrip- (45 day), the number of green spots was evalu- tive statistical analysis. Callogenesis and plant ated. The number of regenerated plantlets was th regeneration data were transformed in arcsin √x assessed on the 60 day. The calli dry weight with the purpose of variance analyses. The anal- (g) was obtained by weighing the dried calli fol- ysis of variance procedures and mean separation lowing to 7 days in a laboratory oven at 50 ºC. were done using SISVAR statistical package The number of germinated embryos was also (Ferreira 1999). quantified. Results Table 1: Concentrations of 2,4-D and KIN (mg In vitro germination L-1) used in treatments for calli induction and plant regeneration from mature embryos of Coated seeds (experiment I) did not germi- Brugmansia suaveolens. nate under in vitro conditions until 42 days, showing a physical dormancy through the pres- Growth Regulators -1 -1 ence of the rough cover. Uncoated seeds (exper- Treatments 2,4-D (mg L ) KIN (mg L ) iment II), required at least 14 days for the be- Induction ginning of the germination process, for lower T1 0.0 0.0 periods any germination was observed. Control T2 0.0 0.5 treatment composed only by the MS medium, T3 0.0 1.0 T4 0.5 0.0 showed itself as an inductor for germination, T5 0.5 0.5 probably through the presence of water and nu- T6 0.5 1.0 trients. However, the maximum germination rate for control in all the times assayed was 75% T7 1.0 0.0 o T8 1.0 0.5 (Figure 1). Results obtained for the effect of 4 C T9 1.0 1.0 exposure (Figure 1A) over different periods Maintenance showed that B. suaveolens seeds had a positive T2 0.0 0.25 correlation between germination and period as- T3 0.0 0.50 sayed, the best responses occurred with 42 days T4 0.25 0.00 of culture; however, at 28 days, control had got- T5 0.25 0.25 ten the highest germination rate (70%).
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