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

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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 uncoated 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%). In all T6 0.25 0.50 periods, there were no statistical differences T7 0.50 0.00 among the treatments applied. The same conclu- T8 0.50 0.25 sion was observed in Group II (seeds submitted T9 0.50 0.50 to 50 ºC for different exposure times) where the greatest germination rate occurred at the 28th day, in spite of among all treatments did not dif- The percentage of callus induction was ob- fer statistically (Figure 1B). tained as regenerative indexes. This was calculated according to the number of calli induced The effect of exposure to sulfuric acid based on the number of embryos available. The (50%) (Figure 1C) showed a tendency of reduc- ratio between the number of plantlets obtained tion in germination probably due to the embryos and the number of calli induced was also calcu- injuries. Only the exposure for 5 min presented lated (Ali et al. 2007). Regeneration efficiency responses statistically different in 28, 35 and 42 was calculated by multiplication between the germination days compared to control. percentages of induction and ratio of plantlets Gibberellic acid (GA ) is a potent growth obtained per treatment. Five embryos per Petri 3 regulator, inducer of dormancy’s breaking. In dish (90 x15 mm) and 10 replicates in each this experiment, germination was not observed treatment were used at the callogenesis and re- in any of the concentrations assessed (data not generation experiments. shown). Such results show the gibberellic acid

http://www.openaccessscience.com Montanucci et al. [email protected] 399 Int. J. Med. Arom. Plants Seed germination and plant regeneration in Brugmansia suaveolens may be promoting the inhibition of development Soaking for 24 hours let to a rapid germina- of embryos at the dosages used in this experi- tion (period of 14 days). However, as the days ment. went by, the effect was stabilized and the treatments did not differ among them (Figure 1D).

Figure 1: Germination rates of uncoated seeds under different conditions. A - Group I: Temperatures of 4oC; B - Group II: Temperatures of 50 oC; C - Group III: Treatments with 50% Sulfuric Acid; D - Group IV: Soaking in water. Mean values within the column followed by the same letter are not significantly different at p>0.05

Somatic embryogenesis and plant regeneration centages of callus induction (100%); however, The stages of callus induction and plant re- only treatments 6 and 7 exhibited the best regeneration are shown in Figure 2. The explants generation efficiencies (32%). Treatment 9, with used were mature embryos (2 to 3 mm) (Figure the highest levels of auxin and cytokinin, exhib- 2A). Some embryos germinated after 7 days of ited a low percentage of induced calli (21%), darkness (Figure 2B). The beginning of calli and proportionally, the highest ratio for number th of plantlets per induced callus (0.84). formation was observed at the 30 day (Figure 2C). Embryogenic calli were observed after 40 Control treatment presented 58% of induced days in the maintenance media (Figure 2D). calli, but it was not observed regenerated plant- Calli were soft, white and friable. Plantlets with lets, which provided evidence for the important a well developed root system were observed af- effect of the plant growth regulators on plant ter 55 days (Figure 2E). Plants were regenerated regeneration in this species. and acclimatized in pots (Figure 2F). Germination indexes are shown in Figure Data related to the regenerative indexes are 3A. Data among different treatments were sta- exhibited to different treatments (Table 2). tistically similar and varied from 78 to 100%. Treatments 2, 3, 6 and 7 exhibited the best per- http://www.openaccessscience.com Montanucci et al. [email protected] 400 Int. J. Med. Arom. Plants Seed germination and plant regeneration in Brugmansia suaveolens Table 2: Regenerative indexes obtained for the of KIN) was statistically different from others, different concentrations of 2,4-D and KIN (mg. originating the largest calli (230 mm) (Figure L-1) used on calli induction and plant regenera- 3B). Treatment 9, with the higher concentration tion from mature embryos of B. suaveolens of auxin and cytokinin exhibited an inhibiting Treatment I C (%)* N P R C** ER (%)*** effect on the calli size (40 mm). In relation to T1 58 0 0 calli dry matter, treatment 6 generated the high- T2 100 0.17 17 er dry matter (0.23 g) and treatments 1 and 9 T3 100 0.13 13 (0.041 and 0.044 g) had the smaller ones. T4 74 0.24 18 In relation to the number of embriogenic T5 66 0.22 15 calli (Figure 3C), the best combination of T6 100 0.32 32 growth regulators was 0.5 mg L-1 of 2,4-D and 1 T7 100 0.32 32 mg L-1 of KIN (Treatment 6) with the highest T8 96 0.20 19 scores although statistically similar to the oth- T9 26 0.84 22 ers. The values of 1.0 mg L-1 of 2,4-D and 1.0 *IC: Callus Induction; -1 **NPRC: Number of regenerated plants / Calli Induced; mg L of KIN (Treatment 9) promoted a de- ***ER: Efficiency of Regeneration crease in the number of green spots, showing the sensitivity of the explants to doses greater The mean size of calli was 94 mm and than 1.0 mg L-1 of auxins combined to treatment 6 (0.5 mg L-1 of 2,4-D and 1.0 mg L-1 cytokinins.

Figure 2: In vitro propagation of B. suaveolens (A) embryo (Bar ~ 3 mm); (B) Geminated embryos; (C) Embryogenic callus; (D) Calli exhibiting green spots; (E) Plantlet after being transferred to regeneration medium; (F) Plant regenerated after acclimatization.

The percentage of regenerated plantlets per spite of getting calli induction, auxins and callus was showed in Figure 3D and the treat- cytokinins are involved in cellular programming ments 5, 6 and 7 regenerated 30, 35 and 45 and differentiation, without which there is no plantlets respectively. Control calli did not pre- formation of a normal plantlet. sent regenerated plantlets, manifesting that, in

http://www.openaccessscience.com Montanucci et al. [email protected] 401 Int. J. Med. Arom. Plants Seed germination and plant regeneration in Brugmansia suaveolens

Figure 3: Effect of the different treatments on the tissue culture of B. suaveolens at different concentrations of 2,4-D and KIN. (A) Germinated Embryos (%). (B) Mean size of calli (mm). (C)- Green Spots (%). (D) Regenerated plantlets (%). Mean values within the column followed by the same letter are not significantly different at p>0.05

Discussion at 55 oC had the germination inhibited; however, 50ºC, proved to be adequate for germination in The knowledge about botanic germination this study. Carneiro et al. (2010) related that performance is the first step to the adoption of biotechniques in any species (Mazandarani et al. seeds treated with hot water at 50 ºC for 10 min 2007). Up to now, references were not found promoted a greater percentage of germination about the influence of environmental factors and greater uniformity in Capsicum baccatum. over germination rates in B. suaveolens. It is Sarker et al. (2000) observed positive effect in well documented that germination studies are germination of Sesbania rostrata by boiling fundamental to any plant multiplication scheme seeds to different periods (0-18 sec). These results would be explained by the fact that high (Cho 2010; Idu et al. 2007). In order to test pos- temperatures could interact with growth regula- sible injuries caused to embryos in uncoated seeds, data showed that hot water bath effect tors, promoting changes in their endogenous over B. suaveolens germination are in agree- levels and, consequently, influencing the germi- ment with Lêdo (1977) who verified the use of nation process (Gualtieri and Moraes 1990; Paul hot water to overcome the dormancy of et al. 1973). Schizolobium parahybum. Franco and Ferreira (2002) observed death On the other hand, Perez and Moraes (1990) of seeds in Didymopanax morototoni after sulfu- observed that Prosopis juliflora seeds incubated ric acid treatment. The same authors mentioned http://www.openaccessscience.com Montanucci et al. [email protected] 402 Int. J. Med. Arom. Plants Seed germination and plant regeneration in Brugmansia suaveolens the use of sulfuric acid would be more adequate mation and regeneration as it was used in treat- for seeds in which there is impermeability to ments 7 (2,4-D) and 2, 3 and 6 (2,4-D and KIN). water and not those there is resistance to embryo Other authors, checking the effect of these growth. Sulfuric acid would act in weakening regulators in vitro culture of Datura stramonium cover´s seed by cuticle removing and the disso- mature embryos, assayed concentrations varying lution of its macrosclereid cells (Martins- from 1-3 mg L-1 (2,4-D) (Parsaemehr and Conder et al. 1999). Freitas and Cândido (1972) Alizadeh 2010) and 0-1 mg L-1 (KIN) and 1-2 found positive results in increasing germination mg L-1 (2,4-D) (Amiri et al. 2011); others, rate in Schizolobium parahyba seeds and over a moreover, assessed the effect of other growth papaya´s seeds (Tachigalia mutlijuge). Alt- regulators: 2.3-3.9 mg L-1 (2,4-D) and 3.4 mg L- hough, Idu et al. (2007) observed in Hura 1 of 6-Benzylaminopurine (BAP) in Datura crepitans that 5 min treatment provoked 42% de stramonium (Sundar and Jawahar 2010); com- abnormal seedling compared to zero percent in binations of 1-4 mg L-1 BAP, IAA and GA in longer period (15 and 25 min). 3 Datura metel (Nithiya and Arockiasamy 2010), In the literature, low concentrations of 3 mg L-1 of 1-Naphthaleneacetic acid (NAA) in gibberellic acid had a positive effect on devel- Brugmansia cândida (Niño et al. 2003) and 0.5 opment of coffee plant embryos (Carvalho et al. and 1 mg L-1 of BAP and indole-3-acetic acid 1998). However, Moreira et al. (2010) working (IAA), respectively (Zayeda et al. 2006). with Physalis sp (solanacea) observed an inhibi- Amiri et al. (2011) observed that in Datura tion effect doses of 1500 ppm of GA3, which stramonium, dosages of 2 mg L-1 of 2,4-D com- agrees with the results obtained for B. bined with 0.25 and 0.5 mg L-1 KIN were the suaveolens. In this experiment, the concentra- best for callus formation and higher dosages of tions were probably high and had an inhibiting -1 KIN, up to 0.5 and 1 mg L inhibited it. Data effect on germination. obtained in this study disagree with Brasileiro The most important observation was that and Carneiro (1999) that did not observe MS medium was enough to promote seed ger- callogenesis and plant regeneration in tomatoes mination on B. suaveolens and the treatments (Lycopersicon esculentum) under the same con- applied to check possible injuries to embryos ditions. were not sufficient to increase or decrease ger- Several authors in the literature reported dif- mination rates. ferent responses of callus induction: Parsaemehr One of the basic requirements of an efficient and Alizadeh (2010) affirmed that the presence protocol is its quickness in obtaining regenerat- of 2,4-D is essential for callus formation in ed plantlets. Data showed that B. suaveolens Datura stramonium. Miskat et al. (2003) ob- required 60 days (~ 8 weeks) to achieve a plant- served that dosages of 2 mg L-1 (2,4-D) present- let. In related species, Datura innoxia, Rahman ed the best responses to callus induction from et al. (2008) were observed 6 weeks as a mini- stem fragments of Datura metel. Amiri and mum period; as observed in Scopolia porniflora Kazemitabar (2011) studied in vitro plant regen- (Yong et al. 2008). Zayeda et al. (2006), ob- eration in Datura stramonium, observed that served a need of 10 months without the addition concentrations of 2.0 mg L-1 of 2,4-D were suf- of hormones. ficient to enhance calli induction and plant regeneration. The results obtained agree with those found by Amiri et al. (2011) who observed that in or- Although, Sundar and Jawahar (2010) ob- der to maintain regeneration of plants in Datura served that only the 2,4-D presence in the medi- stramonium, the most important effect was um is not by itself an inductor for callogenesis presence of 2,4-D and of 2,4-D and KIN togeth- and regeneration of plants The authors reported er. It could be explained by the fact that the ki- that the best performances were obtained with netin would promote the effect of 2,4-D, but by concentrations of 3.9 mg L-1 and 3.4 mg L-1 of itself, it would not be capable of callus for- BAP. Otroshy et al. (2011) observed in another solanaceae (Capsicum annuum) no callogenesis

http://www.openaccessscience.com Montanucci et al. [email protected] 403 Int. J. Med. Arom. Plants Seed germination and plant regeneration in Brugmansia suaveolens and low ratio of plant regeneration using BAP ciency from embryo cultures of Datura and IBA as growth regulators and cotyledons as stramonium by adjusting carbon sources explants, showing that 2,4-D and KIN can be and concentrations. African Journal of Bio- considered as potential inducers. technology, 10(50):10101-10107. Differences in callus induction, regenerative Amorim I.L., Dadive A.C., Chaves M.M.F. capacity and plant regeneration among the 1997. Morphology of fruit, seed and germi- treatments suggests inductive activity in cellular nation patern of Trema micrantha (L.) growth through the presence/absence of these Blum. Cerne, 3(1):138-152. growth regulators in the culture medium. Con- Benesi I.R.M., Labuschagne M.T., Herselman trol exhibited formation of organogenic and L., Mahungu N. 2010. Ethnobotany, mor- embryogenic calli, manifesting that there is no phology and genotyping of cassava need of growth regulators for calli formation, germplasm from Malawi. Journal of possibly explained by the fact that an endoge- Biologcal Science, 7:616-623. nous plant growth regulator imbalance, allied to a stress condition (imposed by in vitro condi- Brasileiro A.C.M., Carneiro V.T.C. 1999. Man- tion) would act as inductor (Perez et al. 2002). ual de transformação genética de plantas. Ed. Embrapa-SPI / Embrapa-Cenargen, The use of mature embryos certainly has the Brasília: Embrapa-SPI / Embrapa- advantages of time reduction by being available Cenargen. throughout the year, being able to be stored without the need of planting stock plants, which Carneiro G.G., Barbosa J.A., Silva E.O., Gois certainly favor the use of biotechnology in this G.C., Lucena H.H., Alves E.U. 2010. Ger- species. mination of pepper seeds subjected to dormancy breaking in hot water. Bioscience Journal, 26(6):882-885. Conclusions Carvalho G.R., Pasqual M., Guimarães R.J., The MS medium for uncoated seeds was Mendes A.N.G., Antunes L.E.C., Silva sufficient to induce germination with satisfacto- A.T. 1998. Effect of the gibberellic acid ry performance. The in vitro propagation proto- and benzilaminopurine on the in vitro col obtained from mature embryos as explants growth of embryos of coffee plants cv. originated healthy plants with a reasonable sur- Acaiá. Pesquisa Agropecuária Brasileira, vival rate. This protocol will be helpful in the 33(6):1-5. establishment of other biotechniques in order to Cho Y.S. 2010. Germination characteristics of produce trophane alkaloids in industrial scale. Korean and southeast asian redrice (Oryza sativa L.) seeds as affected by temperature. Asian Journal of Plant Science, 9:104-107. References Ali G., Hadi F., Ali Z., M. T., Khan M.A. 2007. Corrêa M.P. 1984. 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