Evaluation of seed germination and plant regeneration in Brugmansia ...

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 1

Universidade Estadual do Oeste do Paraná-Brazil

2

Universidade 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 Brugmansia suaveolens, a member of solanaceae family, native to tropical South America, is an ornamental and medicinal plant, considered toxic, popularly known as angel trumpet (Corrêa 1984; Zayed and Wink, 2004). Brugmansia suaveolens was formely described as Datura suaveolens and in 1823, Bercht & Presl reclassified this species in Brugmansia suaveolens. Brugmansia plants are woody trees or bushes, with pendulous, not erect, flowers, without spines on their fruit while Datura species are herbaceous bushes with erect (not pendulous) flowers, and presenting spines on their fruit (Smith and Downs, 1966). Species from the genera Brugmansia and Datura produce the atropine and scopolamine alkaloids. Both are organic esters formed by the combination of an aromatic acid, tropanic acid

and a complex of bases tropine and scopine, exhibiting hallucinogenic, antispasmodic, diaphoretic and diuretic activities (Iranbakhsh et al. 2007; Pitta-Alvarez et al. 2000). Considering its pharmaceutical importance, knowledge about germinative behavior and establishment of an efficient protocol for tissue culture and plant regeneration is a pre-requisite for biotechnological processes (Valizadeh and Valizadeh 2009) and for breeding programs (Benesi et al. 2010). Among these processes, genetic transformation using the Agrobacterium system for insertion of genes and obtaining of hairy roots in high and commercial scale is important (Kieran 2001; Robins et al. 1991; Sheludko 2010). Seed is the main way of reproduction for the majority of woody species, and its storage and 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-NCND 3.0) License (http://creativecommons.org/licenses/by-nc-nd/3.0)

Int. J. Med. Arom. Plants

397 Seed germination and plant regeneration in Brugmansia suaveolens

germinative capacity (Amorim et al. 1997). Studies regarding germination are also important for obtaining in vitro tissue culture (Verpoorte 2000). In vitro procedures and plant regeneration are used to some degree in almost every major plant species. The success of such biotechnology requires an efficient protocol for plant regeneration from different explants (Sultana and Bari Miah 2003). Many studies in the literature assessed the performance of the explant in regard to tissue culture and plant regeneration: anthers (Datura stramonium) (Sundar and Jawahar 2010); adventitious stems and leaves (Datura metel) (De 2003); stems (Datura innoxia) (Zayeda et al. 2006); hypocotyls (Datura metel) (Muthukumar et al. 2000); buds (Datura insignis) (Dos Santos et al. 1990), mature embryo (Datura stramonium) (Amiri et al. 2011). However, in the literature, there are no studies that have checked the plant regeneration in B. suaveolens The purpose of this study was to carry out the evaluation of seeds germination behavior and the establishment of a regeneration protocol from mature embryos from Brugmansia suaveolens. Materials and methods In vitro germination Twenty two accessions of B. suaveolens were collected on rural and urban areas in Palotina city, Paraná, Brazil. Coated (Experiment I) and uncoated seeds (Experiment II) were submitted to 17 different conditions to check germination viability. MS culture medium (Murashige and Skoog 1962) was used with half concentration of macro and micronutrients, 1% sucrose, activated charcoal at 1.0 g L-1, pH 5,8 and 7.0 g L-1 agar without growth regulators. Group I treatment consisted of submitting the seeds to 4 ºC for 24, 48 and 72 h. To verify the effect of high temperatures on the embryo and its germination, B. suaveolens seeds were submitted to 50 ºC in a water bath for 5, 10 and 15 min (Group II). Group III treatment consisted to keep seeds under sulfuric acid at 50% for 5, 10 and 15 min, followed by 3 rinses of autoMontanucci et al.

claved and distilled water. The effect of gibberellic acid (GA3) was assessed by adding 20, 30, 40 and 50 mg L-1 to the MS medium (Group IV). The soaking effect was evaluated keeping seeds immersed in 50 mL of autoclaved and distilled water for 24, 48 and 72 h at room temperature (Group V). After the application of the treatments, coated and uncoated seeds were submitted to the aseptic treatment in a laminar flow. They were submersed for 10 min in 70% alcohol, followed by 2% sodium hypochlorite with two drops of Tween 80 for 20 min. Seeds were subsequently washed three times with distilled and autoclaved water. After asepsis, the seeds were dried on filter paper and inoculated in jars (600 mL volume) containing 50 mL of MS medium. Each treatment consisted of 4 jars containing 5 seeds each. Seeds remained 7 days in the dark and then were transferred to the culture growth room (16 h light /8 h dark) with temperature at 23 ± 2 ºC. The germination experimental design was randomized and the accumulated germination was measured after 7, 14, 21, 28, 35 and 42 days. In vitro calli induction and plant regeneration Brugmansia suaveolens seeds were collected from the same plant (white biotype) and their coats were mechanically removed. Asepsis was performed as described above. The embryos were rescued with the help of a tweezers and a scalpel in a stereomicroscope. The MS medium (Murashige and Skoog 1962) was used for induction, maintenance and regeneration with half the concentration of macro and micronutrients, 30 g L-1 of sucrose, pH 5.8, 8.0 g L-1 of agar and 1.0 g L-1 of activated charcoal with decreasing concentrations of 2,4-D and KIN, (Table 1). Regeneration phase consisted of absence of growth regulators. All medium was adjusted to. At induction phase, the embryos remained in the dark for 7 days, and, at the maintenance and regeneration phases, exposed to 25 ± 2 ºC with a photoperiod (16 light /8 hours darkness). After 30 days of culture at the induction medium, the embryos were evaluated according to their calli induction competence. They were http://www.openaccessscience.com [email protected]



subcultured every 7 days and the number and size of calli were gotten. At maintenance phase (45th day), the number of green spots was evaluated. The number of regenerated plantlets was assessed on the 60th day. The calli dry weight (g) was obtained by weighing the dried calli following to 7 days in a laboratory oven at 50 ºC. The number of germinated embryos was also quantified.

Statistical analysis Germination data were submitted to descriptive statistical analysis. Callogenesis and plant regeneration data were transformed in arcsin √x with the purpose of variance analyses. The analysis of variance procedures and mean separation were done using SISVAR statistical package (Ferreira 1999). Results

Table 1: Concentrations of 2,4-D and KIN (mg L-1) used in treatments for calli induction and plant regeneration from mature embryos of Brugmansia suaveolens. Treatments T1 T2 T3 T4 T5 T6 T7 T8 T9 T2 T3 T4 T5 T6 T7 T8 T9

Growth Regulators 2,4-D (mg L-1) KIN (mg L-1) Induction 0.0 0.0 0.0 0.5 0.0 1.0 0.5 0.0 0.5 0.5 0.5 1.0 1.0 0.0 1.0 0.5 1.0 1.0 Maintenance 0.0 0.25 0.0 0.50 0.25 0.00 0.25 0.25 0.25 0.50 0.50 0.00 0.50 0.25 0.50 0.50

The percentage of callus induction was obtained as regenerative indexes. This was calculated according to the number of calli induced based on the number of embryos available. The ratio between the number of plantlets obtained and the number of calli induced was also calculated (Ali et al. 2007). Regeneration efficiency was calculated by multiplication between the percentages of induction and ratio of plantlets obtained per treatment. Five embryos per Petri dish (90 x15 mm) and 10 replicates in each treatment were used at the callogenesis and regeneration experiments. Montanucci et al.

In vitro germination Coated seeds (experiment I) did not germinate under in vitro conditions until 42 days, showing a physical dormancy through the presence of the rough cover. Uncoated seeds (experiment II), required at least 14 days for the beginning of the germination process, for lower periods any germination was observed. Control treatment composed only by the MS medium, showed itself as an inductor for germination, probably through the presence of water and nutrients. However, the maximum germination rate for control in all the times assayed was 75% (Figure 1). Results obtained for the effect of 4oC exposure (Figure 1A) over different periods showed that B. suaveolens seeds had a positive correlation between germination and period assayed, the best responses occurred with 42 days of culture; however, at 28 days, control had gotten the highest germination rate (70%). In all periods, there were no statistical differences among the treatments applied. The same conclusion was observed in Group II (seeds submitted 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 differ statistically (Figure 1B). The effect of exposure to sulfuric acid (50%) (Figure 1C) showed a tendency of reduction in germination probably due to the embryos injuries. Only the exposure for 5 min presented responses statistically different in 28, 35 and 42 germination days compared to control. Gibberellic acid (GA3) is a potent growth regulator, inducer of dormancy’s breaking. In this experiment, germination was not observed in any of the concentrations assessed (data not shown). Such results show the gibberellic acid http://www.openaccessscience.com [email protected]



may be promoting the inhibition of development of embryos at the dosages used in this experiment.

Soaking for 24 hours let to a rapid germination (period of 14 days). However, as the days 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 The stages of callus induction and plant regeneration are shown in Figure 2. The explants used were mature embryos (2 to 3 mm) (Figure 2A). Some embryos germinated after 7 days of darkness (Figure 2B). The beginning of calli formation was observed at the 30th day (Figure 2C). Embryogenic calli were observed after 40 days in the maintenance media (Figure 2D). Calli were soft, white and friable. Plantlets with a well developed root system were observed after 55 days (Figure 2E). Plants were regenerated and acclimatized in pots (Figure 2F). Data related to the regenerative indexes are exhibited to different treatments (Table 2). Treatments 2, 3, 6 and 7 exhibited the best perMontanucci et al.

centages of callus induction (100%); however, only treatments 6 and 7 exhibited the best regeneration efficiencies (32%). Treatment 9, with the highest levels of auxin and cytokinin, exhibited a low percentage of induced calli (21%), and proportionally, the highest ratio for number of plantlets per induced callus (0.84). Control treatment presented 58% of induced calli, but it was not observed regenerated plantlets, which provided evidence for the important effect of the plant growth regulators on plant regeneration in this species. Germination indexes are shown in Figure 3A. Data among different treatments were statistically similar and varied from 78 to 100%. http://www.openaccessscience.com [email protected]



Table 2: Regenerative indexes obtained for the different concentrations of 2,4-D and KIN (mg. L-1) used on calli induction and plant regeneration from mature embryos of B. suaveolens Treatment I C (%)* N P R C** ER (%)*** T1 58 0 0 T2 100 0.17 17 T3 100 0.13 13 T4 74 0.24 18 T5 66 0.22 15 T6 100 0.32 32 T7 100 0.32 32 T8 96 0.20 19 T9 26 0.84 22 *IC: Callus Induction; **NPRC: Number of regenerated plants / Calli Induced; ***ER: Efficiency of Regeneration

The mean size of calli was 94 mm and treatment 6 (0.5 mg L-1 of 2,4-D and 1.0 mg L-1

of KIN) was statistically different from others, originating the largest calli (230 mm) (Figure 3B). Treatment 9, with the higher concentration of auxin and cytokinin exhibited an inhibiting effect on the calli size (40 mm). In relation to calli dry matter, treatment 6 generated the higher dry matter (0.23 g) and treatments 1 and 9 (0.041 and 0.044 g) had the smaller ones. In relation to the number of embriogenic calli (Figure 3C), the best combination of growth regulators was 0.5 mg L-1 of 2,4-D and 1 mg L-1 of KIN (Treatment 6) with the highest scores although statistically similar to the others. The values of 1.0 mg L-1 of 2,4-D and 1.0 mg L-1 of KIN (Treatment 9) promoted a decrease in the number of green spots, showing the sensitivity of the explants to doses greater than 1.0 mg L-1 of auxins combined to 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 callus was showed in Figure 3D and the treatments 5, 6 and 7 regenerated 30, 35 and 45 plantlets respectively. Control calli did not present regenerated plantlets, manifesting that, in Montanucci et al.

spite of getting calli induction, auxins and cytokinins are involved in cellular programming and differentiation, without which there is no formation of a normal plantlet. http://www.openaccessscience.com [email protected]



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 The knowledge about botanic germination performance is the first step to the adoption of biotechniques in any species (Mazandarani et al. 2007). Up to now, references were not found about the influence of environmental factors over germination rates in B. suaveolens. It is well documented that germination studies are fundamental to any plant multiplication scheme (Cho 2010; Idu et al. 2007). In order to test possible injuries caused to embryos in uncoated seeds, data showed that hot water bath effect over B. suaveolens germination are in agreement with Lêdo (1977) who verified the use of hot water to overcome the dormancy of Schizolobium parahybum. On the other hand, Perez and Moraes (1990) observed that Prosopis juliflora seeds incubated Montanucci et al.

at 55 oC had the germination inhibited; however, 50ºC, proved to be adequate for germination in this study. Carneiro et al. (2010) related that seeds treated with hot water at 50 ºC for 10 min promoted a greater percentage of germination and greater uniformity in Capsicum baccatum. Sarker et al. (2000) observed positive effect in germination of Sesbania rostrata by boiling seeds to different periods (0-18 sec). These results would be explained by the fact that high temperatures could interact with growth regulators, promoting changes in their endogenous levels and, consequently, influencing the germination process (Gualtieri and Moraes 1990; Paul et al. 1973). Franco and Ferreira (2002) observed death of seeds in Didymopanax morototoni after sulfuric acid treatment. The same authors mentioned http://www.openaccessscience.com [email protected]



the use of sulfuric acid would be more adequate for seeds in which there is impermeability to water and not those there is resistance to embryo growth. Sulfuric acid would act in weakening cover´s seed by cuticle removing and the dissolution of its macrosclereid cells (MartinsConder et al. 1999). Freitas and Cândido (1972) found positive results in increasing germination rate in Schizolobium parahyba seeds and over a papaya´s seeds (Tachigalia mutlijuge). Although, Idu et al. (2007) observed in Hura crepitans that 5 min treatment provoked 42% de abnormal seedling compared to zero percent in longer period (15 and 25 min). In the literature, low concentrations of gibberellic acid had a positive effect on development of coffee plant embryos (Carvalho et al. 1998). However, Moreira et al. (2010) working with Physalis sp (solanacea) observed an inhibition effect doses of 1500 ppm of GA3, which agrees with the results obtained for B. suaveolens. In this experiment, the concentrations were probably high and had an inhibiting effect on germination. The most important observation was that MS medium was enough to promote seed germination on B. suaveolens and the treatments applied to check possible injuries to embryos were not sufficient to increase or decrease germination rates. One of the basic requirements of an efficient protocol is its quickness in obtaining regenerated plantlets. Data showed that B. suaveolens required 60 days (∼8 weeks) to achieve a plantlet. In related species, Datura innoxia, Rahman et al. (2008) were observed 6 weeks as a minimum period; as observed in Scopolia porniflora (Yong et al. 2008). Zayeda et al. (2006), observed a need of 10 months without the addition of hormones. The results obtained agree with those found by Amiri et al. (2011) who observed that in order to maintain regeneration of plants in Datura stramonium, the most important effect was presence of 2,4-D and of 2,4-D and KIN together. It could be explained by the fact that the kinetin would promote the effect of 2,4-D, but by itself, it would not be capable of callus for-

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mation and regeneration as it was used in treatments 7 (2,4-D) and 2, 3 and 6 (2,4-D and KIN). Other authors, checking the effect of these regulators in vitro culture of Datura stramonium mature embryos, assayed concentrations varying from 1-3 mg L-1 (2,4-D) (Parsaemehr and Alizadeh 2010) and 0-1 mg L-1 (KIN) and 1-2 mg L-1 (2,4-D) (Amiri et al. 2011); others, moreover, assessed the effect of other growth regulators: 2.3-3.9 mg L-1 (2,4-D) and 3.4 mg L1 of 6-Benzylaminopurine (BAP) in Datura stramonium (Sundar and Jawahar 2010); combinations of 1-4 mg L-1 BAP, IAA and GA3 in Datura metel (Nithiya and Arockiasamy 2010), 3 mg L-1 of 1-Naphthaleneacetic acid (NAA) in Brugmansia cândida (Niño et al. 2003) and 0.5 and 1 mg L-1 of BAP and indole-3-acetic acid (IAA), respectively (Zayeda et al. 2006). Amiri et al. (2011) observed that in Datura stramonium, dosages of 2 mg L-1 of 2,4-D combined with 0.25 and 0.5 mg L-1 KIN were the best for callus formation and higher dosages of KIN, up to 0.5 and 1 mg L-1 inhibited it. Data obtained in this study disagree with Brasileiro and Carneiro (1999) that did not observe callogenesis and plant regeneration in tomatoes (Lycopersicon esculentum) under the same conditions. Several authors in the literature reported different responses of callus induction: Parsaemehr and Alizadeh (2010) affirmed that the presence of 2,4-D is essential for callus formation in Datura stramonium. Miskat et al. (2003) observed that dosages of 2 mg L-1 (2,4-D) presented the best responses to callus induction from stem fragments of Datura metel. Amiri and Kazemitabar (2011) studied in vitro plant regeneration in Datura stramonium, observed that concentrations of 2.0 mg L-1 of 2,4-D were sufficient to enhance calli induction and plant regeneration. Although, Sundar and Jawahar (2010) observed that only the 2,4-D presence in the medium is not by itself an inductor for callogenesis and regeneration of plants The authors reported that the best performances were obtained with concentrations of 3.9 mg L-1 and 3.4 mg L-1 of BAP. Otroshy et al. (2011) observed in another solanaceae (Capsicum annuum) no callogenesis http://www.openaccessscience.com [email protected]



and low ratio of plant regeneration using BAP and IBA as growth regulators and cotyledons as explants, showing that 2,4-D and KIN can be considered as potential inducers.

ciency from embryo cultures of Datura stramonium by adjusting carbon sources and concentrations. African Journal of Biotechnology, 10(50):10101-10107.

Differences in callus induction, regenerative capacity and plant regeneration among the treatments suggests inductive activity in cellular growth through the presence/absence of these growth regulators in the culture medium. Control exhibited formation of organogenic and embryogenic calli, manifesting that there is no need of growth regulators for calli formation, possibly explained by the fact that an endogenous plant growth regulator imbalance, allied to a stress condition (imposed by in vitro condition) would act as inductor (Perez et al. 2002).

Amorim I.L., Dadive A.C., Chaves M.M.F. 1997. Morphology of fruit, seed and germination patern of Trema micrantha (L.) Blum. Cerne, 3(1):138-152.

The use of mature embryos certainly has the advantages of time reduction by being available throughout the year, being able to be stored without the need of planting stock plants, which certainly favor the use of biotechnology in this species. Conclusions The MS medium for uncoated seeds was sufficient to induce germination with satisfactory performance. The in vitro propagation protocol obtained from mature embryos as explants originated healthy plants with a reasonable survival rate. This protocol will be helpful in the establishment of other biotechniques in order to produce trophane alkaloids in industrial scale. References Ali G., Hadi F., Ali Z., M. T., Khan M.A. 2007. Callus induction and in vitro complete plant regeneration of different cultivars of tobacco (Nicotiana tabacum L.) on media of different hormonal concentrations. Biotechnology, 6:561-566. Amiri S., Kazemitabar K., Ranjbar, G.A., Azadbakht M. 2011. In vitro propagation and whole plant regeneration from callus in Datura (Datura stramonium. L). African Journal of Biotechnology, 10(3):442-448. Amiri S., Kazemitabar K. 2011. Enhancement of callus induction and regeneration effiMontanucci et al.

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