Evaluation of seed germination and plant...

Int. J. Med. Arom. Plants, ISSN 2249 – 4340RESEARCH ARTICLE

Vol. 2, No. 3, pp. 396-405, September 2012

*Corresponding author: (E-mail) vendruscolo <@> ufpr.br http://www.openaccessscience.com© 2012 Copyright by the Authors, licensee Open Access Science Research Publisher. [email protected]

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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-Brazil2Universidade 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 evaluateseed germination under different treatments and the establishment of a plant regeneration protocol. In vitro germinationcapacity of coated and uncoated seeds under different conditions was evaluated. Calli induction and plant regenerationwere 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 hoursreduced germination. The plant regeneration protocol was established from mature embryos and 0.5 mg L-1 of 2,4-D and1.0 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 ofsolanaceae family, native to tropical SouthAmerica, is an ornamental and medicinal plant,considered toxic, popularly known as angeltrumpet (Corrêa 1984; Zayed and Wink, 2004).Brugmansia suaveolens was formely describedas Datura suaveolens and in 1823, Bercht &Presl reclassified this species in Brugmansiasuaveolens. Brugmansia plants are woody treesor bushes, with pendulous, not erect, flowers,without spines on their fruit while Daturaspecies are herbaceous bushes with erect (notpendulous) flowers, and presenting spines ontheir fruit (Smith and Downs, 1966).

Species from the genera Brugmansia andDatura produce the atropine and scopolaminealkaloids. Both are organic esters formed by thecombination of an aromatic acid, tropanic acid

and a complex of bases tropine and scopine, ex-hibiting hallucinogenic, antispasmodic, diapho-retic and diuretic activities (Iranbakhsh et al.2007; Pitta-Alvarez et al. 2000).

Considering its pharmaceutical importance,knowledge about germinative behavior and es-tablishment of an efficient protocol for tissueculture and plant regeneration is a pre-requisitefor biotechnological processes (Valizadeh andValizadeh 2009) and for breeding programs(Benesi et al. 2010). Among these processes,genetic transformation using the Agrobacteriumsystem for insertion of genes and obtaining ofhairy roots in high and commercial scale is im-portant (Kieran 2001; Robins et al. 1991;Sheludko 2010).

Seed is the main way of reproduction for themajority of woody species, and its storage andgrowing methods may cause the losing in

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397Int. J. Med. Arom. Plants Seed germination and plant regeneration in Brugmansia suaveolens

Montanucci et al. http://[email protected]

germinative capacity (Amorim et al. 1997).Studies regarding germination are also im-portant for obtaining in vitro tissue culture(Verpoorte 2000).

In vitro procedures and plant regenerationare used to some degree in almost every majorplant species. The success of such biotechnolo-gy requires an efficient protocol for plant regen-eration from different explants (Sultana and BariMiah 2003). Many studies in the literature as-sessed the performance of the explant in regardto tissue culture and plant regeneration: anthers(Datura stramonium) (Sundar and Jawahar2010); adventitious stems and leaves (Daturametel) (De 2003); stems (Datura innoxia)(Zayeda et al. 2006); hypocotyls (Datura metel)(Muthukumar et al. 2000); buds (Daturainsignis) (Dos Santos et al. 1990), mature em-bryo (Datura stramonium) (Amiri et al. 2011).However, in the literature, there are no studiesthat have checked the plant regeneration in B.suaveolens

The purpose of this study was to carry outthe evaluation of seeds germination behaviorand the establishment of a regeneration protocolfrom mature embryos from Brugmansiasuaveolens.

Materials and methods

In vitro germination

Twenty two accessions of B. suaveolenswere collected on rural and urban areas inPalotina city, Paraná, Brazil. Coated (Experi-ment I) and uncoated seeds (Experiment II)were submitted to 17 different conditions tocheck germination viability. MS culture medium(Murashige and Skoog 1962) was used with halfconcentration of macro and micronutrients, 1%sucrose, activated charcoal at 1.0 g L-1, pH 5,8and 7.0 g L-1 agar without growth regulators.

Group I treatment consisted of submittingthe seeds to 4 ºC for 24, 48 and 72 h. To verifythe effect of high temperatures on the embryoand its germination, B. suaveolens seeds weresubmitted to 50 ºC in a water bath for 5, 10 and15 min (Group II). Group III treatment consistedto keep seeds under sulfuric acid at 50% for 5,10 and 15 min, followed by 3 rinses of auto-

claved and distilled water. The effect ofgibberellic acid (GA3) was assessed by adding20, 30, 40 and 50 mg L-1 to the MS medium(Group IV). The soaking effect was evaluatedkeeping seeds immersed in 50 mL of autoclavedand distilled water for 24, 48 and 72 h at roomtemperature (Group V).

After the application of the treatments, coat-ed and uncoated seeds were submitted to theaseptic treatment in a laminar flow. They weresubmersed for 10 min in 70% alcohol, followedby 2% sodium hypochlorite with two drops ofTween 80 for 20 min. Seeds were subsequentlywashed three times with distilled and autoclavedwater. After asepsis, the seeds were dried onfilter paper and inoculated in jars (600 mL vol-ume) containing 50 mL of MS medium. Eachtreatment consisted of 4 jars containing 5 seedseach. Seeds remained 7 days in the dark andthen were transferred to the culture growth room(16 h light /8 h dark) with temperature at 23 ± 2ºC. The germination experimental design wasrandomized and the accumulated germinationwas measured after 7, 14, 21, 28, 35 and 42days.

In vitro calli induction and plant regeneration

Brugmansia suaveolens seeds were collectedfrom the same plant (white biotype) and theircoats were mechanically removed. Asepsis wasperformed as described above. The embryoswere rescued with the help of a tweezers and ascalpel in a stereomicroscope. The MS medium(Murashige and Skoog 1962) was used for in-duction, maintenance and regeneration with halfthe concentration of macro and micronutrients,30 g L-1 of sucrose, pH 5.8, 8.0 g L-1 of agar and1.0 g L-1 of activated charcoal with decreasingconcentrations of 2,4-D and KIN, (Table 1). Re-generation phase consisted of absence of growthregulators. All medium was adjusted to.

At induction phase, the embryos remained inthe dark for 7 days, and, at the maintenance andregeneration phases, exposed to 25 ± 2 ºC with aphotoperiod (16 light /8 hours darkness).

After 30 days of culture at the induction me-dium, the embryos were evaluated according totheir calli induction competence. They were





subcultured every 7 days and the number andsize of calli were gotten. At maintenance phase(45th day), the number of green spots was evalu-ated. The number of regenerated plantlets wasassessed on the 60th day. The calli dry weight(g) was obtained by weighing the dried calli fol-lowing to 7 days in a laboratory oven at 50 ºC.The number of germinated embryos was alsoquantified.

Table 1: Concentrations of 2,4-D and KIN (mgL-1) used in treatments for calli induction andplant regeneration from mature embryos ofBrugmansia suaveolens.

Growth RegulatorsTreatments 2,4-D (mg L-1) KIN (mg L-1)

InductionT1 0.0 0.0T2 0.0 0.5T3 0.0 1.0T4 0.5 0.0T5 0.5 0.5T6 0.5 1.0T7 1.0 0.0T8 1.0 0.5T9 1.0 1.0

MaintenanceT2 0.0 0.25T3 0.0 0.50T4 0.25 0.00T5 0.25 0.25T6 0.25 0.50T7 0.50 0.00T8 0.50 0.25T9 0.50 0.50

The percentage of callus induction was ob-tained as regenerative indexes. This was calcu-lated according to the number of calli inducedbased on the number of embryos available. Theratio between the number of plantlets obtainedand the number of calli induced was also calcu-lated (Ali et al. 2007). Regeneration efficiencywas calculated by multiplication between thepercentages of induction and ratio of plantletsobtained per treatment. Five embryos per Petridish (90 x15 mm) and 10 replicates in eachtreatment were used at the callogenesis and re-generation experiments.

Statistical analysis

Germination data were submitted to descrip-tive statistical analysis. Callogenesis and plantregeneration data were transformed in arcsin √xwith the purpose of variance analyses. The anal-ysis of variance procedures and mean separationwere done using SISVAR statistical package(Ferreira 1999).

Results

In vitro germination

Coated seeds (experiment I) did not germi-nate under in vitro conditions until 42 days,showing a physical dormancy through the pres-ence of the rough cover. Uncoated seeds (exper-iment II), required at least 14 days for the be-ginning of the germination process, for lowerperiods any germination was observed. Controltreatment composed only by the MS medium,showed itself as an inductor for germination,probably through the presence of water and nu-trients. However, the maximum germinationrate for control in all the times assayed was 75%(Figure 1). Results obtained for the effect of 4oCexposure (Figure 1A) over different periodsshowed that B. suaveolens seeds had a positivecorrelation between germination and period as-sayed, the best responses occurred with 42 daysof culture; however, at 28 days, control had got-ten the highest germination rate (70%). In allperiods, there were no statistical differencesamong the treatments applied. The same conclu-sion was observed in Group II (seeds submittedto 50 ºC for different exposure times) where thegreatest germination rate occurred at the 28th

day, in spite of among all treatments did not dif-fer statistically (Figure 1B).

The effect of exposure to sulfuric acid(50%) (Figure 1C) showed a tendency of reduc-tion in germination probably due to the embryosinjuries. Only the exposure for 5 min presentedresponses statistically different in 28, 35 and 42germination days compared to control.

Gibberellic acid (GA3) is a potent growthregulator, inducer of dormancy’s breaking. Inthis experiment, germination was not observedin any of the concentrations assessed (data notshown). Such results show the gibberellic acid





may be promoting the inhibition of developmentof embryos at the dosages used in this experi-ment.

Soaking for 24 hours let to a rapid germina-tion (period of 14 days). However, as the dayswent by, the effect was stabilized and the treat-ments did not differ among them (Figure 1D).

Figure 1: Germination rates of uncoated seeds under different conditions. A - Group I: Temperaturesof 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 sig-nificantly different at p>0.05

Somatic embryogenesis and plant regeneration

The stages of callus induction and plant re-generation are shown in Figure 2. The explantsused were mature embryos (2 to 3 mm) (Figure2A). Some embryos germinated after 7 days ofdarkness (Figure 2B). The beginning of calliformation was observed at the 30th day (Figure2C). Embryogenic calli were observed after 40days in the maintenance media (Figure 2D).Calli were soft, white and friable. Plantlets witha well developed root system were observed af-ter 55 days (Figure 2E). Plants were regeneratedand acclimatized in pots (Figure 2F).

Data related to the regenerative indexes areexhibited to different treatments (Table 2).Treatments 2, 3, 6 and 7 exhibited the best per-

centages of callus induction (100%); however,only treatments 6 and 7 exhibited the best re-generation efficiencies (32%). Treatment 9, withthe highest levels of auxin and cytokinin, exhib-ited a low percentage of induced calli (21%),and proportionally, the highest ratio for numberof plantlets per induced callus (0.84).

Control treatment presented 58% of inducedcalli, but it was not observed regenerated plant-lets, which provided evidence for the importanteffect of the plant growth regulators on plantregeneration in this species.

Germination indexes are shown in Figure3A. Data among different treatments were sta-tistically similar and varied from 78 to 100%.





Table 2: Regenerative indexes obtained for thedifferent concentrations of 2,4-D and KIN (mg.L-1) used on calli induction and plant regenera-tion from mature embryos of B. suaveolensTreatment I C (%)* N P R C** ER (%)***

T1 58 0 0T2 100 0.17 17T3 100 0.13 13T4 74 0.24 18T5 66 0.22 15T6 100 0.32 32T7 100 0.32 32T8 96 0.20 19T9 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 andtreatment 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) (Figure3B). Treatment 9, with the higher concentrationof auxin and cytokinin exhibited an inhibitingeffect on the calli size (40 mm). In relation tocalli dry matter, treatment 6 generated the high-er 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 embriogeniccalli (Figure 3C), the best combination ofgrowth regulators was 0.5 mg L-1 of 2,4-D and 1mg L-1 of KIN (Treatment 6) with the highestscores although statistically similar to the oth-ers. The values of 1.0 mg L-1 of 2,4-D and 1.0mg L-1 of KIN (Treatment 9) promoted a de-crease in the number of green spots, showingthe sensitivity of the explants to doses greaterthan 1.0 mg L-1 of auxins combined tocytokinins.

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 re-generation medium; (F) Plant regenerated after acclimatization.

The percentage of regenerated plantlets percallus was showed in Figure 3D and the treat-ments 5, 6 and 7 regenerated 30, 35 and 45plantlets respectively. Control calli did not pre-sent regenerated plantlets, manifesting that, in

spite of getting calli induction, auxins andcytokinins are involved in cellular programmingand differentiation, without which there is noformation of a normal plantlet.





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

Discussion

The knowledge about botanic germinationperformance is the first step to the adoption ofbiotechniques in any species (Mazandarani et al.2007). Up to now, references were not foundabout the influence of environmental factorsover germination rates in B. suaveolens. It iswell documented that germination studies arefundamental to any plant multiplication scheme(Cho 2010; Idu et al. 2007). In order to test pos-sible injuries caused to embryos in uncoatedseeds, data showed that hot water bath effectover B. suaveolens germination are in agree-ment with Lêdo (1977) who verified the use ofhot water to overcome the dormancy ofSchizolobium parahybum.

On the other hand, Perez and Moraes (1990)observed that Prosopis juliflora seeds incubated

at 55 oC had the germination inhibited; however,50ºC, proved to be adequate for germination inthis study. Carneiro et al. (2010) related thatseeds treated with hot water at 50 ºC for 10 minpromoted a greater percentage of germinationand greater uniformity in Capsicum baccatum.Sarker et al. (2000) observed positive effect ingermination of Sesbania rostrata by boilingseeds to different periods (0-18 sec). These re-sults would be explained by the fact that hightemperatures could interact with growth regula-tors, promoting changes in their endogenouslevels and, consequently, influencing the germi-nation process (Gualtieri and Moraes 1990; Paulet al. 1973).

Franco and Ferreira (2002) observed deathof seeds in Didymopanax morototoni after sulfu-ric acid treatment. The same authors mentioned





the use of sulfuric acid would be more adequatefor seeds in which there is impermeability towater and not those there is resistance to embryogrowth. Sulfuric acid would act in weakeningcover´s seed by cuticle removing and the disso-lution of its macrosclereid cells (Martins-Conder et al. 1999). Freitas and Cândido (1972)found positive results in increasing germinationrate in Schizolobium parahyba seeds and over apapaya´s seeds (Tachigalia mutlijuge). Alt-hough, Idu et al. (2007) observed in Huracrepitans that 5 min treatment provoked 42% deabnormal seedling compared to zero percent inlonger period (15 and 25 min).

In the literature, low concentrations ofgibberellic acid had a positive effect on devel-opment of coffee plant embryos (Carvalho et al.1998). However, Moreira et al. (2010) workingwith Physalis sp (solanacea) observed an inhibi-tion effect doses of 1500 ppm of GA3, whichagrees with the results obtained for B.suaveolens. In this experiment, the concentra-tions were probably high and had an inhibitingeffect on germination.

The most important observation was thatMS medium was enough to promote seed ger-mination on B. suaveolens and the treatmentsapplied to check possible injuries to embryoswere not sufficient to increase or decrease ger-mination rates.

One of the basic requirements of an efficientprotocol is its quickness in obtaining regenerat-ed plantlets. Data showed that B. suaveolensrequired 60 days (∼8 weeks) to achieve a plant-let. In related species, Datura innoxia, Rahmanet al. (2008) were observed 6 weeks as a mini-mum period; as observed in Scopolia porniflora(Yong et al. 2008). Zayeda et al. (2006), ob-served a need of 10 months without the additionof hormones.

The results obtained agree with those foundby Amiri et al. (2011) who observed that in or-der to maintain regeneration of plants in Daturastramonium, the most important effect waspresence of 2,4-D and of 2,4-D and KIN togeth-er. It could be explained by the fact that the ki-netin would promote the effect of 2,4-D, but byitself, it would not be capable of callus for-

mation and regeneration as it was used in treat-ments 7 (2,4-D) and 2, 3 and 6 (2,4-D and KIN).

Other authors, checking the effect of theseregulators in vitro culture of Datura stramoniummature embryos, assayed concentrations varyingfrom 1-3 mg L-1 (2,4-D) (Parsaemehr andAlizadeh 2010) and 0-1 mg L-1 (KIN) and 1-2mg L-1 (2,4-D) (Amiri et al. 2011); others,moreover, assessed the effect of other growthregulators: 2.3-3.9 mg L-1 (2,4-D) and 3.4 mg L-

1 of 6-Benzylaminopurine (BAP) in Daturastramonium (Sundar and Jawahar 2010); com-binations of 1-4 mg L-1 BAP, IAA and GA3 inDatura metel (Nithiya and Arockiasamy 2010),3 mg L-1 of 1-Naphthaleneacetic acid (NAA) inBrugmansia cândida (Niño et al. 2003) and 0.5and 1 mg L-1 of BAP and indole-3-acetic acid(IAA), respectively (Zayeda et al. 2006).

Amiri et al. (2011) observed that in Daturastramonium, dosages of 2 mg L-1 of 2,4-D com-bined with 0.25 and 0.5 mg L-1 KIN were thebest for callus formation and higher dosages ofKIN, up to 0.5 and 1 mg L-1 inhibited it. Dataobtained in this study disagree with Brasileiroand Carneiro (1999) that did not observecallogenesis and plant regeneration in tomatoes(Lycopersicon esculentum) under the same con-ditions.

Several authors in the literature reported dif-ferent responses of callus induction: Parsaemehrand Alizadeh (2010) affirmed that the presenceof 2,4-D is essential for callus formation inDatura stramonium. Miskat et al. (2003) ob-served that dosages of 2 mg L-1 (2,4-D) present-ed the best responses to callus induction fromstem fragments of Datura metel. Amiri andKazemitabar (2011) studied in vitro plant regen-eration in Datura stramonium, observed thatconcentrations of 2.0 mg L-1 of 2,4-D were suf-ficient to enhance calli induction and plant re-generation.

Although, Sundar and Jawahar (2010) ob-served that only the 2,4-D presence in the medi-um is not by itself an inductor for callogenesisand regeneration of plants The authors reportedthat the best performances were obtained withconcentrations of 3.9 mg L-1 and 3.4 mg L-1 ofBAP. Otroshy et al. (2011) observed in anothersolanaceae (Capsicum annuum) no callogenesis





and low ratio of plant regeneration using BAPand IBA as growth regulators and cotyledons asexplants, showing that 2,4-D and KIN can beconsidered as potential inducers.

Differences in callus induction, regenerativecapacity and plant regeneration among thetreatments suggests inductive activity in cellulargrowth through the presence/absence of thesegrowth regulators in the culture medium. Con-trol exhibited formation of organogenic andembryogenic calli, manifesting that there is noneed of growth regulators for calli formation,possibly explained by the fact that an endoge-nous plant growth regulator imbalance, allied toa stress condition (imposed by in vitro condi-tion) would act as inductor (Perez et al. 2002).

The use of mature embryos certainly has theadvantages of time reduction by being availablethroughout the year, being able to be storedwithout the need of planting stock plants, whichcertainly favor the use of biotechnology in thisspecies.

Conclusions

The MS medium for uncoated seeds wassufficient to induce germination with satisfacto-ry performance. The in vitro propagation proto-col obtained from mature embryos as explantsoriginated healthy plants with a reasonable sur-vival rate. This protocol will be helpful in theestablishment of other biotechniques in order toproduce trophane alkaloids in industrial scale.

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