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Acta Scientiarum 


ISSN printed: 1679-9283 
ISSN on-line: 1807-863X 
Doi: 10.4025/actascibiolsci.v39i3.36312 


Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 381-388, July-Sept., 2017 

Production of pigments in Alternanthera sessilis calli mediated by 

plant growth regulators and light  

Cristini Milech


, Simone Ribeiro Lucho, Alítcia Moraes Kleinowski, Débora Berwaldt Dutra, 

Mariana Mühlenberg Soares


and Eugenia Jacira Bolacel Braga 


Laboratório de Cultura de Tecidos, Programa de Pós-Graduação em Fisiologia Vegetal, Departamento de Botânica, Instituto de Biologia, 

Universidade Federal de Pelotas, Capão do Leão, 96010-900, Pelotas, Rio Grande do Sul, Brazil. *Author for correspondence. E-mail: 

ABSTRACT. Among the compounds produced by plants, pigments such as betalains have received 

attention from both food and pharmaceuticals industries. The Alternanthera sessilis species produces these 
pigments, though in small quantities, and so it is necessary to increase production. Thus, many studies use 
elicitors that are capable of triggering physiological or morphological responses in plants. The objective was 
to establish callus production in A. sessilis grown under different combinations of growth regulators and 
light qualities and to assess whether these factors can increase betalain and flavonoid production. Leaf and 
internodal explants in MS medium with different growth regulators were used to obtain calli, which were 
subsequently transferred to a betacyanin induction medium remaining for 40 days under different light 
qualities (white, blue, red, and dark). The most suitable treatment for callus formation and subsequent 
betalain and flavonoid induction was to combine a medium containing 6.7 μmol L


 2,4-D and 9.0 μmol L



BAP and blue light. Physical elicitation by light combined with appropriate concentration of growth 
regulators on calli can increase production of commercially important metabolites.


Keywords: betalain, flavonoids, secondary metabolites, medicinal plants. 

Produção de pigmentos em calos de Alternanthera sessilis mediados por reguladores de 
crescimento e luz 

RESUMO. Dentre os compostos produzidos pelas plantas, os pigmentos, como as betalaínas, vêm 
recebendo destaque tanto pela indústria alimentícia como farmacêutica. A espécie Alternanthera sessilis 
produz esses pigmentos, porém em pequenas quantidades, sendo necessário incrementar a produção. Para 
isso, muitos estudos utilizam elicitores que são capazes de desencadear respostas fisiológicas ou 
morfológicas nas plantas. O objetivo do trabalho foi estabelecer a produção de calos de A. sessilis crescidos 
quando submetidos a diferentes combinações de reguladores de crescimento e qualidades de luz, e avaliar 
se esses fatores são capazes de incrementar a produção de betalaínas e flavonoides. Foram utilizados 
explantes foliares e internodais em meio MS com diferentes reguladores de crescimento para obtenção dos 
calos que, posteriormente, foram transferidos para meio de indução de betacianina, onde permaneceram 
por 30 dias sob diferentes qualidades de luz (branca, azul, vermelha e escuro). O tratamento mais propício 
para formação de calos e consequente indução de betalaínas e flavonoides foi a combinação do meio 
contendo 6,7 μmol L


 2,4-D e 9,0 μmol L


 BAP e a luz azul. Conclui-se que a elicitação física pela luz em 

conjunto com a concentração adequada de reguladores de crescimento em calos é capaz de incrementar a 
produção de metabólitos de interesse comercial. 

Palavras-chave: betalaína, flavonoides, metabólitos secundários, plantas medicinais. 


Light is one of the most important 

environmental factors for plants, it is a source of 

energy affecting their growth and development. 

However, excessive light can result in the 

accumulation of reactive oxygen species (ROS) and 

create disorders in plants, leading to death. In the 

evolutionary process, in order to protect themselves 

against  the  harmful  ROS  effects,   plants   develop 

protection mechanisms, including alterations in the 
production of secondary metabolites, such as 
pigments (anthocyanins, carotenoids, betalains) and 
antioxidants (Tariq, Ali, & Abbasi, 2014). 

Tissue culture techniques - including callus 

formation - are widely used as alternative method 

for producing and accumulating secondary 

metabolites (Al-Jibouri, Abed, Ali, & Majeed, 2016). 

Calli can be induced in response to organogenetic 

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Milech et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 381-388, July-Sept., 2017 

stimuli using different growth regulators and 
environmental conditions and, in general, they 

present varied forms and sizes and a certain degree 

of cellular wall thickening (Carvalho et al., 2011). 

One example of natural products receiving 

attention in the pharmaceutical market is betalains. 

A class of water soluble nitrogen containing plant 

pigments of the order Caryophyllales which consists 

of the yellow betaxanthins and the violet betacyanins 

(Gandia-Herrero, Escribano, & Garcia-Carmona, 

2005; Yusuf, Shabbir, & Mohammad, 2017). 

Within the Caryophylalles order, the 

Alternanthera genus stands out in terms of betalain 
production and Alternanthera sessilis species is quite 
well known for its medicinal properties, thus being 
of interest to associate its possible pharmacological 
effects with the optimization of pigment production 
(Hossain, Faisal, Rahiman, Jahan, & Rahmatullah, 

Due to environmental fluctuations in nature, 

these plants cannot provide sufficient amount of 
such compounds. Therefore, the use of 
biotechnological tools is common to meet the 
demand to try producing and increasing the 
quantities of these metabolites, such as practicing in 
cultivation associated with using elicitors 
(Pérez-Alonso et al., 2014). 

Elicitors are chemical, physical, or biological 

products or stimuli that are able to induce 
morphological or physiological alterations in 
organisms (Vasconsuelo & Booland, 2007). As a 
physical factor, light is especially important for 
betalain production, as seen in studies carried out on 
beetroot (Shin, Park, & Paek, 2013) and Suaeda salsa 
(Wang & Liu, 2006), as it acts in reprogramming 
plant metabolism. According to Zhao, Sun, Gao, 
Sui, and Wang (2011), the quality and quantity of 
light affect betacyanin synthesis. 

In this context, the purpose of this paper was to 

use  in vitro cultivation to produce calli in A. sessilis 
grown under different combinations of growth 
regulators and light qualities, aiming at evaluating 
whether these factors can increase betalain and 
flavonoid production. 

Material and methods 

We used three parent plant specimens of 

Alternanthera sessilis  (L.)  R.  Br.  Ex  DC. 

(Amaranthaceae) for in vitro  culture.  Plants  were 

collected in the municipalities of Rio Grande, state 

of Rio Grande dos Sul (2006) grown in the 

greenhouse identified by Élen Nunes Garcia. The 

voucher specimen was deposited in the Herbarium 

of the Botany Department of the Universidade Federal 
de Pelotas
 in RS, under 24.534. 

Leaf and internodal explants originated from 

aerial parts of plants cultivated in vitro in MS 

medium (Murashige & Skoog, 1962) were used to 

induce calli. Therefore, MS medium containing 100 

mg L


of mioinositol, 30 g L


 of sucrose, 7 g L



agar, and pH adjusted to 5.8 was used. The media 

were supplemented with 2,4-D, in 0.0, 4.5, 6.7, and 

9 μmol L


 concentrations, and BAP in 0.0, 4.5, 6.7, 

and 9 μmol L


 concentrations, totaling 16 types of 

cultivation medium. Moreover, combinations of 

2,4-D (9 μmol L


) x IAA (1.4, 2.8, 4.2, and 5.6 μmol 



) were tested, totaling four types of medium. Petri 

dishes were randomly distributed with occasional 

rotation and kept in the dark at 25 ± 2° C for 20 

days and then transferred to light for 10 days where 

they were kept a 16 hours photoperiod at 25° C ± 2 

(Reis et al., 2017). 

After 30 days, the percentage of calli formed in 

the callus inductor medium (CIM) was evaluated. 

The formed calli were transferred to dishes with MS 

medium with 2.2 μmol L


of Thidiazuron (TDZ) 

and 5.3 μmol L


of naphthalene acetic acid (NAA), 

0.5 mg L


of adenine, and 3 mg L


of ascorbic acid 

(AA), known as BIM medium – betacyanin 

induction medium according to Zhao, Sun, Chen, 

and Wang (2010), and kept under white light 

provided by fluorescent bulbs. 

This stage was carried out in order to choose the 

best media for calogenesis induction and pigment 
formation. After choosing, the experiment was 

repeated with the four best callus inductor media 

(CIM), which were called M1, M2, M3, and M4, 

and subsequently distributed between the different 

light qualities (white, blue, red, and dark), in the 

BIM medium for 40 days and 16 hours photoperiod 

at 25° C ± 2. The different light qualities were 

provided by fluorescent tube (Sylvania


 - 40W) for 

white light, blue compact fluorescent lamp for blue 

light (Taschibra


 - 14W - peak emission in 470 nm), 

and red compact fluorescent for red light (G-light



15W - peak emission in 660 nm). The photon flux 

densities for the white, blue, and red lights, 

measured with a Hansatech


 Quantum Sensor 

QSRED light meter were 25, 12, and 22 μmol m




respectively. Dishes exposed to the dark remained in 


In order to extract the flavonoids and 

bethanidine, fresh mass of calli obtained in each 

treatment were used, which were macerated in a 

porcelain mortar using acetate/methanol (70/30%, 

v/v) extraction buffer, pH 5.0, plus sodium ascorbate 

of 10 mM. The homogenized product was filtered 

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Incresing of pigments in Alternanthera sessilis 


Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 381-388, July-Sept., 2017 

in gauze and centrifuged at 10.000 g for 20 minutes, 
at 4° C (Reis et al., 2017). 

The flavonoid concentration was expressed in 

μmol of quercetin per gram of fresh mass and the 
readings were carried out in a spectrophotometer at 
330 nm. For bethanidine the molar extinction 
coefficient used for the calculation was €  = 54000  




 at a wavelength of 536 nm. 

In order to extract the amaranthine, bethanin, 

and miraxanthine, the methodology described above 
was used, substituting the extraction buffer with a 10 
mM phosphate buffer, pH 6.0. The amaranthine, 
bethanin, and miraxanthine concentrations were 
determined using a molar extinction coefficient of  

 = 56600 M




,  € = 65000 M




, and  

 = 48000 M




, respectively. The readings were 

carried out in a spectrophotometer at 536 nm for 
amaranthine and bethanin and at a 480 nm 
wavelength for miraxanthine. 

In order to determine the best callus induction 

medium and type of explant, the experimental 
design was completely randomized, represented by 
20 media types and two explants types. In order to 
choose the best combination of medium and light 
quality, the design was in a 4 x 4 factorial scheme 
(four media and four light qualities). Both 
experiments were conducted in triplicate, each one 
containing five explants. The data were subjected to 
variance analysis (p ≤ 0.05) and the average 
compared by Tukey test with a 5% error probability, 
using the Winstat 1.0 Statistical Program (Machado 
& Conceição, 2007). 

Results and discussion 

As the objective of this work was producing 

pigments in callus culture, we evaluated three main 
aspects in the cultivation (percentage of callus 
formation, color and callus appearance) to select the 
best culture media for experimental sequence as 
shown in Table 1. 

Considering these three aspects, the media 

showing these three desired characteristics were 
those containing the 2,4-D growth regulator at 
concentrations of 6.7 μmol L


 and 9.0 μmol L



association with BAP at these concentrations (M11, 
M12, M15 and M16). Pigments observed in these 
media are shown in Figure 1. 

Although medium 13 showed favorable results 

in relation to callus induction, it formed friable calli 
and, according to Ahmad, Rab, and Ahmad (2015), 
very friable calli accumulate water and tend to 
undergo rapid oxidation, compact calli are more 
desirable. Although exhibiting good callus 
production rate, Media 10 and 14, were not efficient 
in pigment production, suggesting that high 
concentrations of BAP are required to express the 

The use of two auxins combined (2,4-D and 

IAA) was not efficient for callus formation in 
internodal explants however, in leaf explants, the 
calli formation percentage reached 50%, 
corroborating Kakani, and Peng (2011), who found 
that for callus formation a combination of cytokinin 
and auxin is needed. 

Table 1. Percentage of callus formation in callus induction medium (CIM) and callus coloring derived from leaf (L) and internodal (I) 

explants of Alternanthera sessilis after 20 days in the dark and 10 days in the light in betacyanin induction medium (BIM). 

Induction Medium 

% of callus formation 



Leaf Internodal Leaf Internodal Leaf Internodal 

Medium 1 (without regulator) 

Medium 2 (0.0 2,4-D + 4.5 BAP) 

Medium 3 (0.0 2,4-D + 6.7 BAP) 

Medium 4 (0.0 2,4-D +9.0 BAP) 

Medium 5 (4.5 2,4-D + 0.0 BAP) 

Medium 6 (4.5 2,4-D + 4.5 BAP) 




Medium 7 (4.5 2,4-D + 6.7 BAP) 




Medium 8 (4.5 2,4-D + 9.0 BAP) 







Medium 9 (6.7 2,4-D + 0.0 BAP) 







Medium 10 (6.7 2,4-D + 4.5 BAP) 







Medium 11 (6.7 2,4-D + 6.7 BAP) 







Medium 12 (6.7 2,4-D + 9.0 BAP) 







Medium 13 (9.0 2,4-D + 0.0 BAP) 







Medium 14 (9.0 2,4-D + 4.5 BAP) 







Medium 15 (9.0 2,4-D + 6.7 BAP) 







Medium 16 (9.0 2,4-D + 9.0 BAP) 







Medium 17 (9.0 2,4-D + 1.4 IAA) 

Medium 18 (9.0 2,4-D + 2.8 IAA) 




Medium 19 (9.0 2,4-D + 4.2 IAA) 




Medium 20 (9.0 2,4-D + 5.6 IAA) 




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Milech et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 381-388, July-Sept., 2017 


Figure 1. Callus pigmentation of Alternanthera sessilis in leaf (A) and internodal (B) explants growth in different callus inductor media and 

after transferring to a betacyanin inductor medium, after 40 days of cultivation. (A1-B1) – calli originating from medium 11 (6.7 μmol L



2,4-D + 6.7 μmol L


 BAP), (A2- B2) –calli originating from medium 12 (6.7 μmol L


 2,4-D + 9.0 μmol L


 BAP), (A3-B3) – calli 

originating from medium 15 (9.0 μmol L


 2,4-D + 6.7 μmol L


BAP), and (A4-B4) – calli originating from medium 16 (9.0 μmol L



2,4-D + 9.0 μmol L


 BAP). Bar = 1 cm. 

Auxin (2,4-D) alone or combined with 

cytokinins, such as BAP, is widely used to stimulate 

callus formation, however, the concentration of 

these regulators should be defined for each species 

(Castro, Braga, Souza, Coimbra, & Chagas, 2016). 
In a study carried out by Trejo-Tapia et al. (2008), a 

1 mg L


of 2,4-D in Beta vulgaris combined with 1.0 

mg L


 of CIN produced calli in leaf explants with a 

high betalain concentration. When evaluating callus 

formation in Barringotonia racemosa, Dalila, Jaafar, and 

Manaf (2013) observed that the best results were 

obtained with a 1.5 mg L


 concentration of 2,4-D 

together with cytokinin in 0.5 to 1.0 mg L




The plant growth regulator 2,4-D has greater 

mobility and lower oxidation and conjugation rates, 

and so it would be efficient for promoting calli 

together with indole acetic acid (IAA) or 

naphthalene acetic acid (NAA), explaining the 

results obtained in this paper (Naz & Khatoon, 


The media containing the concentrations of 

growth regulators that presented the best pigment 

formation in callus development in white light were 

repeated and called M1 (6.7 μmol L


2,4-D + 6.7 

μmol L


BAP), M2 (6.7 μmol L


2,4-D + 9.0 μmol 



BAP), M3 (9.0 μmol L


2,4-D + 6.7 μmol L


BAP), and M4 (9.0 μmol L


2,4-D + 9.0 μmol L


BAP). The material originating from these media 

was transferred to a BIM medium and exposed to 

different light qualities (dark, red, blue, and white), 

where it remained for 40 more days. 

The results for the concentration of flavonoids 

present in A. sessilis calli (Figure 2A) revealed that 

there was interaction between the light treatments 

and cultivation medium, with it being possible to 

observe that the blue light combined with the M2 

cultivation presented the most significant results, 

reaching an average of  9.32 μmol of quercetin per 
100 g of callus fresh mass, this quantity being greater 

than that found by Reis et al. (2015) where in the 

maximum concentration obtained with this species 

in blue light was 1.6 μmol of quercetin per 100 g of 

callus fresh mass. 

The blue light would be associated with 

increased gene expression at specific points on the 

flavonoid biosynthesis route, such as the FaCHS 

expression, according to Kadomura-Ishikawa, 

Miyawaki, Noji, and Takahasni (2013). Studying 

Kolonchoe pinnata, Nascimento et al. (2013) observed 

accentuated increase in flavonoids when exposed to 

blue light. This increase was also observed by Xu et 

al. (2014), total anthocyanin content of strawberry 

fruits significantly increased after four days of 

treatment with blue light (40 μmol m




) at 5° C 

compared to the control fruits. 

The amaranthine content was also significantly 

greater in the combination of M2 medium and blue 

light, reaching a value of 147.6 mg of amaranthine in 

100 g of callus fresh mass (Figure 2B). This value 

was much higher compared with previous studies 

that used elicitors to increase this pigment in species 

of the Alternanthera genus, in which the maximum 

concentrations obtained were approximately 40 mg 

of amaranthine in 100 g of fresh mass (Perotti et al., 

2010; Reis et al., 2015). 

The bethanidine concentration in M2 combined 

with blue light was 71.42 mg in 100 g of callus fresh 

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Incresing of pigments in Alternanthera sessilis 


Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 381-388, July-Sept., 2017 

mass, this result being significantly greater than in 
the other treatments (Figure 2C). This increase was 

also observed in the betanin concentration, reaching 

an average of 59.34 mg of betanin per 100 g of callus 

fresh mass (Figure 2D). Similar bethanin 

concentrations were obtained by the study from Reis 

et al. (2015), but when A. sessilis and A. brasiliana 

plants were exposed to red light. 

For the betaxanthin levels (Figure 2E and 2F), it 

was possible to observe that the interaction between 

the callus cultivation medium and blue light was not 

significant, with only the isolated parameters being 

significant. The cultivation media known as M1 and 

M2 were more efficient in the production of 

betaxanthins. With regards to light quality, blue light 

was significantly better, having resulted in an 

increase of up to 10 times the miraxantin content 

compared with the white treatment. Bhuiyan, 


  and Adachi (2002), by evaluating cell 

cultures of the Portulaca  sp genus, also observed an 

increase in this parameter when the plants were 

exposed to blue light. 

Analyzing the effect of light on betacyanin 

production in Mesembryanthemum crystallinum, Vogt et 

al. (1999) verified that an increase in photon flux 

resulted in an increase in betacyanin quantity, with it 

being possible to observe with the naked eye that 

younger leaves had a more intense purple coloring 

than the control and those that remained under 

lower flux densities. The increase in betalain 

production in callus of Portulaca  exposed to blue 

light was also observed by Kishima, Shimaya, and 

Adachi (1995). where they justify that this fact may 

have occurred through a signaling system that would 

activate the gene expression given by the blue light 




Figure 2. Level of Flavonoids (A), Amaranthine (B), Bethanidine (C), Bethanin (D), and Miraxanthine (E and F) present in the calli of 
Alternanthera sessilis plants, after elicitation in different light qualities.M1 (6.7 μmol L


 2,4-D + 6.7 μmol L


 BAP), M2 (6.7 μmol L



D + 9.0 μmol L


 BAP), M3 (9.0 μmol L


 2,4-D + 6.7 μmol L


 BAP), and M4 (9.0 μmol L


 2,4-D + 9.0 μmol L


 BAP). Averages 

followed by different uppercase letters differ between each other in relation to the type of light for the media. Averages followed by 
different lowercase letters compare cultivation media in relation to a particular light using the Tukey test (p˂0.05). 

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Milech et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 381-388, July-Sept., 2017 


According to Heo et al. (2012), blue light acts 

directly in the signaling of secondary metabolism 

genes, as well as being important for the formation 

of chloroplasts and pigments, since red is involved 

in the development of the photosynthesis apparatus. 

Kinoshita et al. (2003) reported that blue light is 

responsible for maintaining electric potential in the 

membranes, forcing the stomata to open and 

allowing more CO


 to enter, and consequently 

increasing photo assimilate content, which would 

stimulate the production of secondary metabolites. 

Cryptochromes are blue light receptors linked to 

a flavin and pterine, and responsible for mediating 

various plant responses, such as regulation of 

circadian rhythms, depolarization of membranes, 

anthocyanins production, and other effects (Yu, Liu, 

Klejnot, & Lin, 2010). 

Since these cryptochromes act in the production 

of anthocyanin, we believe that these receptors may 

also be associated with the production of betalains, 

once they are exclusively present in the order 

Caryophyllales, replacing anthocyanins (Gandia-

Herrero et al., 2005). According to Khan and 

Giridhar (2015) in prehistory, these pigments 

coexisted and during evolution, some anthocyanin 

synthesis enzymes were lost and this order evolved 

the formation of pigments for their protection, 

through another metabolic route. They also 

reinforce that the similarity can be explained by 

hegemony analyzes, in which L-DOPA dioxygenase, 

enzyme that leads to the formation of betalamic acid 
precursor of many betalains, bears similarity to 

enzymes of the route of anthocyanin biosynthesis. 

According to Cao et al. (2012), light acts in the 

formation of the dihydropyridine portion of 

betalains, the chromophore, responsible for the 

formation of color, and therefore the absence of this 

could impair biosynthesis. 

The fact that the calli exposed to white light 

presented a lower concentration of betalains in this 

experiment may be explained by the study from 

Hatlestad et al. (2015), who affirm that this light is 

responsible for degrading betalains that are already 

formed, however, it would be fundamental for 

initiating biosynthesis, since white light acts in the 

DOPA-dioxigenase enzyme at the beginning of the 


Lee et al. (2014) reported that light is not a 

limiting factor in betalain synthesis, but that in 

combination with other factors it can alter the 

quantity of product formed. This association 

between different factors supports the results 

obtained in this paper, in which significant increases 

in betacyanins were obtained with the M2 

cultivation medium with a 6.7 μmol L


 2,4-D and 

9.0 μmol L


 BAP, in combination with blue light. 

Other question possible is that different light 

qualities can generate a plant imbalance and form 
oxygen reactive species and may have been the cause 
of this accentuated increase in betacyanins in 
response to blue light, in combination with the M2 
medium, since betacyanins are considered 

In a study with Saueda salsa, Wang and Liu (2006) 

reported an increase in betacyanins in the dark and 
that, probably, the signal for triggering this increase 
had been the accumulation of oxygen reactive 
species. Ramakrishna and Ravishankar (2011), in 
their reviews regarding the effect of abiotic factors in 
increasing secondary metabolites, cite various papers 
that have proven that different colors and spectrums 
are associated with the production of metabolites of 
interest and they also suggest that the production of 
oxygen reactive species induce antioxidant pathways 
such as betalains. 

Lage, Tirado, Vanicore, Sabino, and Albarello 

(2015) emphasize that cell suspensions and calli are 
considered the most efficient biotechnological 
techniques for in vitro production of special 
metabolites, favoring the formation of a more 
homogenous system with high cellular proliferation 
with potential for large-scale production of these 


The use of natural pigments has been gained a 

great deal of commercial appeal, but their 
production is limited, since large mass quantities 
should be used for extraction and purification. The 
use of biotechnological techniques used in this 
work, such as the in vitro culture for callus 
production in A. sessilis grown under different 
combinations of growth regulators and light 
qualities, were efficient in increasing biosynthesis of 
betalains with potential for subsequent commercial 


The authors gratefully acknowledge the CNPq 

(Conselho Nacional de Desenvolvimento Científico e 
) for their financial support and 
researcher's fellowship EJBB, the CAPES 
(Coordenação de Aperfeiçoamento de Pessoal de Nível 
 and the FAPERGS (Fundação de Amparo à 
Pesquisa do Estado do Rio Grande do Sul
) for 
supporting the research. 

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Incresing of pigments in Alternanthera sessilis 


Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 381-388, July-Sept., 2017 


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Received on March 20, 2017. 
Accepted on June 13, 2017. 



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