DOI: 10.14295/CS.v9i2.

2602
Comunicata Scientiae 9(2): 298-302, 2018
Scientific Note
e-ISSN: 2177-5133
www.comunicatascientiae.com

In vitro cultivation of Tamarindus indica L.:

explants obtention and contamination in culture medium

Antonio Flávio Arruda Ferreira1*, Laís Naiara Honorato Monteiro2,

Maria Gabriela Fontanetti Rodrigues3, Natália Batista Oliveira4, Aparecida Conceição Boliani4

1
University of São Paulo, Piracicaba, Brazil
2
São Paulo State University, Botucatu Brazil
3
São Paulo State University , Dracena, Brazil
4
São Paulo State University, Ilha Solteira, Brazil
*Corresponding author, e-mail: [email protected]

Abstract

The tamarind tree (Tamarindus indica L.) is a common tree in tropical countries with a
great exploitation potential due to its high nutritional value and important pharmaceutical
characteristics, justifying its potential as a promising crop. The scarcity of scientific studies of
the species, especially on propagation, hinders its availability and, consequently, the supply of
the product in the market. The aim of this study was to verify the obtainment of nodal segments
via sexual propagation and the in vitro establishment of sweet tamarind in MS culture medium
(25, 50, 75 and 100% of salts) and with or without activated charcoal (2 g.L-1). The experiment
was carried out in a completely randomized design in a 2 x 4 factorial scheme (presence and
absence of activated carbon x salt concentrations), with 25 replicates, each replicate consisting
of a test tube with an inoculated explant. According to the results, it is possible to conclude that
from seedlings with 45 days after sowing, nodal segments of sweet tamarind are obtained for
in vitro establishment. As a precursor of protocol for in vitro formation of healthy seedlings is
indicated the use of MS culture medium with 75 % of the salts and added with 2 gL -1 of activated
charcoal to reduce the contamination index.

Keywords: Tamarind, tissue culture, exotic fruit tree, salt concentration, active charcoal

Pomology is an agricultural sector including its bark, leaves, fruits, seeds and roots,
characterized by the diversification of production as a pharmaceutical ingredient, food for human
and with economic, social and nutritional roles, and animals, etc (Semenzato et al., 2014). The fruit
with special relevance in Brazil, where, given is its most consumed portion, containing vitamins,
its relative extension when compared to other tartaric and malic acids, sugars and due to the
countries, it is possible to grow temperate, tropical bittersweet taste, it is used as spice and as an
and subtropical species. ingredient in food products such as beverages,
Tamarind tree (Tamarindus indica L.) is gravies, hot sauces, and worcestershire’s sauce.
an arboreal fruit tree belonging to the Fabaceae It is also used as wood and animal feed source
family, native from Africa. It is currently cultivated (Pereira et al., 2008).
in several countries in humid and arid tropical Despite its several uses, only a few studies
regions (Ajiboye et al., 2010), with medicinal regarding the tamarind tree seedlings production
importance, mainly due to its high anti-oxidant can be found, which occurs by seeds and sexual
activity (Razali et al., 2015). propagation (Buyinza et al., 2010). However, plant
The tamarind tree is widely used, propagation by seeds presents the disadvantage
Received: 28 March 2017
Accepted: 03 March 2018
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of juvenility, which may lead to more than seven (Koné et al., 2015).
years to start the fruit production. Considering the Tamarind tree as a crop
Techniques aiming at reversing the with potential to expand, the aim of the present
mature stage of woody plants into juveniles, study was to assess the feasibility of obtention of
called rejuvenation, or at returning the plant to nodal segments by sexual propagation for in vitro
a high physiological vigor, named reinvigoration cultivation of sweet tamarind, evaluating the
(Bonga & Von Aderkas, 1983), are employed fungi and bacteria contamination in MS culture
to induce the growth of juvenile buds and to medium, with different salt concentrations and
increase the number of propagules with larger with or without active charcoal.
rooting potential. The experiment was carried on between
Thus, vegetative propagation with the November and December of 2015 in
use of micropropagation is an option to reduce a Pad&Fan greenhouse, with average
the time to start fruit production and to produce temperature and relative humidity of 25.6°C and
healthy seedlings with higher yield (Dantas et 76%, respectively. The sweet tamarind fruits were
al., 2012). The establishment is the stage where harvested at the end of the maturation stage,
the explants are established in vitro for the in Ilha Solteira - SP, Brazil, located at 20°24’04”
subsequent multiplication, rooting, and seedlings of latitude S and 51°20’55” of longitude W,
production experiments. and altitude around 320m. The climate is Aw,
For in vitro establishment beyond according to Köppen-Geiger's classification, with
concentrations of culture media, which influence annual average temperature of 24.5 ±3 °C.
cell growth, activated charcoal, when used, has The flesh of the collected fruits were
a capacity to reduce the oxidation of explants obtained using a sieve (3 mm) and running water.
and has been widely used in tissue culture of The seeds were dried in shade on absorbent
woody plants (Fermino Junior & Scherwinski- paper, at room temperature (26 °C) for two days
Pereira, 2012). (Figure 1a), followed by the sowing in 72-wells
However, one of the main problems polyethylene trays filled with fine grade exfoliated
during this cultivation stage is the contamination vermiculite (Figure 1b). The seeds were sown, one
mainly by endogenous bacteria and fungi, which per well, in a total of 248 seeds, irrigated twice
grow slowly and diffuse on the explants after a day using suspended micro-sprinkler with an
establishment or during the multiplication stage, average flow rate of 1800 cm3 min-1 for 5 minutes.
reducing or impairing the plantlets growth in field The initial emergence index and the
condition (Montovani et al., 2007). emergence 45 days after the sowing (Figure
In this context, studies involving the 1c), the length of the aerial part (cm), the stem
processes of germination and initial development diameter (mm), the number of leaf pairs and
of plantlets for vegetative propagation using the average number of nodal segments were
tissue culture are important to assure high-quality evaluated in an experimental design with four
seedlings and to promote the species cultivation repetitions and five plantlets per repetition.

Figure 1. Tamarind seeds (a) sowed in styrofoam trays filled with expanded vermiculite (fine texture) (b). Seedlings of tamarind
45 days after sowing (c). Ilha Solteira (SP), Brazil, 2016

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The nodal segments were collected randomized design, with a 2 x 4 factorial scheme
with approximately 1 cm of length with one (presence or absence of active charcoal x salt
bud each, from 45 days after sowing. The nodal concentration), with 25 repetitions, represented
segments were immersed in 70% (v:v) alcohol for by a test tube with one inoculated explant. After
one minute, then in a 1% active chlorine solution 30 days, the percent of tubes with microorganisms
(Qboa ) added three drops of neutral detergent
®
contamination was determined, and the
(Tween 20®) for 30 minutes. A triple washing averages were submitted to Tukey´s test at 5% of
was performed in a laminar flow cabinet using significance using the software SISVAR 5.6.
autoclaved distilled water to remove the residue Literature suggests that the average time
of the used products. for the tamarind seed germination is 13 days (El-
Following the triple washing, the nodal Siddig et al. 2006), but it may last up to one month.
segments were vertically inoculated in test tubes It is also reported that the seeds germinative
(2.5 x 15cm) with 20 mL of culture medium, sealed capacity can vary from 35 to 95 % (Costa et al.,
with plastic caps and kept in a growth room 2012). However, Queiroz et al. (2011) noticed
under 16-hours photoperiod, temperature of 25 that the tamarind plantlets emergence process
± 3 ºC, relative humidity of 45 ± 0.5 %, and active begins 6 days after sowing and lasts for 17 days,
photosynthetic radiation of 45-55 µmol m-2 s-1. with an average of 91.60%, corroborating with
The control solution (control) was distilled the results described by Pereira et al. (2008), using
water solidified with 3.5 g L-1 of agar, with pH seed scarification and 24 hours immersion.
adjusted as 5.7 ± 0.3 and autoclaved for 20 From the 248 evaluated sweet tamarind
minutes at 1 Kgf cm-3 and 121 °C. Treatments seeds, 92.7 % of the plantlets emerged at the
consisted of culture media containing 25, 50, 75 7th day after sowing, corroborating the data
and 100 % of salts in MS medium (Murashige and obtained by Queiroz et al. (2011), even without
Skoog 1962) with and without active charcoal the seeds scarification and soaking.
(2 g L-1). The treatments were added of vitamins Regarding the aerial part length and
as suggested by White (1943), 30 g L of sucrose, -1
stem diameter of the sweet tamarind plantlets,
adjusted pH of 5.7 ± 0.3 and solidified with 3.5 g L-1 only the plants between 30 to 40 cm height and
of agar. Then the medium was autoclaved for 20 with a diameter of 4.0 mm after 45 days of sowing
minutes at 1 Kgf cm-3 and 121 °C. are considered viable according to Pereira et al.
The experiment was set in a completely (2008) (Table 1).
Table 1. Shoot length (SL), stem diameter (SD), leaf pairs (LP) and the number of nodes (NN) of sweet tamarind
seedlings. Ilha Solteira (SP), Brazil, 2016

REP SL (cm) SD (mm) LP NN

I 24.40 ns 2.46 ns 11.20 ns 5.00 ns
II 22.70 2.44 11.20 5.00
III 22.50 2.65 12.20 5.40
IV 25.90 2.64 11.20 4.80
Average 23.88 2.55 11.45 5.05
ns
Not-significant according to Tukey´s test at 5% of probability.

After 45 days of sowing, in average, the sweet tamarind contaminated by pathogens

plantlets presented 23.88 cm in length and 2.55 incubated in MS culture medium, with or without
mm of diameter, which is considered below the activate charcoal is presented in Table 2.
adequate for field sowing. However, the average The control treatments (without salt)
numbers of leaf pairs (11.4) and nodal segments and with or without active charcoal addition
(5) were appropriate for the in vitro implantation presented, at 30 days, no regeneration of
of the species. In addition, according to the aerial part or callogenesis in the tamarind
Ferreira (2014), the use of nodal segments in explants, as observed by Ferreira (2014). Thus,
micropropagation is growing, mainly with woody since there was no incidence of pathogens,
species in the Fabaceae family. it is possible to conclude that the explants
The number of nodal segments of the disinfestation was satisfactory.

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Table 2. Contamination (fungi and bacterial) of nodal segments of sweet tamarind inoculated in culture media
with different salt concentrations. Ilha Solteira, SP, Brazil, 2016

CONTAMINATION (%)
Salt Concentration (%)
MS + CA** MS
Control 0 a* 0a
25 28 b 12 c
50 24 b 20 d
75 0a 20 d
100 0a 8b
Contamination Index 50% A 100% B
*Same lower case letters in the same column and uppercase letters in lines: means are not statistically different according to Tukey´s test at 5%
of probability.**MS + CA = MS medium with activated charcoal.

At the 30th day of cultivation, in the Buyinza, M., Senjonga, M., Lusiba, B. 2010.
presence of salt and active charcoal, the Economic valuation of a tamarind (Tamarindus
indica L.) production system: Green money from
nodal segments were contaminated by fungi
drylands of eastern Uganda. Small-scale Forestry
and bacteria in the concentrations of 25 and 9: 317-329.
50% of salt, whereas in the concentrations of 75
Costa, E., Ferreira, A.F.A., Silva, P.N.L., Nardelli,
and 100% of salt, there was no contamination.
E.M.V. 2012. Diferentes composições de
It is possible to observe that the salts included substratos e ambientes protegidos na formação
in the culture media have a nutritional role, and de mudas de pé-franco de tamarindeiro. Revista
influence cellular growth and morphogenesis Brasileira de Fruticultura 34: 1189-1198.

due to the osmotic properties. Dantas, A.C.V.L. Queiroz, J.M.O., Vieira, E.L.,
The nodal segments inoculated in Almeida, V.O. 2012. Effect of gibberellic acid and
the biostimulant Stimulate® on the initial growth
the culture medium without active charcoal,
of tamarind. Revista Brasileira de Fruticultura 34:
regardless the salt concentration, showed 8-14.
contamination by pathogens, showing that
El-Siddig, K., Gunasena, H. P. M., Prasa, B. A.,
both the salt concentration and the presence of
Pushpakumara, D. K. N. G., Ramana, K. V. R.,
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to harmful pathogens. On the other hand, the – Tamarindus indica L. Fruits for the future 1.
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other components, avoiding contamination by Fermino Junior, P. C. P., Scherwinski-Pereira, J.
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With the results obtained it is concluded 1-9.
that 45 days after sowing, seedlings presented an
Ferreira, A.F.A. Propagação vegetativa de
average of 5 nodal segments of sweet tamarind Tamarindus indica L. 2014. 95f. (Dissertação de
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