Indução de calos e regeneração de embriões em anteras de Coffea arabica L. por
nitrato de prata e etileno
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Introduction
Coffee is the second most important commodity in the international market,
coming second only to petroleum. Its industry generates more than 100 million
jobs around the world. Around three-fourths of all coffee grown enters the
international market, and 70% of this coffee is produced by small properties,
each of less than 10ha, in more than 60 mostly developing countries (CENTRO DE
INFORMAÇÕES, 2010). World coffee production in 2010 should be around 124
million 60 kg sacs, and according to estimates from Companhia Nacional de
Abastecimento (CONAB), Brazil will be responsible for 47 million (38%). Within
these 47 million sacs of home production are included 35.3 million sacs of
Arabica coffee and 11.7 million sacs of Robusta coffee; the Arabica type
therefore, representing about 75% of total Brazilian production (EPTV, 2010).
There are about 100 described species in the genus Coffea, but only two, C.
arabicaL.and C. canephora, yield fruits with economic importance in the
international market; those of C. arabicayield a better quality beverage and
represent around 70% of the world's production (MINEIRO, 2006).
Biotechnology offers alternative strategies for generating new and improved
coffee varieties, including those resistant to environmental extremes, pests
and diseases, and with low levels of caffeine, and of uniform fruit maturation.
Large improvements in somatic embryogenesis, the development of haploids, and
scaled-up micro-propagation have been achieved in the last 5 years (BUZZY et
al., 2007). The use of genetically improved cultivars could be in part a
solution for these problems, since breeding programs have presented promising
results for coffee culture, including yield increases and new cultivars.
Conventional breeding techniques have been widely used for this purpose.
A number of attempts have been made to reduce the time needed for embryogenesis
in coffee. Triacontanol, silver nitrate (AgNO3), salicylic acid, thidiazuron
and 2IP are the growth regulators most used in coffee embryogenesis for this
purpose. According to Giridhar, Indu and Vinod (2004), direct somatic
embryogenesis was obtained from hypocotyl explants of in vitro regenerated
plantlets of C. arabica and C. canephora in a modified MS medium containing
AgNO3 supplemented with N6 benzyladenine and indole-3-acetic acid.
Contrasting responses to ethylene have been observed during the somatic
embryogenic process. Ethylene may stimulate the embryogenic response in some
systems, while inhibiting it in others. Both these effects of ethylene have
been observed in the coffee embryogenic response, which is in agreement with
the responses generally observed for growth regulators: a stimulant effect at
optimum concentration and inhibition at supra-optimum levels. (HATANAKA et al.,
1995).
Silver ion is considered one of the most effective inhibitors of this ethylene
action. Although its effect is not well understood, it is supposed that the
silver ion binds a possible ethylene receptor at the plasma membrane, thus
inhibiting the binding of ethylene to this receptor and consequently triggering
the specific action of the hormone (TEIXEIRA et al., 2002).
This study evaluated the effect of AgNO3 and ethylene on anther cultures of
Coffea arabicaL. cv. Catuaí Vermelho 99 in inducing the formation of calli and
regenerating bi-haploid seedlings.
Material and methods
The experiment was done in the Biotechnology laboratory of the Instituto de
Ciências Agrárias at the Federal University of Uberlândia (UFU), Uberlândia MG,
from August 2004 to December 2006. The plant material was Coffea arabica L.
cultivar Catuaí Vermelho LCH-2077-2-5-99, obtained from the experimental area
of Campus Umuarama at the University.
The flower buds were collected between 8 and 9 am, stored in Petri dishes
containing tissue paper moistened with distilled water, placed in a Styrofoam
box to prevent drying, and taken immediately to the laboratory. Flower-bud size
was measured with calipers; 4.5 to 6.0 mm long buds being selected.
The flower buds were wrapped in sterilized cheesecloth and disinfested in 70%
alcohol for a few seconds followed by a 1% sodium hypochloride solution for
fifteen minutes, with constant stirring. Subsequently, the buds were rinsed
three times with sterile distilled water in a flow-hood. The anthers were
removed from the flower buds through a side cut with the aid of a stereo
microscope, tweezers and scalpel, these being flame-sterilized for each bud.
The anthers were dipped in 600 mg L-1ascorbic acid, 0.2% sodium hypochloride
and sterile distilled water for a few seconds before seeding in the medium. The
culture medium had its pH adjusted to 5.9 and was sterilized in an autoclave at
121 ºC at 1 atm. for twenty minutes.
In the first part of the experiment, flower buds of the coffee cultivar Catuaí
Vermelho 99 were collected and disinfested. The anthers were removed with care
to avoid damage, and were seeded in sterilized Petri dishes containing the
medium MS (MURASHIGE; SKOOG, 1962), to which 2 mg L-1 2,4-D had been added. The
experimental design was completely randomized (CRD), with 10 treatments
combining the exposure-time to ethylene (2; 4; 6; 8 days and the control) both
with and without 5 mg L-1 AgNO3in four repetitions, where each experimental
unit consisted of one Petri dish containing 25 anthers. Damaged anthers were
discarded. A drop of ETHREL® was placed in the center of each dish after
seeding, with the aid of a sterilized disposable syringe; subsequently the
plates were wrapped and placed in the dark for different periods (the control,
2; 4; 6 or 8 days). After the first incubation period the plates were opened in
a flow-hood enabling the ETHREL® to evaporate completely from the dishes which
were then rewrapped and maintained in the dark for up to another eight days
before being transferred to the growth room and maintained at 26 ºC with 16
hours of light at a photon flux of approximately 170 µmol m-2 s-1 where they
were kept until the twelfth day after inoculation.
In the second part of the experiment at the end of the twelve days, the anthers
were transferred to a regeneration medium, called medium R by the authors
(Sibi; Dumas de Vaulx; Chambonnet, 1979), supplemented with 0.108 mg L-
1 kinetin, where they were kept for 30 days.
The experimental design was completely randomized (CRD), with 10 treatments
combining the exposure-time to ethylene (2; 4; 6; 8 days and the control) both
with and without 5 mg L-1 AgNO3 in four replications, where each experimental
unit consisted of one Petri dish containing 25 anthers.
Twelve days after inoculation, in the first part of the experiment, the
percentages of anthers that were either oxidized, contaminated or contained
intumescences and calli was determined, this procedure being repeated thirty
days after seeding in the R medium (the second part of the experiment), using
the same variables and additionally the pro-embryoid count. The data were
submitted for statistical analysis using the program PROPHET. When a lack of
normal distribution of the residues and or the absence of homogeneity of
variances were observed, the data were transformed by square root of √x + 1/
2 and run through the program SISVAR (FERREIRA, 2003). The F test was performed
at 5% significance, the averages compared using the Tukey test, and polynomial
regression was used for the study of the effect of ethylene.
Results and discussion
In the first part of the experiment, based on the analysis of variance,
significant differences were found for the variables oxidation, intumescence
and contamination in the presence of ethylene. Only the variable contamination
was significantly affected by Silver nitrate (TAB._1).
As ethylene exposure-time increased, the number of oxidized anthers also
increased, becoming stable (4.0605) after approximately two days (FIG._1),
suggesting that ethylene is capable of oxidizing the anthers in a very short
time after inoculation; incubation in darkness could also have contributed to
this result.
The results obtained in this study are similar to those of Marques (2006).
Although some oxidation was observed, anther development was not jeopardized,
subsequently presenting calli and thus far, possible direct embryogenesis.
Figueira (2005), studying the same cultivar Catuaí Vermelho 99, confirms the
hypothesis mentioned above in relation to seeding time and oxidation, observing
oxidation of all anthers, even in those maintained in darkness for up to 60
days. Fialho et al. (2005), working with anther culture of Anacardium
occidentale L. observed better anther development when these were initially
kept in the dark. Maintaining the anthers in the dark, although favorable to
their development, was not effective in the present study, since even under
that condition they were found to have oxidized in the final evaluation; this
result is different from that of Silva (2003), who states that oxidation tends
to decrease when Coffeaanthers are incubated in the dark.
Another factor that may explain explant oxidation is the fact that coffee is a
woody species and liberates phenols. For this reason, antioxidant substances
such as activated charcoal, polyvynilpyrolidone (PVP), ascorbic acid or citric
acid, should be added to the medium (ANDRADE, 1998).
Development of intumescences was observed while the explants remained in
contact with ethylene for 8 days, reaching the smallest average for this
variable (2.17) corresponding to 13.19%. The explant had better averages of
intumescence in the first periods of contact with ethylene, including the
control, the highest average being observed in the control (3.87) (23.25%) at 2
days of contact (3.65) (22.12%), although there were no significant differences
between 4 and 6 days in the presence of ethylene (FIG._2).
When we consider the two-day period, the ethylene-stimulated intumescence of
the Cultivar Catuaí Vermelho 99 has an average of (3.87). It is assumed that
ethylene induces anther intumescence on first contact; however, as time passes
it has the opposite effect, reducing the number of intumescences and leading to
explant stagnation. This demonstrates that the studied cultivar is responsive
to intumescence and future callus development. The same fact was observed by
Figueira (2005) who obtained high indices of intumescence with Catuaí Vermelho
99 anthers soon after seeding.
Contamination was high in the first days after seeding, when the anthers were
not exposed to ethylene, reaching 34.61%, which is considered relatively high
for plant-tissue culture. No significant differences were found for the other
periods of exposure to ethylene (FIG._3). According to the analysis of
variance, the presence or absence of silver nitrate was significant in the
contamination. Contamination was lowest in the presence of nitrate, in contrast
to the material which had been inoculated in its absence (data not shown).
One of the hypotheses for reduced contamination by fungi and bacteria, is that
silver nitrate acts as a chemical control agent. Another hypothesis is that it
can inhibit anther intumescence, since the explant seeded in its presence
showed low development or did not intumesce, as observed during evaluation
(data not shown). The contamination level could also be related to the plant
genotype, since the anthers were collected from field plants.
In the second part of the experiment, the analysis of variance shows
significant values only for the variables of intumescence and callus formation
(callosity) (TAB._2). Due to contamination, treatment T10 (8 days with
ethylene/no nitrate) was lost; therefore analysis was carried out only for the
remaining treatments.
Anther intumescence was effective in treatments T1, T2, T8 and T9 with averages
of 2.71 (17.12%), 3.02 (19.09%), 2.20 (13.89%) and 2.49 (15.78%) respectively;
whereas in the other treatments no significant differences were seen (TAB._3).
Based on these results, it can be stated that the treatments which originated
from the control batch in the first part of the experiment and those from the
six and eight day contact periods with ethylene, were those that performed best
in medium R (TAB._3). It can therefore be inferred that on first contact with
ethylene, followed by a change in growth medium, the explant intumesces,
favoring callus formation. This is highlighted in the same table, where the
best results for callus formation were also the treatments T1(0/0), T2 (0/1)
and T8 (6/1), although they did not differ from treatments T4 (2/1) and T6 (4/
1); with callus percentages of 19.85%, 18.73% and 10.04%, respectively; in the
last two treatments most of the calli came from those dishes with 5 mg L-
1 nitrate. Treatments T3 (2/0), T5 (4/0), T7 (6/0) and T9 (8/0) were the least
responsive; coming from media without nitrate (TAB._3).
Silver nitrate affected intumescence and callosity. The treatment with silver
nitrate on the sixth day, was the one which gave the best responses for both
variables. According to Kumar, Parvatam and Ravishankar (2009), silver nitrate
is known to promote multiple shoot formation in different plants. In vitroshoot
formation was improved by incorporating silver nitrate into the culture medium.
Giridhar, Indu and Ravishankar (2004) stated that AgNO3, acetylsalicylic acid
(ASA), triacontanol, TDZ and 2iP are commonly used as growth regulators in
coffee embryogenesis studies. Giridhar, Indu and Vinod (2004) reported that
direct somatic embryogenesis was found in hypocotyl explants of C. arabicaandC.
canephora in vitroin MS medium modified with 10-70 mM AgNO3, 1.1 mM N-
benziladenine and 2.85 mM AIA. Kumar, Ramakrishna and Ravishankar (2007),
evaluating the influence of different ethylene inhibitors on somatic
embryogenesis, observed that 35-48% of explants responded for embryogenesis
under the silver nitrate treatment, 38 ± 7 and 153 ± 27 embryos being produced
from each callus mass respectively, whereas only 5% of the control explants
responded in a medium devoid of silver nitrate, cobalt chloride and salicylic
acid.
Ethylene was significant in anther oxidation, as shown by analysis of the
variance (TAB._4), and presented a quadratic effect. According to Figure_4, as
the anther remain in contact with ethylene, oxidation increases, reaching
maximum level around four days after seeding, it then decreases, reaching its
lowest level on the eighth day. It can therefore be inferred that in the
absence of ethylene, or with few days exposure, anther oxidation is observed,
decreasing within four to six days; this possibly occurring due to the
stabilization of the physiologic process in the conditions present in the
medium. According to Pasqual et al. (2002) cited by Figueira et al. (2008),
ethylene is a growth-regulator gas produced by every plant cell, and which
leads to important physiologic effects; however, with in vitro cultivation, the
production and action of this gas in growth containers directly affect explant
response, which can be either negative or positive.
A quadratic effect was also observed for intumescence, similar to that found
for oxidation, since the effect of the ethylene was significant (TAB._4). The
effect of the ethylene on the percentage of intumesced anthers after the first
days of cultivation is shown in Figure_5, with a relatively large number of
intumesced anthers (approximately 3.0) in the absence of ethylene; however,
after about four days in contact with ethylene, this average decreases, rising
again on the eighth day after seeding. Therefore it would appear that anthers
can intumesce in the absence of ethylene or within a few days of contact, and
moreover longer exposures to ethylene demonstrated positive effects on anther
intumescence.
Ethylene was significant in inducing callosity, with the data being better
adjusted by a quadratic regression even though the linear and cubic effects
were also significant (TAB._4). It was possible to observe that callosity
stabilized at about 0.7441 after 3.51 days in contact with ethylene. This
demonstrates that ethylene has a positive effect only in the first few days of
contact, its absence also favoring callus formation (FIG._6). According to the
intumescence observed, the lack of ethylene or few days of contact, as well as
the increase in exposure time, was favorable to callosity, demonstrating that
ethylene affects both intumescence as well as callus formation.
According to Kumar, Naidu and Ravishankar (2006), the known effects of silver
nitrate on somatic embryogenesis support the hypothesis that this product
promotes direct somatic embryogenesis and callus formation in indirect somatic
embryogenesis, which can be attributed to ethylene regulation during the
specific embryogenesis stages of Coffea. According to Feria, Jimenez and Barbon
(2003), exogenous ethylene concentrations and dissolved O2 concentrations
perform an important role in coffee somatic embryogenesis. Jha, Dahleen and
Suttle (2007), studying plant regeneration in barley (Hordeum vulgare L.),
observed that blocking the action of ethylene with silver nitrate during weeks
5-10, almost doubled regeneration in Morex, while Golden Promise increased
regeneration 1.5-fold. Luz et al. (1999) obtained direct regeneration of
haploid seedlings from pepper anthers in a medium to which 2,4-D, kinetin and
AgNO3 had been added, and when exposed to ethylene. In C. arabica, embryogenic
callus was induced after prolonged culture with cytokinin, with subsequent
somatic embryo formation on the embryogenic callus (BRIONES; SOTOMAYOR, 2006).
Nitrate is highly effective in inducing responses for primary and secondary
embryogenesis in comparison to other ethylene inhibitors (KUMAR; NAIDU;
RAVISHANKAR, 2006). Due to its negative action, there are several studies on
ethylene inhibitors, such as silver nitrate (AgNO3) and acetylsalicylic acid
(ASA). Silver nitrate, which is a potent inhibitor of ethylene, promoted
regeneration in Triticum aestivum, Nicotiana plumbaginifolia, Zea mays, in some
Brassicagenotypes and in Daucus carota(HATANAKA et al., 1995). Pereira et al.
(2007) studied the direct somatic embryogenesis in Coffea arabica L. and found
that the combination of kinetin and GA3 promoted embryo induction. the use of
kinetin 8 mg L-1 and GA3 17 mg L-1 not associated in medium, with 8.0 mg L-1 of
ANA promoted higher rates in vitro.
After 30 days of cultivation in medium R to which 0.108 mg L-12,4-D had been
added, possible direct anther embryogenesis was observed in the treatments
T1r2 (MS + 2.0 mg L-1 2,4-D + lacking AgNO3 + the control) and T2r4 (MS + 2.0
mg L-1 2,4-D + 5.0 mg L-1 AgNO3 + the control) (FIG._7; FIG._8), which were not
in contact with ethylene (data not shown).
According to the results, AgNO3 probably together with 2,4-D, caused this
direct embryogenesis, in contrast to the other results, showing that ethylene
is a good embryogenesis- inductor agent, and that silver nitrate would be
beneficial in some cases whilst negative in others. Studies, such as those of
Figueira (2005) and Gurel, Gurel and Kaya (2000) report this disagreement over
the action of silver nitrate on pro-embryoid induction. Greater somatic
induction occurs in media containing silver nitrate, possibly because nitrate
blocks the inhibitory endogenous ethylene action in the embryos (BIDDINGTON;
ROBINSON, 1991).
Marques (2005) found that the longer the explant remains in the culture medium,
the greater is the response in the formation of pro-embryoids, highlighting the
fact that long seeding times lead to the oxidation of the material. That author
obtained 84.5% pro-embryoids from calli in Coffea arabica; in contrast,
Figueira (2005), working with the concentration of 2.0 mg L-1 2,4-D both with
and without silver nitrate, found that 19.8% of the anthers with calli had pro-
embryoids, concluding also that silver nitrate was more effective for this
induction.
Conclusions
1. Ethylene induced an increase in oxidation and intumescence in the first days
after inoculation, with a subsequent stabilization of its effects;
2. The cultivar Catuaí Vermelho 99 responded to callus formation and also to
direct embryogenesis in the presence of ethylene.
