Indução e multiplicação de calos embriogênicos de Coffea arabica L.

Este estudo foi realizado com o objetivo de avaliar a inducao de calos embriogenicos de dois clones de Coffea arabica, selecionados para resistencia a ferrugem e de alta produtividade e comparar a multiplicacao desses calos em dois meios de cultura nos sistemas de cultivo gelatinoso e liquido. Para a inducao de calos foi utilizado o protocolo descrito por Teixeira et al. (2004), em delineamento experimental inteiramente casualizado, sendo cada clone considerado um tratamento. A avaliacao deste experimento foi realizada 180 dias apos a instalacao, por meio da contagem de calos embriogenicos formados. Para a multiplicacao de calos, os tratamentos constituiram-se de dois meios de cultura (meio do estagio dois de Albarran et al. (2004) e meio de multiplicacao de Teixeira et al. (2004)) e dois sistemas de cultivo (gelatinoso e liquido). As avaliacoes foram realizadas aos 21, 42 e 63 dias apos a instalacao do experimento, por meio da pesagem dos calos. Verificou-se que os clones avaliados apresentam o mesmo potencial de formacao de calos embriogenicos. O potencial de multiplicacao de calos embriogenicos e influenciado pelo genotipo. O sistema gelatinoso apresentou maior eficiencia na multiplicacao de calos embriogenicos dos clones estudados quando comparado ao sistema liquido.


Introduction
Somatic embryogenesis has been achieved in a great number of families and species of plants and has been used for basic studies of vegetable physiology and in more practical applications, such as micropropagation and genetic transformation.The potentially important application of embryogenesis for the accelerated multiplication of coffee plants was quickly noticed by the earliest studies, and numerous techniques that simplify this process have been developed that seek to establish large-scale plant production and reduce seedling production costs.
Embryogenic calli can continue to originate somatic embryos for long time periods and after many subcultures.According to Pasqual et al. (1997), continuous somatic embryo production depends on the continuous proliferation of proembryogenic nodules from young somatic embryos during each subculture.Some authors have described protocols seeking to optimize the multiplication of embryogenic coffee calli (BOXTEL;BERTHOULY, 1996;DUCOS et al., 2007a).Studies to establish better protocols were conducted using gelatinous media; however, the multiplication rates of these media were too low to be used in the large-scale production of coffee plants (SÖNDHAL;SHARP, 1977).In view of that result, there have been several attempts, with varying degrees of success, to establish somatic embryogenesis protocols in liquid media (AFREEN et al., 2002;DUCOS et al., 2007a and b;KUMAR et al., 2006).
Based on the published results mentioned above, this work was performed with the objective of evaluating the induction of embryogenic calli from two clones of C. arabica that were selected for high productivity and resistance to rust and comparing the multiplication of those calli in two culture media under gelatinous-and liquid-cultivation systems.

Material and methods
The experiments were performed in the Laboratório de Cultura de Tecidos da Fundação Procafé in Varginha, Minas Gerais State, using clones 3 and 28, which were selected for their resistance to rust and high productivity.The mother plant 3 is from the Siriema (C.racemosa x C. arabica) population and also exhibits resistance to the leaf-miner.The mother plant 28 was generated by crossing Red Catuaí IAC 44 (UFV Control: VER 209-2 = UFV 2144-260 EL 7) x Timor Hybrid CIFC 2750 (UFV Control: VER 209-2 = UFV 439-2).
The formation potential of embryogenic calli was first evaluated with leaf explants from clones 3 and 28.Well-developed leaves corresponding to the third pair were collected from the plagiotropic branches of the medial third of the adult mother plants and were taken to the laboratory.
The asepsis protocol consisted of immersion in 70% alcohol for 1 min.and disinfestation with sodium hypochlorite at a concentration of 2.4% active chlorine for 15 min.Soon afterwards, the leaves were rinsed three times with distilled, autoclaved water.After autoclaving at 121°C and 1 atm for 20 min., the culture medium was poured into 0.9 x 10 cm Petri dishes.
The callus induction protocol described by Teixeira et al. (2004) was used in this study.Initially, the explants were cultivated in primary medium (PM) for 30 days and were then transferred to secondary medium (SM), where they remained for 180 days.The experimental design was entirely random, using 33 replicates with nine explants per dish with each dish considered a replicate.
The analysis was conducted 180 days after the start of the experiment through the quantification of embryogenic callus formation.Statistical analyses were undertaken using two models: the first with no factor (null) and the second considering the main effect of the hybrid.The Deviance Analysis was used.The residue analysis showed that the best adjustment was obtained from the quasibinomial family and the probit connection function.The standard deviation was determined, and being satisfactory, the Scott and Knott test for the comparison of averages was used.The analyses were performed using the GLM (Generalized Linear Models * ) protocol due to the fact that the percentage data do not follow a normal distribution.
The embryogenic calli in this study were multiplied in a previously described multiplication medium (TEIXEIRA et al., 2004), with the medium being refreshed every 21 days over six months until the calli reached the necessary size to be used for a multiplication experiment.The treatments used were two culture media (ALBARRAN et al., 2004) stage two medium and Teixeira et al. ( 2004) multiplication medium), two cultivation systems (gelatinous medium and liquid medium) and evaluation time (21, 42 and 63 days) with five repetitions.
The media were adjusted to pH 5.8 ± 0.1 using 0.1 N NaOH or 0.1 N HCl before the autoclaving process, which was performed at 121°C and 1 atm for 20 minutes.In a laminar-flow chamber, 20 mL of the liquid medium was poured into 125 mL Erlenmeyer flasks, and soon afterwards, 160 mg of embryogenic calli were inoculated into the flasks.These flasks were sealed with laminated paper and were maintained in an orbital agitator at 100 rpm under the absence of light and at a temperature of 25 ± 2°C.The gelatinous media were poured into a 0.9 x 10 cm Petri dish and inoculated with nine embryogenic sections of 60 mg each.The dishes were sealed with PVC film and stored in wooden boxes under the absence of light and at a temperature of 25 ± 2°C.
Measurements were conducted at 21, 42 and 63 days and involved weighing the calli using a precision scale placed in the laminar-flow chamber.At each evaluation, the culture medium was refreshed and the initial quantity of calli was adjusted, i.e., 160 mg of embryogenic calli in the case of the flasks and nine embryogenic sections of 60 mg each in the case of the Petri dishes.
The callus-increase percentage [I (%)] was calculated with the following formula: I (%) = [(FW x 100)/(FW initial )] -100, where FW is the fresh weight (g) measured every 21 days and FW initial is the fresh weight of the embryogenic callus initially inoculated in the flask or Petri dish.The I (%) was calculated with the intention of enabling a comparison between growth in liquid medium and in gelatinous medium, as the two media had different initial concentrations.
The callusi-increase percentage was analyzed by the GLM procedure.The significance of the comparison was determined to a 5% probability by Student's t-test.As significant differences between the treatments and interactions were detected, further testing was undertaken.The regression analysis was used to study increase percentage values of calli in function of time analyzed.The clones' genotypes were analyzed separately in independent experiments, as it was not the objective of this experiment to compare them.

Callus Induction
There was no significant difference between the clones studied (X 1 ) in the chi-squared test, demonstrating that their genotypes had no effect on the formation of embryogenic calli (Table 1).The two studied clones presented similar average percentages of embryogenic call formation (22% for clone 3 and 20.31% for clone 28) despite their different genetic origins.This average percentage of callus formation is in line with results observed in prior studies.In the first work on the somatic embryogenesis of C. arabica, Söndhal and Sharp (1977) obtained 20% embryogenic callus formation using 5.4 μM ANA and 4.6 μM kinetin.Berthouly and Michaux-Ferriere (1996) obtained embryogenic callus formation starting at 20.8% from various genotypes of C. canephora when a primary medium was used containing 2.2 μM 2,4-D, 4.9 μM IBA and 9.8 μM 2-iP.Teixeira et al. ( 2004) obtained embryogenic calli formation in different C. arabica cultivars which values were 8% for cv.Mundo Novo and 100% for cv.Catuai.

Callus Multiplication
We observed that, for the two studied clones, the treatments with the gelatinous medium yielded a higher production of embryogenic calli at the end of 63 days than did the treatments with the liquid medium (Table 2).Due to the initial amount difference of inoculate used in both systems no statistical analyses aiming at comparing the cultivation system calli fresh mass was performed.
In the gelatinous-cultivation system, clone 3 produced, after 63 days, 5.06 g of calli in the Albarran et al. ( 2004) medium and 3.99 g of calli in the Teixeira et al. ( 2004) gelatinous medium (Table 1).For the liquid system, the media described by Albarran et al. ( 2004) and Teixeira et al. ( 2004) presented, after 63 days, 0.27 g and 1.12 g of calli, respectively.Clone 28, after 63 days, yielded a fresh mass of 1.58 g of calli from the Teixeira et al. ( 2004) medium and 0.99 g from the Albarran et al. ( 2004) medium.For the gelatinous cultivation system, the fresh mass of calli at the end of this period was 7.11 g with the Albarran et al. ( 2004) medium and 6.40 g with the Teixeira et al. ( 2004) medium.
The clones studied here presented varying average weight increases.The growth variation of clone 3 was from 1.69-fold to 9.37-fold, whereas clone 28 presented an average weight increase varying from 6.19-fold to 13.16-fold.In the Albarran et al. ( 2004) medium in the gelatinous-cultivation system (which mediated stronger growth), we observed that, at 63 days, clone 28 increased its weight 40.45% more than did clone 3.These results, which indicate that the multiplication potential of embryogenic calli is influenced by the clone's genotype, are in line with the results of Fuentes et al. (2000), which suggested that the somatic embryogenesis process is sensitive to culture conditions such as the composition of the medium, the physical environment of the culture, and the source and genotype of the explants.

Callus percentage increase
These results demonstrated the significance of the percentage increase of callus as functions of cultivation system and duration of cultivation, as well as the significance of the interactions between cultivation system and culture medium, cultivation system and duration, cultivation system and culture and medium and time factors.The significant cultivation system and culture medium interaction suggests differentiated behavior between the levels of the cultivation system factor and each level of the culture medium factor (Table 3).For the Albarran et al. ( 2004) culture medium, there was a significant difference between the liquid-and gelatinous-cultivation systems, with the gelatinous system being statistically superior in the percentage increase of calli when compared with the liquid system.For the Teixeira et al. ( 2004) culture medium, a significant difference was not observed in the behavior of the cultivation systems.In Figure 1, the average percentage increase in callus is presented for clone 3 as a function of time as determined for each culture medium and cultivation system.In callus multiplication with the gelatinous cultivation system, an increase of approximately 17.61% per day was observed using the Albarran et al. ( 2004) medium and 12.92% was observed using the Teixeira et al. ( 2004) medium.For the liquid-cultivation system, the increases were 1.25 and 12.72% for the Albarran et al. ( 2004 These values are in line with those reported by Ducos et al. (2007a), who quantified the multiplication of embryogenic cells from C. canephora in liquid medium and observed a growth rate of 6.6 to 13.3% a day, starting with 1 g of calli and exhibiting a biomass growth of two to three times the initial amount, every 15 days.Teixeira et al. ( 2004) observed that the fresh-matter weight at the end of various cultivation periods varied according to the initial cell density, reaching a maximum value of callus growth when the initial density was 8 g L -1 , which is the same density used in the present work in which the average fresh-weight increase at 63 days was 9.8-fold for clone 28 and 7.0fold for clone 3 when the Teixeira et al. ( 2004) medium was used.The present work, therefore, corroborated the results obtained by those authors, who achieved a cell-density increase of nearly 10-fold after 56 days of cultivation (Table 1).

Clone 28
These results demonstrated the significance of the primary effects of cultivation system and time, as well as the significance of the interaction between cultivation system and time and culture medium and time.The increase in the percentage of calli for the Acta Scientiarum.Agronomy Maringá, v. 34, n. 1, p. 93-98, Jan.-Mar., 2012 gelatinous-cultivation system was superior to that of the liquid-cultivation system (Figure 2).We found an increase in calli of approximately 23.6% per day when the gelatinous system was used and of about 10%, when the liquid cultivation system was used.It is likely that the low growth rate observed in the liquid medium is due to the low level of oxygenation to which the cellular suspensions were exposed.
Calli increase (%) According to Curtis (2005), the liquid cultivation system can provide strongly anaerobic conditions due to the reduced level of oxygen inside the tissue, which is caused by the transfer limitations and physiological limitations of the tissue.De Feria et al. (2003) compared the multiplication of embryos in liquid medium containing 50 and 80% dissolved oxygen and obtained improved multiplication of calli and embryo regeneration at 80% dissolved oxygen, probably due to the enhanced metabolic activity of the cellular suspensions in the medium with increased oxygen content.
Although the liquid cultivation system employed in this experiment was studied under agitation conditions, which allow more penetration of oxygen within the culture medium and improve the distribution of recently-solubilized oxygen, the gelatinous-cultivation system exhibited a higher rate of callus growth.This fact can be explained by the high intensity of oxygenation that occurs on the surface of solid media, because the explants are in direct contact with the air, which results in oxygen being more available for metabolism (PINTO et al., 1995).Therefore, the use of gelling agents creates improved aeration conditions for the base of the explants.It is worth emphasizing that the culture medium used in the present study was a gelatinous medium in which low concentrations of the gelling agent were applied, thereby allowing the diffusion of nutrients in the medium.
In spite of the higher efficiency of the gelatinous-cultivation system observed in the present work, many authors affirm that the use of liquid culture is a prerequisite for the automation and cost-reduction required for commercial propagation systems (DE FERIA et al., 2003;DUCOS et al., 2008).However, the advantages of cultivationure in liquid media are frequently counterbalanced by the disadvantages, such as cell asphyxia, hyperhydricity, the need for complex equipment (ETIENNE et al., 2006) and higher culture medium consumption.
In the present work, for the gelatinous cultivation system, 25 mL of culture medium was employed for the inoculation of 0.54 g of calli, which resulted in an average weight at the end of 63 days of 4.25 g for clone 3 and of 6.75 g for clone 28 (Table 2).To inoculate the same 0.54 g of calli in liquid medium, 67.5 mL of culture medium would be necessary, representing an increase of at least 227.5% in the volume of medium to be used, and the rate of growth under the liquid system remains lower.
In Figure 3, the average percentage increase of calli is presented as function of time as determined for each medium.The percentage increase of callus provided by the Albarran et al. ( 2004  (4.5 μM of 2.4-D) and one cytokinin (4.6 μM of kinetin) are used.Although auxins and cytokinins of different types and concentrations were used, the average percentage increase of calli they provided were quite similar, indicating that embryogenic calli present high plasticity in their response to the culture medium.

Conclusion
The clones analyzed here exhibit similar potential for embryogenic callus formation.Additionally, the embryogenic callus multiplication potential was found to be influenced by the clone's genotype.The gelatinous cultivation system demonstrated greater efficiency for the multiplication of embryogenic calli from the clones studied here when compared with the liquid system.

Figure 1 .
Figure 1.Average percentage increase of calli as a function of time as determined for each culture medium and cultivation system for clone 3.

Figure 2 .
Figure 2. Average percentage increase of calli from clone 28 as a function of time as determined for each cultivation system.

Figure 3 .
Figure 3. Average percentage increase of calli as a function of time as determined for each culture medium for clone 28.

Table 1 .
Deviance analysis for presence of inducted embryogenic calli in leaf explants of clones 3 and 28.

Table 2 .
Fresh mass of embryogenic calli (g) during the cultivation period and weight increase after 63 days for each culture medium and cultivation system under study, as determined for clones 3 and 28.

Table 3 .
Average percentage increase of calli as a function of the cultivation system and the culture medium for clone 3.