Growth pattern of friable calluses from leaves of Capsicum annuum var . annuum cv . Iberaba Jalapeño

The genus Capsicum belongs to the Solanaceae botanical family and is notable for the production of secondary metabolites of medicinal and economical importance. In vitro methods have been successfully utilized for the large scale production of plant secondary metabolites. The objective of this study was to establish a protocol for callus induction from leaves, nodal and internodal segments of the cultivar C. annuum var. annuum cv. Iberaba Jalapeño, and to determine the growth pattern of the calluses, aiming at the identification of the deceleration phase, when the callus cells must be subcultured for the establishment of cell suspensions and the production of secondary metabolites. The explants were inoculated into a medium supplemented with BA and 2,4-D in factorial combination. Percentage of callus induction and the explant area covered by callus cells (ACCC) were evaluated. The procedures that resulted in higher proliferation of callus cells were repeated in order to determine the growth curve of the calluses. The highest callus induction percentage and ACCC were observed with the combination of 2.22 μM BA + 18.10 μM 2,4-D for leaf and internodal explants and 2.22 μM BA + 9.05 μM 2,4-D for nodal explants. The calluses produced were friable and whitish, and their growth pattern followed a sigmoid shape. The deceleration phase started on the 31st day of cultivation for leaf explants, on the 26th for internodal explants, and on the 29th day for nodal explants.


INTRODUCTION
Plant cell and organ cultures have emerged as potential sources of secondary metabolites, which are used as pharmaceuticals, agrochemicals, flavors, fragrances, coloring agents, biopesticides, and food additives (MURTHY;LEE;PAEK, 2014).
The medicinal importance of the Capsicum genus has been confirmed by studies involving the detection of capsaicinoids, secondary metabolites of the alcaloid chemical group, and their numerous benefits for human health (SANATOMBI;SHARMA, 2008).The abundance of compounds of agricultural interest such as flavonoids, cumarins, saponins and essential oils have promoted the study of Capsicum species as alternatives in the control of parasites (LUZ, 2007).
The concentration of secondary metabolites in a plant varies according to the interactions; plant-animal, plant-plant, and nutritional stresses and, in spite of the existence of a genetic control, the expression can be modified as a result of the interaction of biochemical, physiological and ecological processes (GERSHENZON;ENGELBERTH, 2013).
Secondary metabolites can be efficiently produced in vitro.Research to date has succeeded in producing a wide range of valuable secondary phytochemicals in disorganized callus or suspension cultures (HUSSAIN et al., 2012).The major advantages of a cell culture system over the conventional cultivation of whole plants are; (1) useful compounds can be produced under controlled conditions independent of climatic changes or soil conditions; (2) cultured cells would be free of microbes and insects; (3) the cells of any plant could easily be multiplied to yield their specific metabolites; (4) automated control of cell growth and rational regulation of metabolite processes would reduce the labor costs and improve productivity; (5) organic substances are extractable from callus cultures (VANISREE et al., 2004).
To date, no study has described the development of protocols for callus induction in Capsicum annuum var.annuum cv.Iberaba Jalapeño.The determination of the procedures for callogenesis and the subsequent study of the callus growth pattern are necessary for the establishment of cell suspensions and also to subsidize studies regarding the bioactivity of its secondary metabolites.
The focus of this study is the establishment of a protocol for callogenesis in leaf, nodal and internodal explants of Jalapeño plants, evaluating the effects of different concentrations and combinations of the growth regulators 6-benzylaminopurine (BA) and 2,4dichlorophenoxyacetic acid (2,4-D), and the determination of the callus growth curve, aiming at the identification of the deceleration phase, when the callus cells must be subcultured for the establishment of cell suspensions and the production of active principles of agricultural interest.

Callus induction
The experiments were carried out at the Plant Tissue Culture Laboratory at Embrapa (Brazilian Agricultural Research Corporation).Seeds of C. annuum var.annuum cv.Iberaba Jalapeño were purchased at the local market and submitted to disinfestation procedures by washing with running tap water and a detergent agent for five minutes, immersion in 70% ethanol for one minute and in a 1.5% (v/v) sodium hypochlorite solution for 15 minutes, and then rinsed three times with sterile water.In an aseptic hood, the seeds were individually inoculated into test tubes with 10 mL of an MS (MURASHIGE; SKOOG, 1962) basal culture medium supplemented with 30 g L -1 sucrose and 6 g L -1 agar, pH 5.8, autoclaved at 121 °C for 20 minutes.After 45 days of cultivation, the plants were approximately 8 cm tall.Under aseptic conditions, the explants were produced; the leaves were cut in explants of 1.0 cm 2 , the internodal segments in explants of 1.0 cm length and the nodal segments in explants of 0.03 cm 3 , all inoculated individually into test tubes with 10 mL of an MS basal culture medium containing 30 g L -1 sucrose and 6 g L -1 agar, pH 5.8, autoclaved at 121 °C for 20 minutes.The medium was supplemented with BA (0, 0.44, 2.22 and 11.10 µM) and 2,4-D (0, 4.52, 9.05 and 18.10 µM) in a factorial combination.The explants were incubated in a growth chamber at 26±1 °C under light provided by cool white fluorescent tubes (50 µmol.m -2 .s - ) 16 hours a day.Callus formation was evaluated weekly until the 49 th day, by assessing the number of callus induced per treatment and the explant area covered by callus cells (ACCC), according to Mendonça et al. (2013).Variance analyses, test F (P<0.05) and Scott-Knott test (P<0.05)were performed by using the Assistat 7.5 statistical program.

Determination of the growth curve
The explants were individually inoculated into test tubes containing an MS basal culture medium and the hormonal combinations that resulted in the highest callus cell proliferation; 18.10 µM 2,4-D + 2.22 µM BA Growth pattern of friable calluses from leaves of Capsicum annuum var.annuum cv.Iberaba Jalapeño for leaf and internodal explants and 9.05 µM 2,4-D + 2.22 µM BA for nodal explants.The explants were incubated in a growth chamber at 26±1 °C under light provided by cool white fluorescent tubes (50 µmol.m -2 .s - ), 16 hours a day.In the subsequent 49 days, every seven days, three calluses from each treatment were carefully separated from culture medium and weighed on a precision scale in order to determine their fresh weight.These calluses were kept in an oven at 50 °C until reaching constant weight and then weighed again to determine their dry mass.From these data sets the lag, exponential, linear, deceleration and stationary phases of callus growth were determined.

Callus induction
Callus induction became apparent on the fifth day of culture, with a swelling of the explants.On the 14 th day callus formation was observed in all the explants and in all the hormonal combinations, except for the leaf and internodal explants cultivated without BA or 2,4-D; however, nodal explants gave origin to calluses even in the absence of the growth regulators.The calluses produced  SOUZA et al., 2011).This kind of callus can be used to initiate cell suspension cultures, for the cells can easily disperse in the liquid medium.
The variance analyses for callus induction percentage and ACCC are summarized in the Tables 1  and 2. The effect of BA and 2,4-D on callus induction and ACCC were highly significant in the three explants, as well as their interaction.
Callus induction occurred in all the explants where BA or 2,4-D were supplemented, alone or in combination, but did not occur in the absence of growth regulators.The interaction between these two growth regulators was positive for callus induction, what was expected, because in general an adequate balance between auxins and cytokinins is needed to the differentiation of specialized cells (SANTOS; SOUZA, 2016).

Explant
Source  The percentages of callus induction (CI) and percentages of the explant area covered by callus cells (ACCC) in leaf, internodal and nodal explants are summarized in Table 3.The highest percentage of callus induction in leaf explants was observed with combinations of 2,4-D from 4.52 to 18.10 µM and BA from 4.44 to 11.10 µM, or BA in isolation at the concentration of 11.10 µM, which resulted in formation of callus in 100% of the explants.The treatment with 2.22 µM BA + 18.10 µM 2,4-D resulted in the highest ACCC, with 100% of the explant area covered by callus cells.
On the internodal explants, several treatments reached 100% callus induction; 4.52 to 18.10 µM 2,4-D in combination with 0.44 and 2.22 BA, or BA in isolation, at 11.10 µM.Khan et al. (2011) studied the effects of 2,4-D and BA on internodal explants of C. annuum L. cv.Pusa Jwala and observed 70% callus induction with the use of 2,4-D in isolation at the concentration of 10.0 µM or, in combination to 1.78 µM BA, 95% callus induction.The combination of 18.10 µM 2,4-D + 2.22 µM BA resulted in the highest ACCC on internodal explants.
In relation to nodal explants, callus induction and ACCC were higher in the concentrations ranging from 9.05 to 18.10 µM 2,4-D in combination with 0.44 to 2.22 µM BA, in the combination of 4.52 µM 2,4-D + 0.44 µM BA, or 2,4-D in isolation at the concentrations of 4.52 and 9.05 µM, all of them leading to 100% induction.
The concentration of 11.10 µM BA, without 2,4-D, resulted in shoot formation in addition to callus induction.This is in accordance with Otroshy, Moradi and Khayam Nekouei (2011), who regenerated plantlets of C. annuum from nodal explants by using 11.10 µM BA.Ebida and Hu (1993) observed shoot formation in C. annuum L. cv.Early California Wonder by using concentrations of BA ranging from 4.44 to 44.44 µM in combination with 0.44 µM 1-naphthaleneacetic acid (NAA).In general, the presence of cytokinins is essential for shoot formation, and even can be satisfactory in isolation, as observed by Singh and Tiwari (2012).
The highest ACCC (100%) on the nodal explants was achieved with the combination of 9.05 µM 2,4-D + 2.22 µM BA.The formation of callus on the nodal explant without the addition of growth regulators can be an effect of a preexisting hormonal balance in the explants (POZO et al., 2005) or also of the physical and chemical injuries to which in vitro cultivated tissues commonly pass through (DALPONTE; VALLE; VALLE, 2010).Evaluating simultaneously the two variables, callus induction and ACCC, the treatments with the highest level of callus cell proliferation were 18.10 µM 2,4-D + 2.22 µM BA for leaf and internodal explants, and 9.05 µM 2,4-D + 2.22 µM BA for nodal explants.Umamaheswari and Lalitha (2007) tested several kinds of explants; young leaves, buds, pericarp tissue, nodal segments, and placental region, cultivated in an MS medium with gibberellic acid (GA 3 ), indole-3-acetic acid (IAA), NAA, 2,4-D and KIN in diverse combinations.The placental region submitted to the combination of 9.05 µM 2,4-D + 2.32 µM kinetin (KIN) surpassed all the other treatments in relation to the production of callus and this tissue is being used for the commercial production of capsaicin.The authors also recorded the formation of large and friable calluses in leaf explants of C. annuum L. by using a combination of 9.05 µM 2,4-D + 2.32 µM KIN.
Although there was callus induction in the absence of BA or of 2,4-D, the ACCC had the lowest values in these treatments in all the explants, evidencing the necessity of supplementation of the medium with combinations of the two growth regulators for an effective callus cell proliferation, as an outcome of the hormonal balance between cytokinins and auxins.
Callus induction can be dependent on cytokinins and auxins or only one of these classes of growth regulators (SANTOS et al., 2014).Barbosa et al. (1994) observed different morphogenic responses to the same concentrations of BA, Thidiazuron (TDZ) and adenine in apical, cotyledonary and hypocotyledonary explants of C. annuum.In general, TDZ and BA promoted callus formation; BA in isolation resulted in axillary bud formation.
The growth regulator 2,4-D is the most often used auxin in callogenesis and has been referred to as essential in some cases (SANTOS et al., 2014).The auxins are able to start cell division and to control the processes of growth and cell elongation (NOGUEIRA et al., 2008).In general, slightly similar concentrations of auxins and cytokinins in the culture medium promote callus induction, but the responses to interactions of these classes of growth regulators can vary according to the regulator, explant and genotype peculiarities (CORDEIRO et al., 2007).They can act together in synergistic interaction or not, leading to dedifferentiation.These interactions have been used and tested in different forms to establish and to refine the exact concentrations in each situation (SANTOS et al., 2014).

Callus growth
The growth curves of the calluses of the three types of explants followed a sigmoid pattern with six distinct phases; lag, exponential, linear, deceleration, stationary and decline (Figure 1).
The leaf calluses presented a lag phase from the day of inoculation to the 12 th day of cultivation, exponential phase from 12 th to the 25 th , linear from the 25 th to the 31 st , deceleration from the 31 st to the 34 th , stationary from the 34 th to the 36 th , and decline from the 36 th to the 42 nd day.
On the internodal calluses, the lag phase occurred from the inoculation to the 11 th day, exponential phase from 11 th to the 22 nd , linear from the 22 nd to the 26 th , deceleration from the 26 th to the 29 th , stationary from the 29 th to the 31 st , and decline from the 31 st to the 35 th day.
The nodal calluses showed a lag phase from the day of inoculation to the 7 th day of cultivation, exponential phase from 7 th to the 29 th , linear from the 29 th to the 32 nd , deceleration from the 32 nd to the 40 th , stationary from the 40 th to the 50 th , and decline from the 50 th to the 56 th day.
In the scientific literature there were not found studies regarding the determination of callus growth curves for the genus Capsicum, and so this is an innovative study.The pattern of the callus curve is dependent on the species and explant under consideration (FEITOSA et al., 2013) and the sigmoid pattern is peculiar to dedifferentiated tissues (PEIXOTO; CRUZ; PEIXOTO, 2011).The growth curves of the three types of explants followed this sigmoid pattern with six phases; lag, exponential, linear, deceleration, stationary and decline.Nogueira et al. (2008) identified the same six phases, lasting 120 days.Santos et al. (2003) identified only the lag, exponential and linear phases for coffee plants, due to the low growth rate characteristic of the species.
Callus growth curves in general are established to identify the stages or phases of fundamental growth processes, in order to determine the exact moment to subculture the calluses into a new medium (SANTOS; FERREIRA; SARUBO, 2010).These stages are; 1) lag phase: metabolite mobilization starts and synthesis of proteins and specific metabolites occurs, without cell multiplication; 2) exponential phase: cell division reaches the maximum; 3) linear phase: cell division reduces; 4) deceleration phase: cell division decreases and cell expansion occurs -this is when the cells have to be transferred to a new culture medium due to the reduction of nutrients, agar dryness and accumulation of toxic substances; 5) stationary phase: no cell division or weight increase occur, but the secondary metabolites accumulation reaches the maximum; and 6) decline phase: loss of weight due to cellular death (CASTRO et al., 2008;NOGUEIRA et al., 2008;SANTOS;FERREIRA;SARUBO, 2010).
The deceleration phase occurred from the 31 st to the 34 th day for leaf explants, from the 26 th to the 29 th for internodal explants, and from the 32 nd to the 40 th day for Growth pattern of friable calluses from leaves of Capsicum annuum var.annuum cv.Iberaba Jalapeño nodal explants.In general the focus of the callus growth curves is the beginning of the deceleration phase, which is the exact moment to subculture the calluses into a new liquid medium in order to establish cell suspensions (SANTOS; FERREIRA; SARUBO, 2010).In this case, the adequate moment to subculture callus cells of Jalapeño plants into a liquid medium is on the 31 st , 26 th and 32 nd day, respectively, for leaf, internodal and nodal explants.Similarly, Balbuena et al. (2009) used callus cells of P. solmsianum on the 24 th day of culture to initiate cell suspension cultures.Santiago (2003) studied the callus growth in P. hispidinervium and identified the deceleration phase starting from the 42 nd day, from which there was a decrease in the dry mass of the calluses.Valle (2003), studying the callus growth of the same species observed the deceleration phase starting on the 40 th day.
Iberaba Jalapeño it is recommended to utilize an MS basal culture medium supplemented with 2.22 µM BA + 18.10 µM 2,4-D for leaf and internodal explants and 2.22 µM BA + 9.05 µM 2,4-D for nodal explants, which result in formation of calluses in all the explants with 100% of their area covered by callus cells; 2. The growth curves of these calluses follow a sigmoid pattern, with identifiable lag, exponential, linear, deceleration, stationary and decline phases.The adequate times to subculture callus cells into a new liquid medium in order to establish cell suspensions are on the 31 st , 26 th and 32 nd days of cultivation for leaf, internodal and nodal explants, respectively.

Figure 1 -
Figure 1 -Growth curve of calluses of Jalapeño plants produced from leaf, nodal and internodal explants

Table 1 -
Results of the analysis of variance for callus induction in explants of Jalapeño plants submitted to different combinations of BA and 2,4-D in the culture medium 28 days after inoculation ** -significant at 1% probability by F test

Table 2 -
Results of the variance analyses for explant area covered by callus cells in explants of Jalapeño plants submitted to different combinations of BA and 2,4-D in the culture medium, 28 days after inoculation ** -significant at 1% probability by F test

Table 3 -
Growth pattern of friable calluses from leaves of Capsicum annuum var.annuum cv.Iberaba Jalapeño Percentages of callus induction (CI) and percentages of the explant area covered by callus cells (ACCC) in leaf, internodal and nodal explants of Jalapeño plants submitted to different combinations of BA and 2,4-D in the culture medium 28 days after inoculation *Averages followed by the same capital letter do not differ in the same row by Scott-Knott test at 5% probability; averages followed by the same lower case letter do not differ in the same column by Scott-Knott test at 5% probability