Induction and growth pattern of callus from Piper permucronatum leaves

Piper permucronatum is a perennial shrub, a medicinal plant native to the Amazon Rainforest. Traditionally, the tea of its leaves is used to combat menstrual and intestinal cramps, stomach pain, digestive problems, diarrhea, hemorrhage, and nausea. Its leaf’s essential oil is effective against Aedes aegypti larvae; its flavones and flavanones have a fungicidal effect against Clamidosporium cladosporioides and C. sphaerospermum; its hexanic extract is effective against Leishmania amazonensis. The objective of this study was to provide a protocol for callus induction from P. permucronatum leaves and an identification of the callus growth pattern, focusing on the deceleration phase, when the callus cells must be subcultured into liquid medium in order to produce a cell suspension cultures. Leaf explants were inoculated in a solid MS medium supplemented with factorial combinations of 2,4-D, BA, NAA and GA3. Callus formation was evaluated weekly until the 49th day. Subsequently, new explants were inoculated at the hormonal combination that resulted in the highest callus cell proliferation and, every seven days during a period of 70 days, samples were dried and weighed to determine the callus growth pattern. NAA and GA3 were not effective for callus induction. Combinations of 2,4-D and BA resulted in callus induction and proliferation. The highest percentage of callus induction was observed with the combination of 4.52 μM 2,4-D and 4.44 μM BA. The calluses thereby produced were friable and whitish. The callus growth pattern followed a sigmoid shape. The deceleration phase started on the 56th day of culture.


INTRODUCTION
Nature has developed an enormous diversity of species during several billion years of evolution and all these species coexist in ecosystems and interact with each other in several ways in which chemistry plays a major role (Verpoorte, 1998). It is well established that secondary metabolites play an important role in plant chemical defense (Morais et al., 2007), besides those attractants that promote pollination and seed dispersal (Vanin et al., 2008).
Several species of the Piper genus are native to the Amazon Rainforest and are notable producers of chemical compounds which have several biological effects in insects, fungi, bacteria, Trypanossoma cruzi (Navickiene et al., 2003;Dyer et al., 2004;Danelutte et al., 2005;Balbuena et al., 2009) and can also affect human health, as analgesic, anti-depressant, cytoprotective, anti-ulcer, anticonvulsant, anti-inflammatory, and antioxidant agents (Ahmad et al., 2010).
Piper permucronatum Yuncker is a perennial shrub, a medicinal plant native to the Amazon Rainforest. Traditionally, the tea of its leaves is used to combat menstrual and intestinal cramps, stomach pain, digestive problems, diarrhea, hemorrhage, and nausea (Anjos Júnior, 2007). The bioactivity of the species has been demonstrated; its leaf's essential oil (dilapiol, myristicin, elemicine, and asaricine) is effective against Aedes aegypti larvae (Morais et al., 2007); its flavones and flavanones have a fungicidal effect against Clamidosporium cladosporioides and C. sphaerospermum (Lemeszenski, 2013); its hexanic extract is effective against Leishmania amazonensis and Artemia salina (Anjos Júnior, 2007). Sesquiterpenes and monoterpenes have also been identified (Torquilho et al., 1999).
The effects of P. permucronatum compounds in diverse organisms indicate their potential to be used as replacement for, or simultaneously with, agrochemicals for the control of pests and diseases. The reduction in the use of agrochemicals is a current and topical challenge, considering the toxicity of these products for the environment, the demand for healthy food, and the development of resistance to fungicides and insecticides (Khan et al., 2011, Speck-Planche et al., 2011. Biotechnological approaches, more specifically plant tissue cultures, have potential as a supplement to traditional agriculture in the industrial production of bioactive plant metabolites (Rao & Ravishankar, 2002). Cell suspension culture systems are used for large scale culturing of plant cells from which secondary metabolites are extracted. The advantage of this method is that it can ultimately provide a continuous, reliable source of natural products, which can be produced at a rate similar or superior to that of intact plants (Vanisree et al., 2004). These cultures offer the possibility of obtaining desirable amounts of compounds as well as ensuring sustainable conservation and rational utilization of biodiversity (Coste et al., 2011). Besides, in vitro production of secondary compounds under controlled conditions prevents fluctuations in concentrations due to geographical, seasonal, and environmental variations (Murthy et al., 2014).
This research is part of a project in which in vitro produced secondary metabolites from Piper species will be tested against agricultural pests and diseases. As such, the study provides a protocol for callus induction from P. permucronatum leaves and an identification of the callus growth pattern, focusing on the stationary phase, when the callus cells must be subcultured into liquid medium in order to produce a cell suspension culture.

MATERIALS AND METHODS
Plant materials and sterilization. Young leaves were excised from flowering, healthy, and disease free Piper permucronatum Yuncker Piperaceae (traditional name: elixir paregórico) stock plants from Embrapa (Brazilian Agricultural Research Corporation), in Porto Velho, RO, Brazil. Taxonomic identification was carried out at the herbarium of INPA (National Institute of Amazon Researches) by Dr. J. Gomes, Voucher Number 211711, by using branches with leaves and flowers collected in January 2005 in Porto Velho, Rondonia State, Brazil. The plants were six months old and approximately 90 cm tall, maintained in a shaded greenhouse under a maximum photosynthetic photon flux density of 300 µmol.m -2 .s -1 and average temperature of 25.6°C. After washing with running tap water and a detergent agent for five minutes, the leaves were surface-sterilized in 70% (v/v) ethanol for 1 minute and soaked in a 1.0% (v/v) sodium hypochlorite solution for 1 minute, and then rinsed three times with sterile water. Explants were produced by cutting the leaves into 1 cm 2 pieces in sterile petri dishes.
Callus induction and growth. Leaf explants were individually transferred with the adaxial surface up, to test tubes ( (Table 1) each one with three replicates of three explants. The growth regulators, their concentrations and combinations tested were based on successful studies on callus induction in Piper species; P. cernuum, P. crassinervum (Danelutte et al., 2005), P. solmsianum (Balbuena et al., 2009), P. aduncum (Delgado-Paredes et al., 2013), and P. hispidinervum (Santiago, 2013). The pH of the medium was adjusted to 5.8 before the addition of agar followed by autoclaving at 121°C for 20 minutes.
The percentage of explants where callogenesis occurred was evaluated weekly until the 49 th day, when all the explants were dried at 50°C for 24 hours and subsequently weighed to quantify callus cell proliferation where it occurred. After that, new explants were inoculated at the hormonal combination that resulted in the highest callus cell proliferation at the same conditions described and, every seven days during a period of 70 days, three samples were dried and weighed to determine the callus growth pattern.
Experimental design and evaluation. All the cultures were incubated in a growth chamber at 25±2°C under light provided by cool white fluorescent   Table 2). The highest percentage of callus induction was observed with the combination of 4.52 µM 2,4-D and 4.44 µM BA. The calluses thereby produced were friable and whitish.

Callus
Callus growth. The growth pattern of P. permucronatum callus followed a sigmoid shape (Figure 1). It was possible to identify a lag phase from the day of inoculation until the 21 st day, an exponential phase from the 21 st to the 49 th day, a linear phase from the 49 th to the 56 th day, a deceleration phase from the 56 th to the 63 rd day, and a decline phase from the 63 rd to the 70 th day.

Callus induction
There is a great variation of in vitro responses among Piper species. In the present research it was observed that NAA, isolated or in combination with BA, was not efficient for callus induction in leaf explants of P. permucronatum. Differently, Delgado-Paredes et al. (2013) observed callus induction in leaf explants of both P. cernuum and P. aduncum in MS medium supplemented with a combination of 5.37 µM NAA and 0.89 µM BA. This combination was tested in this study, but did not result in callus induction.
Santiago (2003) evaluated the effect of combinations of 2,4-D, BA, and NAA in MS medium to induce callus in leaf explants of P. hispidinervium and observed the highest percentage of friable green callus, 98.3%, with the combination of 27.14 µM 2,4-D, 8.88 µM BA, and 2.69 µM NAA. Different results were found by Valle (2003), who observed that higher concentrations of these growth regulators were effective for callus induction in leaf explants of P. hispidinervium, with a pro-cytokinin hormonal balance; the highest callus formation was achieved in MS medium supplemented with 22.62 µM 2,4-D and 45.29 µM BA, or 26.85 µM NAA and 45.29 µM BA.
The use of 2,4-D and GA 3 together did not promote callus induction in P. permucronatum leaf explants. Danelutte et al. (2005), aiming at the establishment of cell suspensions from leaves of P. crassinervium and P. cernuum, found the highest callus induction by using MS medium supplemented with 0.11 µM Indoleacetic acid (IAA) and 0.06 µM GA 3 in P. crassinervium and 0.91 µM 2,4-D and 1.44 µM GA 3 in P. cernuum. The last combination was also tested in the current approach, without success.
Lower concentrations of 2,4-D (0.45, 0.91, and 1.81 µM) were not efficient in this study. Contrastingly, Delgado-Paredes et al. (2013) observed callus induction in leaf explants of both P. cernuum and P. aduncum in MS medium supplemented with 0.91 µM 2,4-D. Also using lower concentrations of 2,4-D, Balbuena et al. (2009), aiming at the establishment of cell suspension culture of P. solmsianum, evaluated the induction of callus in leaf and petiole explants cultivated in an MS medium supplemented with combinations of 2,4-D, BA, NAA, and IAA. The authors observed that the highest proliferation of callus cells occurred in the combination of 0.91 µM 2,4-D and 8.88 µM BA, for both explants. Dominguez et al. (2006) used combinations of IAA with NAA or 2,4-D with Kinetin (Kin) in an MS medium to promote callus induction in P. auritum leaves aiming at the subsequent regeneration of plantlets from the calluses. They observed that *Means followed by the same capital letter in the rows or small letter in the columns do not differ significantly at 5% probability by Tukey's test.  Kelkar et al. (1996), who reached 90, 75, 75, and 67% of callus induction in P. colubrinum leaf explants, utilizing 2. 22,4.44,8.88 and 13.32 µM BA,respectively. In the present research the use together of 2,4-D (4.52, 9.05, and 18.10 µM) and BA (4.44,8.88,and 17.76 µM) at relatively higher concentrations was, in general, adequate to promote callus induction in leaf explants of P. permucronatum, reaching 100% with 4.52 µM 2,4-D and 4.44 µM BA. Similarly, Kelkar et al. (1996) observed that the combination of 2.22 µM BA with 2.26 µM 2,4-D resulted in 100% callus induction in P. colubrinum. Ahmad et al. (2010) also tested 2,4-D, BA, GA 3 and NAA in the induction of callus in P. nigrum leaves in order to regenerate plantlets from the calluses. The authors found the highest callogenic response in explants cultured on MS medium supplemented with either 2.22 µM BA or 4.44 µM BA in combination with 5.37 µM NAA, resulting in 93 and 90% of callogenesis, respectively. GA 3 alone did not result in relevant callus induction, as observed in the present study. However, in combination with BA, GA 3 enhanced the callogenesis to relatively high levels.
According to Li et al. (2012) only auxins and not cytokinins induce callogenesis by initiating explant cell dedifferentiation through auxin-responsive gene activation, while cytokinins wold be more related to differentiation, by stimulating the division of the newly formed meristematic cells. All the diverse responses found among Piper species in relation to the adequate hormonal balance needed for callus induction can be attributed to the interaction of growth regulators added to the medium with dissimilar endogenous levels of auxins and cytokinins preexistent in the tissues. As stated by Jiménez (2001), very little is known about the possible interactions of an endogenous phytohormone system with the exogenous growth regulators supplied to the nutrient medium; it seems probable that the observed responses of cell culture systems, after a growth regulator supplement, are related to such interactions. The levels of endogenous hormones are dependent on characteristics such as age of the plant, its physiological and nutritional status, and mainly genotype, for these levels are postulated to be the main difference among genotypes with various grades of competence (Bhaskaran & Smith, 1990). As was mentioned by George et al. (2008), due to genotypic specificity, media and cultural environment often need to be varied from one genus or species of plant to another and even closely related varieties of plants can differ in their cultural requirements.

Callus growth
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 et al., 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 et al., 2010).
In the current approach, the growth pattern of P. permucronatum callus followed a typical sigmoid shape with the deceleration phase between the 56 th and the 63 rd day of culture. Differently, 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. Balbuena et al. (2009) used callus cells of P. solmsianum on the 24 th day of culture to initiate cell suspension cultures. The appropriate time to subculture the calluses of P. permucronatum in order to establish cell suspension cultures is in the beginning of the deceleration phase, on the 56 th day of culture, 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.

CONCLUSION
Callus induction in leaf explants of P. permucronatum can be achieved in MS medium supplemented with 4.52 µM 2,4-D and 4.44 µM BA; callus cells at the 56 th day of culture are appropriate to start a cell suspension culture.