PLANT TISSUE CULTURE IN MUNTINGIA CALABURA IN VITRO CLONAL PROPAGATION, CALLUS INDUCTION AND GERMPLASM CONSERVATION OF ‘CERECILLO’ MUNTINGIA CALABURA L. (MUNTINGIACEAE)

© 2020 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 277 PLANT TISSUE CULTURE IN MUNTINGIA CALABURA IN VITRO CLONAL PROPAGATION, CALLUS INDUCTION AND GERMPLASM CONSERVATION OF ‘CERECILLO’ MUNTINGIA CALABURA L. (MUNTINGIACEAE)

was performed on the methanol extract of M. calabura stem bark and isolation of chalcones and flavonoids from the leaves, and prior to the isolation process the methanol extract of leaves was partitioned using several compounds as H 2 O-CHCl 3 , H 2 O and n-BuOH. A comprehensive review of phytochemical constituents and properties of M. calabura and several phramacological studies as acute toxicity and cytotoxic, antiproliferative, quinone reductase, antiplatelet aggregation, antibacterial, antioxidant, insecticidal, antinociceptive, anti-inflammatory, antipyretic, antiulcer, antidiabetic, antihypertensive, and cardioprotective activities it was done by Mahmood et al. (2014).
In physiological studies, the antityrosinase activity of the several extracts (aqueous, ethanol, hydroethanol and petroleum ether) from various parts of the plant (leaf, flower and fruit) from M. calabura was determined, and it was found that the hydroethanolic extract of leaves possessed the maximum tyrosinase inhibiting potential among the various parts examined; likewise, the antioxidant activity of leaf extract of the plant was also ascertained by using 2, 2-diphenyl 1-picryl hydrazil (DPPH) scavenging assay (Balakrishnan et al., 2011). Likewise, chromatographic fingerprinting and free-radical scavenging activity of ethanol extracts of M. calabura leaves and stems at 4.0 mg/mL exhibited 2,2-diphenyl-1-picrylhydrazyl inhibition of more than 90% relative to gallic acid, a very high antioxidant activity (Cruiz et al., 2017). Two types of extracts, ethanol and hexane extracts, from M. calabura flowers and fruits, at concentrations ranging from 0.25 to 30.0 mg/mL, were tested them against Plutella xylostella (Lepidoptera) larvae and pupae using leaf disc immersion assay, and all extracts were reported to be toxic to the larvae and pupae stage (Bandeira et al., 2013).
Studies in tissue culture of M. calabura are very scarce. In a pioneering study, somatic embryogenesis and subsequent plantlet regeneration were achieved in callus cultures from immature zygotic embryos using MS semisolid basal medium supplemented with different concentrations of auxins (4.5 μM 2,4-D) and cytokinins (11.6 μM Kn) (Rout et al., 1996). In a recent paper, in vitro germination and seedlings growth of M. calabura in different culture medium: MS ½ macronutrients, and Hyponex® Scotts (NPK 6.5-9-19) and Kristalon laranja® Yara (NPK 6-12-36) at 2.0 g L -1 , respectively, were studied (Pierine et al., 2019). Therefore, the aim of this study was to formulate an efficient method for micropropagation and in vitro rooting, callus induction and germplasm conservation of 'cerecilllo' Muntingia calabura L., an important arborea species of the seasonally dry tropical forest (SDTF) from the north coast of Peru.

PLANT MATERIAL, FRUITS DESINFESTATION AND CULTURE MEDIUM
Plants of Muntingia calabura L. were colected from the Cerro Tres Puntas, a SDTF of Pilasca (Salas, Peru). Mature fruits with red or yellow pericarp were surface-sterilized with 70% ethanol for 60 s followed by 5% sodium hypochlorite (solution of commercial bleach Clorox®) for 5 min, rinsed thoroughly with sterile distilled water to remove traces of sodium hypochlorite. The plants have been previously identified by Dr. Guillermo E. Delgado-Paredes, botanist at Universidad Nacional Pedro Ruiz Gallo (UNPRG), Lambayeque-Peru, and a voucher specimen (No 18825) has been preserved at the Herbarium Pedro Ruiz Gallo (PRG).
Aseptic seeds per se were inoculated on MS basal medium (Murashige and Skoog, 1962), without plant growth regulators.

MICROPROPAGATION, CALLUS INDUCTION, ROOTING AND GERMPLASM CONSERVATION
The laboratory experiments were conducted at the Plant Tissue Culture and Genetic Resources Laboratory (Facultad de Ciencias Biológicas) and General Biotechnology Laboratory (Vicerrectorado de Investigación), Universidad Nacional Pedro Ruiz Gallo, Lambayeque, Perú.
Shoot-tips or nodal segments of 6-wk-old seedlings were used as a source of explants for in vitro micropropagation, callus induction, rooting and germplasm conservation. All experiments were carried out on MS basal medium with various concentrations and combinations of plant growth regulators. It was used 6benzylaminopurine (BAP), 2iP (N 6 -(Isopentenyl adenine), Kinetin (KIN) and thidiazuron (TDZ) for micropropagation, 2,4-D (2,4-dichlorophenoxyacetic acid) and NAA (1-naphthaleneacetic acid) for callus induction, IBA (indole-3-butyric acid) and GA 3 (Gibberellic acid)-NAA for rooting and ABA (abscisic acid)-mannitol for germplasm conservation. MS medium was supplemented with 100 mg L -1 myo-inositol, 1.0 mg L -1 thiamine-HCl and 2.0% sucrose (w/v). In all these morphogenic processes each explant (shoot-tips or nodal segment) was implanted in the culture medium formulated for each case. Glass culture tubes (25 x 150 mm) containing 15 mL of medium were used for seeds culture, micropropagation, rooting and germplasm conservation, whereas that 60 x 50 mm culture flasks containing 25 mL medium with aluminum foil caps were used for callus induction.
The pH of the medium was adjusted to 5.8, before autoclaving, with 0.1 N HCl or 0.1 N NaOH and gelled with 0.7% (w/v) bacteriological agar powder (HYMEDIA®) for aseptic explants. All media were autoclaved at 121 o C and 108 kPa pressure for 20 min. The cultures were established at 25±2 o C and maintained in culture room under a 16 h photoperiod of 45 μmol m -2 s -1 irradiance provided by white fluorescent tubes and with relative humidity of 80%.

STATISTICAL ANALYSIS
Each experiment was repeated three times with 15 explants per replicate. All data were subjected to one-way analysis of variance test (ANOVA) and performed with versión 3.4.0 R software. Tukey's test was used to compare the mean values at the 5% level.

FRUITS STERILIZATION AND OXIDATIVE BROWNING
The efficiency of fruits surface sterilization was 100% and the germination rate was also 100%, for seeds with 0, 2 and 4 months of the fruit collection. In seeds grown, 12 months after collection, the germination rate was 0.0% ( Table 1). The asepsis of the seeds is considered per se; however, it is still premature to ensure that M. calabura is an orthodox or recalcitrance (or non-ortodox or desiccation sensitive) species (Obroucheva et al., 2016). A peculiarity in the culture of M. calabura seeds was that due to their small size and high mucilage content it was difficult to separate the seeds individually; therefore, the culture was carried out at a rate of 15 to 25 seeds per 150 x 25 mm test tube.  Recently, in vitro germination and seedlings growth of M. calabura in different culture medium, MS ½ macronutrients (control), and Hyponex® Scotts (NPK 6.5-9-19) (HY) and Kristalon laranja® Yara (NPK 6-12-36) (KR) at 2.0 g L -1 , respectively, were studied, and the culture media MS ½ macronutrients proved to be the most efficient for the in vitro development seedlings, with the highest seed germination (96.54%) and speed germination (6.43), after 45 days of culture (Pierine et al., 2019). These authors attributed their results to the greater efficiency of the intermediate concentrations of the nitrogenous salts MS and the higher concentrations of P and K in relation to the KR medium and lower in relation to the HY medium, which would influence the osmotic relationships and the mobility of the nutrients between the culture medium and the growing seedlings. In the study presented, the germination rate was 100%, even in seeds grown four months after collection.
Seeds explants in MS medium produced slightly browning exudation within a few hours, resulting no toxic and no leading to the death of the tissue as was reported with several species (Deberg and Reed, 1991).

SHOOT ELONGATION AND NODES FORMED OF SEEDLINGS
After 90 days of culture the seedlings reached a height of 3.02 cm and an average of 3.8 nodes formed by seedlings, without observing the development of lateral shoots (Table 2); likewise, a profuse roots system was induced. Therefore, this method of obtaining seedlings can be used for the mass propagation of the species, considering that in greenhouse conditions difficulties have been observed in its propagation, where it was found significative interaction between substrate and temperatura (Lopes et al., 2002) and no information on the stem cuttings propagation is found. Own preliminar observations have confirmed the difficulty of obtaining plants from seeds in greenhouse conditions.

SHOOT-TIPS ELONGATION, SHOOT FORMED AND CALLUS INDUCTION IN NODAL SEGMENTS
The age of the in vitro plantlets used for shoot-tips elongation and nodes formed varied from 2 to 4 weeks and the shoot size from 5 to 10 mm. Obtained results indicated that nodal segments cultured on medium with 2iP added in the lowest concentration (0.5 mg L -1 ) had the highest shoot elongation rate (3.11 ± 0.5) and the difference to the control plants (0.38 ± 0.1) was statistically significant ( Table 3). The lowest shoot elongation rate (0.13 ± 0.2) was observed for the medium supplemented with the highest used concentration of TDZ (1.0 mg/L) and the difference in comparison to other concentration was significant. However, in this concentration of TDZ the highest nodes formed rate (16.50 ± 0.6), together with the concentration of KIN (0.5 mg L -1 ) (18.00 ± 2.2) were observed, and the difference in comparison to other cytokinins and concentrations was also significant (Table 3). Increasing the cytokinins BAP, KIN, 2iP or TDZ concentrations, from 0.5 to 1.0 mg L -1 , led to limiting growth of the shoots, but no the nodes formed with 2iP and TDZ. Only in the medium supplemented with BAP and TDZ callus induction was observed.

CALLUS INDUCTION AND SHOOT PROLIFERATION IN STEM WITH NODAL SEGMENTS
The highest callus induction (++ and +++, in empirical scale) was observed in medium supplemented with 0.5 to 2.0 mg L -1 2,4-D, 2.0 mg L -1 NAA and 2.0 mg L -1 + 0.1 or 0.5 mg L -1 BAP. Multiple shoots (> 10) were observed in medium supplemented with 0.1 to 2.0 mg L -1 NAA and the greater elongation of the shoot (4.5) in medium supplemented with 0.1 mg L -1 NAA. In contrats, 2,4-D alone and 2.0 mg L -1 NAA + 0.5 mg L -1 BAP did not produce any shoots (Table 4).   Auxins are important in many plant species to induce callus and somatic embryogenesis, and 2,4-D and NAA are the plant growth regulators more used for this purpose. However, 2,4-D can produce epigenetic and genetic changes in the cells, such as methylation and mutations in the DNA (Fehér, 2015), and even, 2,4-D has been related to abnormal somatic embryos formation (Garcia et al., 2019), and somaclonal variation during indirect organogenesis (Krishna et al., 2016), because in high concentration it disrupts normal genetic and physiological cellular processes.
In Cinnamomum camphora (camphor tree) callus growth was optimized in terms of medium type concentration of plant growth regulators, revealing that MS culture medium containing 2.0 mg L -1 NAA plus 1.0 mg L -1 BAP yielded the best combination for callus growth, optimal for the establishment of cell suspensions (Abd et al., 2019). In Isodon amethystoides it was observed that a combination of auxin (IAA or NAA) and cytokinin (BAP) positively influenced callus formation, and the combination of 4.4 μM BAP and 2.7 μM NAA achieved better callus induction and callus quality (Duan et al., 2019). In all these studies, as in the study conducted with M. calubura, only the auxin supplement and in other cases the auxin-cytokinin combination were necessary.

ROOTING OF REGENERATED BUDS
The highest rooting rate was observed in culture media supplemented with 0.5 mg L -1 IBA (92.8%) and the combination 0.01 GA 3 -0.01 mg L -1 NAA (83.3%), after 30 days of culture ( Table 5). The root system by explant contained more than 30 roots formed with a length between 1.0 to 3.0 cm, optimal condition for its successful transfer to greenhouse conditions. In the rooting process the formation of basal callus was not observed, which is known rooting with basal callus formation is not desirable and adversely affects the survival of plants during the acclimatization.

IN VITRO GERMPLASM CONSERVATION
Among the various treatments tested for ABA (0.5 and 1.0 mg L -1 ), mannitol (2.0 and 4.0%) and the ABAmannitol combination, using nodal segments as explants, with or without adventitious roots, only in treatment with 2.0% mannitol in explants without roots, the highest in vitro conservation rate (50%) was reached, after 6 months of culture, while in the control treatment in culture medium without ABA and mannitol, but supplemented with 0.02 mg L -1 GA 3 -0.02 mg L -1 IAA, the conservation rate reached 100% (Table 6), although after 9-12 months of conservation it was necessary to subculture them in fresh culture medium. Likewise, plantlets conserved in 2.0% mannitol showed a poor physiological condition.
In vitro medium-term conservation of tropical plant germplasm, as in our study with SDTF species, is used routinely in many laboratories, with growth reduction by modifying various parameters, such as temperature, culture medium and gaseous environment (Engelmann, 1991). However, methods of conservation of seedless and heterozygous orthodox tropical plant species and those with recalcitrance seeds are still limited (Cha-um and Kirdmanee, 2007). In Roberts' (1973)  seeds. Recalcitrant seeds contain high moisture and are unable to withstand much desiccation, therefore, they remain viable only for a short time (weeks or months) (Engelmann, 1991).  Among the various factors studied that limit growth in in vitro conditions are the substances with osmotic properties as mannitol, sucrose and sorbitol. These osmoticum reduce the hydric potential and restrict the water availability to the explants, and reduce mineral uptake by cells through differences in osmotic pressures thereby retarding plant growth (Shibli et al., 2006). In pioneering studies, the addition of mannitol reduces significantly the growth of Colocasia esculenta and Xanthosoma brasiliense shoots (Staritsky et al., 1985). The growth of Solanum tuberosum shoots at 25 o C is reduced by the addition of 4.0% mannitol (Espinoza et al., 1985). However, cassava (Manihot esculenta) shoots deteriorate in presence of 0,1% mannitol and with a storage temperature lower than 20 o C (Roca et al., 1982). In embryogenic cultures of Phoenix dactylifera, addition of 4.0% or 6.0% mannitol and 2.0% sorbitol showed the highest percentage of survival in Bartamoda cultivar (El-Bahr et al., 2016). However, in another Arecaceae species, Elaeis guineensis and E. oleifera, mannitol at 1.0, 2.0, and 3.0% after 12 months, the temperature of 20 o C reduced both plant growth and plant survival (Camillo and Scherwinski-Pereira, 2015).
Growth retardants as ABA also can be added for in vitro germplasm conservation. In order to reduce the growth of shooots of S. tuberosum, ABA was supplemented in the culture medium (Espinoza et al., 1985); however, those authors indicate that ABA was detrimental to some varieties. In sweet potato (Ipomoea batatas) the best results of survival, growth decrease and green but small leaves were obtained in the treatments that contained 1.0, 1.5 and 2.0% mannitol; however, growth of the explants was not achieved with 5.0 and 10 mg L -1 ABA, and even the explants died, after 8 months of culture (Rayas et al., 2019). The application of ABA to the culture media was effectively applied to inhibit the growth of cedar shoots (10 mg L -1 ABA) of Cedrela atlantica and C. libani (Renau-Morata et al., 2006). Likewise, a large number of buds from seed explants of Polygonum multiflorum were preserved in MS basal media supplemented with 2.0% mannitol and 5.0 mg L -1 ABA, and the survival rate was over 70% with optimal growth and genetic stability (Huang et al., 2006). In in vitro conservation of Piper aduncum and P. hispidinervum shoots the supplemented with mannitol (1.0, 2.0 and 3.0%) or ABA (0, 0.5, 1.0, 2.0 and 3.0 mg L -1 ), negatively affected shoot growth, which was evidenced by the low rate of recovery shoots (da Silva and Scherwinski-Pereira, 2011).
These contradictory results seem to be confirmed with the study that shows that even the minimum ABA concentrations were detrimental, while only the lowest concentration of mannitol (2.0%) allowed the conservation of M. calabura germplasm, although these results cannot be conclusive and further investigation is needed.

CONCLUSION
It has been estimated at around 300 species of trees and shrubs that inhabit seasonally dry forest of Peru and Ecuador. Some preliminar studies on the physiology of seed germination of these species indicate that with exception of Fabaceae, the seeds gradually lose their viability. In 'cerecillo' (Muntingia calabura) in vitro germination was 100% in seeds collected after four months, and 0% for the ones collected after 12 months. Added to this is the increasing deforestation, natural disasters, increased agriculture and urban planning in natural environments and the dramatic effects of Climate Change. For all these reasons, in vitro plant tissue culture is the most effective tool for the micropropagation of the best genotypes and the conservation of germplasm by limiting growth at minimal rates, in explants (apical shoots) without roots; however, more research is needed.

SOURCES OF FUNDING
None.