Establishing-a-Callus-Culture-of-Cnidoscolus-chayamansa-McVaugh-A-Species-with-Ethnopharmacological-Value40 establishing a callus culture of cnidoscolus chayamansa mcvaugh a species with ethnopharmacological value

Advances in Biochemistry and Biotechnology (ISSN: 2574-7258)

Article / Research Article

"Establishing a Callus Culture of Cnidoscolus chayamansa McVaugh: A Species with Ethnopharmacological Value"

Mariana Zuleima Pérez-González1,5, Aurelio Nieto-Trujillo1, Ignacio García-Martínez2, María Elena Estrada-Zúñiga3, Antonio Bernabé-Antonio4, María Adelina Jimenez-Arellanes5, Francisco Cruz-Sosa1* 

1Department of Biotechnology, Autonomous Metropolitan University-Iztapalapa, Mexico

2Department of Chemical Engineering and Biochemistry, TESE, Mexico

3Faculty of Sciences, Autonomous University of the State of Mexico, Mexico

4Department of Wood, Pulp and Paper, University of Guadalajara, Mexico

5Unit of Medical Research in Pharmacology, Hospital of Specialties, México 

*Corresponding author: Francisco Cruz-Sosa, Departamento de Biotecnología, UAM-Iztapalapa Ciudad de México, México. Tel: +525558044714; Fax: +525558044712; Email: cuhp@xanum.uam.mx 

Received Date: 28 February, 2018; Accepted Date: 03 April, 2018; Published Date: 11 April, 2018

1.                   Abstract 

Cnidoscolus chayamansa is a plant widely used as food with potential medicinal applications. Plant tissue culture enables the production of secondary metabolites from plants in a sustainable manner. The aim of this work was to establish a callus culture of C. chayamansa using leaf explants, which, in the future, can be used to obtain whole extracts or specific compounds of interest. Different concentrations and combinations of 6-Benzylaminopurine (BAP) and 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Murashige and Skoog (MS) culture medium were evaluated for their ability to support callus induction from immature leaf explants. Highest callus yield (100%) was achieved when leaf explants were incubated in MS medium supplemented with BAP (5.0 mg L-1) and 2,4-D (2.5 or 5.0 mg mL-1). A reduced rate of callus induction (75%) was observed when leaf explants were exposed to lower concentrations (0.5 mg L-1) of BAP or 2,4-D. The calluses obtained in this work can be used as a biotechnological alternative for in vitro propagation of C. chayamansa and for obtaining extracts or compounds with a possible pharmacological application. 

2.                   Keywords: Chaya, In vitro culture, Medicinal plants, Plant growth regulators.

3.                   Introduction 

Cnidoscolus chayamansa, commonly known as “tree spinach” or “chaya” in Central and South America, is a semi-perennial and semi-woody shrub. C. chayamansa is an important staple leafy vegetable for the autochthonous people of Mesoamerica. The nutritional value of C. chayamansa [1] and traditional medicinal and food use has been established in the literature [1,2]. As a food for freshwater fish, partial substitution of balanced feed with this leaf has been determined to be viable for tilapia (Oreochromis niloticus L.) culture in the integrated agricultural–aquaculture systems in Yucatan, Mexico [3]. The antioxidant capacity and phenolic content of the leaf extracts, in addition to anti-inflammatory and cardioprotective properties, has been extensively described in the literature [4-6]. Furthermore, the antioxidant and antimutagenic activity of a methanolic extract from the edible leaves of C. chayamansa has been detailed, in addition to its antidiabetic effects in diabetic mouse [7,8]. A phytochemical profile, including flavonoids, phenolic acids, saponins, and alkaloids, and the antioxidant properties of fresh leaves were recently reported [8]. Antimycobacterial (MIC 50 µg mL-1), Antibacterial (P. aeruginosa and E. coli), Antiprotozoal (IC50 50 µg mL-1), and Anti-Inflammatory (ED50 = 1.66 mg ear-1 for the TPA model and 467.73 mg kg-1 for the carragenine model) activities of the leaf extract (CHCl3: MeOH, 1: 1) have been published. Also, kaempferol-3,7-dimethyl ether, 5-hydroxy-7-3’,4’-trimethoxyflavanone, moretenol, and, ‘moretenol acetate were isolated and characterized. Toxic acute and subacute effects (LD50 of extract > 2 g kg-1) were estimated in a sub-acute toxicity test using diabetic rats; the extract was also administered at 1 g kg-1 for 28 days and did not cause lethality or any alteration in hematological and biochemical values; in addition, histological analysis of the liver, kidney, and spleen revealed no structural changes [9]. 

As mentioned, several works have reported on the properties and benefits of C. chayamansa; however, biotechnological tools have not been used to exploit this species in a sustainable manner. In these regard, plant cell culture represents an advantageous system for the study of different cellular processes, since the conditions can be strictly controlled, thereby allowing the effects of a single factor on a given process to be monitored [10]. In addition, this technique could serve as an invaluable tool for agricultural and pharmaceutical companies around the world to propagate these plants and produce secondary metabolites [11]. Toward this end, callus induction is the necessary first step, as in many tissue culture experiments, including cell suspension culture or indirect organogenesis [12]. According to the literature, a callus culture of C. chayamansa has not yet been reported. The aims of this study were to evaluate the influence of different combinations and concentrations of Plant Growth Regulators (PGRs) on callus induction in C. chayamansa leaf explants and to monitor the phenotypic characteristics of the callus culture, such as color, friability, and proliferation rate. 

4.                   Materials and Methods 

4.1.              Plant Material 

Cnidoscolus chayamansa (Mc Vaugh) was collected in Mexico City, Mexico, in June 2016. The plant was identified by M.Sc. Abigail Aguilar of the Instituto Mexicano del Seguro Social (IMSS) and a voucher specimen (16252) was deposited at the IMSSM Herbarium.

 

4.2.   Aseptic Conditions

 

Immature leaf explants of 1-2 cm in length were excised from the plant, washed with a soap solution for 10 minutes, and rinsed with running tap water. Then, leaf explants were immersed into an aqueous solution with Ampicillin (100 mg mL-1), Tetracycline (200 mg mL-1), and Cefotaxime (15 mg mL-1) for 30 min; followed by another immersion in an aqueous mixture with Shogum® (0.01 mL mL-1) and Fungoxyl® (0.01 g mL-1) for 20 min. Later, explants were immersed into a 70% (v/v) ethanol for 45 s; 0.9% (v/v) sodium hypochlorite with Tween-20 (three drops per 100 mL of solution) at low shaking for 20 min. Afterwards, under aseptic conditions, leaf explants were rinsed three times in sterile deionized water, followed by rinsing and segmenting in a sterile antioxidant solution (100 mg citric acid and 150 ascorbic acid mg L-1) for 15 min. 

4.3.              Culture Medium and Incubation Conditions

The basal culture medium consisted of Murashige and Skoog (MS) [12], supplemented with 3% (w/v) sucrose, 100 mg L-1 citric acid, and 150 mg L-1 ascorbic acid. For callus induction from immature leaf explants, the culture medium was supplemented with different combinations and concentrations of the cytokinin 6-Benzylaminopurine (BAP) and the auxin 2,4-Dichlorophenoxyacetic Acid (2,4-D). Both PGRs were used in concentrations of 0.00, 0.5, 1.0, 2.5 and 5.0 mg L-1 at pH 5.8. Phytagel (0.2% w/v) was used for solidifying the culture medium. 20 mL of culture medium were poured into culture tubes (150 X 20 mm). Culture medium were sterilized in an autoclave at 121ºC and 15 psi for 18 minutes. Cultures were incubated at 26 ± 2ºC under a photoperiod with 16 hours of light using white florescent lighting (50 µmol m-2 s-1). Eight tubes with an explant were used to evaluate callus induction for each treatment. The explants exhibiting a callus or morphogenic responses were expressed as a percentage of the total explants, which was determined after 30 days of culture. The treatments that induced the highest percentages were sub-cultured every 30 days on fresh media. 

5.                    Results 

Although immature leaves were used for callus induction, problems with explant contamination and oxidation were observed, which drastically hindered their growth. To resolve these problems, an antioxidant solution (100 mg citric acid and 150 mg ascorbic acid L-1) was added to the culture medium. Furthermore, this antioxidant solution was used during the rinsing and cutting of the explants. An antifungal solution containing Shogún® 0.01 mL mL-1 and Fungoxyl® 0.01 g mL-1 was also used during the disinfection process. These treatments reduced the stress and contamination caused by wounding and unwanted microorganisms and, thereby, enhanced explant growth. 

Control treatment (CDB0) and the treatments from CDB1 to CDB5, CDB7, and CDB9 to CDB22 were not able to produce callus or induce morphogenic responses. Treatments CDB6 (0.5 mg L-1 BAP with 0.5 mg L-1 2,4-D) and CDB8 (0.5 mg L-1 BAP with 2.5 mg L-1 2,4-D) yielded 75% callus induction with a compact greenish appearance for B6 and a compact brown appearance for B8. Highest callus induction (100%) was achieved when leaf explants were supplemented with CDB23 (5.0 mg L-1 BAP and 2.5 mg L-1 2,4-D) or CDB24 (5.0 mg L-1 BAP and 5.0 mg mL-1 2,4-D) (Table 1). The CDB23 and CDB24 treatments produced friable greenish callus (Figure 1).

6.                    Discussion 

To the best of our knowledge, this is the first study to establish a callus culture from the C. chayamansa leaf. Similar studies have described callus induction with BAP and 2,4-D on hypocotyl, stem, node, and leaf explants of Lopezia racemosa, in which, the leaf explants showed the highest callus formation with 0.5 mg L-1 BAP along with 1.0 mg L-1 2,4-D; from this, the 6-O-palmitoyl-3-O-β-D-Glucopyranosylcampesterol compound was isolated, which exhibited anti-inflammatory activity [13]. Other similar species from the Euphorbiaceae family, such as Manihot esculenta, produced callus at 100% efficiency when shoot apical meristems with one to two leaf primordia were cultured on MS medium supplemented with 0.1 mg L-1 BAP, 0.2 mg L-1 Naphthaleneacetic Acid (NAA), and 0.25 mg L-1 Gibberellic Acid (GA3) [14]. Phyllanthus stipulatus showed callus formation and growth when nodal segments of explants were cultured under light conditions on MS medium supplemented with 5.0 mM NAA or under dark conditions in MS medium supplemented with 5.0 mM NAA or BAP or N6-(2-isopentenyl) adenine (2iP) (1.25 - 5.0 mM) [15]. 

7.       Conclusion 

Different concentrations and combinations of BAP and 2.4-D in MS medium were evaluated in immature leaf explants of C. chayamansa to establish callus cultures. In future studies, this callus can be used to initiate cell suspension cultures and obtain extracts and pure compounds. 

8.       Acknowledgement 

This work was partially supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT) from Mexico, who awarded a doctoral fellowship for the first author, and the support through the project FIS/IMSS/prot/g17-2/1739 to carry out her PhD thesis. The authors declare no conflict of interest.


Figure 1: Effect of BAP and 2,4-D on leaf explants of C. chayamansa after 30 days of culture.


Treatment code

PGRs (mg L-1)

Callus induction (%)

Appearance of the callus

BAP

2,4-D

CDB0

0

0

0

CDB1

0

0.5

0

CDB2

0

1

0

CDB3

0

2.5

0

CDB4

0

5

0

CDB5

0.5

0

0

CDB6

0.5

0.5

75

compact greenish callus

CDB7

0.5

1

0

 

CDB8

0.5

2.5

75

compact brown callus

CDB9

0.5

5

0

CDB10

1

0

0

CDB11

1

0.5

0

CDB12

1

1

0

CDB13

1

2.5

0

CDB14

1

5

0

CDB15

0

2.5

0

CDB16

0.5

2.5

0

CDB17

1

2.5

0

CDB18

2.5

2.5

0

CDB19

5

2.5

0

CDB20

5

0

0

CDB21

5

0.5

0

CDB22

5

1

0

CDB23

5

2.5

100

friable greenish callus

CDB24

5

5

100

friable greenish callus

PGRs: Plant Growth Regulators; BAP: 6-Benzylaminopurine (BAP); 2,4-D: 2,4-Dichlorophenoxyacetic Acid.

 

Table 1: Callus induction (%) on immature explants from C. chayamansa leaves under different concentrations and combinations of BAP and 2,4-D, after 30 days of culture. 

1.       Kuti JO, Kuti HO (1999) Proximate composition and mineral content of two edible species of Cnidoscolus (tree spinach). Plant Food Hum Nutr 53: 275-283.

2.       Bautista-Cruz A, Arnaud-Viñas MR, Martínez-Gutiérrez GA, Sánchez- Medina PS, Pérez-Pacheco R (2011) The traditional medicinal and food uses of four plants in Oaxaca, México. J Med Plants Res 5: 3404-3411.

3.       Poot-López GR, Hernández JM, Gasca-Leyva E (2010) Input management in integrated agriculture-aquaculture systems in Yucatan: Tree spinach leaves as a dietary supplement in tilapia culture. Agr Syst 103: 98-104.

4.       Jimoh OF, Bavalola AS, Yakubu TM (2009) Assessment of the antioxidant potential of Cnidoscolus chayamansa. Pharm Biol 47: 903-909.

5.       Kuti JO, Konuru HB (2004) Antioxidant capacity and phenolic content in leaf extracts of tree spinach (Cnidoscolus spp). J Agric Food Che 52: 117-121.

6.       García-Rodrguez RV, Gutiérrez-Rebolledo GA, Méndez-Bolaina E, Sánchez-Medina A, Maldonado-Saavedra O, et al (2014) Cnidoscolus chayamansa McVaugh, an important antioxidant, anti-inflammatory and cardioprotective plant used in Mexico. J Ethnopharmacol 151: 937-943.

7.       Loarca-Piña G, Mendoza S, Ramos-Gómez M, Reynoso R (2010) Antioxidant, antimutagenic, and antidiabetic activities of edible leaves from Cnidoscolus chayamansa Mc Vaugh. J Food Sci 75: 68-72.

8.       Ramos-Gómez M, Figueroa-Pérez MG, Guzmán-Maldonado H, Loarca-Piña G, Mendoza S, et al. (2017) Phytochemical profile, antioxidant properties and hypoglycemic effect of chaya (Cnidoscolus chayamansa) in stz-induced diabetic rats. J Food Biochem 41: e12281

9.       Pérez-González MZ, Gutiérrez-Rebolledo GA, Yépez-Mulia L, Rojas-Tomé IS, Luna-Herrera J, et al. (2017) Antiprotozoal, antimycobacterial, and anti-inflammatory evaluation of Cnidoscolus chayamansa (Mc Vaugh) extract and the isolated compounds. Biomed Pharmacother 89: 89-97.

10.    Loyola-Vargas VM, Ochoa-Alejo N (2012) An introduction to plant cell culture: The future ahead. In: Loyola-Vargas VM, Ochoa-Alejo N (eds) Plant cell culture protocols, Methods in Molecular Biology. Vol. 877, Humana Press, Heidelberg, pp 29-40.

11.    Galaz-Ávalos RM, Aguilar-Díaz S, Xool-González PA, Huchín-May SM, Loyola-Vargas VM (2012) Callus, suspension culture, and hairy roots. Induction, maintenance and characterization. In: Loyola-Vargas VM, Ochoa-Alejo N (eds) Plant cell culture protocols, Methods in Molecular Biology. Vol. 877, Humana Press, Heidelberg, pp 29-40.

12.    Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plantarum 15: 473-497.

13.    Salinas R, Arellano-García J, Perea-Arango I, Álvarez L, Garduño-Ramírez ML, et al. (2014) Production of the anti-inflammatory compound 6-O-Palmitoyl-3-O-β-d-glucopyranosylcampesterol by callus cultures of Lopezia racemosa Cav.(Onagraceae). Molecules 19: 8679-8690.

14.    Acedo VZ (2006) Improvement of in vitro techniques for rapid meristem development and mass propagation of Philippine cassava (Manihot esculenta Crantz). J Food Agric Environ 4: 220-224.

15.    Catapan E, Otuki MF, Viana A (2001) In vitro culture of Phyllanthus stipulatus (Euphorbiaceae). Braz J Bot 24: 25-34.

Citation: Pérez-González MZ, Nieto-Trujillo A, García-Martínez I, Estrada-Zúñiga ME, Bernabé-Antonio A, et al. (2018) Establishing a Callus Culture of Cnidoscolus chayamansa McVaugh: A Species with Ethnopharmacological Value. Adv Biochem Biotehcnol: ABIO-164DOI: 10.29011/ABIO-164. 000064