Available online at www.ijpab.com ISSN: 2320 – 7051 Int. J. Pure App. Biosci. 3 (2): 8-18 (2015) Research Article INTERNATIONAL JOURNAL OF PURE & APPLIED BIOSCIENCE Micropropagation, Callus Induction and Indirect Organogenesis of Maytenus octogona (L'Hér.) DC. (Celastraceae), a Shrub of Peruvian Desert Magali Sánchez-Chuquicusma 1, Guillermo E. Delgado-Paredes 1* and Consuelo Rojas-Idrogo 1 1Facultad de Ciencias Biológicas, Universidad Nacional Pedro Ruiz Gallo, Ciudad Universitaria, Juan XXIII No 391, Lambayeque, Perú *Corresponding Author E-mail: [email protected] ABSTRACT A micropropagation protocol for Maytenus octogona, a medicinal shrub of Peruvian desert, was established using in vitro germinated 3 to 4-week-old seedlings as a source of explants. Shoot elongation was obtained from apical buds and nodal segments cultured in MS medium supplemented with 0.01 mg/L IAA – 0.25 mg/L BAP and 0.01 mg/L IAA – 0.01 mg/L GA 3, with 37.0 and 36.7 cm, respectively, after 60 days of culture. Isolated individual shoots were cultured on full strength MS medium containing IBA 25 mg/L for 24 h in the dark for induction of roots, followed by transfer to gelled hormone-free full strength MS basal medium, with 100% of shoots rooted. Callus induction was achieved from various explants in MS medium supplemented with 5.0 mg/L 2,4-D + 1.0 mg/L KIN + 25 mg/L cysteine +510 mg/L KH 2PO 4, 2.0 mg/L 2,4-D + 0.2 mg/L KIN, and 5.0 mg/L 2,4-D + 0.2 mg/L KIN. Indirect organogenesis was observed in callus induced from hypocotyls in MS medium supplemented with 1.0 mg/L BAP and 2.0 mg/L 2iP. This study is the first attempt to standardize the shoot elongation and shoot prolifer ation from apical buds and nodal segments, callus induction from various explants as zygotic embryos, seeds, leaves, hypocotyls and roots, and indirect organogenesis from hypocotyls. Keywords : Callus induction, in vitro propagation, Maytenus octogona, shoot elongation, rooting. INTRODUCTION The order Celastrales consists of 12 families and a little more than 2000 species. The Celastraceae (800), Hippocrateaceae (300), Aquifoliaceae (300+), and Icacinaceae (400) are fairly closely related and make up the bulk of the order 11 ; however, in the APG classification, the order Celastrales consists of the few families such as the Celastraceae, Lepidobotryaceae and Parnassiaceae 3. The family Celastraceae encompasses 98 genera comprising approximately 1210 species that are widely distributed throughout Asia, Africa and the Americas 8,32 ; these species are trees and shrubs and sometimes climbing or vining. In the Peru the Celastraceae family comprises 8 genera and 27 species, of which the genus Maytenus is the largest, with 16 species described 4, and in the world Maytenus is a genus of over 200 species, mostly in the tropical and subtropical Africa, Asia and Australia. Maytenus octogona (L´Her.) DC. known as “mude” or “realengo” is a shrub or small tree to 1-5 m tall, commonly found in western South America (Ecuador, Peru and Chile) in the coastal zone and arid lowlands. The Celastraceae family has shown the accumulation of an interesting class of bioactive compounds, the quinone-methide triterpenes (QMTs). These QTMs are secondary metabolites that occur only in species of this family, therefore are considered chemotaxonomic indicators 5,10 . These triterpenes have been intensively studied by several important research groups due their biological activities, such as anti- Copyright © April, 2015; IJPAB 8 Sánchez-Chuquicusma et al Int. J. Pure App. Biosci. 3 (2): 8-18 (2015) ISSN: 2320 – 7051 inflammatory 19 , antioxidant 13 , antifungal 17 , antitrypanosomal 14 , antimicrobial 31 , and recently, attention has been focused on the QMT maytenin, because it is highly toxic to a panel of cancer cell lines 2. In in vitro tissue culture, suspension cell cultures of Maytenus hookeri , collected from Xishuangbanna (Yunnan, China), were established from the calli induced from the leaves and young stems on MS media with the supplement of 2.0 mg/L 2,4-D and 0.1 mg/L KIN, and the ethyl acetate extract of the cultures did not yield maytansine instead of affording nine compounds including one novel triterpenoid, named 2,3- diacetoxyl maytenusone, and eight known ones including squalene, salaspermic acid, maytenonic acid, and others 20 . In another study, cell culture of M. ilicifolia was established with callus induced from leaf explants in order to produce and to quantify the antitumoral and antioxidant QMTs, 22 β- hydroxymaytenin and maytenin, and the QMTs showed maximum accumulation in the logarithmic phase growth of cell culture 6. Likewise, explants of M. aquifolium were induced to form callus and, subsequently, suspension cultures, and the isolation of natural products from callus led to the identification of the cytotoxic triterpene quinonemethides, maytenin and 22 β-hydroxymaytenin by reverse phase high performance liquid chromatography 9. Recently, cell suspensions of Maytenus ilicifolia , derived from the long-term cell line retained their capacity to synthesize and accumulate QMTs, and presented levels of maytenin, 22 β-hydroxymaytenin, celastrol and pristimerin that were, respectively, 1.96-, 2.48-, 8.85- and 3.29-times higher than those in the roots of 5-year old plants 7. In the other hand, in some Maytenus species was observed in vitro clonal propagation. In a pioneer study, an in vitro method for cloning and mass multiplication of Maytenus emarginata , a highly drought resistant tree of the Indian Desert, was developed; in this study, shoot segments harvested from a “plus” tree of 30-year-olds to produce multiple shoots on MS medium containing 0.1 mg/L IAA and 2.5 mg/L BAP 30 . Axillary buds of M. aquifolium were cultured in MS medium, and shoot proliferation induced with 3.0 mg/L BAP was greater than in MS medium with KIN at the same concentration; addition of 0.2 mg/L IAA to the medium contributed to an increase in plantlet height, and rootless plantlets were rooted successfully in vitro or ex vitro 26 . In M. ilicifolia , micropropagated shoots were obtained from axillary buds cultured in MS medium supplemented with 3.0 mg/L BA, and the addition of 0.2 mg/L IAA to the medium increased shoot elongation 27 . In this same species, in the micropropagation of nodal and apical segments, the solution with 0.05% ascorbic acid showed the best regeneration rates and the slowest oxidation rates, and 2.0 mg/L BAP was more effective than KIN with regard to percentage of explants regenerative 15 . Mature plants of M. canariensis , an endemic species to the Canary Islands, Spain, were established for apical meristems cultures using branches of two-month-olds collected during the first seven weeks of spring, and the most satisfactory results were obtained using a modified WPM.2 macronutrients medium and MS micronutrients medium supplemented with 0.5 mg/L GA 3, 0.5 mg/L BAP and 0.2 mg/L IAA 23 . In this species, in vitro seed germination occurred only after treatment of the seeds with H 2SO 4, and adventitious shoot regeneration was achieved employing axillary or apical buds taken from 2 – 2.5 months old seedlings, following culture on nutrient media supplemented with BAP, KIN and NAA 18 . Likewise, a micropropagation protocol for M. senegalensis was established using in vitro germinated 6-week-old seedlings as a source of explants; in this study was observed that low levels of BA (0.5 or 1.0 mg/L) were optimal for shoot induction and growth, and generally promoted the average number of shoots produced per explant, while KIN promoted the mean length of shoots 22 . At the present are not known studies in tissue culture of Maytenus octogona , therefore the present investigation focused on factors affecting the efficient micropropagation, callus induction for the establishment of cell suspensions, and indirect organogenesis, of this important species of the Peruvian desert. MATERIALS AND METHODS Plant material Seeds of Maytenus octogona (L´Hér.) DC. (Celastraceae) were collected from the Dunas La Virgen, Lambayeque, Peru. Botanical identification was performed by Doctor Guillermo E. Delgado Paredes from Universidad Nacional Pedro Ruiz Gallo (UNPRG) based on taxonomic description realized by Macbride 21 . The botanic specimen vouchers were deposited at same herbarium of the institution (HPR). Copyright © April, 2015; IJPAB 9 Sánchez-Chuquicusma et al Int. J. Pure App. Biosci. 3 (2): 8-18 (2015) ISSN: 2320 – 7051 Establishment of aseptic cultures Seeds surface were sterilized in a solution of 50% commercial bleach (2.0 – 2.5% active chlorine) for 15 min plus one drop of detergent Tween 20, followed by 1 min immersion in 70% ethanol. The material was rinsed three times with sterile, distilled water in a clear air cabinet, one to three surface-sterilized seeds were placed into test tubes (18x150 mm) containing the nutrients of the MS formulation 24 supplemented with 3% sucrose, 100 mg/L myo-inositol, 1.0 mg/L thiamine.HCl and 8 g/L agar-agar. Plantlet mass propagation Prior to the study of M. octogona morphogenesis, in vitro mass propagation was carried out through axillary bud propagation. First and second axillary buds emerging were excised from germinated plantlets, vertically placed onto the shoot multiplication MS medium (shoots and roots induction) with sucrose, vitamins and several plant growth regulator interactions, and sub-cultured after 60 days of growth. In order to investigate M. octogona clonal propagation potential, explants were cultured in a different combinations (0.01, 0.05 and 0.1 mg/L) of the auxin indole-3-acetic acid (IAA) and 0.25, 1.0 and 2.5 of the cytokinin 6-benzylaminopurine (BAP) and 0.01 and 0.02 mg/L IAA with 0.01 and 0.02 mg/L GA 3 (gibberellic acid); likewise, some treatments were supplemented with Plant Prod (PP) fertilizer (N 100%, P 2O2 52%, K 2O 10%).