HORTSCIENCE 52(7):1000–1005. 2017. doi: 10.21273/HORTSCI11942-17 survival; therefore, an action is required to protect this species (Gomez-Aiza, 2011). Cell and tissue culturing are valid alter- Efficient In Vitro Plant Regeneration natives for in vitro production of secondary metabolites with biological activity because from Internode Explants of Ibervillea they are independent of seasonal factors. A standardized protocol for in vitro micropro- sonorae: An Antidiabetic Medicinal pagation represents a suitable option for the conservation of endangered species or prop- agation of variants with a desired phenotype Plant (Elias et al., 2015). Many of these protocols Ilse-Yazmín Arciniega-Carreon, Carmen Oliver-Salvador1 have been developed for regeneration of and María-Guadalupe Ramírez-Sotelo cucurbitaceous species, like cucumber í (Cucumis sativus) (Kim et al., 2010; Kumar Laboratorio de Biotecnolog a Molecular, Unidad Profesional Interdisciplinaria et al., 2003a), winter squash (Cucurbita de Biotecnología del Instituto Politecnico Nacional (UPIBI-IPN), Av. maxima Duch.) (Lee et al., 2003), ash gourd Acueducto s/n C.P. 07340, Colonia La Laguna Ticoman, Ciudad de Mexico, (Benincasa hispida) (Thomas and Sreejesh, Mexico 2004), summer squash (Cucurbita pepo L.) (Kathiravan et al., 2006), spiny gourd Carlos Edmundo Salas (Momordica dioica Roxb.) (Devendra et al., Departamento de Bioquímica, Instituto de Ciencias^ Biologicas, 2009), athalaikai and kakrol (Momordica Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901, tuberosa) (Aileni et al., 2009), melon gubat (Melothria maderaspatana Linn.) (Baskaran MG, Brasil et al., 2009), fig-leaf gourd (Cucurbita ficifo- Additional index words. micropropagation, cucurbitaceae, callus culture, internode explant, lia Bouche) (Kim et al., 2010), balsam apple hypoglycemic (Momordica balsamina) (Thakur et al., 2011), telakuch (Coccinea cordifolia) (Roy Abstract. Ibervillea sonorae is a medicinal plant mainly used to treat diabetes, ulcers, and et al., 2012), ridge gourd (Luffa acutangula other metabolic disorders. A regeneration protocol using internode segments containing L. Roxb.) (Zohura et al., 2013), and bitter axillary buds grown on Gamborg medium (B5) supplemented with 0.5 mg·LL1 melon (Momordica charantia L.) (Sammaiah a-naphthalene-acetic acid (NAA), 0.5 mg·LL1 N6-benzyladenine (BA), and 1.0 mg·LL1 et al., 2014). Moreover, micropropagation in indole-3-acetic acid (IAA) successfully regenerated shoots in I. sonorae explants. The vitro has been reported using shoot and nodal induction of organogenic calli attained 100% efficiency. The highest percent shoot explants of cucurbitaceous Cucumis sativus, production was 87.5% with a mean of 9.17 shoots per explant on day 15, and the Trichosanthes dioica (Ahmad and Anis, maximum length of 5.8 cm was observed on day 21. Regenerated shoots induced roots 2005; Kumar et al., 2003b). Despite this in B5 medium supplemented with 0.5–3.0 mg·LL1 indole-3-butyric acid (IBA). The profuse account on Cucurbitaceae, no records maximum rooting frequency observed in the medium containing 2.0 mg·LL1 IBA was are available for regeneration of I. sonorae 83.3% which promoted long, thick roots on day 21. The plantlets with emerging roots except for one report on calli induction using grown at the culture facility attained 50% survival after acclimatization for 30 d. The leaf explants (Estrada-Zuniga~ et al., 2012). account describes a simple and efficient protocol for in vitro plant regeneration, and this Therefore, the aim of this work is to establish micropropagation procedure offers an alternative for preservation of this medicinal a protocol for in vitro regeneration of I. plant. sonorae to serve as a plant repository, and to define the growth conditions for subse- quent establishment of cell-suspension cul- From ancient times, florae are source of metabolic disorders (Johnson et al., 1996; tures aiming the production of secondary compounds for the treatment of diseases. It is Xolalpa, 2002). It belongs to the Cucurbita- metabolites as well. estimated that around 75% of the world’s ceae family, and it is native to arid areas from population currently depends on plants as northern Mexico (Lira and Caballero, 2002; Materials and Methods source of traditional medicine (Arias et al., Xolalpa and Aguilar, 2006). It produces 2009; Rao and Ravishankar, 2002). Medici- secondary metabolites such as alkaloids, Plant material. I. sonorae (S. Watson) nal plants contain therapeutic molecules, tannins, saponins (Alarcon-Aguilar et al., Greene plants (750 g) were obtained at the whose active principles also serve as pre- 2005), flavonoids, phenols, (Zapata-Bustos local Sonora market, Mexico City, Mexico. cursors for drug synthesis (Loraine and et al., 2014) and cucurbitacins (Achenbach The plant internode containing axillary Mendoza-Espinoza, 2010). et al., 1993; Jardon-Delgado et al., 2014). buds (NXB) was selected as the source of Ibervillea sonorae (S. Watson) Greene is Pharmacological studies show that plant root explants. a medicinal wild perennial plant usually extracts display hypoglycemic and anti- Explant decontamination was done accord- known as ‘‘wereke’’ or ‘‘guareque,’’ tradi- inflammatory (Alarcon-Aguilar et al., 2005; ing to the procedure modified by Estrada- tionally used to treat diabetes, ulcers, and Jardon-Delgado et al., 2014; Rivera-Ramírez Zuniga~ et al. (2012). The explants were washed et al., 2011; Zapata-Bustos et al., 2014), with 1% (v/v) Extran detergent solution antioxidant (Estrada-Zuniga~ et al., 2012), (Merck) for 10 min and rinsed with distilled antimicrobial (Robles-Zepeda et al., 2011), water, followed by immersion in 70% (v/v) Received for publication 20 Mar. 2017. Accepted and antifungal activities (Ruiz-Bustos et al., ethanol for 30 s, then transferred to a 1.2% (v/v) for publication 1 June 2017. 2009). A recent study conducted on human sodium hypochlorite solution diluted from The authors would like to acknowledge the finan- preadipocyte cells showed that aqueous root a 5% commercial stock (CloralexÒ)for cial support by Instituto Politecnico Nacional, extracts from I. sonorae stimulate glucose 10 min and finally rinsed four times with Mexico SIP 20140775, SIP 20141244, 20151038. uptake by a PI3K independent pathway sterile-distilled water. The explants were C. Oliver-Salvador is the recipient of a fellowship from Comision de Operacion y Fomento de Acti- (Zapata-Bustos et al., 2014). This evidence excised into 0.4–0.8 cm length fragments that vidades Academicas-IPN. I. Y. Arciniega-Carreon supports the antidiabetic properties attributed were separately cultured in flasks contain- is the recipient of a scholarship from Consejo to I. sonorae roots in traditional medicine. ing Murashige and Skoog (MS) (Murashige Nacional de Ciencia y Tecnología, Mexico. The widespread demand of I. sonorae roots and Skoog, 1962) or Gamborg medium 1Corresponding author. E-mail: [email protected]. for therapeutic purposes threatens their (B5) (Gamborg et al., 1968)supplemented 1000 HORTSCIENCE VOL. 52(7) JULY 2017 with various concentrations and combinations of B5 and MS media efficacies to induce calli from green calli (Fig. 1C) and white calli plant growth regulators (BA, IAA, and NAA). from root explants supplemented with IAA, (Fig. 1D). Organogenic callus induction and NAA, and BA. The B5 medium was 3.8- Furthermore, under this condition, orga- adventitious shoot regeneration. The ex- fold more efficient than the MS medium to nogenic calli sprouted on day 5 at the ends of plants were grown on basal media comprising induce calli under similar conditions. In addi- internodes in contact with the medium. White MS (4.3 g·L–1 or B5 3.1 g·L–1) supplemented tion, calli grown on B5 increased their mass friable calli were 2.7 times more abundant with 25 g·L–1 sucrose, 150 mg·L–1 ascorbic 4.5-fold, whereas MS-grown calli exhibited than green calli and their masses increased acid, 6 g·L–1 agar-agar, and growth regula- a 2-fold mass increase after 15 d in culture 4-fold by day 15 (Table 2) regardless the tors. The cultures were then incubated in (data not shown). color of calli (green calli, Fig. 1A; white calli, a growth chamber maintained at 25 ± 2 °C Following optimization of calli induction Fig. 1B). After one week, it was observed that under 16 h photoperiod (50 mmol·m–2·s–1, from root explants, a screening for the best regenerated shoots emerged more frequently daylight fluorescent tubes). To determine conditions to optimize shoot induction was (3.5x) in friable white calli than in green calli the best concentrations of growth regulators implemented using NXB explants. In these (Table 3). for shoot induction, we applied a two level experiments, B5 or MS medium required On emerging white-calli, the first shoots full factorial design (2k =23) using the growth supplementation with growth regulators appeared between 5–8 d (Fig. 1B). After regulators BA, IAA, and NAA as the three (IAA, NAA, and BA) to stimulate shoot culturing for 15 d, internodes with axillary factors (Gardiner and Gettinby, 1998). In this formation. The absence of any of them buds emerged, and the frequency of shoot study, the most efficient auxin/cytokinin ratio obliterated this effect (Table 1). Overall, B5 induction was 87.5 ± 14.4% (Fig. 1D; Ta- in MR medium for the production of organo- medium was more efficacious than MS as ble 3). On the other hand, green calli showed genic callus and regenerated shoots derived shoot inducer in I. sonorae NXB explants. At lower frequency of induced shoots (25 ± 4.0%) from NXB explant was investigated. Besides, low concentration (0.5 mg·L–1) of each and their shoots emerged around day 13 the frequency of shoot induction and the growth regulator, only B5 medium induced (Fig. 1C; Table 3). At the end of the elongation number of regenerated shoots per explant shoots (23.3 ± 7.5%). The highest shoot period (15–20 d), the regenerated shoots were recorded after 15 d of culture. induction (96.7 ± 4.7%) was observed in B5 attained 4–6 cm length.