Plant material.

I. sonorae (S. Watson) Greene plants (≈750 g) were obtained at the local Sonora market, Mexico City, Mexico. The plant internode containing axillary buds (NXB) was selected as the source of explants.

Explant decontamination was done according to the procedure modified by Estrada-Zuñiga et al. (2012). The explants were washed with 1% (v/v) Extran detergent solution (Merck) for 10 min and rinsed with distilled water, followed by immersion in 70% (v/v) ethanol for 30 s, then transferred to a 1.2% (v/v) sodium hypochlorite solution diluted from a 5% commercial stock (Cloralex^®) for 10 min and finally rinsed four times with sterile-distilled water. The explants were excised into 0.4–0.8 cm length fragments that were separately cultured in flasks containing Murashige and Skoog (MS) (Murashige and Skoog, 1962) or Gamborg medium (B5) (Gamborg et al., 1968)supplemented with various concentrations and combinations of plant growth regulators (BA, IAA, and NAA).

Organogenic callus induction and adventitious shoot regeneration.

The explants were grown on basal media comprising MS (4.3 g·L⁻¹ or B5 3.1 g·L⁻¹) supplemented with 25 g·L⁻¹ sucrose, 150 mg·L⁻¹ ascorbic acid, 6 g·L⁻¹ agar-agar, and growth regulators. The cultures were then incubated in a growth chamber maintained at 25 ± 2 °C under 16 h photoperiod (50 µmol·m⁻²·s⁻¹, daylight fluorescent tubes). To determine the best concentrations of growth regulators for shoot induction, we applied a two level full factorial design (2^k = 2³) using the growth regulators BA, IAA, and NAA as the three factors (Gardiner and Gettinby, 1998). In this study, the most efficient auxin/cytokinin ratio in MR medium for the production of organogenic callus and regenerated shoots derived from NXB explant was investigated. Besides, the frequency of shoot induction and the number of regenerated shoots per explant were recorded after 15 d of culture.

Conditions for root induction.

To establish the culturing conditions for root induction, proliferating shoots (4–6 cm length and 21-d-old) with one or two leaves were excised and transferred to B5 medium supplemented with IBA. Different IBA concentrations were assayed to determine the optimal condition for root induction. These conditions were chosen by factorial design [one factor multilevel, equation: Y = 3.62 + 20.8A, where Y is the response (root induction), and A is the IBA factor] (Gardiner and Gettinby, 1998). The culturing conditions were similar to those described in section “Organogenic callus induction and adventitious shoot regeneration.” The number of emerging roots and the root length of explants bearing shoots were recorded by day 21. Shoots containing roots were removed from the culture and after rinsing with a 1% (v/v) aqueous nystatin were separately placed inside plastic pots containing a commercial potting soil mix pH 6.0 (Hydro-Environment) containing 20% perlite, 20% vermiculite, 20% humus, and 40% peatmoss. The pots were covered with transparent polythene bags and irrigated with sterilized tap water. The plantlets were incubated in an ambient with 33% humidity under conditions similar to those described in the section “Organogenic callus induction and adventitious shoot regeneration.”

Statistical analysis.

Statistical analysis of the effect of each growth regulator on the frequency of regenerated shoots and rooting efficacy were determined with Statistical Analysis System (SAS) and online University Edition and Minitab (Statistical Software Version 16), respectively. Each experiment was repeated three times. Data were analyzed using analysis of variance, and the significance of differences between means was determined via Tukey’s test at 5% significance level.

Callus Induction and shoot regeneration.

Experiments were initially designed to compare B5 and MS media efficacies to induce calli from root explants supplemented with IAA, NAA, and BA. The B5 medium was 3.8-fold more efficient than the MS medium to induce calli under similar conditions. In addition, calli grown on B5 increased their mass 4.5-fold, whereas MS-grown calli exhibited a 2-fold mass increase after 15 d in culture (data not shown).

Following optimization of calli induction from root explants, a screening for the best conditions to optimize shoot induction was implemented using NXB explants. In these experiments, B5 or MS medium required supplementation with growth regulators (IAA, NAA, and BA) to stimulate shoot formation. The absence of any of them obliterated this effect (Table 1). Overall, B5 medium was more efficacious than MS as shoot inducer in I. sonorae NXB explants. At low concentration (0.5 mg·L⁻¹) of each growth regulator, only B5 medium induced shoots (23.3 ± 7.5%). The highest shoot induction (96.7 ± 4.7%) was observed in B5 when concentration of IAA was 1 mg·L⁻¹, whereas the concentration of BA and NAA were kept constant at 0.5 mg·L⁻¹. Besides, the same treatment enhanced shooting in MS medium to a lower degree (30.0%). The optimal shooting effect in MS medium (40 ± 8.1%) occurred when increasing BA to 1 mg·L⁻¹ while keeping IAA and NAA at 1.0 and 0.5 mg·L⁻¹, respectively. Interestingly, at 0.5 mg·L⁻¹ IAA and 1.0 mg·L⁻¹ NAA no shoot induction in B5 or MS medium was evident, regardless the concentration of BA (Table 1).

Table 1.Effect of medium and concentration of growth regulators on shoot induction in I. sonorae NXB explants.

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Therefore, we adopted the optimized conditions as shown on Table 1 (BA 0.5 mg·L⁻¹, IAA 1.0 mg·L⁻¹, and NAA 0.5 mg·L⁻¹) and named the MR medium in subsequent experiments to induce organogenic calli and regenerate shoot in NXB explants. The dynamic of these events is summarized in Fig. 1A–D. Figure 1A shows green organogenic calli on day 5 of culture and white organogenic calli from day 7 of culture (Fig. 1B). On day 15, they are shown regenerating shots from green calli (Fig. 1C) and white calli (Fig. 1D).

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Furthermore, under this condition, organogenic calli sprouted on day 5 at the ends of internodes in contact with the medium. White friable calli were 2.7 times more abundant than green calli and their masses increased ≈4-fold by day 15 (Table 2) regardless the color of calli (green calli, Fig. 1A; white calli, Fig. 1B). After one week, it was observed that regenerated shoots emerged more frequently (3.5x) in friable white calli than in green calli (Table 3).

Table 2.Frequency and weight of organogenic calli in NXB explants from I. sonorae.

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Table 3.Frequency of regenerating shoots from calli in NXB I. sonorae explants.

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On emerging white-calli, the first shoots appeared between 5–8 d (Fig. 1B). After culturing for 15 d, internodes with axillary buds emerged, and the frequency of shoot induction was 87.5 ± 14.4% (Fig. 1D; Table 3). On the other hand, green calli showed lower frequency of induced shoots (25 ± 4.0%) and their shoots emerged around day 13 (Fig. 1C; Table 3). At the end of the elongation period (15–20 d), the regenerated shoots attained 4–6 cm length.

Figure 1E displays the elongating shoots. Using the optimized MR medium, the addition of fresh growing medium to stimulate elongation of regenerated shoots was not necessary. After 20 d in MR culture, 70.8 ± 19.1% of the regenerated shoots grew up, averaging 9.17 ± 2.9 shoots per explant, exhibiting normal appearance and no signs of hyperhydricity (Fig. 1F). These shoots were selected for further rooting experiments.

Root induction.

Cultures of organogenic white calli from NXB explants, displaying the largest number of regenerated shoots (10 ± 1.9 shoots per explant) and averaging 4.44 ± 0.3 cm (data not shown) were used to evaluate the rooting ability. In the absence of IBA, shoots survived up to 10 d after transplantation without emergence of roots. Successful rooting was observed in regenerated shoots grown in B5 medium supplemented with IBA (Table 4). Between 0.5 and 1.0 mg·L⁻¹ IBA, rooting attained 6.6% to 20%, respectively. Most shoots failed to generate roots and did not survive beyond day 15. Survivors at 0.5 mg·L⁻¹ IBA exhibited thin primary roots without lateral roots (0.5 ± 0.47 roots) on day 21 (Table 4). Meanwhile, root induction gradually increased to 20% in the medium supplemented with 1.0 mg·L⁻¹ IBA attaining 1.25 ± 0.94 roots per regenerated shoot. At 2.0 mg·L⁻¹ IBA and 21 d of culture, the plantlets displayed fasciculate thin roots (Fig. 2A) and primary thick taproot with lateral roots (Fig. 2B) and yielded the highest rooting frequency of 83.3% (Table 4). The result of the specimen selected for acclimatization on day 25 (Fig. 2C) shows a robust increase in root induction accompanied by larger number of roots (4.25 ± 0.98). At 3 mg·mL⁻¹ IBA, emergence of thin primary fasciculate roots declined by 40% compared with 2 mg·L⁻¹ IBA (Fig. 2D; Table 4). On day 25, regenerated plantlets with primary and secondary roots were transferred to small plastic pots with sterilized soil (Fig. 2E), and 50 ± 0.5% (Table 4) of these plantlets survived after culturing for 30 d (Fig. 2E and F).

Table 4.Effect of IBA concentration on root induction of regenerated shoots from I. sonorae.

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Callus induction and shoot regeneration.

Micropropagation is a tissue culture technique to propagate species with slow growth in their natural habitat, production of plant free of pathogens, year around nursery of plantlets, clonal propagation of parental stocks, production of germplasm and implicit with this, preservation of endangered species. The aim of this study was to establish a protocol for micropropagation using NXB explants from I. sonorae. Initially, we investigated the most efficient culture media and auxin/cytokinin ratio for callus and shoot induction in I. sonorae NBX explants. We found that B5 medium was 3.2-fold more efficacious to induce callus than MS medium under equivalent conditions (unpublished data). Similarly, B5 medium induced 2.2-fold more shoots than MS medium (Table 1). Possibly, this organogenic efficacy of B5 medium is due to its higher content in vitamins (Smith, 2013).

We now demonstrate that optimized MR medium comprising 1.5 mg·L⁻¹ auxin (IAA + NAA) and 0.5 mg·L⁻¹ cytokinin (BA) with molar ratio 3.8:1 induced high percentage of organogenic friable calli in I. sonorae NXB explants on day 15 (Fig. 1B) and promoted shoot induction as well (Fig. 1C and D). Inclusion of IAA was based on the notion that it participates during the developmental and growing stages, coordinating plant metabolism (synthesis, conjugation, hydrolysis, oxidation, and transport) in cucurbitaceous, more efficiently than NAA or 2,4-D (Lee et al., 2010; Normanly et al., 1995). The auxins IAA and NAA have been reported to promote callus formation and new shoots in tissue culture of cucurbitaceous Cucumis metuliferus (Compton and Gray, 1993). Prior reports showed that high auxin/cytokinin ratio enhances root formation in explants of Melothria maderaspatana and during generation of organogenic callus in Cucumis sativus (Baskaran et al., 2009; Kakani et al., 2009; Selvaraj et al., 2007), while low auxin/cytokinin ratio privileges shoot formation, although in this study we observe an increase of shoot production with high auxin/cytokinin values.

In previous studies, the stimulatory effect of BA combined with IAA in Cucumis melo was shown to be more efficient for calli formation than BA plus NAA or 2,4-D (Tabei et al., 1991). However, Valdez-Melara and Gatica-Arias (2009) demonstrated that the superior effect elicited by BA-IAA in C. melo depended on the genotype used. Also, the authors noted that regardless the genotype, supplementation of medium with BA alone without auxins was able to sustain shoot emergence. These results depart from what is observed in I. sonorae where IAA, NAA, and BA are essential for shoot emergence. Differences in the requirement of growth regulators highlight the relevance of variations between species that must be taken into account during micropropagation studies.

Results of induction of organogenic calli or shoot induction, similar to those reported here were described on cultures using axillary buds of nodal explants in cucurbitaceous Momordica charantia L. (0.5 mg·L⁻¹ BA and 2.0 mg·L⁻¹ NAA) (Agarwal and Kamal, 2007), in hypocotyl and leaf explants of Cucumis anguria L. (0.5 mg·L⁻¹ BA and 1.5 mg·L⁻¹ of 2,4-D, 1.5 mg·L⁻¹ IAA) (Ju et al., 2014) during propagation in Momordica balsamina using 1.0 mg·L⁻¹ BA (Thakur et al., 2011) and callus induction and plantlet regeneration of Citrullus colocynthis (Cucurbitaceae) with 0.5 mg·L⁻¹ IAA, 0.5 mg·L⁻¹ 2,4-D, and 1 mg·L⁻¹ BA (Satyavani et al., 2011).

The role of cytokinins and auxins as agents for organogenesis has been discussed earlier. Agarwal (2015) proposed that cytokinins activate totipotent cells in callus for shoot organogenesis whereas in the case of direct organogenesis, these molecules activate preexisting machinery pertaining to somatic cells. Cytokinins in shoots stimulate growth because of the presence of meristematic cells located at the tip of explants, whereas auxins regulate or influence diverse responses at the whole-plant level, by mechanisms such as tropisms, apical dominance and root initiation, and by triggering cellular responses such as cell enlargement, division, and reactivation of differentiated cells to promote additional vascular tissue development and regulating lateral organ formation (Hagen and Guilfoyle, 2002; Mockaitis and Estelle, 2008).

Although the mean number of shoots per explant obtained in I. sonorae was lower when compared with Citrullus colocynthis (Meena et al., 2013) or Momordica charantia (Thiruvengadam et al., 2012), their inception within two weeks is somewhat earlier than in most regenerating systems. Interestingly, most examples describing regeneration of organogenic callus include two regulators (1 auxin and 1 cytokinin), while in this work we maximize calli production by using 2 auxins and 1 cytokinin.

The rationale for using axillary buds as vehicle for micropropagation rests on the notion that they are able to preserve the genetic traits due to the presence of meristematic tissue (Souza et al., 2006). This notion is supported by studies, showing that many of the propagated plants in Echinocereus cinerascens and Momordica dioica when derived from axillary buds displayed higher genetic stability and uniformity (Elias et al., 2015; Thiruvengadam et al., 2006). Our results showed that the optimized MR medium enhanced the growth of regenerated shoots, and preserved them in fair condition without damage or loss during the 15 d experimental period, obviating the need for further passage to an elongation medium.

Root induction.

The efficacy of root induction seen in I. sonorae regenerating shoots at 2.0 mg·L⁻¹ IBA was also demonstrated in Stackhousia tryonii, M. dioica, Citrullus lanatus, Momordica cymbalaria, and Cucumis sativus (Bhatia et al., 2002; Selvaraj et al., 2007; Thiruvengadam et al., 2006). In S. tryonii, Bhatia et al. (2002) also reported that IBA was more effective than IAA and NAA for rooting, thus justifying its application as root inducer in many cucurbitaceous. In M. balsamina, a dose dependent effect on rooting has been demonstrated with maximal activity at 1.5 mg·L⁻¹ of IBA (Thakur et al., 2011).

IBA has been used in commercial agriculture as well for its capacity in plants to promote cuttings, root initiation/growth, inhibition of primary root elongation, and stimulation of lateral root formation (Zažímalová et al., 2014). In I. sonorae, regenerating shoots in B5 medium supplemented with 2.0 mg·L⁻¹ IBA there was a strong induction of roots attaining 83.3% (Table 4). In contrast, Yan et al. (2010) observed no significant difference on rooting rate in vitro and ex vitro in Siratia grosvenorii. However, in vitro developed roots in S. grosvenorii were thick, fragile, and easily broken while handling. Because of this difference, ex vitro rooting was preferable as it promoted higher percent survival rate. In I. sonorae, we detected 50% survival for plantlets regenerated following treatment with 2.0 mg·L⁻¹ IBA and acclimatized in small pots for 30 d.

Interestingly, tuberous root of I. sonorae has been demonstrated to possess pharmacological activity. To date, no report exists on micropropagation or induction of tuberous root of I. sonorae in vitro or even the procedure to modulate its growth in soil. Rooting similar to that observed in this study is found in field specimens of I. sonorae; hence, investigating the presence of biologically active metabolites in in vitro grown roots is essential to confirm the presence of hypoglycemic activity. Fan et al. (2011) reported that in Manihot esculenta combinations, auxins and cytokinins are necessary to induce tuberous roots in vitro, and the absence of one of them in the medium led to their decrease or full absence. According to our results, IBA alone induced thick taproots and moderated the number of fasciculate roots; thus, obviating the need for cytokinins during root development. Future studies aim to investigate if the regenerated roots arising in culture and roots developed in ex vitro plantlets contain the metabolites responsible for the medicinal properties in I sonorae.