Introduction

Physalis spp. (Solanaceae) are distinguished by an additive and inflated calyx, which expands completely around the fruit (Soares et al. 2009). The genus includes more than 90 species distributed throughout the American continent (Stehmann et al. 2015); approximately 70 of these species grow in Mexico where this country is the place of origin, diversity, and distribution of the taxon (D'Arcy 1991; Martinez 1998; Vargas-Ponce et al. 2011). Recently, P. angulata has been widely cultivated in an artisanal manner in central Mexico due to its nutritional and medicinal properties (Valdivia-Mares et al. 2016, Vargas-Ponce et al. 2016). Conversely, P. chenopodifolia has several uses, taking advantage of the whole plant; roots, stems, leaves, and fruit calyces are used in traditional medicine, and their sweet-bitter fruits are highly consumed (Valdivia-Mares et al. 2016).

Germplasm conservation through in vitro techniques is commonly used for species with high pharmaceutical and commercial potential, such as P. angulata and P. chenopodifolia (Romo-Paz et al. 2021). This technique allows germplasm to multiply, conserves genetic variability, and allows the study of the species’ properties under controlled conditions without affecting wild populations (Bertoni et al. 2010).

Recent studies in different plant species where micropropagation techniques have been used have shown that this technique can be a solution for commercial exploitation, propagation, and conservation of selected species, improved genotypes, or elite specimens (Afroz et al. 2010; Delgado-Aceves et al. 2021). The biotechnological interest in the Physalis genus has increased in recent years due to it being rich in nutrients and compounds of pharmacobiological interest, such as phytosterols, withanolides, carotenoids, phenolic compounds, and physalins (Puente et al. 2011; Salcedo-Pérez et al. 2015; Lima et al. 2020). It also shows important antibiotic, antioxidant, anticancer, and anti-inflammatory properties (Reyes-Reyes et al. 2012; da Silva et al. 2015). However, there are few studies on the management, conservation, micropropagation, and plant regeneration of selected species of the Physalis genus with high commercial potential. Escobar-Guzmán et al. (2009) described the capacity of P. ixocarpa Brot. to regenerate embryos and plants from anther cultures treated with different levels of 2,4-D and vitamin B12 was tested. In the case of P. minima L., Mungole et al. (2011) reported the induction of callus in vitro and the regeneration of shoots from leaves with different concentrations of 2,4-D, Kinetin, and BAP, either alone or in combination. In P. peruviana L., rhizogenesis and organogenesis were induced from cultured leaves and nodes with treatments containing BAP and Kin (Otroshy et al. 2013). For P. angulata L, various methods have been used to generate propagation of clones in vitro including the use of phytohormones, such as BAP, Zeatin, Gibberellic acid (GA3), IAA, Kin (Kumar et al. 2015; 2016), or different concentrations of sucrose and light spectra (Santos et al. 2020). On the contrary, for P. chenopodifolia Lam., there is no published information on the in vitro management of the species to the knowledge of the present authors.

Despite the great potential for use in the pharmaceutical industry, the commercial extraction of these chemical compounds is still difficult mainly because of two factors: the high genetic variation caused by the cross-pollination that predominates within Physalis genus and the low production of secondary metabolites in wild plants (Trillos et al. 2008). Thus, in vitro propagation techniques are promising tools for large-scale multiplication of selected varieties with a higher content of bioactive compounds and for in vitro production of high added-value compounds (de Oliveira et al. 2013).

Organogenesis is an important in vitro tissue culture technique that induces genetic changes and increases the plant multiplication rate for conservation (Su and Zhang 2014). Organogenesis is the ability to regenerate entire organs or plants from cells that rearrange to develop a meristem upon stimulation (Hnatuszko-Konka et al. 2021). It includes the formation of buds, shoots, or radical meristems directly from explants (direct organogenesis) or after cell proliferation and the generation of a callus (indirect organogenesis) (Lardon and Geelen 2020; Shin et al. 2020). In morphogenetic competition, explants develop the ability to respond to organ-inducing hormonal signals, a process known as dedifferentiation (Hnatuszko-Konka et al. 2021). Auxins and cytokinins are the main phytohormones involved in this process. These plant growth regulators (PGRs) need to be applied simultaneously or sequentially because they coordinately guide the formation of organ-generating structures through complex intracellular crosstalk (Duclercq et al. 2011; García-Pérez et al. 2020). This process also depends on the capability of the cells and tissues to respond to these changes (Sugiyama 2014). This study aimed to determine an effective procedure for mass multiplication of P. angulata and P. chenopodifolia through whole-plant regeneration from callus induction of leaf segment explants.

Materials and methods

Plant material and culture conditions

The biological material was established in vitro from dried seeds of Physalis angulata L. (Voucher OVP 544) and P. chenopodifolia Lam. (Voucher OVP 539). Seeds are deposited at the University of Guadalajara, at the National Laboratory for Plant Identification and Characterization (LaniVeg).

The plant material (50 seeds per species) was disinfected with 70% ethanol for 1 min, and then commercial sodium hypochlorite (Cloralex®, Monterrey, Mexico) (25%, v/v) was applied (1.5% active chlorine), plus two drops of liquid detergent for 15 min in constant agitation, followed by washing three times with sterile distilled water. Plant material was established on basal Murashige and Skoog (MS) medium (25%) (Sigma-Aldrich, St. Louis, MO) (Murashige and Skoog 1962) supplemented with L2 vitamins (Sigma-Aldrich) (Phillips and Collins 1979), 3% (w/v) sucrose (Golden Bell®, Zapopan, Mexico), and 0.8% (w/v) agar (Sigma-Aldrich); pH was adjusted to 5.8 ± 0.02 (Mascarenhas et al. 2019). The medium was sterilized by autoclaving at 121 °C for 15 min. The seeds were kept in dark conditions for 1 d, then they were maintained under diffuse light conditions (27 µmol m−2 s−1) with a 16-h photoperiod, at 27 ± 2 °C (Silva et al. 2016).

Micropropagation

Aseptic explants of P. angulata and P. chenopodifolia (axillary buds) were established for shoot proliferation in basal MS medium, supplemented with L2 vitamins (Phillips and Collins 1979), and complemented with different combinations of PGRs, 0.0, 2.22, 4.43, and 6.65 μM BAP (Sigma-Aldrich), combined with 0.0, 2.32, 4.64, and 6.96 μM Kin (Sigma-Aldrich). Three stem segments (1 to 2 cm) with one axillary meristem each were established per container, seven replications per treatment for a total of 16 treatments. The culture media were enriched with 3.0% (w/v) sucrose (Golden Bell®) and 0.8% (w/v) agar (Sigma-Aldrich); pH was adjusted to 5.8 ± 0.02. Shoot proliferation of axillary buds’ data were documented and analyzed 4 wk after culture initiation. These in vitro propagation experiments were replicated in three cycles. Data from all three cycles were pooled, and average proliferation rates over time were determined. For indirect organogenesis, explants (leaf sections) of approximately 0.5 cm in length were used; these explants were obtained from the 4-wk-old plants propagated in vitro. Explants were individualized and established in sterile plastic Petri dishes, containing 20 mL of basal MS medium supplemented with L2 vitamins (Phillips and Collins 1979) and several combinations of PGRs (cytokinins and auxins): 0.0, 2.22, 4.43, 6.65, and 8.86 μM BAP (Sigma-Aldrich) combined with 0.0, 1.13, 2.26, and 3.39 μM 2,4-D (Sigma-Aldrich). Three leaf segments were established per container, and seven replicates per treatment were inoculated for a total of 20 treatments. Sucrose (Golden Bell; 3.0% w/v) and agar (Sigma-Aldrich) (0.8% w/v) were added to all culture media. The pH was adjusted to 5.8 ± 0.02. The medium was sterilized by autoclaving at 121 °C for 15 min. The explants were maintained under diffuse light conditions (27 µmol m−2 s−1) with a 16-h photoperiod at 27 ± 2 °C. Percentage of explants with callus, percentage of explants forming shoots, and number of shoots per explant were recorded and analyzed 4 wk after culture initiation.

Histological analysis

Fresh organogenic calluses samples were dissected; to fix the samples, the plant material was treated with 70% v/v ethanol. Subsequently, the tissue was placed in a solution of polyethylene glycol (BioReagent®, Toluca, Mexico) (PEG, 1450 molecular weight) and deionized water in a 1:4 proportion, as described by Delgado-Aceves et al. (2021). Subsequently, the plant tissue was embedded in the PEG solution, and cuts were made with a rotary microtome (YIDI® Jinhua, China) to obtain 15 μm sections of the samples; then, in order to observe structures, the tissues were double stained, as described by Gupta and Durzan (1987), applying 0.5% (1:1 w/v) acetocarmine and 0.5% (1:1 w/v) Evan’s blue. The samples were then analyzed under an optical microscope (Carl Zeiss® White Plains, N Y).

Rooting and acclimatization

Micropropagated accessions of P. angulata and P. chenopodifolia were established in a rooting experiment, in basal MS medium supplemented with L2 vitamins and auxins in the following concentrations: 0.0, 1.07, 2.68, and 5.37 μM NAA (BioReagent®, Toluca, Mexico) or 0.0, 1.41, 2.85, and 7.70 μM IAA (Sigma-Aldrich). Three healthy shoots from the indirect shoot multiplication (2 cm long) were established per container; seven replications by treatment were placed in the culture medium for a total of nine treatments. Sucrose (Golden Bell; 3.0% w/v) and 0.8% w/v agar (Sigma-Aldrich) were added to all culture media. The pH was adjusted to 5.8 ± 0.02. The medium was sterilized by autoclaving at 121 °C for 15 min. The explants were maintained under diffuse light conditions (27 µmol m−2 s−1) with a 16-h photoperiod at 27 ± 2 °C. Number of roots present per explant and their corresponding length were recorded 4 wk after treatment. Healthy plantlets with well-developed shoots and roots were transferred to plastic pots containing sterile perlite and peat moss (1:1). Potted plantlets were covered with transparent domes to ensure high humidity and were maintained under diffuse light conditions (27 µmol m−2 s−1) with a 16-h photoperiod at 27 ± 2 °C. The plantlets were watered every 3 d with 1/8-strength MS basal salt solution supplemented with L2 vitamins without sucrose. The plastic dome was removed after 2 wk to acclimatize plants to greenhouse conditions. After 4 wk, plantlets were maintained in the greenhouse under full sunlight (Swartwood and Van-Eck 2019).

Statistical analysis

A completely randomized design was used to carry out all experiments. The significance of differences among factors in micropropagation (levels BAP-Kin), indirect organogenesis (levels BAP-2,4-D), and rooting (levels NAA-IAA) experiments was determined by two-way analysis of variance (ANOVA). Fisher’s LSD test was performed when significance was detected to differentiate among means (P < 0.05). Statistical analyses were carried out using the Statgraphics Centurion XVI® statistical software 16.2.4.

Results and discussion

Plant material and culture conditions

The seeds of P. angulata exhibited 92% germination and those of P. chenopodifolia 96% germination after 4 d of inoculation on the medium supplemented with 25% MS basal salts supplemented with L2 vitamins. These results demonstrated the high capacity of Physalis spp. to develop under reduced in vitro conditions, as reported by Rodrígues et al. (2013) and da Silva et al. (2021) who performed in vitro culture of P. peruviana, P. minima, and P. ixocarpa explants with 25, 50, and 75% MS basal salts.

Micropropagation

Micropropagation of P. angulata and P. chenopodifolia via multiplication of axillary buds on MS basal medium supplemented with L2 vitamins and different combinations and concentrations of PGRs was tested. In all treatments, both species showed shoot proliferation (Fig. 1).

Figure 1.
figure 1

Effects of 6-benzylaminopurine (BAP) and Kinetin (Kin) on micropropagation of Physalis angulata L. and P. chenopodifolia Lam. via multiplication of axillary buds (data recorded after 4 wk). The means followed by the same letter(s) are not significantly different according to the Fisher’s LSD test (P. angulata, lowercase letter; P. chenopodifolia, capital letter). Data represent means ± standard deviation (SD).

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The ANOVA showed a significant difference (P ≤ 0.05) among factors (BAP and Kin) and a positive interaction between PGRs (P ≤ 0.05) in P. angulata culture. The highest average number of new shoots in this species was found in the treatment with 4.43 μM BAP and 2.32 μM Kin in which 8.47 ± 2.91 shoots per explant were counted. The interaction between BAP and Kin has been favorable for bud multiplication of various plant species (Mungole et al. 2011; Handique and Bhattacharjee 2000). Conversely, P. chenopodifolia showed the best result with the addition of Kin alone; 10.47 ± 2.25 shoots per explant were obtained in basal medium supplemented with 4.64 μM Kin only (P < 0.05).

Cytokinins are known to induce axillary shoot formation (Jafari et al. 2011; de Oliveira et al. 2019). Indeed, PGRs, such as BAP or Kin, induce multiplication in solanaceous plants like Solanum lycopersicum (= Lycopersicon esculentum), Capsicum annum, and S. tuberosum (Harish et al. 2010; Kumar et al. 2011; Kazemiani et al. 2018). The results in the present study were similar to those of previous reports (Romo-Paz et al. 2021), demonstrating the multiplication capacity of the Physalis after several micropropagation cycles.

In contrast, the highest shoot length was obtained without PGRs in both species (P < 0.05). These results confirmed the findings of Huetteman and Preece (1993) and Brassard et al. (1996), stating that cytokinins commonly inhibit shoot elongation. P. angulata and P. chenopodifolia had the maximum lengths of 15.14 ± 1.2 and 18.09 ± 1.1 cm, respectively, on media without cytokinins; furthermore, the length of the shoots decreased as the concentration of the PGRs increased (Fig. 2).

Figure 2.
figure 2

Effects of 6-benzylaminopurine (BAP) and Kinetin (Kin) on the stimulation of the length of the micropropagated shoots of Physalis angulata L. and P. chenopodifolia Lam. (data recorded after 4 wk). The means followed by the same letter(s) are not significantly different according to the Fisher’s LSD test (P. angulata, lowercase letter; P. chenopodifolia, capital letter). Data represent means ± standard deviation (SD).

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Additionally, the effect of 2,4-D combined with BAP in the micropropagation of P. angulata and P. chenopodifolia via indirect organogenesis was investigated. Leaf explants cultured on MS basal medium formed a yellow-to-green friable callus (Fig. 3), followed by numerous shoot buds in most treatments with both PGRs (Table 1). This result was similar to those reported for other species, such as P. minima (Sandhya and Rao 2016) and P. peruviana (Hernández-Villalobos and Chico-Ruíz 2020). The highest number of new indirect shoots was found in P. chenopodifolia; the treatment with 1.13 μM 2,4-D and 6.65 μM BAP generated 37.14 ± 3.54 shoots per explant (P < 0.05). For P. angulata, the best result was observed with 1.13 μM 2,4-D and 4.43 μM BAP in the basal medium; this treatment generated 22.71 ± 2.5 shoots per explant (P < 0.05). In both species, adding both BAP and 2,4-D to the medium stimulated the induction of shoots, and the interaction between these PGRs was significant (P < 0.05). These results contrast with those of Gupta and Chandra (1987), who found that the formation of shoot buds in P. ixocarpa leaf explants was induced with BAP (0.5 to 5.0 mg L−1) addition alone.

Figure 3.
figure 3

Anatomical characterization and histological analysis of organogenic callus of Physalis angulata L. and P. chenopodifolia Lam.). (a) Leaf explant (1 wk). (b) Callus formation with friable characteristics (2 wk). (c) Development of meristematic structures from organogenic callus (3 wk). (d) Shoots of P. angulata from leaves treated with 1.13 μM 2,4-D and 4.43 μM BAP (4 wk). (e) Shoots of P. chenopodifolia from leaves treated with 1.13 μM 2,4-D and 6.65 μM BAP (4 wk). (f) Histological section of a P. angulata callus with signs of cell development and proliferation prior to the development of shoots (yellow arrows). (g) De novo meristem from organogenic callus of P. chenopodifolia. (h) Section of callus with shoots of P. angulata before obtaining rooting material. (i) Section of callus with shoots of P. chenopodifolia before rooting experiments. (j) Rooted and acclimatized P. angulata plantlets (6 wk after acclimation). (k) Rooted and acclimatized P. chenopodifolia plantlets (6 wk after acclimation).

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Table 1. Effects of Murashige and Skoog medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D) and 6-Benzylaminopurine (BAP) on indirect organogenesis — stimulation from leaf explants of Physalis angulata L. and P. chenopodifolia Lam. (data analyzed after 4 wk)
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Leaf explants of P. angulata and P. chenopodifolia barely exhibited sprout regeneration when the auxins or cytokinin were added alone in MS basal medium, supplemented with L2 vitamins. Callus initiation was not obtained in all treatments. According to Phillips and Garda (2019), the production of a pluripotent callus is closely linked to the addition of 2,4-D to the culture medium. Moreover, a high cytokinin concentration promotes shoot regeneration from callus cells because cytokinins mediate early loss of root identity, root primordia disorganization, and initiation of shoot development (Shin et al. 2020). Thus, hormone equilibrium and crosstalk between auxins and cytokinins are crucial for determining cell fate and patterning (Cheng et al. 2013).

Established explants on media supplemented with low concentrations of 2,4-D and different BAP concentrations showed the highest frequency of shoot formation. These findings agreed with those of Ramar et al. (2014) and Rezanejad and Hosseini (2019), who observed that, due to cellular pluripotency, the regenerated explant of other solanaceous species (S. americanum and P. alkekengi) dedifferentiated the tissues, returning them to their proliferative condition (Greb and Lohmann 2016).

Indirect shoots were induced along the entire surface of the explants (Fig. 3, d and e). Shoots induced by these PGR interactions at different concentrations did not show morphological alterations. The shoot buds obtained on BAP and 2,4-D medium were excised and grown to complete plants on 100% MS medium supplemented with L2 vitamins.

Histological analysis

Histological analysis of the organogenic callus originating from disorganized leaf explants allowed the present authors to characterize the tissues anatomically and structurally. The shoots obtained were presented asynchronously and abundantly, which allowed the present authors to observe differentiation zones and meristem formation of both species (P. angulata and P. chenopodifolia) (Fig. 3, f and g).

In the early stages of the culture, the organization of the callus with large asymmetric vacuolated cells was observed. The cells that comprise it show continuous proliferation and high mitotic activity where many differentiation zones were identified on the periphery of the callus (Fig. 3, f). Subsequently, in advanced stages of the crop, it was identified that the formation of apical meristems begins from these differentiation zones, presenting a central zone, procambium, protoderm, and defined leaf primordia (Fig. 3, g).

Rooting and acclimatization

Root formation of P. angulata and P. chenopodifolia was observed in all the treatments with NAA or IAA (Table 2). The results showed a significant difference (P ≤ 0.05) between the two factors. These auxins had already been used for rooting in Physalis plants (Ramírez-Malagón and Ochoa-Alejo 1991). The first roots emerged from the basal segment of the shoot 5 to 6 d after culture with no callus formation. The highest number of roots was observed in regenerated Physalis shoots when cultured on MS medium (Murashige and Skoog 1962) supplemented with L2 vitamins (Phillips and Collins 1979) and with NAA. The highest number of roots was registered on P. chenopodifolia 32.19 ± 2.5 (P ≤ 0.05) with a media containing 5.38 μM NAA; P. angulata showed a maximum average of 27.47 ± 2.01 (P ≤ 0.05) roots per shoot in the medium with 1.07 μM NAA. Conversely, IAA treatments yielded a lower number of roots; P. angulata generated 19.57 ± 1.85 roots per explant in the treatment with 2.85 μM IAA, and P. chenopodifolia generated 31.3 ± 2.93 roots in the treatment with 5.70 μM IAA.

Table 2. Effect of NAA (1-naphthaleneacetic acid) and IAA (Indoleacetic acid) on root induction of micropropagated plantlets of Physalis angulata L. and P. chenopodifolia Lam. (data analyzed after 4 wk)
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Most studies using NAA and IAA for root induction have achieved a high rooting frequency (De Klerk et al. 1997; Ozel et al. 2006; Afroz et al. 2010). These auxins stimulate cell division; however, although they are essential for the initial induction of roots, their presence at certain concentrations can inhibit root development (Mills et al. 1997; Singh et al. 2008), as observed for the highest concentrations of NAA and IAA (Table 2). Thus, the balance between the substances added to the medium should be standardized to optimize the rooting of the explant (Borchetia et al. 2009), and the association between the rooting percentage and the endogenous-exogenous auxin levels in the plantlets must also be considered (Blakesley et al. 1991).

The highest root length was obtained without PGRs in P. chenopodifolia (17.85 ± 1.73 cm) while P. angulata recorded a maximum average of 13.6 ± 1.05 cm. The roots observed in the treatments with the highest concentration of NAA (8.05 μM) and IAA (8.55 μM) were small, thick, yellow–brown, and devoid of root hairs. Rooted shoots were transferred to plastic pots and successfully established and grown in greenhouse conditions (Fig. 3, j and k) with a survival rate of 90% for P. angulata and 94% for P. chenopodifolia.

Conclusion

The interaction between BAP and Kin was favorable for the development of P. angulata, stimulating the proliferation of buds and inhibiting their elongation. For P. chenopodifolia, the best result was observed with the addition of Kin alone. Moreover, leaf explants were shown to be effective for shoot regeneration via indirect organogenesis. The histological analysis demonstrated this process and allowed the multiple-cellular origin of buds to be defined. The successful production of multiple indirect shoots and the formation of roots in vitro depended on the effect of different PGRs (2,4-D and BAP and NAA or IAA, respectively) and growing conditions. The standardized method for mass multiplication of these two Physalis species through callus induction and plant regeneration could be used to produce and isolate medicinally important secondary metabolites, such as physalins and withanolides. To the best of the present authors’ knowledge, this is the first report regarding in vitro whole-plant regeneration by indirect organogenesis of P. angulata and P. chenopodifolia from leaf segment explants.