Introduction

The advance in the knowledge of in vitro hormonal regulation has allowed the development of micropropagation protocols in species and cultivars of economic importance, such as in hybrid rose Mimosa pudica L. (^{Bianchetti et al., 2017}); (^{Aggarwal et al., 2018}), even in red algae (^{Villanueva et al., 2013}). The effect of different concentrations of phytohormones on the micropropagation of species of the family Cactaceae has been studied in germplasm of interest for ornamental horticulture at risk status, such as: in several species of the genus Coryphanta spp. (^{Pérez-Molphe-Bach et al., 1998}), the old man cactus Cephalocereus senilis (Haw.) Pfeiff. (^{Choreño et al., 2002}), swallow nest biznaga Epithelantha micromeris (Engelm.) F. A. C. Weber ex Britt. & Rose (^{Villavicencio et al., 2012}), mammillaria Mammillaria voburnensis Scheer (^{Ordoñez, 2003}), artichoke cactus Obregonia denegrii Frič & A. Berger (^{Cardarelli et al., 2010}), various mini cactus of the genus Turbinicarpus spp. (^{Mata-Rosas et al., 2001}; ^{Dávila et al., 2005}; ^{Villavicencio et al., 2011}) and pitaya Stenocereus stellatus (Pfeiff.) Riccob. (^{Martínez-Villegas et al., 2011}), where in vitro shoots have been obtained using cytokinins alone or combined with auxins.

There are results with the interaction of two cytokinins 6-benzyl aminopurine (BAP) and 2-isopentenyl-adenine (2-iP), in combination with low levels of auxins; in other cases, with the interaction of cytokinin-auxin such as: BAP-α-naphthaleneacetic acid (NAA), BAP-indole-3-butyric acid (IBA), 6-furfuryl aminopurine (KIN)-NAA or indole-3-butyric acid (IBA). This variety of responses is a function of the genotype and culture medium used. ^{Gonçalves et al. (2016)} mention that cytokinins (CK) and auxins (AUX) have been used to regulate the growth and development of plant tissues, but it is not yet clear how these compounds influence the metabolic activity of species in vitro, where CK concentrations from 0 to 20 μM can influence the functionality of the photosystem (PS) II, leaf anatomy, stomatal density, production of shoots from meristems or axillary buds.

Considering that the in vitro culture of plant tissues has become a very useful tool to accelerate plant production processes, its feasibility will depend on the management of the factors that favor this process, such as the culture medium used, where the concentration of nutrients, hormones, hours of light, temperature, pH, relative humidity, oxygen supply, among others, influence the development of plants; so the morphogenetic response of the explant will depend on the standardization of the micropropagation protocol.

With the purpose of carrying out ex situ conservation actions, promote the use and enhancement of native material of ornamental interest (^{Espinoza-Flores et al., 2003}; ^{Granada, 2014}; ^{Gámez et al., 2016}), such as the ornamental biznaga ‘inverted cone of Viereck’ Turbinicarpus viereckii subsp. major (Glass & R.A. Foster) Glass, native to northern Mexico, belonging to the subfamily Cactoideae and Tribe Cacteae (^{Guzmán et al., 2003}; ^{Hunt, 2006}; ^{Tropicos, 2019}) and that it is in special protection risk status (Pr) according to NOM-059-ECOL-2010 (^{SEMARNAT, 2010}), it became necessary to evaluate other unconventional propagation schemes to regenerate this species through in vitro culture, proposing as an objective of this research to define the type and dose of cytokinins, as well as the cytokinin-auxin ratio that influence the multiplication stage, to select the ratio of phytohormones that promotes the greatest number of shoots in vitro without losing the stability of the explant and standardize the micropropagation protocol to regenerate this species and produce plants in sufficient quantities.

Materials and methods

The research was carried out in the facilities of the Plant Tissue Culture Laboratory (LCTV) of the Saltillo CIRNE-INIFAP Experimental Field, located in the municipality of Saltillo, Coahuila, Mexico.

Plant material. In the first stage, vitroplants germinated in vitro from seed collected in the summer of 2016 in the municipality of Guadalcázar in San Luis Potosí were used.

Explants. When the vitroplants reached a height of 10 mm ±1, two cuts were made to remove the distal and proximal area of the stem, using the intermediate segments of the stem (SIT) as explants. This type of explant was used in all subsequent subcultures (Figure 1).

Figure 1 Obtaining explants and establishing treatments in the multiplication of the ornamental biznaga ‘inverted cone of Viereck’ (Turbinicarpus viereckii subsp. major (Glass & R. A. Foster) Glass). 

From the second subculture, the SITs obtained from the distal area of the stem (ZDT) (apical) were used, which were subcultured and grown in the Murashige-Skoog medium (^{MS; Murashige and Skoog, 1962}) as a base medium, halving (50%) its concentration of macrosalts and without growth regulators, to obtain again intermediate segments of the stem (SIT) and produce the required explants in the following subcultures and continue with multiplication.

Multiplication of shoots. The intermediate segments of the stem (SIT) used as explants were established in the Murashige-Skoog medium (^{MS; Murashige and Skoog, 1962}) at 50% of its macrosalt concentration, supplemented with 100 mg L^-1 of myo-inositol (SIGMA ^®), 0.4 mg L^-1 of thiamine-HCl (SIGMA^®, T-3906), 1 mg L^-1 of pyridoxine-HCL (SIGMA^®, P-8666), 30 g L^-1 of sucrose (SIGMA^®, K-0753) and 7 g L^-1 of agar (SIGMA^®, A-1296), with 200 mg L^-1 of activated charcoal. The pH of the medium was adjusted to 5.7 and previously sterilized in a Felisa brand autoclave at 1.2 kg cm^-2 pressure at 121 °C for 15 min.

Using a completely randomized experimental design with factorial arrangement, two cytokinins were evaluated: BAP and KIN at four concentrations 2.5, 5, 7.5 and 10 mg L^-1 in interaction with two auxins: NAA and IBA in a cytokinin-auxin ratio of 10:1. Five SITs per bottle were established, with 10 repetitions per treatment and a total of 16 treatments were evaluated. This evaluation was repeated five times by successive subcultures from 2017 to 2019 in the LCTV, evaluating the number of shoots per explant (Nb) and the shoot height (Ab, mm) every ten weeks.

Statistical analysis. The evaluated variables were analyzed using the PROC GLM procedure of the statistical analysis system SAS, (Version 9.4) (^{SAS, 2019}), performing an analysis of variance (Anova) to determine the effect of phytohormones (cytokinins and auxins), influence of concentration and a Tukey multiple range test (p≤ 0.05) considering the treatments as independent effects to select the best treatment.

Incubation conditions. The vegetative material was established in the incubation room of the LCTV considering a photoperiod of 14 h light by 10 of darkness, with a luminous intensity of 9-10 μmol sec^-1 m^-2 and a temperature of 25 ±2 °C.

Acclimatization and transfer to soil. The shoots obtained were individualized and subcultured in a MS medium at 50% of its components, adding 1 g L^-1 of activated charcoal to induce rooting and then passed to their acclimatization. The rooted vitroplants were washed and treated with 1 g L^-1 of commercial systemic fungicide with active ingredient N-(trichloromethylthio) cyclohex-4-ene-1,2-dicarboximide prior to transplantation in pots of 0.15 L capacity (6.5 x 5.6 x 5.6 cm). As a substrate, a mixture sterilized in an autoclave (Sumi Brand) at a temperature of 120 °C and 1.5 pounds of pressure, composed of sand + peat moss + agrolite in a 3:2:1 ratio, was used.

Results and discussion

In vitro culture stability. Although, there were significant differences (F= 60.31, p= 0.0001) between evaluations in the Anova, the statistical parameters corresponding to a high coefficient of determination (R²) of 0.72, lower coefficient of variation (CV) of 26.4 and minimum significant difference (MSD) of 0.44 showed that the intermediate segments of the stem (SIT) used as an explant can express a morphogenetic response under the influence of the treatments.

Under in vitro conditions, 1 to 22 shoots per explant can be obtained, with subcultures III and IV where, on average, an Nb of 9.22 ±1 shoots per explant was obtained (Figure 2a). Although there were differences between the first and last of the five subcultures, the results were relevant for a mass production system, which can be maintained for more than three years and decrease its production from the fifth clonal multiplication, an issue that must be considered in the programming of production on a commercial scale.

Figure 2 Number of shoots per explant1 (Nb) and shoot height (Ab) in the in vitro multiplication of the ornamental biznaga ‘inverted cone of Viereck’ (Turbinicarpus viereckii subsp. major (Glass & R. A. Foster) Glass). a) influence of the stability of the in vitro subculture; b) influence of the type of cytokinins; c) influence of the type of auxins; and d) influence of concentration. 

In shoot height (Ab), significant differences between evaluations (F= 427.45, p= 0.0001) were also found, registering a coefficient of determination (R²) of 0.50, a coefficient of variation (CV) of 32.65 and a minimum significant difference (MSD) of 0.25. Under in vitro conditions, the Ab ranges from 1 to 18 mm, with subcultures III and IV where, on average, an Ab of 7.56 ±1 mm was obtained; however, the size of the shoots obtained in all the evaluations carried out could be manipulated by the operator when performing the subculture (Figure 2a).

The results recorded in each of the evaluations carried out showed that the SIT explants are stable in in vitro culture, being an organized plant tissue with axillary buds, from which the biznaga ‘inverted cone of Viereck’ T. viereckii subsp. major can be regenerated.

Influence of the type of cytokinins. When analyzing the total of the subcultures, Anova does not show significant differences (p≤ 0.05) between the cytokinins evaluated in Nb; however, with the culture medium MS at 50% with KIN, the greatest response was obtained, registering on average 8.69 shoots per explant in Nb (Figure 2b).

The multiplication rate for the biznaga T. viereckii subsp. major with both cytokinins exceeds that obtained in M. craigii (4.65), M. formosa (4.42), M. obscura (4.78) and M. uncinata (5.25) (^{Pérez-Molphe-Balch et al., 1998}), M. oteroi (5.3) (^{Castro-Gallo et al., 2002}) and is like that reported for biznaga ‘inverted cone of Knuth’ (Turbinicarpus knuthianus (Boed.) John and Říha) (^{Villavicencio et al., 2011}). In Ab, there was a statistically significant difference (p≤ 0.05) between cytokinins, where BAP is the phytohormone that influences this variable with an Ab of 6.27 mm, exceeding in size the shoots obtained with KIN (Figure 2b).

In other species of cacti such as Epithelantha micromeris var. micromeris, Strombocactus disciformis, and T. schmiedickeanus var. klinkerianus (^{Soltero and Portillo, 2015}) and T. knuthianus (^{Villavicencio et al., 2011}), KIN has also been used for in vitro culture, as in other ornamentals, such as orchids Laeliocattleya (Orchidaceae) (^{Gonçalves et al., 2016}) and coffee (Coffe arabiga) (^{Cantos-Cevallos et al., 2018}), showing that the morphogenetic response depends on the cytokinin used and the genotype. KIN is of natural origin, derived from purines or adenines, and can produce an effect on cell division and induce a morphogenic response in the explant, activating the axillary buds or areolar meristems that induce the formation of shoots of the biznaga T. viereckii subsp. major as has also occurred in Mammillaria schiedeana schiedeana (^{Soria-Campos et al., 2013}).

Influence of the type of auxins. There were significant differences (p≤ 0.05) between the auxins evaluated, with indole butyric acid (IBA) being the one that influenced morphogenetic response in both Nb and Ab.

With this auxin, a greater response in Nb was obtained, registering on average 8.92 shoots per explant, with an Ab of 6.20 mm, exceeding the effect generated with naphthaleneacetic acid (NAA), where both variables registered a lower average, with an Nb of 7.3 shoots per explant and an Ab of 4.5 mm (Figure 2c).

IBA has also been used for the induction of shoots of Epithelantha micromeris var. micromeris, where up to 13 shoots per explant have been obtained (^{Villavicencio et al., 2012}), while the use of NAA has had an effect on other cacti of the genus Mammillaria spp. (^{Pérez-Molphe-Balch et al., 1998}; ^{Castro-Gallo et al., 2002}; ^{Giusti et al., 2002}; ^{Ramírez-Malagón et al., 2007}; ^{Soria-Campos et al., 2013}), registering a height similar to that reported in this paper.

The greater number of shoots with smaller size is an effect that frequently occurs in micropropagation, as reported by ^{Mata-Rosas et al. (2001)}; ^{Pérez-Molphe-Balch et al. (2015)}, so it is always appropriate to evaluate various cytokinin-auxin combinations to determine the balance between the response variables that are sought in the in vitro multiplication stage of the species of interest. This to ensure intensive propagation, give continuity to the multiplication stage and ensure survival during acclimatization.

Influence of concentration. Regardless of the type of cytokinin used, there were highly significant differences between concentrations (p≤ 0.01). The concentrations of 2.5 and 5 mg L^-1 were statistically equal, surpassing the rest of the concentrations evaluated, with an Nb of 8.99 shoots/explant and an Ab of 6.24 mm (Figure 2d).

Influence of the type of concentration and cytokinin. The 50% MS base medium added with 2.5 mg L^-1 of KIN and with 5 mg L^-1 of BAP were statistically equal, surpassing the rest of the concentrations evaluated. Both cytokinins and concentrations promoted shoot induction with an Nb of 9.19 and 9.09 shoots per explant, respectively (Table 1).

The difference between both concentrations and type of cytokinins was in the height of shoots, where the greatest response was obtained with 5 mg L^-1 of BAP, registering an Ab of 6.55 mm. These results showed that SITs were more compatible with KIN, a natural phytohormone, where a lower concentration in plant tissue generated a greater response in the induction of shoots, unlike SITs that were exposed with BAP, which is a synthetic cytokinin, where the greatest induction of shoots was obtained by doubling the concentration (Table 1).

The effect of the type of cytokinin and concentration on the number and height of shoots has also been reported in globose-type biznagas, where 3 to 10 mg L^-1 of KIN (^{Soltero and Portillo, 2015}) and from 0.5 to 2 mg L^-1 have been applied when BAP has been used (^{Soria-Campos et al. 2013}).

The results show that hormonal control influences the differentiation of the explant, as reported by ^{Pérez-Molphe-Balch and Dávila-Figueroa (2002)}; ^{Dávila et al. (2005)}; ^{Ascough and Van Staden (2010)}; ^{De la Rosa-Carrillo et al. (2012)}. This control had a positive effect on in vitro culture, making ex situ conservation feasible for both the cactus under study T. viereckii subsp. major, and for other species, such as the old man cactus Cephalocereus senilis (Haw.) Pfeiff. (^{Choreño et al., 2002}), pitaya Stenocereus stellatus (Pfeiff.) Riccob. (^{Martínez-Villegas et al., 2011}), mammillaria Mammillaria voburnensis Scheer (^{Ordoñez, 2003}), artichoke cactus Obregonia denegrii Frič & A. Berger (^{Cardarelli et al., 2010}) and mini cactus Turbinicarpus spp.

Influence of treatments. When performing the Tukey mean test (p≤ 0.05) to the treatments as independent effects, considering the interaction between the type of cytokinin and auxin with their different concentrations evaluated, it was determined that there are two treatments that can be used in the multiplication stage. The 50% MS medium added with 5 mg L^-1 of BAP + 0.5 mg L^-1 of IBA and the one added with 2.5 mg L^-1 of KIN + 0.25 mg L^-1 of IBA were statistically equal, surpassing the rest of the treatments evaluated in Nb, registering an Nb of up to 9.25 shoots per explant; however, with the second treatment, larger shoots are obtained, with an Ab of 6.73 mm, which facilitates a better manipulation of these when performing the subculture (Table 2).

These results show that the cytokinin:auxin interaction in a 10:1 ratio influences the morphogenesis of the explant of this species, regulating the cell division and growth of the axillary buds for the induction of shoots (Figure 3).

Figure 3 Multiplication of the ornamental biznaga ‘inverted cone of Viereck’ (Turbinicarpus viereckii subsp. major (Glass & R. A. Foster) Glass). a, b c) induction of shoots; d) vitroplants; and e) acclimatized plants. 

The KIN:IBA interaction is similar to that reported by ^{Finti et al. (2012)}; Pacheco (2015); ^{Velázquez and Soltero (2001)}; ^{Villavicencio et al. (2020)}, who found that the cytokinin source has significant effects on the production of shoots in the biznaga ‘swallow nest’ Epithelantha micromeris var. micromeris (Engelm.) F. A. C. Weber ex Britton & Rose, cactus pear Opuntia ficus-indica (L.) Mill. and ‘sacasil’ Echinocereus poselgeri Lem., so this aspect must be considered in the micropropagation of cacti, since the efficiency of the treatment for the induction of shoots, such as the budding coefficient, are important in the in vitro multiplication stage to achieve the final results in the micropropagation, because the number of explants that are established, number of shoots that are generated and vitroplants that are produced will depend on this stage.

As the concentration in the BAP:IBA and KIN:IBA interactions increases, the multiplication rate decreases. The lowest morphogenetic response in the different concentrations evaluated was obtained with the BAP:NAA and KIN:NAA interactions.

This response indicates that naphthaleneacetic acid (NAA) is an auxin that poorly stimulated growth in the axillary buds of T. viereckii subsp. major. In other species, such as Mimosa pudica (^{Bianchetti et al., 2017}), the BAP:NAA association also registered low shoot induction, as the effect reported in the present research.

In cacti such as Turbinicarpus pseudomacrochele and Strombocactus disciformis, ^{Soltero and Portillo (2015)} report that the KIN:NAA interaction can present a significant effect when applied at a concentration of 3 to 10 mg L^-1 of KIN, combined with a low concentration of 0.02 mg L^-1 of NAA, so it is necessary to consider these factors in this process, since the morphogenetic response of the explant will depend significantly on this interaction and the rest of the components of the culture medium.

Conclusions

The regeneration of this species in risk status, as well as its ex-situ conservation can be carried out with in vitro multiplication, where the cytokinin-auxin ratio influences the morphogenetic capacity and stability of the explant, from which the proliferation of axillary shoots can be induced.