Vegetal material

Healthy and vigorous seedlings grown under nursery conditions provided by the cactus management and utilization unit, Tepetlaoxtoc, Estado de México, with the UMA MX/VIV-CO-194.MX-SEMARNAT registration, were used.

Culture medium and incubation conditions

The basic culture medium used was ^{Murashige and Skoog (MS, 1962}) with complete inorganic salts, supplemented with sucrose (30 g L^-1), myoinositol (100 mg L^-1), thiamine (1 mg L^-1) and solidified with agar (Merck^®, 9 g L^-1). The pH was adjusted to 5.7 with NaOH or HCl 1N and sterilization was performed in a vertical autoclave (AESA® Model 300) at 121 °C and 1.5 kg cm^-2 pressure for 20 min. Cultures were maintained at 25 ±2 °C in photoperiod of 16 h and light intensity of 45 µmol m^-2 s^-1.

Aseptic culture establishment

Young explant donor plants, of 4 to 6 cm in diameter, were washed with detergent and tap water for 25 min, then rinsed six times with sterile distilled water. Immediately, a surface disinfection treatment was applied, which consisted in the immersion of the plants in a commercial sodium hypochlorite (NaOCl, cloralex^®; 30% v/v) with stable colloidal silver (Microdyn^®; 1.5% v/v) for 10 min and then rinsed six times with sterile distilled water. Subsequently, the plants were immersed in ethanol (70%) for 5 min and again six rinses with sterilized distilled water were applied.

Induction of shoots

Segments of 5-8 cm tuber length with areola were seeded in 45 mL capacity flasks with 15 mL of basic culture medium MS (1962) added with 2,4-D (9, 13.5 and 18 μM) combined with Kinetin (4.6, 9.3 and 13.9 μM) and medium without growth regulators as a control. After four weeks of culture, the formation of calli (%), sprouting (number of explants with shoots) and number of shoots per explant were quantified. The experimental design was completely random, where each treatment was represented by 10 replicates and the experimental unit was an areolar explant per flask.

Calli proliferation and shoot multiplication.

In order to promote the proliferation of shoots, the explants (shoots and calluses) were transferred to the same treatments used in the induction stage of shoots for 12 weeks, with subcultures every four weeks. At 4, 8 and 12 weeks the callus growth (measured as weight of fresh matter (mg), the number of novo organogenic shoots by explant; the number of sprouts per areolar activation per explant; novo shoot length and areolar activation (mm), the number of roots and root length (cm) were quantified. The experimental design was completely random where each treatment was represented by 10 repetitions and the experimental unit was an areolar explant per flask.

Rooting and acclimatization of plants

Shoots of 1-1.5 cm diameter were subcultured on MS medium (1962) to half concentration of mineral salts supplemented with sucrose (15 g L^-1) for six weeks. When roots lengthened and constituted a root system with 3 to 5 roots, 40 plants with an average diameter of 1-1.5 cm were selected for acclimatization. Plants were extracted from the flasks; roots were washed with sterile distilled water and planted in polystyrene containers of 150 mL capacity. Two types of sterile substrates were evaluated: peat + river sand (1:1) and peat + leaf soil (1:1). The plants were covered with a polyethylene bag and placed in a growth chamber with photoperiod of 12 h light; temperature of 26 ±2 °C and irrigation with sterile distilled water every third day. At 30 and 60 days after transplantation, the survival percentage was quantified.

Histological analysis

The purpose of this analysis was to study the origin of the shoots and callus obtained during the induction and multiplication stages; at 10 weeks samples were taken from explants with organogenic areolar shoots and callus tissue. Samples were fixed in FAA; dehydrated in a series of ethanol gradients and processed for inclusion in paraffin (^{Ruzin, 1999}). Using a rotary microtome (Spencer^®) transverse and anticlines samples were obtained (10 μm) which were stained with safranin 0 and solid green FCF (^{Zavaleta and Engleman, 1994}). Observations were made under a microscope (Axioskop 2 Plus, ZEISS^®) and photomicrographs were taken with a digital camera (Axioskop MRC5, ZEISS^®).

Statistic analysis

The obtained data were subjected to ANOVA with SAS statistical analysis package (^{SAS Institute, 2003}) and for comparison of means Tukey test (p≤ 0.05) was used.

Establishment of aseptic culture

The disinfection process used for the establishment of aseptic cultures of M. plumosa was efficient, since the survival rate of the explants was 90% (Figure 1a-b). The effectiveness of the disinfection process and eventual tissue damage of the explants are directly related to exposure time, disinfectant concentration, phytosanitary condition of donor plants, type, size and physiology of the explants and are critical factors during early stages of crops (^{George and Debergh, 2008}) so that in M. plumosa finding a balance in these aspects was achieved. This stage is considered fundamental, since the presence of pollutants and the blackening of tissues negatively affect the morphogenic responses (^{Smith, 2013}).

Figure 1 In vitro morphogenesis and histological analysis of M. plumosa . a) adult plant; b) areolar explant; c) regenerated callus and bud of apical areola with 2,4-D (18.0 μM) and kinetin (9.30 μM) at 60 days of culture; d) organogenic callus, areolar and organogenic bud after 90 days; e) initial stage 1, green callus; f) bud emergence at 60 days; g) intermediate stage 2 of the shoot; h) longitudinal section of the intermediate shoot at 90 days; i) stage 3 of the shoot; j) longitudinal section of the differentiated shoot at 120 days. ca= organogenic callus; bra= areolar shoot; bro= organogenic shoot; br= shoot; alm= starch; pa= parenchyma; proc= procambium; mab = apical meristem of bud; x= xylem; f= phloem; em= modified spines. 

Shoots induction

The concentrations of growth regulators in the culture medium had a significant effect on the observed morphogenic responses (p≤ 0.05). All combinations of growth regulators promoted indirect organogenesis in the explants, whereas direct organogenesis was only obtained in three treatments (Table 1).

Table 1 Effect of 2,4-D and kinetin in direct and indirect organogénesis of M. plumosa after four weeks of cultivation. 

¶= Medias con la misma letra en cada columna no son significativamente diferentes (Tukey, 0.05).

In indirect organogenesis the explants generated several types of calli, which varied in their coloration, texture and growth rate. The coloration varied from white to green and the consistency was firm and compact to friable (Figure 1c-d). Friable green calli originated and grew on the cut surface at the base of the explants, which is a response that matches observations in Mammillaria carmenae, since callus grown in the presence of 2,4-D showed a better appearance (^{Mata et al., 2001}). In general, a marked tendency was observed for callus growth as the 2,4-D concentration in the culture medium increased. Treatments with 13.5 and 18 μM induced calli formation in 100% of the explants; however, the best results were obtained with the combination of 18 μM of 2,4-D and 9.3 μM of kinetin (Figure 1e-j).

These responses are similar to those reported in in vitro morphogenesis of other species of cactus, as Notocactus magnificus (De Medeiros et al., 2006), Opuntia spp. (^{Juárez and Passera, 2002}; ^{Estrada et al., 2008}); Coryphantha retusa (^{Ruvalcaba-Ruiz et al., 2010}); Turbinicarpus spp. (^{Dávila et al., 2005}) and Mammillaria albicoma (^{Wyka et al., 2006}). In vitro propagation of these species and various cacti have been successfully employed in different culture media (^{Vidican and Cachita, 2010}) but the MS medium (1962) has been the most satisfactory. However, the desired morphogenic responses depend mainly on the type and concentration of growth regulator used, and this agrees with what was obtained in this research.

In callus differentiation in Mammillaria albicoma and Coryphantha retusa the best results were achieved with naphthaleneacetic acid with a concentration range of 2.6 and 26μM (^{Wyka et al., 2006}; ^{Ruvalcaba et al., 2010}). The individual action of auxins or the combination with cytokinins and its concentrations determine the success of the organogenic responses observed during various propagation techniques (^{Quiala et al., 2004}).

The shoots production at this stage of the process was the result of the activation of axillary buds present in the explants; however, it was only obtained with the combination of 2,4-D (18 μM) and kinetin (9.3 and 13.9 μM). These concentrations favored the regeneration of one to three shoots per explant, while the doses of 13.5 μM of 2,4-D and 4.6 μM of kinetin induced two shoots on average (Table 1). At this time the regeneration of shoots in indirect organogenesis was not observed. Although not all treatments that included kinetin achieved bud regeneration, it was observed that mean doses were able to break the latency of the axillary buds and activate the meristems of the areolas.

Calli proliferation and shoot multiplication

The buds and calli obtained in the callus and bud induction stage were subcultured three times (30, 60 and 90 days) in the same culture media to promote indirect organogenesis and shoot multiplication. All doses of auxin promoted callus proliferation (p≤ 05). The highest growth of calli with friable appearance was achieved with 18 μM of 2,4-D and 9.3 μM of kinetin; at 30 days of the subculture, its initial weight was tripled from 2 to 6 g. At 60 and 90 days of growth the growth rate was reduced and the callus reached a final average weight of 6.9 g (Table 1 and 2).

Table 2 Effect of 2,4-D and kinetin in the callus proliferation of M. plumosa after 30, 60 and 90 days culture. 

¶ = Medias con letra diferente entre columnas son diferentes; Tukey (p≤ 0.05).

Regarding to sprouting, it was observed that the regeneration of shoots took place from areolar activation, as happened in the induction cultures, and the differentiation of primordia shoot from indirect adventitious organogenesis derived from dedifferentiated cells. None of the treatments showed damage by hyperhydration, or darkening of tissue, and the new shoots showed a normal appearance.

The best response in areolar budding was obtained with 18 μM of 2,4-D and 9.3 μM of kinetin where the largest number of explants that generated shoot outbreaks was expressed and the average number of shoots per explant was 11.4 at 90 days (Table 3). As for organogenic outbreaks, these same concentrations of growth regulators favored the higher bud regeneration response in the three subculture periods (Table 4).

Table 3 Effect of 2,4-D and kinetin in areolar outbreaks multiplication of M. plumosa at 30, 60 and 90 days culture. 

¶= Medias con letra diferente entre columnas son diferentes; Tukey (p≤ 0.05).

Table 4 Effect of 2,4-D and kinetin in the multiplication of organogenic shoots of M. plumosa at 30, 60 and 90 days culture. 

¶= Medias con letra diferente entre columnas son diferentes; Tukey (p≤ 0.05).

The shoot regeneration responses obtained in this research are different from those reported for other cactus species. In Coryphantha elephantidens indirect organogenesis of shoots was achieved with 2.3μ M 2,4-D and 6.9 μ M kinetin (^{Wakhlu and Bhau, 2000}), while in Opuntia ficus-indica regeneration was obtained with 2.26 µM of 2,4-D and 2.21 µM of BA with values of 2 to 3 shoots per explant (^{Angulo-Bejarano y Paredes-López, 2011}), significantly lower amount than what was obtained in this research.

Activation of growth of areolar axillary buds of each explant of Mamillaria plumosa promoted by 2,4-D and kinetin can be considered high; however, in the regeneration of Pelecyphora aselliformis and P. strobiliformis cactus 13.7 and 12.3 shoots per explant were obtained, respectively, with 8.8 µM of BA (8.8) (^{Pérez and Dávila, 2002}). Also, ^{Mata et al. (2001)} determined that higher shoot multiplication in Turbinicarpus laui was obtained with BA (8.8-13.32 µM) and naphthaleneacetic acid (0 - 2.68 µM).

In all morphogenic processes, the produced outbreaks number is genetically determined and is considered the main indicator of the multiplication potential of a species.The shoots amount reflects the number of potential adult plants and in this research, the average number of shoots that can be regenerated from an explant of M. plumosa is 537 approximately after 120 days of culture.

Histological analysis

The histological analysis allowed to characterize the origin and development of the shoots from the organogenic calli. Three stages identified as stage 1 (initial; 60 days); Stage 2 (intermediate, 90 days) and stage 3 (differentiated, 120 days) were observed. In the initial, the callus was formed by parenchyma cells, isodiametric thin-walled cells with starch grains. Subepidermal meristematic activity was also observed, which promoted the lifting of a globular protuberance, characterized by the differentiation of procambium in the central part (Figure 1e-f). In the intermediate stage, the callus turned a greener color and the number of shoots with a greater differentiation degree increased.

The formation of an apical meristem, differentiation of a juvenile areola, with spines primordia, irrigated by a xylem beam and differentiated and functional phloem was observed (Figure 1g-h). In the differentiated stage a callus appeared with numerous clearly differentiated green shoots (Figure 1i); the callus parenchyma developed numerous vascular bundles of anficribal type (external phloem and internal xylem) with branches oriented towards the buds. These shoots had modified spines in the center of the areola with mature vascular bundles (Figure 1j). Finally, the shoots grew and the base of the new shoot became thinner and detached from the callus and regenerate a new individual.

Rooting

In the multiplication stage, the areolar and organogenic novo shoots of Mammillaria plumosa naturally generated roots after 90 days of culture. Root formation in regenerated shoots throughout the morphogenic process represents an advantage compared to other species where a rooting stage with specific growth regulators is required to induce or improve the number and length of roots. However, the shoots were grown in MS basic medium (1962) without growth regulators to improve response. At four weeks of cultivation the shoots constituted a radical system with 2 to 4 roots on average.

In cactaceae it has been reported that, in general, are easy to root species both in vitro and in vivo conditions which, in the same way, can show differentiated adventitious roots without the presence of auxin. ^{Davila-Figueroa et al. (2005)} indicated that the in vitro rooting of buds of Turbinicarpus schmiedickeanus subsp. flaviflorus and T. subterraneus regenerated cacti (54-94%) was achieved in basic medium MS (1962) without growth regulators. In other species as Pilocereus robinii (^{Quiala et al., 2009}) and Turbinicarpus laui (^{Mata et al., 2001}) in vitro rooting (94-100%) was also effective in the basic medium MS (1962) but with half nutrient concentration. For Pilocereus aselliformis and P. strobiliformis the best rooting response (87-89%) was obtained with indole butyric acid or indoleacetic acid (^{Pérez and Dávila, 2002}).

Acclimatization of plants

Fort the acclimatization stage, in vitro rooted shoots of Mammillaria feathery were used and planted in pots containing a proportional mixture (1:1) of peat and river sand. At 30 days the survival rate of the plants was 85%, which is an acceptable percentage. Acclimatization of plants of in vitro regenerated cacti, survival rates can be variable; 70% in Schlumbergera truncata, 88% in Pilocereus aselliformis and P. strobiliformis (^{Pérez and Dávila, 2002}) and 94-100% in Turbinicarpus laui, Coryphantha elephantidens and Mammillaria carmenae (^{Mata et al., 2001}; ^{Coca et al., 2007}).