In vitro formation of adventitious shoots on caulinary tissue of physiologically contrasting Agave angustifolia plants

*Corresponding author: Gerardo Rodríguez-Ortiz, División de Estudios de Posgrado e Investigación-Instituto Tecnológico del Valle de Oaxaca (ITVO), Nazareno, Xoxocotlán, Oaxaca, Zip Code 71230, México. E-mail: grodriguez.itvo@yahoo.com Received: 01 January 2017; Accepted: 07 January 2018 Ríos-Ramírez, et al. 50 Emir. J. Food Agric ● Vol 30 ● Issue 1 ● 2018 Zeng et al., 2007). This technology permits designing intensive plant production systems with duly controlled propagation environment in which somaclonal variation is minimized (Fortes et al., 2010). The technique consists of aseptic culture of different explants in culture medium and incubation to induce cell division, organogenesis and plant development (Morales et al., 2009; Kehie et al., 2012; Siddique et al., 2015). For this purpose, it is necessary to select plants in the field that are outstanding for their productivity and harvest quality to obtain clonal populations for establishment in plantations (Domínguez-Rosales et al., 2008a; Koné et al., 2013; Sagare and Mohanty, 2015). Since 1986, research in Oaxaca has been conducted to develop a methodology of in vitro propagation of A. angustifolia. In 1987, the first 12 thousand micropropagated plants were established in a nursery, and in 1988 in the field. Now, we have a methodology that is technically viable but can be improved. Data have been published regarding the stages of propagule multiplication: shoot rooting in preparation for transplanting to soil (Enríquez-del Valle et al., 2005), acclimatization of plants in the greenhouse (Enríquezdel Valle et al., 2009), and development in the nursery (Enríquez-del Valle et al., 2012). However, during stage I of the methodology, establishment of aseptic cultures, at least 50% of the explants are contaminated by bacteria and fungi. Mroginski and Flaschland (2010) mention the difficulty of quantifying the impact of contamination, but they estimate that, on average, in laboratories dedicated to micropropagation, around 10% of the cultures are lost. It is of utmost importance to establish aseptically the highest percentage possible of explants that are viable for cell division and morphogenesis to occur. The physiological condition of the stock plant influences the morphogenetic capacity of its somatic tissues when they are established in vitro (Styer and Chin, 1984). For this reason, it is recommended that in the nursery the stock plant, from which somatic tissues are to be extracted, be subjected to a treatment to improve its nutrient and physiological condition before tissue extraction for in vitro culture. Therefore, to implement Stage I, a high percentage of explants in in vitro culture must be aseptic but also viable to assume cell division and morphogenesis (Niubó et al., 2004). In this regard, there are no data on A. angustifolia Haw. and for this reason the objective of this study was to evaluate the effect of the physiological condition of Agave angustifolia Haw. stock plants on the organogenic response of their somatic tissues when they were established under similar conditions of culture medium. And the hypothesis was that the physiological condition of the stock plant from which the explants were obtained had effect on the stem tissue capacity to assume a process of organogenesis. MATERIALS AND METHODS The study consisted of two stages, each with its respective experiment. Experiment 1 evaluated growth and condition of the stock plants subjected to different nutrient supplies in the nursery. Experiment 2 assessed the organogenic response in vitro of explants obtained from stock plants that exhibited different growth vigor in the nursery. Experiment 1. Evaluated growth and condition of the stock plants Experiment 1 initiated with 40 plants obtained from bulbils from A. angustifolia Haw. inflorescences. The plants were 18 months old and had been established in the nursery in individual 4 dm3 pots with sandy loam textured soil. Four groups were prepared, each of which received a different irrigation treatment twice a week for seven months. Treatment 1 was irrigation with only water. The other treatments were nutritive solution (NS) at different dilutions of the Steiner formulation (1984): Treatment 2) NS-50%, Treatment 3) NS-75%, and Treatment 4) NS100%. The NS-100% contains in mg/L: 166.42 N, 30.68 P, 276.44 K, 182.34 Ca, 49.09 Mg, 111.15 S, 1.25 Fe, 0.21 Mn, 0.025 Zn, 0.076 B, 0.005 Cu y 0.021 Mo. Also, all of the groups were sprayed with N-trichloromethylthio4-cyclohexene-1,2-dicarboximide fungicide (Bayer®) and a systemic antibiotic agricultural terramycin, 5% oxitetracyclin (Zoetis®), twice a week. After seven months under these conditions, the plants were harvested, and height and number of leaves were quantified and leaf area determined with a Laser Area Meter (CID Bio-Science, Copyright ©2016 CID Bio-Sciencie Inc., Camas, WA, USA). Root and leaf volume (cm3) were determined by submerging them in a known volume of water in a graduated test tube. Leaf and root fresh weight (g) were determined on a triple bar balance (IROSA® 700 PPW, Mexico) with 0.1 g precision. The leaves and roots were then placed separately in labeled paper bags and dried in a convection oven (Felisa® Fabricantes Feligneo, S. A. de C.V. Mexico) for two weeks at a temperature of 65 oC. After this time, leaf and root dry weights were determined on a balance. The experiment was established under a completely randomized design. The experimental unit was one plant, and there were 10 replications per treatment. The data were subjected to analysis of variance and comparison of means. Experiment 2. Organogenesis in stem tissues The 40 stock plants that in experiment 1 were separated into groups of 10 subjected to different dosages of fertigation were harvested to evaluate their leaves and root. The stem was taken to the laboratory and superficially disinfected using the following sequence: 1) rinsing with running water for 5 min; 2) immersion and Ríos-Ramírez, et al. Emir. J. Food Agric ● Vol 30 ● Issue 1 ● 2018 51 washing in soapy water for 15 min, 3) immersion in a 2.5 g/L solution of N-trichloromethylthio-4-cyclohexene1,2-dicarboximide fungicide (Bayer®) for three min; 4) three rinses with distilled sterilized water; 5) immersion for 2 min in 90% ethanol; 6) immersion for 15 min in a 0.6% solution of sodium hypochlorite; 7) five rinses with distilled sterilized water. Step four and thereafter of the disinfection process were conducted in aseptic conditions in a horizontal laminar flow bell. The disinfected stems were placed in sterilized glass Petri dishes of 10 x 100 mm and, with sterilized forceps and scalpel, cut into 1.5×1.5 cm and 0.3 cm thick fragments. Each fragment was placed in a 145 cm3 culture recipient containing 20 mL sterilized geled culture medium prepared with MS (Murashige and Skoog, 1962) mineral salts, 1 mg/L thiamine-HCl, 100 mg/L de myo-inositol, 30 g/L sucrose, 1.5 mg/L N6bencylaminopurine, 1 mg/L indol-3-acetic acid, 40 mL/L coconut milk, 5.6 g/L agar, and 250 mg/L chloramphenicol. After establishing the stem tissues in vitro, the recipients were closed with a polypropylene top and sealed with adherent polyethylene. The cultures were placed in the area of incubation, exposed to white fluorescent lighting 35 μmol/m2/s in 16 h photoperiods/ 8h darkness with temperature ranging from 15 to 29 oC. The number of tissue segments (explants) obtained from each stem was counted. During the incubation period, qualitative changes occurring in the tissue fragments in the culture media were observed: cell proliferation (callus), change in pigmentation, and shoot formation. After the eleven weeks, the number of viable uncontaminated stem segments achieved were quantified, as well as the total number of adventitious shoots and the average number of shoots per stem segments (explants) obtained from one stock plant. The experiment was established according to a completely randomized design. The experimental unit was one stem obtained from each stock plant, with 10 replications per treatment. The variables contaminated explants, viable uncontaminated explants, and percentage of contamination were transformed to log10(x) to comply with the assumptions of normality and homogeneity of variances. The data were subjected to analysis of variance and comparison of means (Tukey, 0.05). The statistical analysis routines of each experiment were performed using SAS® Statistical software, version 9.0.

. This technology permits designing intensive plant production systems with duly controlled propagation environment in which somaclonal variation is minimized (Fortes et al., 2010).The technique consists of aseptic culture of different explants in culture medium and incubation to induce cell division, organogenesis and plant development (Morales et al., 2009;Kehie et al., 2012;Siddique et al., 2015).For this purpose, it is necessary to select plants in the field that are outstanding for their productivity and harvest quality to obtain clonal populations for establishment in plantations (Domínguez-Rosales et al., 2008a;Koné et al., 2013;Sagare and Mohanty, 2015).
Since 1986, research in Oaxaca has been conducted to develop a methodology of in vitro propagation of A. angustifolia.In 1987, the first 12 thousand micropropagated plants were established in a nursery, and in 1988 in the field.Now, we have a methodology that is technically viable but can be improved.Data have been published regarding the stages of propagule multiplication: shoot rooting in preparation for transplanting to soil (Enríquez-del Valle et al., 2005), acclimatization of plants in the greenhouse (Enríquezdel Valle et al., 2009), and development in the nursery (Enríquez-del Valle et al., 2012).However, during stage I of the methodology, establishment of aseptic cultures, at least 50% of the explants are contaminated by bacteria and fungi.Mroginski and Flaschland (2010) mention the difficulty of quantifying the impact of contamination, but they estimate that, on average, in laboratories dedicated to micropropagation, around 10% of the cultures are lost.It is of utmost importance to establish aseptically the highest percentage possible of explants that are viable for cell division and morphogenesis to occur.
The physiological condition of the stock plant influences the morphogenetic capacity of its somatic tissues when they are established in vitro (Styer and Chin, 1984).For this reason, it is recommended that in the nursery the stock plant, from which somatic tissues are to be extracted, be subjected to a treatment to improve its nutrient and physiological condition before tissue extraction for in vitro culture.Therefore, to implement Stage I, a high percentage of explants in in vitro culture must be aseptic but also viable to assume cell division and morphogenesis (Niubó et al., 2004).In this regard, there are no data on A. angustifolia Haw. and for this reason the objective of this study was to evaluate the effect of the physiological condition of Agave angustifolia Haw.stock plants on the organogenic response of their somatic tissues when they were established under similar conditions of culture medium.And the hypothesis was that the physiological condition of the stock plant from which the explants were obtained had effect on the stem tissue capacity to assume a process of organogenesis.

MATERIALS AND METHODS
The study consisted of two stages, each with its respective experiment.Experiment 1 evaluated growth and condition of the stock plants subjected to different nutrient supplies in the nursery.Experiment 2 assessed the organogenic response in vitro of explants obtained from stock plants that exhibited different growth vigor in the nursery.: 166.42 N,30.68 P,276.44 K,182.34 Ca,49.09 Mg,111.15 S,1.25 Fe,0.21Mn, 0.025 Zn, 0.076 B, 0.005 Cu y 0.021 Mo.Also, all of the groups were sprayed with N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide fungicide (Bayer®) and a systemic antibiotic agricultural terramycin, 5% oxitetracyclin (Zoetis®), twice a week.After seven months under these conditions, the plants were harvested, and height and number of leaves were quantified and leaf area determined with a Laser Area Meter (CID Bio-Science, Copyright ©2016 CID Bio-Sciencie Inc., Camas, WA, USA).Root and leaf volume (cm 3 ) were determined by submerging them in a known volume of water in a graduated test tube.Leaf and root fresh weight (g) were determined on a triple bar balance (IROSA® 700 PPW, Mexico) with 0.1 g precision.The leaves and roots were then placed separately in labeled paper bags and dried in a convection oven (Felisa® Fabricantes Feligneo, S. A. de C.V. Mexico) for two weeks at a temperature of 65 ºC.After this time, leaf and root dry weights were determined on a balance.The experiment was established under a completely randomized design.The experimental unit was one plant, and there were 10 replications per treatment.The data were subjected to analysis of variance and comparison of means.

Experiment 2. Organogenesis in stem tissues
The 40 stock plants that in experiment 1 were separated into groups of 10 subjected to different dosages of fertigation were harvested to evaluate their leaves and root.The stem was taken to the laboratory and superficially disinfected using the following sequence: 1) rinsing with running water for 5 min; 2) immersion and washing in soapy water for 15 min, 3) immersion in a 2.5 g/L solution of N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide fungicide (Bayer®) for three min; 4) three rinses with distilled sterilized water; 5) immersion for 2 min in 90% ethanol; 6) immersion for 15 min in a 0.6% solution of sodium hypochlorite; 7) five rinses with distilled sterilized water.
Step four and thereafter of the disinfection process were conducted in aseptic conditions in a horizontal laminar flow bell.The disinfected stems were placed in sterilized glass Petri dishes of 10 x 100 mm and, with sterilized forceps and scalpel, cut into 1.5×1.5 cm and 0.3 cm thick fragments.Each fragment was placed in a 145 cm 3 culture recipient containing 20 mL sterilized geled culture medium prepared with MS (Murashige and Skoog, 1962) mineral salts, 1 mg/L thiamine-HCl, 100 mg/L de myo-inositol, 30 g/L sucrose, 1.5 mg/L N 6 -bencylaminopurine, 1 mg/L indol-3-acetic acid, 40 mL/L coconut milk, 5.6 g/L agar, and 250 mg/L chloramphenicol.After establishing the stem tissues in vitro, the recipients were closed with a polypropylene top and sealed with adherent polyethylene.The cultures were placed in the area of incubation, exposed to white fluorescent lighting 35 µmol/m2/s in 16 h photoperiods/ 8h darkness with temperature ranging from 15 to 29 ºC.The number of tissue segments (explants) obtained from each stem was counted.During the incubation period, qualitative changes occurring in the tissue fragments in the culture media were observed: cell proliferation (callus), change in pigmentation, and shoot formation.After the eleven weeks, the number of viable uncontaminated stem segments achieved were quantified, as well as the total number of adventitious shoots and the average number of shoots per stem segments (explants) obtained from one stock plant.The experiment was established according to a completely randomized design.The experimental unit was one stem obtained from each stock plant, with 10 replications per treatment.The variables contaminated explants, viable uncontaminated explants, and percentage of contamination were transformed to log10(x) to comply with the assumptions of normality and homogeneity of variances.The data were subjected to analysis of variance and comparison of means (Tukey, 0.05).The statistical analysis routines of each experiment were performed using SAS® Statistical software, version 9.0.

RESULTS
The substrate was sandy loam in texture so this did not supply enough nutriments to the plants.The physiological condition of the stock plants was determined by the magnitude of their growth, observing that the nutrient supply did influence plant growth; fertigated plants grew larger than unfertilized plants.Later, it was observed that the condition and different growth vigor of the stock plant did indeed affect the magnitude of organogenic response of the in vitro cultured stem tissue obtained from these plants.

Experiment 1. Condition of stock plants in response to the level of nutrient supply in the nursery
Initially, the Agave angustifolia Haw.plants had 14 to 20 leaves and were 25 to 35 cm tall.After seven months, the plants supplied with different concentrations of nutrients exhibited different sizes in accord with the dosages of fertigation they received.In our study, the analysis of variance showed that the dosage of fertigation had significantly different effects (P ≤ 0.05) on leaf number and length, root and leaf volume, and monthly root and leaf volume increase, and a highly significant effect (P≤ 0.01) on leaf area (Table 1).
The plants that received 75% nutrient solution, compared with those that did not receive NS, had increments of 7.8 and 3.8 in total number of leaves and 10.5 and 6.5 cm in length of largest leaf, 1136.1 and 695.5 cm 3 leaf volume, and monthly increases in leaf volume of 162.3 and 99.3 cm 3 (Table 2).These values were statistically different (Tukey, 0.05).Most of these characteristics in plants fertigated with 100% NS did not show additional growth, relative to the plants supplied with 75% NS; the exception was leaf area (4401.4cm 2 ), which was 2.73 time larger and significantly different (Tukey, 0.05) than the leaf area of unfertigated plants.It was also observed in all of the groups that the plants began rhizome shoot development with 1.6 to 2.0 shoots per plant, numbers that were not significantly different (Tukey, 0.05) among groups.

Experiment 2. Organogenesis in stem tissue
Data on time and percentage of contamination reveal that 75.6% of the explants from unfertigated A. angustifolia stock plants became contaminated during the first 9.6 days of incubation.In contrast, 69.5% of the explants from stock plants that received 75% NS became contaminated during the first 6.2 days of incubation.
During the organogenesis process of A. angustifolia, the explants established in vitro were initially cream colored and smooth.In the first 8 to 15 days of incubation, the explants increased in size by 10%, relative to their initial size, and their surfaces acquired a granular texture.Also, as of day 15 up to day 40 of incubation, the explant showed beginnings of green coloring, initially pale green and changing gradually to lime green.On day 50 small shoots could be observed.The analysis of variance showed that the condition of vigor of A. angustifolia stock plants that received different dosages of nutrient solution had a significant effect (P ≤ 0.05) on stem tissue capacity for organogenesis, formation of adventitious shoots, and a highly significant effect (P≤ 0.01) on total number of shoots formed on the viable explants obtained from each mother plant and on days to shoot formation (SF) (Table 3).
Stem tissue in the culture medium exhibited different levels of organogenic response, depending on the physiological condition of the stock plants from which they were obtained.Comparing explants obtained from unfertigated stock plants with those from plants fertigated with 100% nutrient concentration, of the total viable explants, 14.6 and   a SV: source of variation, CSn: concentration of nutrient solution.b DF: degrees of freedom.c EC: contaminated explant, UCVE: uncontaminated viable explant, TSVE: total number of shoots in viable explant from each donor plant, ASVE: Average number of shoots on each viable explant, DC: days to appearance of contamination, DS: days to appearance of shoots.d ns: non-significant F value (P>0.05),*: significant F value (P≤0.05),**: highly significant F value (P≤0.01)(Tukey test, P<0.05) 32.7 shoots were formed, visible at 84.6 and 47.7 days of in vitro culture, respectively.In both cases, the differences were significant (Tukey, 0.05) (Table 4).
Nutrient supply to stock plants in the nursery had an effect on their physiological condition, evaluated by several characteristics of size and later, during in vitro culture, by the number of shoots formed on the explants.Table 4 shows that differences between the explants obtained from each of the unfertigated stock plants and those from plants fertigated with 100% NS were significant (Tukey, 0.05): a total of 14.6 vs 32.7 adventitious shoots and averages of 4.5 and 8 shoots per explant.There was a positive relationship between formation of a larger number of total shoots and average number of shoots per explant and the dosage of fertigation that the stock plant received in the nursery.

DISCUSSION
Experiment 1.The results show that agave plants supplied with an appropriate quantity of nutrients reached bigger size than the unfertilized plants, coinciding with (Enríquezdel Valle et al. 2000Valle et al. , 2009Valle et al. , 2013)).According to Zúñiga (2013), the nutrients form part of the plant's metabolic compounds and increase their capacity for photosynthesis and synthesis of structural compounds, thus increasing rates of formation and growth of their organs.
In our study, after seven months of fertigation, the plants were of different sizes, positively related to the quantity of nutrients they received.This is in agreement with data reported by Enríquez-del Valle et al. (2016), who demonstrated the positive effect of fertigation on growth of A. potatorum plants to which Steiner (1984) NS was administrated at different dilutions (1, 20, 40, 60, 80 and 100%).Plants fertigated at 1% NS increased the number of leaves by 9.8 and leaf area, LA, by 125.53 cm 2 , while those fertigated at 100% increased the number of leaves by 17 and leaf area by 392.35 cm 2 .The data obtained on leaf area, in agreement with Warnock et al. (2006), suggest that the magnitude of LA has a positive relationship to nutrient supply and, in turn, LA is related to the capacity to intercept photosynthetically active radiation (PAR) and fixing CO 2 to synthesize carbohydrates and accumulate biomass.
Experiment 2. The evidences show the difficulty in obtaining high percentages of aseptic cultures during the first stage of in vitro culture of Agave stem tissue since the tissues are close to the substrate and to populations of microorganisms, originating a high degree of endogenous and exogenous contamination.For this reason, for this type of plant some of the management conditions should be modified in the nursery stage, as described by Deberg and Maene (1985).However, when the stage of establishing aseptic cultures in vitro is over and explants that responded to organogenesis stimulus are obtained, in the next stage of propagule multiplication it is expected that the levels of contamination decrease drastically and the number of shoots on each explant increase notably.During the in vitro establishment of aseptic cultures of node segments of the woody species Legrandia concinna (Phil.),Uribe-Moraga and Cifuentes ( 2004) reported that of all explants, 81% died and 19% were viable.In species such as Musa spp, during in vitro establishment of aseptic cultures, Ortega et al. (2012) obtained 6.6% contaminated and 93.4% aseptic cultures when they tested six methods of disinfecting explants.Niubó et al. (2004) described a methodology for decontaminating sugarcane of endophyte and exophyte microorganisms in in vitro culture, combining a surfaceaction biocide with a broad-spectrum systemic biocide.They obtained 90% of the explants free of exophyte contamination by using mercury chloride (0.005 % for 10 min) and 92% explants free of endophyte contamination three months after removing gentamicin sulfate (30 mg/L for 14 d).Rache and Pacheco (2010) applied fungicides to explant donor Vaccinium meridionale plants to reduce levels of contamination in the stage of aseptic culture establishment in vitro.When the explant donors were not sprayed with fungicides, 80-100% of the in vitro explants became contaminated, while only 20% of the explants from sprayed donors became contaminated.
In our study explants obtained from nursery stock plants with the highest nutrient supply formed more shoots in a shorter time than the explants obtained from unfertigated plants or plants supplied with nutriments under the optimum level.No previous studies had shown differences in in vitro organogenic response of explants from Agave plants that varied in physiological condition.It is much The process of organ formation on somatic tissues was described in Nicotiana (Thorpe and Murashige, 1968;Bertrand-Garcia et al., 1992) and Pinus sp.(Villalobos et al., 1984;Thompson and Thorpe, 1997).The process occurs through a sequence of events: parenchima cells are induced to division to form groups of dediferenciated cells, meristemoids formation, meristems and shoots.This process consume a great quantity of energy and occurs active synthesis of nucleic acids, and proteins.At present work the differences in magnitude of organogenesis in Agave angustifolia explants cultured in vitro probably could be affected by the physiological condition of the stock plants from which these were taken.

CONCLUSIONS
Agave angustifolia plants that were fertigated in the nursery for seven months had better growth than unfertigated plants.Plant vigor was positively related to the quantity of nutrients received in fertigation.When stem tissue explants taken from the more vigorous plants were established in vitro, they produced a larger number of adventitious shoots in a shorter time than explants taken from the less vigorous plants.
technique.G.R.O: Experimental design planning and statistical analysis of data.J.R.L and V.A.V.V: Revision and writing design.

Experiment 1 .
Evaluated growth and condition of the stock plants Experiment 1 initiated with 40 plants obtained from bulbils from A. angustifolia Haw.inflorescences.The plants were 18 months old and had been established in the nursery in individual 4 dm 3 pots with sandy loam textured soil.Four groups were prepared, each of which received a different irrigation treatment twice a week for seven months.Treatment 1 was irrigation with only water.The other treatments were nutritive solution (NS) at different dilutions of the Steiner formulation (1984): Treatment 2) NS-50%, Treatment 3) NS-75%, and Treatment 4) NS-100%.The NS-100% contains in mg/L

Table 1 : Summary of the analysis of variance of the characteristics of Agave angustifolia Haw. plants fertigated with different concentrations of nutrients for seven months in the nursery SV a DF b Mean Squares and significance
SV: source of variation, CSn: solution concentration.b DF: degrees of freedom.c IL: increase in number of leaves, LL: length of largest leaf, DL: diameter of the largest leaf, NS: number of shoots, VR: root volume, MIVR: monthly increases in root volume, VL: leaf volume, MIVL: monthly increases in leaf volume, LFW: leaf fresh weight, MILW: monthly increases in leaf weight, RFW: root fresh weight, MIRFW: monthly increases in root fresh weight, LA: leaf area.d ns: non-significant (P>0.05),*: significant effects (P≤0.05),**: highly significant effects (P≤0.01)(Tukey test, P<0.05) a

Table 2 : Characteristics of Agave angustifolia Haw. plants fertigated with different concentrations of nutrients for seven months in the nursery Treatments, dilutions (%) of Steiner nutrient solution b Variable a
a IL: increase in number of leaves, LL: length of largest leaf, DL: diameter of largest leaf, NS: number of rhizome shoots, RV: root volume, MRV: monthly increase in root volume, LV: leaf volume, MLV: monthly incrase in leaf volume, LFW=leaf fresh weight, MILFW: monthly increase in leaf fresh weight, RFW: root fresh weight, MIRFW: monthly increase in root fresh weight, LA: leaf area.b In each column, values with the same letter are not statistically different (Tukey test, P<0.05), mean±standard deviatio.

Table 4 : Percentages of contamination, viable explants, total number of shoots, and average number of shoots on in vitro cultured explants from stem tissue obtained from Agave angustifolia that received different nutrient supply in the nursery NS (%) a
Domínguez- Rosales et al. (2008a), Ramírez-Malagón et al. (2008) plant, C Exp: contaminated explants, DOS: days to observation of shoots, DC: days to occurrence of contamination, TSE: total shoots on the explants from one plant, SE: shoots on each explant.cIneachcolumn,meanswith the same letter are not significantly different (Tukey test, P<0.05), means are ± the standard deviation more frequent to find assessments of variations in the composition of the culture media, mostly of growth regulators.Thus, in A. tequilana different number of shoots were formed from explants that were subjected to pulsesThe efficiencies per species in a single proliferation cycle of 50 to 60 days were from 4.0 shoots per explant in T. hoferi and up to 26.3 per explant in T. pseudomacrochele subsp.lausseri.Martínez-Palacios et al. (2003)assessed indirect organogenesis in A. victoriae reginae, in which regeneration of multiple shoots was induced from axillary buds from stem segments cultured in MS medium with 2,2-4,4 μM BA.Arzate-Fernández and Mejía-Franco (2011) used zygotic embryonic axes from mescal agave (Agave angustifolia Haw.) as explants and achieved formation of 40 to 216 somatic embryos in vitro.Summarizing, the results obtained in our study and in those we mentioned conducted with diverse species show that levels of organogenic response depend on several factors: species, genotype within the species, type of explant, physiological condition of the stock plant, composition of the culture medium and physical conditions of incubation, coinciding withPierik (1987),Domínguez- Rosales et al. (2008a), Ramírez-Malagón et al. (2008).
Rosa-Carrillo et al. (2012) explants of Cucurbita pepo, variety round zucchini, produced 2 shoots per explant, and Cucurbita maxima, variety Brujita, produced 2.7 shoots after 22 days in similar culture medium.Rosa-Carrillo et al. (2012)evaluated the capacity of regeneration and in vitro propagation of 14 species of Turbinicarpus (Cactaceae).