Disinfestation

The pre-disinfestation was to select twigs of 10 to 15 cm in length in optimum conditions and with large number of axillary buds, considering the measurement of the apical side down. The braces were washed with liquid soap and rinsed with potable water. They were then sectioned into smaller units approximately 2-3 cm. In order to decrease the percentage of phenols, the plant material is placed in a soap solution for three periods of 10 min c/u and constant stirring. Followed by three washes with double distilled water to proceed to place them in a fungicide-bactericidal solution consisting of 2 g L^-1 Agry-Gent Plus 800 (Gowan Mexicana), 1.5 mL L^-1 Amistar Gold (Syngenta), 2 g L^-1 Antrak PH 500 (Trident Agrochemistry), 800 mg L^-1 citric acid, 800 mg L^-1 of ascorbic acid and 30 g L^-1 sucrose stirring for 1.5 hours, ending with a rinse with bidistilled wáter. Under these conditions the plant material was taken to the laminar flow hood for disinfestation.

Disinfestation consisted of immersing the plant material in 100% ethanol for 1 min, followed by immersion in a solution with commercial bleach 50% v/v plus 0.2% Tween-20 for 20 min. It concluded the time was rinsed three times with sterile distilled water. Immediately the plant material is placed in a solution of PVP (polyvinylpyrrolidone) 400 mg L^-1 to reduce oxidation. Finally the material was sectioned into explants of 1 cm in length to be subsequently used in the setting means.

Establishing in vitro

The type of explants used were internodal segments with one or two axillary buds (micropiles), which are sown on media MS culture medium (^{Murashige and Skoog, 1962}) with macroelementos 50%, DCR (^{Gupta and Durzan, 1985}) Yasuda (^{Yasuda et al., 1985}) supplemented with vitamins, 30 g L^-1 sucrose, 2.0 mg L^-1 BAP, 0.3 mg L^-1 of AIB, 2 g L^-1 of oxytetracycline, 2 g L^{- 1} of benomyl and 4.4 g L^-1 Phytagel™. The pH was adjusted to 5.7 and Yasuda on MS media, while 6 DCR was then sterilized at 1.2 kg cm^-2 pressure at 121 °C for 15 min. After the sowing, the test units were incubated at a temperature of 26 ± 2 °C under a 16 hour photoperiod light for two weeks. The variables evaluated were contamination, oxidation and viability of the explants.

Seedlings induction

After 14 days of establishing in vitro, viable microcuttings were transferred to the same media modified crop, which was 50% MS with macroelements, DCR and Yasuda containing added vitamins, 30 g L^-1 sucrose and solidified with 4.4 g L^-1 Phytagel™; also supplemented combinations: 20% coconut water (AC), 2.0 mg L^-1 BAP, 0.01 mg L^-1 IAA and 0.5 mg L^-1 AG₃ (^{Sandoval-Prando et al., 2014}); and the second combination of 2 g L^-1 casein hydrolyzate (CH), 1 mg L^-1 BAP and 0.3 mg L^-1 of AIB (Table 1). The pH of the culture media was adjusted to 5.7 (MS and Yasuda) and 6.0 (DCR) subsequently sterilized at 1.2 kg cm^-2 pressure at 121 °C for 15 min. The micropiles were incubated under the same conditions described above. the number of seedlings s per explant, seedlings length and number of leaves at 60 and 90 days after subculture were evaluated.

Statistic analysis

For the establishment phase, qualitative variables were: pollution, oxidation and viability of the explants to 14 days after sowing in the three culture media of said stage with eight repetitions, which were incubated under a completely randomized design under homogeneous conditions. These variables were analyzed using contingency tables and chi-square test. number of seedlings s, seedlings length and number of leaves at two and three months after the establishment of the explants: while in the seedlings induction stage, variables were evaluated. These variables were established under a completely randomized design with a factorial arrangement 2 x 6, where the factor A were the cultivars and factor B were the induction means, obtaining a total of 12 treatments with six repetitions. An analysis of variance with the data previously processed with X + 1 and the differences between treatment means was conducted were compared by Tukey test at 5% probability. Data from both stages were analyzed using the Statistical Package for the Social Sciences (SPSS) version 21.

Establishment aseptic

Microbial contamination and darkening of explants are major difficulties in establishing in vitro cultivars avocado Mexican race (^{Cortés-Rodríguez et al., 2011}). The process of disinfestation of plant material had different effects on the variables evaluated.

Pollution for the media used in both cultivars established 14 days the experiment was over 50%, except in the growing medium Yasuda Huevo de Toro with 26.66% (Table 2). Importantly, this variable was affected by the presence of fungi and bacteria, which prevented the favorable development of at least 50% of the explants. Similar results shown ^{Cooper (1987)}, with 70% contamination in the grown Duke 7, reducing the effect to 16% using 100% ethanol and a solution of sodium hypochlorite (NaClO) 0.5% for 30 min. For its ^{Dalsaso and Guevara (1988)} part, they noted the high percentage of bacterial contamination in micropiles cultivar Fuerte, where the use of bactericide disinfestation and treatment of the mother plant, were not effective in reducing pollution, so they suggest that the pathogen is endogenously, possibly located in the vascular tissues of the stem portion. In contribution to the above, ^{Vidales-Fernández (2002)}, recommended combining benomyl Agrimycin and pre-disinfestation explants and disinfestation of up to 40% v/v commercial bleach and plant explants amid I grow benomyl added to 2 g L^-1. ^{Zulfiqar et al. (2009)} report up to 72% contamination by fungi and bacteria in micropiles Fuerte cultivar, recommending that 1% NaClO is sufficient for disinfestation of plant material without damaging severely the explant.

Oxidation

For oxidation, it was present only in the MS medium with poor results did not exceed 7% in both cultivars (Table 2). The low percentage of explants oxidation may be due to use of different antioxidants disinfestation processes such as washing the plant material with water for 30 minutes, reported by ^{Vidales-Fernández (2002)} and using PVP reported by ^{Dalsaso and Guevara (1988)}. Recent research suggests the addition of ascorbic acid and activated carbon to the culture medium to reduce to 70% the oxidation process (^{Cortés-Rodríguez et al., 2011}).

Viability

The viability of the explants showed values of 22-37% in mass MS and DCR crop, cultivars used. Unlike the above, the culture medium showed higher viability Yasuda explants, to 73.33% in case of growing Huevo de Toro. Note that this variable is affected by the contamination process was developed in the time after the aseptic establishment of plant material (Table 2).

Contingency tables

The contingency tables with tests chi-square (Table 3) showed that in the mass MS and DCR cultivation in the set time, the variables contamination, oxidation and viability were not be related to the cultivars used, so no there is significant relationship between the variables and cultivars (p≤ 0.05). The opposite happened for the same variables in the middle of Yasuda culture, where they show dependence cultivars, ie, if there is significant relationship between pollution, oxidation and feasibility cultivars (p≤ 0.05).

Seedlings induction

Viable explants from step aseptic establishing both cultivars were transferred on seedlings induction media and variable number of seedlings s, seedlings length (cm) and leaf number at 60 and 90 days subculture were evaluated.

Number of seedlings

The interaction of the factors cultivar for induction medium showed that in the case of Huevo de Toro, M1 and M2 means favorable for the development of buds, with values of 1.32 and 1.5 seedlings s per explant respectively (sig. p≤ 0.05) at 12 weeks of subculture. The M3, M4, M5 and M6, led means no seedlings development in the growing egg Toro. While in the case of butter cultivar, the average M3, was the most favorable seedlings growth in the times evaluated, 1.89 and 2.14 seedlings s per explant (sig. p≤ 0.05). In significance they continued M2 and M6 means with values of 1.13 to 1.5 Seedlings formation in the media M1, M4 and M5 farming Butter were not presented (Table 4; Figure 1). Similar results were obtained in the same type of explant, but with changes in the amount of plant growth regulators used in different cultivars, time of development and type and concentration of salts in the culture medium; Whereas all are consistent in the use of BAP (0.1 to 5 mg L^-1) added to the culture medium (^{Cooper, 1987}; ^{Dalsaso and Guevara, 1988}; ^{Barceló-Muñoz et al., 1999}; ^{Vidales-Fernández, 2002}; ^{Zulfiqar et al., 2009}; ^{Cortés-Rodríguez et al., 2011}). ^{Sandoval-Prando et al. (2014)} mention that the growth regulators play a key role in the qualitative and quantitative development of seedlings.

Barra: 1 cm.

Figure 1 Response avocado cultivars used in vehicles seedlings induction to 8 weeks. a-b: cultivating Huevo de Toro; c-d: Butter grow. a: M1; b: M2; c: M2; d: M3. 

These results as well as previous investigations indicate that BAP, plays an important role in the induction of seedlings s and explants length avocado (^{Barceló-Muñoz and Pliego-Alfaro, 2003}; ^{Zulfiqar et al., 2009}; ^{Cortés-Rodríguez et al., 2011}; ^{Sandoval-Prando et al., 2014}). However, ^{Barceló-Muñoz and Pliego-Alfaro (2003)} and ^{Cortés-Rodriguez et al. (2011)} mention that high concentrations of this controller not only reduce the number of buds formed, but also stops the growth thereof.

^{Sandoval-Prando et al. (2014)}, indicate that the use of coconut water in the culture medium is usually not enough to promote a satisfactory multiplication, so suggest combination with BAP and AG₃.

Seedlings length

According to the interaction cultivar through induction in the growing Huevo de Toro, M1 and M2 media is conducive to the growth of seedlings s, appearing in securities of 1.07 to 1.09 cm in the days evaluated (sig. p≤ 0.05). But not seedlings growth in M3, M4, M5 and M6 media in this cultivar was presented. Butter in the cultivar, the average M3 was the most successful in the growth of seedlings s in the times evaluated, 1.23 and 1.21 cm respectively (sig. p≤ 0.05). In the M6 and M2 means lower results were observed in the securities of 1.02 to 1.13 cm; while the M1, M4 and M5 means not seedlings growth (Table 4; Figure 1) was presented. These results are consistent with previous work where growth is reported 0.89 to 2.5 cm in different avocado cultivars using MS culture media with modified macro and DF (^{Dixon and Fuller, 1976}); both in the presence of BAP in concentrations from 0.5 to 5.0 mg L^-1 (^{Dalsaso y Guevara, 1988}; ^{Rodríguez et al., 1999}; ^{Zulfiqar et al., 2009}; ^{Cortés-Rodríguez et al., 2011}). ^{Vidales-Fernández (2002)} mentions that the reduction of macroelements in MS medium is beneficial for the growth of seedlings s, but induces a reduction of the same and consequently a smaller amount of axillary buds. This may suggest the low growth of buds on the treatments used, as the MS medium was used with macroelementos 50%, while Yasuda is a modification of macro-MS with 25% and vitamins Gamborg (Gamborg et al., 1985); DCR medium differs in the amount of ammonium nitrate and potassium nitrate and further supplemented with the addition of calcium nitrate.

Number of sheets

The number of sheets developed in the growing Huevo de Toro was a greater degree in the M1 and M2 media, up 1.21 leaves per seedlings (sig. p≤ 0.05). Likewise, the M3, M4, M5 and M6 media showed no sheet formation in the explants used. Regarding the growing butter, the average Yasuda + AC, BAP, GA3 and IAA, showed the highest values in the formation of leaves, in the evaluated times, 2.28 and 2.38 respectively (sig. p≤ 0.05). In the middle Yasuda continued significance + CH, BAP and AIB, with values up to 1.45 leaves per seedlings. Lower results were presented in the middle M2, 1.21 leaves per seedlings . M1, M4 and M5 media did not cause induction of leaves in this cultivar (Table 4; Figure 1). These results are low compared with those reported by ^{Zirari and Lionakis (1994)}, for Topa Topa, Fuerte, Hass and Duke cultivars with values of 3.9 to 8.25 leaves per explant, using etiolated etiolated material and not in MS dual phase. Similarly, ^{Viña et al. (2001)}, values obtained until 4.8 leaves microestaca in cultivating RR-86, using the MS medium with 50% macroelements and 1.3 µM BAP. ^{Dalsaso and Guevara (1988)}, provided that microcuttings cultivar Fuerte, present highly developed fully extended foil sheets, unlike using axillary buds, which separate stem portion, are free of these had correlations with tissue and initiate a different morphogenetic process, modulated by the internal capacity and the culture medium.