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*Corresponding Author: J. L. Rodríguez-De La O, Departamento de Fitotecnia, Universidad Autónoma Chapingo, Km. 38.5 Carretera México Texcoco. Chapingo C.P. 56230. México.
Citation: J. L. Rodríguez-De La O, Arellano-Durán L, M. Serrano-Covarrubias, (2024), Obtaining and Propagation In Vitro Of Plants of Beaucarnea Recurvata Lem, J, Biotechnology and Bioprocessing, 5(1); DOI:10.31579/2766-2314/113
Copyright: © 2024, J. L. Rodríguez-De La O. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received: 17 August 2023 | Accepted: 18 September 2023 | Published: 31 January 2024
Keywords: asepsis; H2 O2; in vitro propagation; growth regulators
Beucarnea recurvata or "Elephant's Leg," is an ornamental plant with enormous potential, both in green areas and in decorative gardening. In order to promote seed germination and in vitro release of new shoots, tissues of newly germinated seedlings were planted. The seeds, once disinfested, were placed for 0, 24 and 48 h in H2O2 (hydrogen peroxide); incubating under light and dark conditions. From the germinated seedlings, tissues were taken from three regions of the stem, grown in a medium with the inorganic salts of Murashige and Skoog (1962), (MS) at 100%, supplemented with 0.40 mg. L-1 thiamine, 100 mg. L-1 of myo inositol, 3% sucrose, pH 5.7 +-0.1 and 7.0 gr. L of agar, evaluating the effect of cytokinins. Benzyladenine (BA), kinetin (KIN), and 2-isopenteniladenine (2iP); at 3.0 mg.L-1 and 10 mg.L-1 combining the effect with indoleacetic acid (AIA), indolbutyric acid (AIB) and naphthaleneacetic acid (ANA), at 0.1 and 0.3 mg∙L-1. The effect of hydrogen peroxide during 48 and 24 h, inhibited the presence of contaminating organisms such as fungi and bacteria, so germination was promoted 100% at 32 days. The best response for the emission of new shoots was achieved by culturing the stem tissues of the Intermediate and basal region, with the medium MS (1962), and 10 mg∙L-1 of BA with 0.3 mg∙L-1 AIA, successfully obtaining up to 10 new shoots of 3.0 cm in length at 12 weeks of culture.
The species Beaucarnea recurvata Lem. known as "elephant's foot", originally from Mexico, it has taken importance as an ornamental, for its colorful bearing in gardens and collections of succulent plants, because of this they are over-collected extracting them from many regions of our country illegally. The reduction of their natural populations, coupled with their slow growth, puts these species in danger of extinction (Semarnat, 2002). The use of in vitro culture of plant tissues, represents an excellent option for the rescue of multiplication and conservation of important species, this biotechnological tool allows to have procedures to carry out its efficient and massive propagation, of It is proposed to have an in vitro protocol as a viable method of propagation of elephant foot, being able to benefit its reincorporation into different protected areas as well as its conservation. In this research was raised as an objective, to establish a methodological process in vitro to promote both seed germination as well as obtaining new sprouts or shoots of B. recurvata Lem. This species usually appears within the family of Liliaceae, Nolinaceae and other authors cite it within the Agavaceae, families in which several studies have been reported where favorable results were obtained with in vitro propagation techniques. The proliferation of axillary buds is the most reported method in micropropagation in Agavaceae, which is based on the phenomenon called apical dominance, which is the regulatory control of growth over the remaining meristematic regions (Rodríguez et al., 1996), if the apical bud is cut, the source of inhibitory hormones is interrupted, beginning to grow one or more axillary buds that will give formation to a new outbreak. It has been observed that cytokinin’s, mainly benzyladenine (BA), is very effective in removing apical dominance (Pierik, 1990), in some cases the use of a high concentration of cytokinins and low concentration of auxins is favorable. Regeneration of adventitious branches from intact dicotyledonous stems is relatively rare; however, in culture media it is possible to induce the formation of adventitious shoots in separate parts of plants with high multiplication rates (Harmann and Kester, 1981).
The research was carried out in the Plant Tissue Culture Laboratory of the Department of Plant science of the Autonomous Chapingo University, México. The seeds of B. recurvata Lem. They were provided by the PLANTEC COMPANY nursery, located in Amacuzac, Morelos, from trees in San Luis Potosi. Mexico.
The research was divided into two parts, the first: to establish the conditions for the establishment and in vitro germination of seeds and the second: to develop a methodology to promote the obtaining of news sprouts or shoots from sections of stem tissue.
Seed disinfection.
Initially, the seeds were washed vigorously with commercial detergent powder plus a few drops of tween 80 (surfact agentante) dissolved in water; they were then placed in a 70% ethanol solution (v / v) for 3min and finally immersed in sodium hypochlorite (6
The percentages of contamination and in vitro in vitro were evaluated. Data collection for germination was performed at eight days after establishment (DDE), so on until 48 DDE after germination of seeds grown in vitro no longer occurred. The length of emitted shoots, the number and length of the roots emitted were evaluated, the data were taken eight days after germination.
Treatments were distributed using a completely randomized experimental design with factorial arrangement 3 X 2. In the first of them the seeds were exposed to different stirring times (0, 24, 48 h) in hydrogen peroxide (3 % V / V) (Factor TA) and then lighting conditions of 16 h light and darkness, (LU Factor) were placed. The total treatments generated were 6 which are indicated in Table 1
Table 1: Procedures for in vitro seed establishment and germination.
The experimental unit was to place one seed per tube, establishing 15 repetitions per treatment. Frequency tables were obtained under the chi-square test for the variable contamination and germination.
Establishment of stem of seedlings obtained in vitro.
Within the laminar flow chamber, from the seedlings obtained from in vitro germination, three different regions of the thickened stem zone were isolated and cultivated; Making cross-sections located in: basal zone, intermediate and the apical or terminal zone, which were placed in the culture medium maintaining their normal polarity.
Culture medium
In all treatments the culture medium included: A basic medium including inorganic salts of MS (1962) 100%, myo-inositol (100 mg∙L-1), thiamine-HCL (0.40 mg∙L-1), adenine sulfate (80 mg∙L-1), sucrose (3%) and agar (7g. L-1), with different growth regulator types and concentrations. The cytokinins used were: benzyladenine (BA), kinetine (KIN) and 2-
isopentyladenine (2iP); auxins: indoleacetic acid (AIA), indolebutyric acid (AIB) and nahthalenacetic acid (ANA).
Incubation Conditions
The experiments were held in a room with a controlled environment at a temperature of 25 ± 2 ° C, under a light intensity of 3000 lux, photoperiod 16h light and 8 h darkness.
The variables evaluated were: number and length of outbreaks, The experimental design was a completely random with factorial design 3 X 9, where the first refers to the type of explant used (basal, intermediate or terminal), in different combinations of cytokinin-auxin; thus, in a design of treatments of complete factorial type 27 treatments were generated, as shown in table 2.
Table 2: Distribución de tratamientos, para promover las respuestas hacia la brotación in vitro de de B. recurvata Lem.
The different stem sequences were taken as experimental units, establishing five repetitions for each treatment. For the quantitative variables, an analysis of variance was performed using the Statical Analysis System (SAS) package, and the comparison of Tukey's means (α=0.05). For the qualitative variables, frequency tables were obtained under the chi-square test.
Establishment and in vitro germination of seeds
Light and dark effect
The presence and absence of light during the experimental incubation period did not influence promoting the presence of either fungi and bacteria in vitro, since the process for their control and cleaning was similar in both cases. (Figure 1).
1: Light 0: Darkness I 5% error bars
Figure 1: Percentage of clean seeds of Beaucarnea recurvata Lem. in light and dark conditions. Blue=clean, Red=Contamination percentage,
The percentages of seed germination under the light and dark condition, at the end of the experimental period, and in the data collection, was not statistically different for both conditions, reaching similar percentages (Figure 2 and 5); however, during the incubation time the trend was slightly higher with the presence of light.
I 5 percent error bars
Figure 2: Percentage of in vitro germination of Beaucarnea recurvata Lem. seeds in the presence of light and dark. Ligh=green with ligh, Dark= green dark.
Agitación in hydrogen peroxide (H2 O2)
The seeds stirred in H2O2 in the presence and absence of light at the beginning, presented a higher percentage of contamination (30 %), while after 24 h of stirring only 3.3 % were contaminated and those that were stirred for 48 h, were completely cleaned, free of the presence of contaminating organisms (Figure 3). In most cases, the contamination was mainly due to the presence of fungi.
Figure 3: Percentage of contaminated and clean seeds of Beaucarnea recurvata Lem. in 0, 24 and 48 h of stirring in H2O2. Blue=clean, Red=contaminants presence.
The seeds of the treatments of 24 and 48 h in agitation in H2O2 showed a percentage of germination of 100 and 96.67, respectively; while the seeds that were not stirred in the presence of H2O2 78.26 % did not germinate; so the real percentage for this treatment was 59.9 (Figure 4); it should be noted that seeds with agitation of 24 h, germinated from 16 DDE; however, while seeds that were 48 h in stirring began to germinate a week later, but reaching almost the same percentage at 48 DDE.
I 5% error bars
Figure 4: Percentage of germination of Beaucarnea recurvata Lem. According to the stirring time in H2 O2
Figure 5: In vitro germination of Beaucarnea recurvata Lem seeds.
Stem tissue responses of germinated seedlings in vitro.
Type of explant
A greater number of outbreaks was obtained in the intermediate segment (I); however, there are no statistically significant differences between Intermediate (I) and Basal (B), but there are differences with respect to Terminal (Figure 6, 7 and 8). While the terminal stem tissue was the one that presented a greater length of shoots compared to the basal and intermediate showing significant difference according to the comparison de means (Table 3).
Figure 6: Promotion of sprouts of basal region (b) intermediate (i) and terminal (t) at 30 DDE in medium MS (1962) added with 10 mg· L-1 BA plus 0.3 mg· AIA L-1.
Figure 7: Sprouts obtained in basal region (B) intermediate (I) and terminal (T) at 60 DDE in medium MS (1962) added with 10 mg·L-1 BA plus 0.3 mg·L-1 AIA.
Figure 8: Promotion of sprouts in vitro of basal region (B) intermediate (I) and terminal (T) at 90 DDE in medium MS (1962) added with 10 mg·L-1 BA plus 0.3 mg·L-1AIA.
Figure 10: Plantlet of Beaucarnea recurvata establishment in soil.
Cytokinin-auxin effect.
The medium with the cytokinin-auxin 1 combination (10 mg∙L-1 BA- 0.3 mg∙L-1 AIA) was the one with the highest number of outbreaks 80 days after its establishment (Figure 8). The longer shoots were obtained in the medium with a combination of 10 mg∙L-1 2ip - 0.3 mg∙L-1 AIA; however, the results showed no significant differences between the combinations of growth regulators (Table 3).
CUADRO 3. Number and lenght of sprouts obtained according to the type or region of explants and cytokinin -auxin combination in the culture medium.
z Values with the same letter within factor in each column are equal according to Tukey's test with a P ≤ 0.05.
Establishment and in vitro germination of seeds
The works reported in Beaucarnea recurvata Lem. By Atta-Alla (2003) showed that the best responses towards germination was under light conditions, on the other hand Benítez et al. (2004) in four species of Mammilaria (Cactáceae). They showed that light was not a factor that conditioned germination, other results reported in Tillandsia eizii were between 76 and 88% seed germination (Kimberly et al., 2003). Hartman and Kester (1988) and Salisbury and Ross (1992), reported that in some species seed germination is stimulated by light; however, in other plant species germination may be inhibited; Salysbury and Ross (1992) reported that the seeds of many plant species whose light-responding seeds have not been domesticated.
Effect of hydrogen peroxide (H2 O2)
Flores (2005a) in Nolina parviflora, reported having immersed the seeds for 24 h in stirring in 3% diluted hydrogen peroxide, which did not show the presence of contaminating organisms such as fungi or bacteria, other similar cases have been reported for Abies religiosa (Alvarez et al., 2008.). In Pinus palustris Mill. and P maximartinezii (Rzedowski) the use of H2O2 in the disinfestation process also proved to be very effective, other similar results were reported to control harmful organisms, it was when commercial fungicides were used in the soaking (Barnett and McGilvary 2001, Ojeda et al., 2006). Hydrogen peroxide (H2O2), is described as an inorganic compound, which acts by oxidizing the components of the membrane and enzymes of microorganisms (Mateos, 2002 cited by Flores 2005a), because of that it has been widely used as a desinfestant for its high oxidizing power (Tom, 1998).
The results reported with seed germination by Osorio and Mata (2005). in Beaucarnea gracilis and B. recurvata, were between 89.8 and 95.3
The best disinfestation process for the establishment of in vitro seeds of B. recurvata Lem. was the application of hydrogen peroxide, under stirring for 24 and 48 hours; We can conclude that germination is not conditioned by the effect of darkness or presence of light; The periods of agitation with the effect of hydrogen peroxide for 24 hours, accelerated the times to activate seed germination. And finally, the best explants used to promote the emission of new sprouts were those isolated and sown from the intermediate and then basal region of the stems of the seeds newly germinated in vitro in the media with the inorganic salts of the MS medium and the combination of growth regulators10 mg· L-1 BA plus 0.3 mg·L-1 AIA
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