Ciencia e investigación agraria
versión ISSN 0718-1620
Cienc. Inv. Agr. vol.38 no.1 Santiago abr. 2011
Cien. Inv. Agr. 38(1):107-115. 2011
ENVIRONMENT AND ECOLOGY
In vitro rooting of Beilschmiedia berteroana, endemic to the South Central area of Chile
Enraizamiento in vitro de Beilschmiedia berteroana, endémica de la zona Centro-Sur de Chile
Matilde Uribe1, Marian Sandoval2, Amado Méndez2, Freddy Mora3, and Catherine Delaveau1
1Facultad de Ciencias Forestales y Centro de Biotecnología, Universidad de Concepción, Chile. Barrio Universitario s/n, Concepción, Chile.
2Depto. Forestal, Universidad de Concepción, Campus Los Ángeles. Juan Antonio Coloma 0201. Los Ángeles, Chile.
3Facultad Ciencias Forestales, Universidad de Concepción. Victoria 631, Casilla 160-C, Concepción, Chile.
Beilschmiedia berteroana ("belloto del sur") is a screrophyllous tree of the family Lauraceae that is endemic to the Central-South region of Chile. It is an endangered species; considered a second priority in the "Extinction Danger" category. This species is known for its timber, fruits and lots of mucilages, also has great potential for its size and ornamental evergreen foliage. This study had as a main objective to develop an in vitro rooting protocol of microshoots, obtained from the proliferation phase, by adding auxin: indole 3-butyric acid (IBA), naphthaleneacetic acid (NAA) and indole 3-acetic acid (IAA) in order to contribute to the recovery of the species. Results are discussed in the rooting phase, where this species is induced or maintained in different trials with exogenous auxin. Rooted microshoots were obtained when 250 μM of IBA were applied for 24 hours, obtaining a 40% rate of rooting, whereas no response was observed with the hormone-free treatment. Therefore, it can be concluded that Beilschmiedia berteroana can be vegetatively propagated in vitro through microcutting induced by IBA or a combination of exogenous auxin (IBA + NAA and IBA + IAA).
Key words: Auxin, Beilschmiedia berteroana, endangered species, Lauraceae.
Beilschmiedia berteroana (belloto del sur), es un árbol esclerófilo de la familia Lauraceae endémica de la zona Centro-Sur de Chile. Es una especie amenazada, considerada en segundo lugar de prioridad ubicada en la categoría "En Peligro". La especie es interesante por su madera, frutos y gran cantidad de mucílago; además tiene gran potencial ornamental por su porte y follaje siempreverde. Este trabajo tuvo por objetivo establecer un protocolo de enraizamiento in vitro de microtallos provenientes de la fase de proliferación mediante la aplicación de auxinas: ácido indolbutírico (AIB), ácido naftalenacético (ANA) y ácido indolacético (AIA), con la finalidad de contribuir a la recuperación de la especie. Se discuten los resultados obtenidos en la fase de enraizamiento, donde esta especie es inducida o mantenida en diferentes ensayos con auxinas exógenas. Microtallos enraizados se obtuvieron con AIB 250 μM aplicado por 24 horas, donde se logró obtener un 40% de enraizamiento, mientras que el ensayo libre de hormonas no indujo respuesta. Se concluye que Beilschmiedia berteroana puede ser propagada vegetativamente in vitro a través de microestacas inducidas con AIB o con una combinación de auxinas exógenas (AIB + ANA y AIB + AIA).
Palabras clave: Auxina, Beilschmiedia berteroana, especie en peligro, Lauraceae.
Belloto del sur, Beilschmiedia berteroana (Gay) Kosterm. is a threatened species considered a second priority in the "Extinction Danger" category (Benoit, 1989; CONAMA, 2007). It is an endemic evergreen tree from Central Chile that belongs to the family Lauraceae (Rodríguez et al, 1983). It has been reported the presence of only 8 subpopulations inhabiting between the Metropolitan Region (Melipilla province) (34°10'S), to the Bio Bio Region in the Ñuble province (36°43'S) (Hechenleitner et al., 2005). It is estimated that the whole population of this species is formed by no more than 2,000 individuals; almost all the forests surrounding the subpopulation to the North of La Rufina were destroyed in a fire in 1999 (Hechenleitner et al., 2005).
This species is protected in the Los Bellotos del Melado and Roblería del Cobre de Loncha National Reserves; the largest known subpop-ulation lives in Roblería del Cobre de Loncha (Hechenleitner et al, 2005; Ricci et al, 2007). Belloto del sur grows in the altitudinal range from 60 m to 1,800 m in Andes, Central Valley and Coast Range. In Andes, it can be found in rocky sidehills close to watercourses, associated to vegetation formed by a mixture of sclero-phyllous -forest and Roble-Hualo forest vegetal species (Hechenleitner et al., 2005). San Martín et al. (2002) indicated that the patterns of vegetative growth, flowering and fruiting of this species are adapted to temperate climate conditions. This species is interesting because of timber, fruits and large amounts of mucilages; it additionally has a great ornamental potential because of its height and evergreen foliage (Rodríguez et al., 1983).
The micropropagation techniques have been widely used as a powerful tool for propagation of plants which are difficult or impossible to obtain through conventional techniques (Fay and Clemente, 1997; Thorpe, 2007; García-Gonzáles, 2010). A fast production of selected material and availability of pathogen-free mi-croplants is induced by an adequate management of the process, whose usefulness extends to numerous activities, among them, safeguarding biodiversity, as the germplasm from species close to extinction and with conservation problems are multiplied (Teixeira, 2003; Uribe and Cifuentes, 2004; Jordan et al., 2005; Hami-doghli et al., 2007; Uribe et al., 2008).
Plant rooting is of great importance in the case of plants that are difficult to reproduce sexually, or in those plants where the resource is degraded (Caro et al, 2003; Martínez-Pastur et al., 2005). In this context, there is information on in vitro rooting in Chilean microprogated species such as Nothofagus alessandrii, N. glauca and N. leonii (33, 93.3 and 56.7% respectively) (Cardemil, 2000). Studies performed by Martínez Pastur and Arena (1997), in N. pumilio, reported rooting rates between 60 to 70% in microshoots obtained from secondary forest shoots and an adult tree, respectively. In Gomortega keule, root formation occurred in presence of 24.6 μM IBA in approximately 46% of the sub-cultivated nodal segments after a three months period (Jordan et al., 2005). Studies performed by Martínez-Pastur et al. (1998) in Nothofagus obliqua showed that it is possible to obtain acceptable rooting percentages in microshoots from apical and basal portions (48.6 and 27.0%, respectively). An induction of adventitious roots was made in Drymis winteri in a MS medium, from stem apices from 6-month-old plants, obtaining rooted plants in MS medium, with half diluted macronutrients, in presence of 26.58 μM NAA and 0.5 mg L-1 of calcium pantothenate (rooting percentages are not provided) (Jordan, 1999). In regard to the species Beilschmiedia berteroana, there are no reports on in vitro rooting; only one study covers the in vitro propagation of this species, using terminal and axillary buds with nodal segment from young plants as seedlings (Calderón-Baltierra and Rotella, 1998).
In vitro rooting represents a difficult stage during the micropropagation process of forest species; therefore, the aim of this work was to determine a protocol of in vitro rooting of belloto del sur, studying the influence of auxins on the rhizogenic potential of the species influence and whether they affect positively the obtaining of complete plants.
Materials and methods
The material used corresponded to seedlings maintained in a nursery during 3 years, obtained from germinated seeds. The source plants were treated with a broad spectrum systemic fungicide (Benomil, Bayer® and Mancozeb, Bayer®) in a 10 g L-1 concentration applied every seven days during seven consecutive weeks. After this period, lateral branches were isolated and the material was washed under tap water in order to eliminate dust remains completely. Subsequently, they were submerged in a fungicide solution (Captan, Bayer® 2.5 g L-1) during 2 hours. Then, they were rinsed in sterile distilled water and the nodal segments longer than 1.5 cm were cut with at least two internodes.
The surface asepsis of the nodal segments was initiated under sterile conditions in laminar flow cabinet and continuous agitation by immersion in alcohol at 70% (v/v) during 5 minutes, followed by four successive rinses with sterile distilled water for 2, 5, 10 and 15 minutes, respectively. Then, they were submerged in a solution of commercial chlorine at 50% (v/v) with 2.5% of active chlorine during 15 minutes. After that, four washes with sterile distilled water were applied during 5 minutes each.
In the establishment phase, two seedlings were placed per 5.5 cm diameter x 6.5 cm high-glass recipient, with 30 ml from the MS culture medium (Murashige and Skoog, 1962), with complete macronutrients and diluted at 25% (MS and MS7) and DKW (Driver and Kuniyuki, 1984), with total macronutrients or diluted at 50% (DKW and DKW14). The media were supplemented with sucrose (30 g L-1) and 0.8% of bacteriological agar (Merck®). The pH was fitted at 5.8 and sterilized in autoclaving during 20 minutes at 1 pressure atmosphere and 121 °C of temperature.
Thirty repetitions were made by treatment. The quantification was made at 45 days, when the percentages of viables, non-viable and contaminated seedlings were evaluated.
The rooting tests were made with microshoots obtained from the second subculture, longer than 1.5 cm, testing the effect from different concentrations and types of auxins. One hundred microshoots of 1.5 of length with at 4 leaves were used, to which an incision was made in the lower portion in order to allow the absorption of the rooting hormones by the seedling. A first test consisted on checking the effect of IBA 24.6 μM (Merck®), for 5 minutes of induction. The effect of AIB applied on a solution of 250 μM during 12 and 24 hours was checked in a second test. Finally, two hormonal combinations were applied: IBA (49 LM) plus NAA (5.37 μM, Merck®) and IBA (49 μM) plus indole-3-acetic acid (1 μM, Merck®). The hormones used in combination were added to the basal medium in order to evaluate the responses generated after 30 days.
The microshoots, once induced in the corresponding hormone, were introduced in DKW17; basal medium supplemented with sucrose 20 g L-1 and bacteriological agar 0.8% (manifestation medium), fitting the pH to 5.8. Then, they were incubated in a growth chamber under controlled conditions, at 25 ± 1oC of temperature, 55% of relative humidity, under a photoperiod of 16 hours of cold light and luminous intensity of 40 μmol m-2 s-1, for a minimum period of 30 days.
Ten microshoots with their respective control treatment were used in all the tests, and the following parameters were evaluated at the end of the period: callus percentages, radicular pri-mordia and rooted microshoots.
The methodology of Generalized Linear Model was used for the statistical analysis, according to the formula by Nelder and Wendderburn (1972). The variables of interest were considered binary; therefore, the binomial distribution (absence or presence of an event or variable) and the canonical link function (logit) were considered in the GLM fit, according to Mora et al. (2007). The procedure GENMOD by SAS was used for the analysis of deviance (ANDEVI) with the option of generalized orthogonal contrasts in order to determine the differences between treatments (Dos Santos and Mora, 2007).
Results and discussion
In the establishment stage, the analysis of deviance indicated significant differences among the media used for the variables evaluated (Table 1). The high percentage of establishment in both culture media (Table 2), at over 70% and a pathogenous contamination that does not exceed 10% in the cases, where the saline concentration was reduced, indicated that the treatment applied to the source plants before the extraction of the initial material, provided an excellent alternative to introduce contamination-free tissues. These results are similar to those from other studies made on native species, as Noth-ofagus pumilio, which presented 80% of contamination-free seedlings (Martínez-Pastur and Arena, 1997), as well as the results obtained by Cardemil (2000) in Nothofagus glauca, N. ales-sandrii and N. leonii.
Establishing pathogen-free material in this stage is the base to obtain a successful in vitro culture (Pérez, 1998); which shows partly the good sanitary status of the mother plant and the effectiveness of the surface asepsis applied (Figure 1).
On the other hand, the results suggest that the belloto del sur tissues established in vitro may be susceptible to the salts concentration of the medium, as the best results were obtained in these media (MS1/4 and DKW1/2), with statistically significant differences between them, reaching the best survival result (90%) in medium DKW1/2. Media with low salt concentration have been used successfully in the propagation of Nothofagus nervosa and various Fagaceae (Vieitez et al., 1993; Jordan et al., 1996).
It is observed in medium DKW1/2, that there is a low percentage of seedlings loss due to their brownishing. Literature reports that this brown-ishing may be caused by enzyme oxidases, like polyphenoloxidases and tyrosinases, which are released when the tissues are wounded. The growth inhibition of seedlings, on the other hand, occurs by phenols oxidation and the subsequent formation of quinonic compounds, which are highly active (Espejo et al., 1990; Marks and Simpson, 1990; Seemann, 1993; Alvarado, 1998).
During the phase of root induction, the in vitro microshoots rooted when indole 3-butyric acid or an auxin combination were applied to the DKW1/4 (Figure 2). Therefore, the results from the analysis of deviance indicate that there are significant differences among the tests for the variables radicular primordia and rooted microshoots (Table 3). In this regard, both IBA (49 μM) + NAA (5.37 μM) applied to the culture medium during 30 days, and IBA 250 μM applied during 24 hours, produced one or various roots, with lateral roots and shoots and leaves development after 3 to 5 months of culture in manifestation medium, obtaining between 30 and 40% of rooting respectively (Table 4, Figure 2A and B). Regardless belloto del sur has been categorized as a recalcitrant species to propagation (Reyes, 1997), as acceptable rooting percentages were obtained in this study.
Similar rooting results (46%) were obtained in Gomortega keule in seedlings from nodal sections from 2-year material, with the application of IBA (24.6 |jM) in the culture medium during three months (Jordan et al, 2005). In Quercus rubra, using a hormonal concentration of IBA of 25 mg L-1 during 24 hours (San-Jose et al., 1996), a 37.3% of rooting was reached. Higher rooting percentages (59%) were obtained from seedlings of Quillaja saponaria, with the addition of IBA (0.49 |jM) during one month (Prehn et al, 2003).
The low rhizogenic response (10%) obtained in the concentration of 24.6 LiM of IBA (Table 4, Figure 2C) may be attributed to the reduced immersion time of the microshoots in the growth hormone, which did not allow its absorption by the seedling and the low concentration used.
Similar studies made ex vitro in Gomortega keule (Peña, 1995), provided similar results with the application of IBA 6000 mg L-1 (3.3% of rooting). If only the total of treatments applied in queule are considered, only 0.26% of rooted cuttings were obtained.
However, higher percentages have been observed in other wood-like species. For example, Peralta (2001) obtained 84.38% of rooting in Nothofagus alpina with IBA 500 mg L-1 for a 7-minute-in-duction time, from shoots from seedlings cultivated in vitro, while San-Jose et al. (1996), using the same concentration for 2 minutes, obtained 45% of rooting in Quercus rubra.
In regard to the number of radicular primordia obtained, it must be considered that, during the manifestation stage and radicular development, other problems reducing the efficiency of the process become evident. Also, a portion of the centers of radicular initiation creates nodular structures that never evolve to root formations, which might cause a problem to the sites of origin and tissues implied (Hartmann and Kester, 1999).
In general, there is a low percentage of formation of basal callus in all the tests, an important aspect to be mentioned, as the presence of callus partly reduces root production and greatly impedes the transference of the vitroplants to the consolidation field (Ríos et al., 2005).
A difference was recorded in the time required for the manifestation of rhizogenic responses among the different test; a faster response was seen when hormonal combinations were used in the culture medium (IBA/NAA and IBA/IAA), in a period of three months. Potentially, it was necessary to wait for five months for results for the treatment IBA 250 μM 24 hours.
The results obtained were powered by the low number of subcultures (2) which had the source material, as the number of subcultures is increased, the response capacity of the tissues is affected, which is reflected in a lower rooting capacity (Ríos et al., 2005).
On the other hand, the hormone-free tests did not provoke any response of rhizogenic induction. This might indicate the absence of preformed structures for the emergence of radicular primordia in the species; structures found easily induced in other species like Salix sp., Populus sp., Ribes nigrum or Fagus sylvatica (Haissing, 1972; Fink, 1982,), where these pri-mordia may develop roots, depending on the culture conditions. Similar observations were seen in a study made with Nothofagus nervosa, where the lack of pre-radicular structures was evident (Martínez-Pastur et al., 2005). Therefore, the exogenous application of inducing agents as auxins and other regulators of vegetal growth becomes relevant, as they play an important role in this response (Martínez-Pastur et al., 2007).
Acclimation of the rooted plants was made in a peat-perlite mixture (80%: 20%) and maintained inside the culture chamber for one month. Subsequently, they were transferred to the laboratory of tissue culture in fall-winter, under uncontrolled conditions; this is, at temperatures lower than 15 °C and with high humidity. A permanent irrigation with macronutrients from the medium DKW diluted at 25% was made, reaching 50% of survival (Figure 2 D).
The present study is the first report on in vitro rhizogenic induction for the species Beilschmie-dia berteroana, from nodal segments of three-year-old plants, by the application of IBA 250 llM during 24 hours and IBA/NAA in media DKW at a quarter of the normal macronutrient concentrations. Between 30 and 40% of rooted microshoots in IBA 49 μM + NAA 5.37 μM and IBA 250 μM were obtained, respectively. These results will allow a continuous improvement of the rhizogenic process, and will represent the base for the development of further studies.
Study financed by Universidad de Concepción, Project DIUC N° 203.412.003-1.0. The authors thank Forestal y Agrícola Monteáguila (Los Ángeles) for providing the vegetal material used in this study.
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Received October 15, 2009. Accepted February 12, 2011.
Corresponding author: email@example.com