RESEARCH WORK
Effect of explant type and concentration of 2,4-dichlorophenoxyacetic acid on callus formation in Morus alba L.
A. Espinosa1, J. Silva1, Sandra Sariego1, L. Cholo Masapanta2 y Hugo Delgado2
1Centro de Estudios de Biotecnología Vegetal, Facultad de Ciencias Agrícolas. Universidad de Granma, Carretera a Manzanillo km 17½, Bayamo, Cuba. CP 85100
2Universidad Técnica de Cotopaxi. La Maná, Ecuador
E-mail: aespinosar@udg.co.cu
ABSTRACT
The objective of the work was to evaluate the effect of explant type and concentration of 2,4-dichlorophenoxyacetic acid on callus formation, in Morus alba L. As explant sources leaf blades, stems and petioles were used , obtained from cuttings which produced growths under laboratory conditions. The basal culture medium was constituted by MS salts and vitamins (1962), with different concentrations of 2,4-D (0; 0,5; 1,0 and 2,0 mg.L-1). A completely randomized experimental design was used. Callus formation in all explant types started in the first 15 days after the in vitro establishment. Callogenesis only occurred in the culture media with 2,4-D. The callus formation zone, callus color and texture depended on the explant type used. The increase of 2,4-D concentration in the culture medium increased callus size, with the best results in the concentrations 1,0 and 2,0 mg.L-1.
Key words: tissue culture, Morus alba, explants.
INTRODUCTION
Mulberry (Morus alba L.) is a forage tree of Asian origin, perfectly adapted to tropical conditions. It belongs to the Moraceae family and has 24 species grouped in the Morus genus. Among them, the diploid species Morus alba is the most extended (Machii, Koyama and Yamanouchi, 2000).
The main use of mulberry worldwide is to feed the silkworm (Milera, 2010). Depending on the place, it is also appreciated for its fruit, which can be consumed fresh, in juice or preserved. In some countries its leaves and fresh stems are used as food, and it is also acknowledged for its medicinal properties and as forage (Cepeda, 1991).
Mulberry, which is used as shrubby plant since the 1980's, constitutes an alternative for ruminant feeding in countries of Central America, and it is recommended for the quality of its forage (Medero, Montoya and Osa, 1999). Its leaves might have more than 46% of essential aminoacids (Machii, 1992), more than 20% of crude protein, and between 70 and 80% of in vitro dry matter digestibility (Benavides, Lachaux and Fuentes, 1994); in addition it has a high mineral content (up to 17% of ash), with high calcium and phosphorus values (Sánchez, 1999).
Nevertheless, it is amazing that a plant which has been used and bred to feed an animal with high nutritional requirements, has received limited attention by livestock raisers, technicians and researchers (Tikader and Roy, 1999).
The initial reports on the micropropagation of mulberry were made by the Japanese scientist Ohyama (1970), who obtained the first whole seedlings from axillary growths; since then many reports have been elaborated about tissue culture in mulberry using different explants (Lu, 2002; Thomas, 2003; Kavyashree, 2007; Vijayan, 2010). The in vitro induction of callogenesis has been studied in different crops, in order to establish protocols for the propagation by indirect organogenesis and somatic embryogenesis, which can be used for breeding or massive propagation. Many factors influence the process of in vitro callogenesis, among which the explant type and hormonal composition of this crop stand out. In this work the effect of explant type and concentration of 2,4-D on callus formation during the somatic embryogenesis process in M. alba L was evaluated.
MATERIALS AND METHODS
The study was conducted at the Center of Plant Biotechnology Studies (CEBVEG), belonging to the School of Agricultural Sciences of the University of Granma, Cuba. Mulberry cuttings of the Acorazonada variety were used as plant material, with a size of 10-12 cm; they were placed in flasks with water, under semi-controlled laboratory conditions, to propitiate the emergence of axillary buds. When the growths reached a size of 8-10 cm they were cut, and from them the leaf blades, stems and petioles were selected as explant sources for callus formation.
For their disinfection, the explants were carefully and separately- washed with water and detergent; afterwards, they were kept in continuous agitation for 30 minutes, in a dilution of water with detergent at 3%, and they were rinsed four times with sterile distilled water. Then, in the laminar flow cabinet, they were dipped in a solution of sodium hypochlorite at 1% of active chlorine, during 15 minutes, and they were rinsed four times with sterile distilled water.
The leaf blades were divided into 0,5-cm2 portions and the central zone was selected to be placed in the culture medium. The petioles and stems were cut in 1,0 cm-long segments and the parts which suffered damage during disinfection were eliminated.
The basal culture medium was composed by Murashige and Skoog (1962) salts and vitamins, myo-inositol (100 mg.L-1), thiamine (1,0 mg.L-1) and saccharose (30 g.L-1). The pH was adjusted at 5,7 with a pH meter (CRISON)- before placing it in the autoclave, and it was solidified with agar 6 g.L-1. Different concentrations of 2,4-D in the culture medium were evaluated, with which the following treatments were formed:
Ten mL of culture medium were added in each test tube and they were sterilized in vertical autoclave, at 121ºC of temperature and 1,2 kg cm-2 of pressure, during 20 minutes. The combination of explant type and culture medium with different concentrations of 2,4-D added up to a total of 12 treatments.
The design was completely randomized. Twenty test tubes were used for each treatment, each tube was considered a repetition and in each one of them an explant was cultivated. The incubation was made under darkness conditions, at a temperature of 25ºC and relative humidity of 60-65%.
After 15 and 30 days the following variables were evaluated: disinfection (%), onset of callus formation (days), percentage of formed calluses (%), root formation (%), and callus coloration and texture.
Statistical analysis. A proportion comparison analysis was applied to all data, through the statistical program STATISTICA version 6.0 for Windows®, with 5% error probability.
RESULTS AND DISCUSSION
In all treatments 100% of disinfected explants were achieved. These results indicate that the use of sodium hypochlorite at 1% active chlorine, combined with the use of explants from cuttings which emerged under semi-controlled laboratory conditions, turned out to be an efficient disinfection method, because it allowed obtaining high values of contamination-free explants, during the in vitro establishment of mulberry.
The studies conducted by Salas et al. (2004) showed values higher than 80% of disinfection of explants obtained from mulberry leaves using sodium hypochlorite at 1%, during 20 minutes, which coincides with the results of this study. García et al. (cited by Salas et al., 2004) used a mixture of mercury bichloride with low concentrations of sodium hypochlorite for the disinfection of M. alba L. explants, with good results.
Fifteen days after the in vitro establishment, callus formation began in all the explant types cultivated in media which contained 2,4-D, independently from its concentration, which was proven by changes in explant coloration and structure, as well as by the development of small protuberances of undifferentiated tissue.
Table 1 shows the callus percentages in the different explant types, 30 days after in vitro establishment. In the treatment without 2,4-D no calluses were formed; while in the treatments in which 2,4-D was used no significant differences were found because calluses were formed in 100% of the explants. These results confirmed the important role of 2,4-D in callus formation, and the strong auxinic activity of this growth regulator was shown, as well as its capacity to produce a disorganized growth in the explant cells.
The 2,4-D is a strong auxin which is used for in vitro cultivation in callus formation and the induction of somatic embryos (Rivero et al., 2008). It has been used as growth regulator for this first purpose in diverse plant species, with good results (Silva, 2006; Medero, 2006; Paneque, 2006) in the cacao, cassava and sweet potato cultivation.
In leaf explants, the onset of callus formation became evident when they started to wrinkle, got separated from the culture medium and turned light green in color; likewise, the formation of small protuberances on the edges and the on main nervature could be observed.
Callus formation on the zone where the cutting was made and on the main nervature could be associated to the fact that in the latter less differentiated cells are found, as compared to the others from the explant, which respond more rapidly to the effect of growth regulators and favor callus formation (Michaux-Ferriere and Carron, 1989).
The callus tissue was shown as groupings of undifferentiated tissue, cream in color, with friable consistency. Callus size increased as the concentration of 2,4-D increased, and they practically covered the whole explant in the treatments where the higher concentrations were used (1,0 and 2,0 mg.L-1 of 2,4-D).
Santana (1993), when using leaf segments in coffee cultivation to induce callus formation, reported that they started in the zones where the leaves were cut and afterwards an engrossing occurred of the perivascular cells of the leaf bundle, with a later growth in all directions; this propitiated the callus to become noticeable and to be projected towards the exterior through the epidermis, results which coincide with the ones obtained in this work.
In the petiole explants, callus formation began with the engrossing of the central part and the emergence of undifferentiated tissue in the zone where the cuttings were performed. Afterwards, callus growth extended all over the surface and on the explant ends. The callus mass was higher as the concentration of 2,4-D increased and covered the whole explant in the treatments in which concentrations of 1,0 and 2,0 mg.L-1 were used. The calluses showed light brown coloring and a spongy consistency.
Callus formation on the stem explants started on the zone where the cuttings were made, that is, from the scar tissue. The calluses were dark brown in color and had spongy consistency. In the treatments in which 0,5 and 1,0 mg.L-1 of 2,4-D were used, small-size calluses were obtained; while when using 2,0 mg.L-1 of 2,4-D, the calluses were higher and practically covered the whole explant.
Sanginés et al. (1999) studied callus formation from the petioles, leaf blades and axillary buds of M. alba L., in a culture medium in which 5,0 mg.L-1 of 6-BAP was combined with 0,25 mg.L-1 of 2,4-D. The formed calluses showed good appearance, with embryogenic characteristics.
On the other hand, Bhau and Wakhju (2001) showed that callus formation in M. alba depended on the genotype, explant type and growth regulators used. The leaves were the best explants, and with the combination of 1,0 mg.L-1 of 2,4-D and 0,5 mg.L-1 of BA the highest percentages of formed calluses were obtained (95-100%).
Root formation was directly related to the concentration of 2,4-D (table 2). In leaf explants root growth started from the calluses, which indicates that indirect organogenesis occurred. Root formation increased as the auxin concentration increased, with significant differences among the evaluated concentrations. The highest values (100%) were reached when using 2,0 mg.L-1 of 2,4-D, followed by 68% of explants with roots in the treatment of 1,0 mg.L-1 of 2,4-D. The number of roots per explant was abundant.
In petiole and stem explants, root formation was lower as compared to the leaves and values lower than 10% of explants with roots were reached. In the petioles roots were formed in 7,14% of the explants, when the culture medium with 2,0 mg.L-1 of 2,4-D was used. The roots originated from the callus, and they were thin and short. The root number per explant was low.
In the stems, when using 1,0 and 2,0 mg.L-1 of 2,4-D, roots were formed (one or two per explant) in 7,69 and 9,38% of the explants, respectively. They were thick and long, and did not seemingly emerge from the callus.
The results of the study showed that the use of leaf, petiole and stem explants in the in vitro mulberry cultivation allowed high values of callus formation, and that 2,4-D in the culture medium was essential.
González, Morejón and Portilla (2007) stated that the differences in the response to in vitro cultivation, when using different types of explants, may occur due to variations in the endogenous content of hormones and their anatomical characteristics.
On the other hand, Suksa-Ard et al. (1999) considered that callus formation and characteristics depend, remarkably, on the organ used as explant. Shu, Ying-Cri and Hong-Hui (2005) showed the best results in obtaining embryogenic calluses, when using stem segments of Dioscorea zingiberensis as explants and including in the culture medium 0,5 mg of benzylaminopurine and 2,0 mg.L-1 of 2,4-D, with which they achieved a callus formation frequency of 87%.
CONCLUSIONS
The use of 2,4-D concentrations in the culture medium, equal to or higher than 0,5 mg.L-1, propitiated callus formation in leaf (100%), petiole (100%) and stem explants (100%) in mulberry cultivation, and bigger calluses were obtained as the concentration increased.
In the leaf explants roots were formed in all the concentrations of 2,4-D, and 100% of rooted explants was reached when using 2,0 mg.L-1 of 2,4-D. In the petiole and stem explants roots were formed when using concentrations of 2,0 mg.L-1 of 2,4-D, with values of 7,14 and 9,38%, respectively.
ACKNOWLEDGEMENTS
The authors thank the Government of Álava from the Basque Country and the Euskadi-Cuba Association, for the funds granted to conduct this study.
