Abstract: The optimum sucrose concentration and pH level were estimated for in vitro shoot regeneration from callus of Aralia elata Seem in a Broad-leaved tree (BT) medium with different concentrations of sucrose (0.0-25.0 g L-1) and a wide range of pH levels (4.5-6.8). The highest rate of shoot production was achieved in a shoot-regenerating medium with 15 g L-1 sucrose and a pH of 5.8. Shoot multiplication and growth were significantly affected by sucrose. Shoots were not formed in a sucrose-free medium. When the pH was adjusted below or above 5.8, the growth rate of the shoot significantly decreased.
INTRODUCTION
Tissue culture techniques have become an attractive field of biotechnological research. The benefits of these studies are particularly valuable in the areas of large-scale clonal propagation, crop improvement, the production of important plant compounds and the conservation of genetic resources. The development of shoot regeneration efficiency requires a better understanding of the influence of culture conditions on shoot regeneration.
Sucrose is the main source of carbon energy for in vitro cultures. Plant cells and tissues in a culture medium lack autotrophic ability and therefore, need external carbon for energy (Razdan, 1993). The addition of an external carbon source to the medium enhances the proliferation of cells and regeneration of green shoots (Nowak et al., 2004; Gurel and Gulsen, 1998). The optimal sucrose concentration in a medium should be sufficient to satisfy the basic energy requirements for cell division/ differentiation and not impose any negative osmotic effects on shoot formation (Stavarek et al., 1980). This indicates that sucrose acts not only as a carbon energy source in a medium but also as an osmotic (Nowak et al., 2004) and that different concentrations of sucrose are one of the factors controlling the induction and growth of shoots (Gibson, 2000; Gurel and Gulsen, 1998).
Plant cells and tissues require an optimum pH for growth and development in cultures. The pH affects nutrient uptake as well as enzymatic and hormonal activities in plants (Bhatia and Ashwath, 2005). The optimal pH level regulates the cytoplasmic activity that affects cell division and the growth of shoots and it does not interrupt the function of the cell membrane and the buffered pH of the cytoplasm (Brown et al., 1979). The pH also influences the status of the solidifying agent in a medium: a pH higher than 6 produces a very hard medium and a pH lower than 5 does not sufficiently solidify the medium (Bhatia and Ashwath, 2005). The change of pH in cells or organs is due to the ions absorbed from the medium (Sakano, 1990). Therefore, it is necessary to optimize the sucrose concentration and pH level for maximum shoot regeneration because the sucrose concentration and pH level directly influence shoot regeneration.
In the present study, calli of Aralia elata, which has fresh edible buds that are used in traditional folk medicine, were cultured in a Broad-leaved tree (BT) medium (Chalupa, 1984) with various sucrose concentrations and pH levels. Based on the obtained results, optimum sucrose concentration and pH level were determined that affected to the efficient shoot regeneration from a callus in A. elata.
MATERIALS AND METHODS
Petiole and leaf explants were used for callus induction. They were collected from mature and field-grown Aralia elata Seem. trees. A callus was induced in a BT medium with 5.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) according to the method of Karim et al. (2007). Petiole-derived calli were used for examining the effects of the sucrose concentration on shoot regeneration from calli. Six different concentrations (0.0, 5.0, 10.0, 15.0, 20.0 and 25.0 g L-1) of sucrose in a medium were employed for this experiment. On the other hand, leaf-derived calli were used to examine the effects of pH on shoot regeneration from calli. The pH of the medium was varied from 4.5 to 6.8 (4.5, 5.3, 5.8, 6.3 and 6.8) by adding 1.0 or 0.1 M HCl or NaOH aqueous solution to the medium. The percentage of shoot formation, total number of shoots per culture and average length of shoots per culture were recorded after 7 weeks of culture. The flasks containing explants and proliferating cultures were incubated on culture racks in the growth chamber. The cultures were maintained at 25± 2°C under the cool white fluorescent lights for 16 h photoperiod with a photon flux density of about 60 μ mol m-2 sec-1, which were described in previous papers (Karim et al., 2006, 2007). For statistical analysis, ten replicates per treatment with three repetitions were done for this experiment. The statistical analysis was conducted by Tukey’s Multiple Comparison Test with JMP Statistical Discovery Software (SUS Institute, USA). The analysis showed significant variation among different sucrose concentrations and pH levels. This experiment was studied at the Laboratory of Forest Products, Utsunomiya University, Japan in 2005.
RESULTS
No shoots regenerated in a BT medium without sucrose (Table 1). On the other hand, the percentage of shoot formation, total number of shoots per culture and average length of shoots per culture increased gradually with the increase of the sucrose concentration in the medium up to 15 g L-1. However, the performance of shoot development gradually decreased with an increase in the sucrose concentration to above 15 g L-1. The medium with 15 g L-1 sucrose showed the highest percentage of shoot formation (93.3%), total number of shoots per culture (28.3 shoots) and average length of shoots per culture (6.1 cm).
The percentage of shoot formation increased with an increase in the pH from
4.5 to 5.8 (Table 2), whereas pH levels above 5.8 significantly
decreased the shoot growth efficiency. In this study, the highest percentage
of shoot formation was 90.0% and the total number and average length of shoots
per culture, 26.7 shoots and 5.9 cm, respectively, were recorded when the medium
was adjusted to pH 5.8.
| Table 1: | Adventitious shoot regeneration from petiole-derived callus cultured in BT media containing different concentrations of sucrose |
| Values represent means± standard errors of 10 replicates per treatment in three repeated experiments. Means followed by the same letter(s) are not significantly different by Turkey’s test at 0.05 probability level | |
| Table 2: | Adventitious shoot formation from leaf-derived callus cultured in BT media at different pH levels |
| Values represent means± standard errors of 10 replicates per treatment in three repeated experiments. Means followed by the same letter(s) are not significantly different by Turkey’s test at 0.05 probability level | |
DISCUSSION
The optimum sucrose level for shoot development from calli may vary among species and genotypes (Nowak et al., 2004). The optimum sucrose concentration as an efficient carbon source has been examined in tissue cultures of some plant species, such as Paederia foetida (Amin et al., 2003) and Elaeocarpus robustus (Rahman et al., 2004), in which 30 g L-1 sucrose enhanced shoot growth and development. In the present study, a shoot-proliferating medium with 15 g L-1 sucrose showed the best result for shoot formation in A. elata (Table 1). Shoot elongation of alfalfa leaflets regenerated from a callus was promoted when the sucrose concentration in the medium was 10 to 30 g L-1 (Stavarek et al., 1980). Lower concentrations of sucrose have been shown to be less effective for adventitious shoot formation. On the other hand, the detrimental effect of a high sucrose concentration on shoot formation implies that the osmotic level in the medium may be inhibitory to further shoot development. Thus, high concentrations of sucrose seem to inhibit shoot growth and development. These observations are supported by an other report (Nowak et al., 2004).
The pH of the culture medium is an important factor for proliferating shoots in vitro. In the absence of pH regulation, the ionization of acidic and basic groups causes considerable changes in structure that affect their function at the cellular level (Sakano, 1990). Every species requires an optimum pH which can promote maximum shoot formation. In the present study, a better performance in all parameters on shoot development was found at pH 5.8 in a BT medium containing 0.5 μM BAP. The proliferation of Azadirachta indica shoots significantly increased when the pH of the culture medium was adjusted to 5.8 before autoclaving (Gautam et al., 1993). Nair and Seeni (2003) also found the best shoot multiplication at pH 5.8 level in a medicinal plant, Calophyllum apetalum. Lower and higher pH levels showed low performance for the induction and elongation of shoots from leaf-derived calli of A. elata. The main reason for these results seems to be the solid status of the medium: a higher pH level resulted in a hard medium, while a lower pH resulted in unsatisfactory solidification of the agar. This also agrees the results of a study of Amygdalus communis (Gurel and Gulsen, 1998). In this study, the pH level played an effective role in enzyme and growth regulator activities that affect the function of cells as well as whole plants. The role that optimum pH plays in enhancing the activities of growth regulators and enzymes has been suggested earlier by Faisal et al. (2006).