Document Type : Full Paper
Authors
1 Department of Horticultural Sciences, Faculty of Agriculture, Tabriz University, Tabriz, Iran
2 Department of Horticultural Sciences, Faculty of Agriculture, Guilan University, Guilan, Iran
3 Department of Horticultural Sciences, Faculty of Agriculture, Guilan University, Guilan, Iran.
Abstract
Keywords
Main Subjects
Extended Abstract
Introduction
Gotu kola (Centella asiatica L.) is one of the important medicinal plants that are used in traditional medicine for the treatment of diseases and in the cosmetics industry. In Iran it grows in wetlands of Anzali city. Due to the excessive exploitation of natural resources, as well as the reduction of surface water and the increase of agricultural toxins, the biodiversity of Gotu kola is at risk in the whole world. This is why its name has been registered as a threatened plant species by the International Union for Conservation of Nature and National Resources (IUCN). Considering the low seed germination of this plant, it seems necessary to use in-vitro micropropagation methods for producing a large number of plants in the shortest time.
Materials and methods
In this research, in order to control fungal and bacterial contamination and reduce the use of disinfectants, first the disinfected runner tips were cultivated in MS culture medium, and when transformed into whole plants, their leaves were used as explants for embryogenesis. Leaf explants were used to induce embryonic calli on MS solid media containing 0, 0.25, 0.5, 1.5 and 2 mg/l 2,4-D alone or in combination with 0,0.25,0.5 and 1 mg/l BAP. The obtained calli were transferred to MS medium containing different concentrations of 2,4-D, including 0, 0.1, 0.2 and 0.3 mg/l for induction of somatic embryos. BAP growth regulator was used at concentrations of 0, 0.25, 0.5 and 1 mg/l to germinate the formed embryos.
Results and Discussion
The results of inducing embryonic calli by plant growth regulators showed that the concentration of 2 mg/l 2,4-D treatment had the highest percentage of callus formation (96.61%), callus fresh weight (3.96g) and callus mean diameter (3.56cm) in dark conditions. The effect of 2,4-D treatment on somatic embryos formation showed that concentration of 0.1 mg/L 2,4-D had the highest mean number of embryos formed at different stages of spherical-, heart- and torpedo-shaped. Also, the 0.1 mg/l 2,4-D treatment had the highest percentage of embryogenesis (55.27%) which was not significantly different from 0.2 mg/l 2,4-D treatment. BAP growth regulator was used at concentrations of 0, 0.25, 0.5 and 1 mg/l to germinate the formed embryos. The results showed that maximum percentage of embryos germination (56.8%) and maximum mean number of seedling production (12.4%) were obtained at the concentration of 1 mg/l BAP. It has been reported that culture medium containing 2,4-D is one of the most suitable mediums for inducing embryogenic callus in many plant species because this medium increases internal auxin in plants.
In the examined treatments where the concentration of BAP was higher than 2,4-D, the fresh weight of the callus also increased with the increase in the amount of BAP. Adding cytokinin to the auxin-rich medium in the present study increased callus formation and also the average of callus diameter. By increasing the concentration of 2,4-D from zero to 2 mg/liter, the percentage of callus formation increased. The concentration of 0.1 mg/liter of 2,4-D had the highest percentage of embryogenesis. The results of other studies also showed that by reducing the amount of growth regulators, the stages of fetal development are improved. Although the process of embryo induction from cells in the callus is not completely understood, today it is generally believed that the continued presence of auxin causes a change in gene expression and is probably associated with an increase in DNA demethylation of the embryo masses. Under these conditions, the embryonic masses inside the calli synthesize the genes necessary to complete the globular stage of embryogenesis. Somatic embryos of many species are capable of germination in the induction medium. In a few cases, it is necessary to transfer the embryos to a fresh culture medium with different concentrations of growth-regulating compounds in which auxin has been removed. The use of 2,4-D growth regulator as auxin in the creation of embryogenic calli had good results. Induction of somatic embryos with lower concentrations of 2,4-D was more effective. For the germination of embryos, the use of BAP growth regulator was successful. In this research, the embryos formed roots after germination, and it took a long time for the roots to grow, which probably would have shortened the rooting time if growth regulators such as IBA and NAA were used after germination. Acclimatization of the seedlings in laboratory conditions resulted in a good process, but in strong light conditions, the plants suffered from sunburn, which, due to the nature of the Gotu kola plant, which is a shade-loving plant, the use of a cover as a shade solved the problem of sunburn.
Conclusion
Considering that Gotu kola is a creeping plant, it is difficult to control its various surface and internal contaminations in the conditions of in vitro cultivation. Using the tip of the creeping stem as the initial explant to obtain plants free from pollution and disease was extremely satisfactory and made the next steps of the work, i.e., the use of the free of contamination leaf as explant for callus generation, very easy. The use of 2,4-D regulator as auxin in creating embryogenic callus had good results. Induction of somatic embryos with lower concentrations of 2,4-D was more efficient, especially in the spherical and heart-shaped stages, but in these treatments, the torpedo-shaped stage was not formed, which by transfer to MS culture medium without growth regulator and also light conditions, torpedo-shaped stage was also observed. For the germination of embryos, the use of BAP growth regulator was successful.
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