Document Type : Research Article
Authors
Department of Horticultural Science and Landscape Architecture, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
Abstract
Introduction
Hypericum perforatum is a perennial plant that has been used in traditional medicine. H. perforatum have several types of medicinal compounds including antiviral compounds, antioxidants, flavonoids and also has valuable compounds such as Hypericin, Hyperforin, Pseudo-hypericin and xanthones that have effect on human physiology. Aerial parts of H. perforatum are dotted with dark glands that appear as black nodules. Black glands were known as localization of secondary metabolites. As a consequence of the commercial potential of this species attempts have been at plant improvement by application of in vitro culture methods. Among seedling explants of H. perforatum, it appears that roots are superior for shoot regeneration. It is generally accepted that explants source is an important factor r successful establishment of tissue culture in many cases. Production of secondary metabolites via plant cell and tissue culture yields various advantages, including standardization and quality. These criteria are also valid for the main economically important chemical in st. John’s wort, namely hypericin, pseudo hypericin and hyperforin. The aim of this study was to evaluate the effect of some tissue cultures on plant callogenesis, regeneration and also, study the effect of cytokinin and auxin on rooting rate and shoot multiplication.
Materials and Methods
This research included two experiments; first experiment was a completely randomized design in the form of factorial research. Second experiment was completely randomized design. First part of experiment: this part was conducted with two explants type, leaf and shoot which, maintained in light and dark condition. Shoot explants were derived from sterile seedlings that was obtained from seeds which were cultured on MS medium. Seeds were decontaminated by NaClO 20% (V/V) for 20 min and were washed with sterile deionized water. Leaf explants were derived from seedlings in the in vivo condition and decontaminated by NaClO 20% (V/V) for 20 min then washed with sterile deionized water. Both of the explants cultured on MS medium supplemented with BA (3 and 4 mg.l-1) and 2,4-D (0, 1 and 2.5 mg.l-1). Callogenesis and regeneration were measured after 4 weeks. Second part of experiment: shoot of indirect regeneration for rooting study, were cultured on ½ MS medium supplemented with BA (0 and 0.5 mg.l-1) and IBA (0 and 1 mg.l-1). Proliferation, shoot and root length were measured after 4 and 8 weeks.
Results and Discussion
Effects of the factors on first part of experiment; calluses of shoot and leaf explants were induced after 4 weeks. Shoot explants in medium supplemented with 4 mg.l-1 BA and 2.5 mg.l-1 2, 4-D showed 95% callogenesis. Leaf explants in medium supplemented with 3 mg.l-1 BA and 2.5 mg.l-1 2, 4-D showed 98% callogenesis. Shoot explants in medium supplemented with BA (3 mg.l-1) showed 62% regeneration and leaf explants in medium supplemented with BA (4 mg.l-1) showed 8% regeneration. For second part of experiments; root induction on half strength medium without hormone and medium supplemented with 0.5 mg.l-1 BA and 1 mg.l-1 IBA had the highest rooting frequencies. Average of root length was registered as 5.25 cm. half strength medium supplemented with 0.5 mg.l-1 BA had 100% and also, medium with 0.5 mg.l-1 BA and 1 mg.l-1 IBA had 86% shoot multiplication and were not appeared any roots. Average shoot length on medium without hormone and medium supplemented with 0.5 mg.l-1 BA and 1 mg.l-1 IBA was registered as 6.24 cm and in media with 0.5 mg.l-1 BA and 1 mg.l-1 IBA was registered as 1.8 cm. Based on the result of this experiments, the concentration levels of the two hormones BA and 2, 4-D in the induction of calli formation and regeneration of the H. perforatum have been effective. In the second experiment, hormone BA, in the absence of IBA did not cause rooting and increased the degree of shoots and ultimately proliferation was effective. Also, in treatment with 0.5 mg.l-1 BA and 1 mg.l-1 IBA, although the concentration of cytokinin was less than auxin, rooting was not done.
Conclusion
The goal of this study was to introduce the suitable medium for callogenesis and regeneration of H. perforatum for production and breeding aims. Tissue and cell culture methods are used for various purposes. The result of each section can be used effectively in research topics, including corrective tests, processes for increasing secondary metabolites, as well as increasing commercial products. Therefore, by introducing efficient and effective methods, it is possible to increase the production of plants by micropropagation in less time.
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