Document Type : Research Article
Authors
1 University of Tehran
2 Ferdowsi University Of Mashhad
3 Central Agriculture and Natural Resource of Fars
Abstract
Introduction Hypericum perforatum L. is an important medicinal plant that used for depression treatment. In vitro regeneration has been successfully achieved for many Hypericum species from a range of explants sources with different growth regulators. Recent studies have demonstrated that in vitro culture is an option for multiplication of different Hypericum species. With consideration this notice that tissue culture can provide an affordable alternative method for propagation with high speed to production of intensive plant material as well as suitable materials for breeding programs of H. perforatum, the objective of this study was to investigate the effects of explant types (leaf and stem), plant growth regulators (2,4-Dichlorophenoxyacetic acid (2,4-D), Naphthalene Acetic Acid (NAA), Benzyl Adenine (BA) and Kinetin (Kin)), light (dark and weak light), media culture (MS medium and MS medium with B5 vitamins) and poly vinyl pyrrolidone (PVP) on callus induction in H. perforatum at in vitro culture condition.
Materials and Methods For preparation of sterile plantlets, the seeds of Iranian H. perforatum (Azadshahr population) were cultured in 1.2 MS growth regulator free-media. For investigation the callus induction of Iranian H. perforatum the leaf and stem explants of in vitro obtained plantlets were used. Explants were cultured in different concentrations of 2,4-D (0.2, 0.5 and 1 mg l-1) with two kinds of cytokines BA and Kin (0.2, 0.5 and 1 mg l-1) as well as 1 mg l-1 NAA. For browning control of calli the effects of light (dark and weak light), media culture (MS medium and MS medium with B5 vitamins) and four concentrations of poly vinyl pyrrolidone (0, 50, 100 and 200 mg l-1) were also surveyed.
Results and Discussion Callus cultures could be used for cell suspension initiation, studying of their morphogenetic potential and screening of secondary metabolite profile. In present study the response of two H. perforatum explants (leaf and stem) to different levels and combinations of auxins and cytokinins were tested. The callus induction in both studied explants (leaf and stem) was just observed in supplemented media with plant growth regulators. The calli of leaf explants were showed better growth in dark and the highest callus fresh weight was obtained in 0.25 mg l-1 2,4-D + 1 mg l-1 Kin and 0.5 mg l-1 2.4-D + 1 mg l-1 BA. Of the various concentrations and combination of growth regulators, the minimum response about callus induction was observed in the presence of 1 mg l-1 2, 4-D in combination with 1 mg l-1 NAA. The obtained calli got browning shortly after induction. The investigation of light effect on callus quantity and quality showed that not only light did not affect the callus induction and callus browning but also reduced the callus growth. The highest callus fresh weight was obtained in 100 mg l-1 poly vinyl pyrrolidone in leaf explant. Few species within the genus Hypericum have been used to produce callus. In H. perforatum seedling, different explants such as shoot apical meristem, stem segments and leaves were used for callus induction. In H. erectum callus induction was obtained by culturing seedlings in the presence of Indole Acetic Acid (IAA) and BA under darkness. The combination of cytokinins and auxins did not support callus growth of H. brasiliense and callus of nodal explants was only obtained in the presence of 2,4-D or NAA using either MS or B5 medium. These differences among literatures can be due to different cultivars, culture conditions, explant type and medium composition.
Conclusion The plant growth regulators is necessary for callus induction in Iranian H. perforatum and leaf is suitable for this purpose. The light intensity and poly vinyl pyrrolidone did not control the browning of of H. perforatum calli.
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