Medium Optimization for Callus Induction, Shoot Regeneration and Rooting of Hypericum perforatum from Stem and Leaf Explants

Document Type : Research Article


Ferdowsi University of Mashhad



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, flavonoides 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 or red 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 advantage es, 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 effectof 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 plan was a completely randomized in the form of a factorial. Second experiment plan was completely ranom.

first part of experiment : this part were conducted with two explants, leaf and shoot ,maintained in light and dark condition. Shoot explants were derived from sterile seedlings that was obtained from seeds 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 explants cultured on MS media supplemented with BA(3 and 4 mg/l) and 2,4-D (0, 1 and 2.5 mg/l). Callogenesis and regeneration was measured after 4 weeks.

Second part of experiment: shoot of indirect regeneration for rooting study, were cultured on ½ MS media supplemented with BA (0 and 5/0 mg/l) and IBA (0 and 1 mg/l). 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 Medium supplemented with 4 mg/l BA and 2.5 mg/l 2, 4-D showed 95% Callogenesis. Leaf explants Medium supplemented with 3 mg/l BA and 2.5 mg/l 2, 4-D showed 98% Callogenesis. Shoot explants Medium supplemented with BA 3 mg/l showed 62% regeneration and leaf explants Medium supplemented with BA 4 mg/l showed 8% regeneration.

For second part of experiments; root induction on half strength medium without hormone and medium supplemented with 0.5 mg/l BA and 1 mg/l IBA had highest rooting frequencies. Average of root length was registered 5.25 cm. half strength medium supplemented with 0.5 mg/l BA had 100% and also, medium with 0.5 mg/l BA and 1 mg/l 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 BA and 1 mg/l IBA was registered 6.24 cm and in media with 0.5 mg/l BA and 1 mg/l IBA was registered 1.8 cm. based on result of this experiments, the concentration levels of the two hormone 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 BA and 1 mg/l IBA, although the concentration of cytokinin was less than auxin, rooting was not done.


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.

Main Subjects


Articles in Press, Accepted Manuscript
Available Online from 05 April 2021
  • Receive Date: 28 November 2017
  • Revise Date: 15 February 2023
  • Accept Date: 04 April 2020
  • First Publish Date: 05 April 2021