M.A. Thesis:

The effect of chitosan on callus induction and shoot regeneration in Hypericum perforatum L.

To evaluate the effect of concentration of chitosan on callus induction and shoot regeneration in St. John'swort, two separate factorial experiments based on completely randomized design with 3 replications were carried out in biotechnology laboratory of Islamic Azad University of Sabzevar. At the first, effect of different concentrations of chitosan (0, 10, 20, 40 and 60 mg/l) and NAA (0, 0.5 and 1 mg/l) on callus induction in stem explants of Hypericum perforatum L. was evaluated. After selection of the best medium composition for callus induction in stem explants, effect of different concentrations of chitosan (0, 10, 20, 40 and 60 mg/l) and BAP (0, 0.5 and 1 mg/l) on shoot regeneration from callus were studied. The MS medium containing 30 g/l sucrose was used in both experiments. The results showed that NAA application increased callus induction percentage, callus diameter and callus fresh weight. The highest percentage of callus induction, callus diameter and fresh weight were observed at 0.5 mg/l NAA concentration, which had a significant increase compared to control treatment. The application of all chitosan concentrations in the culture medium significantly increased the callus induction percentage, callus diameter and fresh weight compared to the non-use of chitosan. There was no significant difference between the concentrations of 10, 20, 40 and 60 mg/l of chitosan in relation to the percentage of callus induction. Concentration of 40 mg/l of chitosan produced the highest diameter and callus fresh weight, which did not have a significant difference with the concentration of 60 mg/l. In the absence of BAP growth regulator in culture media, no shoots regeneration was performed in calluses. The use of BAP in the culture medium caused shoots regeneration in callus explants. The highest percentage of shoot regeneration, number of shoots in explants and shoot lengths were observed at 1 mg/l BAP concentration, which was significantly higher than the concentration of 0.5 mg/l. In the culture media containing BAP, application of 20 and 40 mg/l chitosan increased the percentage of shoot regeneration compared to concentrations of 0, 10 and 60 mg/l of chitosan. Using the concentrations of 10, 20, 40 and 60 mg/l of chitosan significantly increased the number of shoots in the explant and shoot length compared to the non-use of chitosan. The highest number and length of shoots were obtained at a concentration of 20 mg /l of chitosan. There was no significant difference between the concentrations of 20 and 40 mg/l of chitosan in relation to shoot length. According to the results, the use of MS medium containing 0.5 mg/l NAA and 40 mg/l chitosan is recommended for induction of callus in stem explants of Hypericum perforatum. Also, use of MS medium containing 1 mg/l BAP and 20 mg/l chitosan is recommended for producing the highest shoots number under in vitro conditions.

Keywords: Hypericum perforatum, callus, shoot regeneration, explant, auxin, cytokinin, chitosan.