In Vitro Shoot Proliferation of Hypericum Perforatum L. Through Indirect and Direct Plant Regeneration
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Journal of Medicinal Plants and By-products (2017) 1: 81-89 Original Article In vitro shoot Proliferation of Hypericum perforatum L. through Indirect and Direct Plant Regeneration Manizhe Abdollahpoor1*, Siamak Kalantari1, Majid Azizi2 and Yusef Ali Saadat3 1Department of Horticultural Science, Tehran University, Karaj, Iran. 2Department of Horticultural Science, Ferdowsi University of Mashhad, Mashhad, Iran. 3Central Agriculture and Natural Resource of Fars Province, Shiraz, Iran. Article History: Received: 04 December 2016 /Accepted in revised form: 15 March 2017 © 2013 Iranian Society of Medicinal Plants. All rights reserve Abstract Hypericum perforatum L. (St. Johns’ wort) is the most commercially important species of the genus Hypericum and contains a wide range of components including naphthodianthrones, phloroglucinols, tannins, xanthones, phenolic acids and essential oil. In order to establish an efficient protocol for regeneration, the effects of explant type and plant growth regulators on direct and indirect shoot regeneration in H. perforatum were evaluated. According to obtained results the media supplemented with 0.1 mg l-1 Benzyl Adenine (BA) was effective for shoot proliferation from shoot tip explants of H. perforatum that showed the highest shoots number (15.5 shoots per explant) and shoot height (2.07 cm). In second experiment a method for rapid micro propagation of H. perforatum through indirect plant regeneration from calli has been developed. The results demonstrated that a combination of auxin and cytokinin was needed for optimum callus induction and leaf segments were suitable explant for callus induction in H. perforatum. Callus induction was observed in most studied treatments but the highest callus volume (1.43 cm3) was obtained by leaf segments in media supplemented with 0.25 mg l-1 2,4- Dichlorophenoxyacetic acid (2,4-D)+1 mg l-1 Kinetin. Successful shoot regeneration from callus was observed in MS medium containing 0.5 mg l-1 BA, which resulted the highest shoot proliferation rate (61.75%) and maximum number of induced shoots (9 shoots per explant). All of shoots formed root in media with 0.5 mg l-1 Indole-3-Butyric Acid (IBA) on which 100% of the regenerated shoots developed roots with an average number of 5 roots per shoot. The plantlets were acclimatized and transferred to the greenhouse with 80% survival. This in vitro propagation protocol should be useful for conservation as well as mass propagation of H. perforatum plant. Keywords: Hypericum perforatum L., Shoot formation, Callus induction, Micro propagation Introduction neuralgia, sciatica, menopausal neurosis, anxiety and depression [4]. Nowadays H. perforatum is the Hypericum perforatum L. commonly called St. most economically important natural anti John's wort, is an important perennial herb depression source [2,3,5]. The steadily increasing belonging to the family Hypericaceae [1]. This market interest in antidepressants effect of H. plant is a natural source of active compounds perforatum is one of the factors that have including naphthodianthrones (hypericin and contributed to the economic value of this plant in pseudo hypericin), phloroglucinols, tannins, herbal industry. This economic value of H. xanthones, flavonoids, phenolic acids and essential perforatum has stimulated the development of oil [2,3]. St. John's wort is stated to possess programs aimed to improve high-speed sedative and astringent properties, and has been propagation, breeding and selection of this plant used traditionally for the treatment of excitability, [6]. Corresponding author: Department of Horticultural Science,Faculty of Agriculture, Tehran University, Tehran, Iran Email Address: [email protected] Journal of Medicinal Plants and By-products (2017) 1: 81-89 82 Propagation of H. perforatum occurs by means of pretreatments. This experiment was done in the runners or from seeds. In vitro regeneration has base of completely randomized design with three been successfully achieved for many Hypericum L. replications. species from a range of explants sources with Shoot Proliferation different growth regulators. Recent studies have demonstrated that in vitro culture is an option for For preparation of sterile plantlets, the seeds of St. multiplication of different Hypericum species such John’s Wort were pretreated according to results of as H. foliosum [7], H. brasiliense [8], H. first experiment and then culture in 1.2 MS [12] canariense [9] and H. perforatum [10]. With the growth regulator free-media. For shoot induction, exception of H. canariense and H. foliosum, using when plantlets attained to height of 5-10 cm, whole seedlings or their excised parts as initial aseptically the shoot tips of each plantlet were explants resulted in the formation of multiple excised and cultured on MS medium with different shoots. Aseptically germinated seeds of H. concentrations of Benzyl Adenine (BA) included 0, perforatum and H. maculatum have been used as 0.1, 0.25, 0.35 and 0.45 mg l-1 as well as high initial material for establishing cell cultures, which concentration of BA (0.5, 1 and 1.5 mg l-1). have been analyzed for the presence of hypericin, Cultures were maintained at temperature of 25±1 flavonoids, procyanidines and tannins [11]. °C under a 16/8 h light regime provided by white With consideration this notice that tissue culture fluorescent tubes. After 30 days the shoots number can provide an affordable alternative method for per explant, fresh weight and height of shoots were propagation with high speed to production of recorded. This experiment had four replications intensive plant material as well as suitable with three explants in each replication and was materials for breeding programs of H. perforatum, done in the base of completely randomized design. the objective of this study was to determine According to results of this experiment, the optimum explant and plant growth regulators for application of 0.1 mg l-1 BA resulted the highest direct and indirect regeneration of H. perforatum shoot induction so was choose for next experiment. for developing a high frequency of the plant In the experiment three, the effect of cytokine and multiplication system of this plant. auxin combination on shoots induction of H. perforatum was investigated. For this purpose the Material and Methods shoot tips of plantlets were cultured on supplemented media with 0.1 and 0.05 mg l-1 BA in cted from combination with three concentrations (0, 0.01 and The seeds of St. John’s Wort were colle -1 Iranian native population in Azadshahr at Golestan 0.05 mg l ) of IBA (Indole-3-Butyric Acid). province (37° 08ʹ latitude, 55° 17ʹ longitude and Cultures were maintained at temperature of 25±1 143 m altitude). Voucher from this plant have been °C under a 16/8 h light regime provided by white deposited at the herbarium of the Tehran fluorescent tubes. After 30 days, shoots number per University. explant, fresh weight of shoot and black glands number on leave were recorded. This experiment Seed Germination had four replications with four explants in each In first experiment due to low germination rate of replication and was done in the base of completely H. perforatum seeds, the effect of pretreatments to randomized design. improve seed germination of this species was Callus Induction investigated. The seed pretreatments concluded: A) washing seeds with tap water for three days, B) For callus induction the young leaves of 8-10 mm soaking seeds in distilled water with some drops of long and stem segments with 1-3 node of obtained detergent at 4 °C in darkness for three days, and C) in vitro H. perforatum plantlets from previous soaking seeds in solution of hypochlorite sodium experiments were excised and cultured on media 60% for 20 minutes. The pretreated seeds were with different concentrations of 2,4- washed by water and set in petri-dishes (9 cm) with Dichlorophenoxyacetic acid (2,4-D) (0.25, 0.5 and -1 -1 top paper method (25 seeds put in each petri- 1 mg l ), BA (0.2, 0.5 and 1 mg l ), Kinetin (Kin) -1 -1 dishes). Then 4 ml distilled water was added in (0.2, 0.5 and 1 mg l ) and 1 mg l Naphthalene petri-dishes. Finally, the germination percentage Acetic Acid (NAA). All the cultures were then was calculated for determination of best maintained at 25±1 °C in the dark. The callus induction, days to callus induction and callus 83 Abdollahpoor et al. volume were recorded after 27 days. This Basal Medium and Culture Conditions experiment was done as a factorial in the base of All media contained MS mineral salts and vitamins completely randomized design with 4 replication [12], 3% (w v-1) sucrose and solidified with 0.8% and 4 explant in each replication. According to agar. The pH of the media was adjusted to 5.7 obtained results, leaves were the best explants for before autoclaving for 20 min at 121 °C. 2,4-D, callus formation; therefore, the next experiment NAA, BA, Kin and IBA were added to media was done using leaves derived calli as initial before autoclaving,. explants. Data Analysis Shoot Regeneration from Callus Results were analyzed statistically using the For shoot regeneration, the leaves derived calli Statistical Analysis Program (SPSS ver.16.0). The were transferred to supplemented medium with 0.5 mean values were calculated and compared by and 1 mg l-1 of BA and Kin as well as the Duncan’s multiple range tests (P < 0.05). combination of 0.5 mg l-1 of BA and Kin with 0.1 mg l-1 IBA. The media contained macro and micro Results and Discussion elements of MS and vitamins of B5 [13] medium. Cultures were maintained at temperature of 25±1 °C under a 16/8 h light regime provided by white Seed Germination fluorescent tubes. After 6 weeks the shoots number, The seeds of Hypericum perforatum due to mean growth of shoots and black glands on leaves inhibitor compounds that secrete from seed’s were recorded.