In Vitro Achievement of Ferutinin from Root of Ferula ovina with Emphasis on Germplasm Resources Conservation

There is a growing body of the literature that recognizes the importance of ferutinin (C22H30O4) as one of the natural phytoestrogens with potency to treat osteoporosis and some kind of cancers. One of the greatest challenges is availability of ferutinin that is found just in root of some plants of the genus Ferula (Apiaceae), which is the reason of high price of it in global markets. Ferula ovina , an endemic plant of Iran, is known as one of the greatest sources of ferutinin. Unfortunately, access to ferutinin requires uprooting Ferula ovina especially older plants with more secondary metabolites content. There is a large volume of published studies describing the importance of tissue culture in propagation of endangered plants including secondary metabolites without possibility of chemical production and with deep dormancy seed exactly like the characters of F. ovina. Up to now, far too little attention has been paid to importance of tissue culture in accessing ferutinin without degrading the germplasm resources of it. The main aim of this study was to find an approach to access ferutinin associated F. ovina germplasm conservation. In this experiment, the aerial parts of F. ovina were collected from Zoshk area (Mashhad, Iran). The plants were recognized as F. ovina by the Institute of Plant Sciences (Ferdowsi University of Mashhad). Tissue culture part was performed with preparation of sterilized media and explants. Therefore, the MS salts and vitamins was applied as basic medium, however MS salt was decreased to half strength for rooting of shoots. The node (root junction) explants were cultured on 24 callus induction/shooting media with different combinations of plant growth regulators (BAP, IAA, Kin, NAA and IBA). The shoots from direct and indirect regeneration were then transferred to media with different combinations of PGRs (BAP, IBA, IAA and NAA) in order to find rooting medium. Following these treatments, unbalanced ANOVA for analysis of data were performed using IBM SPSS 19. The final stage of the study comprised a TLC test for the purpose of finding ferutinin in samples which resulted from tissue culture. For this purpose, air-dried parts of samples were powdered and extraction was done after being in 3-5 times dichloromethane for 24hours. Then after optimizing solvent system was done with selection the ratio that presented bands in middle of TLC paper. The results indicated that from 24 tested media, just 8 of them had potency of callus formation, but just L6 on MS medium containing 1 mg/l BAP and 1.5 mg/l IAA showed significant difference for percentage of callus induction at 5% level with compact green and the heaviest calluses. Although direct and indirect shoot regeneration was observed in this study, L9 on MS medium along with 1.5 mg/l IAA and 3 mg/l kin demonstrated significant difference for percentage of shooting at 5% level. Moreover, R6 on ½MS with 1 mg/l IBA showed significant difference for percentage of rooting of shoots at 5% level. The most surprising observation to emerge from the data comparison was L24 on half strength MS medium containing 0.2 mg/l BAP and 3 mg/l IBA with potency of callus induction, shooting and rooting of shoots. Regarding to the results of TLC test, ferutinin positive bands were observed in some samples. The aim of the present research was to examine possibility of achieving ferutinin without need to uproot F. ovina because there are several problems to achieve this valuable sesquiterpene: 1) Chemical production of ferutinin is impossible, 2) It could be accessible just from roots of genus Ferula, 3) Propagation with seeds of F. ovina is limited because of morphophysiological dormancy of them, 4) Natural habitat of this plant in Iran is going to be destructed, 5) Access to natural habitat is difficult, 6) Time of access is limited with short growth season, 7) Maintaining F. ovina in greenhouse condition is impossible. The results of this research support the idea that producing ferutinin in Iran without any harmful effect on germplasm resources of F. ovina is possible. This is the first study of high scale commercial production of ferutinin which examine associations between tissue culture and ferutinin production.