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Asafetida), an Endangered Medicinal Plant Trakia Journal of Sciences, Vol. 8, No. 1, pp 11-18, 2010 Copyright © 2009 Trakia University Available online at: http://www.uni-sz.bg ISSN 1313-7050 (print) ISSN 1313-3551 (online) Original Contribution CALLUS INDUCTION AND PLANT REGENERATION IN FERULA ASSA FOETIDA L. (ASAFETIDA), AN ENDANGERED MEDICINAL PLANT A. R. Zare 1*, M. Solouki2, M. Omidi3, N. Irvani2, N. Mahdi Nezad2, Sh. Rezazadeh 1 1Institute of Medicinal Plants, ACECR, Karaj, Iran 2Department of Agronomy and Plant Breeding, Faculty of Agriculture, Zabol University, Zabol, Iran. 3Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Tehran, Karaj, Iran. ABSTRACT Plant regeneration in Ferula assa-foetida L. (Asafetida), a medicinally important vulnerable species, was achieved through the mediation of callus derived from root, hypocotyl and cotyledon. Murashige and Skoog (MS) medium with 1 mg l-1 α-naphthaleneacetic acid (NAA) and 2 mg l-1 N6-benzyladenine (BA) was most effective (100%) for the proliferation of callus for root explants. MS medium containing 1-3 mg l-1 BA or KIN alone or in combination with 0.2 or 0.5 mg l-1 NAA facilitated indirect organogenesis. BA was superior to KIN in shoot morphogenesis. Hypocotyl- derived callus subcultured on medium with 1 mg l-1 BA and 0.2 mg l-1 NAA showed maximum percentage of cultures regenerating shoots (81.1%), with a mean of 7.4 shoots per callus. Root- derived callus was inferior, which yielded a mean of 2 shoots on medium with 2 mg l-1 BA. Rooting of in vitro raised shoots was best the on half-strength MS medium containing 2.5 mg l-1 indole-3-butyric acid (IBA) with a mean of 7.23 roots per shoot measuring a mean length of 5.34 cm. The plantlets acclimatized and transferred to soil exhibited 90% survival. Key words: Ferula assa foetida L., seedling explants, shoot regeneration, Murashige and Skoog medium, vulnerable species. INTRODUCTION Ferula assa-foetida (Apiaceae), exploiting the wild populations. Indiscriminate commonly known as Asafetida is an collection of this plant from the wild has posed herbaceous, perennial medicinal plant a serious threat to its existence in the wild indigenous to Iran (1) Asafetida is a very populations, especially when the plants are effective medicinal herb that acts mainly on harvested well before seed set. The plant is the digestive system, cleansing and conventionally propagated through seed but is strengthening the gastro-intestinal tract. The hampered by the seed dormancy. Further, the pungently flavoured gum-resin that is obtained methods of extraction employed are almost from the root by incisions is alterative, invariably crude and unsystematic (16). anthelmintic, antiperiodic, antispasmodic, Consequently, Asafetida is recorded as a carminative, deobstruent, deodorant, vulnerable species in the Red Data Book of expectorant, laxative, sedative and stomachic (9). Iran (8). On the other hand, improvement of The commercial and pharmacological Apiaceae plants following the classical need of Asafetida is achieved usually by breeding procedure is generally slow, laborious _____________________________ and time consuming (18). Hence, there is a * Correspondence to: Amir Reza Zare strong need for proactive understanding in the Gmail: [email protected], Tel: conservation, cultivation and sustainable usage 00989121097826, Fax: 00982614764021 of this species for future use. Submission date: April, 2009 Trakia Journal of Sciences, Vol. 8, No. 1, 2010 11 A.R. ZARE, et al. Recent years witnessed an increased (KIN) for callus induction. After 6 weeks of interest in tissue culture techniques which offer culture, callus formation was evaluated. All the a viable tool for mass multiplication and calluses obtained were subcultured onto germplasm conservation of rare, endangered medium containing the same or lower and threatened medicinal plants (6, 11, 17, 20). concentrations of same growth regulators after Further, indirect organogenesis offers the every 6 weeks for callus proliferation. induction of variability and thereby the improvement of the crops. In vitro propagation 3. Regeneration has been accomplished in many Apiacean species, The shoot regeneration was attempted by including Eringium transferring 6-week-old calluses on MS Centella asiatica (19, 13), -1 -1 foetidum (7, 12), Cuminum cyminum L (3). medium with 1-3 mg l BA or 1-3 mg l KIN and in combination with lower levels of NAA Despite being a valuable medicinal plant, -1 very little work has been done on tissue culture (0.2 and 0.5 mg l ). After 6 weeks of culture, of Ferula assa foetida. There has been only data were recorded for percent of shoot one report on somatic embryogenesis via formation and number of shoots. hypocotyl explants of this plant (5). Here we describe the first methods for plant 4. Root formation regeneration through the mediation of callus Healthy shoots (2 cm height) excised at the end derived from seedling explants of F. assa- of 6th week were transferred onto rooting medium consisting of half strength MS medium with 1-5 mg foetida, an endangered medicinal plant. l-1 NAA, IAA or IBA. The data were recorded on MATERIAL AND METHODS percent root formation, number and length of roots after 4 weeks. 1. Preparation of plant materials The mature seeds of F. Assa-foetida 5. Culture conditions collected in July 2007 from a biological All the media were supplemented with 3% reserve (Nasr Abad) in the nearby county (w/v) sucrose and medium was gelled with Yazd, Iran (1900 and 2050 m above sea level) 0.8% (w/v) agar. The pH of all media was were washed thoroughly under running tap adjusted to 5.8 before the addition of agar. The water for 3 days. The soaked seeds were media were sterilized under 1.5 kg cm-2 and treated with 70% ethyl alcohol for 2 min, then 121 ˚C for 20 min. All the cultures were grown at rinsed with sterile water for three times. The 23 ± 2 ˚C under 16 h photoperiod supplied by two seeds were then surface sterilized in 2.5% Philips TL 40W fluorescent tubes. sodium hypochlorite solution for 20 min and was followed by three washes in succession 6. Acclimatization with sterile water. The surface sterilized seeds Plantlets gently washed in distilled water were cultured on 1/4MS (Murashige and to remove agar from their roots were planted in Skoog, 1962) medium supplemented with 30 g plastic cups (upper dia 6 cm × height 8 cm) l−1 sucrose and 7 g l−1 agar. To overcome containing sand, fertile soil and vermiculite dormancy, the plates were kept in a refrigerator (1:1:1) and placed in a glasshouse with 90% (4 ˚C) for 30 days. After cold stratification pre- humidity. After 2 months, the plants were treatment, the cultures were incubated at 23 ± transferred to glasshouse and placed in shade 2 ˚C under 16 h photoperiod of 80 µ mol m-2 s- (approximately 60% shade) under natural 1 irradiance provided by cool fluorescent conditions. The plants transplanted to pots lamps. The cultures were used as a source of (upper dia 20 cm × height 30 cm) after 2 plant material for establishment of explants before months were transferred subsequently to the field. initiating the experiments. 7. Experimental design and data analysis 2. Callus induction The treatments were arranged in factorial Aseptically excised root, hypocotyl and experimental based on Completely cotyledon explants (root and hypocotyl of 10 Randomized Design (CRD) with three mm, cotyledon of 5 mm2) from 20-days-old replicates per treatment and ten explants per seedlings were cultured on sterile MS medium replicate. Data given in percentages were fortified with 0-4 mg l-1 α-naphthaleneacetic subjected to arcsine transformation before acid (NAA) or 0-4 mg l-1 2,4- statistical analysis. Data were analyzed dichlorophenoxyacetic acid (2,4-D) alone and statistically using MSTATC software. The mean or in combination with 0-2 mg l-1 values of different treatments were compared using benzyladenine (BA) or 0-2 mg l-1 kinetin Duncan's multiple range tests (at 5% level). 12 Trakia Journal of Sciences, Vol. 8, No. 1, 2010 A.R. ZARE, et al. Table.1 Callus induction from different explants of F. assa-foetida on MS medium with different growth regulators. Growth Regulators (mg l-1) Callus induction (%) 2,4-D NAA BAP KIN root hypocotyl cotyledon MS basal media 15wx 0y 0y 1 37.1f-u 28.6m-x 24q-x 1 1 50d-n 50.4d-m 39.6f-t 1 2 60.5c-g 57.5d-h 43.8e-q 2 45.2e-q 26.3o-x 18.6u-x 2 1 63.3b-f 29.2l-x 25.4p-x 2 2 59.6c-g 30.4k-w 19.2u-x 4 27.1o-x 0y 0y 4 1 28.3n-x 0y 0y 4 2 23.8t-x 16v-x 0y 1 1 46e-s 42.3e-s 34.6k-w 1 2 46.8d-p 46e-q 33.4h-v 2 1 53.3d-k 42.3e-s 0y 2 2 60c-g 37.4f-u 31.7j-w 4 1 22.4s-x 19.1u-x 0y 4 2 23.6q-x 0y 0y 1 63.6b-f 47.5d-o 36.9f-u 1 1 90.2a-c 62.4b-f 56d-j 1 2 100a 64b-f 52.5d-l 2 69.6b-e 57.7d-h 33.1h-w 2 1 92.8ab 63.6b-f 43.1e-r 2 2 89.5a-c 74.9a-d 62.4b-f 4 57.5d-i 27.1o-x 24.3q-x 4 1 57.7d-i 24.9q-x 0y 4 2 64.6c-g 34.8h-v 26.5o-x 1 1 50.8d-o 42.4f-s 33.8h-v 1 2 54d-k 46e-q 40f-s 2 1 40f-s 30k-w 34h-v 2 2 43.1e-r 28.2m-x 20.9t-x 4 1 33.1i-w 22.9r-x 12.9x 4 2 38g-u 29.8l-x 18u-x Means followed by the same letters within columns are not significantly different at the 5% level.
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