Micropropagation of Bunium Persicum (Boiss.) B. Fedtsch. via Indirect Somatic Embryogenesis

Transcription

1 Micropropagation of Bunium Persicum (Boiss.) B. Fedtsch. via Indirect Somatic Embryogenesis Mahmoud Otroshy *1, Vajihe Pakravan 2, Shokoufeh Enteshary 2, Arash Mokhtari 1, Shirin Roozbeh 1 1 Department of Tissue Culture, Branch of Central Region of Iran, Agricultural Biotechnology Research Institute of Iran (ABRII), Isfahan, Iran 2 Department of Plant Physiology, University of PayameNur, Najaf Abad, Isfahan, Iran *otroshy@yahoo.com Abstract- Bunium persicum (Boiss.) B. Fedtsch., a member of the Apiaceae family, is one of the oldest and most economically important species. Because of seed dormancy and lack of hypocotyl growth when cotyledon emerged, In vivo cultivation of Bunium persicum has not succeeded. In order to investigate the effects of some plant growth regulators on somatic embryogenesis of Bunium persicum in tissue cultured condition, we have used indirect embryogenesis technique. Explant harvested from root, hypocotyl and cotyledon (leaf) were investigated on MS media combined with 2, 4-D, BAP, NAA (each of those with 6 concentration levels: 0, 0.5, 1, 2, 3 and 4 mg/l) and furthermore Kin (using 3 concentration levels: 0, 0.2 and 0.5 mg/l). Calli developed into different media and after 3 or 4 weeks some of them regenerated embryos. The best indirect somatic embryogenesis was obtained in a medium treated with root explants and the highest indirect embryogenesis was resulted from media treated with 2 mg/l 2, 4-D, 0.5 mg/l BAP or 1 mg/l BAP Kin. Also the highest percentage of callogenesis was observed on MS media containing 0.5 mg/l NAA+0.5 mg/l Kin, 4mg/l NAA or 2 mg/l 2, 4-D mg/l Kin. Keyword- Bunium persicum (Boiss.) B. Fedtsch.; Somatic Embryogenesis; Callus; Hypocotyls I. INTRODUCTION Black Zira (Bunium persicum (Boiss.) B. Fedtsch.) is an important aromatic plant which belongs to the Apiaceae family. This plant originates from central Asia to north India. According to the literature Zira s fruits contain nearly 9% essential oil [1]. The plant shows several remedial effects on digestive and urinary tract disorders and is well-known as an anti-convulsion, anthelmintic, anti-asthma and anti-dyspnea in Iranian folk medicine. B. persicum oil is capable of suppressing the initial stage of an inflammatory process [2]. Improvement of Apiaceae plants following the classical breeding procedure is generally slow, laborious and timeconsuming [3]. Production of this plant is limited due to seed dormancy and several biotic stresses of which wilt disease is the most serious. Only cold treatments are effective in seed germination. Other treatments such as gibberllic acid, cytokinin, potassium nitrate, washing and light treatments are not useful [4]. The propagation of this species in its natural habitats occurs rarely evidenced by close field observations. Micropropagation through somatic embryogenesis can be extremely applied to shorten the long sexual cycle and other problems like limited seed availability [5]. Embryogenesis may provide alternative plant micro propagation [6]. The formation of somatic embryos in callus cultures, which was first reported in the Apiaceae member, carrot (Daucus carota L.), is a typical process of embryogenesis on the same medium or after transferring it to a second medium lacking plant growth regulator (PGR) supplements [7]. Subsequently, the somatic embryos germinate regenerating plants. This phenomenon has been observed to be similar with other Apiaceae members such as fennel (Foeniculum vulgare Mill.) [8] and celery (Apium grageolens L.) [9]. Callus formation from mericarps of Bunium persicum on MS medium supplemented with 2 mg/l 2, 4-D and 4 mg/l Kin obtained by [10]. In this report, small white clump of compactly packed cells developed on the callus on a medium containing 1 mg/l 2, 4-D. These cell clumps differentiated into numerous globular embryos on the same medium. Callus induction in hypocotyl and cotyledon explants of Bunium persicum, on B5 medium containing 2 mg/l NAA and 2mg/l Kin and somatic embryogenesis were higher on MS supplemented with 0.5 mg/l 2,4-D [11]. In addition to the results obtained it has been shown that the maximum somatic embryogenesis occurred in MS medium combined with NAA and Kin [12]. The main objective of this study is to achieve a suitable method for in vitro regeneration of Bunium persicum via indirect embryogenensis technique. II. MATERIAL AND METHODS The experiment was carried out in the Tissue Culture laboratory of Agricultural Biotechnology Research Institute of Central region of Iran (ABRICI) in The first seed of black Zira was obtained from the medicinal Plant Collection of Agriculture research center. A. Seed Sterilization, Breaking Dormancy and Plantlet Formation For seed sterilization, the seeds were pre-washed 10 times with tap water and then with sterile distilled water. They were soaked in 70% (v/v) ethanol for 1.5 minutes. After the seeds were kept in 100 ml of sodium hypochlorite with one drop tween 20 and finally rinsed 3 times with sterile distilled water in a laminar flow cabinet, and were cultured in petri dishes containing

2 breaking dormancy medium (0.25 mg/l BAP and 7 g/l agar). The cultures were incubated at 5 C and full darkness for a period of 1 month. Then, the germinated seeds were transferred to the petri dishes containing MS medium for plantlet formation. After 2 weeks incubation at 25 C, and the light intensity of 42 (µmoles/m 2 /s) and a photo phase of 16 hours light per day, plantlets were produced. B. Preparing Explants and Treatments This experiment was conducted by three types of explants: root, hypocotyls and cotyledons (Leaf) and various combinations of plant growth regulators: 2, 4-D, BAP, NAA (each of those with 6 concentration level: 0, 0.5, 1, 2, 3 and 4 mg/l) and furthermore Kin (using 3 concentration level: 0, 0.2 and 0.5 mg/l). First media were prepared and the ph was adjusted to 5.8. Immediately after autoclave process required dose of growth regulators were added to the autoclaved nutrient media before it was allowed to solidify. To take explants, the plantlets were separated into 3 parts: root, hypocotyl and cotyledon (leaf). Each of them was separately cut into 0.5 to 1 cm pieces. The explants were cultured in sterilized petri dishes containing 30 ml of the mentioned media combined with the required dose of plant growth regulators and without growth regulators (as control). The petri dishes were closed with a cap and sealed with household plastic foil and were randomly placed in a growth chamber set at 25 C and 16 hours per day photoperiod; with the light intensity of 21 (µmoles/m 2 /s) for a period of 1 month. Then the explants were transferred to the Petri dishes containing MS media without any growth regulators for embryogenesis and development. C. Data Collection Petri dishes were incubated in the condition as above mentioned in a period of 1 month. After that period, the percentage of callogenesis was recorded and then transferred to next media. At this stage following parameters were measured and recorded after 2 weeks: The percentage of embryogenic callus, number of producing embryos in different stages (Globular, torpedo and cotyledonary) and the percentage of conversion of embryos to seedlings. D. Experimental Design and Statistical Analysis Each experiment was conducted as a completely randomized factorial design with two factors and three replications. Data were analyzed using the SAS version 9.1 statistical computer program. When the ANOVA indicated significant treatment effects (5 or 1%) based on the F-test, the Duncan s multiple range test (P<0.5) was used as a Post Hoc method to determine which treatments were significantly different from other treatments. A. Percentage of Callogenesis III. RESULT Plant growth regulators had highly significant effects on the percentage of callogenesis (Data were not shown). The highest percentages of callogenesis were observed on MS medium containing 0.5 mg/l NAA+0.5 mg/l Kin, 4mg/l NAA without Kin or 2 mg/l NAA+0. 5 mg/l Kin (Table 1). The kind of explant had a highly significant effect on callogenesis. The explants of roots and hypocotyl showed maximum and minimum of callogenesis respectively (Fig. 1, Fig. 2a and 2b). In all stages of the experiment explant of root was the best; thus, results of hypocotyl and leaf were ignored. Figure. 1 The effect of explants on indirect somatic embryogenesis, the number of total, globular, torpedo, cotyledonary and conversion embryos, percentage of callogenesis and embryogenic callus

3 Concentr ation Journal of Agricultural Engineering and Biotechnology Nov 2013, Vol. 1 Iss. 3, PP a b c d B. Percentage of Embryogenic Callus Figure. 2 Callogenesis in (a) root explants, (b) hypocotyls, (c) embryogenic callus, and (d) converted embryo Highly significant effects of plant growth regulators on the percentage of embryogenic callus were found. 2 mg/l 2,4-D, 0.5 mg/l BAP without Kin and 1 mg/l BAP+0.2 mg/l Kin resulted in maximum percentage of embryogenic callus (Table 1, Fig. 2c). The kind of explants had highly significant effect on the percentage of embryogenic callus production. For the effect of explants, root showed the best results. Nevertheless, there was no significant difference between explants of hypocotyls and leaves (Fig. 1). C. Number of Total Embryos and Embryos at Different Embryogenic Stage Growth regulator had a highly significant effect on the total number of embryos and the number of globular and cotyledonary embryos. But, no significant effect on the number of torpedo embryos was observed. The maximum number of globular and cotyledonary embryos were obtained from 2 mg/l 2, 4-D without Kin (Tables 1-2). TABLE 1 THE EFFECT OF DIFFERENT CONCENTRATION OF SOME PLANT GROWTH REGULATORS ON THE PERCENTAGE OF CALLOGENESIS, THE NUMBER OF EMBRYONIC CALLUS AND TOTAL EMBRYOS IN ROOT EXPLANTS Growth regulators 2,4-D BAP NAA Callus Embryogenic Callus Total Embryos Kinetin Kinetin Kinetin MS=0 z 17.2r-u 11.67t-y MS=0. 0p 2.08j-m 4.17h-k MS=0.0h 0.11g 0.86g c-f 53.33c-g 57.78b-f 12.06b-e 3.06i-l 0.39k-o 8.50ab 4.56cd 0.89g b-i 42.78i-n 39.72j-p 10.99c-f 3.18i-l 5.65g-i 5.39c 1.81fg 1.81fg b-h 61.11b-e 64.17ab 18.94ab 2.50j-m 7.68f-h 9.29a 1.78fg 4.01cd n-s 32.28l-q 33.61l-q 10.00c-f 4.74h-k 4.44h-k 3.44c-e 0.39g 1.11fg p-r 31.39n-r 49.44e-j 5.79g-j 7.00f-h 4.31h-k 4.39cd 1.07fg 2.72ef 0 MS 17.50r-u 14.72s-v MS 0.00p 4.17h-k MS 0.00h 0.86g t-y 12.78t-x 22.22p-r 19.49a 7.13f-h 9.72c-f 3.36c-e 1.28fg 1.27fg p-r 20.00q-s 14.44s-v 9.88c-f 18.84ab 12.94b-e 1.67fg 3.11ce 2.00ef l-q 20.00q-s 18.02r-t 8.79e-g 9.41c-f 13.50b-d 0.58g 0.56g 1.67fg s-v 14.72s-v 13.06s-w 2.36j-m 1.39j-n 8.79e-g 2.58ef 0.33g 2.83ef t-y 10.83u-z 10.56u-z 5.56g-j 12.04b-e 1.39j-n 0.89g 0.53g 0.36g 0 MS 15.83s-v 13.89s-w MS 0.56k-o 2.50j-m MS 0.39g 1.00fg d-i 56.39b-g 65.83a 3.31i-l 4.98h-k 1.03j-n 4.06cd 0.29g 3.78c-e j-o 62.22b-d 62.78b-d 4.22h-k 2.13j-m 0.56k-o 5.16c 0.78g 0.14g d-h 48.33f-k 46.94g-l 6.98f-i 3.40i-l 0.00p 4.00cd 3.11c-e 0.00h j-o 41.94j-o 63.91bc 16.08b 8.92e-g 1.88j-n 4.14cd 2.00ef 0.33g ab 45.28h-m 45.00h-m 3.98i-l 1.28j-n 3.33i-l 1.31fg 1.56fg 4.00cd ** Significant at p 0.01, * Significant at p Values followed by a similar letter are not statistically significantly different

4 Concentration Journal of Agricultural Engineering and Biotechnology Nov 2013, Vol. 1 Iss. 3, PP TABLE 2 THE EFFECT OF DIFFERENT CONCENTRATION OF SOME PLANT GROWTH REGULATORS ON THE NUMBER OF TOTAL, GLOBULAR, COTYLEDONARY AND CONVERTED EMBRYOS, PERCENTAGE OF CALLOGENESIS AND EMBRYONIC CALLUS Globular Embryos Cotyledonary Embryos Conversion Embryos Growth Regulators Kinetin Kinetin Kinetin ,4-D BAP NAA 0 MS=0 d 0.00d 0.00d MS=0 f 0.11e 0.33e MS=0 f 000f 0.53cd b 0.06bc 0.00d 356bc 0.22e 0.83e 0.14de 0.17de 0.06ef d 0.00d 0.25b 340bc 1.64de 1.47de 1.64a 0.17de 0.08ef a 0.00d 0.33b 646a 1.78de 2.39cd 0.61bc 0.00f 0.03ef b 0.00d 0.33b 217cd 0.28e 0.75e 0.44cd 0.11de 0.03ef bc 0.07bc 0.14b 428b 0.72e 1.89de 0.06ef 0.03ef 0.31cd 0 MS 0.00d 0.00d MS 0.00f 0.33e MS 0.00f 0.53cd bc 0.00d 0.03bc 206cd 1.44de 1.44de 0.97b 0.14de 0.25de d 0.25b 0.00d 144de 1.53de 1.92de 0.14de 1.25ab 0.08ef d 0.00d 0.00d 044e 0.31e 1.06de 0.14de 0.25de 0.63bc d 0.22b 0.00d 247cd 0.06e 2.64cd 0.11de 0.00f 0.19de d 0.00d 0.00d 061e 0.33e 0.08ef 0.28cd 0.19de 0.00f 0 MS 0.00d 0.00d MS 0.11e 0.36e MS 0.00f 0.64bc bc 0.25bc 0.00d 344bc 0.53e 3.44bc 0.33cd 0.14de 0.33cd d 0.00d 0.00d 469b 0.75e 0.14e 0.81b 0.03ef 0.00f b 0.00d 0.00d 322bc 2.97cd 0.00f 0.03ef 0.17de 0.00f bc 0.22b 0.00d 3.58bc 1.47de 0.28e 0.44cd 0.56cd 0.06ef bc 0.03bc 0.03bc 072e 0.78e 3.78bc 0.06ef 0.69bc 0.19de ** Significant at p 0.01, * Significant at p Values followed by a similar letter are not statistically significantly different. D. Number of Converted Embryos A highly significant effect of growth regulator on the number of converted embryos was found. The treatments of 1 mg/l 2, 4-D without Kin and 1 mg/l BAP mg/l Kin caused the highest rate of embryo conversion into seedling (Table 2, Fig. 2d). The kind of explants had a highly significant effect on embryo conservation. The best result was obtained from root explants. Nevertheless, there was no significant difference between explants of hypocotyls and leaves. IV. DISCUSSION Among 54 various combinations of plant growth regulators exerted on black Zira, some treatments could induce callogenesis and somatic embryogenesis in this plant. The result of this experiment showed that in treatments containing the highest concentration of NAA (4 mg/l) and 0.5 NAA mg/l mg/l Kin leads to increase of callogenesis. The same result is also reported on Bunium persicum (Boiss.) [12] and Eryngium foetidum [13]. An increase in the NAA amount leads to enormous callus production [14]. In C. asiatica, using higher concentrations of NAA resulted in increasing the amount of callus production [15]. The highest percentage of callus production was observed in MS medium containing 0.1 mg/l NAA callus production increased. With respect to the obtained results, treatment 2 mg/l 2, 4-D+0. 5 Kin produced a high callogenesis value. The highest callus frequency observed in the medium contained 0.1 mg/l 2, 4-D or 1 mg/l 2, 4-D as well as 2 mg/l 2, 4-D and 0.5 mg/l Kin [16]. According to performed studies on callus production equal concentrations of auxin and cytokinin were used [17]. Also reported that 2,4-D is one of the best and most effective auxins for induction of callogenesis [18]. The result of this experiment showed that treatment of 2 mg/l 2, 4-D, 0.5 mg/l BAP and 1 mg/l BAP+0.2 mg/l Kin caused the most somatic embryogenesis. Previous study also reported that 2, 4-D has induced somatic embryogenesis [19]. The similar result also reported by [20]. In Tylohora indica species, the embrogenic callus was obtained from the medium containing 2, 4-D and embryogenesis was induced by Kin [21]. The explant of Foeniculum vulgare cultured in MS medium containing 2, 4-D succeeded to produce callus; and the transition of these calluses to medium without hormone caused somatic embryogenesis [22]. Somatic embryogenesis was obtained from hypocotyl segments on MS medium Supplemented with BAP [23]. Somatic embryogenesis was higher in the MS medium supplemented with 0.5 mg/l 2, 4-D [11]. The best treatment for somatic embryogenesis was obtained from the MS medium containing 2 mg/l 2, 4-D [12]. Explants of roots showed highest indirect somatic embryogenesis and also had the maximum of callogenesis. Indirect somatic embryogenesis in explant of root in Peucedanum palustre reported [24]. Also the maximum callogenesis in root explants reported by [25]. The previous study suggested that callus transferred to media supplemented with 1 mg/l 2, 4-D [10] or 0.5 mg/l 2, 4-D [4] leads to embryogenesis. However, a suitable combination of auxins and cytokinins is important for embryogenesis [26]. In

5 other species, induced somatic embryos might need a little cytokinin or other plant growth regulators for growth [27]. In this research, embryogenesis was not observed until the calli were in induction medium, and embryogenesis occurred in induced explants after transferred to hormone free media. These results confirmed by [28] as well as [29] reported that in many plants such as carrot, somatic embryogenesis has taken place in MS hormone-free medium after induction in a medium containing 2, 4-D. Removal of auxin from the culture medium is recommended for the successful completion of embryonic development [30]. REFERENCES [1] Azizi, M. (2005). Bunium persicum Essential Oils Suppressed Potato Sprouting in Storage. Proceedings of the 36th International Symposium on Essential Oils, 4 7 September, Budapest, Hungary. [2] Salehi P, Mohammadi F, Asghari B. (2008). Seed essential oil analysis of Bunium persicum by hydro distillation headspace solvent micro extraction. Chem. Nat. Compounds., 44: [3] Hunault, G., Desmarest, P., Manoir, J.D. (1989).Foeniculumvulgare Miller: cell culture, regeneration and the production of anethole In: Bajaj YPS (Ed) Biotechnology in Agriculture and Forestry 7: Medicinal and Aromatic plants II. Springer-Verlag, Berlin, Germany, pp [4] Bonianpoor, A. (1995). Investigation of Sexual Propagation of Bunium persicum and Cuminum cyminumand Cumin Callus Induction. M. Sc. Thesis, College of Agriculture, Shiraz University, Iran. [5] Chandrasekhar T., Mohammad hussain, T., Rama Gopal G., SrinivasaRao, J.V. (2006). Somatic embryogenesis of Tylophora indica (Burm.F.) Merril., an important medicinal plant. Int. J. Appl. Sci. Eng. 4: [6] Ammirato, P.V., Sommers, D.A., Hackett, W.R. and Biesboer, D. (eds). (1987). Organizational events during somatic embryogenesis. In: Green Plant Tissue and Cell Culture, [7] Hunault, G. (1984) In vitro culture of Fennel tissue(foeniculum vulgare Miller) from cell suspension to mature plant. ScientiaHorticulturae, 22: [8] Steward F. C.; Mapes M. O.; Mears K. (1958). Growth and organized development of cultured cells. II. Organization in culture grown freely suspended cells. Am. J. Bot. 45: [9] Saranga Y., J. Janick. (1991). Celery somatic embryo production and regeneration: improved protocols. Hort Science 26:1135. [10] Wakhlu, A. K., Nagari, S. and Barna, K.S. (1990). Somatic embryogensis and plant regeneration from callus cultures of Bunium persicum Boiss. Plant Cell Rep., 9: [11] Sharifi, M. (1995). Comparitive investigation of essences of Bunium persicum and Cuminum cyminum seed and explant fragments. M.Sc. thesis, Agricultural College, Tehran University. [12] Valizadeh, M., A. Safarnejad, G.A. Nematzadeh, and S.K. Kazemitabar. (2006). Regeneration of plantlets from embryo explants of Bunium persicum (Boiss.) B. Fedtsch. Indian J. Crop Science, 1(1-2): [13] Martin, K. P. (2004) Efficacy of different growth regulators at different stages of somatic embryogenesis in Eeryngium foetidum L. A rare medicinal plant. In Vitro Cell. Dev. Biol. Plant, 40: , [14] Tiwari, K.N., N.Ch. Sharma, V. Tiwari, and B.D. Singh. (2000). Micropropagation of (Centella asiatica L.), a valuable medicinal herb. Plant Cell, Tissue and Organ Culture, 63: [15] Banerjee, S., M. Zehra, S. Kumar. (1999).In vitro multiplication of Centella asiatica, a medicinal herb from leaf explants. Curr. Sci. 76: [16] Valizadeh M. andkazemitabar S. K. (2009) Investigation of Plant Growth Regulators Effects on Callus Induction and Shoot Regeneration of Bunium persicum (Boiss.) B. Fedtsch. J. Agr. Sci. Tech. 11: [17] Slater, A., N.W. Scott. (2003). Fowler, M.R., Plant biotechnology: The genetic manipulation of plants, Oxford University Press [18] McKersie, B.D., D.C.W. Brown. (1996). Somatic embryogenesis and artificial seeds in forage legumes, Seed Sci. Res., Vol. 6, pp [19] Mizukami, M., T. Takeda, H. Satonaka and H. Matsuoka. (2008). Improvement of propagation frequency with two-step direct somatic embryogenesis from carrot hypocotyls. Biochemical Engineering Journal, 38: [20] Dudits, D., B. L., gre and G. J. rgyey. (1991). Molecular and cellular approaches to the analysis of plant embryo development from somatic cells in vitro. J Cell Sci. 99: [21] Tawfik, A.A., Noga, G. (2002). Cumin regeneration from seedling derived embryogenic callus in response to amended kinetin. Plant Cell Tiss. Org. Cult. 69: [22] Anzidei, M., A. Bennici, S. Schiff, C. Tani, B. Mori. (2000). Organogenesis and somatic embryogenesis in Foeniculum vulgare: histological observations of developing embryogenic callus. Plant Cell, Tissue and Organ Culture, 61(1): [23] Hussein, M. A. and Batra A. (1998). In vitro embryogenesis of cumin hypocotyl segments. Adv. Plant Sci. 11: [24] Vuorela, P., K. M. Oksman-Caldentey, J. Lipponen, and R. Hiltunen. (2004). Spontaneous somatic embryogenesis and plant regeneration from root cultures of Peucedanum palustre. Plant Cell Reports, 12: [25] Petru, E. (1970). Development of embryoids in carrot root callus culture (Daucus carota L.). BiologiaPlantarum, 12 (1):1-5 [26] Guohua, M. (1998). Effects of cytokinins and auxins on cassava shoot organogenesis and somatic embryogenesis from somatic embryo explants. Plant Cell Tiss. Org. Cult. 54:1-7 [27] Kumar, S. A., Gamborg, O. L. and Nabors, M. W. (1988). Plant Regeneration from Long-term Cell Suspension Cultures of Tepary Bean (Phaseolus acutifolius). Plant Cell Rep., 7:

6 [28] Hassani, B., Saboora, A., Radjabian, T. and Fallah Husseini, H. (2008). Somatic Embryogenesis of Ferula assa-foetida. JUST, 33(4), p [29] Cho, D. Y., E. K. Lee, S. Lee, W. I. Chung and W. Y. Soh. (2003). Enhanced somatic embryogenesis and plant regeneration in leaf explants cultures of Ostericum koreanum on medium of varying ph. Plant Cell, Tissue and Organ Culture, 75: [30] Zimmerman, J. L. (1993). Somatic embryogenesis: A model for early development in higher plants. Plant Cell