IN VITRO CALLUS INDUCTION AND PLANTLET REGENERATION IN FIG (FICUS CARICA L.)

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1 Journal of Cell and Tissue Research Vol. 12(3) (2012) (Available online at ISSN: ; E-ISSN: Original Article IN VITRO CALLUS INDUCTION AND PLANTLET REGENERATION IN FIG (FICUS CARICA L.)? DHAGE, S. S., PAWAR, B. D., CHIMOTE, V. P., JADHAV, A. S. AND KALE, A. A. State Level Biotechnology Centre, Mahatma Phule Krishi Vidyapeeth, Rahuri , Maharashtra. E. mail: Received: October 7, 2012; Revised: November 25, 2012 Accepted: November 30, 2012 Abstract: In vitro plant regeneration was tried in four fig cultivars namely Brown Turkey, Conadria, Deanna and Poona Fig using leaf explants obtained from in vitro established shoots Best callusing was observed on MS medium supplemented with 2.0 mg/l thidiazuron (TDZ) and 4.0 mg/l 2-iso-pentenyl adenine (2 ip). Among genotypes, Brown Turkey showed maximum response to callusing (85.8 %) and showed shooting on same medium. Earliest callus induction was observed on MS medium supplemented with 2.0 mg/l TDZ and 2.0 mg/l IBA (Indole-3- butyric acid). Shooting was induced from callus on transfer to MS medium supplemented with 0.5 mg/l α-naphthalene acetic acid (NAA) and 7.0 mg/l TDZ. Shoots developed roots following transfer to half strength MS medium supplemented with 1.0 mg/l IBA and 2 g/l activated charcoal. Key words: In vitro, callus culture, Ficus carica INTRODUCTION Fig (Ficus carica L.), is a monoecious, deciduous tree bearing infruitescences. Fig fruit is consumed both as a fresh or processed. Fig fruit have attractive taste, high nutritive and pharmacological value due to its antioxidant properties [1,2]. It is indigenous to wide areas ranging from Asiatic Turkey to Northern India, and its local varieties are cultivated in most Mediterranean countries [1]. Genus Ficus has more than 800 species and are vegetatively propagated. The propagation by conventional method of cuttings grafting and layering is slow and limited. As those materials can be obtained only from upright branches, which results in poor rooting and only 20 30% of the cuttings survive [3]. Hence, in vitro micropropagation of Ficus species has been widely studied as an alternate method for mass-scale production of high quality planting material [4]. Only a limited number of reproducible regeneration methods have been published, and again most of the successful results were obtained from using shoots tip and apical buds [3,5]. Yakushiji et al. [6] reported induction of organogenesis from leaf explants of F. carica, however frequency of adventitious bud differentiation from leaf fragments was relatively low and no adventitious buds were observed without the addition of phloroglucinol. The effective use of tissue culture techniques, such as in vitro selection, exploitation of somaclonal variation, and genetic transformation, depends upon the ability to initiate and establish stable callus cultures capable of plant regeneration [7]. Once genotypes with a desirable culture response have been identified, transfer of such traits to elite germplasm can be attempted. The present study was, therefore, undertaken to generate information on four fig cultivars for organogenic potential induced by using leaf explants on variable cultural conditions and to develop an efficient in vitro regeneration protocol. 3395

2 J. Cell Tissue Research MATERIALS AND METHODS Axillary shoot tips of four fig cultivars, viz. Brown Turkey, Conadria, Deanna and Poona Fig were collected in morning hours from healthy mature plants from experimental orchard of the All India Coordinated Research Project Arid Zone Fruits, Mahatma Phule Krishi Vidyapeeth, Rahuri. Shoot tips were sterilized with 0.1 %, 0.2 % and 0.3 % (w/v) mercuric chloride (HgCl 2 ) or 4% (w/v) sodium hypochlorite (NaClO) solution for 3, 5, 7 and 9 min. They were further rinsed five times with sterile distilled water. Sterilized shoot tips were inoculated on Murashige and Skoog (MS) basal medium [8] supplemented with 2.5 mg/l 6- benzyl-amino-purine (BAP), 0.5 mg/l gibberellic acid (GA 3 ), 100 mg/l ascorbic acid and 150 mg/l citric acid. They were initially kept in dark for a week and then incubated at 25 C temperature with 16 h light period and 8 h dark period. Leaves from such 2-3 week old in vitro established shoots were used for callusing. Leaves from in vitro cultured shoots were inoculated on MS medium supplemented with different combinations of TDZ either with 2iP or IBA along with 100 mg/l ascorbic acid and 150 mg/l citric acid. They were initially kept in dark for a week and then incubated at 25 C temperature with 16 h light period and 8 h dark period. After forty days of culture the leaf calli were transferred to shooting medium i.e. MS supplemented with 7 mg/l TDZ, 0.25 mg/l NAA, 100 mg/l ascorbic acid, 150 mg/l citric acid. All treatments of regeneration experiments had three replicates and with 40 explants in each replication. Shoots derived from the regeneration stage were transferred to MS medium with 1.0 mg/l IBA, 2g/l activated charcoal. Plantlets thus produced were transferred to pots containing coco peat and manure (2:1), and irrigated with water at regular intervals. They were initially covered with plastic bags for a week, and kept in polycarbonated polyhouse. RESULTS AND DISCUSSION Contamination of axillary shoot tip explants was major problem, during establishment of in vitro culture in Table 1: Effects of disinfectant on shoot tip sterilization in Poona fig. Disinfectant Duration (min.) Sterilization (%) HgCl 2 (0.1 %, w/v) HgCl 2 (0.2 %, w/v) HgCl 2 (0.3 %, w/v) 7 Complete explant browning 9 Complete explant browning NaClO (4%) Complete explant browning Table 2: Effects of disinfectant on shoot tip sterilization Genotype HgCl 2 (0.2 %, w/v) for 7 min Sterilization (%) HgCl 2 (0.2 %, w/v) for 9 min Brown Turkey Conadria Deanna Poona Fig fig. Initially shoot tip of cultivar Poona Fig was used for standardization of sterilization treatment. In preliminary experiments darkening or browning of the culture medium hindered establishment of shoot tips. Therefore, antioxidants such as ascorbic acid and citric acid were used in all medium. In all the genotypes tested 0.2 % HgCl 2 for 7 min was found to be optimum for shoot tip sterilization (Table 1, 2). Though 0.2 % HgCl 2 for 9 min gave highest sterilization but explant establishment was low. Decrease in concentration of disinfectant and duration of treatment resulted in high percentage of contamination and while increase in concentration leads to browning of shoot tip. Previously leaf explants obtained from in vitro cultured shoots have been used for regeneration in fig [2,6,9]. Accordingly in vitro shoot tip culture derived leaves were used as explants for in vitro regeneration via callus. Combination of TDZ with either 2iP or IBA, supplemented with antioxidants (100 mg/l ascorbic acid and 150 mg/l citric acid) was used for this purpose. Figs. A-C: A(i): Shoot tips 1 day after inoculation; B(i),B(ii): 10 days after inoculation: C(i),C(ii): 30 days after inoculation on MS medium supplemented with 2.5 mg/l BAP and 0.5 mg/l GA 3. Figs. D-E: D(i),D(ii) and E): Callusing on MS medium supplemented with 2.0 mg/l TDZ and 4.0 mg/l ip 3396

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4 J. Cell Tissue Research Table 3: Effect of fig regeneration medium on callus induction from leaf explants. CM1: MS mg/l TDZ mg/l IBA mg/l ascorbic acid +150 mg/l citric acid. CM2: MS mg/l TDZ mg/l ip mg/l ascorbic acid +150 mg/l citric acid. CM3: MS mg/l TDZ mg/l ip mg/l ascorbic acid +150 mg/l citric acid. Genotype Treatment % % Days to callus % callusing Days to callus % callus indu- Shooting on Browning leaf folding initiation at petiole at petiole initiation at leaf ction at leaf callusing media CM1 28.3± ± ± ± ± ± Poona Fig CM2 6.7± ±0.48 0±0.06 0±0.56 0±0.03 0± CM3 100± ± ± ± ± ± Brown Turkey Conadria Deanna CM1 29.2± ± ± ± ± ± CM2 12.5± ± ± ±0.50 0±0.05 0± CM3 28.3± ± ± ± ± ± ±0 CM1 26.7± ± ± ± ± ± CM2 33.3± ±0.48 0±0.04 0±0.85 0±0.11 0± CM3 55.8± ± ± ± ± ± CM1 55.8± ± ± ± ± ± CM2 20.8± ±0.50 0±0.12 0± ± ±0.7 0 CM3 20.8± ± ± ± ± ±0.5 0 CD at 5% SE CV 4.24% 1.97% 0.47% 2.41% 0.75% 2.52% - The results of callus cultures induced from leaf explants at different hormone concentrations and combinations are given in Table 3 and Figures A-H. Browning of explant was observed in all four genotypes. Minimum explant browning was observed in Brown Turkey. Leaf folding was high in Brown Turkey and Poona Fig. Among all treatments, callus induction frequency varied from 0 % to 85.8 % (Table 3). Highest callus induction was observed, when leaf explants were cultured MS medium supplemented with 2.0 mg/l TDZ and 4.0 mg/l 2iP. Brown Turkey exhibited highest callus induction (85.8 %) followed by Conadria (78.3 %), Poona fig (63.3 %) and Deanna (34.2 %). On MS medium supplemented with 2.0 mg/l TDZ and 2.0 mg/l 2iP callus induction (14.2%) was observed only in Denna. Whereas, moderate callus induction was observed in Deanna (56.7%) followed by Conadria (51.7 %), Brown Turkey (50.8 %) and Poona Fig (28.3 %) on MS medium with 2.0 mg/l TDZ and 2.0 mg/l IBA; clearly indicating genotypes varies significantly in their response to growth regulators. Quickest callus initiation was observed in Brown Turkey (16.1 and 16.6 days) as compared to Poona fig (18.0 and 20.9 days), Conadria (18.0 and 21.4 days), and Deanna (19.3, and 24.4 days) when cultured on MS medium with 2.0 mg/l TDZ and 2.0 mg/l IBA and MS medium with 2.0 mg/l TDZ and 4.0 mg/l 2iP, respectively. Callusing at petiolar region was observed earlier than leaf region. Brown Turkey (100 %) exhibited highest callus induction at petiolar region followed by Poona Fig (71.7 %), Deanna (58.4 %) and Conadria (48.3 %). TDZ has been reported to be efficient in stimulating adventitious shoot production in several recalcitrant woody plants including fig [10]. The mechanism of TDZ action is partly related to the inhibition of cytokinin degradation by cytokinin oxidase, resulting in increased levels of endogenous cytokinin [11]. TDZ is a synthetic phenylurea derivative with cytokinin activity, known to show higher activity and to promote better shoot organogenesis in comparison to adeninetype cytokinins such as BA and kinetin [12]. Soliman et al. [9] reported optimum direct somatic embryogenesis using 2 mg/l TDZ and 4 mg/l 2iP and indirect somatic embryogenesis on medium comprising 7 mg/l TDZ and 0.25 mg/l NAA. Earlier there are reports of fig shoot regeneration on MS medium having combination of TDZ and IBA [2,14]. Shoot development was observed only in Brown Turkey (21.66 %) on MS medium supplemented with 2.0 mg/l TDZ and 4.0 mg/l 2iP. In genotypes other than Brown Turkey the calli failed to induce shoot. Therefore these calli were transferred to MS with 7 mg/l TDZ and 0.25 mg/l NAA which resulted in Figs. F: F(ii) and F(iii): Shoot initiation from callus on MS medium supplemented with 7 mg/l TDZ and 0.25 mg/l NAA. Figs. G- H: G(i),G(ii) and G(iii): Shoot initiation from callus on MS medium supplemented with 7 mg/l TDZ and 0.25 mg/l NAA; H: Root induction on MS medium supplemented with 1 mg/l IBA and 2 g/l activated charcoal. 3398

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6 J. Cell Tissue Research shooting in 4-5 weeks. However, callus turns brown which may cause due to toxic effect cause due to prolonged culture of callus at higher TDZ concentration. Garcia et al. [13] reported development of non-morphogenic calluses in cultures of Pelargonium suberosa maintained in the presence of TDZ only. Regenerated plantlets were rooted on full-strength MS medium with 1mg/l IBA and acclimatized into pots containing cocopeat and manure in polycarbonated polyhouse. [14] Flaishman, M.A, Yablovich, Z., Golobovich, S., Salamon, A., Cohen, Y., Perl, A., Yancheva, S.D., Kerem, Z. and Haklay, E.: Acta. Hort., 798: (2008). The present study indicates the genotype specificity for callus induction and plant regeneration. Optimum callus induction was observed on when leaf explants were cultured on MS with 2.0 mg/l TDZ and 4.0 mg/ l 2iP. Shooting from these calli was obtained after transfer to MS with 7 mg/l TDZ and 0.25 mg/l NAA. Regenerated plantlets were rooted on full-strength MS medium with 1mg/l IBA. The present investigation may be helpful for commercial micropropagation and development of transformation protocol in fig. REFERENCES [1] Freiman, Z.E., Rodov, V., Yablovitz, Z., Horev, B. and Flaishman, M. A.: Sci. Hort.,138: (2012). [2] Kim, K.M, Kim, M.Y., Yun, P.Y., Chandrasekhar, T., Lee, H.Y. and Song, P.S.: J. Plant Biol., 50(4): (2007). [3] Kumar, V., Radha, A. and Chitta, S. K.: Plant Cell Rep., 17: (1998). [4] Rout, G.R., Mohapatra, A. and Mohan Jain, S.: Biotechnol. Adv., 24: (2006). [5] Hepaksoy, S. and Aksoy, U.: Biol. Plantarum, 50: (2006). [6] Yakushiji, H., Mase, N. and Sato, Y.: J. Hort. Sci. Biotech., 78: (2003). [7] Powell, W. and Caligari, P.D.S.: Heredity 59: (1987). [8] Murashige, T. and Skoog, F.: Physiol. Plantarum, 15: (1962). [9] Soliman, H.I., Gabr, M. and Abdallah, N.: GM Crops 1: (2010). [10] Huetteman, C.A. and Preece, J.E.: Plant Cell Tiss. Org. Cult., 33: (1993). [11] Hare, P.D. and Van Staden, J.: Plant Cell Physiol., 35: (1994). [12] Khurana-Kaul, V., Kachhwaha, S. and Kothari, S.L.: Biol. Plantarum, 54: (2010) [13] Garcia, R., Pacheco, G., Falcao, E., Borges, G. and Mansur, E.: Plant Cell Tiss. Org. Cult., 106: (2011) 3400