Scientia Horticulturae 79 (1999) 175±183 In vitro propagation of pomegranate (Punica granatum L. cv. Ganesh) through axillary shoot proliferation from nodal segments of mature tree Soumendra K. Naik, Sitakanta Pattnaik, Pradeep K. Chand* Plant Tissue and Cell Culture Facility, Post-Graduate Department of Botany, Utkal University, Bhubaneswar, Orissa, India Accepted 31 July 1998 Abstract A rapid and efficient procedure is outlined for in vitro clonal propagation of an elite cultivar of pomegranate (Punica granatum L. cv. Ganesh) using nodal stem segments of a mature tree. Bud break and axillary shoot proliferation was induced in the nodal segments on Murashige and Skoog (1962) (MS) medium supplemented with benzyladenine (BA), zeatin riboside (ZR) or thidiazuron (TDZ), with ZR being most effective. Maximum number of shoots were developed on a medium containing 2.0 mg lÿ1 ZR. However, the shoots failed to elongate. This problem was overcome by transferring the shoot clumps to a medium with a lower level of ZR. TDZ was least effective of all the cytokinins tested. Rooting was induced in 86% of the regenerated shoots in a half-strength MS medium supplemented with 1.0 mg lÿ1 indole-3-butyric acid (IBA). Upon transfer of the rooted shoots to an auxin-free half-strength MS medium the primary roots elongated further and additionally new roots developed. The plantlets were acclimatised and established in soil. # 1999 Elsevier Science B.V. All rights reserved. Keywords: In vitro propagation; Nodal segment; Punica granatum Abbreviations: BA, N6-benzyladenine; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; MS, Murashige and Skoog's (1962) medium; TDZ, thidiazuron; ZR, zeatin riboside * Corresponding author. Tel.: +91-674-581529; fax: +91-674-409259. 0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 9 8 ) 0 0 2 1 8 - 0 176 S.K. Naik et al. / Scientia Horticulturae 79 (1999) 175±183 1. Introduction Pomegranate (Punica granatum L.) is an important tree of the tropical and subtropical regions of the world which is valued highly for its delicious edible fruits. In addition, the tree is also cultivated for its pharmaceutical and ornamental usage. The powdered flower buds are used in bronchitis and the fruit rind, the decoction of the bark, and the juice of the leaves and young fruits are used for the treatment of gastrointestinal disorders. Moreover, the fruit rind, stem and root bark and the leaves are a good source of tannins. The flowers yield a light red dye which is used for dyeing the cloth. The seeds along with the fleshy portions are dried and used as a condiment (Anon., 1982). Conventionally, pomegranate is propagated by stem cuttings including both hard wood and soft wood cutting, the former being more commonly used. However, this method is not very efficient as it requires about one year to raise the saplings (Anon., 1982). During the past years, micropropagation techniques have been widely used for the propagation of several plant species. Protocols have also been developed for in vitro propagation of a number of fruit trees (Bajaj, 1986; Hutchinson and Zimmerman, 1987). Regeneration of plantlets in vitro through either organogenesis from callus derived from leaf segments (Omura et al., 1987) or anther wall (Moriguchi et al., 1987), or through embryogenesis from various seedling explants (Jaidka and Mehra, 1986) have been reported in pomegranate. However, to date, there is no report on in vitro clonal propagation of this fruit tree directly using nodal stem segments from mature trees. We report here a rapid and efficient method for in vitro clonal propagation of an elite cultivar of pomegranate (Punica granatum L. cv. Ganesh) through axillary shoot proliferation from nodal stem segments collected from a mature tree. 2. Materials and methods 2.1. Explant source A 10-year-old elite tree of Punica granatum L. cv. Ganesh that has been indexed and maintained in the garden of the Department of Horticulture, Government of Orissa, Bhubaneswar provided the explants for the present study. Nodal stem segments (1.0±1.5 cm) collected from that tree were washed with running tap water for 1 h, treated with an aqueous solution of 5% liquid detergent Laboline (Qualigens, India) for 3 min and rinsed with distilled water for 5±6 times. The explants were surface-sterilized with an aqueous solution of 0.1% HgCl2 for 5 min and rinsed 5±6 times with autoclaved distilled water. S.K. Naik et al. / Scientia Horticulturae 79 (1999) 175±183 177 2.2. Culture medium and conditions Murashige and Skoog (1962) (MS) medium containing 3% sucrose, 100 mg lÿ1 myo-inositol was used. The medium was further supplemented either with 0.5± 3.0 mg lÿ1 zeatin riboside (ZR) or benzyladenine (BA) or 0.01±0.5 mg lÿ1 thidiazuron (TDZ). The pH of the medium was adjusted to 5.8 before gelling with 0.8% agar (BDH, India). Fifteen ml of media were dispensed into 25 ml Erlenmeyer flasks. Flasks were capped with aluminium foil. All media containing culture vessels were autoclaved at 104 kPa and 1218C for 15 min. The sterilized nodal segments (1 explant/flask) were planted vertically in each culture flask. Each cytokinin treatment consisted of 9 replicate flasks with 1 explant in each. Cultures were maintained at 25  18C, 60% relative humidity and a particle (photon) flux density of 35 mE mÿ2 sÿ1 provided by white fluorescent tubes. The experiment was conducted three times. 2.3. Rooting of the shoots Shoots with 4±6 expanded leaves (3.5±4.0 cm) developed in vitro were excised and transferred to half-strength MS medium containing 50 mg lÿ1 myo-inositol, 1.5% sucrose and 0.2% phytagel (Sigma, USA). The medium was further supplemented with 0.5±2.0 mg lÿ1 indole-3-butyric acid (IBA) or indole-3-acetic acid (IAA). After 8±10 days the rooted shoots were transferred to an auxin-free half-strength medium for further elongation of the roots. Each auxin treatment consisted of seven replicate tubes with one shoot in each and repeated twice. 2.4. Acclimatization and transfer of plants to soil Well rooted plantlets were removed from the culture medium, the roots were washed gently with tap water to remove phytagel and then transferred to small plastic pots (7.5 cm diam) containing autoclaved vermi-compost (Ranjan's Agrotech, Bhubaneswar). The pots were covered with polyethylene bags to maintain a high humidity and kept at 25  18C and a particle (photon) flux density of 50 mE mÿ2 sÿ1 provided by white fluorescent tubes for 3 weeks. Plants were then transferred to larger pots (18 cm diam) containing garden soil (soil : compost, 1 : 1); kept under shade for another 2 weeks and then transferred to a nursery under full sun. 3. Results 3.1. Bud break and shoot proliferation The surface-sterilization procedure followed in the present study yielded 95% of the explants free of apparent microbial contamination. The culture medium 178 S.K. Naik et al. / Scientia Horticulturae 79 (1999) 175±183 Table 1 Influence of different cytokinins on bud break and shoot proliferation from nodal segments of P. granatum Concentration (mg lÿ1) Shoot number Shoot length (cm) ± ± ± 71.0  2.3 93.0  2.5 41.0  2.3 29.0  2.1 1.0  0 2.1  0.1 1.2  0.1 1.0  0 3.2  0.1 3.7  0.2 1.6  0.1 ± ZR 0.5 1.0 2.0 3.0 ± 63.0  2.1 85.0  2.1 22.0  2.3 ± 2.5  0.2 5.2  0.1 1.2  0.1 ± 2.9  0.2a 3.9  0.1a 1.5  0.2a TDZ 0.01 0.05 0.1 0.5 ± 63.0  2.3 52.0  2.5 37.0  2.3 ± 1.0  0 1.0  0 1.0  0 ± 3.6  0.2b 3.2  0.1b 2.1  0.2b 0.0 BA 0.5 1.0 2.0 3.0 % Bud break   SE) from three independent experiments each with nine replicates. Data (X a Shoot length was recorded following transfer to a medium containing 1.0 mglÿ1 ZR. b Shoot length was recorded following transfer to a medium containing 0.5 mglÿ1 BA. turned brown within 3 days of inoculation of the explants. The browning secretion was first noticed at the cut ends of the explants which gradually spread into the surrounding medium. Shoot development was completely inhibited and the explants died within a week. The browning problem was more intense with explants collected from the older shoots than those from the younger shoots. This problem was overcome by transferring the nodal segments to a fresh medium of the same composition at 24 h interval. The browning secretion was stopped after two consecutive transfers and the explants exhibited normal shoot development. Shoot development could not be induced in the nodal stem segments on a growth regulator-free MS medium. Addition of a cytokinin to the medium was essential to induce bud break and multiple shoot formation in the explants (Table 1). MS medium containing BA at an optimum concentration of 1.0 mg lÿ1 induced bud break in 93% of the explants in 12±15 days (Fig. 1). Each explant developed 2.1 shoots averaging 3.7 cm length in 30 days (Table 1). On the other hand 85% of the explants exhibited bud break within 10±12 days on a medium containing 2.0 mg lÿ1 ZR and from each explant an average of 5.2 shoots were developed in 22±25 days (Fig. 2). These shoots elongated within 4±6 days upon transfer to a medium containing a lower concentration of ZR (1.0 mg lÿ1) and S.K. Naik et al. / Scientia Horticulturae 79 (1999) 175±183 179 Fig. 1. Shoot development from a nodal segment of P.granatum after 15 days of culture on MS ‡ 1.0 mglÿ1 BA. Fig. 2. Development of multiple shoots from a nodal segment of P. granatum on MS ‡ 2.0 mglÿ1 ZR after 25 days of culture. attained an average height of 3.9 cm. Concentrations of BA or ZR higher than the optimum level had an inhibitory effect on shoot development (Table 1). In another experiment TDZ at 0.01±0.5 mg lÿ1 was used to induce shoot development from the nodal explants (Table 1). The optimum concentration of TDZ, at which maximum number of explants exhibited bud break, was 0.05 mg lÿ1. The frequency of bud break was 63% and from each explant a single shoot was developed in 15±18 days. However, the shoot failed to elongate. This problem was overcome by transferring the shoot to a medium containing 0.5 mg lÿ1 BA. Within 3±4 days of transfer the shoot elongated and attained an average height of 3.6 cm in 10 days. The frequency of shoot development was reduced markedly at higher concentration of TDZ. 180 S.K. Naik et al. / Scientia Horticulturae 79 (1999) 175±183 Table 2 Influence of different auxins on rooting of the in vitro formed shoots of P. granatum Auxin (mg lÿ1) % rooting Root numbera 1/2 MS basal ± ± IAA 0.5 1.0 2.0 ± 43.0  1.2 28.0  1.5 ± 3.1  0.2 1.8  0.2 IBA 0.5 1.0 2.0 38.0  1.3 86.0  1.5 42.0  1.3 3.9  0.1 6.8  0.1 2.5  0.2   SE) from three independent experiments each with seven replicates. Data (X Root number was recorded following transfer to an auxin-free half-strength MS medium a 3.2. Rooting of shoots Rooting could not be induced in the excised shoots in an auxin-free halfstrength MS medium even after 30 days. Of the two different auxins tested, IBA was more effective (Table 2). Half-strength MS medium containing IBA at 1.0 mg lÿ1 induced rooting in 86% of the shoots in 8±10 days and from each shoot 3±4 roots were developed (Fig. 3(a)). The rooted shoots were then transferred to an auxin-free half-strength MS medium in which the primary roots Fig. 3. (a) Root development from an excised shoot of P. granatum on half-strength MS ‡ 1.0 mg lÿ1 IBA after 10 days of culture. (b) A well-rooted shoot of P. granatum on an auxin-free half-strength MS medium at day 7 of culture. S.K. Naik et al. / Scientia Horticulturae 79 (1999) 175±183 181 Fig. 4. Regenerated plants of P. granatum in soil. elongated within 5±7 days and a few more new roots were developed (Fig. 3(b)). In 21 days the number of roots per shoot was 6.8 (Table 2). The rooting percentage was markedly reduced at higher concentration of IBA. 3.3. Establishment of plants in soil The percentage survival of the plantlets was 68% after transfer to vermicompost. Eighty percent of the plants transferred to soil survived (Fig. 4). The regenerated plants did not show any detectable variation in morphology when compared with the donor plant. 4. Discussion Establishment of in vitro cultures of woody plants is greatly affected by the browning of the medium and necrosis of explants. Browning is generally considered to result from the oxidation of phenolic substances released from the cut ends of the explants by polyphenol oxidases or peroxidases. The methods commonly employed to overcome the harmful effect of browning include the use of adsorbing agents such as activated charcoal or polyvinylpolypyrrolidone, inclusion of antioxidants in the medium or soaking the explants in antioxidant solution, transfer of explants to fresh medium at frequent intervals or sealing the cut ends of the explants with paraffin wax (Broome and Zimmerman, 1978; Weatherhead et al., 1978; Lloyd and McCown, 1980; Gupta et al., 1980; Amin and Jaiswal, 1988; Bhat and Chandel, 1991). In the present study the browning problem was overcome by repeatedly transferring the explants to fresh medium of the same composition. 182 S.K. Naik et al. / Scientia Horticulturae 79 (1999) 175±183 In P. granatum, highest number of shoots per explant was recorded only on a medium containing ZR. This was unlike that reported for several other fruit trees including guava (Amin and Jaiswal, 1988) and mulberry (Pattnaik et al., 1996) in which BA was most effective among the cytokinins in inducing bud break and shoot proliferation in the explants. Thidiazuron is now being widely used for micropropagation of several woody plants because of its tremendous ability to enhance axillary shoot proliferation (Huetteman and Preece, 1993). In the present study, however, it was less effective than the adenine type cytokinins such as BA or ZR. Shoots developed on a TDZcontaining medium failed to elongate. The inhibition of shoot elongation by TDZ may be consistent with its high cytokinin activity (Huetteman and Preece, 1993). This has been reported in several fruit tree species including Malus spp. (van Nieuwkerk et al., 1986) and Populus (Russel and McCown, 1986). To overcome this problem, transfer of shoot cultures to a medium often lacking TDZ or with a different growth regulator has been suggested. The use of a primary and secondary media has been successful in Populus (Russel and McCown, 1986). In P. granatum, shoots developed on TDZ medium elongated following transfer to medium containing BA. In the present study of the two auxins tested for rooting (IAA and IBA), the latter was more effective. IBA was reported to have favoured root induction in several tree species including Artocarpus heterophyllus (Rahman and Blake, 1988) and Morus nigra (Yadav et al., 1990). The in vitro propagation of pomegranate via axillary shoot proliferation from nodal segments producing rooted plantlets in 12 weeks as described in this paper is a rapid method as compared to the conventional method of using hard wood cutting. 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