Effects of explant, media and growth regulators on in vitro regeneration and antioxidant activity of Juniperus phoenicea

Abstract

Juniperus phoenicea is an ornamental shrub that is also used to flavor food and to supply medicines and timber. Its micropropagation is of industrial concern and can occur by axillary shoot multiplication. Microcuttings of J. phoenicia were established in vitro on Murashige and Skoog (MS) and Rugini Olive (OM) media in glass tubes (25 mm x 150 mm). Factors studied were explant length (0.5 or 1.5 cm) and orientation (horizontal or vertical), media strength (OM, ½OM, MS, ½MS) and the following growth regulators: the anti-auxin 2,3,5-triiodobenzoic acid (TIBA), the cytokinins 6-benzyladenine (BA) and thidiazuron (TDZ), and the growth retardant daminozide (DM). Microcuttings placed vertically on the surface of OM, ½OM and ½MS media without hormones exhibited axillary bud differentiation, but they were swollen and turned brown one month later when placed horizontally on the medium surface. The number of shoots, averaged across OM, ½OM and ½MS media, was significantly higher from longer (1.5 cm) than shorter (0.5 cm) microcuttings (4.11 versus 1.57 shoots microcutting -1 ) after 60 days of culture. TIBA or DM at 0.1 mg l -1 included in OM medium enhanced leaf differentiation, callus induction and formation of adventitious shoots over three months from 0.5 cm long microcuttings taken from in vitro shoots. The formation of adventitious shoots was sporadic and occurred at a rate of 1 shoot microcutting -1 in the presence of 0.1 mg l -1 DM. OM supplemented with 0.1 mg l -1 TIBA resulted in significantly the highest leaf differentiation (55 leaves microcutting -1 ), with a rooting rate of 40%. Contents of phenols, flavonoids and antioxidants were compared for cuttings from young seedlings, callus, in vitro shoots, and seeds. Antioxidant activity was significantly the highest for shoots grown on hormone-free OM medium and callus maintained on OM medium containing 0.1 mg l -1 2,4-D (94.7 and 94.3% inhibition of DPPH free radicals, respectively). Thus, different routes for in vitro regeneration of J. phoenicea can be of potential use for many biotechnological, pharmaceutical and food industry applications.

Propagation of Juniper Species by Plant Tissue Culture: A Mini-Review

“…The authors obtained 5.2 shoots per explant using shoot apices and nodal segments, which rooted at 7%-10% frequency (Table 1). Microcuttings with axillary buds [2,4,7,19,22] or shoot tips [3,9,15,22] were the most often used explants for establishment of axillary shoot cultures. The efficiency of induction of shoots from these types of explants varied among Juniperus species from 4 to 12.9 per explant for J. navicularis and J. thulifera, respectively [4,22].…”

“…Successful growth of the axillary shoots of various juniper species was obtained on different media, such as SH [36], OM [2,7,9,19], WPM [3,15,22], GD = DCR [4,48] and DKW [2]. It was demonstrated that the content of total nitrogen in the media may affect the morphological responses of the cultured explants [9].…”

“…Callus and adventitious buds were induced from various explant types, including leaves from shoots growing in vitro [16], mature zygotic seeds [17], cotyledons from germinating seedlings [18], shoots with apical or lateral buds and needles [49], spring buds [23] or microcuttings from branches of 3-year-old seedlings [19]. Adventitious shoots of J. oxycedrus were also obtained from single-cell-derived calluses [54].…”

“…Adventitious shoots of J. oxycedrus were also obtained from single-cell-derived calluses [54]. The induction of this type of bud has been reported on MS or modified MS, SH, 1 2 QP, half strength Quoirin and LePoivre (1977) [65], OM and Eriksson and Eriksson (1965) media [16][17][18][19]23,49,53,66]. Adventitious buds of J. communis were induced from spring buds on full-strength MS medium supplemented with various concentrations of BA and 2,4-D [23].…”

“…Similar to J. cedrus, shoots of J. excelsa grew very quickly on the medium without PGR. Recently, sporadic adventitious shoot induction was reported for J. phoenicea [19]. The authors applied OM medium supplemented with daminozide (DM) or TIBA at 0.1 mg L −1 .…”