Factors influencing axillary shoot proliferation and ... - Tree Physiology

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480 RENAU-MORATA, OLLERO, ARRILLAGA AND SEGURA twice. A hierarchic analysis of variance (nested ANOVA, Sokal and Rohlf 1995) design was also used to estimate variance components for the morphogenic parameters recorded, partitioning the variation among seed lots and among seedlings within lots. All analyses were performed with the Super- ANOVA program (Abacus Concepts, Berkeley, CA). Results Axillary shoot proliferation from juvenile explants Nutrient medium, growth regulators and agar brands Fewer than 5% of apical and nodal explants of C. atlantica and C. libani sprouted during 40 days of culture, although most explants formed a callus. The frequency of sprouting was unaffected by pulse treatments with BA or TDZ , nutrient medium (MSH, MBF or MSBN/2), the presence of BA in the culture medium or the agar brand (Difco-Bacto, Phytagel, Pronadisa or A-1296) (data not shown). Agar A-1296, which has been used previously for C. libani cultures (Piola and Rohr 1996), was selected for subsequent experiments. Maximum callus formation (80–90%) was observed when apical explants were first dipped in BA or TDZ solution and then transferred to BA-supplemented medium. Although some calli underwent necrosis, others differentiated adventitious buds and needles. These organogenic responses were mainly observed in apical explants from C. atlantica and C. libani growing in the presence of 9 µM BA. Organogenic calli were subcultured on their respective basal media with or without activated charcoal at either 26 °C or at 30 °C (Piola and Rohr 1996), but the treatments were without significant effect on bud elongation. Explant size, temperature and growth regulators Shoot apices and microcuttings, isolated from 3-month-old C. libani and C. atlantica seedlings, were cultured at 30 °C on MSBN/2 medium with or without 9 µM BA. Most of the shoot apices grown in the presence of BA produced a callus. Culture survival percentages (85–100%) were not significantly affected by explant size in either species; however, axillary bud proliferation oc- TREE PHYSIOLOGY VOLUME 25, 2005 curred only when microcuttings were used as primary explants (Figure 1). In both species, sprouting percentages and mean number of axillary shoots per explant were significantly increased in microcuttings grown on medium without BA (80% without BA versus 60% with BA, P = 0.05; 3.0 versus 1.5, P = 0.05). In another experiment, entire or decapitated C. libani and C. atlantica microcuttings were cultured on MSBN/2 medium with or without BA or Z and kept for 45 days at 30 or 26 °C. In C. libani cultures, survival and sprouting percentages ranged from 85 to 100%, and these responses were not significantly affected by incubation temperature or the BA and Z treatments (P > 0.05) (data not shown). However, decapitation of C. libani microcuttings significantly reduced the mean number of shoots formed per explant (3.0 versus 4.1, P = 0.05; Table 1). Basal MSBN/2 medium or Z was more effective than basal medium + BA in promoting axillary shoot proliferation from cultured C. libani microcuttings (4.0 or 4.5 versus 2.1 shoots per explant, respectively; P = 0.05, Table 1). In C. atlantica cultures (Table 2), an incubation temperature of 30 °C and explant decapitation both reduced microcutting survival percentages (69.4 versus 87.8%, P = 0.05; 87.8 versus 93.1%, P = 0.05; respectively). Incubation temperature, decapitation and growth regulators all significantly influenced sprouting percentages, the best results being obtained at 26 °C (82.3 versus 66.7%, P = 0.05), with entire explants (84.9 versus 64.1%, P = 0.05) and basal or Z-supplemented medium (83.8 or 77.1% versus 62.7%, P = 0.05). The detrimental effect of incubation at 30 °C versus at 26 °C was particularly evident in decapitated microcuttings. The mean number of axillary shoots formed per cultured explant was higher in entire microcuttings (2.6 versus 1.3, P = 0.05) and the presence of BA reduced this response (1.2 versus 2.4 and 2.1 shoots per explant in Z-supplemented medium or basal medium, respectively; P = 0.05). Significant interactions between explant type and incubation temperature or presence of cytokinin in the medium were also evident, with the highest mean number of shoots per explant obtained when entire microcuttings were Figure 1. Axillary shoot proliferation from C. atlantica (A) and C. libani (B) microcuttings cultured on MSBN/2 without growth regulators. Downloaded from http://treephys.oxfordjournals.org/ by guest on June 5, 2013
Table 1. Effects of temperature, explant type and cytokinins on shoot proliferation from microcutting cultures of juvenile C. libani. Values are combined means from two experiments of 10 observations each. Cultures were established on MSBN/2 medium and culture time was 60 days. Values followed by different letters are significantly different according to Tukey’s test at P < 0.05. Microcutting Temperature Shoots per explant (°C) Cytokinin (µM) cultured at 30 °C in either basal medium or Z-supplemented medium (Table 2). Based on these results, entire microcuttings of C. atlantica and C. libani were cultured at both 26 and 30 °C in the presence of Z or TDZ (Table 3). In both species, survival percentages were lower when microcuttings grown on cytokinin- supplemented medium were kept at 30 °C. This negative effect of the higher temperature was especially evident in C. atlantica microcuttings cultured in the presence of TDZ (40%). Sprouting percentages ranged from 80 to 100% and neither incubation temperature nor cytokinin type significantly influenced this response (P > 0.05, data not shown). In contrast, mean number of shoots formed per explant was higher at 30 °C than at 26 °C (4.1 versus 2.9, P = 0.05), and Z was generally more effective than TDZ (4.1 versus 2.7, P = 0.05). Under the culture conditions tested, shoot proliferation rates were higher in C. libani than in C. atlantica microcuttings (4.7 versus 2.4, P = SHOOT PROLIFERATION IN CEDAR CULTURES 481 0.0 2.2 BA 2.2 Z Mean 1 Entire 26 4.4 2.3 5.3 30 5.3 3.1 3.9 Decapitated 26 3.6 1.3 4.6 30 2.8 1.6 4.1 Mean 2 1 Effect of explant type. 2 Effect of cytokinin type. 4.0 a 2.1 b 4.5 a 4.1 a 3.0 b TREE PHYSIOLOGY ONLINE at http://heronpublishing.com 0.05). The maximum number of shoots formed per explant (7.0) was obtained when C. libani microcuttings were cultured on basal medium at 30 °C. This temperature effect was less evident in microcuttings isolated from shoot-proliferating cultures. In all experiments, and irrespective of the treatments, the sprouted buds reached a final length of 1 to 2 cm. These experiments were performed with seedlings from different seed stocks obtained from two commercial companies. To determine the variance attributable to the seed lots and to the individuals within each lot, the results were subjected to a nested ANOVA, which indicated that there was no significant effect of seed lot on survival or sprouting percentage. In contrast, variations in shoot yield were due to differences among seed lots (23 and 35% in C. atlantica and C. libani, respectively) and among seedlings within seed lots (77 and 65% for C. atlantica and C. libani, respectively). Axillary shoot proliferation from explants of mature origin None of the culture conditions tested promoted axillary shoot proliferation from apical shoot meristems of mature trees of C. atlantica or C. libani. Most of the responding explants (60–70%) formed calli. Neither the culture medium (SH or MS/2) nor growth regulators (BA or NAA, or both) affected this morphogenic response. Meristem-derived calli were maintained in culture for almost 6 months, but attempts to induce organogenesis failed. Based on the data obtained with seedlings, shoot apices (0.6 cm) from 30-year-old C. libani were surface sterilized with either NaClO or HgCl2 and cultured on MSBN/2 medium with or without BA or NAA or both. Although the effectiveness of the two sterilizing agents was similar (an 80% yield of axenic explants), NaClO caused generalized chlorosis in some explants. Survival percentage was 50% and neither the sterilizing agent nor the presence of growth regulators significantly affected this result. Explant elongation and subsequent axillary bud sprouting was observed only in cultures established on basal medium. Under these conditions, 15% of the explants elongated and showed bud sprouting (1–3 buds per explant) within 2–3 months of culture. Explants grown in the presence of BA with or without NAA formed calli. Table 2. Effects of temperature, explant type and cytokinins on shoot proliferation from microcutting cultures of juvenile C. atlantica. Values are combined means from two different experiments of 10 observations each. Cultures were established on MSBN/2 medium and culture time was 60 days. Microcutting Cytokinin (µM) Survival (%) Sprouting (%) Shoots per explant Mean 1 26 °C 30 °C 26 °C 30 °C 26 °C 30 °C Entire 0.0 100.0 100.0 92.3 91.7 1.9 3.8 2.9 a 2.2 BA 100.0 83.3 66.7 75.0 1.1 1.6 1.4 b 2.2 Z 91.7 83.3 91.7 91.7 2.5 4.4 3.5 a Mean 2 83.6 a 86.2 a 1.8 b 3.3 a Decapitated 0.0 76.9 41.7 84.6 66.7 1.3 1.4 1.4 b 2.2 BA 83.3 75.0 75.0 33.3 1.8 0.4 1.1 b 2.2 Z 75.0 33.3 83.3 41.7 1.5 1.2 1.4 b Mean 2 81.0 a 47.2 b 1.5 b 1.0 b 1,2 Interaction of explant type with cytokinins or temperature, respectively. For each interaction, values followed by different letters are significantly different according to Tukey’s test at P ≤ 0.05. Downloaded from http://treephys.oxfordjournals.org/ by guest on June 5, 2013
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