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

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482 RENAU-MORATA, OLLERO, ARRILLAGA AND SEGURA Table 3. Effects of temperature and cytokinins on shoot proliferation from microcutting cultures of juvenile C. libani and C. atlantica. Values are combined means from two experiments of 12–14 observations each. Cultures were established on MSBN/2 medium and culture time was 60 days. Within a column, values followed by different letters are significantly different according to Tukey’s test at P ≤ 0.05. Species Temperature (°C) Cytokinin (µM) Survival (%) Shoots per explant C. libani 26 0.0 90 ab 4.2 bcde 2.2 Z 80 ab 4.8 ab 0.45 TDZ 90 ab 3.0 bcdef 30 0.0 90 ab 7.0 a 2.2 Z 60 bc 4.6 abc 0.45 TDZ 80 ab 4.5 abcd C. atlantica 26 0.0 100 a 1.7 ef 2.2 Z 100 a 2.7 bcdef 0.45 TDZ 100 a 1.5 e 30 0.0 100 a 2.0 def 0.45 TDZ 40 c 2.1 cdef Under the most favorable conditions of those tested (sterilization with HgCl2 and culture on basal MSBN/2 medium), the percentage of microcuttings from 10–15-year-old C. libani showing axillary bud sprouting ranged from 15 to 20%. To obtain enough material for further experiments, sprouted buds were routinely isolated and subcultured on basal MSBN/2 medium. Excised shoots from these proliferating cultures were used to study the effects of nutrient medium (WPM and MSBN/2) and cytokinin type (TDZ and Z) on axillary bud proliferation (Table 4). Survival percentages ranged from 90 to 100%, except when microcuttings were cultured on TDZ-supplemented MSBN/2 medium (survival = 60%), as found for explants of juvenile origin. Both nutrient medium and cytokinin type significantly influenced sprouting percentage and mean number of axillary buds formed per explant and there was a significant interaction between these factors. In cultures established on MSBN/2, all microcuttings showed axillary bud sprouting and the mean number of developed buds per explant (3.7–6.2) was not significantly influenced by the presence and type of cytokinin. In contrast, cytokinin enhanced axillary bud proliferation in microcuttings grown on WPM, the best results (100% sprouting and 7.3 buds per explant) being obtained in the presence of Z. TREE PHYSIOLOGY VOLUME 25, 2005 Although initial multiplication rates were low, they increased during the first few subcultures, providing enough material for mass multiplication of adult C. libani. When this protocol was used for the in vitro propagation of 200-year-old C. libani trees, we achieved successful in vitro establishment in three out of the six sampled trees (genotypes AL1, AL2 and AL3), suggesting that in vitro establishment was genotype-dependent. The initial multiplication efficiency of the three genotypes (as measured by the frequency of axillary bud breaking after 2 months of culture) was 3.0, 7.6 and 9.3%, respectively. Multiplication efficiency of the three genotypes increased with number of subcultures, and within 6 months, mean sprouting percentage reached 70% for all three genotypes and this rate was maintained during subsequent subcultures, yielding 20–30 clones of each genotype after 2 years. Adventitious bud differentiation from cultured embryos of C. atlantica Based on a previous report of successful adventitious bud induction from cultured leaves of mature Juniperus oxycedrus (Gómez and Segura 1994), needles from C. atlantica and C. libani seedlings were cultured on MSH medium with 0.5 µM BA. Although some needles formed a callus in the presence of BA (23 and 13% in explants from C. atlantica and Table 4. Effects of nutrient media and cytokinins on shoot proliferation from 10–15-year-old C. libani microcuttings. Explants were first cultured for 60 days on media with or without cytokinin and then transferred to their respective basal media. Values are combined means from two different experiments of 10 observations each and culture time was 90 days. Within a column, values followed by different letters are significantly different according to Tukey’s test at P ≤ 0.05. Nutrient medium Cytokinin (µM) Survival (%) Sprouting (%) Shoots per explant WPM 0 90 a 10 a 0.2 c 0.45 TDZ 100 a 100 b 3.4 bc 2.2 Z 100 a 100 b 7.3 a MSBN/2 0 100 a 100 b 4.4 b 0.45 TDZ 60 b 100 b 3.7 b 2.2 Z 100 a 100 b 6.2 ab Downloaded from http://treephys.oxfordjournals.org/ by guest on June 5, 2013
C. libani, respectively), none of the cultured explants differentiated adventitious buds. In subsequent experiments, mature C. atlantica embryos were cultured on MSBN/2 medium with or without Z and BA. Within the first week of culture, embryos elongated and the cotyledons and hypocotyls became green. Embryos on basal medium produced no callus or adventitious buds, developing as normal seedlings. In the presence of cytokinins, hypocotyls and cotyledons in contact with the medium proliferated quickly, producing callus. Usually the radicles did not show a response either in color or in cell proliferation. The first adventitious buds were directly induced from the upper surface of the hypocotyls (Figure 2A) after 20–30 days of culture. Further bud differentiation occurred on the surface of the previously induced calli. Indirect needle primordia differentiation was also observed, especially when embryos were cultured in the presence of BA. After 50–60 days, the entire upper surface of the responding embryos was covered with adventitious buds and needles, although the latter did not develop into shoots. Table 5 summarizes the bud differentiation process in cultured C. atlantica embryos. The bud-forming capacity of the explants depended on cytokinin type and concentration, the best results being obtained when the embryos were cultured in the presence of 9 µM Z (47% of caulogenic explants and 4.2 adventitious buds per embryo), whereas 9 µM BA induced a smaller response (13% and 0.3, respectively). Embryo subculture to medium with 4.4 mM Z enhanced the development of adventitious buds (Figure 2B). Further shoot elongation was achieved following excision and transfer of these shoots to hormone-free medium. Transfer of embryos bearing adventitious buds to basal medium did not promote bud elongation (data not shown). Although WPM provided a greater percentage of embryos with buds than MSBN/2 medium (49.0 versus 40.0%, P = 0.05), the mean number of buds formed per embryo in the presence of 9 µM Z was similar in the two media (3.1). Irrespective of the nutrient medium, 18 µM Z enhanced acicular primordia differentiation and reduced adventitious bud differentiation. Rooting Rooting experiments were carried out with axillary and adventitious shoots isolated from proliferating cedar cultures of ju- SHOOT PROLIFERATION IN CEDAR CULTURES 483 TREE PHYSIOLOGY ONLINE at http://heronpublishing.com Table 5. Effects of cytokinin type and concentration on the differentiation of adventitious buds from embryos of C. atlantica. Values are combined means of results from two experiments of 20 observations each. Cultures were established on MSBN/2 medium and culture time was 60 days. Within a column, values followed by different letters are significantly different according to Tukey’s test at P ≤ 0.05. Cytokinin Concentration Explants with Buds per (µM) buds (%) explant Z 0.0 0.0 0.0 2.2 13.3 0.8 4.4 33.3 1.3 6.6 33.3 0.8 9.0 46.7 4.2 Mean 25.3 a 1.4 a BA 0.0 0.0 0.0 2.2 6.7 0.1 4.4 6.7 0.1 6.6 13.3 0.1 9.0 13.3 0.3 Mean 8.0 b 0.1 b venile and adult origin. None of the experimental variables tested promoted rooting. Discussion We observed in vitro axillary bud proliferation of explants from juvenile and adult C. atlantica and C. libani trees. In both species, explant size was an important factor affecting axillary bud proliferation in in vitro culture. Piola and Rohr (1996) reported that axillary and apical buds from in-vitro-grown microcuttings of C. libani sprout at 30 °C but not at 24 °C. Subsequently, Piola et al. (1998) demonstrated that needle removal, but not microcutting decapitation, substituted for the higher temperature requirement to break bud dormancy. Abscisic acid (ABA) concentrations in microcuttings with dormant buds were higher than in microcuttings bearing sprouted buds, leading these authors to suggest that ABA accumulation in needles caused bud dormancy of C. libani microcuttings at 24 °C. Therefore, we always used defoliated microcuttings, to Figure 2. Induction and development of adventitious buds from cultured embryos of C. atlantica. Adventitious bud differentiation on MSBN/2 medium with 9 µM Z (left) and adventitious bud development on MSBN/2 medium with 4.4 µM Z (right). Bar = 0.3 cm. Downloaded from http://treephys.oxfordjournals.org/ by guest on June 5, 2013
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