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

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478 RENAU-MORATA, OLLERO, ARRILLAGA AND SEGURA seedlings and surface sterilized in 3% NaClO with 0.1% Tween-20 for 15 min or 0.1% HgCl2 for 3 min, followed by four 5-min washes in sterile distilled water. Needles were removed with a razor blade at 1–2 mm above the needle–node junction, and these initial explants were cultured on different nutrient media. Freshly isolated C. atlantica embryos were used as primary explants in some experiments. Seeds were imbibed for 4 h in sterile distilled water, surface-sterilized for 30 min in 70% ethanol and rinsed in sterile distilled water; the mature embryos were then dissected out of the seedcoat and female gametophyte. Only undamaged embryos that were firm and white to pale yellow in color were cultured. Mature explants Actively growing shoots (3–5 cm long) were collected from several trees growing outdoors, including trees in a 20–30year-old forest plantation of C. libani and C. atlantica located in Arbúcies, Girona, Spain; 15–20-year-old C. libani trees in a natural forest located in El Shouf, Lebanon; and 200-year-old C. libani trees growing at the Reading University campus, Reading, U.K. All material was collected in the spring (May to early June). After the leaves were removed, stems were immersed in 1% NaClO for 20 min, rinsed for 8 h in running tap water, submerged for 15 min in an aqueous solution of 1% benomyl, and kept at 4–6 °C for 1–10 days. Shoots were then surface sterilized with 0.1% HgCl2 as described for the juvenile explants. Initial explants, meristematic domes plus one or two pairs of leaf primordia (0.6–1.0 mm), apical shoots (6– 7 mm) and microcuttings were isolated under sterile conditions and cultured on different nutrient media. Unless otherwise stated, initial explants were chosen at random from different donor plants. Media and culture conditions The basal media tested were: Murashige and Skoog (1962) medium, with macronutrients reduced to a half (MS/2) or a quarter (MS/4) of their original concentration; modified SH medium (MSH), with Schenk and Hildebrandt (1972) macronutrients and MS micronutrients and vitamins; modified BF medium (MBF), with Boulay and Franclet (1977) salts and MS vitamins; MSBN/2 medium, with half-strength MS salts and half-strength Bourgin and Nitsch (1967) vitamins and Skoog (1944) amino acids; MSBN/4 (half-strength MSBN/2 medium); and WPM medium (Lloyd and McCown 1980). All media contained 3% sucrose except for MSBN/2 which contained 2% sucrose. Culture media were supplemented with growth regulators (indoleacetic acid (IAA), naphthaleneacetic acid (NAA), benzyladenine (BA), zeatin (Z) and thidiazuron (TDZ)) as specified. In some experiments, conifer-derived activated charcoal (Sigma, St. Louis, MO), calcium gluconate, triacontanol, paclobutrazol, pectimorf (a mix of oligopectins from citric cell walls, kindly provided by Dr. Juan Carlos Cabrera, INCA, Cuba), coumarin or phosphoric acid were used. Several agar brands were tested: Difco-Bacto (0.7%; Sparks, MD), Sigma phytagel (0.2%), Sigma A-1296 (1%) and Pronadisa (0.8%; Torrejón de Ardoz, Madrid, Spain). The TREE PHYSIOLOGY VOLUME 25, 2005 pH was adjusted to 5.8 before autoclaving for 20 min at 120 °C (10 5 Pa). Except for TDZ and Z which were sterilized by filtration, growth regulators and ancillary compounds were added to the medium before autoclaving. Culture vessels included 15 × 100-mm petri dishes and 150 × 25-mm glass tubes covered with polypropylene closures (Bellco, Vineland, NJ), each containing 25 ml of agar-solidified nutrient medium; Sigma jars with 100 ml of medium and glass jars with 50 ml of medium, both covered with sun-cap closures (Sigma); and 100-ml pots. In some experiments, explants were cultured, with continuous agitation (110 rpm), in 125-ml Erlenmeyer flasks (containing 25 ml of liquid medium) capped with Bellco stainless steel closures. Unless otherwise specified, cultures were kept in growth chambers at 26 ± 2 °C and a 16-h photoperiod provided by fluorescent lamps (70 µmol m –2 s –1 irradiance at culture level). Axillary shoot proliferation from juvenile explants Nutrient media, explant type, growth regulators and agar brands Shoot apices and three types of nodal explants (upper (containing buds of the first verticil, immediately below the apex); central (second verticil); and basal (third verticil, immediately above the cotyledonary node)) were initially dipped for 3 or 12 h in a sterile aqueous solution of 100 µM TDZ or BA, and then transferred individually to glass tubes containing agar-solidified MSH, MBF or MSBN/2 nutrient medium with or without BA (2.2 or 9 µM), 0.45 µM TDZ, 10 –14 µM IAA or combinations of the three types of growth regulators. Control explants were maintained for 3 or 12 h in sterile distilled water. The following brands of agar were tested: Difco-Bacto, Phytagel, Pronadisa and A-1296. Explant-derived organogenic calli were isolated, transferred to their respective media without growth regulators and kept at 26 or 30 °C. At least 13 apical or nodal explants were cultured for each treatment. Total culture time was 40 days. Explant size, incubation temperature and cytokinin type In a first experiment, shoot apices and microcuttings were cultured on medium with and without 9 µM BA and maintained at 30 °C. In a second series of experiments, both entire and decapitated microcuttings were cultured on medium without growth regulators or supplemented with 2.2 µM BA or 2.2 µM Z or 0.45 µM TDZ. Cultures were kept at 26 or 30 °C. In all experiments, microcuttings were placed vertically in glass tubes containing MSBN/2 medium solidified with A-1296 agar. In addition, microcuttings from C. libani proliferating cultures were isolated and cultured on the same medium with or without 2.2 µM Z and kept at 26 °C. Between 10 and 14 replications were prepared for each treatment. Cultures were examined for survival and sprouting percentages and mean number of axillary shoots per sprouted microcutting. Culture time was 40– 60 days. Axillary shoot proliferation from mature explants In a first experiment, meristematic domes of C. atlantica and C. libani trees from Arbúcies, Spain, were cultured for 30 days on Difco-Bacto agar-solidified MSH or MS/2 medium supple- Downloaded from http://treephys.oxfordjournals.org/ by guest on June 5, 2013
mented with BA (0, 2.2, 4.4 or 8.8 µM), alone or in combination with NAA (0, 0.5, or 5.4 µM). Subsequently, explants were transferred to their respective media without growth regulators for another 30 days. Twenty explants were cultured per treatment. In a second experiment, shoot apices from C. libani trees growing in Arbúcies were cultured for 45 days in glass tubes containing A-1296 agar-solidified MSBN/2 medium supplemented with BA (0, 0.44, 2.22 and 4.44 µM), NAA (0 and 0.05 µM), or combinations of both types of growth regulators. Subsequently, explants were transferred to their respective media without growth regulators for another 45 days. Twentysix explants were cultured per treatment. In a third experiment, microcuttings from C. libani trees growing in El Shouf, Lebanon, were cultured in glass tubes containing A-1296 agar-solidified MSBN/2 medium without growth regulators. To obtain enough material for further studies, developing axillary shoots were routinely isolated from the elongated microcuttings and subcultured on the same medium. Shoots isolated from these proliferating cultures were first cultured for 60 days in glass tubes containing WPM medium or MSBN/2 medium supplemented with 0 or 0.45 µM TDZ or 2.22 µM Z. Subsequently, explants were transferred to their respective media without growth regulators. At least 10 microcuttings were cultured per treatment. To test the effect of genotype, 50 to 140 microcuttings from six 200-year-old C. libani trees (genotypes AL1 to AL6), growing in Reading, U.K., were cultured on A-1296 agar-solidified MSBN/2 medium without growth regulators. Axillary shoots that formed on these explants were subcultured on the same medium to promote their elongation and the proliferation of new buds. This procedure was repeated about every 2 months for 2 years. In all experiments, cultures were examined for survival and sprouting percentages and the mean number of axillary shoots per sprouted explant. Adventitious bud differentiation from explants of C. atlantica In a first experiment, excised needles from 3-month-old C. libani and C. atlantica seedlings were cultured (adaxial surface to the medium) in petri dishes containing MSH medium with or without 0.5 µM BA. Each treatment contained 20 replicates (20 dishes with five explants each) and culture time was 60 days. In a second experiment, freshly isolated C. atlantica embryos were cultured in petri dishes containing MSBN/2 medium supplemented with BA or Z (0, 2.2, 4.4, 6.6 or 9.0 µM). After 60 days, embryos were transferred to the same medium with or without 4.4 µM Z. Proliferating buds were isolated and transferred to fresh medium without growth regulators. Embryos were also cultured for 60 days on MSBN/2 or WPM medium supplemented with Z (4.4, 9.0 or 18.0 µM), and then subcultured as described previously. Each treatment contained five replicates (five dishes with five embryos each). In all experiments, media were solidified with A-1296 agar and cultures were examined for percent of explants with callus or buds and number of adventitious buds per explant. SHOOT PROLIFERATION IN CEDAR CULTURES 479 TREE PHYSIOLOGY ONLINE at http://heronpublishing.com Rooting Both axillary and adventitious shoots (longer than 1 cm) isolated from C. atlantica and C. libani proliferating cultures, previously maintained for 1 month on MSBN/2 without growth regulators, were used for rooting experiments. Unless otherwise stated, shoots with trimmed basal needles were cultured in glass tubes containing MBN/2 medium solidified with A-1296 agar and cultures were maintained in a 16-h photoperiod. In a first series of experiments, shoots were either cultured for 7, 15 or 30 days on medium containing IBA or IAA (0, 0.3, 2.5, 5, 10, 25 or 50 µM), or dipped in IBA (2.5 or 5 mM) in the presence or absence of 1 mM NAA for 1 s and 5 min, respectively. Alternatively, the basal ends of the shoots were treated with rooting powders (2.5 mM IBA in talcum or RAIFORT SF from RIBA Quimiagra SL, Granollers (Barcelona), Spain, a commercial formulation containing 53.7 mM NAA and fungicides). Sterile support systems (filter paper bridges on liquid medium, a mixture (1:1) of peat moss and perlite or vermiculite) were also tested. When appropriate, light was reduced from the rooting zone by covering the bases of vessels with aluminum foil. In all cases, shoots were transferred to the respective auxin-free support system for a 90-day culture period. In a second series of experiments, the effects of the addition of several ancillary compounds to media containing IBA or NAA (0, 6 and 12 µM) were tested: (1) 0.25 mg l –1 paclobutrazol, an inhibitor of gibberellin biosynthesis; (2) 3 or 6mMCa 2+ (as calcium gluconate); (3) 10 µM coumarin; (4) 2, 5, 10 and 20 µg l –1 triacontanol; (5) 1, 3, 5, 7 and 10 mg l –1 pectimorf; (6) 15 and 60 g l –1 sucrose; (7) 150 or 300 mg l –1 H3PO4 and 500 mg l –1 glucose; and (8) 0.5 and 1% conifer-derived activated charcoal. Shoots were cultured for 30 days in their respective media and subsequently transferred to basal medium for 60 days. Cultures including coumarin were kept at 26 and 20 °C. In a last experiment, shoots were infected with Agrobacterium rhizogenes LBA9402 strain. Basal shoot infection was performed either by dipping the basal end of the explants directly into the bacterial suspension or after the shoot had been wounded with a blade. In some cases, the bacterial suspension was injected into the basal cut surface of the shoots. After infection, explants were blot-dried between sterile filter paper and placed on medium with or without auxins (10 µM IAA or IBA), and incubated at 28 ± 1 °C in darkness for 24 h. Subsequently, the explants were transferred to medium containing 200 mg l –1 cefotaxime. In all experiments, rooting percentage and the number of roots per rooted shoot were recorded. Each treatment contained 10–12 replicates. Statistical analysis Significance of treatment effects was assessed by analysis of variance employing a completely random design. Percentage data were subjected to arcsine transformation before analysis. Variation among treatment means was analyzed by Tukey’s (1953) procedure. All experiments were conducted at least Downloaded from http://treephys.oxfordjournals.org/ by guest on June 5, 2013
Page 1: Tree Physiology 25, 477-486 © 2005
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Page 7 and 8: C. libani, respectively), none of t
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