Abstract
Douglas maple (Acer glabrum Torr. var. douglasii Hook. Dipple [Aceraceae]) is a potentially valuable landscape species with significant propagation issues. Micropropagation has potential to resolve these issues and allow production of marketable plants at commercial quantities. A research project was initiated to refine procedures for successful tissue culture multiplication of Douglas maple. A series of experiments were designed to optimize factors that impact propagation efficiency and efficacy, including: explant establishment, cytokinin type and concentration for enhancing multiplication, length of subculture period, explant density in culture, light intensity, nutrient formulation of culture medium, rooting methods and their effect on acclimatization, and auxin type and concentration for rooting explants. Establishment (decontamination and initiating shoot growth) was accomplished by surface sterilizing explants with a 20% bleach solution (v/v) and then culturing on medium made with DKW medium salts and zeatin. This surface sterilization method resulted in 46% contamination-free explants and many shoots. Cytokinin compound and concentration optimization was conducted to maximize shoot growth during multiplication. Three experiments were conducted, in which, benzyl-adenine (BA), kinetin, meta-topolin (MT), thidiazuron (TDZ) and zeatin were all tested at various concentrations. The cytokinin concentration and compound producing the longest average shoots (39 mm (1.54 in) in a 31 day subculture was 2 µM MT. An experiment testing shoot growth rates was conducted to optimize subculture length. Two different genotypes were tested over 6 weeks in culture. The shoot growth of the fastest growing genotype was modeled linearly by Y = (1.456) X – 7.937, where X = days in culture, and Y = shoot length (mm), indicating that a subculture of 6+ weeks is best for resource and time efficiency. An explant density experiment was conducted to maximize resource efficiency. Explant densities at 4, 6 and 8 explants per tissue culture vessel (GA-7) failed to significantly affect shoot length, number of nodes or dry weights, meaning 8 explants per vessel is likely more efficient. A light intensity experiment tested 3 light intensities (7, 17 and 37 µmols•m-2•s-1 (0.65, 1.58 and 3.44 µmols•ft-2•s-1) photosynthetic photon flux) for their effect on shoot health. The highest and lowest light intensities were significantly different from each other in average dry weight (lowest light treatment had lowest weight), and plants exposed to the highest light intensity exhibited interveinal chlorosis. Deficiency symptoms such as chlorosis and shoot tip necrosis of Douglas maple plantlets grown on Driver Kuniyuki Walnut (DKW) medium salts prompted nutrient experimentation. Informal experimentation with 2x iron (relative to Fe content in DKW medium salts) remediated the chlorosis issue. Standard commercial salts (DKW, Murashige and Skoog (MS) and Woody Plant Medium (WPM) medium salts) with supplemental iron were tested for their ability to rapidly grow shoots, increase node number, and increase dry weights. The DKW medium was statistically superior at promoting shoot growth and the most number of nodes. Another experiment tested various fortifications to DKW medium with supplemental iron and was primarily focused on remediating the shoot tip necrosis. Additional boric acid (12.0 mg/l (0.0016 oz/l)) significantly improved shoot length (51 mm (2.01 in)) in comparison to control DKW medium with supplemental iron (29 mm (1.14 in)), and also reduced necrosis issues over time. A final nutrient experiment tested whether nutrient ratios within Acer glabrum sap were appropriate for formulating medium. The sap nutrient ratio medium performed slightly worse than the other control treatments included in this experiment, but not statistically worse. Sap-based nutrient ratio formulation showed potential, though much experimentation is still needed to improve the methods and strategies. In vitro and ex vitro rooting procedures were tested for their ability to root high percentages of explants, and to efficiently acclimatize plantlets. The in vitro rooting factors tested were: auxin type (indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or both in combination), light vs dark, apical vs nodal explants, half vs full strength DKW medium salts, IBA concentration (0, 1, 2, 4 and 8 µM), and the length of the explant (2.0 to 3.0 cm (0.79 to 1.18 in), or 3.5 to 4.5 cm (1.38 to 1.77 in)). The best in vitro rooting treatment, which was 1 µM IBA in half-strength DKW regardless of explant length, yielded 75% survival after rooting and acclimatization. Ex vitro rooting treatments included explant length as a covariate and a treatment (concentrations of IBA (0.1 and 0.3%) in talcum based rooting powders, and a control (water) group). The most successful treatments were 0.1 and 0.3% IBA talcum powder, both yielding a 92% survival rate after rooting and acclimatization. Ex vitro rooting methods were deemed better because of better time and resource efficiency (1 month of vitro culture subtracted), and higher survival rates after rooting and acclimatization. A second ex vitro experiment involving another Douglas maple genotype further validated the effectiveness of ex vitro rooting using 0.1% IBA talcum powder. Overall, these experiments have optimized micropropagation methods such that an estimated 12 fold multiplication of explants can be achieved every 6 week subculture, and only 8% of plantlets fail to survive after the rooting and acclimatization stages.