A two-stage micropropagation system was devised for cranberries (Vaccinium macrocarpon Ait.). Shoot-tip explants taken from four cultivars of greenhouse-grown plants were placed on media composed of Anderson's major salts, Murashige and Skoog's (MS) minor salts and organics, plus various concentrations of 2iP, IBA, and GA3. In other experiments, explant source, salt formulations for media, and rooting treatments were studied. Optimal multiplication and shoot quality occurred when nodal explants taken from greenhouse-grown or micropropagated plants were placed on medium containing 150 μm 2iP, 1.0 μm IBA, and no GA3. Histological examination revealed that the initial response of nodes to culture is axillary bud proliferation, but adventitious shoot formation occurred after 4 to 6 weeks. Cultures that contained only axillary shoots were not evident unless low levels of 2iP were used, at which point only axillary buds present on the explants were released. Proliferated shoots could be rooted ex vitro without auxin treatment. Optimal rooting occurred under high-light conditions. Plants were transplanted to the field for comparison to conventionally propagated material. Chemical names used: gibberellic acid (GA3), N-(3-methyl-2-butenyl)-1H-purin-6-amine (2iP), 1H-indole-3-butanoic acid (IBA).
Michael Marcotrigiano and Susan P. McGlew
Jim Ault* and Sandy Siqueira
Shoot, root, and callus induction were examined in the North American lily, Lilium michiganense, in response to treatment with four auxins. Seed from controlled crosses were aseptically excised from slightly immature capsules and cultured in vitro on Murashige and Skoog basal medium and vitamins with 30 g/l sucrose, 7.0 g/l agar, and a pH = 5.7. Seed were maintained at 20 °C with a 14-h photoperiod. After 5.0-5.5 months, leaves and roots were removed from seedlings, the bulbs transversely sectioned, then the bulb sections cultured cut-surface down on the identical medium supplemented with 0.0, 1.0, 2.0, 4.0, or 8.0 μm dicamba, picloram, K-NAA, or 2,4-D. PGRs were added to medium prior to autoclaving except dicamba which was dissolved in 50% ethanol and added after medium autoclaving. 16 explants were utilized for each treatment. The experiment was conducted three times. Morphogenetic response (# of shoots produced, % of explants forming roots, and % of explants forming callus) was tabulated 4 months after treatment. Shoot formation was promoted by treatment with dicamba, picloram, and K-NAA in comparison to the control (2.5 shoots/explant). Shoot formation varied significantly in response to individual dicamba, picloram, and 2,4-D concentrations. A maximum of 7.9 shoots per explant was promoted by 4.0 μm K-NAA and 1.0 μm dicamba, respectively. Root and callus formation also varied significantly between auxin treatments. Root formation was inhibited by dicamba, picloram, and 2,4-D treatments in comparison with the control (100% rooting); callus formation was promoted by dicamba, picloram, and K-NAA treatments in comparison with the control (15% callusing).
Phillip A. Wadl, Timothy A. Rinehart, Adam J. Dattilo, Mark Pistrang, Lisa M. Vito, Ryan Milstead, and Robert N. Trigiano
Pityopsis ruthii is an endangered species endemic to the Hiwassee and Ocoee Rivers in Tennessee. As part of a recovery effort focused on P. ruthii, vegetative propagation and in vitro multiplication and seed germination techniques were developed. Plants were vegetatively propagated using greenhouse stock plants and wild-collected stems. Rooting occurred with and without auxin treatments but was greatest when 0.1% indole-3-butyric acid (IBA) talc was applied to the vegetative cuttings; rooting was lowest when flowering stems were used. Pro-Mix BX substrate provided the most consistent rooting. In vitro multiplication was accomplished by the removal of lateral shoots from in vitro-grown plants that were rooted on Murashige and Skoog (MS0) basal medium with 270 clones produced from a single individual after 4 months. Nineteen clones were transplanted and secured with bonded fiber matrix into their natural habitat and 14 survived for 1 year. To avoid genetic swamping of native populations with the introduction of large numbers of genetically identical individuals through clonal propagation, seed-based propagation efforts were explored. Open-pollinated seeds were collected, disinfested and germinated, and seedlings established on MS medium. Seeds were submersed in 70% ethanol for 1 minute and briefly flamed. Seeds were surface-sterilized in a range [10% to 50% (v/v)] Clorox® bleach solutions with vigorous shaking for 20 minutes, rinsed three times in sterile water, and germinated on MS0. Removal of pappus from seeds was required for successful disinfestations, but the bleach concentration was not critical. Successful propagation is a step toward the conservation and recovery of P. ruthii and should allow future reintroduction projects.
Alan G. Smith and Elizabeth S. Zimmermann
Euonymus alata is an attractive landscape plant that has been reported to be an invasive species. Genetic modification through transformation is a method of reducing its invasiveness by producing sterile cultivars having limited or no seed production. A critical step in Agrobacterium-mediated gene transfer is the production of adventitious shoots. E. alata internodes and leaves from in vitro cultures were tested for adventitious shoot production on 16 plant growth regulator combinations: four levels of 6-benzylamino purine (BA) and three auxin treatments [0.5 or 0.25 mg·L-1 indole-3-butyric acid and 0.1 mg·L-1 naphthaleneacetic acid (NAA)], as well as no auxin. The optimal BA levels were found to be 0.5 or 1.0 mg·L-1 for maximizing the number of explants forming shoots and for producing the greatest number of shoots per explant. Culturing on NAA gave the greatest number of shoots per explant with both 0.5 and 1.0 mg·L-1 BA. Shoot production from internode segments was markedly superior to leaves. An initial dark treatment of 10 days did not influence shoot production. Using 1.0 mg BA with 0.1 mg·L-1 NAA, E. alata internodes were transformed with A. tumefaciens EHA105 carrying Kanamycin resistance and β-glucuronidase genes. Transformed shoots were selected on 30 mg·L-1 Kanamycin. Of the 36 shoots produced, 16 were confirmed to be transformed by β-glucuronidase histochemistry. Treatment with rooting powder containing indole-3-butyric acid did not aid rooting of shoots, but after 3 months in soil in high humidity, 21 of 24 E. alata shoots from tissue culture were rooted and acclimated.
Christopher L. Rosier, John Frampton, Barry Goldfarb, Farrell C. Wise, and Frank A. Blazich
Two experiments were conducted to develop a protocol for rooting stem cuttings from 3-, 5-, and 7-year-old fraser fir [Abies fraseri (Pursh) Poir.] Christmas trees. The first experiment tested the effect of stumping treatments and tree age on shoot production and subsequent adventitious rooting. One auxin concentration [4 mm indole-3-butyric acid (IBA)] and a nonauxin control were tested. Stock plants were stumped to the first whorl (trees in the field 3 and 5 years) or the first, third, and fifth whorls (trees in the field 7 years). Intact (nonstumped) controls were also included for each age. The second experiment was designed to create a quantitative description of the effects that crown (foliage and above ground branches of a tree) position have on the rooting of stem cuttings collected from stumped and nonstumped trees. The exact position was determined by measuring the distance from the stem, height from the ground, and the degrees from north. Crown positions were recorded as cuttings were collected and then cuttings were tested for rooting response. The rooting traits assessed in both experiments included rooting percentage, percent mortality, number of primary roots, total root length, root symmetry, and root angle. In the first experiment, rooting percentage, primary root production, and total root length increased as the age of the stock plant decreased and the severity of the stumping treatment increased. Auxin treatment significantly increased rooting percentage, root production, root lengths, and root symmetry while decreasing mortality. Overall, the highest rooting percentages (51%) and the greatest number of primary roots (8.1) occurred when 3-year-old stock plants were stumped to the first whorl and treated the cuttings with 4 mm IBA. The greatest total root lengths (335 mm) occurred in cuttings from the 3-year-old stock plants. In the second experiment, rooting percentage was significantly affected by the position from which the cuttings were collected. Cuttings collected lower in the crown and closer to the main stem rooted more frequently than cuttings collected from the outer and upper crown.
Tracy S. Hawkins, Nathan M. Schiff, Emile S. Gardiner, Theodor Leininger, Margaret S. Devall, Dan Wilson, Paul Hamel, Deborah D. McCown, and Kristina Connor
. Analysis of data collected from the 0% auxin treatment and that of the initial rooting protocol (10% auxin treatment) showed that rooting percentages were significantly greater ( P < 0.0001) in the absence of auxins, than in the initial 10% auxin treatment
by commercial nurseries for landscape use in USDA hardiness zones 5b to 8a. An auxin treatment traditionally has been recommended for rooting cuttings. Blythe and Sibley (p. 771) found that terminal stem cuttings taken in winter rooted readily both
successfully without auxin treatment, thus eliminating one step in the propagation process and reducing chemical use. Rooting percentages of cuttings receiving no auxin treatment were comparable to cuttings treated with 2500 ppm IBA + 1250 ppm NAA in trials
winter. Blythe (p. 476) found that treatment of cuttings with a 1-second basal quick-dip in an auxin solution enhanced rooting percentage and total root length in comparison with nontreated cuttings. Auxin treatment produced no inhibitory effect on
equal or greater root growth compared to basal application of 1000 or 3000 ppm talc-diluted IBA. Both auxin treatments yielded greater root growth than the controls. Soil Carbon Reduces Toxicity of Topramezone to St. Augustinegrass Topramezone was