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Thomas Yeager, Claudia Larsen, and Gisele Martins

Multiple branched liners of Ilex vomitoria Ait. `Nana' were greenhouse-grown in 3-L containers with a 2 pine bark: 1 Canadian peat: 1 sand substrate. Plants were fertilized weekly with a solution of 50 N, 10 P, and 30 K (mg·L–1) for either 5, 10, or 15 weeks. Then plants for each of the three fertilizer durations were fertilized weekly with a solution of either 50, 150 or 300 N, 10 P, and 30 K (mg·L 1) for an additional 15 weeks, at which time root and shoot dry weights were determined. A control group of plants was fertilized weekly with 300 N (mg·L–1) for 30 weeks. Shoot dry weight increased linearly as fertilizer rate or duration of fertilization increased. Root dry weights increased linearly as fertilizer duration increased while root dry weights were not different due to fertilizer rate. These data indicate that duration of fertilization is important in promoting root and shoot growth; however, the largest amount of root and shoot dry weight resulted from the highest N application rate (300 mg·L–1) for the longest duration (30 weeks).

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Thomas H. Yeager

Multiple branched liners of `Mrs. G. G. Gerbing' azaleas (Rhododendron L.) were greenhouse-grown for 16 weeks in 3-liter containers with a common nursery medium. The growth medium of each plant was amended with either 0.5, 1.5, or 2.5 g N from Osmocote 14N-6P-11.6K and irrigated with either 920 ml water twice a week or evapo-transpiration (ET) plus 10%, 30%, or 50%. Shoot dry weights (35 and 35 g, respectively) for plants irrigated with ET plus 30% or 50% and fertilized with 1.5 g of N were larger than plants fertilized with 0.5 or 2.5 g N and irrigated with ET plus 10%, 30%, or 50%. Shoot dry weights of plants irrigated with ET plus 30% or 50% were similar to plants irrigated with 920 ml twice a week when plants received 1.5 g N. Plants that received 920 ml twice a week and 2.5 g N had larger shoot dry weights than plants irrigated with ET plus 10%, 30%, or 50% and fertilized with 2.5 g N. Shoot dry weights increased from 17 to 46 g for the 0.5 and 2.5 g N treatments, respectively, when plants were irrigated with 920 ml.

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Leigh E. Towill and Philip L. Forsline

The dormant vegetative bud method for cryopreservation has been successfully applied to many lines of apple. We examined this method for five cultivars (Kentish, Montmorency, Meteor, North Star, Schatten Morelle) of sour cherry (Prunus cerasus L.) with the aim of developing long-term storage at NSSL. Singlebud nodal sections (35 cm) were desiccated to 25%, 30%, or 35% moisture before cooling at 1°C/hour to –30°C and holding for 24 hours. Sections were then directly placed in storage in the vapor phase above liquid nitrogen (about – 160°C). Warmed samples were rehydrated and patch budded at Geneva to assess viability. Sections that were either undried, dried but unfrozen, or dried and cooled to –30°C survived very well. For samples then cooled to –160°C, highest viabilities for each line occurred with the 25% moisture level, although fairly high viabilities also were observed at 30% and 35% moistures. Cryopreserved buds from four lines directly developed into a single shoot; buds from Montmorency formed a shoot from a lateral within the bud, suggesting that the terminal meristem died but that axillary meristems within the bud survived and formed a shoot or multiple shoots. Nineteen lines were harvested in January 1996 for long term storage of sour cherry germplasm under cryogenic conditions.

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Charlotte M. Guimond, Preston K. Andrews, and Gregory A. Lang

Young sweet cherry (Prunus avium) trees are typically upright, vegetatively vigorous, and nonprecocious, taking 5 to 6 years to come into production. To produce fruit in high-density orchards by year 3 or 4, development of lateral shoots for potential fruiting is critical in year 2 or 3. An experiment was designed to promote lateral branching on 2-year-old trees. The experiment was conducted in a commercial orchard in Roosevelt, Wash., with `Bing' and `Van' on the vigorous rootstocks Mazzard and Colt. The trees were planted at 415 trees per acre with three scaffolds trained into a “V” canopy design. The experimental variables were treatments with and without Promalin (1.8% BAP plus 1.8% GA4+7), applied at a ratio of 1:3 in latex paint at green tip stage; superimposed on these treatments were either heading cuts of each scaffold to 2 m long (or tipping the scaffold if it was <2 m), removing four to five buds subtending the terminal bud, a combination of heading and bud removal, or controls. On trees that were not treated with Promalin, three additional treatments included either removing subtending buds at budbreak, or removing buds at multiple locations along the scaffold at green tip or at budbreak. New lateral shoots were counted 4 weeks after budbreak, and the quality of the shoots (shoot diameter and angle of emergence) was measured at the time of summer pruning. Interactions between Promalin, bud manipulation, and pruning will be discussed in relation to development of canopy structure.

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Narumi Matsuda*, Kanji Isuzugawa, Mei Gao, Tadashi Takashina, and Koichi Nishimura

The transformation of pears such as `Conference', `Doyenne du Comice' and `Passe-Crassane' has been attributed to the high regeneration frequency from leaf discs (71% to 97%; Leblay et al. 1991). However, it has been difficult to transfer desirable genes into cultivars with low-regeneration frequency such as `Silver bell' (35.4%) and `La France' (10.7%), which are the two major pear cultivars in Japan. In this study, we developed an Agrobacterium-mediated transformation system for `Silver bell' and `La France'. For `Silver bell', leaf discs derived from in vitro shoots were used as explants. The antibiotics for selection of transformants and elimination of Agrobacteria were investigated. In the most optimum condition, which is 30 mg·L-1 Kanamycin and 500 mg·L-1 Sulbenicillin, a 3.2 % transformation efficiency was obtained. However, no success was recorded in an effort to transform `La France' using leaf disc explants because of very poor regeneration frequency. Therefore, axillary shoot meristems were used as explants for transformation of `La France'. The conditions for antibiotic selection and elimination of Agrobacteria were also investigated. In 5 mg·L-1 Kanamycin and 375 mg·L-1 Carbenicillin, transformed shoots were produced at 4.8% efficiency. No chimera was observed in the transgenic shoots during a 2-year subculture period. Since the inoculated explants developed into multiple shoots during selection, it was thought that the problem of chimera might have been overcome. Therefore, this transformation method using axillary shoot meristem may be applicable to pear cultivars recalcitrant to regeneration from leaf disc. To the best of our knowledge, this is the first report of a transformation system in pear cultivars with low regeneration efficiency.

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Rebecca E. Scoville and Todd P. West

The objective of this study was to investigate the effects of multiple nutrient salt formulations and different plant growth regulator concentrations on initiation and proliferation of axillary shoot culture of tropical hibiscus (Hibiscus rosa-sinensis L.). Combinations of five thidiazuron (TDZ) concentrations (0, 10-6, 10-7, 10-8, or 10-9 M) in conjunction with two 6-benzylaminopurine (BA) concentrations (0, 10-5 M) and two indole-3-butryic acid (IBA) concentrations (0, 10-5 M) were compared to determine which plant growth regulator combination(s) would stimulate the proliferation of the most viable axillary shoots. Also, five nutrient salt formulations (MS, 1/2 MS; Macro MS, WPM, LP, or DKW) ranging from high to low salt formulations were studied to determine a suitable nutrient medium formulation for axillary shoot proliferation. Nodal explants that were 2 cm in length were used to initiate cultures and were maintained on the various medium treatments plus 30 g·L-1 sucrose and 7 g·L-1 agar at a pH of 5.8. Explants were incubated about 30 cm beneath cool-white fluorescent lamps that provide a photon flux of about 40 μM·m-2·s-1 for a 16-hour photoperiod at 25 ± 3 °C. Nodal explants were transferred every 3 weeks for a total culture period of 12 weeks. At each transfer date data were collected on node number, axillary shoot number and length. Initial results indicate that high nutrient salt formulations coupled with low TDZ concentrations performed better at axillary shoot initiation. Poor shoot elongation was observed and further research needs to be performed to address this issue.

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Eugene K. Blythe, Jeff L. Sibley, Ken M. Tilt, and John M. Ruter

In five experiments, singlenode cuttings of `Red Cascade' miniature rose (Rosa) were treated with a basal quick-dip (prior to insertion into the rooting substrate) or sprayed to the drip point with a single foliar application (after insertion) of Dip `N Grow [indole-3-butyric acid (IBA) + 1-naphthaleneacetic acid (NAA)], the potassium salt of indole-3-butyric acid (K-IBA), or the potassium salt of 1-naphthaleneacetic acid (K-NAA); a single foliar spray application of Dip `N Grow with and without Kinetic surfactant; or multiple foliar spray applications of Dip `N Grow. Spray treatments were compared with their respective basal quick-dip controls {4920.4 μm [1000 mg·L-1 (ppm)] IBA + 2685.2 μm (500 mg·L-1) NAA, 4144.2 μm (1000 mg·L-1) K-IBA, or 4458.3 μm (1000 mg·L-1) K-NAA}. Cuttings sprayed with 0 to 246.0 μm (50 mg·L-1) IBA + 134.3 μm (25 mg·L-1) NAA, 0 to 207.2 μm (50 mg·L-1) K-IBA, or 0 to 222.9 μm (50 mg·L-1) K-NAA resulted in rooting percentages, total root length, percent rooted cuttings with shoots, and shoot length similar to or less than control cuttings. Exceptions were cuttings sprayed with 0 to 2.23 μm

(0.5 mg·L-1) K-NAA, which exhibited shoot length greater than the control cuttings. Addition of 1.0 mL·L-1 (1000 ppm) Kinetic organosilicone surfactant to spray treatments resulted in greater total root length and shoot length. Repeated sprays (daily up to seven consecutive days) had no or negative effects on root and shoot development.

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Hak-Tae Lim, Haeng-Soon Lee, and Tage Eriksson

Plant regeneration ability of ginseng (Panax ginseng) via organogenesis was studied morphologically and anatomically. Compact callus was introduced from four different types of explants—leaf, petiole, flower stalk, and root—of in vitro-grown plantlets. Petioles were found to be the best material for callus induction. Calli induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (1.0 mg·L–1) and kinetin (0.1 mg·L–1) were conditioned for 2 weeks on the same medium. These calli differentiated into adventitious shoots when cultured on half-strength MS basal medium plus kinetin at 1.0 mg·L–1 and STS at 2.5 mg·L–1. An addition of GA3 (1.0 mg·L–1) and BA (1.0 mg·L–1) to MS basal medium, however, induced high-frequency in vitro flowering (86.1%) and multiple shoot budding, which affected the normal, complete development of plantlets. Plantlets with well-developed root systems were obtained 6 weeks after regenerated shoots had been transplanted to half-strength MS20 medium containing IBA at 0.25 mg·L–1. Nuclear DNA content was measured to check the stability of their ploidy level. Based on DNA flow cytometric analysis, all of the regenerants were typically diploids as were the mothers plants, indicating that nuclear DNA content remained stable during cell differentiation.

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Veronique Declerck and Schuyler S. Korban

Leaf segments of Prunus persica L. (peach) collected from greenhouse-grown plants and from micropropagated shoots were cultured on a basal medium containing half-strength Murashige and Skoog (MS), Staba vitamins, sucrose (30 g/1) and agar (6.5 g/l); medium adjusted to pH 5.6. The influence of 6 different growth regulators at 3 concentrations (5, 10, 15 μM) were investigated using leaf explants from proliferating shoots of 'Elberta Queen' peach. With thidiazuron (TDZ), compact and multiple green calli were obtained; with benzyladenine and zeatin, lower numbers of small sized calli were obtained; with kinetin, no callus development was observed. Among auxin treatments, both Dicamba and 2,4-D resulted in friable white and yellow calli. Most of the calli produced in all treatments were formed along the cut margins of the explants. In an another experiment, leaf explants of' Bellaire' (greenhouse) and `Elberta Queen' (in vitro shoots) were used to determine the influence of a large scale concentration of TDZ (3 to 23 |iM). Explants from greenhouse and in vitro leaves resulted in higher levels of callus development at TDZ concentrations of 8-13 μM. Higher TDZ levels resulted in necrosis of leaf explants. The-influence of different carbon sources on callogenesis was investigated. We observed more green and compact calli with glucose than with sucrose and fructose at 100 mM. The influence of the glucose at 10 different concentrations (30 to 300 mM) was also investigated.

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Leighan Howard, Philip Stewart, Amit Dhingra, Craig Chandler, and Kevin Folta

Cultivated strawberry (Fragari×ananassa) is a valuable crop, yet has benefitted little from recent advances in biotechnology and genomics. A high-throughput system for transformation and regeneration would hasten elucidation of gene function for strawberry and possibly the Rosaceae in general. In this report, a protocol for high-frequency octoploid strawberry transformation and regeneration is presented. The protocol uses leaf, petiole, and stolon as explants from a newly selected genotype, `Laboratory Festival #9'. This genotype was selected from progeny of a `Strawberry Festival' self-cross exclusively for its rapid regeneration and robust growth in culture. Direct organogenesis has been achieved from the leaf or from prolific callus with multiple shoots being visible in as few as 14 days. Over 100 viable regenerants may be obtained from a single leaf explant of about 3-cm2 size. This laboratory-friendly genotype allows high-throughput, statistically relevant, studies of gene function in the octoploid strawberry genetic background as well as generation of large transgenic populations.