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Charles F. Forney, Sharon J. Peterson, and Preston Hartsell

Callus tissue grown from `Marsh' grapefruit (Citrus paradisi Macf.) albedo tissue was grown at 30C for ≈ 40 days. Calli were preconditioned in normal air for 5 days at 10 or 30C before being fumigated for 2 hr with 0, 32, or 48 g of methyl bromide (MB)/m 3. Calli were then held at 10C and K+ leakage was measured after 1, 10, 20, and 30 days. The amount of K+ leaked from MB-fumigated calli was greater than that for nonfumigated calli and increased with higher MB dose. Leakage also increased with time following fumigation. Leakage of calli preconditioned at 30C and fumigated with 48 g MB/m3 was 140%, 196%, and 260% greater than leakage from nonfumigated calli 10, 20, and 30 days after fumigation, respectively. Leakage from calli preconditioned at 10C for 5 days before MB fumigation was less than that from calli held at 30C. MB doses of 32 and 48 g·m-3 increased leakage of calli preconditioned at 10C by 6% and 43% and for those preconditioned at 30C by 99% and 140%, respectively, 10 days after fumigation. In addition to K+ leakage, MB induced the development of a tan to orangish-brown discoloration.

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Ryutaro Tao and Akira Sugiura

Callus cultures were initiated in the dark from leaf primordia, stem internodes, and young leaves of adult Japanese persimmon (Diospyros kaki L.) to induce adventitious buds. A high frequency of regeneration occurred on Murashige and Skoog medium (MS) with half the normal NH4NO3 and KNO3 concentration (1/2N) and containing 10 μm zeatin or 1 μm 4PU-30 in combination with 0.1 μm IAA, or MS(1/2N) medium containing 0.03 to 0.1 μ m IAA or 0.01 to 0.03 μm NAA combined with 10 μm zeatin. No significant differences in the capacity of regeneration were observed among the calli from different explant sources. Only eight of 16 cultivars formed adventitious buds on MS(1/2N) medium containing 10 μm zeatin and 0.1 μm IAA, with the percentage of explants forming adventitious buds ranging from 2% to 72%. Chemical names used: indole3-acetic acid (IAA); 1-naphthaleneacetic acid (NAA); N-phenyl-N'-(2-chloro-4-pyridyl)urea (4PU-30).

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Erik C. Fargo and Jeffrey A. Adkins

Development of a reliable method for Hydrangea macrophylla (Thunb.) Ser. organogenesis is critical for developing an in-vitro mutagenesis protocol. Container-grown (11.8 L) H. macrophylla `Nikko Blue' plants were maintained in a controlled environment greenhouse, with supplemental lighting (1600 hr to 2400 hr mercury vapor lamp), fertilized with 65 g Nutricote total (18N–2.6P–6.6K, Agrivert, Inc., New York, N.Y.) and hand-watered. To reduce fungal contamination, stock plants were sprayed to run-off biweekly with Alliette WDG (375 mg·L-1, aluminum tris), Bayleton (250 mg·L-1, triadimefon), and Heritage (25 mg·L-1, azoxystrobin). Leaf explants were sterilized with 0%, 10%, 15%, or 20% bleach (5.25% sodium hypochlorite) (by volume) for 10 or 15 min, and stem explants were sterilized with 0%, 10%, 25%, or 50% bleach (5.25% sodium hypochlorite) for 10 or 15 min. About 97% of fungal contaminates were eliminated from leaf and stem explants when treated with 10% bleach for either 10 or 15 min. Leaves were plated on Gamborg B5 media at pH 5.7 containing 0, 2.5, 5, or 10 μM 2,4-D and 0, 0.25, 0.5, or 1.0 μM BAP and placed in a controlled environment growth room under a 14-h photoperiod or in a dark growth chamber. Callogenesis followed by root organogenesis was observed on explants treated with a variety of concentrations and combinations of 2,4-D and BAP. Strongest callogenesis was observed on media supplemented with 10 μM 2,4-D. A greater callus concentration was observed along the edges of dark cultured leaf discs. Indirect root induction was greatest on 10 μM 2,4-D.

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Wayne A. Mackay, Timothy J Ng, and Freddi A. Hammerschlag

Studies examining exposure methods and callus type were conducted to develop an in vitro selection system using roridin E as a selection agent. Vacuum infiltration of callus with the toxin solution was the only successful selection method at the concentrations tested. Primary callus (callus originating directly from the explant) was not sensitive to roridin A or E at the concentrations used. Secondary callus (callus produced from primary callus) exhibited a differential response to roridins A and E similar to that of detached-leaf assays. Electrolyte leakage studies of callus were not conclusive in establishing the membrane as the site of toxin action or useful for screening tolerance in vitro. A small percentage of callus from tolerant and susceptible cultivars survived repeated exposure to roridin E at 50 μg·ml-1.

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Chiu-Yueh Hung and Jiahua Xie

observed in some of their early subcultures, although they had been subcultured and maintained for several years ( Ziebur and Shrift, 1971 ). Synthetic auxin, 2,4-D, is a strong hormone that is widely used to induce callus formation ( Khanna and Raina, 1998

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Hiroko Sato, Tadashi Takamizo, Tsutomu Shimizu, Kiyoshi Kawai, and Koichiro Kaku

ranged from one to five ( Fig. 2 ). Some of the transgenic plants originated from the same callus showed the similar hybridization patterns, suggesting that they originated from the same transgenic cell. Fig. 1. Polymerase chain reaction (PCR

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M.M. Jenderek and A.J. Olney

The ability of Hibiscus syriacus explants to produce regenerable callus was investigated. Fragments of cotyledons, hypocotyl, and roots were cultured on MS media supplemented with two different auxins (2,4-D and NAA, both 0.3 mg/L) and three different cytokinin (BA, 2iP and kinetin, all 0.1 mg/L). Plants were regenerated on McCown media with three different cytokinins at two different concentrations (0.1 and 1.0 mg/L). The biggest volume of callus was produced on medium containing 2,4-D/2iP (2,821 mm3/explant). The smallest mass of callus was induced on medium with NAA/kinetin (120 mm3/explant). On BA-supplemented media, the auxin type had no significant influence on the amount of callus produced. The highest number of shoots and leaves was produced on callus induced on NAA/BA supplemented media and regenerated on medium with 0.1 mg/L BA (4.2 shoots and 20 leaves/explant). Callus cultured on medium with 1.0 mg/L of BA produced significantly less shoots (2.4 shoots/explant). The lowest number of shoots was observed on callus originating from NAA/kinetin and NAA/2iP callus media and grown on medium with kinetin or 2ip (both 1.0 mg/L). The highest number of roots was produced by cultures originating from NAA/BA callus medium grown on the BA regeneration medium, irrespective of the cytokinin level. The longest shoots were observed on medium supplemented with 1.0 mg/L BA (18 mm). In this study, the best plant regeneration results were obtained for callus initiated on NAA/BA supplemented medium and regenerated on medium with 0.1 mg/L BA, however, the highest production of callus was observed on medium with 2,4-D/2iP.

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Song Ping and Ellen B. Peffley

Callus of five onion genotypes representing two species. Allium cepa and A. fistulosum, and their interspecific hybrid were used for establishing suspension cultures. Cultures were derived from callus that had been maintained on solid media and routinely subcultured for four years and from callus induced within six months of this experiment. Long-term callus from which plants were routinely regenerated and newly-induced callus were composed of cells which were, for the most-part, meristem-like with higher mitotic indices than cells from long-term callus which had been maintained as callus but had lost us capability to regenerate plants, these cells were large with small nuclei. Callus from newly-induced and long-term regenerable cultures were selected for further studies. Eight liquid media with factorial combinations of plant growth regulators were tested. Cells cultured in BDS liquid medium supplemented with 0.5 mg/l ABA and 1.0 or 2.0 mg/l 2,4-D without e-BA had higher mitotic indices and plant regeneration percentages than did cells cultured in the same media without ABA and with 6-BA. Suspension cultures from A. fistulosum and interspecific hybrids with A. fistulosum produced the highest numbers of plants regenerated.

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Raymond P. Chée, Daniel I. Leskovar, and Daniel J. Cantliffe

Embryogenic callus growth of sweetpotato [Ipomoea batatas (L.) Lam.] was selectively enhanced by subculture on basal callus proliferation medium modified to contain 15 mm NH4NO3. Embryogenic callus production was doubled on basal callus proliferation medium modified to contain 60 mm K+, while nonembryogenic callus production was reduced 40%. Additions of up to 40 mm NaCl to basal callus proliferation medium did not affect callus proliferation. The development of embryos from calli subculture to embryo production basal medium was unaffected by the KCl or NaCl treatments of the callus proliferation phase. However, embryo production was increased by subculturing callus from callus proliferation medium containing 20 mm NH4 + to embryo production medium containing 10 mm NH4 + Our results demonstrate that changes in mineral nutrition, in addition to growth regulator differences between callus proliferation and embryo production media, are important factors in sweetpotato somatic embryogenesis.

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David C. Zlesak*, Corinne M. Radatz, and Neil O. Anderson

Haploid (2x) roses derived from modern tetraploid breeding lines would allow for crosses to diploid species at the diploid level. In addition, inheritance studies are easier at the diploid level, using diploids derived from tetraploids possessing economically important traits. Haploidization of 4x roses through anther culture has not been successful due to challenges in callus induction and shoot regeneration. This study investigates rose anther responses to recently reported methods that optimize in vitro adventitious shoot regeneration in rose leaves. Anthers of three cultivars (Akito, Grand Gala, and Orlando) were put in a two-step callus induction (CI) and shoot regeneration procedure with varying CI factors. Experiment one (E1) compared continuous light/dark and silver nitrate (0,30,60 mg·L-1) and experiment two (E2) used the optimal E1 treatment comparing two and four weeks on CI media. Twenty-five anthers per treatment per cultivar were used in E1 and n = 100 for E2. Although no adventitious shoots were generated, callus formed on anther tissue and frequency of formation was variable across treatments. Continuous light resulted in 100% lethality. Darkness and silver nitrate (30 or 60 μm) favored callus generation and significant differences for callus generation were found among cultivars. Darkness and 30 μm silver nitrate were used in E2. Two and four weeks on initiation media were not significantly different for generation of anther-derived callus. Identification of factors which optimize callus formation on rose anthers is a positive step toward reliably generating rose haploids.