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Nevena Mitić, Mariana Stanišić, Jelena Milojević, Ljiljana Tubić, Tatjana Ćosić, Radomirka Nikolić, Slavica Ninković, and Rade Miletić

regeneration from leaf explants of the apple cultivar Golden Delicious and to establish an efficient in vitro shoot regeneration system for ‘Melrose’ as a prerequisite for successful genetic transformation aimed at incorporating genes for insecticidal proteins

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Josephina G. Niederwieser and Bela M. Vcelar

The physiological stage of donor plants determined to a great extent the morphogenic potential of Lachersalia (Jacq.) hybrid leaves, but the optimal stage for various cultivars was different. Contact of the bud-forming adaxial epidermal cells with the medium did not significantly stimulate in vitro bud formation on Lachenalia leaf explants, but resulted in the formation of callus from the buds of certain hybrids. Wounding on either the adaxial or the abaxial side of leaves had a stimulating effect on certain hybrids, but others did not respond significantly. A reduction in the length of explants from 10 to 3.3 mm resulted in an increase in the total number of buds formed by a specific amount of explant tissue (width of explant = 15 mm).

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Abraham Cruz-Mendívil, Javier Rivera-López, Lourdes J. Germán-Báez, Melina López-Meyer, Sergio Hernández-Verdugo, José A. López-Valenzuela, Cuauhtémoc Reyes-Moreno, and Angel Valdez-Ortiz

., 2005 ), zeatin ( Dan et al., 2006 ) and timentin ( Costa et al., 2000 ) were evaluated. Leaf explants (0.5 cm 2 ) from 4-week-old in vitro plantlets were placed in abaxial orientation (downside of the leaf touching the medium) on petri dishes with four

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Seong Min Woo and Hazel Y. Wetzstein

-grown material collected from mature specimens of different populations. Adventitious structures are induced on leaf explants placed on a medium with 10 μ m thidiazuron (TDZ) + 5 μ m indole-3-acetic acid (IAA), and shoot expansion proceeds upon subculture to a

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Aisu Gu, Wenfang Liu, Chao Ma, Jin Cui, Richard J. Henny, and Jianjun Chen

this study were to establish a new method of regenerating A. andraeanum cultivars from leaf explants and to study different light wavelengths (red, blue, yellow, and red + blue) emitted from LEDs for rooting and growth of adventitious shoots under in

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Manoj Singlachar, Robert R. Tripepi, and Mary W. George

Attempts to regenerate plants from leaf explants of Rosa xhybrida L. on Murashige and Skoog medium have met with limited success. We report improved regeneration of somatic embryos and adventitious shoots from leaves of `Golden Emblem' on N6 medium. Leaf explants were obtained from microshoots that had been in culture for 4 years. Leaves were placed on N6 medium containing various combinations of 0, 0.4, or 1.0 μM 2,4-D and kinetin for 20 days with an initial dark treatment of 12 days, then transferred to a medium without plant growth regulator. Adventitious shoots and somatic embryos were observed 3 weeks after transferring to medium without plant growth regulators. Leaf explants placed on media without 2,4-D failed to form embryos or shoots. The best combinations of 2,4-D and kinetin (0.4 and 0.4 μM, 0.4 and 1.0 μM, or 1.0 and 1.0 μM) induced regeneration percentages ranging from 21% to 39%. N6 appears to improve regeneration of somatic embryos and adventitious shoots from `Golden Emblem' leaf explants, but the interaction between media formulation and duration of exposure to 2,4-D and kinetin needs to be examined.

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Karim H. Al-Juboory and Jabar H. Al-Niami

Leaves of wild apple (Malus domestica Borkh) were excised from in vitro grown shoots transversely cut into halves and plated onto petri dishes containing regeneration media. Cultures were kept in the dark for three weeks before adventitious shoots were observed. Callus from leaf explants produced adventitious shoots after 3 months of in vitro culture. Callus were cultured on Nitsch and Nitsch medium supplemented with a range of BA (0.0–2.0 μm) and NAA (0.0–10 μm). BA at 10 μm combined with NAA (0.5 μm) proved most effective for stimulating shoot proliferation of cultured apple. Plantlets from tissue culture were easily transferred to the greenhouse environment.

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Mary W. George and Robert R. Tripepi

Previous reports of somatic embryogenesis on rose tissues involved an embryogenic callus stage with either a complicated multi-step process or low numbers of embryos being produced. We have produced somatic embryos without a callus stage from leaf explants of the cut rose cultivar `Golden Emblem' by using a two step process. Explants were obtained from microshoots of `Golden Emblem' that had been in culture for three years. All experiments were repeated twice. When explants were maintained on Murashige and Skoog (MS) with 0.4 μM NAA and 0.4 μM kinetin for 10 weeks, 10% or less of the explants produced somatic embryos. Keeping the explants on the NAA/kinetin medium for two weeks, then switching to medium with 0, 0.5, 1.0, or 10.0 μM kinetin for the remaining 8 weeks failed to increase embryo production. Decreasing the time the explants were on the NAA/kinetin medium to 8 or 12 days, and then placing explants on MS medium with 1.0 μM kinetin increased somatic embryo production to a maximum of 25%. By limiting the length of time the rose leaf explants were exposed to auxin, direct somatic embryo production was increased.

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X. Cao and F.A. Hammerschlag

As part of a program to develop transgenic highbush blueberry (Vaccinium corymbosum L.) cultivars, studies were conducted to determine optimum conditions for high efficiency shoot regeneration from leaf explants of shoots propagated in vitro. The effects on shoot organogenesis of age of explant source, length of dark treatment, the addition of either thidiazuron (TDZ) at 1 or 5 μm, or zeatin riboside at 20 μm to the regeneration medium, and a photosynthetic photon flux (PPF) of either 18 ± 5 or 55 ± 5 μmol·m–2·s–1 were investigated. A maximum of 13.0, 13.0, 12.6, and 4.6 shoots regenerating per explant for cultivars Duke, Georgiagem, Sierra, and Jersey, respectively, occurred on regeneration medium with zeatin riboside and under a PPF of 55 ± 5 μmol·m–2·s–1. `Duke' regenerated equally well on medium with either zeatin riboside or 1 μm TDZ, whereas the number of shoots per explant for `Georgiagem' and `Sierra' was significantly higher on zeatin riboside. Regeneration of `Duke', `Jersey', and `Sierra' on zeatin riboside was significantly better under a PPF of 55 ± 5 μmol·m–2·s–1 than under 18 ± 5 μmol·m–2·s–1, but the higher PPF inhibited regeneration of `Duke' on 5 μm TDZ. There were no significant differences in percentage of regeneration or the number of shoots per explant from leaf explants derived from either 1-, 2-, or 3-week-old shoot cultures, or when either 1 week or 2 weeks of darkness preceded light treatments. Chemical names used: 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ); 9-(-β-ribofuranosyl)-6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin riboside).

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James A. Kapaun and Zong-Ming Cheng

Four aminoglycoside antibiotics were evaluated for their effects on shoot regeneration from leaf explants of Siberian elm (Ulmus pumila L.) seedlings and their potential use as selective agents in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene. Kanamycin at 100 mg·L–1 or higher concentration reduced shoot regeneration, with complete inhibition at 225 mg·L–1, and was considered a suitable selective agent. Neomycin completely inhibited shoot regeneration at 450 mg·L–1, but all explants remained green; therefore, it may also be used as a selective agent. Geneticin significantly inhibited shoot formation at 1 mg·L–1 and completely killed the explants at 4 mg·L–1 after 1 week. Geneticin was too toxic for direct selection, but may be useful in a delayed selection scheme or for confirmation of transformation. Paromomycin was least effective in inhibiting shoot formation; 13% of explants still regenerated shoots on the medium with the highest concentration tested (400 mg·L–1). Both neomycin and paromomycin precipitated in media containing Phytagel as a gelling agent if antibiotic stock solutions were added to the medium without adjusting their pH. Precipitation was prevented by adjusting the pH of the stock solutions from 6.2 (neomycin) or 6.9 (paromomycin) to above 9, or by using agar as a gelling agent. The precipitation was not affected by the concentrations of salts in the media.