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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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Jin Cui, Juanxu Liu, Jianjun Chen, and Richard J. Henny

Flash’ produced in 15-cm pots from liners regenerated using the indirect shoot organogenesis method established in this study, in which each pot has only one shoot and no distorted leaves. As a result of its unique color and adaptation to low light

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Andrea Swanberg and Wenhao Dai

regeneration. Periwinkle organogenesis was first reported in the late 1970s by Dhruva et al. (1977) followed by Ramavat et al. (1978) and Abou-Mandour et al. (1979) . However, the shoot regeneration rate was low. In 1989, Mollers and Sarkar induced

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Marjana Galperin, Aaron Zelcer, and David Kenigsbuch

BU-21/3 is an inbred melon (Cucumis melo L.) genotype that exhibits superior in vitro capabilities for adventitious organogenesis. In order to characterize the inheritance of the regeneration competence in BU-21/3, this line was crossed with PMR45 or `Ananas-Yokneam'—two reticulatus cultivars virtually incompetent for organogenesis. F1, F2, and backcross generations were produced and evaluated for adventitious regeneration competence. Our results indicate that the regeneration trait in BU-21/3 is controlled by a single dominant locus, without cytoplasmic interactions. This locus may become a valuable tool for imperative improvements in future protocols for transgenic breeding of commercial melon genotypes.

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Mark P. Bridgen, Masood Z. Hadi, and Madeleine Spencer-Barreto

A laboratory exercise on direct and indirect organogenesis from leaf explants is presented for students of plant tissue culture or plant propagation. Torenia fournieri, the wishbone flower, is used for this laboratory exercise because the in vitro production of adventitious shoots from Torenia is easy to control, seeds are easy to obtain, and plants are easy to grow. Direct shoot organogenesis results from leaf explants without an intervening callus phase, and indirect shoot organogenesis is possible after 4 to 6 weeks of callus production from leaf explants. The basal medium for all forms of organogenesis contains Murashige and Skoog (MS) salts and vitamins, 30 g sucrose/liter, and 7 g agar/liter at pH 5.7. To obtain direct shoot organogenesis, leaf explants should be placed on the MS basal medium with 1.1 μM (0.25 mg·liter-1) 6-benzylaminopurine (BAP) and 0.25 μM (0.05 mg·liter-1) indole-3-butyric acid (IBA). If leaf explants are placed on MS medium with 2.3 μM (0.5 mg·liter-1) 2,4-dichlorophenoxyacetic acid (2,4-D), callus formation will occur. Callus can be subcultured onto a MS medium with 8.88 μM BAP (2.0 mg·liter-1) plus 2.5 μM IBA (0.5 mg·liter-1) for indirect shoot organogenesis to occur.

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James S. Busse, M. Figueroa-Cabanas, and D.P. Stimart

Developmental anatomy of adventitious shoot formation in vitro from snapdragon (Antirrhinum majus L.) hypocotyls was investigated using two inbred lines, the most (R) and least (NR) regenerative lines selected from screening (Schroeder and Stimart, 1999). Time course analysis indicated cell division in the most regenerative line occurred first in one or a small number of epidermal cells with periclinal and anticlinal divisions within 2 days of placing hypocotyls on induction medium. Subsequently, cortical then vascular cells were recruited beneath the dividing epidermal cells. Once shoots formed, their vascular system was continuous with the original hypocotyl explant. The least regenerative line had no cell division directed towards organogenesis through 6 days. Shoot formation on snapdragon hypocotyls was adventitious in origin, by direct organogenesis and genotype dependent.

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Sharon Bates and John E. Preece

At three different times throughout the growing season, white ash seeds representing different maturity levels were transversely dissected, surface sterilized, and placed on agar-solidified MS medium containing 10 μM thidiazuron (TDZ) and a factorial combination of 0, 1, and 5 μM 2,4-D and 0, 0.5, and 1 μM glyphosate. After four weeks, explants were transferred to MS medium containing 5 μM BA and 5μM NAA. After 12 weeks in vitro, the greatest percent (60%) of explants with organogenesis (adventitiously produced leafy structures that could grow into shoots) was when seeds were exposed to 1 μM 2,4-D. Organogenesis occurred on explants only exposed to TDZ (31%), therefore auxin was not necessary for, but enhanced organogenesis. Seed explants were not consistently stimulated, or inhibited by these levels of glyphosate. Compared to immature-seeds, mature see explants were the most organogenic (67%) and had the greatest amount of adventitious shoots (14%).

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S.O. Park, L.H. Chen, S.K. Dhir, and A.S. Bhagsari

The objective was to determine optimum conditions for embryogenic callus, embryo, organogenesis, and embryogenesis developed from leaf, petiole, stem, and tip tissues of the sweetpotato `Jewel' cultivar and from subcultured callus. Embryogenic callus was developed from stem and tip tissues on MS medium containing combinations of BA and NAA only under light conditions. Plant regeneration via organogenesis was developed from stem and tip tissues on medium including 1, 3 and 4 mg/L BA under dark and light conditions, while no plant regeneration via organogenesis was developed from leaf and petiole tissues. Frequencies for plant regeneration via organogenesis from the tissues were very low. No plant regeneration via embryogenesis was developed from the four tissues on medium having any combinations of BA+NAA and of kinetin+NAA. Embryogenic callus was observed in the subculture of callus developed from petiole and tip tissues on medium containing 0.2 and 2 mg/L 2,4-D only under dark conditions. Embryo was found in the subculture of callus from the tissues on medium containing 0.2 mg/L 2,4-D only under both conditions. Plant regeneration via embryogenesis was obtained in the subculture of callus from the tissues. Plant hormones and other factors affecting plant regeneration from the four tissues of the `Jewel' cultivar and other elite cultivars are currently being investigated at our lab for its application in transformation.

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Paula P. Chee

A procedure for the regeneration of muskmelon (Cucumis melo L.) cv. Topmark via shoot organogenesis from cotyledon explants is described. The best induction medium for a morphogenic response was MS salts and vitamins medium with BA at 1.0 mg·liter-1. Further vegetative bud development was completed by transferring organogenic tissue to MS medium containing BA at 0.05 mg·liter-1 . The shoots were rooted in MS medium containing NAA at 0.01 mg·liter-1. Morphologically normal plantlets were obtained. Chemical abbreviations used: 6-benzylaminopurine (BA); indoleacetic acid (IAA); naphthaleneacetic acid (NAA).

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Sheila M. Colby, Adrian M. Juncosa, James A. Stamp, and Carole P. Meredith

The developmental anatomy of direct shoot organogenesis from in vitro leaves of Vitis vinifera L. cv. French Colombard was studied by light microscopy. Regenerating petiole stubs of leaf explants were fixed at intervals and were sectioned longitudinally to determine the developmental sequence of direct shoot organogenesis. After 6 days, three distinct regions of meristematic activity were apparent within expanding petiole stub: the wound-response, organogenic, and vascularization regions. In the organogenic region, divisions of vacuolate outer cortical cells formed nodular bumps that sometimes became adventitious leaves. Promeristems, which had the potential to become adventitious shoot meristems, were also initiated asynchronously in the organogenic region. Promeristem initiation occurred by two or several synchronous cell divisions occurring in the epidermal and subepidermal cell layers. Adventitious shoots and leaves developed new vascular bundles that connected to the pre-existing vascular bundles of the explant.