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Rida A. Shibli and M.A.L. Smith

Ohelo (V. pahalae Skottsb.) and bilberry (V. myrtillus L.) shoots were regenerated via direct organogenesis from whole leaves and leaf sections and also from hypocotyl explants of bilberry. Explants preincubated for 1 to 2 weeks in darkness yielded ≈75% regeneration frequencies and the highest number of regenerating shoots/explant on TDZ-supplemented media (0.9 to 2.7 μm). When 2iP or zeatin were substituted as the cytokinin source, frequencies of regeneration and shoot productivity were significantly lower. Explants held under constant illumination (no dark pretreatment) had significantly lower regeneration frequencies in all tested cytokinin-supplemented media. 2,4-D stimulated callus formation, but did not support regeneration from vegetative explants. Cells from callus and suspension cultures did not exhibit regeneration in any of the media that supported organogenesis from leaves. Regenerants were successfully micropropagated, although callus formation caused by zeatin and high 2iP levels interfered with shoot proliferation. Zeatin induced hyperhydricity in shoots from both species, but more severely in ohelo. Ex vitro rooting after treatment with 4.9 μm IBA or 5.4 μm NAA was 95% and 60% successful for bilberry and ohelo, respectively, and plants were readily acclimatized after an interval in a fog chamber. Bilberry microshoots also rooted in vitro in the absence of growth regulator treatment. Chemical names used: 1H-indole-3-butanoic acid (IBA); N-(3-methyl-2-butenyl)-1-H-purine-6-amine (2iP); 6-furfurylaminopurine (kinetin); 1-naphthaleneacetic acid (NAA); thidiazuron=1-phenyl-3-(1,2,3-thiadiazio-5-yl)urea (TDZ); 2,4-dichlorophenoxyacetic acid (2,4-D); 6-(4-hydroxy-3-methylbut-2-enylamino) purine (zeatin).

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Kullanart Obsuwan, Wayne B. Borth, John Hu, and Adelheid R. Kuehnle

A Cymbidium mosaic virus movement protein gene with a site-specific mutation (mut11) under control of a ubiquitin promoter was inserted using biolistics into two Dendrobium varieties with the intention of creating CymMV-resistant orchids. Presence of the transgene in regenerated plants of D. × Jaquelyn Thomas `Uniwai Mist' and D. x Jaq–Hawaii `Uniwai Pearl' was confirmed by PCR using genomic DNA, and mut11-positive plants were potted ex vitro. Forty-two transgenic plants and four non-transgenic control plants at the 4- to 6-leaf stage were inoculated with a 1:1000 dilution of CymMV obtained from infected orchids. Plants were analyzed for systemic infection using tissue blot immunoassay (TBIA). Seventeen plants from at least six independent transformations remained virus-free, whereas all control plants were infected with CymMV within 1 month. Further analysis by RT-PCR showed that the mut-11 mRNA was detectable in only two of these 17 plants. All plants were challenged again with a second CymMV inoculation as above, followed by TBIA analysis after 1 month. Thirteen of 17 plants remained free from virus. A third challenge of these plants with CymMV as above was followed by TBIA analysis at 1 week, 2 weeks, 1 month, 3 months, 6 months, and 12 months after challenge. Results at 2 weeks post-inoculation showed that all six controls and four individual transgenic plants, including the RT-PCR-positive plants, became systemically infected. Nine transgenic plants from both varieties remained free from CymMV 12 months after the third challenge. Lack of detectable mut11 mRNA in these resistant lines suggests that a post-transcriptional gene silencing (PTGS) mechanism may be conferring resistance to CymMV.

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Youping Sun, Donglin Zhang, and John Smagula

room. The results presented for multiple shoot proliferation were the means of 36 explants with ± se , whereas those for rooting experiments were the means of three individual experiments with ± se 36 explants per experiment. Because ex vitro rooting

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Elise A. Konow and Yin-Tung Wang

Presently, there are no standards for producing Phalaenopsis Blume (the moth orchids) as a flowering, potted crop. Determining optimal irradiance for in vitro and greenhouse production will help optimize growth and flowering. Four-month-old, aseptically propagated Phalaenopsis Atien Kaala seedlings with 1.0 cm leaf spread were transferred to a sterile agar medium in November 1995. They were placed under 10, 20, 40, or 80 μmol·m-2·s-1 photosynthetic photon flux (PPF) from cool-white fluorescent lamps. In June 1996, plants grown under 40 or 80 μmol·m-2·s-1 in vitro PPF had 38% greater fresh weight (FW), wider leaves, and more roots than those under the two lower PPF levels. Plants from each in vitro PPF were then transplanted and grown ex vitro in a greenhouse (GH) under high, medium, or low PPF, representing 12.0%, 5.4%, or 2.6% of full sunlight, respectively. Full sunlight at this location was 2300 and 1700 μmol·m-2·s-1 in August 1996 and January 1997, respectively. In November 1996 and June 1997, plants that had received 40 μmol·m-2·s-1 in vitro PPF and then grown under the high or medium GH PPF had the greatest FWs. Overall, plants under the high, medium, or low GH PPF had average FWs of 61, 36, or 17 g, respectively, in June 1997. By mid-September 1997, plants had increasingly larger leaves and higher concentrations of malic acid, sucrose, and starch as GH PPF increased. Leaf glucose and fructose concentrations remained constant as GH PPF increased; however, sucrose level doubled and malic acid concentration increased by nearly 50% from the low to high GH PPF. Each doubling in GH PPF more than doubled plant FW. Plants grown under the high, medium, or low GH PPF had 98%, 77%, or 2% flowering, respectively, in Spring 1998. Anthesis occurred 2 weeks earlier under the high GH PPF. Plants grown under the high GH PPF had twice as many flowers and larger flowers than those grown under the medium PPF.

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Doina Clapa, Claudiu Bunea, Orsolya Borsai, Adela Pintea, Monica Hârța, Răzvan Ştefan, and Alexandru Fira

selected plantlets (five jars per treatment per cultivar) were measured. Ex vitro rooting and acclimatization. The shoots obtained in the multiplication stage were acclimatized in a nonsterile mixture of peat [Klasmann-Deilmann, Lithuanian Peat Moss Special

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Celina Gómez, Megha Poudel, Matias Yegros, and Paul R. Fisher

, 2020 ). Gibson et al. (2020) found that industry stakeholders are particularly interested in acclimating tissue culture (TC) transplants indoors. That is because the ex vitro acclimation process requires TC transplants to adjust to rapid changes in

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Marija Perić, Slavica Dmitrović, Suzana Živković, Biljana Filipović, Marijana Skorić, Ana Simonović, and Slađana Todorović

the critical step for ex vitro survival and preservation of this endangered species has been efficiently overcome. The acclimatized plants were normal without any morphological abnormalities or variations ( Fig. 1H ). Supplementation of sugars to

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Juan Bernardo Pérez-Hernández and María José Grajal-Martín

blackening (not shown). Under liquid incubation, on the other hand, both static and agitated culture systems ( Fig. 1B–E ) promoted embryo growth and consequent complete in vitro plantlet formation ( Fig. 1F–H ), allowing the ex vitro acclimatization of

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Alejandro Martínez Palacios, Raúl Cárdenas Navarro, Diana Beatriz Hernández Ortega, and Víctor Chávez Avila

experiment. Four weeks after establishment, root formation was evaluated considering the number of shoots with at least one visible root (≈3 mm). Ex vitro plantlet establishment. Two weeks after the evaluation of root formation, the plantlets were established

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Ilse-Yazmín Arciniega-Carreón, Carmen Oliver-Salvador, María-Guadalupe Ramírez-Sotelo, and Carlos Edmundo Salas

with primary thin roots induced on B5 medium plus 2 mg·L −1 IBA, and ( C ) Roots from plantlets 5–6 cm length, ( D ) Plantlet with primary thin roots induced in B5 medium plus 3 mg·L −1 IBA, ( E ) ex vitro in soil acclimatization of regenerated