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Guochen Yang, Carl Niedziela, and Zhongge (Cindy) Lu

The goal of this study was to expedite galax seed germination in vitro. Galax seeds were collected from Yancey County, N.C., at an elevation of about 1100 m. Aseptic cultures were established using the tiny rust-colored seeds. In vitro seed germination was achieved under different pH conditions (4.2, 5.0, and 5.8). Seeds cultured in the medium with pH 4.2 tended to germinate early with a better rate than those cultured with a higher pH of 5.0 or 5.8 at the very beginning. Gradually, seeds from media with pH 5.0 and 5.8 caught up in germination. Eventually, seeds from all pH treatments produced a very similar germination rate. Attempts to use the matted and scaly rhizomes and very tender new growth as explant materials to establish aseptic cultures were not successful, due to severe contamination. However, our observations suggested that the very tender new growth could be a good source of explants once the optimum sterilization time is established.

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Ikuo Miyajima, Adriana Kato, Juan Carlos Hagiwara, Diego Mata, Gabriela Facciuto, Silvina Soto, Alejandro Escandón, Marcela Mori, and Nobuo Kobayashi

2 Current address:Lab. Plant breeding, Fac. Life and Environmental Science, Shimane University, Matsue 690-8504, Japan. This research was funded by INTA–JICA project `The Horticulture Development Project in

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Erika Szendrák and Paul E. Read

The temperate native terrestrial orchids are endangered species. Their propagation from seeds poses specific problems. It is well known that orchid seeds are devoid of endosperm and in nature they need microscopic fungi in a symbiotic relationship for germination. We developed a successful asymbiotic in vitro culture method for germinating seeds of several temperate orchid species and for maintaining the cultures of young plantlets. The medium used for both germination and seedling culture was a modified FAST medium. Seeds were surface-disinfested for 10 minutes in a 10% calcium hypochlorite solution. After sowing, the cultures were kept under dark condition at 10–12°C for 4 weeks. After that the cultures remained in the dark, but the temperature was raised to 25–26°C until germination occurred. Thereafter cultures required alternating seasonal temperatures: 25–26°C from the beginning of April to the end of September and 17–19°C from October to March. For the development of the young plantlets natural dispersed light and prevailing day-length was favorable. After 2 years of aseptic culture they were suitable for transfer ex vitro. Different stages of seed germination and plant development were observed using a scanning electron microscope and will be included in this presentation. Further observation of the effects of different environmental factors is currently under investigation.

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Amy J. Moberg, James J. Luby, Carl J. Rosen, and Peter D. Ascher

Accessions of Vaccinium species (deliciosum, ovalifolium, membranaceum, parvifolium, scoparium) were evaluated for tolerance to higher pH in the root zone using an in vitro screening procedure. Seeds were germinated on media containing all essential nutrients with nitrogen in the nitrate form at pH 5 and pH 6 and evaluated for 21 weeks. Excess EDTA was used to buffer the micronutrients and pH was buffered by MES and succinic acid. Germination varied among species with V. ovalifolium being highest and V. parvifolium not germinating at all. Mortality was lower at pH 5. At pH 6, V. ovalifolium and V. membranaceum exhibited variation for growth while all other species suffered complete mortality.

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Ing-Jiun Tom Wu, G.L. Wheeler, and F.H. Huang

Scarification treatments (a control, a 10-minute vacuum, or a 1.5-minute ultrasound), different media (modified Norstog and Van Waes) and growth regulators [benzyladenine (BA) at 0, 1, 1.5, or 2 mg·L-1 and 6-(r,r-dimethylallylamino)-purine riboside (2iPR) at 0, 1, 1.5 or 2 mg·L-1] were used in combination to increase seed germination of Cypripedium calceolus var. parviflorum. Seeds treated with ultrasound had higher germination (58.0%) than those treated with vacuum (27.4%) or controls (19.2%). Germination rates increased with 2iPR level and reached a maximum between 1.5 and 2 mg·L-1. Seeds on Van Waes medium, which were not transferred to fresh medium after germination, had a severe browning problem causing many protocorms to die. Those on Norstog medium continued to grow into seedlings with less browning. Germination rates of Calopogon tuberosus × Calopogon `Adventure' and Liparis liliifolia were determined on the different media and growth regulator treatments. Multiple shoots of Calopogon developed from single seeds on media containing growth regulators. Flower buds formed in vitro on Calopogon in media containing 1 mg·L-1 or higher BA 5 months after germination. L. Iiliifolia seeds in Norstog medium had a higher proportion of germination than those in Van Waes medium.

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Marìa Andrade-Rodrìguez, Angel Villegas-Monter, and M. Alejandra Gutièrrez-Espinosa

Polyembryony is an important characteristic for citrus that allows them to be propagated clonally through seed. Even when it is genetically controlled by a quantitative trait, the environment in which the seed is developed can affect it. The aims of this investigation were to evaluate polyembriony in two citric rootstocks in two harvest cycles and embryo germination of polyembrionic seeds. Embryos of 300 seeds of Citrus volkameriana and C. amblycarpa were counted and measured in Summer-Fall and Winter 1998 and 1999, respectively; embryo of 50 seeds of both rootstocks were germinated in vitro. The number of embryos per seed was 1.9 and 1.6 in C. volkameriana and 4.7 and 5.7 in C. amblycarpa. In C. volkameriana, we observed 42% of monoembryonic seeds during summer-fall and 67% in winter, whereas in C. amblycarpa 5.0 and 4.1% were detected, respectively. Only embryos that were larger than 1 mm long germinated. Even when germination takes similar time (5 to 6 days), further growth is faster in larger embryos (5 to 10 mm) than smaller ones. Therefore, size of embryos would need to be considered for propagation purposes.

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S. Kukkurainen, A. Leino, S. Vähämiko, H.R. Kärkkäinen, K. Ahanen, S. Sorvari, R. Rugienius, and O. Toldi

The occurrence of bacteria in different tissues was studied using field-grown strawberries, in vitro-grown strawberries, wild strawberries, and aseptically germinated strawberry seedlings. Strawberry has a number of endophytic bacteria in its the internal tissue, most of which appear to be nonpathogenic. In the in vitro-grown strawberries, all identified isolates were in the genus Pantoea. In field-grown garden and wild strawberries the most common genera were Pantoea and Pseudomonas. Location of eubacterial inhabitants within strawberry tissue sections was studied by in situ hybridization. Bacteria were detected in flower stalks, leaf stalks, leaves, stolons, berries and aseptically germinated seedlings. The existence of bacteria in seeds and seedlings suggests that bacteria are able to move up to the generative tissue and, ultimately, to the next generation, forming a symbiosis-like chain of plant-bacteria coexistence.

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M. L. Meyer and F. A. Bliss

Kiwifruit (Actinidia deliciosa) is a functionally dioecious plant where fruit size is dependent on number of seeds set. Pollen fertility was estimated in 1990 and 1991 by percentage stainability and percentage germinability in vitro. Profiles of the isozymes AAT, GPI and PGM were used to assess if any large differences in pollen fertility could be attributed to genotypic variation. Based on these three isozymes, eight different genotypes were discovered. Although significant differences were found among vines within orchards and among orchards, all vines can be considered good pollenizers (stainability > 87%). A positive correlation was found in 1991 between percentage stainability and percentage germination.

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Michael E. Compton

Several methods have been published on shoot regeneration from watermelon cotyledon explants. The major differences in regeneration protocols include the light environment in which seeds are germinated and the cotyledon region used. The purpose of these experiments was to compare the two main protocols for plant regeneration and develop one general procedure. To fulfill this objective, seeds were germinated in vitro in darkness or 16-hr light photoperiod for 7 days. Cotyledon explants from four watermelon cultivars (`Crimson Sweet', `Minilee', `Sweet Gem', and `Yellow Doll') were prepared from both dark- and light-grown seedlings. Apical and basal halves were obtained by making a cut across the cotyledon width. Apical and basal quarters were made, for comparison, by cutting apical and basal halves longitudinally. All explants were incubated on shoot regeneration medium for 6 weeks followed by a 3-week cycle on shoot elonga-tion medium. The percentage of cotyledons with shoots was 1.7-fold greater for cotyledons derived from seedings incubated in darkness than those germinated in light. Shoot formation was about 10-fold greater for explants from cotyledon basal halves and quarters than apical halves and quarters. According to these results, the best watermelon regeneration protocol should consists of basal explants from in vitro-germinated seedlings incubated in the dark for 7 days.

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M.R. Pooler and R. Scorza

publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact.