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Hak-Tae Lim, Haeng-Soon Lee, and Tage Eriksson

Plant regeneration of ginseng has been known to be difficult, and there are a few reports on plant regeneration of ginseng via somatic embryogenesis. In vitro flowering has, however, been one of the major drawbacks in these regeneration systems in which BA and GA3 were included in germination and shoot multiplication media. Multiplication of adventitious shoots from a single somatic embryo, abnormal morphology, and vitrified shoots were also observed. All these facts have made successful acclimatization of ginseng plantlets difficult. The purposes of this study were 1) to establish the plant regeneration system via organogenesis, 2) to improve normal plant regeneration via somatic embryogenesis, 3) to improve the efficiency of plant regeneration from protoplast culture, 4) to understand the acclimatization process, 5) to develop effective genetic transformation protocol. Data in relation with all these studies are presented in detail.

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Lynn M. Long, John E. Preece, Gerald R. Gaffney, J.W. Van Sambeek, and David A. Lightfoot

Genetic transformation studies are aided by use of selection agents, such as antibiotics or herbicides. To determine the level of kanamycin to be used as a selection agent, cotyledonary stage somatic embryos from J. nigra lines J26 and J28, J. nigra × J. hindsii line S11, and J. regia line SU2 were placed on gelrite solidified WPM with 1 g/liter casein hydrolysate and 250 mg/liter cefotaxime and 3% (w/v) sucrose. Dosages for inhibiting secondary embryogenesis were 40 mg/liter kanamycin for J. nigra and J. nigra × J. hindsii and 100 mg/liter for J. regia. For the bialaphos experiments, somatic embryos of J. nigra lines J26 and J28 and J. nigra × J. hindsii line S11 were cultured on gelrite solidified LP medium with 0.5 g/liter casein hydrolysate and 3% (w/v) sucrose. Between 0.1 and 1.0 mg/liter bialaphos, inhibited secondary embryogenesis.

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Jaime A.Teixeira da Silva

Filter paper types significantly affected the growth, development and differentiation of chrysanthemum and tobacco stem thin cell layers (TCLs) from in vitro plantlets. Three different filter paper types, normally with varied uses in plant biology, showed varying morphogenic-altering and antibiotic-buffering capacities. Advantec #2 and Whatman #1 significantly stimulated root, shoot and callus formation while Whatman #3 inhibited them, as compared to TCLs placed directly on agar. Filter paper buffered the phytotoxic effect of antibiotics kanamycin and cefotaxime, substances commonly used in genetic transformation experiments, up to as much as 50%, independent of species or genotype. In both `Lineker' and `Shuhou-no-chikara' chrysanthemum cultivars, Advantec #2 and Whatman #1 filter papers stimulated embryogenesis but in tobacco all three filter paper types significantly reduced embryogenesis and explant survival.

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Ahmad Khalighi, Manijeh Mohammadi Dehcheshmeh, Esmaeil Ebrahimie, Manoochehr Sardari, Rohangiz Naderi, and Rohangiz Naderi

Fritillaria imperialis and Fritillaria persica are important medicinal and ornamental plants that are native to Iran. Wild populations of Fritillaria are at risk of extinction. For the first time, capability of developmental stages in respect to induction of different morphogenesis pathways from petal tissue was studied in detail. The developmental stages were green unopened flowers, colored unopened flowers, and open flowers. Direct bulblet regeneration and direct somatic embryogenesis were observed from green unopened flowers in both F. imperialis and F. persica. More bulblet regeneration was produced in F. imperialis in contrast with F. persica in colored unopened flowers. Somatic embryogenesis via callus was established in green unopened flowers of F. persica with cold pretreatment. The effect of light on induction of different morphogenesis pathways was nonsignificant except for green unopened flowers of F. persica with cold treatment. Our results showed that the developmental stages of petal explants play a significant role in micropropagation of Fritillaria and induction of different morphogenesis pathways.

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M.A. Norton and D.R. LaBonte

Somatic embryogenesis in sweetpotato is highly genotype dependent. Unfortunately, many desirable agronomic varieties do not produce embryos capable of germination when published protocols are followed. Using one responsive and three recalcitrant cultivars, we examined the effect on embryogenesis of auxin, nitrogen, and carbon; explant source; and desiccation. All cultivars formed proembryonic masses on medium supplemented with either 2,4-D or picloram; picloram favored the growth of nonembryogenic callus. Twenty mm each of ammonium and nitrate promoted the best proembryo formation in all cultivars. Ammonium was essential for embryogenesis; replacing ammonium with proline, glutamine, asparagine, glycine, or casein hydrolysate resulted in poor or no proembryo formation. More proembryos formed on medium supplemented with sucrose than with glucose, fructose, or maltose. Leaf discs from the first fully expanded leaf produced more embryos than younger leaves for all cultivars; discs taken from the lamina produced more embryos than discs including portions of the midrib. Proembryos matured and germinated only after at least 3 weeks on medium containing 5% w/v polyethylene glycol 8000, greater than 3.3 mm myo-inositol, and 1 or 10 μm abscisic acid. More whole plants were obtained from the responsive cultivar Jewel than from the recalcitrant genotypes.

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Khalid M. Ahmad, Syed M. A. Zobayed, Praveen K. Saxena, and David M. Hunter

Dionaeamuscipula Ellis commonly known as Venus fly trap is an important carnivorous plant with medicinal importance. It contains certain secondary metabolites like naphthoquinones and is used in anti-aid and anti-cancer drugs and other medicines like Cornivora. Increasing interest and use as an ornamental and medicinal plant, and dietary supplement have put it in an endangered state. Development of in vitro techniques for the preservation of germplasm that is on the brink of extinction is highly demanded. A regeneration protocol for the multiplication and micropropagation of Dionaeamuscipla Ellis was established. In vitro regeneration potential of leaf explants in different concentrations and combinations of plant growth substances was investigated in this study. Seeds were grown and leaf disc explants were excised and cultured under aseptic conditions on nutritional medium containing half strength Murashige and Skoog (MS) mix with combinations of 1.0–20.0 μm BA, 2.5.0 μm IBA, 1.0–10.0 μm 2iP and 0.1–0.5μm TDZ. The cultures were kept in growth cabinet with cool white light (40–60 μmol·m-2·s-1) under 16-h photoperiod. Regeneration was recorded after 60 days with the intervals of 15 days based on the degree of shoot organogenesis and somatic embryogenesis. 1/2 MS + 0.1 TDZ appeared to be efficient for somatic embryogenesis and simple MS for direct shoot organogenesis. 1/2 MS combined with 2iP appeared to be efficient for regeneration either by direct shoot organogenesis or by somatic embryogenesis. Plants were rooted well in Cape Cundew medium. These investigations will aid in the development of a model system for clonal mass propagation and in vitro regeneration of Dionaeamuscipla Ellis.

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Jing-Tian Ling, Nobumasa Nito, Masao Iwamasa, and Hisato Kunitake

Embryos were obtained from unfertilized and undeveloped seeds of satsuma (Citrus unshiu Marc.) cultured on a modified Murashige and Tucker (MT) medium. Embryogenic callus was induced from the hypocotyl region of the embryos. The callus was successfully maintained through subculturing on MT medium with 185 μm ade-nine, 2.8 μm GA3 and 400 mg malt extract/liter, solidified with Gelrite. Somatic embryogenesis occurred from callus subculture on medium containing 50 g lactose/liter and in the absence of plant growth regulators. Somatic embryos developed into plants on medium with sucrose and GA3. Protoplasts isolated from this callus produced somatic embryos through colony formation: subsequently, normal, entire plants were regenerated.

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Hazel Y. Wetzstein and Choong-Suk Kim

Although somatic embryogenesis in vitro has been carried out successfully in a number of plants, a limiting factor in many somatic embryogenic systems is that plantlet regeneration is not obtainable or restricted to low frequencies. We have developed a repetitive, high frequency somatic embryogenic system in pecan (Carya illinoensis) and have identified effective treatments for improved somatic embryo conversion. A 6 to 10 week cold treatment followed by a 5 day desiccation, promoted enhanced root germination and extension, and epicotyl elongation. Light and transmission electron microscopic evaluations of somatic embryo cotyledon development will be presented and related to conversion enhancing treatments and their possible roles in embryo maturation.

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Hazel Y. Wetzstein and Choong-Suk Kim

Although somatic embryogenesis in vitro has been carried out successfully in a number of plants, a limiting factor in many somatic embryogenic systems is that plantlet regeneration is not obtainable or restricted to low frequencies. We have developed a repetitive, high frequency somatic embryogenic system in pecan (Carya illinoensis) and have identified effective treatments for improved somatic embryo conversion. A 6 to 10 week cold treatment followed by a 5 day desiccation, promoted enhanced root germination and extension, and epicotyl elongation. Light and transmission electron microscopic evaluations of somatic embryo cotyledon development will be presented and related to conversion enhancing treatments and their possible roles in embryo maturation.

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Hazel Y. Wetzstein and Adriana P.M. Rodriguez

Somatic embryogenic cultures of pecan (Carya illinoinensis) were induced on medium with either naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Percent embryogenesis, embryo development, and subsequent performance were assessed. Cultures induced on medium with NAA were more zygotic-like, with a higher frequency of embryos that had well-defined shoot apices. In contrast, cultures induced with 2,4-D exhibited more extensive callusing and more fused and/or abnormal embryos. Adjustment of the auxin used during induction may be a means of obtaining higher quality embryos, that have higher rates of conversion into plants.