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Lurline Marsh

Four cowpea [Vigna unguiculata (L). Walp] genotypes; IT 82E-18, IT 82E-16, Pinkeye Purple Hull, and Coronet were tested for somatic embryo formation and embryogenesis. Explants were 3-week-old cotyledons from which the embryonic axes were removed. Cotyledons were cultured in eight media combinations representing modifications of two media, one containing Murashige and Skoog Basal salt with B5 vitamins (MSB), 500 mg/L casein-hydrolysate (CS), 500 mg/L sodium chloride, 3% sucrose, 0.7% agar, 2mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L benzylamino purine, and the other containing (MSB), 3% sucrose, 40 mg/L 2-4-D and 0.2% gellan gum. After 1 month, 40% to 100% of explants produced calli and few produced shoots. Subcultured shoots in MS with 0.1 mg/L indole-3-butyric acid (IBA) or with IBA and 0.5mg/L kinetin (KT) failed to produced roots. The only green cotyledonary stage embryo was produced on this latter medium. Subculture of calli in MSB containing CS, mannitol, sucrose, agar, indoleacetic acid, and KT produced cream-colored globular embryos, roots, and a few leaves.

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Sudeep Vyapari and Houchang Khatamian

Somatic embryogenesis was successfully achieved in chinkapin oak (Quercus muehlenbergii Engelm.) and pin oak (Quercus palustris Muenchh.) when surface disinfested zygotic embryo explants were cultured on MS or WPM containing BA or kinetin (1.0 or 2.0 mg 1-1) plus IBA (1.0 mg 1-1). Immature embryos resulted in greater callus induction than the mature ones. Two weeks of dark, proved to be superior to 4 weeks or no dark in callus induction. Somatic embryos of pin oak distinctly showed globular, heart and cotyledonary stages.

Maturation and germination of pin oak somatic embryos was done in growth regulator free WPM by increasing levels of agar (7 - 15 g 1-1). Somatic embryos cultured at various levels of agar were then maintained in incubator under standard conditions, desiccated by air-drying or subjected to chilling temperature for 4 weeks to enhance germination of somatic embryos. Root or shoot formation was observed in some cultures, and medium with 9 g 1-1 agar induced plantlet production in 7% of the cultures.

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Mohammad Sadat-Hosseini, Kourosh Vahdati, and Charles A. Leslie

, 1990 ). Somatic embryogenesis plays an important role in micropropagation, genetic manipulation, cryopreservation, induced mutagenesis, and germplasm conservation in plants ( Sharma et al., 2012 ; Vahdati et al., 2008 ). In general, somatic

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I.E. Yates and C.C. Reilly

The influence of stage of fruit development and plant growth regulators on somatic embryogenesis and the relation of cultivar response on somatic embryogenesis and subsequent plant development have been investigated in eight cultivars of pecan [Carya illinoensis (Wangenh.) C. Koch]. Explants from the micropylar region of the ovule were more embryogenic when removed from fruits in the liquid endosperm stage than were intact ovules from less-mature fruits or from cotyledonary segments of more-mature fruits. Explants conditioned on medium containing auxin alone or auxin + cytokinin produced more somatic embryos than medium containing cytokinin alone. Under the conditions of this study, frequency of embryogenesis, as well as the germination of somatic embryos leading to plant development, indicated appreciable variation among cultivars. Plant development was greatest by far from somatic embryos of `Schley' than other cultivars studied.

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Whei-Lan Teng, Yann-Jiun Liu, Yu-Ching Tsai, and Tai-Sen Soong

A bioreactor was used to establish a scale-up system for somatic embryogenesis in `Scarlet' carrot (Daucus carota L.). At a cell density of 1–2 × 106 cells/ml, mature and germinating embryos could be observed within 4 to 5 weeks. As cell density exceeded 2 × 106 cells/ml, the culture turned darker yellow, and embryo development was inhibited. Cell densities below 106 cells/ml resulted in abnormal embryos. Bioreactor design had a critical impact on somatic embryogenesis due to various types and the strength of shear forces generated. In this study, an air-lift bioreactor was selected from three different types (spinner flask, screen column bioreactor, and air lift) because it resulted in the highest biomass production and somatic embryogenesis. Foaming was eliminated by preculture of embryogenic cells in flasks; cells were then sieved on a 60-μm polyester screen and thoroughly rinsed with distilled water before being transferred to the bioreactor. Such preculture for at least 10 days significantly increased the regeneration of somatic embryos. During somatic embryogenesis, dissolved O2 concentrations decreased to 33% of saturation, and then increased up to 80% when embryo development approached maturity and mature embryos germinated. Bioreactor-cultured embryos germinated with relatively short cotyledons and long roots, whereas flask-cultured embryos germinated with relatively long cotyledons and short roots.

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Lianghong Chen*, Ramana Gosukonda, and Johnny Carter

To improve somatic embryogenesis of Daylilies (Hemerocallis hybrid); six types of explants namely immature seeds, immature embryos, mature embryos, young inflorescence, ovary-sections and filaments of daylily flower buds were investigated as source of explants. Explants from field grown plants were surface sterilized and followed by culturing on MS medium supplemented with 1.5 mg/L NAA and 0.5 mg/L BAP for four weeks. Explants were scored for development of embryogenic tissue and formation of somatic embryos with/without the formation of an intermediate callus stage. Both mature and immature embryo explants produced direct development of embryogenic tissue followed by somatic embryos. Young inflorescence explants developed compact calli and produced roots around cut ends and showed no somatic embryogenesis. Ovary explants exhibited swelling and not produced embryogenesis. Production of embryogenic callus and formation of somatic embryos in filament explants depended on the sizes of flower buds. Explants from 0.5 to 1.2 mm size flower buds produced calli and formed somatic embryos while explants from sizes over 1.2 mm flower buds only non-embryogenic calli. Immature seeds failed to grow. The results indicated that immature and mature embryos and filaments from young flower buds responded better than other explants for developing somatic embryos in daylily.

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R.A. May and R.N. Trigiano

Somatic embryogenesis from leaf midrib explants of Dendranthema grandiflora Tzvelev. `Iridon' cultured on modified Murashige and Skoog basal medium (MSB) containing 1.0 mg 2,4-D and 0.2 mg BA/liter was influenced by light and sucrose concentration. Somatic embryos formed directly from explants when cultured on medium containing 9% to 18% sucrose and incubated first in the dark for 28 days, followed by 10 days in light, and then returned to the dark for 14 days. Embryogenesis did not occur in continuous darkness and was drastically reduced when explants were incubated in light only. The most embryos were formed on medium containing either 12% or 15% sucrose; lower concentrations stimulated shoot and root development. Light also mediated embryogenesis from leaf explants of 'other cultivars. White-opaque or occasionally light-green cotyledon-stage somatic embryos germinated on MSB medium without growth regulators but containing 3% sucrose. Twelve of the 23 cultivars evaluated produced somatic embryos, but plants were recovered from only five. Regenerated plants were phenotypically similar to parent plants in growth habit, leaf morphology, and flower color. Chemical names used: N- (phenylmethyl)-1 H- purine-6-amine (BA); (2,4-dichlorophenoxy) acetic acid (2,4-D).

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Nancy A. Reichert and Nancy J. Zimmerman

Fourteen commercial cucumber (Cucumis sativus L.) cultivars were screened on media designed to induce somatic embryogenesis and shoot organogenesis. Cotyledon explants from mature dry seeds and 7-day-old seedlings also were compared for responses. Seven cultivars (Burpless F1Hybrid, Burpee Hybrid II, Cross Country, Picklebush. Sweet Delight, Tasty Green, and Yard Long) responded best for individual regeneration ability. Within the somatic embryogeneais regeneration protocol, `Picklebush' responded better than most other cultivars, while `Burpless F1 Hybrid' responded best in shoot organogenesis. Also, within each type of regeneration, the developmental stage of the cotyledon explants appeared to influence regeneration ability.

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D.J. Gray, R.N. Trigiano, and B.V. Conger

Orchardgrass is a member of the Poaceae, a family characterized by monocotyledonous-type embryos. This species is unique in that somatic embryogenesis can be induced from the cultured leaves of potted plants, which can be maintained conveniently in the greenhouse. Both embryogenic callus and isolated somatic embryos develop directly from cultured leaf sections. Embryogenic callus can be maintained indefinitely. Somatic embryos exhibit typical embryo morphology and germinate readily into plants. Exercises designed to lead students through aspects of culture initiation, maintenance, and plant regeneration are described.

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V.M. Gingas

Partially expanded male catkins at the pre-pollen shedding stage of Quercus rubra L. and Quercus bicolor Willd. were cultured on MS medium supplemented with BA or 2,4D Explants on 2,4D produced a yellow embryogenic callus, seeming to originate from the pedicels. Subsequent transfers to BA and then, MS without growth regulators, resulted in callus proliferation. After ten weeks in culture, white embryoids developed from the callus of Q. bicolor. Separated and individually cultured embryoids underwent direct, repetitive embryogenesis. Upon transfer to ½-strength MS, embryoid germination and plant regeneration occurred, Callus of Q. rubra degenerated after five months in culture, failing to produce embryogenic structures.