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Annette D. Leege and Robert R. Tripepi

A plant regeneration system that is compatible with recombinant DNA techniques is required before birch can be genetically transformed. The goal of this research is to develop a shoot regeneration system from leaf discs of European birch (Betula pendula Roth), since this tissue culture procedure is compatible with current transformation technology. Leaves from microplants were excised from stems, cut into approximately 25 mm2 pieces, and placed on WPM media containing differing ratios of NAA (0, 3, 6, 9 μM) to BA (0, 7.5, 15, 22.5 μM) in a 4 × 4 factorial design. Four replicates, each containing 4 leaf pieces, were used per treatment. After 4 and 8 weeks, data was taken including the percent leaves forming shoots and the number of shoots per leaf disc. Only a concentration of 15 or 22.5 μM BA without NAA stimulated shoot formation on leaf discs. Data on the effects of light, media formulations and tissue orientation will be presented. With a reliable and efficient shoot regeneration system for European birch, genetic engineering of this species is now possible.

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D. Sankhla, T.D. Davis, N. Sankhla, and A. Upadhyaya

This report describes an efficient in vitro regeneration protocol for H. patens (firebush), a heat-tolerant ornamental shrub native to tropical and subtropical America. Shoot cultures were initially established using shoot tips placed on MS-revised medium containing 2.3 μM 2,4-D, 2.3 μM kinetin, and 0.25% polyvinylpyrrolidone. Other types of explants (nodal and internodal segments, leaf pieces, floral buds) did not regenerate shoots when placed on this medium. Two-month-old plantlets derived from the shoot tips were subcultured on MS medium supplemented with 0.5 μM thidiazuron (TDZ), and within 3 to 4 weeks, some callus was produced at the root–shoot junction. When this callus, with a small portion of the root and shoots, was placed on MS medium with 0.05 μM TDZ and 0.01 μM ABA, prolific shoot formation occurred within 3 to 4 weeks followed by root formation. By regular subculturing every 5 to 6 weeks, hundreds of plantlets have been obtained over the past 3 years with no apparent decline in regeneration potential. Addition of activated charcoal (0.5%) to the culture medium has greatly improved growth of the plantlets.

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Whei-Lan Teng, Chiao-Po Lin, and Yann-Jiun Liu

A scale-up process of lettuce (Lactuca sativa L.) suspension culture in a 2-liter bioreactor was investigated. Factors that influenced cell growth and differentiation, including foaming, the wall effect (inoculum adhering onto the vessel wall above the medium level), aeration, and dissolved oxygen (DO), were tested. The wall effect resulted in severe inoculum loss (10%) in 24 hours. Inoculum loss significantly decreased shoot regeneration. The wall effect was caused by two factors: 1) foaming caused by the interaction between air bubbles and inoculum, and 2) the bubbles produced by aeration. Foaming could be prevented by sieving the inoculum through a 400-pm screen filter and then rinsing the inoculum thoroughly with distilled water to remove single cells, cell debris, and the contents of broken cells. The wall effect caused by air bubbles could be prevented by putting a 150-μm screen column in the center of the bioreactor to isolate the aeration area from the inoculum. After the wall effect was removed, shoot regeneration in the bioreactor increased significantly to a level similar to that in 125-ml flasks at an aeration rate of 1 to 2 vvm (liters air/liters medium per rein). DO for this shoot regeneration level was ≈ 70% to 80%of saturation at the end of bioreactor culture.

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R.N. Trigiano and R.A. May

A tissue culture laboratory exercise illustrating regeneration of whole plants from leaf segments of chrysanthemum by organogenesis is described. Using simple, common media, shoots can be generated in 5 weeks and rooted after an additional 3 weeks. Acclimatization of plants can be accomplished in a simple mistbed in the greenhouse. The exercise is adaptable to depict genotype differences among cultivars, optimization of shoot induction, effects of growth regulators, and experimental design. Callus is typically not formed during shoot formation; however, co-cultivation of leaf segments with a virulent strain of Agrobacterium tumefaciens produces callus with a strain of disarmed A. tumefaciens harboring NPTII construct affects regeneration of plants resistant to kanamycin.

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Jason M. Jaworski and Michael E. Compton

Cotyledon explants of five watermelon cultivars (`Desert King', `Mickylee', `Sangria', `Sweet Princess', and `Male Sterile') were prepared from 7-day-old in vitro-germinated seedlings. Explants were incubated on shoot regeneration medium for 6 weeks, followed by several 3-week cycles on shoot elongation medium. The five cultivars differed in their ability to form shoots within 9 weeks on the selected media. Shoot regeneration frequency was about 1.5to 2.9-fold greater for `Mickylee' (60%) than `Sangria' (47%), `Sweet Princess' (27%), `Male Sterile' (26%), and `Desert King' (24%). Rooting of elongated shoots (>2 cm) occurred within 2 weeks on medium containing 1 μM IBA and ranged from 25% (`Desert King') to 92% (`Sangria'). Plantlets were transferred to six-pack containers filled with soilless medium (1 Sunshine Mix: 1 coarse perlite) and covered with a transparent plastic lid. Plants were acclimatized to ambient conditions by gradually removing the lid over a period of 3 days after new growth was observed. The percentage of acclimatized plants ranged from 50% (`Sweet Princess' and `Mickylee') to 100% (`Male Sterile'). Acclimatized plants were transferred to the greenhouse and grown for at least 4 weeks before screening for ploidy variants. Ploidy of regenerated plants was estimated by counting the number of chloroplasts per guard cell pair. Plants with an average of 18 or more chloroplasts per guard cell pair were declared tetraploids. Plants with fewer chloroplasts per guard cell pair were declared diploids and discarded. Tetraploid plants were transferred to the field, grown to maturity, and self-pollinated for seed increase.

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Laura R. Bahr and Michael E. Compton

Competence for in vitro bulblet regeneration was investigated for eight diverse Lilium genotypes (L. `Citronella', L. `Stargazer', L. `Stones', L. `Lovely Girl', L. pumilum, L. lancifolium, L. lancifolium `Orange Star', and L. speciosum var. rubrum). Explants established from bulb scales were cultured on lily bulblet regeneration medium [Murashige and Skoog (1962) modified by reducing the NH4NO3 to 0.825 g·L-1 and KH2PO4 to 0.170 g·L-1, and adding (per liter) 30 g sucrose, 0.1 g myoinositol, 0.4 mg thiamine·HCl, 80 mg adenine sulfate, 16 μm naphthaleneacetic acid, 2 mL·L-1 Plant Preservative Mixture (PPM), and 4.5 g·L-1 AgarGel at pH 5.7] for 8 weeks before transfer to sphagnum peat moss and 4 weeks refrigeration at 5 °C in darkness. All genotypes produced bulblets in vitro. However, the response rate varied among genotypes. Explants of Lilium `Lovely Girl', L. `Citronella', and L. speciosum var. rubrum were most responsive with ≈90% producing bulblets. The number of bulblets per responding explant ranged from 5.2 (`Stones') to 2.3 (L. lancifolium). When comparing in vitro and greenhouse bulblet production, about twice as many bulblets were produced by explants in vitro compared to halved scales incubated in the greenhouse. The percentage of responding explants ranged from 33% to 360% greater for six of the genotypes tested when propagated in vitro compared to bulb scales propagated in the greenhouse. Following refrigeration, bulblets were transferred to the greenhouse for sprouting. Over 80% of bulblets obtained from L. `Citronella', L. lancifolium `Orange Star', L. lancifolium, and L. `Stones' sprouted in the greenhouse. This study illustrates that a diverse range of lily species can be successfully propagated in vitro using a single medium formulation and is the first report of bulblet regeneration in vitro for L. `Citronella', L. `Stones', L. `Lovely Girl', and L. lancifolium `Orange Star'.

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Brian W. Trader, Hope A. Gruszewski, Holly L. Scoggins, and Richard E. Veilleux

Coreopsis species (tickseed) can be regenerated from leaf segments allowing the possibility to exploit somaclonal variation as a means to develop novel phenotypes. We used true leaf explants from in vitro seedlings of perennial C. grandiflora (A. Gray) Sherff `Domino' and `Sunray' grown on Murashige and Skoog (MS) basal medium. Two of ten seedlings of `Domino' regenerated freely and others were generally recalcitrant. From these two seedlings, designated E2 and H2, shoots were regenerated and acclimatized to the greenhouse. About 175 plants were established and vernalized from which somaclones were selected based on distinct differences in flower orientation and appearance. The selected somaclones were propagated by division and transplanted to the field in August 2001 in a randomized complete block design with three-plant plots and three replications to determine whether novel characteristics persisted through an additional propagation cycle. In the field, plant height, leaf dimension, flowering, and flower dimensions were scored in June and July 2003. Differences were found between somaclones and similarly propagated E2 and H2 for desirable (more petals per flower, greater flowering, shorter plants), undesirable (less flowering, smaller flowers), and neutral (narrower leaves, taller plants) traits. Open-pollinated (OP) seed was collected and germinated and the seedlings from somaclones that differed significantly from E2 and H2 were evaluated. These maternally selected seedlings were overwintered then planted in the field in May 2004. Most traits that differentiated somaclones from E2 and H2 did not persist in the OP seedling population; however variation that was likely introduced through outcrossing resulted in desirable phenotypes with potential for new cultivar development.

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Andrew S. Wang

Friable embryogenic callus of American ginseng (Panax quinquefolium L.) was induced from root pith on Murashige and Skoog medium supplemented with 2 mg 2,4-D and 1 mg KIN/liter. Optimal callus growth occurred on medium containing 1.5 mg dicamba/liter. Plants were regenerated on MS medium supplemented with various concentrations of plant growth regulators (PGRs); the best PGR combination was 0.5 mg IBA and 0.1 mg NAA/liter. Chemical names used: (2,4-dichlorophenoxy) acetic acid (2,4-D); 3,6-dichloro-o-anisenic acid (dicamba); 6-benzylaminopurine (BA); gibberellic acid (GA); indole-3-butyric acid (IBA); kinetin (KIN); and naphthaleneacetic acid (NAA).

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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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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.