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G.G. Ning and M.Z. Bao

the successful regeneration of plants from cultures of P. mume . This article presents the first report of the successful regeneration of plants of P. mume achieved through indirect organogenesis from cotyledons of immature embryos. In the course

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Guochen Yang and Marihelen Kamp-Glass

An efficient and reliable protocol of in vitro shoot regeneration must be first established to have a successful genetic transformation. As a member of legume family, alfalfa is very difficult for direct shoot regeneration. There is no published information on direct shoot organogenesis, although success has been well documented on embryogenesis, which must go through callus stage. Different plant growth regulators at various concentrations were evaluated for callus initiation, development, and direct shoot regeneration. Multiple shoots were produced directly from each individual explant. This will provide an efficient means for production of transgenic alfalfa plants. Therefore, genetic transformation of Medicago germplasm will be significantly expedited.

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Roberto A. Rivas, P.T. Evans, and D.R. LaBonte

Methodology is presented for organogenesis of `Beauregard sweetpotato, a cultivar released in 1987 that is rapidly increasing in commercial use in the U.S. Regeneration of `Beauregard' sweetpotato plantlets was obtained when complete leaves and 10 mm internode explants were cultured in liquid and solid media respectively over a period of 8 weeks. Leaves in liquid Murashige and Skoog medium containing 2 mg/l of IAA placed on a shaker under dark conditions produced white callus at the cut end of the petiole and roots underneath the callus in 4 weeks. Leaves were subsequently transferred to MS medium containing 500 mg/l of Chlorocholine chloride (CCC) and 0, 1, 5 and 10 mg/l of BAP for 4 more weeks. Shoots were regenerated from callus using 1 mg/l of BAP.

The effect of NAA auxin and various concentrations of the cytokinin Thidiazuron on internodes was examined under 16 hr. light and 8 hr dark photoperiod using MS solid medium. Explants on 0.05 mg/l NAA alone produced roots and shoots. The most plantlets however, were regenerated using 0.05 mg/l of NAA and 0.01 mg/l Thidiazuron. Regeneration of plants from leaves and internodes may be a useful system for a clean and rapid propagation of `Beauregard' sweetpotato.

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James A. Stamp, Sheila M. Colby, and Carole P. Meredith

Adventitious shoots developed within 3 weeks from the petiolar stub and, less often, from wounded lamina tissues when leaves excised from nodal cultures of Vitis vinifera L. cvs. French Colombard and Thompson Seedless were cultured on solid Nitsch and Nitsch medium containing BAP at 2 mg·liter-1. The youngest leaf that could be excised, from 1 to 8 mm long, was the most responsive (90% of explants producing shoots compared to 16% for leaf 6). Removal of the lamina from the petiolar stub within the first 3 weeks of culture reduced shoot production. Increase in nodal culture age, without transfer to fresh medium, had no effect on subsequent regeneration from the youngest leaves but did reduce the regeneration frequency of leaves at the next position from 43% to 20%. In regularly subculture nodal cultures, the number of transfers had no effect on subsequent regeneration. Leaves from recently established shoot tip cultures were more responsive than leaves from nodal cultures. The frequency of shoot production was higher in laterally bisected than intact leaves (70% vs. 43%) due to additional regeneration from the distal leaf half at the sites of severed veins. Shoot outgrowth was promoted by the isolation and subculture of regenerating tissue to fresh regeneration medium. Petiolar stub removal promoted de novo shoot organogenesis from the resulting lamina wound. Shoots rooted at a high frequency on Murashige and Skoog medium with 1 mg IA-A/liter and produced morphologically normal plants. Chemical names used: 6-benzylaminopurine (BAP); indole-3-acetic acid (IAA).

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Mohsen Hesami and Mohammad Hosein Daneshvar

, it is necessary to establish an efficient in vitro micropropagation and plant organogenesis protocol to enhance the conservation, development, and utilization of this plant resource. There are only four studies on direct organogenesis of F. religiosa

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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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Victoria E. Rudolph and David W. Burger

The role of N metabolism in organogenesis and growth was studied using tobacco pith callus. Callus was cultured on a solid medium containing 10 μM (1.75 mg/l) IAA and 2 μM (0.43 mg/l) kinetin for 56 days. In the growth experiment, ratios of NH4 +-N to NO3 --N (0:60, 20:40, 30:30, 40:20 and 60:0 mM) were supplied by (NH4)2 SO4 and KNO3. Callus and media were analyzed for inorganic N. Callus supported by 30:30 and 40:20 media removed the highest amounts of NH4 +-N and NO3 --N from the media and exhibited organogenesis. Final dry weight was greatest in callus supported by the 30:30 medium. In the organogenesis experiment, the transfer history of the inoculum source affected N uptake, organogenesis and growth. Inorganic N was supplied by NH4NO3 and KNO3 -. The net uptake of NH4 +-N and NO3 --N was lower in shoot-forming than in root-forming and non-organogenic callus subculture from 7-day-old stock cultures. The final pH of the medium supporting shoot-forming callus was lowest. Growth, on a dry weight basis, was lowest in shoot-forming callus. Callus subculture from 60-day-old stock cultures formed no shoots.

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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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Carol Auer, Jerry D. Cohen, and Todd Cooke

The uptake and metabolism of exogenous tritium-labelled benzyl adenine was studied during the shoot induction period of petunia leaf explants in tissue culture. Transfer experiments with Petunia `MD1' leaf explants (1 cm2) on MS media with 2.2 uM BA show that 27% and 100% of the leaf explants are committed to shoot induction on days 6 and 10, respectively. To study BA uptake and metabolism, leaf explants were placed on media containing tritium-labled BA for 1, 3, 6 and 10 days. BA was taken up from the media on days 1-6. BA metabolizes were analyzed using HPLC, a UV absorbancc detector and enzymatic techniques. Metabolizes produced include: BA, BAdo, BA 7G, BA 9G, BAdoMP, BAdoDP, BAdoTP and 3 unidentified compounds. BA and BAdo were detected on days 1 and 3 but not during day 6-10, the time of shoot induction. The pool of ribotide metabolites decreased from days 1 to 10, from 26.5% of all metabolites to 1.6%. Glucosylated compounds, BA 7G and BA 9G, increased continuously from 24.9% to 69.8% between days 1 and 10. An unidentified compound C increased from 13% on day 3 to 24.8% on day 10. In separate experiments, BA uptake and metabolism were compared in two Petunia hybrida lines, St40 and TLV1, with different shoot organogenic responses in tissue culture. These data show interesting patterns of BA metabolism in relationship to shoot induction and organogenesis.

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

Cotyledon explants were harvested from immature walnut fruits during July and August 1991. Media consisted of either WPM with 0.1 μM 2,4-D, 5.0 μM TDZ and 1.0 g/liter casein hydrolysate or DKW with 4.4 μM BA, 0.05 μM IBA, 9.3 μM Kinetin and 250 mg/liter l-glutamine. Treatments were arranged factorially with 2 gelling agents, 7 g/liter Sigma agar or 2 g/liter Gelrite and were incubated in light or in darkness. After 4 weeks, all explants were placed on basal DKW with no growth regulators and were cultured in darkness. The best treatment tested was from seeds collected 14 weeks post-anthesis on WPM, agar, and incubation in light (22 embryos/explant, 78% embryogenesis). Use of DKW and gelrite in darkness resulted in 1 embryo/explant and 38% embryogenesis. Up to 90% shoot organogenesis also occurred on cotyledon explants from seeds collected 16 weeks post-anthesis and placed on WPM. Shoots elongated on stationary liquid DKW with 10 μM BA.