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Giovanni Iapichino, Tony H.H. Chen, and Leslie H. Fuchigami

An efficient adventitious shoot production protocol has been developed for Rhododendron laetum × aurigeranum. Shoot tips taken from greenhouse-grown plants were cultured on Anderson's medium supplemented with 74 μM 2iP. Axillary shoots were excised and cultured on medium containing 23 μM IAA and 74 μM 2iP. After 6 months, brown callus developed at the cut surfaces of the shoot-tip explants. This callus produced many adventitious shoots (up to 70 per explant). Clusters of adventitious shoots were divided, subculture, and continued to proliferate shoots. An estimated 1600-fold increase in the number of shoots could be readily achieved in 6 months. In vitro rooting of adventitious shoots was accomplished in 4 weeks. Seventy-three percent of shoots rooted on 1/4 strength Anderson's medium supplemented with 28 μm IAA. Plantlet survival was 100%3 weeks after transfer to soil. Chemical names used: 1-H-indole-3-acetic acid (MA); N-(3 -methy1-2-butenyl) -1H-purine-6 amine (2iP).

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A. Raymond Miller and Craig K. Chandler

A protocol was developed for excising and culturing cotyledon explants from mature achenes of strawberry (Fragaria × ananassa Duch.). Cotyledon explants formed callus with multiple shoot buds on agar-solidified Murashige and Skoog media containing several combinations of hormones (1 μm 2,4-D; 10 μm 2,4-D; 1 μm BA + 1 μm 2,4-D; 1 μm BA + 10 μm 2,4-D; 5 μm BA; 5 μm BA + 1 μm 2,4-D; 5 μm BA + 10 μ m 2,4-D; 5 μ m BA + 5 μm NAA; 5 μ m BA + 15 μ m NAA). After three subcultures, only tissues maintained on the medium containing 5 μm BA + 5 μm NAA continued to form shoots. Tissues transferred to other media eventually died (1 μm 2,4-D; 1 μ m BA + 10 μ m 2,4-D; 5 μ m BA; 5 μ m BA + 1 μ m 2,4-D), became unorganized (1 μm BA + 1 μm 2,4-D; 5 μm BA + 10 μm 2,4-D; 5 μm BA + 15 μm NAA), or formed roots (10 μm 2,4-D). Whole plantlets were produced by transferring callus with buds to medium lacking hormones. The rapid regeneration of clonal plantlets from cotyledon explants may be useful for reducing variability in future developmental studies. Chemical names used: N-(phenylmethyl)-1H-purin-6-amine (BA); (2,4-dichlorophenoxy) acetic acid (2,4-D); and 1-naphthaleneacetic acid (NAA).

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J.M. Van Eck and S.L. Kitto

Plant regeneration from callus cultures of mint depended on expiant source, genotype, and culture medium components. Mature embryos, seedling and flower parts, as well as chilled or desiccated immature embryos of peppermint (Mentha piperita L.) and spearmint (Mentha spicata L.) were cultured on a Murashige-Skoog medium containing various combinations of growth factors. Shoots regenerated from callus that developed either on mature peppermint embryos cultured on medium that contained BA at 0.5 mg·liter-1 and NAA at 0.5 mg·liter-1 or on immature peppermint embryos (chilled at SC for 0.6 day or nonchilled) cultured on basal medium containing BA at 1 mg·liter-1 and TIBA at 1 mg·liter-1 Shoots were proliferated, rooted, and acclimated. with 100% survival under greenhouse conditions. Chemical names used: N-(phenylmethyl) -1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA); 2,3,5-triiodobenzoic acid (TIBA).

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P. Gercheva, R.H. Zimmerman, L.D. Owens, C. Berry, and F.A. Hammerschlag

Shoot regeneration from apple (Malus domestica Borkh.) leaf explants following particle bombardment at various acceleration pressures was studied. Basal leaf segments of micropropagated `Royal Gala' apple were bombarded with 1 μm gold particles, accelerated using helium pressures of 4.5, 6.2, 7.6, 9.3, or 13.8 MPa (650–2000 psi), and cultured on shoot regeneration medium consisting of N6 salts supplemented with 10 μM TDZ for 5, 10, or 20 days in darkness. Bombarded and control explants exhibited 63% to 100% shoot regeneration. With a 5-day dark period, average shoot production per explant ranged from 6.1 to 14; bombardments of 4.5 and 6.2 MPa significantly increased shoot production over the controls. With a 10-day dark period, average shoot production per explant ranged from 9.1 to 22 following bombardment at 9.3 and 6.2 MPa, respectively. Following bombardment at 6.2 MPa, 75% of the explants produced more than 20 regenerants per explant. With a 20-day dark period, average shoot production per explant ranged from 8.9 to 19 following bombardment at 13.8 MPa and following no bombardment, respectively. Shoot production per explant was significantly less than the controls following bombardments ranging from 6.2 to 13.8 MPa. Shoot production was highest per explant with particle bombardment at 6.2 MPa followed by incubation in darkness for 10 days. Chemical name used: thidiazuron (TDZ).

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Chiwon W. Lee, Joel T. Nichols, Lijuan Wang, and Shanqiang Ke

Excised leaf sections of lance coreopsis cultured on Murashige Skoog (MS) medium produced adventitious shoots in response to BA. When the combinations of 0, 0.5, 1, or 2 μm NAA with 0, 5, 10, 20, or 40 μm BA were tested, shoots were induced by any of the four BA concentrations used in the medium, regardless of the presence of NAA. The average number of shoots formed per leaf section ranged from 1.4 to 4.3 seven weeks after culture initiation. Roots were induced at the base of individual shoots on the same regeneration medium when cultures were kept longer than 7 weeks. The rooted plants were transferred successfully into soil. The regenerated plants had the same growth and flowering characteristics as the seed-grown plants. Chemical names used: benzyladenine (BA); naphthaleneacetic acid (NAA).

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Mihoko Tamura, Ryutaro Tao, and Akira Sugiura

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Carole H. Saravitz, Frank A. Blazich, and Henry V. Amerson

Cotyledons and hypocotyls of Fraser fir [Abies fraseri (Pursh) Poir.] were excised from seeds treated with H2 O2 for 9 days and placed on bud induction medium containing 10 mg BA/liter and 0.01 mg NAA/liter or medium without growth regulators. Although adventitious buds did not develop, cotyledons exposed to growth regulators responded differently than cotyledons placed on medium lacking growth regulators. Cotyledons and hypocotyls responded similarly to growth regulators during the initial phase in culture, but cell divisions ceased in cotyledons, thus preventing meristemoid and subsequent bud development. After 3 days on medium containing growth regulators cell divisions were localized in epidermal and subjacent layers of hypocotyls, whereas similar cell divisions were' not observed in hypocotyls placed on medium without growth regulators. Cell clusters consisting of two to five cells (promeristemoids) were present after 7 days on hypocotyls placed on bud induction medium. In hypocotyls placed on medium without growth regulators, stomata continued to develop and cells within the cortex became vacuolated during the first 2 weeks in culture. All explants were transferred to secondary medium after 3 weeks. Cell clusters continued to enlarge into meristemoids on hypocotyls initially placed on bud induction medium. Gradually, meristemoids developed into buds and cataphylls were observed covering bud meristems. Chemical names used: N -(phenylmethyl)-1 H -purine-6-amine (BA), 1-naphthaleneacetic acid (NAA).

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Peggy Ozias-Akins and Srini Perera

One cm segments from adventitious roots of sweet potato (Ipomoea batatas (L.) Lam.) will regenerate shoots when cultured on Murashige and Skoog salts and vitamins plus either sucrose (1-3%) or fructose (1-6%). The best source for adventitious roots is sweet potato shoot cultures maintained in Magenta vessels. A low concentration of cytokinin (0.02 mg/liter) promotes shoot formation. Higher levels of cytokinin (0.1-0.5 mg/liter) encourage callus growth. The maximum average number of shoots formed per root segment attained thus far is 0.5. Attempts are being made to increase the frequency of shoot formation. Regeneration of shoots from roots also may be a useful method for obtaining plants from protoplasts of sweet potato. Protoplasts can be isolated from mesophyll tissue and petioles of in vitro grown plants. Plating efficiency of up to 12% routinely can be obtained. Shoot formation directly from callus is sporadic; root formation is more frequent.

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Michael E. Compton and D.J. Gray

Cotyledon explants of four watermelon [Citrullus lanatus (Thunb.) Mataum. & Nakai] breeding lines (F92U8, SP90-1, SP90-2, and SP90-4) were prepared from mature seed or from 2-, 4-, 6-, 8-, or 10-day-old seedlings. Explants were incubated on shoot regeneration medium for 8 weeks followed by 4 weeks on shoot elongation medium. The four genotypes differed in their ability to produce shoots at each explant age. The highest frequency with which F92U8 (66%) and SP90-2 (60%) explants produced shoots was for 2-day-old seedlings. Fewer explants formed shoots when established from mature seed or seedlings older than 2 days. In contrast, the percentage of SP90-4 explants that produced shoots was highest when cotyledons were obtained from 4-day-old seedlings (40%), but the response was less than the optimum for F92U8 and SP90-2. SP90-1 cotyledon explants exhibited the poorest response of the four breeding lines (<11% produced shoots), with little difference in response among the explant ages tested. The number of shoots per responding explant also depended on the age of the explant source. Explants from 2- to 4-day-old seedlings produced the most shoots. Fewer shoots formed on cotyledons from mature seed or seedlings older than 4 days.

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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.