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Sharon A. Bates, John E. Preece, and John H. Yopp

Dissected white ash seeds were placed on an agar-solidified MS medium with 10 μM TDZ and 1 μM 2,4-D (shoot initiation medium). After 4 weeks, explants were transferred to shoot elongation medium (3 μM TDZ, 1 μM BA, and 1 μM IBA) solidified with 0.7% Sigma agar, 0.525% agar + 0.05% gelrite, 0.35% agar + 0.1% gelrite, 0.175% agar + 0.15% gelrite, 0.2% gelrite, or no gelling agent (liquid medium). After 12 weeks in vitro, shoot growth and number were suppressed in cultures containing 0.2% gelrite (9.3 mm and 0.7 shoots) and in cultures containing no gelling agent (6.9 mm and 0.7 shoots). There were no differences in shoot growth and number in cultures containing 0.7% Sigma agar (2.2 mm and 16.5 shoots), 0.525% agar + 0.05% gelrite (2.6 mm and 18.7 shoots), 0.35% agar + 0.1% gelrite (1.6 mm and 17.4 shoots), and 0.175% agar + 0.15% gelrite (2.0 mm and 20.4 shoots). The most vitrification occurred in cultures on medium with the lowest amount of agar, gelrite only, and liquid medium.

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Sharon A. Bates, John E. Preece, and John H. Yopp

White ash has been adventitiously regenerated via organogenesis, somatic embryogenesis, and nodule culture. Explant source genotype, plant growth regulator type and concentration affected the type and/or frequency of regeneration observed. Organogenesis was obtained only when thidiazuron was added to the medium and nodules formed only in liquid culture after exposure to BA. Somatic embryos formed when explants were exposed to 2,4-D regardless of cytokinin used. Although large numbers of somatic embryos and nodules may be obtained through liquid suspension cultures, few plantlets were recovered compared to shoot organogenesis. Elongated adventitious shoots elongated and epicotyls from germinated somatic embryos rooted easily in vitro or under intermittent mist and were then acclimatized to the greenhouse and planted in the field.

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Sharon Bates, John E. Preece, John YOPP, and Robert Trigiano

Dissected white ash seeds were placed on a MS basal medium containing 10 μM TDZ and 1 μM 2,4-D. Adventitious buds formed directly and indirectly on cotyledons and hypocotyls that were in contact with the medium. Histological observations after 7 days from initiation indicated early divisional events originated directly in subepidermal layers on adaxial portions of the cotyledons. As these cells divided, the growth ruptured the epidermis. Bud-like structures were seen at 3 weeks. After transfer to a secondary medium containing 3 μM TDZ, 1 μM BA, and 1 μM IBA, some of adventitious buds elongated. Efforts (gibberellin, etiolation, ABA, and silver nitrate treatments) to increase the number of elongated buds have been unsuccessful. Excised adventitious shoots were rooted under mist and established in the greenhouse.

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Sharon A. Bates, John E. Preece, and John H. Yopp

To increase adventitious shoot formation, we investigated the effects of the number of weeks on medium with high levels of plant growth regulators and seedcoat removal. Dissected white ash seeds were placed on a solidified MS medium containing 10 μM TDZ and 1 μM 2,4-D (shoot initiation medium). After 2, 3, or 4 weeks in vitro, explants were transferred to shoot elongation medium (3 μM TDZ, 1 μM BA, and 1 μM IBA). After 12 weeks, the greatest number (1.8) and longest shoots (18.7 mm) were in cultures incubated on the shoot formation medium for 3 weeks. In a separate experiment, dissected seeds were placed on shoot formation medium. Seedcoats were removed after 10 days in vitro. Explants were transferred to shoot elongation medium after 4 weeks in vitro. There were more shoots (2.5) on 12-week-old explants without seedcoats than on explants with seedcoats (0.9). This result may be related to inhibitors in the testa.

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Sharon A. Bates, John E. Preece, and John H. Yopp

Both greenhouse-grown white ash plants derived from tissue culture and rooted microshoots in high humidity trays were inoculated with 11 tumor-inducing Agrobacterium strains. Eight strains stimulated mutative gall formation. Plants inoculated with strain A281 exhibited a higher frequency of callus formation (greenhouse-22.2%; microshoots-18.8%) than other strains at the site of the wound. Therefore, strain A281 was used to inoculate seed and seedling explants in vitro. Explants were placed on MS medium containiner no plant growth regulators and inoculated at 0, 3, 5, 7, or 10 days after initiation. Plants inoculated at 10 days showed a higher frequency of callus formation (16.4%) than with earlier inoculations. Also, rewounding of the explant at inoculation resulted in a higher frequency of callus formation (11.3%) compared to not rewounding the explant (3.9%).