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Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons

Du, Ningxia, Pijut, Paula M.
Scientia horticulturae 2008 v.118 no.1 pp. 74
Fraxinus pennsylvanica, ornamental trees, nursery crops, micropropagation, adventitious shoots, rooting, explants, culture media, benzyladenine, plant growth substances, hypocotyls, cotyledons, indole butyric acid, indole acetic acid, photoperiod, overwintering, cold storage, acclimation, mortality
An adventitious shoot regeneration and rooting protocol was developed for green ash (Fraxinus pennsylvanica) seedling explants. The best regeneration medium for freshly isolated hypocotyls and cotyledons was Murashige and Skoog (MS) supplemented with 13.3μM 6-benzylaminopurine (BA) plus 4.5μM thidiazuron (TDZ), and 22.2μM BA plus 4.5μM TDZ, respectively. Seventy-six percent of hypocotyl segments and 24% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 2.7±0.5 and 2.3±1.3, respectively. The effect of in vitro-germinated seedling age on adventitious shoot regeneration from hypocotyl and cotyledon explants was also studied. Results showed that hypocotyl and cotyledon explants from freshly isolated embryos exhibited a higher organogenesis potential than 4-15-day-old explants. Adventitious shoots from hypocotyls and cotyledons were established as proliferating shoot cultures following transfer to MS basal medium with Gamborg B5 vitamins supplemented with 10μM BA plus 10μM TDZ. A high rooting percentage (73-90%) was achieved when in vitro shoots were rooted on woody plant medium (WPM) containing 4.9μM indole-3-butyric acid (IBA) and IAA (0, 2.9, 5.7, or 8.6μM) with a combination of 10-day dark culture period followed by a 16-h photoperiod. The highest rooting (90%) of adventitious shoots or the number of roots per shoot (3.0±1.0) was obtained on WPM with 4.9μM IBA plus 5.7μM IAA. Rooted plants were successfully acclimatized to the greenhouse and 100% survived after overwintering in cold storage. This regeneration system using hypocotyls and cotyledons provides a foundation for Agrobacterium-mediated genetic transformation of F. pennsylvanica for resistance to the emerald ash borer.