Cutting propagation plays a large role in the forestry and horticulture industries where superior genotypes need to be clonally multiplied. Integral to this process is the ability of cuttings to form adventitious roots. Recalcitrance to adventitious root development is a serious hurdle for many woody plant propagation systems including black walnut (Juglans nigra L.), an economically valuable species. The inability of black walnut to reliably form adventitious roots limits propagation of superior genotypes. Adventitious roots originate from different locations, and root induction is controlled by many environmental and endogenous factors. At the molecular level, however, the regulation of adventitious root formation is still poorly understood. In order to elucidate the transcriptional changes during adventitious root development in black walnut, we used quantitative real-time polymerase chain reaction to measure the expression of nine key genes regulating root formation in other species. Using our previously developed spatially explicit timeline of adventitious root development in black walnut softwood cuttings, we optimized a laser capture microdissection protocol to isolate RNA from cortical, phloem fiber and phloem parenchyma cells throughout adventitious root formation. Laser capture microdissection permitted high-resolution, site-specific analysis of gene expression that differentiated between participatory and non-participatory root progenitor cells. Results indicated mRNA abundance was altered in all nine rooting-related genes in response to auxin treatment in both juvenile and mature cuttings. SCARECROW LIKE-1 (SCL) had the greatest change in expression in juvenile rooting-competent cells at days 16 and 18, with a 24- and 23-fold increase relative to day 0, respectively. Tissues not linked to root organogenesis had little change in SCL expression at similar time points. AUXIN RESPONSE FACTOR (ARF)6 and ARF8 as well as SHORTROOT expression also increased 2- to 4-fold in rooting-competent tissue. The greatest transcript abundance in rooting-competent cuttings was restricted to root progenitor cells, while recalcitrant cuttings had a diffuse mRNA signal among tissue types.
Pumpkin ash (Fraxinus profunda (Bush) Bush) is at risk for extirpation by an exotic insect, the emerald ash borer (EAB). Pumpkin ash is limited to wetland areas of the Eastern United States, and has been listed as an endangered species because of EAB activity. Pumpkin ash provides many benefits to the ecosystem, and its wood is used in the manufacturing industry. In vitro regeneration provides an integral tool for the mass propagation and genetic transformation of pumpkin ash to combat EAB. Therefore, a plant regeneration protocol was developed for pumpkin ash. Aseptically extracted hypocotyls formed adventitious shoots following 4 weeks on Murashige and Skoog (MS) medium supplemented with 0-22.2 lM 6-benzyladenine (BA) and 0-6.8 lM thidiazuron (TDZ) then transferred for an additional 4 weeks on MS medium with Gamborg B5 vitamins plus 0.2 g L -1 glycine (B5G) containing 6.7 lM BA, 1 lM indole-3-butryic acid (IBA), and 0.29 lM gibberellic acid (GA 3 ). As adventitious shoots developed, these were transferred to a MSB5G medium with 13.3 lM BA, 1 lM IBA, and 0.29 lM GA 3 for shoot elongation. Elongated shoots were successfully micropropagated using MSB5 medium with 10 lM BA and 10 lM TDZ. Adventitious root formation was as high as 94% using woody plant medium supplemented with 4.9 lM IBA with shoots cultured for 10 days in the dark followed by culture under a 16-h photoperiod.Acclimatization to the greenhouse was successful and normal plant growth was observed. This protocol will provide a means for genetic transformation for EAB resistance and mass propagation for conservation.
This transformation and regeneration protocol provides an integral framework for the genetic improvement of Fraxinus profunda (pumpkin ash) for future development of plants resistant to the emerald ash borer. Using mature hypocotyls as the initial explants, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for pumpkin ash (Fraxinus profunda). This transformation protocol is an invaluable tool to combat the highly aggressive, non-native emerald ash borer (EAB), which has the potential to eliminate native Fraxinus spp. from the natural landscape. Hypocotyls were successfully transformed with Agrobacterium strain EHA105 harboring the pq35GR vector, containing an enhanced green fluorescent protein (EGFP) as well as a fusion gene between neomycin phosphotransferase (nptII) and gusA. Hypocotyls were cultured for 7 days on Murashige and Skoog (MS) medium with 22.2 μM 6-benzyladenine (BA), 4.5 μM thidiazuron (TDZ), 50 mg L(-1) adenine hemisulfate (AS), and 10 % coconut water (CW) prior to transformation. Hypocotyls were transformed using 90 s sonication plus 10 min vacuum infiltration after Agrobacterium was exposed to 100 μM acetosyringone for 1 h. Adventitious shoots were regenerated on MS medium with 22.2 μM BA, 4.5 μM TDZ, 50 mg L(-1) AS, 10 % CW, 400 mg L(-1) timentin, and 20 mg L(-1) kanamycin. Timentin at 400 and 20 mg L(-1) kanamycin were most effective at controlling Agrobacterium growth and selecting for transformed cells, respectively. The presence of nptII, GUS (β-glucuronidase), and EGFP in transformed plants was confirmed using polymerase chain reaction (PCR), while the expression of EGFP was also confirmed through fluorescent microscopy and reverse transcription-PCR. This transformation protocol provides an integral foundation for future genetic modifications of F. profunda to provide resistance to EAB.
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