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Transformation of alfalfa using Agrobacterium tumefaciens
Agrobacterium tumefaciens, the causative agent of crown gall
disease, does not readily infect commercial varieties of alfalfa (Medicago sativa). Cloned virulence genes from the megaplasmid pTiBo542 have
been shown to enhance Agrobacterium host range and infectivity on
dicotyledonous plants. To obtain a gene transfer system for commercial
alfalfa cultivars, an Agrobacterium strain containing a disarmed T-DNA, a
cloned virG gene from pTiBo542, and an octapine Ti plasmid TiA6NC was
constructed. This strain, A348 (pToK9, pGA472), was used to inoculate
excised petioles of the alfalfa cultivar "Gladiator. Neomycin resistant callus
tissue grew readily on 100 p.1/mIkanamycin and on appropriate media, leaf
and embryo development was induced. DNA hybridization analysis of the
transformed plant genomic DNA isolated from callus tissue confirmed the
presence of the npt gene. VirG enhanced A. tumefaciens strains can thus
be used in foreign gene transfer and expression in commercial cultivars of
alfalfa
Fast Blue and Cholera Toxin-B Survival Guide for Alpha-Motoneurons Labeling: Less Is Better in Young B6SJL Mice, but More Is Better in Aged C57Bl/J Mice
Fast Blue (FB) and Cholera Toxin-B (CTB) are two retrograde tracers extensively used to label alpha-motoneurons (α-MNs). The overall goals of the present study were to (1) assess the effectiveness of different FB and CTB protocols in labeling α-MNs, (2) compare the labeling quality of these tracers at standard concentrations reported in the literature (FB 2% and CTB 0.1%) versus lower concentrations to overcome tracer leakage, and (3) determine an optimal protocol for labeling α-MNs in young B6SJL and aged C57Bl/J mice (when axonal transport is disrupted by aging). Hindlimb muscles of young B6SJL and aged C57Bl/J mice were intramuscularly injected with different FB or CTB concentrations and then euthanized at either 3 or 5 days after injection. Measurements were performed to assess labeling quality via seven different parameters. Our results show that tracer protocols of lower concentration and shorter labeling durations were generally better in labeling young α-MNs, whereas tracer protocols of higher tracer concentration and longer labeling durations were generally better in labeling aged α-MNs. A 0.2%, 3-day FB protocol provided optimal labeling of young α-MNs without tracer leakage, whereas a 2%, 5-day FB protocol or 0.1% CTB protocol provided optimal labeling of aged α-MNs. These results inform future studies on the selection of optimal FB and CTB protocols for α-MNs labeling in normal, aging, and neurodegenerative disease conditions