Three chrysanthemum (Dendranthema grandiflora) cultivars were cocultivated with 2 Agrobacterium tumefaciens strains in combination with 4 pBIN19 derived binary plasmids, all carrying the Nosnptll selection gene and 35Sgus(intron) reporter gene. All binary plasmids transferred DNA to chrysanthemum explants but only pMOG410 gave good stable expression of GUS. This plasmid differs from the other plasmids in 2 aspects: 1) It carries a restored nptll gene and 2) the selection gene is positioned at the left border side of the reporter gene. Cocultivation with AGLO(pMOG410) yielded up to 13 GUS positive shoots per 100 explants. The presence of the gus and nptll gene in recovered shoots was confirmed by PCR and Southern blot analysis.
Explants from leaves of in vitro-grown chrysanthemum (Dendranthema grandiflora Tzvel.) cultivars regenerated adventitious shoots without an intermediate callus phase. Puncturing explants with a brush increased regenerations, but in combination with cocultivation with Agrobacterium tumefaciens it had an adverse effect on shoot formation. The negative effect of brushing and cocultivation could be overcome by preculturing explants for 8 days. Preculture altered the location of transformed sites but did not inhibit transformation. Regeneration following cocultivation with Agrobacterium is also encouraged if alternative regeneration protocols are used that do not require brushing.
PRELIMINARY NOTES 451tively) are high, and nearly equally so, during the initial intervals. The RNA phosphorus would appear to move as follows: Sp --+ DS --+ RNP. The evidence on the manner in which ribonucleic acid participates in protein synthesis is still fragmentary. If such a participation requires the rapid renewal of the nucleic acid, our observations would suggest that the ribonucleic acid of the RNP particles of the microsomes is not directly involved in the formation of microsomal protein, though the protein precursors, activated in the Sp fraction, may be assembled in the microsomes to form the final polymer. It is perhaps significant that the cytoplasmic supernatant fraction, in which the enzymic activation of amino acids s has been shown to occur ~,7, also contained the ribonucleic acid exhibiting the highest initial uptake of asp.We shall submit more detailed information on these studies in due course. This work, which benefited from the technical assistance of Miss EDITH A. LAWRENCE, has been supported by research grants from the U.S. Public Health Service, the National Science Foundation and the Rockefeller Foundation. The distribution of protease activities on liver cell fractionsUsing hemoglobin as a substrate, DE DUVE and co-workersZ, 2 found the highest cathepsin activity in the light mitochondria fraction of liver homogenates. Earlier MAVER AND GRECO 3 had found that the total mitochondria fraction contains the highest percentage of the total protease activity of liver homogenates not only when hemoglobin, but also when benzoyl-l-argininamide was used as a substrate.As cathepsin is the collective name for the tissue proteinases and other proteases, e.g. dipeptidases and a carboxypeptidase, also occur in the tissues we thought it would be interesting to study the protease activities of the liver cell fractions with synthetic substrates, specific for various proteases. 1:5 rat liver homogenates were prepared in o.25 M sucrose. The homogenate was layered on 0.35 M sucrose and centrifuged for 5-5 rain at 9oo × g in order to sediment the nuclear fraction. The supernatant containing the other subcellular components was centrifuged for 12 min at 18,ooo x g. The total mitochondria sedimented in this way could be separated into the light and the heavy fraction by resuspending them in o.25 M sucrose, centrifuging for 8 rain at ii,ooo × g, and resuspending the slightly pink light mitochondria layer (easily distinguishable from the dark yellow heavy mitochondria layer) by causing a slow rotatory movement in the supernatant by means of a small pestle fitted into the centrifuge tube. The suspension obtained in this way was then transferred to another centrifuge tube and the light mitochondria spun down by centrifuging for 12 min at i8,ooo × g. The whole procedure was carried out at about 2 ° C.The final supernatant obtained by sedimenting the total mitochondria was not fractionated further; thus it still contained the microsomes. In order to avoid damage as much as possible the sedimented fractions were n...
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