A two-component cloning system to transfer foreign DNA into plants was derived from the octopine Ti plasmid pTiB6S3. pGV2260 is a non-oncogenic Ti plasmid from which the T-region is deleted and substituted by pBR322. pGV831 is a streptomycin-resistant pBR325 derivative that contains a kanamycin resistance marker gene for plant cells and a site for cloning foreign genes between the 25-bp border sequences of the octopine T-region. Conjugative transfer of pGV831 derivatives to Agrobacterium and cointegration by homologous recombination between the pBR322 sequences present on pGV831 and pGV2260, can be obtained in a single step. Strains carrying the resulting cointegrated plasmids transfer and integrate T-DNA into the genome of tobacco protoplasts, and transformed tobacco calli are readily selected as resistant to kanamycin. Intact plants containing the entire DNA region between the T-DNA borders have been regenerated from such clones. In view of these properties we present pGV831 and its derivatives as vectors for efficient integration of foreign genes into plants.

To study protein secretion in plant cells, we established and evaluated a model system based on transient synthesis of heterologous proteins in tobacco protoplasts. We show that the nonsecretory enzymes phosphinothricin acetyl transferase, neomycin phosphotransferase II, and beta-glucuronidase are secreted when targeted to the lumen of the endoplasmic reticulum by signal peptide-mediated translocation. These data are consistent with the view that secretion can occur independent of active sorting mechanisms by nonspecific migration through the exocytic pathway. However, the rate of secretion differs significantly among these enzymes. Furthermore, the presence of signal sequences was found to be correlated with a reduction of the levels of the encoded gene products. This is the result of post-transcriptional events that limit either synthesis or stability of the proteins in vivo.

The TL‐DNA in octopine crown galls encodes seven transcripts most, if not all, expressed from individual promoters. Site‐specific deletions and substitutions in the T‐region of the octopine plasmid pTiB6S3 indicate some of the functions of the TL‐DNA transcripts. Two of the seven genes are sufficient to allow tumorous growth. T‐DNA transfer and oncogenicity are controlled by different and independently acting functions. None of the transcripts of TL‐DNA appear to be essential for T‐DNA transfer. Four, possibly five, of the TL‐DNA transcripts act by suppressing organ development. Shoot and root formation are suppressed by the action of different transcripts.

No abstract

To study protein secretion in plant cells, we established and evaluated a model system based on transient synthesis of heterologous proteins in tobacco protoplasts. We show that the nonsecretory enzymes phosphinothricin acetyl transferase, neomycin phosphotransferase II, and 8-glucuronidase are secreted when targeted to the lumen of the endoplasmic reticulum by signal peptide-mediated translocation. These data are consistent with the view that secretion can occur independent of active sorting mechanisms by nonspecific migration through the exocytic pathway. However, the rate of secretion differs significantly among these enzymes. Furthermore, the presence of signal sequences was found to be correlated with a reduction of the levels of the encoded gene products. This is the result of post-transcriptional events that limit either synthesis or stability of the proteins in vivo.