Vic C. Knauf

Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts

SummaryDNA from a bacterial plasmid containing the T-DNA border sequences of Agrobacterium tumefaciens was transferred into the nucleus or the cytoplasm of tobacco mesophyll protoplasts by microinjection. Following culture in hanging drops, some of these protoplasts produced calli containing the foreign DNA sequences. Evidence for the presence of the injected plasmid DNA in these calli was provided by Southern hybridization analysis. The results demonstrated that random portions of the bacterial plasmid were linked to plant DNA and that integration did not occur at the T-DNA borders present on the injected plasmid. The average number of integrated copies ranged from less than one to 1–2 per tobacco genome. The frequency of integration averaged 14% with intranuclear injections compared to 6% with cytoplasmic injections. With further refinement, the use of microinjection may allow the introduction of many different types of genetic elements into plants.

Gene Transfer in Crop Improvement

Transfer of genes between plant species has played an important role in crop improvement for many decades. Useful traits such as resistance to disease, insects, and stress have been transferred to crop varieties from noncultivated plants. Recombinant DNA methods greatly extend (even outside the plant kingdom) the sources from which genetic information can be obtained for crop improvement. Gene transfer systems based on recombinant DNA are available for several crop species and are under development for others. The concerted use of traditional and more recent methods for plant genetic manipulation will contribute to crop improvement.

Transfer of Agrobacterium DNA to Plants Requires a T-DNA Border But Not the virE Locus

Agrobacterium tumefaciens induces tumors in plants by transferring and integrating oncogenes (T-DNA) into the chromosomes of host plant cells. Agrobacterium strains were used to transfer complementary DNA copies of a potato spindle tuber viroid (PSTV) to plant cells at a wound site on tomato plant stems. Subsequently, infectious viroid RNA was found in the leaves of these plants, indicating systemic PSTV infection. This process utilized the T-DNA transfer mechanisms of Agrobacterium since PSTV infection required most virulence genes (vir) as well as one of the DNA sequences that flank either side of the Agrobacterium T-DNA. However, transfer still occurred from virE mutants of Agrobacterium, strains that fail to induce tumors even though a completely functional T-DNA is present. The virE gene seems to be directly involved in the integration of foreign DNA into plant chromosomes.

Triglycerides As Products of Photosynthesis. Genetic Engineering, Fatty Acid Composition and Structure of Triglycerides

In this chapter we will review the contributions of recombinant DNA technology to the study of triglyceride synthesis. The success of this technology will firstly be illustrated by research concerned with fatty acid chain length. Until recently, there was no explanation for the preferential accumulation of fatty acids of a given chain length by certain species. Only through gene cloning and gene transfer technologies was it possible to prove unequivocally that specific thioesterases controlled the elongation of fatty acids. So, the diversity of fatty acids accumulated by different species can now be explained by the diversity of thioesterases active in these species.