Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation.

The visual marker GUS has been utilized in this study to understand the Arabidopsis thaliana vacuum infiltration transformation process by Agrobacterium tumefaciens. High transformation frequencies of up to 394 transgenic seeds per infiltrated plant were achieved. The results showed that the majority of the transgenic seeds from single infiltrated plants were from independent transformation events based on Southern analysis, progeny segregation, distribution of transgenic seeds throughout the infiltrated plants and the microscopic analysis of GUS expression in ovules of infiltrated plants. GUS expression in mature pollen and anthers was monitored daily from 0 to 12 days post-infiltration. In addition, all ovules from a single infiltrated plant were examined every other day. GUS expression frequencies of up to 1% of pollen were observed 3-5 days post-infiltration, whereas frequencies of up to 6% were detected with ovules of unopened flowers 5-11 days post-infiltration. Most importantly, transgenic seeds were obtained only from genetic crosses using infiltrated plants as the pollen recipient but not the pollen donor, demonstrating Agrobacterium transformation through the ovule pathway.

PHA production, from bacteria to plants.

The genes encoding the polyhydroxyalkanoate (PHA) biosynthetic pathway in Ralstonia eutropha (3-ketothiolase, phaA or bktB; acetoacetyl-CoA reductase, phaB; and PHA synthase, phaC) were engineered for plant plastid targeting and expressed using leaf (e35S) or seed-specific (7s or lesquerella hydroxylase) promoters in Arabidopsis and Brassica. PHA yields in homozygous transformants were 12-13% of the dry mass in homozygous Arabidopsis plants and approximately 7% of the seed weight in seeds from heterozygous canola plants. When a threonine deaminase was expressed in addition to bktB, phaB and phaC, a copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate was produced in both Arabidopsis and Brassica.