| The goal of my project was to develop a system for the timed release of specific compounds to protect transgenic crop seedlings from a variety of stresses. As an example, I was attempting to improve flea beetle resistance in canola seedlings through the expression of large quantities of proteinase inhibitors in the first 5–10 days after germination. This pulsed-release of proteinase inhibitors was expected to inactivate flea beetle gut proteinases when the insects fed on the canola seedlings. Specifically, the rice cysteine proteinase inhibitor, oryzacystatin 1, has been-expressed in transgenic B. napus as a fusion with an 18 kDa seed oleosin. Similar fusion peptides have been produced in high levels in the seeds of transgenic plants (van Rooijen and Moloney, 1995b; Parmenter et al., 1995). The fusion site between the oleosin and oryzacystatin contained a recognition sequence for the specific endopeptidase, collagenase, from the marine bacterium, Vibrio alginolyticus. Cleavage of oryzacystatin from the oil body was demonstrated in vitro using a commercial source of collagenase. The highest expressing oleosin-oryzacystatin plants were then crossed to another transgenic line containing the collagenase gene under the control of a B. napus isocitrate lyase promoter, which is germination specific. Double transformants, confirmed by PCR, were then tested for proper spatial and temporal expression of the introduced transgenes. In doubly transgenic seedlings, cystatin was released from its oleosin carrier by collagenase cleavage. This free cystatin was shown to inhibit papain, a cysteine proteinase, and insect gut extracts in in vitro assays. Furthermore, flea beetle feeding trials revealed that transgenic lines containing cystatin are more resistant to flea beetle feeding attack compared to Westar control seedlings. Similar feeding experiments revealed that these transgenic lines also have a negative effect on flea beetle survival. Thus, the presence of cystatin in the cotyledons and embryonic axis appeared to provide protection to the seedling from flea beetle attack. This pulse of cystatin (in 1–7 DAI seedlings) coincides with the most susceptible phase of plant growth. The fact that its appearance is transient may assist in avoiding selection for resistant insects in field situations. |
