Safe Food_ Bacteria, Biotechnology, and Bioterrorism - Marion Nestle [179]
TABLE 17. Highlights of one of the methods used to genetically engineer beta-carotene into Golden Rice*
Obtain the starting vector
Obtain a previously constructed Agrobacterium plasmid vector containing a transfer-DNA (T-DNA) from which the gene segments for crown gall and opines have been removed.
Construct the transfer-DNA
Using enzymes that split and reattach DNA at specific points, introduce into the T-DNA, one step at a time (not necessarily in this order):
• The daffodil gene for one enzyme in the pathway for making beta-carotene
• The gene from bacteria that specifies the other missing enzymes in the beta-carotene pathway
• Genes from peas and bacteria for proteins that will transport the new enzymes to the rice endosperm
• A marker gene for resistance to the antibiotic hygromycin (which blocks protein synthesis in rice and other plants)
• Regulatory DNA segments from cauliflower mosaic virus
• DNA segments that mark places where genes are to be inserted and removed
• Marker genes for resistance to other antibiotics
• DNA regulatory segments that enable the new genes to function in rice endosperm
Construct the new plasmid vector
Insert the plasmid with its new T-DNA “construct” into Agrobacterium by mixing them together in the presence of an electric current (electroporation), a process that makes the bacteria more permeable.
Prepare rice embryos for growth in tissue culture
Grow rice plants until they just set seeds; collect the immature seeds.
Remove the embryos from the seeds, and grow them in tissue culture (a medium containing nutrients and plant hormones).
Remove the sheath (plant material) that surrounds the embryos to make them more permeable; continue growing them in tissue culture.
Transfer plasmid T-DNA into rice embryos
Collect the unsheathed rice embryos growing in tissue culture and immerse them in a suspension of Agrobacterium containing the beta-carotene T-DNA plasmid vector.
Grow the vector-treated embryos in tissue culture.
Select the rare rice embryos able to accept the plasmid T-DNA
Add the antibiotic hygromycin to the growth medium, and continue growing the rice embryos; only those with the T-DNA containing the gene for resistance to hygromycin survive.
Test the surviving rice embryos to make sure they contain the genes for beta-carotene.
Grow the successfully transformed embryos in a rooting medium; grow the plants to maturity in a greenhouse; allow the plants to set seeds to maturity.
Harvest the rice seeds, and test them for beta-carotene. The rice grains that contain beta-carotene are yellow (hence: Golden Rice).
SOURCE: Ye X, et al. Science 2000;287:303–305.
*Refer to figure 13, page 156.
But that is not all. Constructing T-DNA sequences with foreign genes that actually function in plants requires the action of numerous enzymes that break DNA molecules at specific sites (“restriction” enzymes), enzymes that reattach split pieces (ligases), and a great many steps carried out in a specific order. For the system to work in rice, for example, the scientists also must successfully grow rice cells in tissue culture (an artificial medium containing nutrients and growth factors), infect the rice cells, grow them back into rice plants, and have the rice breed true under greenhouse conditions. Each one of these steps presents its own set of technical difficulties. Thus, genetic engineering requires a “feel” for how to make all of the steps work, which transforms the technology into an art as well as a science. The artistic aspects add to the difficulty of explaining the science to nonspecialists.
BRIDGING THE GAP
At issue is what is to be done to bridge the gap in knowledge and outlook between scientists and nonscientists. In a preliminary draft of this appendix (now much revised), I argued that scientists must work