Transplastomic tobacco with three genes for insect resistance. Success was also achieved in tobacco, where more than one genes for a biosynthetic pathway were transferred to plastid genome in a single transformation event. In this exercise, a complete operon (Bt)cry2Aa2,carrying three genes was introduced into the tobacco plastid genome in a single transformation event (the cry2Aa2gene for insecticidal protein is the distal gene of a three-gene operon and the other genes upstream of this gene code for accessory proteins to help in folding).

The operon showed high expression, leading to accumulation of foreign protein up to a level of 45.3% of total soluble protein (TSP), which was so effective that it killed 100% insects (which were recalcitrant to other control methods) fed on leaves of transplastomic plants. This demonstrated that plastid transformation can be efficiently used for transfer of several genes in a single transformation event to produce transplastomic plants having stable expression of all genes belonging to a biosynthetic pathway.