Gene stacking scheme in cotton. A, Schematic representation of strategy I target construct pZH83 with gfp and npt inserted into cotton chromosome from Agrobacterium-mediated random integration; D, Strategy I integration construct with SM (selection marker gene) and TG (trait gene) co-bombarded along with Bxb1 integrase-expressing construct pYQ78 (not shown, see Figure 3B); F, Expected structure from recombination with SM-distal attB and chromosomal attP; G, Structure after Cre recombinase-catalyzed deletion of DNA flanked by directly oriented lox sites. B, Strategy II target construct pZH84 has only two lox sites in opposite orientation. Co-transformation with a cre-expressing construct can delete tandem multicopy insertions, example shows only two copies (C), leading to a single copy structure in (B). E, Strategy II integration construct with an additional lox site distal to SM co-bombarded along with Bxb1 integrase-expressing construct pYQ78 to integrate into expected genome structure (H) from recombination with SM-distal attB and chromosomal attP, and (I) after Cre recombinase-catalyzed deletion of DNA flanked by directly oriented lox sites. Recombinase site symbols as shown in legend, Bxb1 recombinase-mediated attP × attB to yield attL and attR; LB and RB, T-DNA left and right borders, respectively. Promoters and terminators not shown, see “Materials and methods.” Gene transcription from left to right, except for npt indicated by upside down lettering.