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. 2013 May 27;8(5):e64980. doi: 10.1371/journal.pone.0064980

Figure 6. Strategy for testing the empty container hypothesis.

Figure 6

Homozygous ssp1 mutants were transformed with a PHA transgene and single copy insertion lines were selected by Southern blotting (step 1). The primary transgenic lines were selfed and homozygous tester lines were selected by examination of SDS-PAGE profiles of seed extracts (ssp1 pattern) and 100% hygromycin segregation in the T3 lines (step 2). The tester lines were crossed to wildtype with the resulting F1 plants being both heterozygous for CRU3 (ssp1) and for the PHA transgene (step 3). Selfing was conducted and F3 lines screened by SDS-PAGE for normal seed protein profiles, by sequencing of CRU3 to determine homozygosity, and by hygromycin segregation to determine PHA homozygosity (step 4). The resulting tester and backcross lines are homozygous for a single PHA insertion such that reporter gene copy number and context are fixed in both the tester (cru3 background) and the wildtype (CRU3) background.