I have plants (L. japonicus) that have been transformed with an overexpression plasmid. How can I know that these plants are homozygous for the insertion?
One solution is to do a restriction digest of the genomic DNA, dilute the concentration and ligate to form circularized pieces of DNA. You can then use PCR to amplify outwards from the ends of your insert and sequence the product to identify where the insertion is in the genome. You can then design primers to amplify across the insertion locus to allow for genotyping.
Alternatively make several separate lines and sequence a large number of progeny from self fertilization and only keep lines that do not produce any offspring lacking the insert. After two generations you can be confident that it is not heterozygous.