Seeds obtained from heterozygous T0 mutants obtained through CRISPR CAS9 were sown and the plants raised were properly genotyped. Following normal segregation pattern, T1 generation had WT, heterozygous and homozygous plants. The homozygous were not producing normal seeds but strangely few (2 in many) produced more than 50% normal seeds, when those seeds and the plants raised from them were genotyped they were found to be heterozygous. In T1 I ignored this (thinking that T0 plants from CRISPR CAS9 might carry chimeric mutation for the gene).
To get T2 generation, seeds obtained from T1 heterozygous were planted, but T2 homozygous plants also had such plants. The situation even continued to the next generations. I am wondering what might be the cause. I know that cross pollination in rice occurs to small extent but if that is the cause then all homozygous plants must bear small number of such seeds. In this case homozygous usually don’t produce normal seeds at all, unusually few homozygous bear normal heterozygous seeds. Your suggestions will be highly appreciated to explain this situation. Thanks.
RB generates perfectly complementing homozygous parental lines through engineered meiosis.
Any update on this further? Any possibility of outcrossing as you said the homozygous do not prouduce normal seeds (may be defect in male part). This increases the chances of outcrossing.
I would say check for the adulteration. and your selection (may be antibiotic) efficiency (If you're using any). Also when you say heterogeneous T0, i don't understand what you mean by this. How do you confirm their heterozygous or homozygous nature?
Aziz Ul Ikram . When you say heterozygous plants came from HOMOZYGOUS plants, it leads to a lot of questions. Provided your genotyping and phenotyping have been accurate. What trait were you following? Or was it just good/bad seed? Outcrossing in rice is very less. Moreover you might be doing it in the greenhouses etc where pollen contamination should not happen. Again, provided you have accounted for it. The appearance of good seed from a homozygous plant which otherwise is producing only bad seeds could be due to other genes which you might not be following. What markers were you using to genotype the plants?
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