I am interested to know the whole procedure for segregation including population size for segregation analysis to get homozygous transgenic plants.
Plants with flowers are dipped into Agrobacterium solution ---> plants set seeds ---> select seeds on selection agents (ex. kan) ---> individual transgenic plants obtain, and set seeds ---> seeds from individual transgenic plant are used for 3:1 ratio analysis on selection plates (single-copy line screen (to avoid gene silencing)) ---> if in a plate, 3:1 (1AA:2Aa:1aa) resistance:susceptable plantlets are observed, the survived plantlets should have homozygous-plantlets: heterozygous-plantlets (for the transgene) at 1:2 ratio. qPCR can be used to detect which ones are homozygous and which ones are heterozygous.
@Narendra Singh Yadav Yes, you can obtain homozygous transgenic plants in T2 generation following the procedure elaborated by @Yan-Yeu Yau. The minimum population size in T2 generation must be 12. To have greater probability of getting the desired homozygous plants, you should keep population size of 40 individuals.
Yes, you can get homozygous mutants in your T2 plants. But you will need to make sure you are screening for the possibility of multiple insertions (there can be multiple, independent TDNA insertions in one plant lineage).
https://blog.addgene.org/tips-for-arabidopsis-transformation
Yes you can, but to confirm it is real homozygous or the effect of over expression gene in heterozygous plant, you should go up to T3 generation@ . It is best to confirm single insertion in T2 generation (3:1) and select homozygous in T3 generation
I always used 2-3 plants for transformation (2 times with 5 days interval at proper stage) and selected at least 20 transformed lines against antibiotics. T2 seeds of these individual lines were screened for single insertion (1:3 as per the mendel's law) on antibiotic containing media whereas T3 seeds screened for homozygosity. Used 8-10 homozygous T3 lines with 10 replicates to gather the data. It may depend on your gene or studding pathway. just go through the attachment for your reference...
Dear Vijayendra Dalvi
Can you explain a little more about "If using KAN do not count seeds that do not germinate "? Why should it be counted? I count them along with the yellowish sick looking ones and the ones that do not have good roots. Sometimes plants grow and are small but have no roots. What about them?
Ya, its correct Dear Tayebe. The non germinated seeds are not taken in to the count but as most of people using 50-100 seeds for checking 1:3 ratio you rarely get the exact numbers. 10-15 seeds out of 50, may be non germinated, yellow or with out root considered while counting, which is OK and doesn't affect your results in T3 as you are taking data of only those plants which are germinated. do not worry for it.
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