you need to do PCR now in T2

and if you found target gene you selct plant and coolect seed and cultured to get T3

also pls see my thesis have work on arabidopsis plant and trasgenic plant

The easiest way is to grow 8 or 9 T2 plants for each selected T1 line, best grown before on kana so you only bring heterozygous or homozygous plants to soil for seed set. Don't bother trying PCR to select homozygous plants in T2 (real time PCR to detect gene dosage?). Just harvest all T2 plants individually and sow 50-100 seeds each on kana plates ín the T3 Generation. This can be done non-sterile, ie do not add sucrose to the selection medium and simply springle the seeds (no Need to sterlise) on the plates on your normal work bench. Yes, you will see some fungi but you can see the kana resistant plants quick enough. YOu will get segregating and homozygous T3 seed Batches so you can see whether your original T1 was single locus or more. Always go for single locus homozygous lines. Journals require at least 3 individual transgenic lines per constructs best is to start with at least 10 T1 for obtaining T2 and T3.

Depending on the material you need for metabolomics, you might have to bulk your homozygous T3 to obtain grams of seeds to obtain enough biomass.

Agree with Eric. You should use more than 4 T2 from independent T1 lines, and grow them on kanamycin (we assume the mutant line you transformed is not kan resistant, is it?). Then test the T3 to select the homocigous lines. If the construct you transformed with is over-expressing the gene you could probably test the effects in heterozygous lines, but is better to work with homozygous lines. 

Also, it is better to  make a pool of plants for the metabolomic analysis. If you are sending the material to analize to a facility ask them how many material you will need.

Continuing to what Tamara and Eric have explained nicely above, I would like to add some suggestions. To determine whether the transgenic plants you have are homozygous or not, you need to do the "Progeny Test."

Lets assume that you have introduced (nptII + gene of interest) construct and you have single integration of the construct in all of the transgenic lines.To level the ground, I would define the following:

(1) The regenerated transgenic plants are named as T0 generation (T0 transgenic plants). The phenotype of the T0 transgenic plants is Kanamycin resistance (Kan R) and the genotype is hemyzygous (R/0).

(2) The plants grown from seeds, harvested from self pollinated T0 transgenic plants, are named as T1 generation (T1 plants). The phenotypes of the T1 plants are either Kanamycin resistance (Kan R) or Kanamycin sensitive (Kan r). The ratio of phenotype classes are Kan R : Kan r=3 : 1. The genotype of T1 plants will be mixtures of homozygous R/R (Kan R); hemizygous R/0 (Kan R); and homozygous r/r (Kan r). If the T1 seeds are grown on medium containing Kanamycin as Eric suggestion, we can eliminate the r/r (Kan r) individuals from the population.

(3) The plants grown from seeds, harvested from self pollinated T1 transgenic plants, are named as T2 generation (T2 plants). The phenotypes and the genotypes of T2 plants depend on the genotype of the T1 progenitor.

If the T1 progenitor is R/R - then the genotype of T2 plants will all be R/R and the phenotype will all be Kan R. If the T1 progenitor is R/0 - the genotype of T2 plants will be a mixture of R/R, R/0, and r/r (1:2:1) and the phenotype will be a mixture of Kan R and Kan r (3:1). If the T1 progenitor is r/r - the genotype of T2 plants will all be r/r and the phenotype will all be Kan r (but we have eliminated this by growing T1 seeds in medium containing Kan and growing only Kan R to maturity in soil)

Back to your question, if you grow 100 T2 seeds on medium containing Kanamycin (progeny test) and all T2 seedlings are Kan R then most probably the T1 parent of this T2 population is homozygous R/R. You can go back to this particular T2 seed lot and grow as many T2 plants as needed for your metabolomic evaluation.

If you grow 100 T2 seeds on medium containing Kanamycin (progeny test) and you see some Kan r - T2 seedlings, then most probably the T1 parent of this T2 population is hemyzygous R/0. You will not select this seed lot for your metabolomic evaluation.

Words of caution: the more number of integrated construct (multiple copy transgene integration) the more complex the segregation ratios between Kan R vs. Kan r in the progeny. Therefore, make sure you determine the copy number of construct integration in the genome by Southern Blotting first and select one that indicate single integration.

If you did not select for progenitor that are homozygous R/R, probably you will have mixtures of genotype in the progeny population (mixture of R/R; R/0; and r/r) which may probably affect recording of your metabolomic parameters.

Once you establish copy number integration in the T0 plant, you can skip molecular analysis in the T1 generation and just employ selection using media containing Kan, select only Kan R T1 progeny, and grow the T1 seedlings to maturity to harvest T2 seeds. Select T2 seed lots derived from homozygous R/R T1 plant based on progeny test as outline above.

Good luck...

Thank u all for your interesting answers, learning something new from this. Thanks a lot.

Hi Sudarsono, i really appreciate your effort in making me understand the concept with your very nice, simple and clear explaination. Thanks a lot for time.

Even with homozygous lines you may still observe quite a range of differences in metabolite production from your different lines.  It might be a good idea to start growing up seeds from additional lines so you have more than 4 to test. 

Glad to help... I also agree with Amy suggestion. When talking about phenotype (i.e. metabolite production) - It is the results of the genotype by the environment interaction. The genotype may all be R/R but different environment conditions may results in variation of metabolite production. Therefore, the more samples or replicates the lower the environment effects... Good luck.

Homozygous plants are selected at T2 stage only. Following two publications have detail description of selecting honozygous transgenic plants:

1.  Shoot and fruit borer resistant transgenic eggplant (Solanum melongena L.) expressing cry1Aa3 gene: Development and bioassay

2. Expression of rd29A::AtDREB1A/CBF3 in tomato alleviates drought-induced oxidative stress by regulating key enzymatic and non-enzymatic antioxidants.

I'd recommend Eric's and Tamara's suggestion (particularly in coordinating with the facility that's going to .

Also, I'd like to rectify one thing:

"The regenerated transgenic plants are named as T0 generation (T0 transgenic plants). The phenotype of the T0 transgenic plants is Kanamycin resistance (Kan R) and the genotype is hemyzygous (R/0)"

At least in the Arabidopsis community (and AFAIK other organisms, too) the lines described above are T1 plants (primary transformants). This may be important to avoid confusions afterwards...otherwise this was a comprehensive explanation. Just exchange To->T1, T1->T2, T2->T3.

Hope all your experiments work!

Thanks all for your valuable answers, appreciated your time.

@ Marcel: Sudarsano is right when he said T0 plants. In all other crops when we do a transformation the plants obtained after transformation is always named as T0. In Arabidopsis, the transformation is usually done by flower dipping and what you obtain is transformed seeds and not plant, and in this case the seed germinated first generation is actually T1. So it is only for Arabidopsis it is T1 and for other crops it is T0 unless they also follow a direct ovum transformation.

@Beena: Thanks for the Info on crops! That's interesting. Nevertheless the thread opener works with Ariabidopsis. Therefore he should stick to "community standards", don't you think?

Yes of course! as Uday works with Arabidopsis and given that he had done the transformation via floral dip, he should call the seed generated plants as T1 and not T0 :-)

Related to this thread, I have a doubt. If the transgenic plant is a vegetatively propagated one, do we still use the same terminology?

I am working on RICE. so which generation should I consider as homozygous for further studies? (This question is in relation to @Beena's explaining about differences between T0 and T1 calling in Arabidopsis and other crops) Ms. Beena can you please elaborate on this? thanks

Hello Dhananjay,

I am sorry that I did not see your question before. Even if it is too late, I think it is worth answering :-).

To look for Homozygous plants usually you need to go until T3 generation but if you know the sequence and where exactly your gene is integrated, there are PCR methods to know if it is a homozygous or heterozygous plant. Hope this helps.

The number of plants to use for your metabolomics study depends on your study plan. If you want to see the most impact, choose the lines with the highest overexpression. If you want to see the range, choose lines from a broad range of expression, from normal to moderate to very high over expression. Keep in mind that sometimes overexpression can lead to unwanted side effects or unwanted impacts.