the fastest way is what you suggested...mendeleian segregation of the markers.

If you know the point mutation you could try to design a dCAPS primer. The reporters don't not have to be homozygous to know the effect of the transgenic or mutant on the reporter activities. This could save you one generation. Further, stressing the plants (such a high light) in young stage will promote early flowering, you won´t need much seeds in the first stages of genotyping and selection for antibiotic resistance.

@  Eric van der Graaff,

As you mentioned that the reporters don't have to be homozygous, what about the 'mutant allele'? Does it need to be homozygous?  According to Abigail, it is a 'dominant mutation'. Seems like that homozygous for the mutant allele is not necessary or needed neither, since it is not a recessive mutant allele (need homozygous to show phenotype) [see attachment].

@ Stefano D'Alessandro,

It depends on which zygosity (homo- or heterozygous for the mutant allele and the reporter) of the two parental lines, which are used for crossing, carry. Only the offsprings (seeds) from the crossing inherit both alleles should be used.

In principal both the transgenic line and the dominant mutant could be heterozygous to investigate their effect on the reporters. However, gene dosage could affect the results from the reporters so best would be to study the transgene and mutant in homozygous state.

@  Eric van der Graaff,

I am not sure in plants (or any report), but this (dosage effect) has been reported in human beings, that 1 copy of dominant mutation allele or 2 copies of dominant mutation alleles lead to different phenotype (syndrome) presentations. Please see attached paper (yellow highlights). Therefore, probably both plant lines with either heterozygous- or homozygous dominant mutation allele should be tested.

"Dosage effect of a dominant CLCN1 mutation: a novel syndrome."

Selfing or cross pollination of plants raised from seeds of  transformed plant will produce progeny carrying homozygous trait expressed by particular transformed gene.

You can also try using quantitative real-time PCR. It can quickly tell the zygosity (homozygous or heterozygous) of your transgenic plants. If your lab has proper equipment and the knowledge to conduct this type of experiment, it should be faster than progeny test. See attached paper:

"A rapid method for the analysis of zygosity in transgenic plants"

Even when dominant mutants are not labelled as semi-dominant, indeed you might see dosage effects, same goes for ttransgenics. Often it is simply strength of phenotype. Have encountered this in several of my projects. In case the reporter shows a different expression pattern than the wild-type situation due to the dominant mutation or transgene, the heterozygous state is useful to understand the respective effect. For publication you will need the homozygous states.

@ Eric,

Yes, I agree with you that there are many things to concern. But, I am not sure the sentence you put: "when dominant mutants are not labelled as semi-dominant, indeed you might see dosage effects". What does that mean?

 @ Abigail Rubiato Cuyacot,

Attached is an interesting paper (2015). Most likely you will not need the technology now. It just for your future reference:

Authors in the paper used seed-expressed green and red fluorescent transgenes to directly determine homozygous or heterozygous status for a mutation of interest in Arabidopsis seeds. The fluorescent colors and intensity are used for the detection.

In the attached picture: bf: bright fluorescence, if: intermediate fluorescence, nf: no fluorescence, g: green fluorescence, r: red fluorescence

 Hi M. K. Razdan,

Regarding the statement: "cross pollination of plants raised from seeds of transformed plant will produce progeny carrying homozygous trait expressed by particular transformed gene."

It should depend on what kind of plants you are crossed to. The genotypes of the seeds (or the plants derived from these seeds) from a transformed plant can be +/+ or +/- or -/- [unless -/- seeds are removed (ex. by selection)] for the transgene. You won't get progeny with homozygous status for the transgene if cross-pollination occurs between two plants where one or two plants carry -/- genotype for the transgene. Because: +/+ x -/-  = +/-, also +/- x -/- = +/- or -/-, and -/- x -/- = -/-.

Good question and nice discussion. Finding a homozygous transgenic plant while working with crop plants expressing gene of interest is more challenging as far as time is concerned. I endorse qPCR is helpful in this regard as mentioned in article from Israelian fellow researchers. 

Thank you so much for sharing your ideas about this. It help me think. And yes, I intend to design primers and do the genonytping via PCR. But I am just curious how would I do it without the information of the insert (since it's a point mutation). If I can find some references as to where the mutation is, then this will be good I hope. 

 Since you have a point mutation I suggest you PCR the fragment and digest the PCR product with a restriction enzyme sensitive for your point mutation. This way the restriction enzyme will cut only one of two fragments, the dominant or recessive. This method is easy, cheap and can give you good results. You just to have to find a good restriction enzyme, design primers and establish your PCR. Good luck!

With semi-dominant mutations you have difference in phenotype between hetero- and homozygous state. This could simply be gene dosage or also presence of a functional wild-type allele in the heterozygote. When a mutant is called dominant you might still have gene dosage effects, same goes for transgenics (mis- or overexpression). The term semi-dominant is not always used, even when it actually should have.

Hi Eric and all,

1. Yes, I agree @ Eric with your statement "When a mutant is called dominant you might still have gene dosage effects". As it has mentioned in the paper I attached earlier [ "Dosage effect of a dominant CLCN1 mutation: a novel syndrome" ]. 1 copy or 2 copies of that dominant mutation allele does lead to different phenotypes. Some homologous (2 copies) dominant mutation are lethal for the organism.

2. I attached a Molecular and Cell Biology class note from UC-Berkeley below. It gives clearer and more detailed definitions on the Dominant Mutations. It is a pretty good article.

Dominant mutations include (1) haploinsufficient and (2) gain-of-function mutations. In gain-of-function mutation, it further divides into 3 types based on their activities. [see yellow highlights on pages 3-5]

3. I wonder what kind of dominant mutation does Abigail Rubiato Cuyacot 's mutant line has.

I agree with Dr. Yuan-Yeu Yau that crossing with non-transgenic tester will produce only heterozygous. Further selfing of heterozygous plants we can also produce homozygous plants for transgene in the next generation. We will not see further segregation of transgene in further generations if we self the homozygous plants. If the transgene is linked to selectable marker gene, it is easy to identify the homozygous line by antibiotic sensitivity assay. If it is generated by genome editing technology / point mutation, detailed molecular characterization is required to identify the homozygous line. We generally use the terminology T2 for the selfed seeds or plants of T1. Similarly T1 is derived from T0 plants by selfing. If we cross the transgenic plant, then the terminology changes

@  Narendran Madhavan Nair,

I agree with you. Just want to add some elements to it.

1. A simple single transgene insertion is easier to find the homologous progeny in the progeny. However, nowadays, quite often, genetic transformation for multiple genes-of-interest is required. Especially, in the case of studying genes involve in metabolism pathways. Transformation of a whole set of genes from a pathway is needed (Golden rice production and vitamin-enriched GMO production are examples). If a few genes are transferred into a plant separately, tedious work is needed to search for the rare lines with homologous for all those transgenes in the progeny. For example, 6 genes are transferred into 6 unlinked locus (single copy for each transgene) in a diploid, the chance to find an offspring with homologous for all 6 transgenes in a population is (1/4)6 = 1/4096. So, development of strategies for transgene stacking to a single locus as a whole entity in order to co-segregate and inherit together is very useful in this regard.

2. Product produced from Genome-editing technology (as you mentioned above), not only it is important to check whether the right allele(s) are knocked out, it is also very important to check whether off-target mutations are also present. Off-target effects can affect the whole genome stability.

Due to the high efficient mutagenesis (CRISPR/Cas9 for example), some have reported that biallelic homozygous mutations can be achieved at primary transformants T0. One doesn't even have to wait to screen for homozygous mutation genotype from the T1 population (see attached paper, page 6, yellow highlights).

3. This has been discussed in RG and other places. For 'Primary Transgenic Lines', in some plant species, people refer them as T0.  And in others, people refer them as T1. For example, in Arabidopsis community, primary transformants are referred to T1. And, we usually use T0 for tobacco primary transformants.

From my previous advisor's suggestion, in publication, it is better to define them clearly at beginning of the manuscript to reduce the confusion from reviewers and audience.....for example, denote: 'primary transformants (T0 or T1)'........

Just do sequencing and check the chromatogram.