Dear Sutthinut,

Results from work published so far indicate that the molecular changes during in vitro culture and cryopreservation are mainly due to methylation of the DNA and no (or minimal)  point mutations or chromosome aberrations. Therefore, whichever molecular technique you employ, you should use methylation sensitive primers to detect the range of molecular changes.

You say your material is not clonal. So I am guessing these are seed-derived and even so there can be homozygous lines or open pollinated material. So it is important to know the genetic constitution of your material before applying any molecular markers. Not knowing if your material is homozygous or herterozygous, it is difficult to suggest methods.

Regards

Ranjith

I found a recent paper on a seed crop and they use Reversed Phase-HPLC to quantify the DNA methylation. They use DH material so they are again homozygous! PAper just came out in Plant Cell Tissue and Org Culture and I have attached it.

Hello, Greetings.

RAPD, SSR and ISSR are most prominently used methods for detecting genetic variational studies.

ISSR, DAMD,SCoT etc can be used. Concern here is that the tissue cultured/ cryopreserved plants are not clonal, so if you intend to check somaclonal variation then it would be difficult.

For genetic fidelity testing one can use markers like SSR or ISSR (RAPDs are not recommended now a days due to problems of repetability)..but make sure that the sample size as well as the number of primers tested are sufficiently large, say 100 primers for SSR and some 20 for ISSR. Always use parental DNA and totally unrelated or even DNA from other source as controls. The data will tell you two things.... Presence of any variant........and also true to the type plants (if you intended for that)........

Except 'methylation' causes genetic variation mentioned by Ranjith, mutation caused by other genetic factors during tissue culture stress are also reported. Transposable elements (TE) is one of them. TE include transposons and retrotransposons.

TE take a large portion of plant genetic makeup, for example, ~25% rice genome and >50% maize genome. TEs can be activated during different stresses, such as cold, heat, wounds and tissue culture etc (these all have been published and documented). Examples of transposons and retrotransposons which were activated and hopped around in the genome of different plants by tissue-culturing had been reported (see attached papers). The translocations of TE leave footprints all over the genome.  Since you are measuring non-clonal genetic variation, you can also consider the methods involved in genome-wide TE movement detection, after your plants have been grown in tissue-culture medium for a while.