The differences (variations or abnormalities) induced in vitro are generally affected by the type of explant, composition of culture medium (especially, growth regulator regime), length of culture phase (longer the duration more the variation), callus phase (direct regeneration: less the chances of variation), cytogenetic instability (in case of polyploids), etc. Generally, it arise during multiplication phase. At molecular level, endo-reduplication, mitotic irregularities, error by DNA polymerase, etc. are causes.
Dear Ahmed Al mayahi,
Variations occurring during in-vitro culture are more common than exceptional. Many causes have been suggested, the previous answer does list a number of them. But that does not solve your problem. Some general rules apply: use as low as possible of any plant hormone (BP,IAA, nAA) avoid things like 24D like hell. The gelling agent may be of prime influence: use very pure ones like Gelrite or equivalent. Autoclaving of your media may affect its use: autoclave as short as possible, cool down as soon as possible and use directly. Add hormones, vitamins after autoclaving the media, after filter sterilization.
As for plant material: try to use a homogeneous source of your explants. Test them for their ploidy level before putting them into culture by flow cytometry. Many tissues are polyploid and a bad source for explants. Hypocotls can do strange things, try to select for the meristems.
I hope this helps a bit. Feel free to ask for more if you think I could be of help.
kind regards, Harrie Verhoeven
@ Ahmed Al mayahi ,
This is due to 'Somaclonal variations'. During tissue culture (especially long period time of culturing), aberrant chromosomes can be generated, DNA can rearrange..... Somaclonal variation post serious threat for 'true-to-type' plant production through tissue culture propagation.
Please see the attached paper for the "sources of variations detected in plant tissue culture".
The type of growth regulator used is one of the essential reasons.
Apart of the composition of the culture media/environment which can lead to soma clonal variation, mutations can occur in the different cells of the tissue before or during propagation. Know that cells are "totipotent" and no mater how homogeneous your tissue may be, individual cell mutations can lead to different individual genotypes.
Culture age usually affect somaclonal variation besides, auxin type and concentration in the culture media .
Somalonal variations and gametoclonal variations are main factors of the variation occurring in invitro culture of embryo/pollen culture. Somalonal variations occurs due to many reasons like chromosomal rearrangements/ instability, aberrant chromosome containing duplications and deletions. Duration and condition of culturing also leads to variation.
The genetic variations which might be occurred cultured tissues cells are collectively referred to as somaclonal variations.
Somaclonal variations in plants include polyploidy, aneuploidy, chromosomal breakage, deletion, translocation, and gene amplifications, and different mutations. In addition, the presence of several chromosomal aberrations, chromosomal breakage and deletion also considered SV.
Many factors could be count for induction of genetic variations in in vitro cultured tissues including varied nutrients, the conditions of culture and the metabolic products that release and accumulate in the medium. Somaclonal variations due to the auxins and cytokinins concentrations and type have also been reported.
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