Why does my embryogenic cell culture losing its embryogenic potential after several subcultures? I try to maintain it growing fast by frequent subculture, selecting more dispersed cells, maintaining the same media composition, yet after several subcultures it doesn't produce as much embryos as it used to.
Long-term maintenance of the embryogenic potential needs secretion of transcription factors and gene activity is highly essential. Yes it is true that embryogenic cell culture losing its embryogenic potential after several subcultures. Really your media should contain inducers of transcription factors for the cells which are under differentiation, Such cells need evoking of maternal effect genes, and homeotic genes. At least 10-12 important transcription factors mainly proteins such as engrailled, entrappeped, kruppel, osker and TGF, EGF, VDGF are needed in the medium to keep the cells in dynamic state of differentiation.
Hi Ranjith,
The answer added probably more to your second question 'why'--
The induction of somatic embryogenesis (SE) to produce somatic embryos [as appose to 'zygotic embryos'] are also controlled by certain genes. The expression of these genes, such as the carrot DcSERK gene mentioned below, initiate SE. The over-expression of DcSERK gene was observed exhibited a 3- to 4-fold increase in efficiency for initiation of somatic embryogenesis (see attached paper). Therefore, one of the possibilities is that the prolong and frequent subculture of your cells might have somehow 'methylated' these genes and cause the loss of the ability of initiation of somatic embryogenesis in these cells. I wonder if other researchers have looked into this matter.
This is one paragraph from the attached paper:
"In plant tissue culture, a small proportion of single somatic cells can be induced to change fate toward embryogenesis by application of exogenous auxins. These cells are called “competent cells” and can give rise to embryogenic cells from which somatic embryos can develop. The formation of competent cells in culture depends on the presence of certain arabinogalactan proteins produced by non-embryogenic cells in culture (McCabe et al., 1997; Toonen et al., 1997a). One of the genes expressed in competent cells in carrot (Daucus carota L.) tissue culture is the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (DcSERK)."
Hi Ranjith,
The only way to secure embryogenity of your cultures for the long term is through cryopreservation. At least, this is what we do with our banana embryogenic cells suspension. Once we have a good suspension we preserve some aliquots in liquid nitrogen; Currently we have stored more than 1000 tubes of banana cells. Recover after 10 years of storage was identical to 10 minutes of storage.
But Ranjith knowing you, probably you already though at this.
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