Cryopreservation by encapsulation of Gentiana spp cell suspensions maintains regrowth, embryogenic competence and DNA content

A reliable technique for cryopreservation by encapsulation was developed for two suspension cultures of gentian species (Gentiana tibetica and G. cruciata) of different ages and embryogenic potential. The effect of water content, aggregate size and the subculture time on viability was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) test. Regrowth of a proembryogenic mass (PEM) on agar, liquid or agar/liquid media was assayed by measuring the increase in biomass. A water content of 24-30% (fresh weight basis) after 5-6 h dehydration of encapsulated cells of gentians yielded the highest survival (68% for G. tibetica and 83% for G. cruciata) after cryopreservation. Regardless of species, aggregate size and subculture time, the lowest PEM survival was 44%. These parameters did not influence the survival of G. tibetica PEM, but the survival of G. cruciata was higher when the smaller aggregates were cryopreserved on the 5th day of culture. Agar/liquid culture caused the greatest biomass increase. Cryopreservation did not affect the characteristics of suspension cultures and their regrowth after thawing, nor the number and dynamics of somatic embryos formed. Flow cytometry showed that cryopreservation did not change the genome size of the PEMs or regenerants.