Cryopreservation by encapsulation-dehydration affects the vegetative growth of chrysanthemum but does not disturb its chimeric structure

Little is known about the stability of plant chimeras after exposure to liquid nitrogen. The aim of this study was to evaluate the effect of cryopreservation on the cytogenetic and genetic stability, as well as vegetative and generative growth of chrysanthemums that are a solid mutant (‘Richmond’) or periclinal chimeras (‘Lady Orange’ and ‘Lady Salmon’). The following encapsulation-dehydration protocol of shoot tip cryopreservation was applied: 0.09 M sucrose concentration and 10 µM abscisic acid during a two-week preculture, followed by a 4-day osmotic dehydration and 3-hour desiccation. Both, the cryopreservation-recovered and control plants were characterized by the same relative DNA content and chromosome number (ploidy level). By applying RAPD and ISSR markers, 18 and 1 and 0 polymorphic loci within cryopreservation-derived ‘Lady Orange’ and ‘Lady Salmon’ and ‘Richmond’ were detected, respectively. The recovered after cryopreservation and control plants had the same colour, diameter and fresh weight of inflorescences, as well as length of ray florets. Cryopreservation also did not affect the flowering time in chrysanthemum. The biochemical assay did not reveal any alternations in the level of anthocyanins and carotenoids in flowers. It was noted, however, that some of the leaves of the cryopreservation-recovered plants were shorter and/or narrower. They had a reduced chlorophyll content, and their internodes were shorter when compared to the untreated control. The inflorescences of ‘Lady Salmon’ opened slower, but faded faster. In conclusion, these results illustrate the practicability of a cryopreservation method that completely protects the chimera chrysanthemum identity.