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.
Key messageCryopreservation via encapsulation-dehydration technique affects the vegetative growth of chrysanthemum during the first cultivation season but does not disturb its chimeric structure.
The aim of this study was to evaluate the influence of sucrose concentration in the preculture medium, as well as the duration of osmotic dehydration on the efficiency of chrysanthemum 'Richmond', 'Lady Orange' and 'Lady Salmon' cryopreservation by encapsulation-dehydration technique. For all cultivars tested, the best regrowth of cryopreserved shot tips was recorded with 0.25 M sucrose concentration and 10 µM ABA during a two-week preculture, followed by a 4-or 7-day osmotic dehydration. The survival rate ranged from 56.8-58.0% (the Lady group) to 63.6% ('Richmond'). However, the ability to grow was smaller and reached 18.2-50.7%. It was found that higher sucrose concentration during the preculture slowed the growth of chrysanthemum shoot tips and led to an increased formation of multiple shoots (by activating axillary buds) or deformed adventitious shoots (incapable of further growth). The frequency of tissue hyperhydricity also increased, while the rhizogenesis efficiency decreased when higher sucrose concentration in the preculture medium was applied. The influence of osmotic dehydration duration on the explants morphogenetic response was cultivar-dependent.
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