Dariusz Kulus, Monika Rewers, Agnieszka Abratowska and Anna Mikuła
University of Technology and Life Sciences in Bydgoszcz, Faculty of Agriculture and Biotechnology, Department of Ornamental Plants and Vegetable Crops , Laboratory of Biotechnology, Bydgoszcz, Poland
Chrysanthemum is among the most popular species on the ornamental plants market. The number of available cultivars is constantly growing and they constitute a great breeding material source. Furthermore, the species constitute a valuable source of numerous secondary metabolites useful in medicine (e.g. in cancer or AIDS treatment). A fast and easy access to high quality gene banks of a great material variety is the key for ornamental plant producers, as well as for pharmaceutical laboratories and so an efficient method for longtime conservation of the plant material may be extremely valuable for both horticultural production and medicine development. Over time several medium- and long-term plants storage methods have been developed. Traditional storage (cultivation in the ground or in the glasshouse), however, is expensive, work-intensive and threatened with loss due to pests and diseases or bad climate conditions. In vitro storage, on the other hand, may lead to the occurrence of somaclonal variation, decrease in metabolic/embryogenic activities and material loss due to human errors and development of bacterial/fungal contaminations. Today, cryopreservation (i.e. maintenance of biological material at the temperature of liquid nitrogen; -196°C in Dewar flasks), developing rapidly for the last 25 years, is believed to be the most effective long-term storage method. Usually cryopreservation does not influence on the characteristics of the plant material. Still, there are some reports about (epi)genetic disturbance after storage of chrysanthemum in liquid nitrogen, especially with chimeras, which are very popular among the species (about 50% of all available cultivars). These reports emphasize the need to monitor the stability of samples stored in liquid nitrogen by using different markers (molecular, phenotypical and/or biochemical).
The aim of this study was to determine the effect of sucrose concentration during preculture and the time of osmotic-dehydration on the efficiency of chrysanthemum ‘Lady Salmon’ shoot tips cryopreservation by encapsulation-dehydration. In addition, the regenerated plants were verified at the phenotypic, biochemical and molecular levels. Shoot tips were precultured on MS medium supplemented with different sucrose concentrations of 0.09, 0.25 or 0.5 M for 14 days, encapsulated in sodium alginate and then osmotically dehydrated in sucrose gradient for 4 or 7 days. The best explant survival after cryopreservation reaching about 50% was obtained with the lowest (0,09M) sucrose concentration, and 4-day-long osmotic dehydration. It was found that higher sucrose concentrations slow down shoot growth, stimulate their vitrification and conduce to the regeneration via callus, while encapsulation inhibits rooting. Longer dehydration also led to increased formation of multiple shoots. Microscopic analyzes confirmed the protection of not only all of the initial meristem layers, but also of the leaf primordia and even larger leaves. Transmission electron microscopy (TEM) revealed accumulation of starch as a sole alternation observed in the cryopreserved cells. The analysis of the phenotype (inflorescences and leaf colour, diameter and weight, flowering time and plant habit) and biochemical activity (pigment content in ligulate flowers and leaves), as well as, cytogenetic analysis (DNA content, the number of chromosomes) and genetic markers (RAPD and ISSR) confirmed the stability of the plants obtained after liquid nitrogen treatment. However, it was noted, that the leaves of shoot tips cryopreserved-derived plants were smaller and had a reduced amount of chlorophyll, and their internodes were shorter when compared to the control. Furthermore, their inflorescences often opened slower. Finally, these phenotypic changes are positive from the horticultural production point of view. This confirms the validity of utilizing cryopreservation in the protection of valuable plant material.