Cryopreservation of in vitro-grown shoot tips of chrysanthemum by encapsulation-dehydration

Abstract

Chrysanthemums are amongst the most economically important flowers in the world. The protection and storage of these valuable genetic resources is of great importance. Today, cryopreservation, or the storage of biological material at the temperature of liquid nitrogen (-196°C), is believed to be the most promising long-term storage method. To optimise the cryopreservation protocol, the shoot tips of Chrysanthemum × grandiflorum /Ramat./ Kitam. 'Lady Orange' and 'Lady Salmon' mutants were cryopreserved using the encapsulation-dehydration technique. During the experiment, the influence of sucrose concentration (2, 3 and 6%) during preculture and the concentration of kinetin (0.25, 0.5, 0.75 and 1.0 mg dm -3 ) in the regrowth medium were tested. A higher survival rate was observed for 'Lady Salmon'. In general, the media with higher sucrose levels provided the best survival and recovery rates (35-40%). Kinetin had no influence on the survival rate; however, it influenced the morphogenesis of the plants. The lowest number of explants forming multiple shoots was observed on the medium with the lowest sucrose (during preculture) and kinetin (in the recovery medium) concentration. On the other hand, the best rhizogenesis efficiency was observed when 0.25 mg dm -3 kinetin was added. In conclusion, the composition of both preculture and recovery media need to be adjusted to single cultivars. The use of 3% sucrose (preculture) and 0.25 mg dm -3 kinetin (recovery) seems reasonable, since it guarantees a satisfying recovery rate of the explants and at the same time prevents the formation of callus and multiple shoots, stimulating the rooting instead.

Effects of various preculture, pretreatment, and recovery conditions on the morphogenetic response of cryopreserved Lady Orange chrysanthemum shoot tips

“…The explants were placed in cryovials and immersed in LN. After 1 h, the cryovials were removed from the LN and rewarmed in a water bath (as above), and the beads were inoculated on modified MS recovery medium with 3% sucrose and 1.16 µM KIN + 0.54 µM NAA (Zalewska and Kulus, 2013) and sealed with Parafilm. The cultures were kept in the same growth room.…”

“…The survival rate of LN-stored accessions (100% in the best conditions after 90 days in culture) is very high since Zalewska and Kulus (2013) recorded a maximal 60% of viable chrysanthemum shoot tips 30 days after rewarming. The results suggest that even though the evaluation of cryopreservation efficiency based on explant color was often used in the past (Pawłowska, 2008;Kulus, 2013, 2014), it may not be a precise survival indicator due to its variation with time.…”

“…The survival rate of LN-stored accessions (100% in the best conditions after 90 days in culture) is very high since Zalewska and Kulus (2013) recorded a maximal 60% of viable chrysanthemum shoot tips 30 days after rewarming.…”

Evaluation of In Vitro Culture Medium for Conservation of Chrysanthemum Varieties under Different Storage Environment

“…In vitro conservation is a promising tool for preserving chrysanthemum genetic resource (Cruz-Cruz et al 2013). The successful in vitro conservation of chrysanthemum was depend on the use of growth inhibitor (Lan et al 2010), encapsulation-dehydration (Zalewska and Kulus 2013), low-temperature (Lan et al 2015), nutrient modification (Budiarto et al 2007), and the combination of those techniques (Benelli et al 2012).…”

An improved encapsulation protocol for regrowth and conservation of four ornamental species

“…On the other hand, successful long-term conservation can be achieved when plant material is stored at ultra-low temperature as in liquid nitrogen [24]. Among the cryogenic technologies available today, two in particular are used with synthetic seeds: encapsulation-dehydration and encapsulation-vitrification [25][26][27].…”