Droplet vitrification technique for cryopreservation of a large diversity of blackcurrant (Ribes nigrum L.) cultivars

Abstract

The aim of plant gene banks is to preserve genetic resources selected based on their phenotypic, agronomic, historical or other cultural values for future utilization. In the present study the modified PVS2 droplet vitrification technique was tested and optimized for cryopreservation of a large diversity of blackcurrant (R. nigrum L.) accessions propagated in vitro and selected into a national gene bank core collection. Out of four accessions tested to optimize the method, three recovered and regenerated by 89–97% on average, but one recalcitrant in vitro line only by 25%. The tested post-cryopreservation recovery media with different macronutrient and growth regulator levels showed no generalized effect on regenerated shoots, but the effect of recovery media was different between cultivars. When the whole regeneration chain from cryopreservation via micropropagation to greenhouse conditions was tested, shoots at least 1 cm in length were found necessary for successful transfer ex vitro. The long-term cryopreservation of 22 blackcurrant accessions was finally conducted, with practices slightly modified from the tested protocol. The estimated recovery of shoot tips after 9 weeks in vitro was 17–94% with at least 75% recovery in seven accessions and at least 40% recovery in 19 out of 22 accessions. Only one accession had no cryopreservation success. The results demonstrated that the modified droplet vitrification technique is appropriate for a large diversity of blackcurrant accessions. However, cultivar-related differences and recovery procedures are to be considered for success in regeneration and ex vitro adaptation.

Successful Cryopreservation of Dormant Buds of Blackcurrant (Ribes nigrum L.) by Using Greenhouse-Grown Plants and In Vitro Recovery

“…We previously reported a procedure for the cryopreservation of blackcurrant cultivars by using the excised shoot tips of in vitro cultured shoots for freezing procedures [ 41 ]. Dormant bud cryopreservation can be a time-saving method in genebanking, even if the recovery of buds is done in vitro, because the initiation and the multiplication of in vitro cultures prior to cryopreservation can be omitted.…”

“…In the context of the renewal of new field collection, the need to create a cryopreserved backup collection was identified. Blackcurrant can be cryopreserved by using explants excised from tissue cultures [38][39][40][41] or by using dormant buds [42][43][44]. Cryopreservation success of blackcurrant varies depending on cultivar.…”

“…Cryopreservation success of blackcurrant varies depending on cultivar. According to our previous study, the estimated post-cryopreservation recovery of in vitro-derived shoot tips ranged by cultivars from 17 to 94% [ 41 ]. Previously, we also studied the cryopreservation of dormant blackcurrant buds using greenhouse and field-maintained source plants of the cultivar Mortti, and we reported the estimated post-cryopreservation recovery of buds in vitro from 66 to 86% [ 43 ].…”

Optimization of a Cryopreservation Method for the Endangered Korean Species Pogostemon yatabeanus Using a Systematic Approach: The Key Role of Ammonium and Growth Regulators

“…Vavilov All-Russian Institute of Plant Genetic Resources [17]. Modifications of this method have been effective for cryobanking fruit trees, woody shrubs, ornamental and aromatic plants of high economic value [18][19][20]. There is also a growing number of successful examples of D-V application for cryobanking wild flora [6]; however, these cases are few compared to clonal crops.…”

“…Often the conditions at each step should be optimized de novo for every new species to be conserved [23], which require dozens of explants. However, it is not uncommon, that significant variation in survival during the cryopreservation procedure is observed among different cultivars within the same species, which implies that a response of plant material to cryopreservation stress may be genotype-dependent [17,18,20,24]. The need for "reoptimization" of the already established procedure further complicates the development of the effective cryopreservation methods for endangered plant species with limited source material available for the experiments [7].…”