Challenges in implementing plant shoot tip cryopreservation technologies

Cryopreservation is a useful tool for the long-term storage of plant genetic resources, and different cryogenic procedures have recently been developed. The present study focused on the use of the Droplet-vitrification (DV) and V cryo-plate protocol for the cryopreservation of Stevia rebaudiana in vitro-derived apical shoot tips and axillary shoot tips. A preliminary test showed that 90 and 120 min PVS2 (Plant Vitrification Solution 2) treatment significantly reduced the regrowth of the explants before immersion in liquid nitrogen (LN). For both procedures tested, the best osmoprotective condition for obtaining a higher regrowth of cryopreserved explants occurred when explants were PVS2 treated for 60 min. After direct immersion in LN, thawing and plating, the highest regrowth recorded was 80% with DV and 93% with V cryo-plate. Moreover, shoot tips proved to be a more suitable material for Stevia cryopreservation. A satisfactory vegetative regrowth was observed in the subcultures following cryopreservation by DV and V cryo-plate cryogenic procedures.

Section: Discussionmentioning

confidence: 99%

“…[ 52 ], and Vitis spp. [ 29 , 53 , 54 ]. According to our results, applying this method to Stevia, shoot regrowth percentages of 80% and 50% were observed for cryopreserved AST and AxST, respectively.…”

Section: Discussionmentioning

confidence: 99%

Two Advanced Cryogenic Procedures for Improving Stevia rebaudiana (Bertoni) Cryopreservation

Benelli̇

Carvalho

Merzougui

et al. 2021

“…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. Moreover, a dormant bud protocol may be easier to implement compared to in vitro-based protocols, which may need considerable optimization before they can be applied in practice [39,53,54]. If the recovery of cryopreserved buds is possible by grafting or direct rooting, the whole cryopreservation process can be done without laboratory facilities [9,20].…”

Section: Discussionmentioning

confidence: 99%

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

Rantala

Kaseva

Nukari

et al. 2021

The cryopreservation of dormant buds can be a feasible method for preserving germplasm of cold-tolerant woody plants. In the present study, we evaluated the effects of pre-desiccation, thawing method, and the rehydration of bud sections on the post-cryopreservation recovery of dormant blackcurrant buds in vitro. The estimated recovery of small- and medium-sized buds was 80.1 and 62.7% respectively for desiccated buds and 67.8 and 72.3% respectively for non-desiccated buds. The pre-desiccation of bud sections enhanced the number of the shoots regenerated from vegetative buds (2.3 vs. 4.7). The estimated recovery of fast-thawed buds was better after 14-day than after 7-day rehydration (85 vs. 59%). In slowly thawed buds the difference between 14-day and 7-day rehydration was not significant (73 vs. 62%). The estimated recovery of vegetative and flower buds was 77.7 and 41.1% respectively after 7-day rehydration, and 95.2 and 43.6% respectively after a 14-day rehydration period. The rehydration of bud sections was not necessary for the in vitro recovery of non-desiccated, fast-thawed buds. Of the 23 blackcurrant cultivars cryopreserved using non-desiccated dormant buds collected from a greenhouse, the estimated recovery of 22 cultivars ranged between 42 and 90%.

“…Droplet-vitrification is a modification of vitrification [ 79 ]; treating explants with loading (usually 2 M glycerol and 0.4 M sucrose) and vitrification solutions; cooling them ultra-rapidly in a droplet of vitrification solution either PVS2 or PVS3 placed on an alfoil strip [ 49 ] with a droplet of cryoprotectant added before immersion in LN. The alfoil strip helps with the ultra-rapid cooling (about 4000–5000 °C min −1 ) and re-warming (3000–4500 °C min −1 ) of samples due to the good conductivity of thermal current of aluminum [ 80 ]. The removal of the cryoprotectant is achieved during re-warming stage by using an unloading solution usually with high level of sucrose 1.2 M, then transferred to recovery and regeneration media [ 25 , 55 ].…”

Section: Plant Cryopreservation Of Somatic Embryos and Shoot Tipsmentioning

confidence: 99%

Cryopreservation of Woody Crops: The Avocado Case

O’Brien

Hiti-Bandaralage

Folgado

et al. 2021

Recent development and implementation of crop cryopreservation protocols has increased the capacity to maintain recalcitrant seeded germplasm collections via cryopreserved in vitro material. To preserve the greatest possible plant genetic resources globally for future food security and breeding programs, it is essential to integrate in situ and ex situ conservation methods into a cohesive conservation plan. In vitro storage using tissue culture and cryopreservation techniques offers promising complementary tools that can be used to promote this approach. These techniques can be employed for crops difficult or impossible to maintain in seed banks for long-term conservation. This includes woody perennial plants, recalcitrant seed crops or crops with no seeds at all and vegetatively or clonally propagated crops where seeds are not true-to-type. Many of the world’s most important crops for food, nutrition and livelihoods, are vegetatively propagated or have recalcitrant seeds. This review will look at ex situ conservation, namely field repositories and in vitro storage for some of these economically important crops, focusing on conservation strategies for avocado. To date, cultivar-specific multiplication protocols have been established for maintaining multiple avocado cultivars in tissue culture. Cryopreservation of avocado somatic embryos and somatic embryogenesis have been successful. In addition, a shoot-tip cryopreservation protocol has been developed for cryo-storage and regeneration of true-to-type clonal avocado plants.