Improvement to the D Cryo-plate Protocol Applied to Practical Cryopreservation of <i>In Vitro</i> Grown Potato Shoot Tips

Abstract

We modified the D cryo-plate protocol by paper mounting to the cryo-plate with alginate gel and shoot tips, and cryopreserved shoot tips of 13 potato genotypes (potato genetic resources in Mexico) using the revised and optimized D cryo-plate protocol. There were no significant differences in regrowth of cryopreserved shoot tips by addition of the paper mounting step to the base D cryo-plate protocol, besides a reduction in dropping shoot tips in steps during the whole procedure. Some steps of revised D cryo-plate protocol were optimized or reconfirmed, and the effect of optimizations such as cold-hardening, loading solution treatment, and postrewarming treatment on the regeneration of shoot tips was studied. This optimized protocol was successfully applied to 'B-71-240-2' and 12 additional potato genotypes, resulting in 70.0%-93.3% regrowth with an average of 82.8% and stable storage for 1 year. When introducing new cryopreservation techniques, modification and optimization of the method are required to adjust to each laboratory's circumstances. This optimized D cryo-plate method will facilitate cryobanking of potato and other plant genetic resources in Mexico for long-term preservation in a genebank.

A large-scale viability assessment of the potato cryobank at the International Potato Center (CIP)

“…Results of new and promising cryopreservation methods, like the dehydration (D)- and vitrification (V)-plate protocols, show high recovery rates of 70–93.3% (13 accessions; D cryoplate) and 93.3–100% (16 accessions; V cryoplate) (Yamamoto et al 2015; Valle et al 2017), but the assessment of these methods on a larger scale (for example, > 500 randomly selected accessions) has not yet been performed. It is also important to highlight that CIP’s potato cryobank exclusively conserves landraces, while other collections contain higher quantities of improved potato varieties or breeding material, which tend to be less variable and have a narrower genetic base than landraces.…”

“…Recently, researchers have published other promising methods for potato cryopreservation, such as the D and V cryoplate. In these methods, shoot tips are “stuck” on special aluminum plates with uniform wells using sodium alginate to facilitate uniform sample handling, reduced risk of damage, and reduced loss of shoot tips (Yamamoto et al 2015; Valle et al 2017). …”

An Integrated Approach to Creating Scab-Resistant Apple: Phytopathological Testing and Marker-Assisted Selection

“…С использованием метода дроплет-витрификации созданы криоколлекции многочисленных образцов следующих культур (табл. 1): банана в Бельгии (Panis et al, , 2009; лука и чеснока в Южной Корее (Kim et al, 2006(Kim et al, , 2009 и Германии (Keller et al, 2016); картофеля в Перу (Panta et al, 2015;Vollmer et al, 2016Vollmer et al, , 2017, США (Bamberg et al, 2016;Jenderek, Reed, 2017) и России (Shvachko, Gavrilenko, 2011;Ukhatova et al, 2017b;Gavrilenko et al, 2018); малины и ежевики в США (Reed, DeNoma, 2012;Jenderek, Reed, 2017 Метод Cryo-plate, сочетающий приемы инкапсуляции-дегидратации и дроплет-витрификации, является самым новым (Yamamoto et al, 2011(Yamamoto et al, , 2012(Yamamoto et al, , 2015Valle Arizaga et al, 2017). В этом методе апексы микропобегов in vitro растений прикрепляют тонким слоем альгината кальция к алюминиевым крио-пластинам, обрабатывают их растворами LS и PVS, а затем быстро погружают криопластины в жидкий азот (Yamamoto et al, 2011).…”

Influence of ethylene glycol on Eucalyptus grandis cryopreservation using the V cryo-plate technique

“…In addition to optimizing some of the key parameters for cryopreservation success, this work was the first to use cryoplates for the cryopreservation of eucalyptus. The cryo-plate method unites two different approaches with numerous advantages when compared to other vitrification techniques, including: reduced handling of the explants; faster procedures; explant protection via dehydration with cryoprotectants; cryopreservation of large numbers of samples simultaneously; and high survival and regeneration rates (Yamamoto et al 2012, Engelmann 2014, Arizaga et al 2017, Pettinelli et al 2017.…”