Biophysical Characteristics of Successful Oilseed Embryo Cryoprotection and Cryopreservation Using Vacuum Infiltration Vitrification: An Innovation in Plant Cell Preservation

Abstract

Heterogeneity in morphology, physiology and cellular chemistry of plant tissues can compromise successful cryoprotection and cryopreservation. Cryoprotection is a function of exposure time × temperature × permeability for the chosen protectant and diffusion pathway length, as determined by specimen geometry, to provide sufficient dehydration whilst avoiding excessive chemical toxicity. We have developed an innovative method of vacuum infiltration vitrification (VIV) at 381 mm (15 in) Hg (50 kPa) that ensures the rapid (5 min), uniform permeation of Plant Vitrification Solution 2 (PVS2) cryoprotectant into plant embryos and their successful cryopreservation, as judged by regrowth in vitro. This method was validated on zygotic embryos/embryonic axes of three species (Carica papaya, Passiflora edulis and Laurus nobilis) up to 1.6 mg dry mass and 5.6 mm in length, with varying physiology (desiccation tolerances) and 80°C variation in lipid thermal profiles, i.e., visco-elasticity properties, as determined by differential scanning calorimetry. Comparisons between the melting features of cryoprotected embryos and embryo regrowth indicated an optimal internal PVS2 concentration of about 60% of full strength. The physiological vigour of surviving embryos was directly related to the proportion of survivors. Compared with conventional vitrification, VIV-cryopreservation offered a ∼ 10-fold reduction in PVS2 exposure times, higher embryo viability and regrowth and greater effectiveness at two pre-treatment temperatures (0°C and 25°C). VIV-cryopreservation may form the basis of a generic, high throughput technology for the ex situ conservation of plant genetic resources, aiding food security and protection of species from diverse habitats and at risk of extinction.

A new insight in desiccation tolerance and cryopreservation of mazzard cherry (Prunus avium L.) seeds

“…previous work on Prunus avium seeds [25], Camellia sinensis seeds [30], Corylus avellana seeds [31], Citrus seeds: C. sinensis (sweet orange), C. paradisi (grapefruit), C. reticulata (mandarin) [32] or embryonic axes i.e. : Carica papaya, Passiflora edulis and Laurus nobilis [33].…”

Efficiency of Cryoprotectants for In Vitro and Ex Vitro Germination of Cryopreserved Jatropha curcas Seeds

“…Experiment I: In vitro germination and seedling growth from cryopreserved seeds. Although cryopreservation of plant material in the form of seeds and zygotic embryos is currently widely used (Kaviani, 2011;Nadarajan and Pritchard, 2014;Silva et al, 2012), even the best treatments in this study resulted in low percentage of germination in vitro 7 DAS (T1 and T6) and 14 DAS (T1 and T8), varying between 25% and 37.5%. This could be related to intrinsic traits of the seed, such as composition, moisture content, and tolerance to ultralow temperatures (Chmielarz, 2009;Das Bhowmik et al, 2011;Prada et al, 2015;Salomão et al, 2016).…”

“…The main event that limits the survival of cryopreserved tissues using conventional freezing techniques is the possible ice formation within the extracellular matrix of multicellular tissues (Taylor et al, 2015). Therefore, new protocols have been developed involving the use of cryoprotectants to allow cooling of plant cell contents into a vitrification state without the formation of ice crystals (Engelmann, 2011;Nadarajan and Pritchard, 2014;Taylor et al, 2015). The plant vitrification method involves the use of an extremely concentrated solution (7-8 M) of cryoprotectants [plant vitrification solution (PVS)] to allow plant tissues to reach a vitrification state and prevent ice crystal formation (Niino and Arizaga, 2015).…”

Current issues in plant cryopreservation and importance for ex situ conservation of threatened Australian native species

“…The vitrification protocol is currently the most commonly used cryopreservation technique, and hundreds of plant species have been successfully cryopreserved in this way (Sakai and Engelmann 2007). The vitrification method has continued to develop and improve over time, with approaches such as vacuum infiltration vitrification (VIV) (Nadarajan and Pritchard 2014;Funnekotter et al 2015), cryo-plate protocols (Yamamoto et al 2011(Yamamoto et al , 2012, and a new cryo-mesh (Funnekotter et al 2017a) protocol created to increase success in cryopreservation.…”

“…VIV as an alteration to cryopreservation protocols involves applying a vacuum to plant material during the incubation phases, whether that involves loading solutions, CPA solutions or a combination of both. The vacuum is thought to reduce trapped air bubbles on the surface of plant material, thus increasing the surface interactions between plant membranes and the surrounding solution and resulting in a more uniform infiltration and a potentially greater intake of CPAs (Nadarajan and Pritchard 2014). Using the VIV method, Nadarajan and Pritchard (2014) achieved greater post cryopreservation viability and regrowth in embryos of Carica papaya, Passiflora edulis and Laurus nobilis seeds, with a 10-fold reduction in PVS2 exposure times at both pretreatment temperatures of 0 and 25 C. Funnekotter et al (2015) also investigated the use of VIV as an addition to cryopreservation protocols on shoot tips of species native to Western Australia.…”