Cryopreservation of Plant Genetic Resources: Conventional and New Methods

Matsumoto, T.

doi:10.7831/ras.5.13

Cited by 63 publications

(54 citation statements)

References 30 publications

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“…The droplet vitrification technique was derived from the plant vitrification solution (PVS)-based vitrification technique [9] and the droplet freezing technique [15, 16]. These techniques involve the use of a sterile aluminum cryoplate [17], an important conductor of heat or thermal energy during immersion in LN [18, 19]. …”

Section: Introductionmentioning

confidence: 99%

Cryopreservation of Shoot Tips of Elite Cultivars of Cannabis sativa L. by Droplet Vitrification

Uchendu

Lata

Chandra

et al. 2019

Med Cannabis Cannabinoids

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Cannabis sativa L. (marijuana or hemp) is recognized worldwide for its psychoactive properties as well as for fiber production. This study focused on the evaluation of 3 droplet vitrification protocols for long-term conservation of shoot tips in liquid nitrogen (LN). Shoot tips (∼0.5 mm) were excised from 3- to 4-week-old in vitro-grown shoots of 3 cultivars (MX, VI-20, and B-5: high tetrahydrocannabinol [THC], high cannabidiol [CBD], and intermediate THC∼CBD, respectively) and pretreated on 5% dimethyl sulfoxide agar plates for 48 h. The shoot tips were then vitrified in LN using 3 separate cryoprotectant (plant vitrification solutions [PVS] #2, #3, and #4) droplets on an aluminum cryoplate. There was no significant difference between the regrowth of cryopreserved shoot tips exposed to PVS2 for 15 and 20 min, but regrowth of all 3 cultivars significantly declined after 20 min of exposure. Exposure duration of 15 min was adapted for subsequent experiments. Regrowth of cryopreserved MX was significantly higher with PVS2 (63%) than with PVS3 and PVS4 (≤5%). Regrowth of cryopreserved VI-20 was highest with PVS2 (57%) and significantly higher than with PVS3 and PVS4 (≤25%). The regrowth of cryopreserved shoot tips of B-5 was significantly different between all 3 protocols with PVS2 > PVS4 > PVS3. Both PVS2 and PVS4 produced regrowth above 55%, while regrowth with PVS3 was significantly lower (31%). These results indicate that 15–20 min of exposure to PVS2 are most suitable for cryopreservation of these varieties. This is the first report on protocol development for the cryopreservation of organized tissues of C. sativa L. for germplasm conservation.

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Section: Introductionmentioning

confidence: 99%

Cryopreservation of Shoot Tips of Elite Cultivars of Cannabis sativa L. by Droplet Vitrification

Uchendu

Lata

Chandra

et al. 2019

Med Cannabis Cannabinoids

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“…One of the most commonly used techniques for species with recalcitrant seeds has been the encapsulation of embryos after pre-culture in a solution with high concentration of sucrose, which dehydrates the plant material and removes most or all of the freezable water. Sucrose, when it infiltrates into plant tissues, helps to maintain viability during dehydration and freezing by stabilizing the cellular membranes (Sershen et al, 2016;Matsumoto, 2017). Moreover, by replacing the water content, sucrose maintains phospholipid membranes in the liquid crystalline phase and promotes the vitrification of the cytoplasm (Bandupriya et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

“…The cryopreservation techniques using vitrification that have been discussed in the literature include: encapsulation with dehydration; simple vitrification; encapsulation with vitrification; dehydration; pregrowth; pre-growth desiccation and droplet-freezing (Niino & Arizaga, 2015;Matsumoto, 2017).…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…The method of encapsulation and dehydration is based on production of synthetic seeds, in which embryos are inserted in sodium alginate beads (Niino & Arizaga, 2015;Matsumoto, 2017). Recent development in this protocol has facilitated cryopreservation techniques (Shibli et al, 2016;Matsumoto, 2017). Pre-culturing the plant material by encapsulating it in a medium enriched with sucrose before dehydration enables for greater resistance to dehydration and freezing (Bandupriya et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Viability of embryonic axes of Araucaria angustifolia after freezing using two cryopreservation methods

Frizzo

Quoirin

2018

Pesq. flor. bras.

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Araucaria angustifolia (Bertol.) O. Kuntze é uma das espécies nativas do Brasil com maior importância na Região Sul. No entanto, suas sementes são recalcitrantes, constituindo um obstáculo para a sua conservação em longo prazo, sendo a criopreservação uma alternativa viável para o armazenamento de seu germoplasma. Os eixos embrionários (EE) excisados das sementes de araucária foram encapsulados, desidratados e submetidos a dois métodos de criopreservação: o resfriamento rápido, congelando em nitrogênio líquido (NL) durante 2 h, e o pré-resfriamento a - 40 °C, seguido do congelamento em nitrogênio líquido durante 2 h. Posteriormente, os EE foram descongelados rapidamente e avaliados quanto à integridade do DNA pelo teste bioquímico do tetrazólio, germinação in vitro e ocorrência de oxidação. Tanto o DNA dos EE que não foram criopreservados quanto o daqueles que foram criopreservados mantiveram sua integridade. Os resultados do teste de tetrazólio indicaram que a maioria dos EE desidratados pelo congelamento rápido permaneceu viável. Após 15 dias de cultivo in vitro, os EE não germinaram e apresentaram sinais de oxidação. Os métodos de encapsulamento-desidratação seguidos de congelamento rápido são promissores para a criopreservação de EE de araucária, como demonstrado pelo teste de tetrazólio e da manutenção da integridade total do DNA.

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