Freeze Preservation of Somatic Embryos and Clonal Plantlets of Carrot (<i>Daucus carota</i> L)

Withers, Lyndsey A.

doi:10.1104/pp.63.3.460

Cited by 71 publications

(26 citation statements)

References 17 publications

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“…Removal of this medium (Fig. lb) delayed recovery growth, perhaps by depletion of essential factors released into the medium during freezing and thawing (21). The other regrowth conditions were all deleterious (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…However, cryoprotectant toxicity and rehydration/deplasmolysis injury upon thawing and cryoprotectant removal contribute at present to viability losses which may delay or often preclude recovery (21 (20), prompts the search for other compounds. Proline, which has been implicated in protection against salt stress in halophytes (17,18) and cold and desiccation stress in a wide range of plants (2,12), is an ideal candidate for natural cryoprotection since it has a very high solubility, is neutral, exerts a high osmotic pressure, and is nontoxic at high concentrations (3, 17).…”

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confidence: 99%

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Proline: A Novel Cryoprotectant for the Freeze Preservation of Cultured Cells of Zea mays L

Withers¹,

King²

1979

Plant Physiol.

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151

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Proline is an effective cryoprotectant for the storage of cultured cells of Zea mays L. in liquid N2. Increased freeze tolerance can be achieved by pregrowth for 3 to 4 days in medium containing proline. Cells cryoprotected with proline have an increased recovery potential when compared with cells cryoprotected with dimethylsulfoxide and glycerol. They also show a reduced postthaw viability loss and greater tolerance of a range of postthaw culture conditions. It is suggested that the mechanism of action of proline may be similar to that in its putative role of conferring protection against natural stresses. It may be protecting the cell against solution effects caused by dehydration during freezing. These findings are discussed in relation to other freeze tolerance enhancing treatments.Freeze preservation in LN3 as a means of genome storage can now be applied to a very limited range of plant tissue cultures. The reasons for the present limitations are not fully understood (1,20,24).In biological systems, the two major sources of cryoinjury are ice damage and solution effects, the latter caused by the excessive concentration of intracellular solutes (9, 10). Protection against cryoinjury may be achieved by either freezing slowly to induce dehydration by extracellular freezing, thereby minimizing intracellular ice formation, or alternatively by freezing and thawing very rapidly to prevent dehydration while maintaining a small, innocuous, ice crystal size. The addition of cryoprotective compounds may reduce cryoinjury in either rapid or slow freezing. In the majority of freeze preservation protocols applied to plant cells, cryoprotectants (usually DMSO and/or glycerol) are used in combination with freezing at a slow rate (1, 24). The high water content of plant cells results in an enhanced susceptibility to cryoinjury. At the level of dehydration required to avoid solution effect stresses, it is still normally necessary to thaw very rapidly to minimize ice recrystallization damage (1,20,24).Recovery of cultures after thawing is dependent upon both a high postthaw viability and further survival through a recovery period preceding renewal of normal metabolic functions and cell division (15,20 In the present study, proline has been used as both a cryoprotectant and pregrowth additive (23) for the freeze preservation of cultured cells of maize. It will be demonstrated that this compound is highly effective in both modes of use and is superior in a number of respects to conventional cryoprotectants. MATERIALS AND METHODSA suspension culture of Zea mays L. (a cell line derived from cultivar B73 [inbred] and kindly supplied by Dr. Ingo Potrykus) was maintained in exponential growth under the conditions described by Potrykus et aL (13). Cells were harvested for freezing 3 or 4 days after subculturing. Additionally, a cell suspension was pregrown for 3 or 4 days in medium supplemented with 5 or 10%1o (w/v) L-proline. Cryoprotectants were prepared using analytical grades of L-proline, glycerol and DMSO, at double th...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Proline: A Novel Cryoprotectant for the Freeze Preservation of Cultured Cells of Zea mays L

Withers¹,

King²

1979

Plant Physiol.

Self Cite

151

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“…In any of the cryogenic protocols, the cells and tissues to be cryopreserved must be in a physiologically optimal status for the acquisition of dehydration tolerance and to produce vigorous recovery of growth (Dereuddre et al 1988;Withers 1979). Preconditioning of shoot tips is necessary to obtain uniform and vital shoot tips to start with.…”

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confidence: 99%

Cryopreservation of in vitro-grown shoot tips of carnation (Dianthus caryophyllus L.) by vitrification method using aluminium cryo-plates

Sekizawa

Yamamoto

Rafique

et al. 2011

Plant Biotechnology

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Cryopreservation using an aluminum cryo-plate was successfully applied to in vitro-grown carnation (Dianthus caryophyllus L.) shoot tips. The shoot tips (1-1.5 mmϫ1 mm) were dissected from the shoot and precultured at 25°C for 2 days on MS medium containing 0.3 M sucrose. The precultured shoot tips were placed on the aluminum cryo-plate containing ten wells embedded with alginate gel. Osmoprotection was performed by immersing the cryo-plates in loading solution (2 M glycerol and 1.4 M sucrose) for 90 min at 25°C. Then, dehydration was performed by immersing the cryoplates in PVS2 for 25 min at 25°C. After storage in liquid nitrogen, shoot tips attached to cryoplate, were directly immersed into 2 ml 1 M sucrose solution for regeneration. Using this procedure, the average regrowth level of vitrified shoot tips of 4 carnation cultivars reached 95%. This new method has many advantages and will facilitate the cryostorage of reference cultivars of carnation. (LS) and PVS2 without floating and/or clinging of shoot tips and also with no or low level of injury to those shoot tips. In this paper, this V-Cryo-plate procedure was applied for the cryopreservation of carnation.The carnation plants used in this study were obtained from the Nishinihon Station, NCSS, sub-bank of NIAS Genebank Project, in Japan in April 2010. Four cultivars of carnation, 'Melissa', 'Grana', 'Terra Cotta' and 'Yell', were tested. Apical shoots, excised from in vivo explants, were surface-sterilized successively with 70% ethanol for 1 min, and 0.5% sodium hypochlorite for 10 min, and then rinsed twice with sterilized distilled water. After surface sterilization, shoot tips with basal plates (1 mm longϫ1 mm wide) were excised from the shoots and cultured on 70 ml of 0.4% (w/v) gellan gum-solidified MS medium (Murashige, Skoog 1962) in a culture flask (80 mmϫ100 mm) containing 1 mg l Ϫ1 thidiazuron, 1 mg l Ϫ1 naphthalene acetic acid and 3% (w/v) sucrose (Jose et al. 2010). After 50 days of culture, shoots were transferred onto gellan gum-solidified MS medium devoid of plant growth regulators. Then stock plants were subcultured every two months on MS medium. Cultures were incubated at 25°C with a 16 h photoperiod under white fluorescent light (52 mmol m Ϫ2 s Ϫ1) in the culture flask (standard condition). For cryopreservation of in vitro shoot tips of carnation, the V-Cryo-plate procedure was applied. The size of the aluminum cryoplate used was 7 mmϫ37 mmϫ0.5 mm with ten wells (diameter 1.5 mm, depth 0.75 mm) ( Figure 1B). These plates fit in 2 ml cryotube. The different steps of the VCryo-plate procedure are as follows; 1) Cut shoots (5 mm) with a lateral bud and plate on solidified MS medium and culture for 2 weeks at 25°C in standard conditions ( Figure 1A). Then, dissect shoot tips with basal plate (1-1.5 mm longϫ1 mm wide) from the shoots and preculture for 2 days at 25°C on the MS medium with 0.3 M sucrose. 2) Place an aluminum cryo-plate in Petri-dish and pour 2.0-2.5 ml 2% (w/v) Na-alginate solution with 0.4 M sucrose in calcium-free M...

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“…O tratamento Testemunha criopreservado (sem glicerol) mostrou a maior regeneração, o que pode ser uma resposta a situação de estresse ao qual foi submetido. Withers (1979) identificou vários aspectos da fase de descongelamento que podem influenciar a recuperação das células, incluindo a lavagem e a composição do meio de cultivo. Embora a lavagem das células depois do descongelamento remova crioprotetores potencialmente tóxicos, pode causar efeito adverso na viabilidade das suspensões celulares.…”

Section: Resultsunclassified

Criopreservação De Calos De Arroz

Miranda¹,

Bobrowski²,

Dode³

et al. 2009

RSA

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RESUMOA criopreservação é uma técnica que mantém germoplasma por longo prazo. Objetivou-se verificar o efeito da criopreservação em nitrogênio líquido na regeneração de calos de arroz induzidos a partir de sementes maduras e avaliar o uso do glicerol como agente crioprotetor. Sementes de arroz BRS-7 TAIM, foram descascadas e desinfestadas, distribuídas em frascos contendo meio MS 2,5 mg dm -3 de 2,4 D para indução de calos e mantidos a temperatura de ±25 ºC no escuro. Após doze dias os calos foram criopreservados em nitrogênio líquido por uma hora, descongelados a 37º C e inoculados no meio de cultivo para proliferação. Utilizou-se cinqüenta calos por tratamento: Testemunha, Testemunha criopreservada, Glicerol 500 g dm -3 e Glicerol 500 g dm -3 criopreservado. A avaliação foi realizada aos trinta dias observando-se o tamanho e coloração dos calos. Transferiu-se o material para meio de regeneração MS 10 mg dm -3 sacarose, 0,5 mg dm -3 de ANA e 2,0 mg dm -3 de BAP e incubou-se em fotoperíodo de 16 h. Avaliou-se a regeneração aos trinta e quarenta e cinco dias de incubação, os parâmetros foram: crescimento dos calos, número de gemas/calo, número de calos com broto e número de brotos/calo. Na proliferação os calos da Testemunha criopreservado oxidaram, porém, na regeneração, aos trinta dias, apresentaram maior tamanho e número de gemas, e aos quarenta e cinco dias maior número de brotos que os demais tratamentos. Conclui-se que a criopreservação pode ser empregada em calos de sementes de arroz, não afetando sua capacidade regenerativa.Palavras-chave: Calos; regeneração; Oryza sativa. ABSTRACTCryopreservation is a technique that allows maintaining long-term germoplasma using ultra-reduced temperature. The objective of this study was to evaluate the regeneration seed´s callus of rice cryopreservated. Seeds of rice BRS-7 TAIM, were peeled and desinfested for establishment in vitro, distributed in flasks containing MS (Murashige & Skoog, 1962) 2,5 mg dm -3 of 2,4 D for induction of callus and maintained the temperature of ±25 ºC in the darkness. After twelve days the calluses were cryopreservated in liquid nitrogen for one hour, thawed 37º C and put back in the middle of cultivation for proliferation. It was used fifty callus by treatment: Test, Test cryopreservated, Glycerol 500 g dm -3 and Glycerol 500 g dm -3cryopreservated. The evaluation of the proliferation was accomplished to the thirty days being observed the size and coloration of the callus. The material was transferred for middle of regeneration MS 10 mg dm -3 sucrose, 0,5 mg dm -3 of ANA and 2,0 mg dm -3 of BAP and it was incubated in fotoperíodo of 16 h. The evaluation of the regeneration was accomplished to the thirty and forty five days of incubation, the parameters were: growth of the callus, sprout and callus number, number of callus with sprout and sprout/callus number. In the proliferation the callus of the treatment Test cryopreservated rusted, however it was observed in the regeneration, to the thirty days, that the same ones presented such adult an...

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Freeze Preservation of Somatic Embryos and Clonal Plantlets of Carrot (Daucus carota L)

Cited by 71 publications

References 17 publications

Proline: A Novel Cryoprotectant for the Freeze Preservation of Cultured Cells of Zea mays L

Proline: A Novel Cryoprotectant for the Freeze Preservation of Cultured Cells of Zea mays L

Cryopreservation of in vitro-grown shoot tips of carnation (Dianthus caryophyllus L.) by vitrification method using aluminium cryo-plates

Criopreservação De Calos De Arroz

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