Cryopreservation of plant cells
First Claim
1. A method for cryopreserving a plant cell comprising the steps of:
- a) pretreating the plant cell with a cryoprotective agent and a stabilizer;
b) acclimating the pretreated plant cell to a reduced temperature;
c) loading the plant cell with a loading gun;
d) vitrifying the plant cell with a vitrification solution; and
e) freezing the vitrified plant cell at a cryopreservation temperature.
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Abstract
The present invention relates to methods for cryopreserving plant cells and to methods for recovering viable plant cells from long or short term cryopreservation. Plant cells to be cryopreserved can be grown in culture and pretreated with a solution containing an cryoprotective agent and, optionally, a stabilizer. Stabilizers are preferably membrane stabilizers such as ethylene inhibitors, oxygen radical scavengers and divalent cations. Cells can also be stabilized by subjecting the culture to a heat shock. Pretreated cells are acclimated to a reduced temperature and loaded with a cryoprotective agent such as DMSO, propylene glycol or polyethylene glycol. Loaded cells are incubated with a vitrification solution which, for example, comprises a solution with a high concentration of the cryoprotective agent. Vitrified cells retain less than about 20% water content and can be frozen at cryopreservation temperatures for long periods of time without significantly altering the genotypic or phenotypic character of the cells. Plant cells may also be cryopreserved by lyophilizing cells prior to exposure to a vitrification solution. The combination of lyophilization and vitrification removes about 80% to about 95% of the plant cell'"'"'s water. Cells can be successfully cryopreserved for long periods of time and viably recovered. The invention also relates to methods for the recovery of viable plant cells from cryopreservation. Cells are thawed to about room temperature and incubated in medium containing a cryoprotective agent and a stabilizer. The cryoprotective agent is removed and the cells successfully incubated and recovered in liquid or semi-solid growth medium. The invention also relates to the cryopreserved cells and to viable plant cells which have been recovered from long or short term cryopreservation.
57 Citations
48 Claims
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1. A method for cryopreserving a plant cell comprising the steps of:
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a) pretreating the plant cell with a cryoprotective agent and a stabilizer;
b) acclimating the pretreated plant cell to a reduced temperature;
c) loading the plant cell with a loading gun;
d) vitrifying the plant cell with a vitrification solution; and
e) freezing the vitrified plant cell at a cryopreservation temperature. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method for cryopreserving a plant cell comprising the steps of:
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a) pretreating the plant cell with a cryoprotective agent and a stabilizer;
b) vitrifying the plant cell; and
c) freezing the vitrified plant cell at a cryopreservation temperature. - View Dependent Claims (12)
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13. A method for cryopreserving a plant cell comprising the steps of:
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a) incubating the plant cell in medium comprising a vitrifying agent and a stabilizer at a reduced temperature for a first period of time;
b) incubating the plant cell in medium containing an increased concentration of said vitrifying agent for a second period of time; and
c) freezing the plant cell at a cryopreservation temperature. - View Dependent Claims (14)
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15. A method for cryopreserving a plant cell comprising the steps of:
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a) lyophilizing the plant cell;
b) vitrifying the lyophilized plant cell in a vitrifying solution; and
c) freezing the vitrified plant cell at a cryopreservation temperature. - View Dependent Claims (16, 17, 18, 19, 20)
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21. A method for recovering cryopreserved plant cells comprising the steps of:
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a) thawing the cryopreserved plant cells to a temperature above freezing;
b) incubating the thawed plant cells in a growth medium comprising a cryoprotective agent and a stabilizer;
c) removing the cryoprotective agent; and
d) recovering viable plant cells. - View Dependent Claims (22, 23, 24, 25)
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26. A method for recovering cryopreserved plant cells in suspension comprising the steps of:
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a) thawing the cryopreserved plant cells to a temperature above freezing;
b) incubating the thawed plant cells in suspension; and
c) recovering viable plant cells in suspension.
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27. A method for recovering cryopreserved plant cells comprising the steps of:
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a) thawing the cryopreserved plant cells to a temperature above freezing;
b) incubating the thawed plant cells in a growth medium containing an ethylene inhibitor; and
c) recovering viable plant cells. - View Dependent Claims (28, 29, 30, 31, 32)
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33. A method for recovering cryopreserved plant cells comprising the steps of:
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a) thawing the cryopreserved plant cells to a temperature above freezing;
b) incubating the thawed plant cells in a growth medium containing a divalent cation; and
c) recovering viable plant cells. - View Dependent Claims (34, 35)
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36. A method for cryopreserving a plant cell comprising the steps of:
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a) pretreating the plant cell with an osmotic agent and a divalent cation at greater than about 5 mM;
b) loading the plant cell with a cryopreserving agent;
c) vitrifying the plant cell with a cryopreservation solution; and
d) freezing the vitrified plant cell at a cryopreservation temperature. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43)
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44. A method for cryopreserving a plant cell comprising the steps of:
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a) pretreating the plant cell with an osmotic agent and an ethylene inhibitor;
b) loading the plant cell with a cryopreserving agent;
c) vitrifying the plant cell with a cryopreservation solution; and
d) freezing the vitrified plant cell at a cryopreservation temperature. - View Dependent Claims (45, 46)
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47. A method for cryopreserving a plant cell comprising the steps of:
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a) pretreating the plant cell with a heat shock;
b) vitrifying the acclimated plant cell with a vitrification solution; and
c) freezing the incubated plant cell at a cryopreservation temperature. - View Dependent Claims (48)
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Specification