Tissure Culture Preservation in Liquid Nitrogen (LN2)
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The following steps describe the process of placing totipotent plant cells into long-term storage in the liquid phase of LN2.

blue ball bullet Plant Increase

Plants are increased using micropropagation techniques to ensure adequate numbers of shoot tips for exposure tests and placement into long-term storage.

blue ball bullet Conditioning phase

Some plants may require a cold-conditioning stage (1-6 weeks) prior to shoot tip excision.

blue ball bullet Excise shoot tips

Shoot tips consisting of the apical dome and 2-3 leaf primoridia are dissected with a hypodermic needle or razor blade under a dissecting microscope in preparation for pre-treatment, loading, and cryoprotectant phase.

blue ball bullet Pretreatment, loading, and cryoprotectant phase

Shoot tips are placed in various osmotic and protective solutions (i.e. sucrose, glycerol, DMSO, ethylene glycol or encapsulated in alginate beads) to prepare the cells for LN2 temperatures.

blue ball bullet LN2 cooling phase

Shoot tips are placed into cryovials and immersed in LN2.

blue ball bullet Retrieval/warming phase

Cryovials are quickly placed in warm water, uncapped, and 1.2M sucrose is placed over shoot tips.


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