- Cryopreservation is a method to conserve the plant materials at ultra low temperatures. A cryopreservation process comprises the following successive steps which have to be defined for every species: choice of material, pretreatment, freezing, storage, thawing, post-treatment.
Choice of material
- As a general rule, material will be chosen as young and as meristematic as possible. Indeed, cells in this type of material are the most likely to withstand freezing. The material can be sampled from in vivo or in vitro plants. In vitro material is generally preferable, since the explants are already miniaturised, free of superficial contamination and may also be pathogen free. The physiological stage of the material is very important. With carnation meristems, survival decreases progressively with their rank on the shoot axis, starting from the terminal meristem
- The pretreatment corresponds to a culture of the material for a certain period of time (several minutes to a few days) under conditions which prepare it to the freezing process. It is carried out using various cryoprotective substances like sucrose, sorbitol, mannitol, dimethylsulfoxide, polyethylene glycol, which differ greatly one from the other by
- their molecular weight and their structure.
- Different types of freezing processes can be carried out: ultra-rapid, rapid, or slow freezing. In the latter case , a programmable freezing apparatus will be needed in order to obtain precise and reproducible freezing conditions.
- The maximal storage duration is theoretically unlimited, provided that the samples are permanently kept at or near the temperature of liquid nitrogen.
- In the majority of the cases, thawing is carried out rapidly by immersing the cryotubes containing the samples in a water-bath thermostated at around 40° C. The aim is to avoid fusion during thawing of the ice microcrystals formed during freezing.
- Post-treatment consists of culturing the material under conditions that ensure its optimal recovery in the best possible conditions. Cryoprotective substances are progressively eliminated by rinsing, dilution or diffusion, for they are toxic if kept too long in contact with the material.
- In many cases, regrowth is very slow. The only definitive assessment of viability is regrowth after thawing. However, it is very important to know as soon as possible if material is living after freezing. Two main tests exist in order to measure the viability of the material, which can be applied very rapidly after thawing. These tests are:
- -FDA (fluorescein diacetate): FDA is absorbed by the living cells and transformed into fluorescein, whose fluorescence is induced by UV irradiation. This test is quantitative in that the percentage of fluorescing cells can be counted.
- - TTC (2, 3, 5 - triphenyl tetrazolium chloride): TTC is reduced to formazan, colored red, by respiration in the mitochondria of the living cells. This test is quantitative for cell suspensions but is only qualitative for large tissues and organs.
- The major disadvantage of viability tests is that they are destructive. Moreover, the FDA test is very precise for estimating viability, but gives no information on the capacity of the cells to proliferate.
- Non destructive methods for estimating the viability of the material are sought, such as chromatographic analysis of volatile hydrocarbon production (ethylene, ethane) by cryopreserved tissues.
Fig. 1. Schematic representation of the oil palm somatic embryo cryopreservation process.
- Starting material.
- Production of embryoid clumps for freezing: 2 months on sucrose 0.3 M.
- Dissection of clumps and 7-day pretreatment on sucrose 0.75 M.
- Freezing: a) rapid b) programmed.
- Storage in liquid nitrogen (- 196°C).
- Thawing: 1 min at 40° C.
- 1 wk on sucrose 0.3 M + 2.4-D
- 2 wks on sucrose O.1Mf 2,4-D 10-6M.
- Successive transfers on multiplication medium: resumption
- Transfer to jars to allow cultures to grow