Work shope on plant Cryopreservation

The cryopreservation protocol contains the following stages:

1. Culture selection:  The plant source selected must be of right growth stag, active and should be healthy. The selection could be a key factor in the success of cryopreservation.
2. Pre- growth:  pre growth is the treatments which prolong the viability of cells during cryopreservation at freezing temperature. For this purpose various chemicals are utilised such as sugar, polyols, etc. which is usually osmotically active compounds, which act as dehydration factors. Some researchers have shown that compounds like ABA and trehelose that are produced by plants during stress have been found to enhance the recovery process.
3. Cryoprotection: in this stage the cell is transferred to cryoprotectants mixture (ex. Sucrose, DMSO, glycerol and several other chemicals can be used like sorbitol, praline, polyethylene glycol and ethylene glycol. The time of exposure to these cryoprotectants are usually for short time approx. 1hr. The temperature of Cryoprotection helps in reducing the degree of cryoprotectants toxicity because at 0ºC the enzyme activity becomes low.
4. Freezing and cooling: The rate of freezing represents an important key factor with respect to cryopreservation. It is achieved by slow cooling. The cryoprotectants mixture later on is transferred to vials and placed in the assigned freezer. There have been several rates of cooling been employed during this stage. The initial method of Withers and King (1980) cooling i.e. cooling the cell at -1oC to -30oC/-35oC for 30-40 min has been successful in relation to various range of plant species.
5. Cryopreservation/storage: Depending on the purpose the storage time can vary from 1hr to 20 years.
6. Rewarming and recovery: The rearming (thawing rate ) is a crucial stage in which the plant source is recovered by slow heating. It is as important as cooling rate. The main phenomenon which acts in the recovery of the plant resource from freeze is the rehydration.

EXCERSISE 1

AIM:  Cryopreservation of Ribes (currants) shoots tips using encapsulation dehydration.

MATERIALS:

• 10 Ribes shoot tips
• Forces
• Scissors
• Scalpel
• Sterile filter papers
• Sterile empty petri dishes
• 10X petri dish of MS medium
• Parafilm
• 2 hypodermic needles
• Binocular dissecting microscope
• Wilson sieve
• Solution of 3% Alginate
• Solution of 100mM CaCl2
• 50 or 100 ml beaker
• Weighing bottle (X5)
• Liquid nitrogen in a small bench to Dewar of 1l capacity
• Cryovials, canes, pastures pipettes

METHOD:

1. A clump of ribe shoots were transferred to a sterile Petri dish
2. The microscope was set and the shoot was placed on a sterile filter paper on the stage
3. The side branches and the leaves are removed using forceps and needles allowing the small cotyledon leave covering the meristem to expose.
4. The largest cotyledonary leaf was cut carefully and was placed in a Petri dish on sterile papers which were soaked with liquid MS media and the Petri dish was covered
5. The steps were repeated until 10 shoot tips were collected.
6. 10 shoot tips were transferred into a sterile Petri dish
7. The embryos were transferred to the alginate solution and were gently swirled until the shoots get submerged into liquid.
8. With the aid of the pastures pipettes the embryos were suck along with the alginate.
9. The alginate solution was dropped carefully into calcium solution, eventually a somatic embryo was transferred which on contact with Ca++ polymerised the alginate and the embryo was encapsulated.
10. The excesses medium was drained away using a Wilson sieve and the encapsulated embryos were captured.
11. They were then transferred to 1X sterile Petri dish and were allowed to dry for 2 hrs.
12. 3 embryos were taken from the Petri dish and were placed onto the recover medium and sealed. This Petri dish was labelled as control, 2hrs –LN.
13. Another 3 embryos were taken and placed on cryovial labelled as +LN, this is attached to the cryocane and was given for storage in LN for 5min.
14. Another 3 embryos were taken into the glass bottle provided for moisture content (MC) determination after 2 hrs desiccation.
15. The weight of the empty weighing bottle without cap was determined and was recorded as W1.
16. The encapsulated embryos were placed in the weighing bottle and weight was recorded as W2.
17. This bottle was labelled with cryo marker and was given for oven drying at 103oC for 17 hrs. Oven drying was overnight so dry weight of the samples was recorded as W3 after drying.

Calculation of beads moisture content:

% Bead MC = W2-W1 / W3-W1 x 100

18. Rapid warming will be carried out for cryoreserved embryos.
19. After rapid warming the beads were placed onto recovery medium, sealed and labelled.

EXERCISE 2

AIM: cryopreservation of shoot tips by vitrification.

MATERIALS:

METHODS:

1. The solanum shoot tips were placed in a cryovial containing the lowest concentration of PVS2 solution for about 1-10 min.
2. Almost all the vitrification solution were removed from the tissues and were replaced with a step wise higher concentration of PVS2 solution (50%    60%     70%      80%    90%      100%). All these were performed on ice.
3. Using the same procedure the concentrations of the tissues were increased till the concentration of the solution was 100% and was left for 1-5 min.
4. The cryovial was clipped to the cane provided and was covered with protective sleeve and was plunged directly into liquid nitrogen. It was left in a warm water bath at 45oC for 10 min. Outside of the vial was dampened with 70% ethanol.
5. An excess 100% vitrification solution was removed using a sterile pasture pipette and was replaced with unloading solution containing 1.2M sucrose in standard liquid medium. 2-3 washes were performed in a fresh unloading solution and were maintained in the same solution for about 5 min.
6. The content of the vials were expelled onto a sterile filter paper in a Petri dish and the paper was allowed to soak all the excesses vitrification solution.
7. The shoot tips were transferred to recover medium and the survival and regeneration characteristics were determined after two weeks.  

RESULTS:

EXCERCISE 1

                   A  CONTROL (-LN)                                       B (+LN)

The effect of freezing on encapsulated Ribes (Currant) shoot tips (A – encapsulated, and frozen in liquid nitrogen (+LN) Ribes shoot tips; B encapsulated, and unfrozen (-LN) Ribes shoot tips, control)

Table 1 The effects of desiccation time and freezing on encapsulated Ribes (Currant) shoot tips

EXCERCISE 2

             A CONTROL (-LN)                                        B (+LN)

Fig. 2 The effect of freezing on vitrificated Potato shoot tips (B – vitrificated, frozen in liquid nitrogen (+LN) Potato shoot tips; A vitrificated, and unfrozen (-LN) Potato shoot tips, control)

Table 2 The effects of freezing on vitrificated Potato shoot tips

DISCUSSION:

EXERCISE 1:

In case of +LN there was no growth even though the moisture content was about 117.72%. But there was no contamination even that indicated the safety measures taken during the experiment. It also indicates towards the efficiency of low temperature which does not allow the psychryophiles. Same was in the case of –LN, here the expected reason was that there was no temperature regulated storage as it was dried by oven drying. In second case there was a growth in the one bead shoot tips were developed. This case was related to +LN and more over the moisture content was 39.24%. Here as there was no contamination it indicates towards the liquid nitrogen and safety measures. In case of –LN 2 positive results were obtained out of 3 trials, here also there was no contamination and healthy shoot tips were observed. These results have showed certain deviations from the expected results as there was more growth in –LN than +LN. The expected reason behind this could be experimental error that could be related to personal error, mechanical error and other unobservatory defects.

EXCERSISE 2:

In this case there was no contamination. But there was no growth of the test samples on MS media with respect to both the cases i.e. –LN and +LN. This indicates towards unknown experimental error during experiment. But in second case there was a growth in +LN and no growth in –LN. This showed success as per the expected results because liquid nitrogen has allowed the survival of the tested sample. This indicates towards the efficiency of vitrification. Vitrification solution initially dehydrated the sample. The liquid nitrogen at -196oC is very effective in preserving the vital plant genotypes and resources. Vitification solutions aid in this processes by removing the risk of crystal formation. Ice crystal formation can rupture the cell membrane of the plant cells which ultimately result in the failure in the survival of stored samples. Thus the cryopreservation represents a effective and potential technique for storing the plant tissue resources.

CONCLUSION:

REFERANCE:

• Cryopreservation. 2009. In Encyclopædia Britannica. [online]. Available from : [Accessed 5 April 2009]
• Lambardi, M. and Panis, B. 2005. STATUS OF CRYOPRESERVATION TECHNOLOGIES IN PLANTS (CROPS AND FOREST TREES). [online]. Available from:  [Accessed 5 April 2009]

• Reed, B. M. 2004. Technical guidelines for the management of field and in vitro germplasm collections. [online] Bioversity International. Available from:  [Accessed 7 April 2009]