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Work shope on plant Cryopreservation

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Name- Venkatesh Kolluru Student number- 0803568 Course- MSc. BIOTECHNOLOGY Module- BI1102A Title of the work shop- PLANT CRYOPRESERVATION Workshop tutor- Irene Tierney AIM: To learn about the practical aspects of plant cryopreservation and its current developments in the field of plant biotechnology. INTRODUCTION: Cryopreservation is a process where the cells or the whole tissues are preserved by cooling the temperature to -196C (BP of liquid nitrogen). In these extreme conditions the biological activity even the biochemical reactions which would lead to cell death is effectively stopped. But this technique consists of problems like damage to the cell due to solution effect (used in the solution), dehydration and intra cellular ice formation. At this temperature the salts present in the cell may become crystal which can ultimately lead to fatal conditions. To overcome this problem vitrification solutions (sucrose, DMSO i.e. Di-Methyl sulphoxide, glycerol, etc.) are used. Vitrification solutions prevent the formation of ice crystals. They do it by lowering down the freezing point and increasing the viscosity. The time of exposure of cryoprotectants (i.e. vitrification solutions) is very important because the long exposure of chemicals towards the plant cells may result in to phytotoxicity. Cryopreservation is a technique which is carried out frequently for the conservation of plant genetic resources. This was initially developed by Withers and King in 1980, but the cryopreservation techniques which are used now a day have several modifications in the protocol which was used by the initial developers. ...read more.


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. ...read more.


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 Encyclop´┐Żdia Britannica. [online]. Available from :http://www.britannica.com/EBchecked/topic/1452607/cryopreservation [Accessed 5 April 2009] * Lambardi, M. and Panis, B. 2005. STATUS OF CRYOPRESERVATION TECHNOLOGIES IN PLANTS (CROPS AND FOREST TREES). [online]. Available from: www.fao.org/biotech/docs/panis.pdf [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: http://books.google.co.uk/books?id=QNWf4HW1EnMC [Accessed 7 April 2009] ?? ?? ?? ?? ...read more.

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