Oryza sativa (L. cv. Sasanishki)

(High-Throughput Cryopreservation of Oryza sativa Cell Culture)

Intro/Description:

This protocol was taken from a paper that sought to develop a high-throughput method for cryopreservation of 5 commonly used plant model organisms. Using this protocol, over 100 samples are able to be prepared for cryopreservation prior to freezing without significant loss of cell viability, thereby allowing more efficient prep of larger volumes of samples.

Materials:

• Suspension-cultured O. sativa cells
• 1.2 ml cryogenic vials
• Glycerol
• Sucrose
• Expanded polystyrene tube container (such as 50-well EPS Tube Rack HS4283, Heathrow Scientific or the Tube Holder SD-14, Maruemu Co. Ltd.)
• -30°C Freezer
• Liquid nitrogen & dewar
• 85 mm diameter filter paper
• Shaking water bath

Protocol:

1. Pellet exponential phase O. sativa suspension culture by centrifugation.
2. Re-suspend cells in LSP solution (2M glycerol, 0.4M sucrose, 86.9mM proline) in 1.2 ml cryogenic vials.
3. Place the vials in a tube container made of thick, expanded polystyrene (see materials) and place them in a -30°C freezer for 4-5 hours. The freezing rate should be approximately -0.25°C / min.
4. Plunge the cryogenic vials into liquid nitrogen. Store at cryogenic temperatures.
5. To thaw, place the cryovials in a 35°C water bath for 2 minutes with vigorous shaking (approximately 180 r.p.m.).
6. Drop the thawed cells onto double-layered filter papers (85 mm diameter) on culture medium solidified with 7 g / l agar. To obtain nearly uniform cell density on the filter paper, spread the cells within a 20mm circle when dropping them onto the filter paper.
7. Incubate the cells on the filter paper for 1 d at 27°C.
8. Transfer the upper filter paper into fresh medium for further growth.

References:

Ogawa Y, Sakurai N, Oikawa A, Kai K, Morishita Y, Mori K, Moriya K, Fujii F, Aoki K, Suzuki H, Ohta D, Saito K, Shibata D. High-throughput cryopreservation of plant cell cultures for functional genomics. Plant Cell Physiol. 2012 May;53(5):943-52.

Added by: Carlton Hoyt on July 25, 2012

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