1	Working with Tissue-Culture Bags for Germplasm Storage Barbara M. Reed USDA-ARS National Clonal Germplasm Repository Corvallis, Oregon CRIS 5358-21000-033-00D
2	Using Tissue-Culture Bags Bags are useful for medium-term storage of plant tissue cultures due to their semi-permeable walls and heat-sealed tops. The bags come in 1, 2, or 5 chamber sizes and are not sterile. They can be autoclaved like regular medium containers but should not be processed in metal containers or they will melt.
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4	Use the standard medium without growth regulators and with 1 g/L more agar or the gelrite equivalent, heat and dispense 10 ml per section
5	A peristaltic pump with teflon tubing is convenient for dispensing 10 ml per cell
6	Use teflon coated tubing or a pipette tip to dispense medium at the bottom of each cell. Try not to drip medium up the side of the chamber. Secretarial finger cots help to get a grip on the plastic to insert the tubing.
7	The medium should have an oval upper surface. This will hold the bag open and provide a good planting surface.
8	Place the filled bags into an autoclavable plastic container (not metal).
9	They may be folded or curved to fit the container but they should not be squeezed together so that the oval upper surface is changed.
10	Enclose the basket in a paper bag. Tape any seams that might open during autoclaving. Label with the number of bags, the type of medium, and the date. Attach autoclave indicator tape
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12	Autoclaving Place the paper-bagged tissue culture bags into the autoclave Process at standard pressure and temperature for 20 minutes, slow exhaust Remove from autoclave and dry in laminar flow or well ventilated room Store on shelf for up to 3 months
13	To use the sterile bags, open the paper bag in the laminar flow hood and carefully pull out the container or tear off the bag. Use long forceps for planting so you can reach the bottom of the bag without touching the top edge.
14	Cut shoots into 3 – 4 cm pieces or into clumps of several pieces depending on the plant. Do not overfill the cell and do not use very small or large shoots. Long forceps and finger cots on the thumb and index finger may be useful.
15	Carefully smooth the surface of the upper edge before sealing to avoid air channels in the seal. Set the temperature of the sealer to avoid melting through the plastic. Seal the bag twice with an impulse sealer. Sealing can be done outside the laminar-flow hood.
16	Pear shoots ready for storage. Normally plants should be about 3-4 cm tall and if extra shoots are available several could be planted in each cell
17	Strawberry plantlets ready for storage
18	In vitro plantlets store best when grown for 14 days before cold storage. We keep them in the growth room for 1 week then cold acclimate them with 22 C (8 hr) days and -1 C nights for 1 week. Plantlets can be stored without cold acclimation and 2 weeks in the growth room, but most store longer if acclimated for 1 week.
19	Storage is at 1 or 4 C with 12 hr low light (3 microeinsteins)
20	After storage plants may fill the cell and older leaves may turn brown. Monitor the growth and color of the shoot tip for viability
21	Plants are evaluated at 4 month intervals and can remain in storage for 9 months to 4 years
22	Tissue-Culture Bag Storage Use healthy shoots and transfer to the bags at the normal culture interval Do not overcrowd each cell Do not use very small or large plants as they do not do well in cold storage Do not be concerned at the initial death of lower leaves after a few months of storage as the axillary shoots will grow and thrive Watch the shoot tip condition to know when to repropagate the plant
23	Tissue-Culture Bag Sources StarPacs tissue-culture bags are available through several commercial outlets. Two that we know of are: Garner Enterprises 5358 Montego Cove, Willis, Texas 77378 Phone 936-856-3479 Fax 936-856-3479 email: rick@lcc.net www.garnerusenterprisesinc.com PhytoTechnology Laboratories P.O. Box 13481, Shawnee Mission KS 66282-3481 Phone1-888-749-8682 or 913-341-5343 www.phytotechlab.com