Explant Sterilization – Plant Tissue Culture Protocol

Surface sterilizing plant material is an important step when establishing a plant tissue culture protocol. Commonly used disinfectants for plant tissue culture and methods for disinfecting green capsules, dry seeds, and orchid seeds disinfecting are detailed below.

Commonly Used Disinfectants for Plant Tissue Culture

Disinfectant	Product No.	Concentration (%)	Exposure (min)
Calcium hypochlorite	211389	9-10	5-30
Sodium hypochlorite*	425044	0.5-5	5-30
Hydrogen peroxide	H1009	3-12	5-15
Ethyl alcohol	E7148	70-95	0.1-5.0
Silver nitrate	S7276	1	5-30
Mercuric chloride	M1136	0.1-1.0	2-10
Benzalkonium chloride	B1383	0.01-0.1	5-20

*Commercial bleach contains about 5% sodium hypochlorite, and thus may be used at a concentration of 10-20%, which is equivalent to 0.5-1.0% sodium hypochlorite.

Sterilization procedures may be enhanced by the following actions:

• Placing the material in a 70% ethyl alcohol solution before treatment with another disinfectant solution. The use of a two-step (two-source) sterilization procedure has proven beneficial with certain species.
• Using a wetting agent, such as TWEEN^® 20 or 80 (Product Nos. P1379 or P1754, respectively), can be added to the disinfectants to reduce surface tension and allow better surface contact.
• Conducting the sterilization process under vacuum. This results in the removal of air bubbles and provides a more efficient sterilization process.

Orchid Seed Sterilization

Orchid seeds are very small and contain little to no food reserves. A single seed capsule may contain 1,500 to 3,000,000 seeds. Sowing the seed in vitro makes it possible to germinate immature seed (green pods). It is much easier to sterilize green capsules than individual seeds after the capsule has split open. Lucke (1971) indicated that orchid seed can be sterilized when the capsule is about two-thirds ripe. Listed below are the estimated normal ripening times of capsules for various orchid species.

Orchid Genera	Time to Maturity (months)
Bulbophyllum	3
Calanthe	4
Cattleya	11
Coelogyne	13
Cymbidium	10
Cypripedum	3.5
Dendrobium	12
Epidendrum	3.5
Laelia	9
Masdevallia	3.5
Miltonia	9
Odontoglossum	7
Paphiopedilum	10
Phalaenopsis	6
Stanhopea	7
Vanda	20

Green Capsule Sterilization

1. Soak the capsule in a 100% bleach solution for 30 minutes.
2. Dip the capsule into 95% alcohol, and flame.
3. Under aseptic conditions, open the capsule and scrape out the seed.
4. Carefully layer the seed over the surface of the culture medium.

Dry Seed Sterilization

1. Collect seed and place in either a small flask or bottle, or place in a shortened pipet which has one end sealed with cotton. Seal the other end of the pipet with cotton, once the seed has been placed in the pipet.
2. Prepare a solution containing 5-10% commercial bleach containing a few drops of TWEEN^® 20.
3. Add the bleach solution to the flask, or draw up the solution into the pipet. Swirl the flask containing the seed and bleach or repeatedly draw and aspirate the bleach solution in and out of the pipet.
4. Sterilize the seed for 5-10 minutes.
5. Remove the bleach solution and rinse the seed with sterile tissue culture grade water.
6. Transfer the seed to sterile culture medium.