Propagating Orchids from Mature Seed

Mature seed collected from dry, porous or dehisced capsules must be chemically sterilized prior to being sown on sterile media or bacteria and fungi will spoil the culture. Sterilization also removes dormancy factors that are introduced into the seed by the parent plant to prevent germination during the winter months.

Timing of Mature Seed Collection

For species and hybrids of Cypripedium, seed may be considered mature 12 weeks after pollenation. For most species and hybrids this will mean collection from about the beginning of September is acceptable while for Cypripedium reginae and Cypripedium kentuckiense collection may commence from the beginning of October. If possible, seed should be collected before the capsule has begun to split to avoid loss of seed and to prevent too many fungal spores from collecting amongst the seed.

Once collected, the capsules should be placed on dry blotter paper or coffee filters for several days to dry thoroughly. Then, the capsules may be broken open and the seed allowed to collect onto blotter paper. Visible pieces of the capsule should be removed with forceps or tweezers. Another day or two of drying is beneficial to make sure the seed is completely dry. Then, the seed may be packaged into coffee filters or folded into paper sachets, labeled with species/hybrid, date of seed collection, and specific location data, and stored in the refrigerator for several months - at least 3 - in a jar with tightly fitting lid.

Materials Required for Flasking Mature Seed

-	50 ml of seed sterilizing solution: water* to which has been added two drops of Tween 80 or liquid dishwashing detergent. The detergent assures that the seed will become thoroughly wetted.
-	Chlorox or similar sodium hypochlorite bleach that is sold at 5 - 6 % strength. Make sure you purchase bleach without added fragrances or other substances that may harm the seed. Also make sure you use a fresh bottle of bleach as once the bottle is opened, the bleach loses strength.
-	100 ml of 20% bleach solution prepared by adding 20 ml of Chlorox to 80 ml of water that contains two drops of Tween 80 or liquid dishwashing detergent.
-	50 ml of sterile water
-	two 25 x 100 mm test tubes
-	test tube rack
-	#7 rubber stopper with hole and rigid plastic tubing to attach to hand vacuum pump
-	hand vacuum pump (the type used to bleed automobile brakes is readily obtained for about $20.00 on Ebay) with tubing to attach to the rubber stopper
-	small funnel (we use small funnels obtained from Linens N Things that are intended to pour liquid scented oils)
-	coffee filter cut to fit the small funnel
-	sterile plastic or glass pipets and pipet bulb
-	laminar flow hood or sterile glove box
-	stopwatch
-	mother flasks containing media

Processing Mature Seed

1.	Place the test tube rack into the hood and add a test tube. Place the funnel into the test tube and filter paper into the funnel.
2.	Pipet 5 ml of 20% bleach slowly into funnel to assure that the rim and the filter paper become moist. Every 5-10 minutes during sterilization of the seed, pipet 3 ml of 20% bleach over the filter paper to assure that it stays moist with bleach.

3.	Outside of the hood drop the dry seed into a test tube. Pipet 10 ml of seed sterilizing solution into tube. Place rubber stopper into tube and swirl the seed to make it wet. Much of the seed may float due to air bubbles trapped inside the seed coat.

4.	Attach vacuum pump and pull vacuum (about 500 mmHg) for one minute. Release the pressure over about 15 seconds. If seed is still floating, repeat the cycle one minute later. If necessary repeat a third time.

5.	Bleaching times are indicated for several species. Use these as guidelines. Observe the seed during the bleaching period and stop the bleaching when the seed have lost nearly all their color.
6.	Using a pipet, transfer the seed to the funnel and allow the sterilizing solution to drain into the test tube. Using a sterile pipet, slowly add 5 ml aliquots of sterile water, making sure that all the seed is well rinsed as the water flows through. Five aliquots of sterile water should be used, always allowing the previous aliquot to completely drain before adding a new one.

7.	To transfer the seed to the mother flasks, quickly add 2 ml sterile water and immediately withdraw it into the pipet (it may take some practice to become proficient in drawing back the seed solution before the water drains). Dispense 0.5 ml aliquots of seed into each mother flask.