Plating Media: R medium+ 2 uM MT; 4, 1 cm3 cubes Russet potatoes in 100 ml media; 60 mins chlorox bleaching
Replate Media: R medium + 1 cm3 cube Russet potato per 200 ml of replate media
Number of Days between Plating and Replating: 85 Days
Number of Days Between Replating and Vernalization: 265 days
Number of Days between Start and End of Vernalization: 120 days (4 months)
Date to Start of Vernalization: Dec. 1
2010 The seedling removed from the jars (replating jars) on Sept.27,2010 were in excellent condition with no sign of fungal contamination. The shoots were very nice measuring 1/2 to 1 inch long. Each seedling had 2-4 roots measuring 4-6 inches long. No sign of the roots getting tangled together so it was very easy to separate individual seedlings. The seedlings were originally plated 10-15-09, replated 1-05-10 and removed from the jars on 9-27-10. These seedlings will be vernalized for 6 months which is too long. As noted above need to start vernalization on Dec. 15 to produce vernalize seedlngs ready for planting on March 31 approximately 15 months later. The plating and replating media works very well with this species. Stick with it.
2012. Used the same plating and replating media as above except that 20 ml/l pineapple juice was added to the replate media. Shoot growth was moderate (1/2 inches to 3/4 inches) and the roots were thick. However, the survival rate was less than 50% from replating. It appears that more than half died in reflasks after looking like they did start to grow-- most growing protocorms were about 1/4 inches long when they died. Suggest replating these protocorms at 30-40 protocorms per flask to increase surivival rates (as in Cypripedium pubescens). Note added on 10/22/2012--- Found one jar of acaule which had been plated with 30 protocorms per jar. Recovered 34 seedlings from this jar and did not observe any mortality. Definitely replate with 30 protocorms per jar in the future. Will reduce the number of jars of media.