Plating Media: R medium+ 2 uM metatopolin + 4, 1cm3 cubes Russet potato. Bleaching for 33 mins. in 0.525% chlorox bleach.
Replating Media: R medium + 1, 1cm3 cube Russet potato (Recommended)
Number of Days between Plating and Replating: 1-3 months. Roundtop reached in about 35 days but better replate survival may be achieved in replating after 3 months. Reduce the amount of agar in the replate media to make is less rigid. Should have the consistency of loose jello. I think that this might promote penetration of the media by the roots.
Number of Days between Replating and Vernalization: Approximately 5 months
Number of Days between start and end of Vernalization: 120 days (4 months)
Date to Initiate Plating: March 1
Date to Replate: May 31
Date to start to Vernalize: November or December
Seed collected from Kodiak Island in 2010
The seed germinated very well with the protocorms in the round-top stage in about 35 days. I tried 4 different protocols involving different replate media and different times to start replating. They are as follows:
Plated R medium+ 2 uM metatopolin, 33 mins 0.525% chlorox bleach, 4 potatoes 2/1/11. Replated F522 media + 1 potato + 30 g/l banana 3/5/11 (after 35 days). 10 jars. On 10-8-11 observed the jars and found moderate shoot development but little root development and maybe only a 50% survival rate. The roots appeared not to be able to penetrate the media. Method not recommended. On 11/23/2011 deflasked these 10 jars and got around 70 seedlings. Some seedlings had finally penetrated the media. The roots were fewer in number but thicker.
Plated R meduim + 2 uM metatopolin, 4 potatoes, 33 mins 0.525% chlorox, 2/1/11. Replated F522 media + 1 potato 3/4/11 after about 35 days. 5 jars. On 10-8-11 observed the jars and found moderate shoot growth but little root development and perhaps 50% survival. The roots appeared not to be able to penetrate the media. Method not recommended. On 11/23/2011 examined the seedlings in the jars. A few seedlings had penetrated the media but overall a high mortality. Many seedlings grew on the surface but did not penetrate the media. Considerable brown phenolic compounds produced in the media near the dead seedlings.
Plated R medium + 2 uM metatopolin, 4 potatoes, 33 mins 0.525% chlorox, 2/1/11. Replated R medium + 1 potato 5/12/11 after about 3 months of development. 11 jars. On 10-8-11 observed the jars and found very good shoot development and excellent root growth and very good survival. Some of the roots were 5 inches long and penetrated all the way to the bottom of the jar. The closeness to the potato at the bottom of the jar might be beneficial. Unflasked these jars and got 135 seedlings which began vernalization today on 10-8-11. Noted that the replate media was quite loose and it is possible that I made a much less rigid media than in the other methods.. (Can't remember for sure) . Definitely recommend this method. Don't know for sure if it was the looser media or the fact that I used R media for replating whereas the other two methods used F522 media. The addition of banana to the replate media in one method could be detrimental as well.
Plated R medium + 2 um metatopolin 33 mins 0.525% chlorox 4 potatoes 2/1/11. Replated R medium +1 potato + 30 g banana after 35 days 3/5/2011. 100% mortality on the replated media. Observed on 11/23/2011. Method not recommended.
Results are difficult to interpret uniequivocally. Best guess is that F522 media and banana is not conducive to good replate survival. Also, the best results are obtained by replating after 3 months in R medium + 1 potato media (method 3). Suggest use less agar in the replate media to allow better root penetration.