Harvais medium

Many media have been used for the germination of Cypripedium seed and to support early growth of embryos and protocorms. We have focused on Harvais medium as popularized by William Steele.

In Harvais medium, potato is used to provide unidentified growth factors. We have found in several small experiments that inclusion of potato is indeed nescessary. Another popular medium, Malmgren's medium, uses pineapple instead of potato. We often use Malmgren media for young seedlings following initial germination in Harvais medium. We have not yet done systematic experiments to determine which medium is best for which species. We have, however, observed that many young seedlings do far better in Malmgren's medium than Harvais medium, and we have also observed that seed and green pod embryos germinate and grow much more rapidly in Harvais medium than in Malmgren's medium. Thus, we start seed in Harvais medium and move the protocorms to Malmgren's media as soon as they are 1 mm or so in diameter.

Harvais Medium

Component	Steele (1995)	Phytotech Labs T839	GPH Version
mg/liter			T839 plus
Ca(NO₃)₂ · 4H₂O	400	400
NH₄NO₃	1400^a		table
KH₂PO₄	200	200
MgSO₄ · 7H₂O	200	97.69^b
KNO₃	200	200
KCl	100	100
NH₄Citrate	19	19
FeNH₄Citrate	25	25
H₃BO₃	0.5	0.5
CuSO₄ · 5H₂O	0.025	0.025
ZnSO₄ · 7H₂O	0.5	0.5
Na₂MoO₄ · H₂O	0.02	0.02
Co(NO₃)₂ · 6H₂O	0.025
KI	0.1	0.1
MnSO₄ · H₂O	1.54	1.54
Glucose	20,000	20,000
Casein hydrolysate	0	400
Agar	7000^c	6000
Kinetin	table	0	table
Russet potato	table	0	table
pH	5.8		5.8

^aThis concentration was used for germination and growth of Cypripedium reginae. Currently, Steele uses amino acids (casein hydrolysate), sometimes mixed with NH₄NO₃.

^banhydrous

^cSteele (1995) used 7000 mg/liter agar for germination and 6000 mg/liter for further growth of seedlings.

Procedure

1.	Fill a 1 liter Ehrlenmeyer flask with water at room temperature. Add stirbar. Place on a stirring hotplate. Begin stirring.
2.	Add T-830 media.
3.	Add ammonium nitrate if it is to be used.
4.	Add kinetin solution
5.	Measure pH. Adjust to 5.8 using 1 N NaOH, dropwise. pH paper is adequate.
6.	Heat with stirring until solution turns transparent. This is near boiling.
7.	While heating, cut russet potato into cubes 1 cm on a side. Add to flasks.
8.	Add medium to each flask (25 ml to baby food jar, 100 ml to 1 pint jar).
9.	Add lid to each flask, then cover with aluminum foil.
10.	Place flasks into autoclave or pressure cooker. Sterilize for 20 minutes.
11.	When cycle is complete, remove flasks to cool. Do not remove aluminum foil until ready to place into laminar flow hood or glove box.

Nitrogen Source

Different species of orchids require different nitrogen sources, which can include ammonium, nitrate, or amino acids. In Steele's original work, he used ammonium nitrate at 1400 mg/liter as a primary nitrogen source for Cypripedium reginae. In 1996, Steele noted that lower concentrations of ammonium nitrate are better tolerated by other species. Currently, Steele uses amino acids (casein hydrolysate), and sometimes includes ammonium nitrate.

Potato

Potato has been the primary source of complex, unidentified growth factors used in Harvais medium. Russet potato ("Idaho potato") is the variety most often used. Various species have been reported to grow optimally in the presence of different amounts of potato. Excess amounts have been reported to cause abnormal growth of protocorms. We follow the practice of Steele, who prepares cubes of potato 1 cm on a side and adds them whole to the media. We find that adding 1 of these cubes to 25 ml of media supports excellent germination and production of protocorms with virtually all of the crosses we have attempted.

For some species, the amount of potato supporting germination is greater than the amount used in further growth of seedlings in daughter flasks. It has been reported that keeping the amount of potato too high leads to malformed seedlings.

Kinetin

Kinetin is the growth hormone that has been used most frequently to promote germination in Harvais medium. Kinetin is not added to medium to be used to grow green pod embryos, and is used only to initiate germination of mature seed where it is indicated in the following table. Once protocorms reach 1 mm or so in size, they should be transferred to media that does not contain kinetin.

Preparation of kinetin solution

Like many growth factors, kinetin is not freely soluble in water when added as a powder. Kinetin is prepared as follows (Steele, 1995):

Nitrogen Source, Potato, Kinetin

Species	NH₄NO₃^a	Casein^a	Potato^b	Kinetin^a	Reference
C. acaule	1000	0	1	0	Steele (1996)
C. arietinum	500	0	0.5-1	1.2	Steele (1996)
C. californicum	500	0	0.5	0	Steele (1996)
C. candidum	500	0	2-3	0	Steele (1996)
C. fasciculatum	500	0	2-3	0	Steele (1996)
C. formosanum	0		0.5	0	Steele (2005)
C. guttatum	500	0	2	0	Steele (1996)
C. kentuckiense	500-1000	0	1-2	0	Steele (1996)
C. macranthos	200	200	0.5	0	Steele (2004)
C. montanum	500	0	1-3	0	Steele (1996)
C. parviflorum	500	0	2-3	0	Steele (1996)
C. passerinum	500	0	2	0	Steele (1996)
C. reginae	1400	0	1;0.25	1.2	Steele (1995)
C. yatabeanum	500	0	0.25-2	1.2	Steele (1996)

^aConcentrations are in mg per liter. "Casein" indicates casein hydrolysate.

^bIndicates the number of cubes, 1 cm on a side, added to 25 ml of medium. Where there are two values for amount of potato, e.g. "1;0.25", the first value refers to the amount per liter (in the example, one cube) used in 25 ml in the mother flask, whereas the second indicates the amount to be used in daughter flasks for further development of the seedlings, in the example one-fourth cube per 25 ml or one cube per 100 ml of medium.