An improved culture medium for growing the orchid Cypripedium reginae axenically

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Abstract
A new medium for growing Cypripedium reginae Walt. axenically from seed was designed. Liquid culture proved unsuitable, hence a 1% agar medium supplemented with 5% potato extract was used to investigate optimal mineral element, vitamin, amino acid, sugar, and growth regulator supplements for germination, and subsequent growth. A modified Pfeffer solution with 1400 mg/L NH4NO3 + 19 mg/L ammonium citrate + 2% dextrose + 10 mg/L niacin + 5 mg/L calcium pantothenate + 5 mg/L thiamine HCl + 1 mg/L kinetin + 0.1 mg/L α-naphthaleneacetic acid gave best germination and growth to 2 years with little or no phenolic production. Gamborg's B5 medium and Murashige–Skoog (MS) medium were less than optimal when tested against the above medium. Growth regulators were more active when sterilized by membrane filtration instead of autoclaving. Of the three aminopurines tested, kinetin, benzylaminopurine (BAP), and 6(γ,γ-dimethylallylamino) purine (γγ), the order of activity was initially γγ → BAP → kinetin, but kinetin produced better greening of protocorms and plantlets, and eventually greater survival. Hence, it was chosen for further study. The auxins indole-3-acetic acid (IAA), naphthalene acetic acid (NAA), indole-3-butyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D) were also tested alone and in combination with the aminopurines. They did not stimulate germination, but improved growth and survival when combined with aminopurines. The most active of the auxins were NAA → IAA → IBA → 2,4-D. A kinetin:NAA ratio of 10:1 was very satisfactory.