Orchid culture, Medium formulae.

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Copyrights © Reserved Markus Axelsson, 2005.

Here follow formulae for a few useful media. The concentrations are given in mg/L.

pH

It's important to keep the pH at around 5,5. Different species naturally have different optimal pH-values for growth. The availability of nutrients is however pH dependant!

Comments to the BM-medium

^† BM1-medium is good for seed sowing of terrestrials. Hydrolysed casein [500mg/L] and L-glutamine [100mg/L] substitute the inorganic nitrogen (KNO₃, (NH₄)2SO₄, and NH₄NO₃^)3.

A BM2 medium is produced by modifying a BM1 medium (by adding cytokinin; benzyladenine [0,2mg/L]). PLB are transferred or replated to a BM2 for better growth.

Different media?

1. Seed-sowing media have relatively weak nutrient concentrations and (usually) does not contain hormones (some seeds germinate with the aid of hormones). A seed sowing medium is a standard media with the salt concentrations halved, but with the same amount of vitamins and sugars. Activated carbon is sometimes added.

2. Maintenance, replate. These media are generally media from which other media can be made (by adding substances or by diluting). A replate medium may be modified with hormones; either to produce shoots from cells, or by letting shoots form roots. A hormone free medium is used to let protocorms form seedlings.

3. Multiplication. A multiplication medium is formulated with hormones to keep up a calli-generation. callus cells proliferate to produce a large number of clones. The cells stay as undifferentiated cells.

Differences?

Media don't vary much in composition. MS-, Knudson C - and BM-media are old formulae. The salts in an MS-medium have the proportions of the naturally occurring salts in tobacco (relatively high). Amino acids, instead of inorganic nitrogen, has proven to be good for growth in cultures of terrestrial species.

Phytotech P668 (Sigma P6668) and Orchimax are good media for orchids (principally the same formula). MS is very similar to these, with the salt-concentration as difference (twice the concentration). If it's decided to use MS-salts for culturing orchids, half the concentration should be used. The medium should also contain micronutrients and vitamins.

Seed sowing

P668 is sold as a maintenance- and replate-medium. It also works well for sowing seeds. (For sowing; diluted to half the strength and with 20g/L of sugar added). The same is true for the Orchimax (as it's pretty much the same medium). If modified to half the strength, the medium is almost identical to P723; a medium specially formulated for sowing seeds. Enrichment of vitamins and magnesium (e.g. as magnesium sulphate) should be done (Nickels A., 2004).

Other formulae, such as for a replate medium, are not much different from the P668 and Orchimax either. To make a replate medium, the P668 or Orchimax is modified with banana (80g fresh pulp per litre) (P668 + banana-powder = P748). Adding hormones would make such a medium good for cloning.

Cloning

Cloning can be done with a medium like P748. Hormones are used to regulate the development of the cells. Adding BA/BAP [2,0mg/L] and NAA [0,5mg/L] to a P668 or Orchimax, a medium suitable for cloning from stem-nodes and shoot induction, has been produced (almost identical with P793 and O753.

Sum up

P668 and Orchimax can be used for sowing seeds, for cloning, and as maintenance- and replate-media.

Terrestrial species

^3b Terrestrials (e.g. Dactylorhiza) can often be sown onto an MS medium with a fifth (1/5) of the regular concentration. Jeast-extract [1g/L], sugar [10g/L], and activated carbon [0,5g/L] can also be added. A maintenance medium is formulated with more B-vitamins (jeast-extract [500mg] and banan pulp [50g/L]) and may contain slightly more nutrient-salts.

Home-made medium

A home-made medium can easily be put together.

A well balanced water soluable fertilizer (e.g. 20-20-20)

 

The fertilizer must also contain micro nutrients. 2g/L is generally a good concentration to start with. Liquid fertilizer: use 6-8mL/L.

 

To support the plants, explants, or cells, use 7-8g agar/L

 

Agar can be found in Chinese grocery stores and in health care stores. Alternatively, sand or cotton may be used.

 

Vitamins (B-vitamins)

 

Common vitamin pills with B-complex can be used; a proper dose is something like half a pill to one litre of medium.

 

Activated carbon (a spoonful)

 

Carbo medicinalis, can be bought in a variety of places. Vegetable charcoal may be used. Concentration: ca. 0.5-1g/L. Works as a buffer in media.

 

Banana pulp (80-100g/L)

 

Banana powder is a common component in a variety of media (generally for replate). Make pulp of a banana (easily done if the banana has been pre-frozen). Banana contains vitamins and sugars and can easily be found out in grocery stores....

 

Sugar is important (15-20g/L)

 

Common cane-sugar. Can be found pretty much everywhere.

 

Complex additives.

Complex additives may be added to enhance the growth. Herring semen, tomato juice, coconut water, or similar.

Micronutrients and vitamins are naturally occurring in prettymuch everything. If not using extremely pure chemicals for a medium (e.g. technical graded or pro analysis) there will be some nutrients that can improve the medium.

Nickels Anne (2004) personal correspondance via e-mail.

¹ Sigma-Aldrich (www-site August 2004).

³ The Orchid Seedbank Project. (^b: R.J.A.H. Meijer, originally from J. Wetsteyn.)

4 Rasmussen, H. Terrestrial orchids from seed to mycotrophic plant. Cambridge University Press, Cambridge. 1995.

⁵ PhytoTechnology Labs catalog. Reprinted with permission.