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    Introduction

    The composition of the growing medium is crucial in every experiment containing living organisms. The need for a well-balanced plant medium arose at the beginning of hydroponic cultivation and plant tissue cultures. The first recipes for plant tissue cultures are connected with great names of plant in vitro – Hildebrandt and others (e.g. Hildebrandt et al., 1946). Important research in the field of plant media was done in the 60s and 70s, probably the most important publication is Murashige and Skoog (1962). This medium was originally designed for nicotiana (Tabaco) calli but it is used in altered forms for other plants – for instance, omnipresent Arabidopsis thaliana.

    Naturally, different plants require different compositions of media but MS-based media are widely used for many species nowadays. However, there are also many exceptions as plants are diverse organisms – for example, Knudson media for orchids (Knudson, 1951; Sigma-Aldrich-a). The difference in composition with common alternatives is mentioned below. To sum it up, they differ in quantities of macronutrients (for instance, some plants demand more nitrogen or phosphorus) but also in presence of micronutrients – that determinates some media only for short-term cultivation.

    Applications

    • The principal component of In-vitro plant cultivation.
    • Plant tissue culture media like calli induction, shoot regeneration, and root formation.
    • For the germination of seeds of different plant species like Arabidopsis thaliana, Nicotiana benthamiana, under sterile growth conditions.

    pH

    Acidic solution (pH 5.7-5.8) is used in the original study (Murashige and Skoog, 1962).

    Most known plant tissues require pH 5.2-5.8 (Skirvin et al., 1986).

    The pH will be under constant change during the cultivation, so stable buffering conditions are essential for long-term cultivation. MES is commonly used because it buffers well between pH 5-7 (approximately) and it is not toxic to most plants species.

    Medium is usually adjusted with NaOH/HCl. KOH can also be used instead of NaOH.

    Composition

    The compounds used in the media can be divided into the following categories:

    • Basal salts (macro and micronutrients)
    • Vitamins (and organic essentials and beneficial)
    • Buffering substances, carbon resource (sucrose) and
    • Reinforcing agent (none in liquid, agar, or phytagel in solid)

    For making MS, you can buy different products – a full-fledged MS (Thermofisher), only basal salts (both or separate micro and macronutrients), basal salts with vitamins, and many other combinations (Sigma-Aldrich-b).

    Here we are describing the total composition, so it can be made from basic reagents or complemented to some commercial products. Sometimes the composition of the medium is cross-over – for example, Murashige-Skoog medium with Gamborg vitamins.

    The tables below with a comparison of MS to other used media for In-vitro plant/tissue cultivation (adapted from Misawa 1994).

    Basal salts

    Components (mg/l) Murashige - Skoog (1962) White (1963) Gamborg (1968) Heller (1953) Schenk - Hildebrandt (1972) Nitsch - Nitsch (1967) Kohlenbach - Schmidt (1975)
    (NH4)2SO4 - - 134 - - - -
    MgSO4.7H2O 370 720 500 250 400 125 185
    Na2SO4 - 200 - - - - -
    KCl - 65 - 750 - - -
    CaCl2.2H2O 440 - 150 75 200 - 166
    NaNO3 - - - 600 - - -
    KNO3 1 900 80 3 000 - 2 500 125 950
    Ca(NO3)2.4H2O - 300 - - - 500 -
    NH4NO3 1650* - - - - - 720
    NaH2PO4.H2O - 16.5 150 125 - - -
    NH4H2PO4 - - - - 300 - -
    KH2PO4 170 - - _ - 125 68
    FeSO4.7H2O 27.8 - 27.8 - 15 27.85 27.85
    Na2EDTA 37.3 - 37.3 - 20 37.25 37.25
    MnSO4.4H2O 22.3 7 10 (1 H2O) 0.1 10 25 25
    ZnSO4.7H2O 8.6 3 2 1 0.1 10 10
    CuSO4.5H2O 0.025 - 0.025 0.03 0.02 0.025 0.025
    Fe2(SO4)3 - 2.5 - - - - -
    NiCl2.6H2O - - - 0.03 - - -
    CoC12.6H2O 0.025 - 0.025 - 0.1 0.025 -
    AlCl3 - - - 0.03 - - -
    FeCl3.6H2O - - - 1 - - -
    KI 0.83 0.75 0.75 0.01 1 - -
    H3BO3 6.2 1.5 3 1 5 10 10
    Na2MoO4.2H2O 0.25 - 0.25 - 0.1 0.25 0.25

    *For Arabidopsis thaliana, ammonium nitrate is sometimes omitted.

    Vitamins (essentials and beneficial)

    Components (mg/l) Murashige - Skoog (1962) White (1963) Gamborg (1968) Heller (1953) Schenk - Hildebrandt(1972) Nitsch - Nitsch (1967) Kohlenbach - Schmidt (1975)
    Myo-inositol 100 - 100 - 1 000 100 100
    Nicotinic acid 0.5 0.5 1 - 0.5 5 5
    Pyridoxine HCl 0.5 0.1 1 - 0.5 0.5 0.5
    Thiamine HCl 0.1-1 0.1 10 1 5 0.5 0.5
    Ca-antothenate - 1 - - - - -
    Biotin - - - - - 0.05 0.05
    Glycine 2 3 - - - 2 2
    Cysteine HCl - 1 - - - - -
    Folic acid - - - - - 0.5 0.5
    Glutamine - - - - - - 14.7

    Carbon source

    Components (g/l) Murashige - Skoog (1962) White (1963) Gamborg (1968) Heller (1953) Schenk - Hildebrandt(1972) Nitsch - Nitsch (1967) Kohlenbach - Schmidt (1975)
    Sucrose 30 20 20 20 30 20-30 10

    Buffering substances

    Tris, Tricine, MES, HEPES, PB-74, and CAPS have been used in tissue cultures for buffering. They differ in toxicity for particular plant species. MES is widely used because it has a good buffering capacity in mild acidic pH, exhibits low binding to nutrients, and exhibits low toxicity in most of the plants (except Brassica napus protoplasts – PB-74 is used there). (George et al., 2007).

    0.5 g/l of MES is used.

    Summary of MS composition

    Category of components Components For 1 l of media (mg) Total mass of components (mg)
    Macronutrients MgSO4.7H2O 370 4530 (2880**)
    Macronutrients CaCl2.2H2O 440 4530 (2880**)
    Macronutrients KNO3 1900 4530 (2880**)
    Macronutrients NH4NO3 1650** 4530 (2880**)
    Macronutrients KH2PO4 170 4530 (2880**)
    Micronutrients FeSO4.7H2O 27.8 103.33
    Micronutrients Na2EDTA* 37.3 103.1 - 104
    Micronutrients MnSO4.4H2O 22.3 103.1 - 104
    Micronutrients ZnSO4.7H2O 8.6 103.1 - 104
    Micronutrients CuSO4.5H2O 0.025 103.1 - 104
    Micronutrients CoCl2.6H2O 0.025 103.1 - 104
    Micronutrients KI 0.83 103.1 - 104
    Micronutrients H3BO3 6.2 103.1 - 104
    Micronutrients Na2MoO4.2H2O 0.25 103.1 - 104
    Vitamins Myo-inositol 100 103.1 - 104
    Vitamins Nicotinic acid 0.5 103.1 - 104
    Vitamins Pyridoxine HCl 0.5 103.1 - 104
    Vitamins Thiamine HCl 0.1-1 103.1 - 104
    Vitamins Glycine 2 103.1 - 104
    Carbon resource Sucrose 30000 30000
    Buffering substance MES 500 500
    Reinforcing agent (one of those) Agar 5000-10000 5000-10000
    Reinforcing agent (one of those) Phytagel 1500- 2500 1500- 2500

    *EDTA chelates metal ions and prevent them from hydrolysis or immobilization.

    **Ammonium nitrate can be omitted for Arabidopsis thaliana.

    Reagents

    Basal salts

    Vitamins

    Buffering substance (MES)

    Carbon resource (sucrose)

    Reinforcing agent (agar)

    NaOH/HCl for pH adjustment

    Instruments and other requirements

    Glass beaker

    Weighing balance

    Magnetic stirrer and pellet

    pH meter

    Measuring cylinders

    Pipette

    Bottles for autoclaving

    Procedure

    Using a weighing balance, prepare exact amounts of basal salts, vitamins, sucrose, MES, and agar (for solid medium).

    Note:

    If you are preparing the MS from basic reagents, you can prepare stock powder of basal salts and vitamins (well-mixed). Then just weigh the total mass mentioned in the summary table.

    Prepare ¾ of the final volume of distilled water in a glass beaker.

    Put salts, vitamins, sucrose, and MES to dissolve (Do not add agar yet!). A magnetic stirrer can be used.

    After everything is dissolved, measure and adjust pH.

    Adjust the final volume using a measuring cylinder.

    Transfer the liquid to bottles for autoclaving and add agar (if you have more bottles then add a proportional amount of agar to every single bottle).

    Autoclave the bottles with media. Too long autoclaving causes degradation of some compounds.

    After autoclaving, let the bottles cool down a bit before pouring them into plates. Working with nearly boiling liquid is not pleasant. Moreover, the agar congeals in temperature 30-35 °C, so you can wait a bit after the autoclaving.

    Try to avoid working in a non-sterile environment – use a flow box.

    If you want to add hormones/antibiotics, add them to the cooling medium. (Five seconds rule is useful here. If you can hold your hand for five seconds or more on the bottle, you can add labile compounds.)

    Now you can pour the medium into plates. Mix the bottle before every pouring.

    Preparation of solid media

    For solid medium, use either agar or phytagel.

    Agar: 5-10 g/l

    Phytagel: 1.5-2.5 g/l

    Storage

    The universal storing condition is 4 °C. Store in this temperature the basal salts and vitamins stock, prepared liquid media, plates with solid MS.

    Caution

    MS medium is a safe medium. The major problems could be connected with too long autoclaving (even caramelized sugar) and non-sterile conditions of work (sugar is an optimal nutrient for bacteria and fungi).

    Reference

    1. George, E. F., Hall, M. A., & De Klerk, G. J. (Eds.). (2007). Plant propagation by tissue culture: volume 1. the background (Vol. 1). Springer Science & Business Media.
    2. Hildebrandt, A. C., Riker, A. J., & Duggar, B. M. (1946). The influence of the composition of the medium on growth in vitro of excised tobacco and sunflower tissue cultures. American Journal of Botany, 591-597.
    3. Knudson, L. (1951). Nutrient solutions for orchids. Botanical Gazette112(4), 528-532.
    4. Misawa, M. (1994). Plant tissue culture: an alternative for production of useful metabolites (No. 108). Food & Agriculture Org.
    5. Murashige, T., Skoog, F., 1962. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum 15, 473–497.
    6. Sigma-Aldrich-a. Knudson C Modified Orchid Medium K4003.
    7. Sigma-Aldrich-b. Murashige and Skoog Media Formulations.
    8. Skirvin, R.M., Chu, M.C., Mann, M.L., Young, H., Sullivan, J., Fermanian, T., 1986. Stability of tissue culture medium pH as a function of autoclaving, time, and cultured plant material. Plant Cell Reports 5, 292–294.
    9. Thermofisher, n.d. Murashige and Skoog Complete Medium-50X Concentrate liquid.