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        In vitro growth of seedlings

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        1046

        sterilisation of seeds:

        rinse with 70% EtOH for 30 sec

        put in 1% bleach (sodium hypochlorite, supplemented by a few drops of Tween-20) for 5 min

        wash 2-4x with sterile H2O for 1 min

        plating and growth

        resuspend in sterile 0.1% agarose

        plate 4 ml in petridish (0.8% agarose supplemented with 1/2 MS10) in sterile hood

        let dry for 1-2 h in the hood

        wrap with 3M micropore tape

        keep at 4°C for 4 days

        place the dishes under light

        Solutions

        adjust pH to 5.7 with 1 M KOH, alternatively MES (0.5g/l) can be used to ensure more stable pH during plant growth.

        Remarks

        It is essential that the medium is autoclaved in either a small autoclave or pressure cooker because prolonged exposure to heat will cause complex formation between sucrose and phytoagar which becomes visible as a yellow coloring of the medium. This medium will be softer after hardening than correctly autoclaved medium.

        The MS medium protocol is taken from Murashige and Skoog (1962) Physiologia Plantarum 15: 473-497.

        Materials

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