By Jose M Martinez-Zapater, Julio Salinas
This finished number of present and crucial protocols comprises many simply reproducible tools built to be used with Arabidopsis - a approach for imminent basic questions in plant biology. The tools variety from the fundamentals of transforming into those vegetation to stylish gene cloning suggestions and will, in lots of circumstances, even be utilized to different plant species with minor changes. Sections on genetics, transformation and gene expression research which are in particular precious to scientists occupied with mutant research or generating and reading transgenic vegetation.
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Extra resources for Arabidopsis Protocols (Methods in Molecular Biology (Cloth))
Place the protoplast suspension m screw-capped 12 mL glass tubes and centrifuge for 5 mm at 50g 9. 45Mmanmtol solution and pellet the cells at 60g for 5 mm Wash the protoplast pellet by repeating this step twice (see Note 19) 11 If the protoplasts are bemg cultured m algmate drops, suspend the cells m 1 mL of algmate solutton. If the protoplasts are bemg cultured m liquid medium, resuspend the cells m 1 mL of PM medium 12. Take 5 pL from the protoplast suspension and count the number of cells m a hemocytometer (see Note 20) 13.
Protoplasts embedded m algmate may be slower m dividing by 1 or 2 d. 22 Depending on the quality of protoplast preparation, up to 75% of protoplasts survtve and 2040% of cells will undergo divisions during the first 3-5 d of culture In liquid medmm, an mmal high density may result in aggregation and collapse of protoplasts. Therefore, care should be taken to properly dilute the protoplast suspension. Aggregation of protoplasts may also be observed rf there was too much debars and/or dead protoplasts m the preparation, which may be a consequence of either inadequate removal of the enzyme durmg the washes or sudden osmotic changes.
Hypocotyl and root explants thus provide excellent materials for callus imtiation and regeneration in contrast to stem and leaf explants. The tissue culture protocols include callus induction m auxin contaming media. Shoot regeneration is induced by lowering the auxin content and mcreasing the cytokinin levels m the media. The elongated shoots are usually transferred into root induction media, but even in the absence of roots, flowering and seed setting can take place m test tubes m vitro. From Methods m Molecular Bfology, Vol 82 Arabrdopsrs Protocols Edlted by J Martmez-Zapater and J Salmas 0 Humana Press Inc , Totowa, 37 NJ Ma thur and Koncz 32 The method described here utilizes root explants that can be cultured and efficiently regenerated m large quantities The described technique can be applied equally well for the Arabidopsis ecotypes Columbia, C24, RLD, and Wassilewsklja.
Arabidopsis Protocols (Methods in Molecular Biology (Cloth)) by Jose M Martinez-Zapater, Julio Salinas