Arabidopsis Protocols, 2nd Edition (Methods in Molecular by Julio Salinas, Jose J. Sanchez-Serrano

By Julio Salinas, Jose J. Sanchez-Serrano

This number of effectively reproducible Arabidopsis protocols has been up to date to mirror fresh advances in plant biology, the of completion of the Arabidopsis genome series, that's crucial for learning plant functionality, and the advance of complete platforms techniques that permit international research of gene expression and protein and metabolite dynamics. The authors have integrated approximately all ideas built in Arabidopsis, others lately tailored from the normal paintings in crop species, and the newest ones utilizing Arabidopsis as a version process. Highlights contain the latest methods-transcriptomics, proteomics, and metabolomics - and their novel functions (phosphoproteomics, DNA microarray-based genotyping, excessive throughput metabolite profiling, and single-cell RNA).

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The age and state of leaves is critical (8). Do not use pale, discolored, curled, or vitrified leaves. Do not use leaves from plants that are already flowering 3 In order to decrease the amount of debris m the protoplast preparation, the cut should be clean and the leaf should not be crushed Allow the weight of the blade and handle alone to do the cutting and change the blade between different dishes. 4. The activity of enzyme preparations may differ from batch to batch and thus the optimal time of mcubatlon may vary.

Allow the algtnate to form a gel for 45 min. Transfer the individual droplets with a spatula into 55-mm Petri dishes containing approx 5 mL of PM medium and culture the protoplast in a growth chamber at 25°C under dim light 14. 5 mL of fresh PM medium after 7 and 14 d (see Note 11). 15 Remove 1 mL of PM medium and add 1 mL of MSAR I medium to the protoplast cultures on d 21, 28, and 35 (see Note 12). 2. Root- Derived Protoplas ts 1 Sterthze seeds for 15 mm using 10% sodium hypochlortte solutton and after washing and drymg of the seeds, plate them on 0 5 x BM agar plates.

6 After 4 h of mcubatlon, collect the protoplast suspension and filter the cells through 50- and 25-q sieves Dispense the protoplasts to 12-mL, screw-capped glass tubes (see Note 17) 7 Centrifuge the protoplasts for 5 mm at 50g Collect the band of floating protoplasts from the top of solution (see Notes 7 and 18). 8 Wash the protoplasts twice with 0 45M manmtol solution by centrifuging at 60g for 5 mm and resuspendmg the pellet each time (see Note 19). ) and count the cell number m a hemocytometer (see Note 20) 10 For hquld culture, adjust the dens@ of protoplasts m PM medium to 1 x 1O6cells/ml When the protoplasts start dividing, dilute the suspension with PM medium to 5 x lo5 cells/ml (see Note 22).

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