Write the procedure for observing chloroplast.
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Answer:
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Answer:
Materials and Reagents
1. Plant materials
a. Arabidopsis thaliana transgenic plants expressing GFP-TALIN in wild-type or mutant plants (Kong et al., 2013). Actin probe lines such as LIFEACT-YFP and GFP-FABD2, THRUMIN1-GFP transgenic lines are also useful.
b. Young and fully expanded rosette leaves of 3-4-week-old plants grown under 100 pmol/m2/s white light (16 hy/dark (8 h) cycles at 23°C.
Note: Plants grown on soil are better than those in the plate of MS medium. The reason is that the leaves of the plants grown in the plate are more susceptible to wither during sample handling than
those grown on soil.
2. Red cellophane film (TokyoButaiShowmei, catalog number: No. 20 ) 3. (Optional) 2,3-butanedione monoxime (BDM) (Sigma-Aldrich, catalog number: B0753 ) as an inhibitor of
myosin ATPase that inhibits actin dynamics
4. Evacuation solution containing Silwet L-77 (Bristol-Myers Squibb Company, catalog number: BMS SL7755 ) (see Recipes)
Equipment
1. Syringe (10 ml)
2. Confocal microscope (Leica Microsystems, model: SP5 equipped with a 63x/1.20 W objective lens and
multi-line 100 mW argon laser)
3. Plant growth room or chamber
4. Red safe-light (red LED or fluorescent lamp filtered with red films)
5. Forceps
6. Razor blade
7. Scissors
8. A custom-made cuvette system and ring holder: this system is composed of two steel rings mating
each other with complementary threads, two round cover glasses (22 mm in diameter, No. 1 and No.
5), and a silicon ring (100 um in thickness) (Wada and Kong,