Growth of Plants in Sterile Conditions on Solid Media - Agriculture Assignment Help

Assignment Task:

Growth of plants in sterile conditions on solid media:

It is necessary to grow Arabidopsis in sterile conditions on solid media for specific experimental settings such as a selection of drug-resistant and transformed plants, the examination of early root and shoot phenotypes, identification of homozygous lethal mutants, etc. Liquid bleach sterilization, described here, is a practical method to sterilize a few seed lines at a time.

Various containers such as sterile plastic Petri plates (10 cm or 15 cm diameter), Magenta® boxes, or culture tubes are used, depending on the purpose of the experiment. Below we describe the use of the most commonly employed medium for sterile growth conditions in Petri plates (1x MS agar media). Adaptation to other sterile formats is straightforward, and most experimental additives can be easily incorporated in the preparation.

1. Add 0.43 g of Murashige and Skoog (MS) basal salt mixture, add 1 g of Sucrose (optional) and 0.05 g of 2-(N-Morpholino) ethane sulfonic acid (MES) to a beaker containing 80 ml of distilled water and stir to dissolve. Add distilled water to a final volume of 100 ml. Plants grow more vigorously and quickly on media containing 1-2% of sucrose, however, fungal and bacterial contamination must be rigorously avoided by seed sterilization (chose if adding the Sucrose).
2. Check and adjust pH to 5.7 using 1M KOH.
3. Add 1 g of agar granulated per bottle. Keep the lid loose.
4. Autoclave for 20 min at 121°C with a magnetic stirring device in the bottle.
5. Place the bottles on a stir plate at low speed, and allow the agar medium to cool to 45-50°C (until the container can be held with bare hands).
6. Label the bottom of Petri plates with identification number or name, including the date.
7. Pour enough media into 4 plates to cover approximately half of the depth of the plate.
8. Allow the plates to cool at room temperature for about an hour to allow the agar to solidify.

     
Seeds sterilization

1. Surface sterilize seeds in microcentrifuge tubes by soaking for 20 min in 50% household bleach.
2. Remove all bleach residue by rinsing 5-7 times with sterile distilled water.

 
Planting of individual seeds

1. For the planting of individual seeds at low density, adhere one seed to the tip of a pipette using suction, then release seed onto the agar in the desired location. Use a sterile pipet tip to adjust the distribution and remove excess water. Allow the water or top agar to dry slightly before placing lid onto a plate.
2. Seal with Micropore paper tape to prevent desiccation, while allowing slight aeration.
3. Place the plates at 4°C for 1 day for stratification (Breaking seed dormancy) For many lines this is not necessary. However, lines will normally germinate more uniformly (synchronise) if placed in the cold (4 C) for 1-5 days once sown.
4. Transfer the plates to the growth environment. Illumination of 120-150 µmol/m2 sec continuous light and a temperature of 22-23°C are suitable growth conditions

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