Cultivation of Arabidopsis thaliana the Model Plant for Genetic Modification and Molecular Biology Applications
Authors: N. Chandrashekar1, S. ALI2 and A. GROVER2*
1Division of Microbiology, CCUBGA, Indian Agricultural Research Institute, New Delhi, 110012, India
2National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi, 110012, India
Protocol for the cultivation of Arabidopsis
1. Appropriate quantity of healthy seeds was taken in a sterile eppendorf tube
2. Seeds were washed with sterile distilled water by tapping the tube for one minute, centrifuged and discarded the water
3. It was then washed with 90% ethanol as mentioned above
4. 0.1% autoclaved mercuric chloride (HgCl2) and SDS prepared in combination was added, tapped with finger for one minute and incubated for 5 more minutes, centrifuged and liquid was discarded
5. Seeds were washed 6-7 times with sterile distilled water for one minute each as mentioned in step (2)
6. 0.1% autoclaved agarose solution was added to the tube and mixed to disperse the seeds uniformly
7. With the help of 1ml cut filter tip; seed mixture was drawn from the tube and immersed in 40ml of 0.1% agarose solution taken in 50ml transparent Falcon tube, kept at 40 C for 2-3 days.
8. Three inch pots (holes at the bottom were created and blotting paper above the holes was placed) filled with autoclaved soilrite and kept in plastic trays. One litre of Hoagland's solution was poured in the tray and the tray was wrapped with plastic wrapper for one day to create humid environment.
9. With the help of cut 1ml filter tip seed-agarose mix was drawn and placed uniformly on the surface of all the pots.
10. The trays were covered with plastic wrapper to maintain humidity and were kept in culture room upto at least 2 leaf stage other than cotyledonary leaves.
11. The trays were transfered to the National Phytotron Facilty (IARI New Delhi)
12. Wrapper was removed and distributed plants (two leaf stage) to the other pots in order to increase the number of pots. Two leaf stage is most appropriate to get healthy plants and quicker growth when transferred between the pots.
13. Again covered the wrapper for few days (One week). Made small holes and removed the wrapper.
14. Trays were irrigated at 3 days interval by providing half X Hoagland's solution as it is critical to the development of plants and all prophylactic measures needs to be taken to prevent disease and insect attacks.
15. If all the conditions are appropriate (Viz Humidity, temperature, light duration, light intensity, plant prophylactic measures) one can be sure to get healthy and robust Arabidopsis plants by following this method.
About Author / Additional Info:
Scientist (Agricultural Biotechnology)