Date: 18/12/2021 Objective: to prepare LB broth to culture agrobacterium culture.
1.)Autoclaved :
LB(Luria-Bertani) Media (100ml in two flasks)
Microtips
3.) After autoclaving, Let the broth to cool down.
4.) Used the autoclaved microtips to transfer 100 micro liter of kanamycin and rifampicin.
5.) Picked the colony with the help of Incinerated needle and then put it in the flask containing broth, and Shaked.
6.) Labelled :
LB broth , pCAMBIA2301
Rif 50mg/ml ; Kan 50mg/ml
Date: 18/12/2021
7.) Left in room temperature for incubation.
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Objective: to extract bacterial cell pellet from the from the LB broth culture.
Agrobacterium flourish in LB broth after 3 Days.
On 21nd Dec 2021, developed bacterial culture was centrifuged for 10 mins (30mins) between 6000 to 7000 rpm.
Bacterial pellets were obtained after centrifugation and stored in fridge 4°C.
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Objective: to perform floral dip method in Cardamine plant. Part 1.: Preparation of resuspension.
The bacterial pellet obtained was resuspended in silwet L-77 (5 micro litre) and sucrose solution 0.05g.
Here silwet L-77 is surfactant and sucrose is used for energy source for plant cells
Part 2.: Dipping the cardamine floral buds.
1.Selected the cardamine plant with unopened buds and dipped in the resuspension solution for 1 minute.
2. Then the plants were covered using plastic covering.
3. After 24hours, the covering were removed and left in Normal condition to grow.
4. Water the plants two times in a day.
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Objective: collection of putative seeds.
Seeds started maturing after 40 days after the floral dip. Seed collection was stated from 15th Feb 2022, And onwards.
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The above work was performed by Manisha Rani, Rechel Tirkey and Man Masih Beck under the supervision of Dr Shalini Lal in Ranchi, Jharkhand. ( Dr Shyama Prasad Mukherjee University, Ranchi) from November 2021 to March 2022.
