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.
Date : 28th Jan, 2022
Time : 01:06 pm
Location: Morabadi, RANCHI JHARKHAND
Cardamine plants separately kept after floral dip test ( P5, P6, P7, P8, P9, P10) experiment and another set for the control ( C5, C6, C7, C8, C9, C10) . The white tub was kept in balcony and used to water the plants everyday once.
FLORAL DIP EXPERIMENT : Dipping the floral bud in agrobacterium suspension containing kanamycin resistant gene. Inside the Laminar Air Flow.
Date : 23 Dec , 2021
Location: DSPMU, RANCHI, JHARKHAND
Seed collection from Cardamine plant
Mature Seed pods burst just after touching it. And disperse it’s seeds . So keeping the paper below the pods so that seeds can be collected.
Location: Ranchi Jharkhand
Date : 18 dec , 2021
Location: Dept of Microbiology,
DSPMU RANCHI JHARKHAND
Agrobacterium tumefaciens pCAMBIA2301 was inoculated on Nutrient agar (NA) plate on 05/10/2021. Also Rifampicin and kanamycin ( 50mg/L) was added in the NA plate. To ensure the growth of only kanamycin resistant transformedAgrobacterium tumefaciens .
S OR Z shape streaked line can be seen on the NA plate.
After collecting seeds stored in a plastic zip lock bags. And labelled as P6, P7, P8, P9, P10 ( Here “P” stands for putative seeds as we are supposing that these have transformed) and C6, C7, C8, C9, C10 ( here “C” stands for Control cardamine plants.)
Picture was taken on 4th April, 2022 ( Dept of Microbiology, DSPMU RANCHI JHARKHAND)
Putative seeds ( from P10) were cultured on 14/02/2022 . In Murashige and skoog (MS) Media with kanamycin. Out of 10, 6 seeds are growing and elongating and two were stunt. Two didn’t germinate or not visible in this picture.
We can conclude that there were 10 seeds in total.
6 seeds grew in the presence of Kanamycin. Means 60% were transformed according to this sample size and 2 were able to grow but didn’t elongate. And another two was unable to grow.
Picture was taken on 4th April, 2022 ( Dept of Microbiology, DSPMU RANCHI JHARKHAND)
Normal seeds ( from C10) were cultured on 14/02/2022 . In Murashige and skoog (MS) Media with kanamycin. Out of 10, 5 seeds grew but remained stunt.
We can conclude that 50 % were able to germinate but didn’t elongate and the growth stoped. 5 seeds were not able to grow. ( According to the 10 seeds as sample)
Picture was taken on 4th April, 2022 ( Dept of Microbiology, DSPMU RANCHI JHARKHAND)
Putative seeds ( from P10) were cultured on 14/02/2022 . In Murashige and skoog (MS) Media with kanamycin. Out of 10, 2 seeds are growing and elongating and 4 were stunt.
Note:The sample size of the experiment was so small to tell the accurate or most probable % of transformed cardamine seeds having kanamycin resistant gene after the Floral dip experiment.
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) - CUBE RANCHI from November 2021 to April ,2022. The work was discussed in CUBE chatshala with other collaborators present throughout the india and context to curriculum.
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