Can any one help me solving this problem.
I am facing problem of tobacco seed germination in MS,media 0.4% water agar and filter paper.
first seeds was sterilized with 0.4% sodium hypochlorite solution and after washing with sodium thiosulohate seeds was incubated at 25 degree Celsius in MS,media 0.4% water agar and filter paper. out of 30 seeds only 1-2 seeds was germinating.
But in green house in soil pots germination is good 70-80% seed was germination
My first guess is that the sodium hypochlorite/sodium thiosulohate has not been completely removed from the seeds therefore damaging seeds and preventing germination. I would include at least 3 washes with 0.05% Triton water (Triton helps settle the seeds so easy to remove the liquid). Then personally I do another sterilisation with 70% ethanol, and then leave these seeds in a flow hood on filter paper to completely dry. Once dry I then transfer these to MS plates (1% agar) to germinate
Thanks Alison for reply
Sorry there was spelling mistake in my question it is sodium thiosulphate
we use three time sodium thiosulphate wash after sodium hypochlorite treatment to remove sodium hypochlorite.
I have one doubt
and you use only 70% ethanol for sterilization and for how long.
can you discuss your method in detail if possible.
This is the method I follow;
• Sterilise hands with 70% ethanol before beginning or wear clean gloves
• Add 0.75 ml of bleach to eppendorf tubes containing seeds and flick tube 7 times to mix (if doing more than 7 tubes at a time move straight to the next step, if doing less than 7 tubes leave for 4 minutes before adding water).
• Add 0.75 ml of water (with 0.05% triton added) to tubes, this prevents the seeds from sticking to the sides of the tubes, helps settle the seeds to the bottom when pouring away the liquid and allows seeds to be removed from the tubes at the end of sterilisation.
• Shake gently to allow bleach and water to mix (7 invertions)
• Pour out liquid carefully, leaving a small amount to prevent seeds being lost
• Wash 3-5 times with water (with triton added) (fill to top, shake and then carefully pore out to prevent seeds being lost). Check bleach has been removed by pouring a little bit of liquid onto a blue towel – if bleach is present towel will turn pink).
• Add 1ml of ethanol, shake and carefully pour away. Add 0.5 ml of ethanol and immediately pour onto filter paper where the seeds are left to dry for one hour
• Sterilise scalpel by spraying ethanol and letting it evapourate dry
• Label Petri dishes (name, date, media and plant sown)
• Gently scatter seeds onto the dish using the sterilised scalpel to loosen dried seeds from the paper
• Wrap with micropore tape to prevent contamination
• Place in 4oC fridge for 2 days before putting in growth room.
I will always do a sodium hypochloride/bleach step first before the ethanol step. The ethanol on its own is not enough to remove all the contaminants. I only leave it in ethanol for a couple of minutes.
My guess is that the sodium hypochloride is not completely removed as I always got poor germination before I started to check that the bleach had been completely removed. If you want to follow your procedure, I would suggest doing sodium hyochlorite then sodium thiosulphate steps as normal, and use a pH indicator paper to make sure the pH is back to normal and all the sodium hypochlorite has been removed. Or just then continue with the water washes and follow my protocol.
Even before inaculation . you can give the Gibberilic acid (GA3) treatment.. germination percentage may enhanced.
Don't had problem with tobacco seeds.
Sterilisation in 1% NaOCl for 10 min, then three times wash with bidest. H2O. And sown onto 1/2 MS in tubes or dishes. At 25 or more Celsius and light.
I have been working with tobacco seeds for a while now. I always start by surface sterilising the seeds in 75% ethanol for about 30-60 seconds. I then wash them thoroughly with sterile water (about 3 times). Like what Alison has said, ethanol on its own is not enough to remove all the contaminants and therefore I then treat the seeds with 10% Hydrogen peroxide for about 10 minutes then thoroughly wash them again with sterile water before letting them to dry on filter paper in the flow hood.
I hope this will help you.
Hi Marowa i tried your protocol for tobacco seed surface sterilization and it is working. but seeds are not germinating on M.S, media and water agar but germination is good in green house. i don't know why
for other seed i am facing the same problem
can you or any one put some light on this issue.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction