You can dilute your Agrobacterium solution or shorten your immersion time even to 5 min. Good luck!

Hi, Teh, you are right, OD= 1.0 is too too high. You should use much less bacterial density. I hope you used activated bacteria in acid pH solution to switch from bacterial growth to virulence activation. Good luck!

I agree with previous suggestion the concentration of bacteria is very high and you have reduce the time of immersion explant in bacteria .Good Luck

After inoculation, you can try to wash your explants 3 times with sterile H2O and dry with tissue paper, before put in your culture medium.

Bacterial overgrowth is a common problem in transformation experiments. You can reduce the immersion time to 10 -15 minutes. Also you can reduce the O.D of the culture.Usually 0.1-0.2 OD at 600nm and 0.5 OD at 420 nm is followed.Drying of the explants after co- culture on a sterile filter paper before transfering to the selection medium will also help in reducing the overgrowth.

Hi, Nice try… you should confirm your right strain of Agrobacterium. At first, you should confirm whether it is a true Agrobacterium or not. If it is a true one, it should amplify the Vir gene with Vir specific primers. If yes, I would suggest you should infect your explants at 0.3-0.6 OD for 48 hrs. You may reduce the infection time to 20 min. Washing of agro-infected explants with cefotaxim containing water and drying for few mins is a must and common protocol. You may follow Sahoo et al., 2011, Plant Method for further references (http://www.ncbi.nlm.nih.gov/pubmed/22206592). Thanks and Good Luck.

I use it at an OD. 0.6 for 20-60 minutes and I don't have problems with overgrowth. Maybe your problem is only the bacteria concentration. Good luck!

To Taras Pasternak: What acid solution should be used? How much should the pH be? Thanks.

Centrifuge the overnight grown Agro culture (OD 1) to get palette. Dissolve the palette into double volume of growth medium. Let the dead cells to settle down for 10-15 min. Use the upper 20 ml solution and Incubate the the shoot apex into this medium for 48-72 hr. Dry the incubated shoot apex on filter paper for 5-10 min and transfer on regeneration medium without selection for one week. This will help. Good luck.

I wish to share experience of plant transformation experiments, as similar conditions were observed to me, at higher OD of agro culture for longer infection time.

In this study, virulence of agro strain is important and on that basis we have to decide agro density for infection and infection time.

Some of strains with suitable optical density (not mentioned strain’s details),

LBA – 0.5 to 0.6

GV - 0.6 to 0.8

EHA - 0.2 to 0.5

@ 600nm.

I think there is not any thumb rule, but need to understand relation between virulence of Agro strain, type of explants, infection time, infection medium and co-cultivation time to avoid agro overgrowth and to get responsive explants after 1st wash.

Thank you all for the comments and suggestions. I'm actually using the EHA and LBA strains to investigate the transformation efficiency of these two strains on rice. Will try them out at lower concentrations. Thank you once again.

I realize the rice seedlings are not sterile in vitro culture, but if they were the common practice would be to treat them with antibiotics to kill off all bacteria on the surface of the plant tissue. Well at least this is the common practice during the establishment of so called “hairy root cultures”. The example of it you can see here: www.facebook.com/yaroslav.shevchenko.96

the picture on the right are hairy roots in the flask.

Nothing stands in your way to pretend that you have a sterile culture. So try the treatment with antibiotics. Other options were listed above, but I would go for experimentation with different immersion times.

reduce the concentration of bacterial culture, 0.6-0.8 OD at 600 nm, u can immerse for 30-50 mins, thts not a problem, blot the explants dry in sterile blotting paper just after the immersion and then inoculate it in the respective medium. goodluck

After co-cultivation, rinse the explants with sephotaxime (50 ppm)solution 2 times for 5 minutes followed by sterile water. Then blot dry the explants and then keep the explants in media.

good luck

You might want to consider a more through washing step including antibiotics after co-incubation with the Agro. Immersion time has more of an impact on explant vitality.

cefotaxime at a concentration of 500mg/L works well for washing bacterial overgrowth after co-cultivation for 72 hrs.... add cef in the subsequent medium @ 250mg/l initially.... wash 5-7 times in a shaker, but dont wash for longer period...... check out

In my opinion 10 minutes immersion at 0.8 - 1.0 OD is suufficient. after two days incubation at 25oC give cefotaxime/carbencilin wash(250-500mg/L). To check about contamination with your explants, keep control plates without co-cultivation/immersion. good luck!

Yes, Since you are using shoot apex as primary explants,immerse the the explants in agrobacterium culture 5-10 minutes and dry it on filter paper ,cocultivate for 48 hrs.

Reduce the timings in all steps and dilute the suspension culture.

All the best

You can use OD 0.5-0.6 for infection 0f about 20-25 minutes at 50 rpm at 28'C and co-cultivate for 2 days.

The bacterial concentration, the time of immersion/inoculation, blotting after immersion, the time of co-cultivation, and the antibiotic treatments are factors affecting outgrowth of Agrobacterium in Agrobacterium-mediated transformation. The more concentration, the longer the time of immersion and the co-cultivation will usually positively correlated with bacterial outgrowth.

Moreover, to certain tissues used as explants or to certain crops that are sensitive to the bacteria, such conditions also result in less survival of the tissue explants. In your described case, you may want to go with lower concentration, less time for immersion and for co-cultivation. In my experience, once you have seen visible colony on the surface of media surrounding co-cultivated explant, it is usually difficult to clean up the outgrowth of the bacterium.

Blotting explants after immersion will remove excess of bacteria in the surface of explants and eventually help controlling the outgrowth of Agrobacterium. Treatment with cefotaxim or carbenicillin solutions and combine it with shaking are also useful to reduce outgrowth. However, always blot dry the treated explants after washing with antibiotic.

It may also important to work in sequential manners. Let say you want to transform 1000 seedlings, do not do them in a day or two but split them into 10 batches of 100 seedlings. If it is possible only do one batch or even less than that. It always works in our lab, i.e. We can always have quite a number of "clean" regenerants when we are working that way. Good luck.

I agree with lenbi, reduce concentration of bacteria to 0.6 OD at 600nm, 30 minutes imersion time is enough and the explants should be rinsed with cefotaxime 250mg/l fot ten minutes twice followed by washinf of explants with sterile water and then blott dry the explants with sterile filter paper and shift them to media contaning cefotaxime.

Reducing OD, using cefatoxime and blot drying are procedures that helped me overcome this type of issues. One more thing to watch out is the bacterial growth itself. Depending on the quality of the bacterial stock and culture conditions, there is a possibility of outgrowth of bacteria other than Agrobacterium. You can identify this based on the smell. If this is the case, it is best to work more on the Agroprep than the OD, co-cultivation or after treatment protocols. Best of luck.

For an OD of 0.6-0.8 the infection of agrobacterium with the explants for about 15-20 minutes is more than enough along with Cefatoxime antibiotic at 28'C and co-cultivate for 3 days... I hope you can avoid the overgrowth of bacterium....

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If we add a para daily after one month it will result in a productive item (paper , review, thesis , chapter, etc.etc.) So start from today

Dear All

I am pleased to inform u , after successful publication of two books i have started compiling another one or two books depending upon response from authors and dedicated scientists like you) in the aspect of crop improvement

The themes are

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2. Plant bioticjnology aspects of crop improvement( biotic, abiotic stress, quality improvement,) both Marker assisted selection AND TRANSGENIC APPROACH/ CISGENIC APPROACH/ other in vitro techniques etc.)

3. Molecular breeding/QTLS biotic, abiotic stress, quality improvement

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5. Crop specific breeding approaches like rice , wheat , maize or other horticultural crops

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Interested scientists , students are requested to send in their titles and abstracts from the above themes before 30 September 2013 for finalization. Selection of papers will be on first come first serve basis.

Interested scientists who want to be co editors can also send in their interest

For any further query kindly contact

[email protected]

shabirhwani@[email protected]

Kindest regards

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Thanks & Regards

Shabir Hussain Wani Ph.D, F.S.P.R

Assistant Professor

AICRP NSP (Crops), Division of Plant Breeding and Genetics

SKUAST-K, Shalimar, Srinagar, Jammu and Kashmir, India 190025

Mobile : +91 9419035566/ +91 9797001791

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******************************

*****************************************************

Thanks & Regards

Shabir Hussain Wani Ph.D, F.S.P.R

Assistant Professor

AICRP NSP (Crops), Division of Plant Breeding and Genetics

SKUAST-K, Shalimar, Srinagar, Jammu and Kashmir, India 190025

Mobile : +91 9419035566/ +91 9797001791

http://www.skuastkashmir.a