the experiment:
Sterilized carrots were treated with Agrobacterium rhizogenese ATCC15834 along the vasular cambium on the basal end to induce hairy roots. The sample was then put in the petri dish with MS 2% surcose for 3 days and then transferred to MS with Cefotaxime antibiotic 500g/L to kill Agrobacterium rhizogenese. Incubated at 28 degree in the dark.
The expected result is the formation of hairy roots on the inoculated region.
the result:
After 4 weeks, there are a few different result according to the photos.
I noticed that most of them have the whites forming on top especially the vascular cambium where the bacteria were pipetted along, while the protocol of the final result doesn’t have them. What are they? Are they the Agrobacterium rhizogenese colonies? And are they supposed to be there?
Some samples have the white forming on top but no hairy roots to be seen while some have the white forming on top and hairy roots still grow. What could be the reasons?
And there are some samples which have no changes at all.
Thanks in advance!
The little white clumps look rather callus-like, you could try a bit of auxin in your medium to help with root formation after the callus appears. There is often species and cultivar variation in response to Agro treatment so it is likely that your carrots don't make hairy roots easily in the current conditions. You may need to optimize a few variables, such as length of time for co-cultivation, level/presence of auxin, and size of explant. Your cuttings are (from a tissue culture perspective) very large and quite thick. It may work better to use smaller pieces so you can get more surface area contact between the plant and the bacteria, then between the plant and the medium.
Linh Nguyen I agree to Amy Klocko - It is hard to guess the reason for unexpected result from this experiment. You should use different carrot sources (2-3 cultivars), control (positive)-another strain of Agrobacterium rhizogenes, and control negative - Agrobacterium tumefaciens (the callus on carrot is typical for At inoculation).
In the second picture of yours I do see roots.
As soon as roots develop after infection with Agro.rhiz. you have to transfer ( only them) to medium with antibiotics. Normally ampicillin. And then roots will grow further, if they are truely hairy roots.
Why bother with the callus on the carrot piece!
Linh Nguyen I have used Agrobacterium bv1 ("crazy roots" strains) for carrot inoulation and have got the same results as your test, despite the fact that these strains gave hairy roots after inoculation of cucumber seedlings.
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