What is the best ingredient to grow roots in vitro such as Tobacco or Horseradish root in M&S medium ?
What do you think about ( free hormon M&S medium , which supplemented with 30 g/l sucrose) ?
The composition of the medium, particularly the plant hormones and the nitrogen source (nitrate versus ammonium salts or amino acids).An excess auxin will often result in proliferation of roots under in vitro condition. Willow water at 0.1 to 0.8% concentration also helps more plant rooting.
Do you want roost from seedlings or to establish root cultures?
I have already got root from infect the plant leaves by Agrobacterium bacteria by culture them in MS free hormone medium (30g/l sucrose +4.4 g /l MS) .
And now I would like to transfer it to liquid medium and bulk it up to get as much as I can from the roots .
If you want to induce roots from cut plant shoots, for tobacco (mentioned in your question), we just use regular MS medium (+ sucrose, without phytohormones). The roots grow very well. Some people suggest using half strength (1/2X) MS medium for root induction. You can try that too. I am not sure about horseradish's preference for rooting medium.
@ Maha,
Sounds like that you are using agrobacterium rhizogenes for hairy roots induction? And then harvest those hairy roots and grow them further in the liquid medium, to release the recombinant proteins or metabolites in the liquid medium for harvest? Is that correct?
@ Maha,
If you are using Agrobacterium tumefaciens for transformation, when you get putative transgenic shoots, cut it out, and grow them in the MS medium (as described above). I grew them under normal light/dark photoperiod (not 24-hr in the dark). Since some of these shoots can be 'escapees' (not really a transgenic), so, I also add appropriate antibiotic (with reduced concentration) in the rooting medium for selection.
If you are using Agrobacterium rhizogenes for transformation, the optimum conditions for the hairy roots to grow in the liquid medium might be different.
Auxins are growth hormone used generally as rooting media for invitro culture of plant.
I used to grow roots in vitro for nematode races maintenance; just tomato roots. There are protocols in the literature; I used B5 medium or half MS for tomato roots...
You may use MS medium with out hormone and keep for longer period. Stress will induce root development.
Yes I am using Agrobacterium rhizogenes for transformation , and after the appearance of the roots I cut it and transfer it to liquid MS free hormon medium+ sucrose in the dark condition all the time . But the root does not grow for a month ,do you think if I place it in light /dark not only in the dark it will be better ?
We have found 1 week in the dark then in light is good but also charcoal in the media.
I do not have charcoal now and I am afraid of the contamination I do not have time to try it again , so I will leave the roots in MS free hormone medium , and place the flasks in light /dark condition , what do you think ?
Actually it depends on your experiment and which step but generally you can use auxin hormone to accelerate the elongation of root
@ Luis,
I think that Maha has already mentioned her species-of-interest in her question: 'tobacco' and 'horseradish'.
@ Diane Mary Robinson,
When you said that adding active charcoal into the medium, did you mean: when a 'regular' transformation is performed with Agrobacterium tumefaciens?
However, Maha is using Agrobacterium rhizogenes to induce hairy roots, and then grow those hairy roots to a big mass (see attached picture). If she intends to harvest her 'recombinant protein' or 'metabolites' (released from these hairy roots) from the liquid medium, probably adding active charcoal should not be suggested!? Because how will one purify/separate those substances from the active charcoals later? And how efficient will it be to remove the 'ink-like' color and charcoal?
@ Maha,
1. According to these two attached paper, if you are propagating the hairy roots (generated by Agrobacterium rhizogenes) in the liquid medium, you should culture them in 24-hour dark (2013, page 3, yellow highlights). I think it makes sense, roots are not green photosynthetic tissues.
2. I would like to direct your attention to the MS medium you are using. (A). If you are using a commercial MS powder mix, there are tons of different MS mixes in the market or from the same company. It is confusing. You need to be careful to pick out the correct powder mix. For example, there is MS powder with macro-elements only, there is MS powder with both marcro- and micro-elements. Some, MS powder mix has no vitamins, some with vitamins. If with vitamins, some with MS vitamins, some with B5 vitamins. So, you need to sit down and really check out what ingredients in the mix before you purchase it. If you pick a wrong mix, the tissues are not going to grow. (B). In the two attached papers, both used 1/2x (half-strength) MS medium to propagate A. rhizogenes-induced hairy roots. You probably can give it a try. .
Addition of Auxin to media will often result in proliferation of roots in vitro. As , NAA, or IBA or IAA. with increase concentration of sucrose or use 1/2 MS
@Ahmed Al mayahi
Thank you for your reply .
I have read in the papers ,they have used free hormone medium .
And when I did that it did not work , I have got contamination !!
So do you means hormone can increase the hairy roots growth such as NAA, IBA and IAA?
Because I am not sure .if I can charcoal ,how can I get red of the colour of the charcoal latter ?
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