Cool video! Here are my notes: General Multiplication Tissue Culture Medium: To 800 ml of Distilled water add: 30 grams Sucrose Sugar (for Carbohydrates) 4.54 grams of MS Salts Media (for Micro and Macronutrients) Using a Micropipettor add: 1 ml PPM Plant Preservative Mixture (Biocide to prevent contaminants) Plant Growth Regulators: 1 ml Benzylaminopurine/BA/BAP (cytokinin for shoot initiation) 0.1 ml Naphthaleneacetic Acid / NAA (rooting agent) Then add distilled water to bring total volume to 1L Calibrate the pH to 5.8: (Store your probes in 3 Mol KOH, rinse in distilled water) To go Higher (more basic), use Sodium Hydroxide To go Lower (more acidic), use Hydrochloric Acid Optional; Add gel medium: (more gel medium makes a more firm medium) If using Agar - 7-10 grams depending on firmness desired If using gellan gum 2-4 grams depending on firmness desired 500 ml Half Batch Calculator: Liters of Water: 0.5 Agar (g): 3.5 MS Media (g): 2.27 PPM (ml): 0.5
@@hafsabibi8557 The establishment media is generally free of tissue culture hormones. Whereas the multiplication media contain hormones for organ development. We hope this resolves your query.
@@hafsabibi8557 Check out the concentration and quality of your gelling agent. Read this article to further learn about other factors that cause the situation: www.plantcelltechnology.com/pct-blog/gelling-agents-what-to-look-for/
This is so true that with experience you just hand measure :D When I used to make nutrient water and had to balance pH, I would just pour and do a count "21, 22" measure and be usually spot on!
Generally, your guess develops more accurately while working with the technique. However, we still recommend measuring everything in tissue culture before using it because a small difference can show us as a major mistake sometimes.
In all of your bio-coupler videos, it appears that you are fully sealing the bio-couplers before autoclaving them. Are you loosely connecting them to avoid pressure and breaking in the autoclave?
Hello Fransisco , great videos im consuming them all since several Days. For me as a wannabe hobby wanna be Tissue culture starter i still got several questionmarks. One of them is , what plant material excatly do i add and when can i multiply these , because for me it just sound like you put a node wait till new grow and take that new node , which makes 2-3 Plants. But obviously thats not the quantity people are Producing with Tissue culture so how do they do it ? And also there must be some kind of gathering for atleast some recipes right ? Do you guys have a video or a how to on your side that im missing ? Would be awesome if you could answer my first question in particular. Thank you for your time.
In media preparation you don't need to sterilize equipment, just keep them clean. Because after the media is prepared it will be autoclaved for the culturing processes. Yes, whatever equipment you use during the tissue culture process need to be sterilized.
On the point in the video where you stated now would be the time to add agar, is it safe to assume that it will not interfere with your pH, or should I mix it prior to adjusting pH?
Can you please pinpoint the time for the reference to the question? If you are asking if it's safe to add agar after adjusting the pH of the media, then the answer is yes. However, the pH will definitely fluctuate a bit. If you add agar before adjusting the pH, it can ruin your pH meter.
You can do that. But, normally the pH is adjusted before adding agar. This is because agar has no significant effect on the pH of the culture medium and the method keeps the pH meter clean.
In my experience, the PH prior to autoclave and PH after autoclave varies slightly, and the final PH Pre-inserting the plant and post removing the plant (eg. measurement of leftover media after two months of plant growth). I get if the PH is too high or too low, it would be detrimental, and even affect gelling ability if swung too high or too low, but in general if it's within 4.5-7 pre autoclave, is PH adjustment really necessary? Thanks for your insight.
When you adjust the pH of the media, after autoclave there's might be the possibility of it varying a bit. But. it won't be to that extent that it affects your culture. Generally, 5-6 don't affect much. And, yes you need to adjust the pH pre-autoclave if it's between 4.5-7. It'd better be in the range of 5.5-6. or more specifically 5.8.
@@PlantCellTechnology Thanks for the quick response. Okay makes sense! ! I've seen your video about PH calibration pre and post autoclave before but my results varied when I tried the same. Also I found it weird how overtime, the PH of the media would start dropping. I assume there's some chemical interaction from the plant and the media influencing the PH which caused me to wonder my original thought.
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Cool video! Here are my notes:
General Multiplication Tissue Culture Medium:
To 800 ml of Distilled water add:
30 grams Sucrose Sugar (for Carbohydrates)
4.54 grams of MS Salts Media (for Micro and Macronutrients)
Using a Micropipettor add:
1 ml PPM Plant Preservative Mixture (Biocide to prevent contaminants)
Plant Growth Regulators:
1 ml Benzylaminopurine/BA/BAP (cytokinin for shoot initiation)
0.1 ml Naphthaleneacetic Acid / NAA (rooting agent)
Then add distilled water to bring total volume to 1L
Calibrate the pH to 5.8: (Store your probes in 3 Mol KOH, rinse in distilled water)
To go Higher (more basic), use Sodium Hydroxide
To go Lower (more acidic), use Hydrochloric Acid
Optional; Add gel medium: (more gel medium makes a more firm medium)
If using Agar - 7-10 grams depending on firmness desired
If using gellan gum 2-4 grams depending on firmness desired
500 ml Half Batch Calculator:
Liters of Water: 0.5
Agar (g): 3.5
MS Media (g): 2.27
PPM (ml): 0.5
Wonderful. Thanks for sharing it here in the comments. It will be useful for other culturists like you. :)
@@PlantCellTechnologybut why don't Francisco add BAP or NABP?regulators in media
@@hafsabibi8557 The establishment media is generally free of tissue culture hormones. Whereas the multiplication media contain hormones for organ development. We hope this resolves your query.
With the same proportions my media is not solidifying
@@hafsabibi8557 Check out the concentration and quality of your gelling agent. Read this article to further learn about other factors that cause the situation: www.plantcelltechnology.com/pct-blog/gelling-agents-what-to-look-for/
Can we get more of these? Theyre super informative, maybe one on sterilisation and placing the plants in the media. Very helpful for beginners 😊
We already have some videos on surface sterilization of explants and acclimation. Do check them on our channel.
This is so true that with experience you just hand measure :D
When I used to make nutrient water and had to balance pH, I would just pour and do a count "21, 22" measure and be usually spot on!
Generally, your guess develops more accurately while working with the technique. However, we still recommend measuring everything in tissue culture before using it because a small difference can show us as a major mistake sometimes.
Hey what is the media for monstera / philodendron ?
Can you teach me the protocol for date palm please thank you
Yes
In all of your bio-coupler videos, it appears that you are fully sealing the bio-couplers before autoclaving them. Are you loosely connecting them to avoid pressure and breaking in the autoclave?
Yes, that's correct!
Hello Fransisco , great videos im consuming them all since several Days.
For me as a wannabe hobby wanna be Tissue culture starter i still got several questionmarks.
One of them is , what plant material excatly do i add and when can i multiply these , because for me it just sound like you put a node wait till new grow and take that new node , which makes 2-3 Plants.
But obviously thats not the quantity people are Producing with Tissue culture so how do they do it ?
And also there must be some kind of gathering for atleast some recipes right ?
Do you guys have a video or a how to on your side that im missing ?
Would be awesome if you could answer my first question in particular.
Thank you for your time.
What could you use if you dont have a autoclave?
Instant pot - High Pressure mode...
Should not we sterilize all the equipment before using preparing the medium????
In media preparation you don't need to sterilize equipment, just keep them clean. Because after the media is prepared it will be autoclaved for the culturing processes.
Yes, whatever equipment you use during the tissue culture process need to be sterilized.
On the point in the video where you stated now would be the time to add agar, is it safe to assume that it will not interfere with your pH, or should I mix it prior to adjusting pH?
Can you please pinpoint the time for the reference to the question?
If you are asking if it's safe to add agar after adjusting the pH of the media, then the answer is yes. However, the pH will definitely fluctuate a bit. If you add agar before adjusting the pH, it can ruin your pH meter.
what if i add agfar and sset ph after that?
You can do that. But, normally the pH is adjusted before adding agar. This is because agar has no significant effect on the pH of the culture medium and the method keeps the pH meter clean.
Can you make a video for macroalgae propagation through semi liquid media...
I actually want video Tortorial for it using PES media
Hi Hina,
We aren't working on macroalgae for now in our lab. We are mostly into houseplants, carnivorous plants, and fungi tissue culture.
In my experience, the PH prior to autoclave and PH after autoclave varies slightly, and the final PH Pre-inserting the plant and post removing the plant (eg. measurement of leftover media after two months of plant growth).
I get if the PH is too high or too low, it would be detrimental, and even affect gelling ability if swung too high or too low, but in general if it's within 4.5-7 pre autoclave, is PH adjustment really necessary?
Thanks for your insight.
When you adjust the pH of the media, after autoclave there's might be the possibility of it varying a bit. But. it won't be to that extent that it affects your culture. Generally, 5-6 don't affect much.
And, yes you need to adjust the pH pre-autoclave if it's between 4.5-7. It'd better be in the range of 5.5-6. or more specifically 5.8.
@@PlantCellTechnology Thanks for the quick response. Okay makes sense! ! I've seen your video about PH calibration pre and post autoclave before but my results varied when I tried the same. Also I found it weird how overtime, the PH of the media would start dropping. I assume there's some chemical interaction from the plant and the media influencing the PH which caused me to wonder my original thought.
Ok i learned some new stuff as a complete newbie but the the last ingredients wasnt clear and some links doesn’t work
Last ingredient is 1-Naphthaleneacetic acid (NAA). It's a synthetic auxin-a plant hormone.
And about links: Can you share those that aren't working?
My question is wy did you gave me a none existing address to buy your tissue culture mediums in Johannesburg South Africa
To avoid misinformation, please visit plantcelltechnnology.com/distributors to find the nearest one to you.
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