Simple tissue culture help

[Linkedlight]

Ok, to help me expand and get a more reliable source of clones, I've been trying to use tc techniques. I basically take all the small cuts when I do normal clones and use them so they are .5-2cm long.

I basically use bell jars with the following mix for 1L of medium
5g agar
1ppm IBA
1/2 strength MS medium
20g sucrose

I'd love any advice or insight I could get on this.

Questions:
1. Would coconut water help?

2. How long till the mini-cuttings show roots?

 

[gurnt]

you will be a lot happier if you some ppp in your mix

 

[BagAppeal]

A proper guide on how to do this TissueCulture and a few places you can get the gear would be much appreciated.
The guide should include:
How to do it.
Keep them in storage( for at least 4 months)
Multiplying the TC
And making them grow back to normal clones?
Anybody out there got this guide? Please make that the next post, or find someone to make it!
Much respect to all

 

[Linkedlight]

Thanks for the interest Gurnt and BagAppeal,

For tissue culture gear, I'd suggest phytotechlab.com in the US, not sure about Europe or use himedialabs.com. The gear is going to cost you.

This is a long term investment, which I could very well fail at doing, but considering the research out there, could probably set me up nicely if I could do it correctly every time. Experience is neither a kind, cheap nor fair teacher.

The starting point is agar, IBA, MS nutes+vitamins and a sterile working environment with sterile technique skills.

Now my goal is:
*What makes a good cutting?
*How much leaf material should I trim?
*What formulas work for each cultivar?
*What temps make cuttings work well?
*What pH for the formula?
*Should I add activated charcoal?
*Should I add coconut water?
*How much time between transfers?
*How many small children must be sacrifiecd to the dark gods?

As I get it working or not, I'll post more. If I get it worked out, then I'll do a proper post with pics, formulas, gear, pH, cultivars and temps.

I've done 6 jars and lost one to contamination. I'm seeing signs of callus tissue after 7 days but no roots as of yet. This is basically just attempting to clone very small cuttings.

If this succeeds my next step is to callus stem tissue get shoots, then roots and work from there. That opens a lot of possibilities.

Major drawback is most of the research is limited, paywalled, or Asian*.

*Anyone familiar with the inner workings of Asian universities outside India will understand this point.

 

[BagAppeal]

I would think a laminar flow hood class 2 would guarantee a contaminant free work environment at least.That way, one your TC material is cleansed and ready to start working with it stays sterile.
just a thought..
Will be looking forward to all future information!
Make this info available for everyone.
This would eliminate the need of constant monitored mother rooms I would think??, and at the same time make it much easier for those clone only strains of the new world to get sent with ease into europe!
By the way, couldnt google translate solve your problems with all the info beeing in asian??

 

[Hydro-Soil]

There are a lot of DIY's out there on laminar flow hoods.

We use a lot of fans in growing that would work well in that application. Ducting, couple high-micron furnace filters from the hardware store...

It's easily doable on a budget and makes things a loooooooot simpler.

Stay Safe!

 

[BadMojo]

Adding coconut water can be beneficial. It seems to be a nice mixture of auxin and cytokinin with lots of amino acids.

 

[Linkedlight]

@Bag Apeeal-That the idea of getting rid of mother rooms where each crop provides clones for the next one with tissue culture. I'd still keep a few back up moms because once you find a pheno that just does all you want and then some, you want insurance. Posting clones would also be quite easy as well.

It's also not a matter of translating the articles from Asian Journals since they tend to be in English anyway. It's the fact that they just make the whole thing up. They decide on a project and it's outcome and then fill out the numbers with sometimes never entering a lab at all. Some professors really don't know how to use some of the machines in their labs. The professors make a lot of money and get a lot of prestige and job security out of publishing along with their university. Since almost no one checks, it's rampant fraud all around.

@Hydro-Soil- I'd say if you can make a still air set up and get pressure cookers that hit 15psi then everything becomes really cheap. You'd have a slightlyhigher contam rate as the trade off. It's unfortunately not to be for where I live. I'd strongly recommend using a heated stir plate for agar. It makes everything a lot easier.

@Badmojo- Coconut water is unquantifiable which is why they tend to disdain the use of it when doing research papers. Saying it works but we don;t know why just isn't a big selling point. One of my friend ssuggest adding NAA and dropping the coconut water and seeing what that does.

 

[BadMojo]

Coconut water works by balancing root and shoot growth. Some people use it instead of growing roots and shoots more or less separately. The advantage coconut water has is that you do not have to worry about shoot or root initiation failure later in culture. The Side effect is that shoot/root growth a slower than what either would be independently. Its like anything. There are tradeoffs.

 

[chef]

I just got my copy of "Plants from Test Tubes: An Introduction to Micropropagation. 3rd Edition" the other day as a primer. 4th Edition is out in July.

Building a hood later this week.

Still sourcing the rest of the required products, media, hormones etc.

Cool beans!

 

[MassSuperSkunk]

Subscribed.

 

[BadMojo]

I will vouch for Phytotechlab and Caisson Labs. Caisson is for seriously dedicated people who know exactly what they are doing.

 

[chef]

Been thinking of this to get started...
http://shop.billberryfarmstissueculture.com/BBF-Plant-to-Perfection-Set-1-2-bbfgset-1-2.htm

 

[Linkedlight]

@Badmojo- You're right about using cocnut water. I checked with my guy who advises me and he said 1.5ppm IBA, .5ppm NAA and coconut water. Luckily coconut water is cheap as chips.

Phytotech labs is solid.

Plants from test tubes is a good book. A bit irritating to use. I'd definitely like to see a more hands on approach. Considering how much trial and error you have to do to get it set up.

Hopefull we can crack this in the next couple months and help people.

 

[BadMojo]

I have found that there is no accurate book that describes tissue culture in a laymans terms. Plants from test tubes is a more like an advisory on how to set up a commercial lab.
I found that the information is all there on the net though. You just have to take the oblique approach to finding it. Check out Mycology Forums for information on Cleanliness and laminar flow hooding.

 

[BadMojo]

The biggest problem for amateurs conducting tissue culture is not the culture itself or the contamination. It is the moisture super saturation itself. The tissues become used to 100 percent humidity and do not wish for it to change and end up dehydrating themselves. The actual mechanics are far more technical because it also involves weak cell walls.

 

[gurnt]

I have f... with it but no luck
I am going to try bonsai Mons and try to root just 1" cuttings so I do not have to Wat for 5" cuttings
caned coconut water has been a down fall for me

 

[BadMojo]

I could understand that. You have to use the Fresh or Fresh coconut milk that has been frozen. Heat bad.

 

[gurnt]

do not get rip off

do not get rip off

do not not not buy the kit on E bay for $200 are more you are buying 3 types of bottles in the kit just use baby food bottles with metal caps and get $30 of ppp mpa ..........

 

[BadMojo]

It is better to research even if it takes a long time and piece together a kit from individual items that matches your needs. Yes this method costs more but you do not end up feeling conned in the end...