Growing micropropagated plants larger after rooting, and some general observations

[shonman]

I have been experimenting with micropropagation for a while now...it is quite interesting!!

Much thanks again to those who have lit the path before me, helping guide my steps...

(...Darklight, Carol @ Kitchen Culture Kits, Shaman Australis Forum, and so, Torstein & friends...and the book, 'Plants from Test Tubes.)

Dividing and multiplying the plants is very effective.

They seem to put out roots, in some cases, in 1`.2 strength medium...oir, medium after the nutrients run out.

They can be slowed down bv temperature, I have one batch that has not been transferred for five month!! (at 60F)

Now, I am thinking about growing plants to a certain size, so they can be deflasked by others.

The bigger the better up to a certain point.

What might be the best way to do this part quickly?

How big, is big enough?

Here is what I am thinking:

Divide explants until a certain number is reached.

Root.,...in agar...

Then, transfer to final sales container (any suggestions on these containers would be helpful).

The medium in this final container in which the plant will be sold, would be full strength.

They would have roots when placed in there.

I would keep temperatures maybe at 70-75, and increase light.

Theory being, that the plant would grow faster with light and full strength nutrients.

Any thoughts on this are appreciated!!

Thanks!

[shonman]

Hmm..anyone?

[Darklight]

I have been experimenting with micropropagation for a while now...it is quite interesting!!

Much thanks again to those who have lit the path before me, helping guide my steps...

(...Darklight, Carol @ Kitchen Culture Kits, Shaman Australis Forum, and so, Torstein & friends...and the book, 'Plants from Test Tubes.)

Dividing and multiplying the plants is very effective.

They seem to put out roots, in some cases, in 1`.2 strength medium...oir, medium after the nutrients run out.

They can be slowed down bv temperature, I have one batch that has not been transferred for five month!! (at 60F)

Now, I am thinking about growing plants to a certain size, so they can be deflasked by others.

The bigger the better up to a certain point.

What might be the best way to do this part quickly?

How big, is big enough?

Here is what I am thinking:

Divide explants until a certain number is reached.

Root.,...in agar...

Then, transfer to final sales container (any suggestions on these containers would be helpful).

The medium in this final container in which the plant will be sold, would be full strength.

They would have roots when placed in there.

I would keep temperatures maybe at 70-75, and increase light.

Theory being, that the plant would grow faster with light and full strength nutrients.

Any thoughts on this are appreciated!!

Thanks!

Bigger is not necessarily better with direct sales TC plants.

What you want is a saleable plant which will survive deflasking. A plant with a large surface area is going to be at a disadvantage after a certain size, because it will need to feed so much more of itself and learn to survive contam risks.It is also more likely to damage during shipping

Other points against raising larger plants in TC include- time on the lab shelf which could be occupied by faster growing products, giving you more sales per square foot of grow shelf space. If you leave plants for six months prior to sale and they are six inches tall, you are probably not going to recover that against sale price when compared to the fact that the same space could be used to produce six smallersale plants every two months

Finally, there is a tradeoff between plant size, and the increased risk of contamination when transferring it. More surface area on the plant, more potential for contamination to fall on it, or it to be injured during handling, and longer times in containers which may not be 100% contam proof

I'm a fan on 30ml tubes for individual plant sales. Max 120ml. Polycarbonate, for ease and cost of shipping.

Ta on yr update about cooling plant growth to 60F to maintain longer shelf life. I've tried it in a few species too

[shonman]

Thanks Darklight, you are always an inspiration!

I will get some of those polycarbonate disposable tubes and grow out in those.

They will be sent to a greenhouse I will work with who will harden off, ship, etc to the masses.

[shonman]

Lets say I have 350 plants, ready for division, ...would have to pour medium, etc for about 700 plant after dividing, and I am feeling lazy.

I decide instead, to put 100 directly into magenta GA 7 containers, and divide (or don't) those

placing multiple plants in each GA-7 while a hormone takes effect.

ok, that gets me down to 500 new additional medium needed....

so, say I am needing some cash, as well as feeling lazy and decide to sell 50 that are growing and rooted, or deflask then sell...

.....this eliminates the need for another 100 new medium containers, now down to 400 new units needed, individually speaking....

I decide to divide these, and add hormone to 300, leaving 100 for a source of stable, non mutated functionally sterile plants.....

The hormone effectively grows new shoots from axillary buds. over time, these can be separated and create new plants.

Ok, some things I am wondering:

Any suggestions or thoughts of any kind about these sort of things anyone may be so kind as to share are always appreciated

a) Am I 'screwing myself over' in some way, by removing 50 of the most vigorously growing , rooted, ready plants

from propagation, not multiplying them exponentially as they would have been had I divided them continuously?

c) Is it better to just divide all the plants until reaching a goal of say, 1000 plants,

as opposed to taking a certain number out of the cycle to sell each month??

d) How long can these cultures be divided before needing to take new source material?

e) do the new shoots growing from axillary buds grow faster is left on the main plant, as opposed to dividing right away when big enough?

f) how small of a (micropropagation) plant division, is too small in that it makes the resulting plant grow slower, if this is the case?

Thanks for any thoughts of any kind regarding these sort of thing, which I always greatly appreciate.

I would also add, that combining honey as a sugar source, and coconut milk (from a live green coconut is said to be best)

into the medium seem to increase growth and rooting a bit

Edited April 12, 2015 by shonman

[Tripyamine]

a.) you started from a small amount of stock and now have what you have, so cycling a few out to cover costs etc is just part of it, if you are comfortable with your technique and can keep repeating it then your rate of increase is still higher then your rate of loss

c)like I said you started from a small number now your at 350, doubling 300 is a lot closer to a target goal of 1000 then what you started with

d)until they are broken there is no standard for this, the plants will just start mutating and vital machinery will essentially break, your not really trying to stabilize anything so thats less of a worry

e) there is a latent phase for the plant to re-establish itself

f) if you get the hormones right you can use disk of leaf as the explant, you can even use use the petiole i.e allot smaller parts then the cuttings you're using infact a paper came out on this in 2013 which you can find for free here
http://www.hindawi.com/journals/tswj/2013/209434/ < you will find a great deal of solid info in this paper, but you may struggle going from callus to a new plant as they have not covered that stage, if you wanted to put some decent resources into this, you could get a suspension culture of somatic cells and then possibly devide each one into an individual plant, but then from my basic search it seems the knowledge base is limited on the last few steps for this species, but you maby able to try coffee techniques

[Tripyamine]

have you tried gelrite?

[shonman]

You really know your stuff!

Please elaborate on gelrite?

Dammitt, I broke my Hannah instruments PH electrode taking it back and forth between hot and cold fluid, wont do that again!!

Do you think a plant like the coffee tree, or M. Speciosa, where allowed, could be poropagated from leaf/petiole?

I did get a leaf to root last year, of M. Speciosa, but had been just dividing plants until adding hormone

which causes shoots at the axillary buds this year.

There was an excellent bioreactor ginseng tech, that turned lots of cells or callus into hundred of small rooted plantlets!!

Goldenseal is done from leaves sometimes.

I am starting to work with all three of the above plants...coffee is too common.

Today I am starting some new ginseng (stratified) seeds and goldenseal into culture containing SDICN

(miltons sterilizing tabs in stock solution added to agar).

Although, I am a bit conflicted as to whether I should divide these goldenseal roots into sections with new shoots, and grow those out

or just put the whole root in this medium, then start cloning later with the mostly disinfected shoots naturally sprouting from the roots.

Or just grow in coir, then take cuttings and sterilze those

One actual goldenseal tech, thast I will probably work with, uses leaves.

I am just wanting a good disinfected/ functionally sterile culture to work from.

The ginseng seeds should be easier, I did achieve several growing disinfected clean cultures last year,

but lost them to heat in the culture room.

...sometimes I get overwhelmed, and leave cultures in medium too long.

Questions:

1) How long can these plants stay in medium, before they will stop growing and or suffer in quality in some way?

2) How long does medium made in advance, keep in the refridgerator?

The SDICN medium, says it is good for six weeks storage, and then use.

3) also, if there is a phase where the plant adjusts itself after being divided,

am I further ahead to let it grow while connected, then divide, or just divide right away?

4) what is the best way to make these grow quickly after deflasking?

5) what are some profitable plants to produce via micropropagation?

some suggestions have included: Venus Fly Trap, variegated ginger, and orchids.

and any other info you would share, is much appreciated!!

Thanks!!

Edited April 13, 2015 by shonman

[Darklight]

You really know your stuff!

Please elaborate on gelrite?

I wouldn't bother with gelrite for micropropagation unless you have very particular research needs. It's expensive, not really necessary for most commercial replication, and prone to causing hyperhydricity in many species ( cells swell, appear almost transluscent, and replication becomes unreliable )

If you required absolute control over a media ( gelrite is more chemically consistent between batches, wheras agar is almost a biological indeterminate between agar batches of different origin ) then yeah, go the gelrite if you have a bulletproof protocol and enough material to keep back so hyperhydricity isn't an issue, then yeah, gelrite.

Dammitt, I broke my Hannah instruments PH electrode taking it back and forth between hot and cold fluid, wont do that again!!

I didn't know about that but in hindsight it's logical- ta for the warning!

Do you think a plant like the coffee tree, or M. Speciosa, where allowed, could be poropagated from leaf/petiole?

Theoretically yes, but I've never tried it

...sometimes I get overwhelmed, and leave cultures in medium too long.

Me too. Just make a note of it. And keep enough material back so you don't lose the lot and can start again if needed

1) How long can these plants stay in medium, before they will stop growing and or suffer in quality in some way?

Until they do. There are few hard and fast rules. Most advice is informal from other TC people. I ran generations of Mitragyna til they ran out after about 5 years. Another media formulation could have made them last longer, or a different light and temp regime, or a different starting material. Running regen from generations of callus could have reduced the time or introduced variation and unreliability too

2) How long does medium made in advance, keep in the refridgerator?

The SDICN medium, says it is good for six weeks storage, and then use.

If you've sterilised media in the fridge, the other problem is going to be risking contamination via temp changes, especially if your lids and containers are made from different material and you open the fridge door a lot. Domestic fridges don't have the same temp control as lab grade ones too. My advice, don't store autoclaved media in the fridge and expect it to be reliable

Hormones in media, autoclaved or not, are another storage issue. Some like IAA degrade quickly. Others not so much. IDK the death curve, I have a mate with chemical knowledge I ask about this stuff when in doubt, and try not to store made up media too long at all

3) also, if there is a phase where the plant adjusts itself after being divided,

am I further ahead to let it grow while connected, then divide, or just divide right away?

Eh? I don't understand

4) what is the best way to make these grow quickly after deflasking?

If they are fully acclimatised post deflask, then you need to talk to a nursery person. Sorry, no idea

5) what are some profitable plants to produce via micropropagation?

You keep asking this, you've already got a bunch of species you're working on which look good :D Go and talk to some local specialist nurseries to you.

I would also add, that combining honey as a sugar source, and coconut milk (from a live green coconut is said to be best)

into the medium seem to increase growth and rooting a bit

I did not know that- thank you! Got any data to share for it? Have added coconut water occasionally, but not as a regular ingredient. Never tried honey, have some raw. How much do you add?

Edited April 18, 2015 by Darklight

[Darklight]

...and coconut milk (from a live green coconut is said to be best)

Hehe, it is so so easy, given the infinite numbers of possible media formulations and hormone combinations, to forget that sometimes simple changes offer equally good chances of success

I'd forgotten about coconut water as a possible hormone substitute/ elicitor for rooting, but I have some and I'm going to throw it into my next batch of media for trial rooting mix

Ta for reminder mate

[Tripyamine]

Gelrite is cheap as! Its clear and soft and you can see infection with ease

It also is .3% not 3% so you need very little

http://www.plantculture.com.au/GELRITE_50_Grams/p3017392_13618904.aspx Only $20 for 50grams enough for 150 odd liters of media

[Tripyamine]

This is a good paper - http://www.idosi.org/wasj/wasj2(2)/142-150.pdf

In vitro Propagation and In vivo Acclimatization of Three Coffee Cultivars (Coffea arabica L.) From Yemen Naji Ebrahim, Rida Shibli, Ibrahim Makhadmeh, Mohamad Shatnawi and Abdallah Abu-Ein

Abstract: Micropropagation of Coffea arabica cvs. Oudayni, Hammady and Dawaeiry from Yemen were initiated from seeds. Seeds were surface sterilized and inoculated into media supplemented with different salt strengths and germinated under dark. Seeds germinated on agar medium gave high hypocotyl length, high root length and full cotyledonary leaves expansion after 120 days of culture. Proliferation of these cultivars was experimented on MS media supplemented with different levels (0.0, 2.0, 4.0. 6.0 or 8.0 mg l ) of 1 N6-Benzyladenine (BA), Thidiazuron (TDZ), 6-furfurylaminopurine (Kinetin), 6-(4-hydroxy-3-methyl-2- butenylamino) purine (Zeatin) or 6-( , -Dimethylallylamino) purine (2ip). Highest proliferation for all cultivars was obtained when BA was used at the highest level (8.0 mg l ). Satisfactory proliferation rate in the three 1 cultivars was achieved at 8.0 mg l kinetin and 6.0 mg l TDZ. Zeatin and 2ip were both failed to promote 1 1 proliferation at any used level. Rooting was experimented on half-strength MS media supplemented with different levels (0.0, 2.0, or 3.0 mg l ) of indole-3- butyric acid (IBA), indole-3- acetic acid (IAA) or 1 1-naphthaleneacetic acid (NAA). Highest root number and length was achieved at 3.0 mg l IAA or IBA for 1 all cultivars. Rooted plantlets were transferred to 1 peat: 1 perlite mixture and ex vitro acclimatization gave 100% survival.

[shonman]

Thanks!

Dang it, did my Mitragyna Speciosa thread disappear from the forum, or is it just buried?

I have some pictures of incredible rooting from the combination of honey and coconut..... endosperm shall we say

What is it with sperm and micropropagation? Adding Endosprem, herring sperm.....who thinks up this stuff?

"I know. lets add some HERRING SPERM to the medium, that ought to do it !!"

Next thing you know, I might as well experiment with ancient witches formulas, you know, eye of newt, toad, etc

maybe a few bats boiled in cows milk at the full moon, then bury the medium in a graveyard at Midnight for one lunar cycle, and stuff like that.

HOO KNOWS ? it might work!!

I will write a more scientific post with actual info later

Edited April 20, 2015 by shonman

[Darklight]

Thanks!

Dang it, did my Mitragyna Speciosa thread disappear from the forum, or is it just buried?

Is buried, there are like 3 or 4 good micropropagation threads on here now

What is it with sperm and micropropagation? Adding Endosprem, herring sperm.....who thinks up this stuff?

"I know. lets add some HERRING SPERM to the medium, that ought to do it !!"

Next thing you know, I might as well experiment with ancient witches formulas, you know, eye of newt, toad, etc

maybe a few bats boiled in cows milk at the full moon, then bury the medium in a graveyard at Midnight for one lunar cycle, and stuff like that.

lol yeah I know, I've often wondered stuff like that myself. Some scientific publications read like film scripts once you get used to the flow ( all it takes is practice and experience, you do not need a tertiary education to get to that point )

I remember seeing warfarin added to one media fairly early on, and vegemite to another, and thinking ' poor bastards got desperate, threw in their lunch or the lab rat bait or what was in front of them and hey it worked '- but there is actually some good science behind the reasoning for it. Once something is published that works, no matter how weird, people start trying it themselves and the repetition finesses the known effects of that ingredient and it enters general use.

But who in their right mind was the first person who thought ... yeah, salmon sperm, we need salmon sperm. We have salmon, let's go and wank one until it comes and store it in a jar in the fridge '

Still, formal science publishing etiquette means they take out the bits where you can hear everyone screaming in rage and frustration and fear. Only sometimes will the echoes remain...

Will love to hear of the honey experiments. Was it raw honey or commercial? Honey would certainly be a biological indeterminate, so variable

Edited April 20, 2015 by Darklight

[shroomau5]

Has there been success with TC/MP with genetics that fruit with age rather than being photoperiodic? Is it possible even, or do the clones carry the same hormone that induces fruiting with age?

Or can it be successfully done taking clones while the plant is still in its juvenile growth stage and propagated without the plants fruiting in vitro?

Great work guys btw.

[Darklight]

Has there been success with TC/MP with genetics that fruit with age rather than being photoperiodic? Is it possible even, or do the clones carry the same hormone that induces fruiting with age?

Or can it be successfully done taking clones while the plant is still in its juvenile growth stage and propagated without the plants fruiting in vitro?

It took me a while to work out what you were talking about sry

Bamboo would be a prime example, I have a few colleagues who have worked on it extensively in TC, one said something about flowering at some point but I wasn't paying attention. I'll ask her again next time she is in touch

( Non-photoperiod sensitive pot would be another candidate, but there isn't the same amount of data on it/ I don't know if the same mechanism is involved re. flowering )

[shonman]

Has anyone propagated Mitragyna Speciosa from callus?

I also found some info saying that tryptophan as a precursor, added to the plant, increases alkaloid content...

in this case it was a cell suspension culture, and showed increased alkaloid content after six days

[shonman]

Thanks, all!

Tripyamine...your reference mentioned

"highest proliferation achieved with BA @ 8 MG l (Liter?)

do you think they proliferated from axial buds?

.....put 8 mg into a liter of medium with the other stuff?

also, speaking of weird ingredients and wondering about just how they were obtained, thought up etc,,,

.....has anyone tried female stuff, like salmon eggs in the medium, 'Juice' or estrogen, etc?

I believe women and men are equal but have unique and perhaps complimentary strengths and weaknesses,,, but lets not get into that.

[Tripyamine]

Flowering is a pain in the ass, for most plants the signaling molecule is yet to be elucidated and I believe there are talks of it being an miRNA or a protein

for example if you graft a flower to a plant that is not flowering you can induce flowering so there is signaling involved

Icecube might chime in he has done some work on this

Just forget you ever heard about suspension cultures they just don't yield for alkaloids (mg/l) but you could try upping the levels of tryptophan in your media as it is the precursor in the biosynth but again you kinda wanna be giving it to a big plant not a little tc tube, and then you might have troubles with absorption

[Tripyamine]

I dunno about the weird stuff, some plants have phytoestrogens so I guess it has basis

as for the BAP yerh 8mg/L seems to be where its at but if you read a bit more they talk about 8mg/L BAP with 0.8mg/L IAA and the combination being responsible for the proliferation

If you have the explants to spare you could run a matrix and do like 0.5mg 2mg 4mg and 8mg/L BAP and then 0.5mg/L IAA with 0.5mg 2mg 4mg and 8mg/L of BAP and see what works best

Remember that the paper is for coffee, so its just a rough guideline (it may be wise to sacrifice 1 explant and use the optimal conditions of that paper, possibly half strength too)

[Darklight]

Has anyone propagated Mitragyna Speciosa from callus?

I also found some info saying that tryptophan as a precursor, added to the plant, increases alkaloid content...

in this case it was a cell suspension culture, and showed increased alkaloid content after six days

Nup, never did it back when it was legal and I don't know of anyone who has tried it since

Ax node proliferation was sufficient for the purpose of replicating the numbers of whole sale plants I needed- mucking round finding a suitable media ( there were no other protocols around back then that I could find ) would have been a waste of effort unless there had been an urgent need for more plants

Plus suspension culture, like any liquid culture, is very prone to contamination and can be hard to keep clean, especially for large volume systems where you need to add sterile air

[Darklight]

 

also, speaking of weird ingredients and wondering about just how they were obtained, thought up etc,,,

.....has anyone tried female stuff, like salmon eggs in the medium, 'Juice' or estrogen, etc?

Nup. That's not to say it doesn't happen, or I haven't thought the question through enough to link it to some of the commonly used additives. And don't forget, commercial micropropagation people rarely publish their unique findings- so it's entirely possible

Closest would be coconut water, the liquid endosperm of coconut seed

Ha! Knew there would be something on a rethink. Google Foetal Calf Serum and Foetal Horse serum ( hint- it's ugly ). Not used in plant TC too often, but used in TC regularly. There will be others

Edit: Also add: banana powder, tomato powder, V8 juice. Fruity goodness.

Edited April 22, 2015 by Darklight

[Darklight]

Gelrite is cheap as! Its clear and soft and you can see infection with ease

It also is .3% not 3% so you need very little

http://www.plantculture.com.au/GELRITE_50_Grams/p3017392_13618904.aspx Only $20 for 50grams enough for 150 odd liters of media

Lol gelrite works out at 4x cost the amount for agar. I guess that's a matter of scale. I don't like it's capacity to cause hyperhydricity, but it does have it's uses

Just a quick note- this is *not* a criticism, but where you are typing in decimal amounts for scientific ( or any other ) publication it greatly enhances clarity and translation if you put a 0 in front of the decimal point.

0.3% is clearer visually than .3%, and while they mean the same things, the ease of visual identification the former gives is polite and makes for fewer 3am critical errors- critical where ingredients are toxic when they are increased by that order of magnitude

Am mentioning this because it caught me out once too. I used to think it was trivial. Then a colleague ( a PhD, who should have known better- they ram that ritual into your head early in most science degrees ) scribbled me a media list on a post-it. The decimal point required for one critical ingredient was not visible ( it was listed as 1% ). I did question the note but she said it would be OK. I ended up killing a batch of rare explants

Am a convert to scientific notation. It's a good habit

[shonman]

Thanks again!

I will post more/ask more soon, but wanted to reply to a question asked earler.

Here are some bad photos of the rooting of micropropagated M. Speciosa plants obtained by adding honey and coconut water to the medium.

Honey was from the grocery store, said to be pure raw honey but who knows?

Terrible pictures, really....

I will have to go find the originals and place them on here in higher resolution...

all the pictures are of M. Speciosa from micropropagation projects

In the worst photo, I placed an oval around the roots, which actually lifted these two plants out of the agar

One contaminated looking photo, seems to be proliferation from callus induced by coconut water in the medium,

but that one actually WAS contaminated at that point, so I could never be entirely sure...then went a different direction with propagating.

http://shonman.imgur.com/all/

Edited April 23, 2015 by shonman

[shonman]

One question I have which I did not put very well earlier is this:

Say I have induced shoot proliferation at the axial buds at an increased level with a hormone.

Now, will I get a finished plant quicker, if I let these shoots grow longer attached to the main stem, with the rest of the shoots and the top

and then separate them later, allow them to recover, grow and root.....

Or, would I get them further along/faster by separating these newly proliferated shoots from the main plant when they have a few nodes,

so that they grow into a separate plant on their own, starting as soon as possible, like when they have three nodes perhaps??

I understand there is a recovery time after they are divided, but they would be divided eventually anyway...

do I produce plants faster, by leaving the new branching out plants on the main stem,

or, dividing them sooner, allowing them to recover and grow separately?

Also since this is a 'new' thread as opposed to the buried one about this topic...

I must state for the sake of our all seeing infallible overlords whom we obey at all times without question

that this plant is legal where i AM and should that change, I will not work with it then.

One other thing I have been wondering about...

Is there some way to contaminate plants send out to market, in agar

so they will be fine if 'deflasked' but cannot be used to produce functionally sterile cultures from/ would be contaminated 'in vitro'?

Edited April 26, 2015 by shonman