Micropropagation, tropical trees, Mitragyna Speciosa

[shonman]

Ma Huang seeds, batch one, agar too thick....not seeming to sprout at all...maybe I will place these on the heat mat and see what happens.

 

Edited May 24, 2013 by shonman

[shonman]

Ma Huang stem, from first batch... I guess a couple of things did make it so far, from the first batch, after all

(too hard agar, homemade medium)

Edited May 23, 2013 by shonman

[shonman]

This Sinuichi explant, (batch 2, watery medium) has turned brown under the agar, but is still green on top

Dead?

[shonman]

I was hoping this B. CAAPI explant which started growing axillary buds would make it....I am not sure....but I think it dried out/ died

(first batch, medium too dry, homemade)

[shonman]

Here is how I am sealing these....a jar lid, with a hole from a nail poked through in the middle,

... wrapped with some tinfoil on the lid, to keep out contamination....

then, wrapped in a bit of plastic wrap, not covering the jar....

then sealed weith electrical tape.

Following which I place the whole thing in a small, clear sealable sandwich bag,

place on utility shelf under florescent lights, 16 hour day/ light

(Edit...I did the math, turns out they were set at 18 hours/day light....)

Edited May 24, 2013 by shonman

[shonman]

This B. Caapi explant, batch 2, watery medium, still seems healthy....has axillary buds growing a bit, the top meristem is still alive too....

[shonman]

Here are some T. Peruvianus seeds, (batch 2, watery medium)....the agar has started to solidify a littlke more.

Not sure if the floating particles are contamination, or came from the seeds (maybe both)

[shonman]

And ending todays posting with a happy little tree (as Bob Ross would say) I rooted the way I usually do them.

These are small as it is!

They usually survive fairly well, although since moving to a new place with ancient carpeting,

I have had difficulties with fungus.

Now I use sterile tech. to propagate these, too.

It is going better.

So far, I have 'wasted' perhaps 20 or so cuttings in the name of science, hoping to learn micropropagation.

As some might say, you have to crawl before you can walk,

or, you fall off the bicycle many times, before riding well.

I do enjoy this sort of thing, though!

[Darklight]

Hey wow, very lovely of you to keep the thread running. Looks like you've had some successes

May I suggest next time rather than leaving an air vent and covering the plates, you cover the hole with 3M tape ( it's autoclaveable and can be re-sterilised a few times )? That will stop the contam getting in and you can allow your plates to sit in the light. You can also do this with individual re-usable tubes- it works really well

Venting the lids isn't compulsary and can lead to probs. Ideally you are subculturing every 2-4 weeks at the early stages, well before the plant runs out of useful gasses

OK- feedback- here goes:

You're doing really well and I appreciate the feedback. Your record keeping habit is going to serve you well in the future

The PH was not checked on my first attempt, using the home made TC media with coconut water.

Excellent that you noted that! It doesn't mean it couldn't work anyhow. Do you have any pH paper pH 4-7? If you are prepared to sarcrifice an uncontaminated plate or fresh plate you could lay the paper down on it for a few seconds and get a rough reading for your records ( and ours too )

The plant in post #46 does look a bit dead ;) Looks like bacterial contam, or necrosis, which can happen anyhow post sterilisation. May I recommend that next time you cut almost all the leaves off pre-sterilisation? Extra surface area does tend to make more places for contam to live and increases the likelihood of contam, and the plants usually receive sufficient energy in the TC media as to make losing their leaves OK until they need new ones

The plant in post #47 looks contam with a yellow bacteria, it's hard to see from the pics, but is there any discolour radiating out from the base of that one? ( Taking pics of TC is *hard* like you said, and condensation can make many angles unviable )

The end of the kratom section in post #48 is too long IMO, the norm is to cut internodes about 1/8-1/4" from the node ( bit where the leaves come out )- depending on the size of the stem of course. Longer nodes could also be useful of course, but the extra length does tend to throw results out metabolically in-vitro and can give odd results in different species ( which can of course sometimes be useful ). Kratom tends to propagate well conventionally and IME the conventional cutting of stem sections in this species works well

Post#49, cut it again above the node at the next subculture and it could be fine

Post #50- contaminated with bacteria. Deflask and grow on if it still is viable

Post #51- contaminated, sorry. It happens. Sowing them in smaller groups helps prevent this. It's a pain in the arse but it does work

Post #52- again, contaminated I reckon. I haven't heard of anyone doing Ephedra from ax nodes but that def doesn't mean it isn't worth trying

Post #53- dead. Contam.

Post #56- woohoo, looks good!

Post #57 Can't really tell. Have the bubbles started to expand and move after a few days? If so, bacto. Sow more thinly next time

Are you guys moving into summer over there? Spring and summer ( periods of active growth ) are the absolute best time to start your cultures from nursery or field stock as the plants are metabolically inclined to push out new growth anyhow, ahead of contams

So far, I have 'wasted' perhaps 20 or so cuttings in the name of science, hoping to learn micropropagation.

So far you have shared the results from 20 or so cuttings with thousands of people and encouraged them to understand that this process is for everyone. I reckon you win

I really hope you get at least 2 clean TC cuttings from your first starts. Field and nursery plants are especially difficult to work with and get into culture sterile- and moreso out of season. It isn't unusual to get only 20-30% sterile success rate from ex-vitro field and nursery stock even in spp with successful protocols ( one of my latest efforts with a known recalcitrant spp was 80% contam and 20% necrosis- it happens ). But hey, if you have a good protocol and good tek, you only need one sterile plant to get you going

Good luck and thanks again

[kadakuda]

man, thats pretty cool. i usually give up pretty fast after contam, nice to see it stuck with. i think you have motivated me to get back into it soon. pretty cool area of science. like the ephedra, thats one genus i have had 0% with.

[shonman]

Thanks for the advice Darklight, and the encouragement, Kadakuda

....I will get that tape that can allow the culture to breathe.

I have not sub cultured anything yet, just waited to see what lived, how long.

Will do that too.

Test tubes certainly would be great for this.

It is spring here, going into summer.....last frost was supposedly a week ago, but I never trust that.

The plants have been, and still are, inside under 1000 w HPS on a light rail light mover.....

The excellent chrome plated utility shelves are on each end of where the plants are, under florescent lights.

Timer, 18 hour day....i thought 16, but it is set at 18.

I have been washing all plants (not in jars) by spraying with chlorine solution ,

Then 'pressure washing' with a pump water sprayer,

once a week to set back bugs, fungus etc.

They have been inside for at least six months or more.

It would be good to get some kratom seeds and try that way, as you suggested.

These usually have a viability problem.

I know we are focusing on mitrgyna and a couple others here, but the first step, is just to get a sterile culture of anything!

Edits are usually me correcting typographical errors.

I am a good typographer, but not a fast typist.

Edited May 24, 2013 by shonman

[shonman]

Batch three coming soon....I am going to keep doing this until it works.

Then I am going to do it some more!

[shonman]

Ok, batch three medium has been mixed, placed in jars, and sterilized with the pressure cooker...
Here are the notes.....from mixing up this batch to put into jars.

......
Micropropagation batch 3 notes.....I will work on getting the porous tape and perhaps some test tubes for next time


Media
Prepared 1/2 strength media 
Added a bit too much water
Agar not dissolving entirely,
Might be best to add a bit to each jar one at a time, as per Carol kitchen T.C.
Stirred thoroughly before putting 5 tablespoons solution in each jar.
Stirred each time
Filled them just under one inch full
Made 21 jars
Pressure cooked jars, with tin foil over lid.
Sterilized gallon ziplock bags with alcohol
Swirled jars to mix a bit more while hot
Placed jars into bags after sterilizing jars
Bags are inside plastic container to keep dust etc out
........
Darklight, (or anyone else with suggestions too)
Could you summarize any suggestions regarding taking the next batch of explants,
and any other suggestions?
I will not take ones with leaf this next time, just bud.....close to nodes....maybe with meristem starting
...also, could you please tell me more about subculturing every two weeks?
Maybe this is why some plants from my first batch died....that were not apparently contaminated.
So far, my goal has just been to get things to love without being contaminated.....some have made it....
So far.

Here is a picture of my improvised 'clean box'

Edited September 14, 2017 by shonman

[Darklight]

Ok, batch three medium has been mixed, placed in jars, and sterilized with the pressure cooker...

Here are the notes.....from mixing up this batch to put into jars.

......

Micropropagation batch 3 notes.....I will work on getting the porous tape and perhaps some test tubes for next time

Media

Prepared 1/2 strength ( mccowns) Woody plant media

1 tablespoons sugar per liter

Distilled water, almost 4 cups / one liter

Added ingredients to three cups distilled water, stirred, added more water.

Mixed thoroughly

Did not add agar yet

Rinsed ph meter in distiller water,

Tested ph of nutrient solution.

Ph was 5.6

A range of ph five to six is preferred, so it is ok.

Added a tiny bit more distilled water,

Placed media (without agar) into electric coffee pot.

Heated up media until steaming hot.

Poured into stainless mixing bowl.

Mixed in 8 grams agar

Stirred alot, poured into measuring cup a bit at a time

Added a bit too much water

Agar not dissolving entirely,

Might be best to add a bit to each jar one at a time, as per Carol kitchen T.C.

Stirred thoroughly before putting 5 tablespoons solution in each jar.

Stirred each time

Filled them just under one inch full

Made 21 jars

Pressure cooked jars, with tin foil over lid.

Sterilized gallon ziplock bags with alcohol

Swirled jars to mix a bit more while hot

Placed jars into bags after sterilizing jars

Bags are inside plastic container to keep dust etc out

........

Darklight, (or anyone else with suggestions too)

Could you summarize any suggestions regarding taking the next batch of explants,

and any other suggestions?

I will not take ones with leaf this next time, just bud.....close to nodes....maybe with meristem starting

...also, could you please tell me more about subculturing every two weeks?

Maybe this is why some plants from my first batch died....that were not apparently contaminated.

So far, my goal has just been to get things to love without being contaminated.....some have made it....

So far.

Here is a picture of my improvised 'clean box'

Hey mate thanks for keeping us up to date! Most ppl don't bother, and it is really really helpful of you and encourages others

First thing I'd recommend is getting access to a digital scale, even a cheapie. Even an 0.1g-50g +/- 0.01g can be useful and I gather they're cheap in the US

Tablespoons aren't much god at measuring weight, in case you want your experiments to be replicable next time

Subcultures are done every few weeks to get fresh nutrients to your plants ( and hormones too if you use them ). You can also do frequent subcultures at the start- it's not compulsary- to get your plant moving away from any contamination or phenolics that might leach from the cut ends into the media.

Phenolic leaching is usually visible in the media, a blue/brown fog usually, at the cut end of your explant. If you see it early on, do move your plants into fresh media as they can interfere with nutrient uptake and poison your cutting

You can totally take cuttings for TC with leaves on, however I'd cut the leaves near the end of the petiole ( bit that holds the leaf on ) before sterilising, and cut again close to the stem *after* sterilising and before transplanting. You may have noticed that if you leave the cut ends on after sterilising with bleach solution they can go white from the cut end back. This is called chlorosis.

Leaving the chlorotic/ potentially chlorotic bits on can

• sap the vigour of your cutting, because your plant is still trying to stop the damage
• cause secondary contamination, by weakining your plant's resistance because it is fighting more than one battle and is dealing metabolically with dead tissue
• waste your plant's energy- chlorotic parts rarely regenerate

I'm so, so glad you have some sterile explants ( cuttings ) from your first batch- congratulations and thank you!

[altenate]

Awesome info Darklight & Shonman.

Its inspiring & Iam learning as I go, so Thankyou.

Just a thought & I am just starting out on the whole media batch making thing. (still in the research stage & getting media organised)

The only thing I have managed is deflasking at this stage but its still early days.

& still waiting to see if the plants take to their new media.

(BTW thanks DL for the acacias)

Instead of sugar in Shonmans media mix could that be substituted for say a barley malt extract?

Besides sucrose & some other ose's it contains B vitamins.

I have used this for edible mushroom spore cultures ( shiitakes/oysters etc) in an agar blend & had vigourous response in regards to mycellium growth.

I am guessing there can be some cross over of receipe ingredients.

I guess I will find out soon.

Thanks for the info in this thread.

[shonman]

Thanks Darklight and Alternate!

Will do.

I have been removing the chlorine affected pieces in the way you mentioned the last couple times.

I also so this with my cuttings now.....

I have noticed, on some cuttings in the past, the stem died and turned brown up to almost the last node,

Where the stem meets my rooting mix (coco coir, sand, perlite)

Could that be due to chlorination?

It doesn't seem as bad recently....a bit off topic, but still relative....

Have acquired a good digital scale, so in the future I can use/ give precise measurements as opposed to tablespoons.

Also, I have noticed that the 1970s "shag" style carpeting is truly a hideous jungle of small mites,

Fungi, and things I cannot even see....I am disgusted but it is difficult to move now.

I am taking my plants back out side and hardening them off for summer here.

The HPS light is making the stems elongate a bit much, but also branching our in incredible ways.

I like to keep nodes close together.

The sun is truly a force to be reckoned with...God? Might as well be, for all practical purposes.

It also adds mich vigor to my cuttings when they root.

Back to the point of micropropagation....

Will my explants be affected much by putting the motherplants outside in a makeshift greenhouse?

They take up a space about 6 x 10 ft.

That is maybe 2 meters by 3.33 meters...for you scientific metric people who use measurements that make sense, based on units of ten as opposed to base 12 measurements.

The motherplants are onside now, but inside is disgusting in it's own right,

From a sterile tech point of view...should I keep a couple inside to take explants?

Bugs are actually MORE of a problem indoors, too, surprisingly.....

Edited June 2, 2013 by shonman

[shonman]

I am transferring some plants from batch 2, and starting more jars as well....

Here is a B. Caapi, before transfer......I cut off some of the stem...after I transferred it,...it started to turn brown.

[shonman]

hit the send button twice on some of these, got duplicate posts...cant seem to delete, sorry!

Edited June 19, 2013 by shonman

[shonman]

.....

Edited June 17, 2013 by shonman

[shonman]

This time, I changed the lids, used the micropore tape as suggested.

I think this does make a big difference in the amount of light the jars get, inside.

Excellent suggestion, Darklight!

I dont know how anything lived before, now that I see how well this works.

I am a bit concerned about things drying out more this way, though....

Edited June 17, 2013 by shonman

[shonman]

Here is a kratom plant that grew roots, and a top.....

It was a bit long, the new jars were not as wide,

so I cut the middle of the stem out, keeping root and top...

Then, here it is after transfer to a new jar....I tried to submerge it in the agar as much as possible....

Shown next to it, are the roots it grew.

Edited June 17, 2013 by shonman

[shonman]

Will add more photos very soon, have to go for now......

[shonman]

Here is one I am also counting as a success, (so far)...

It has three branches and tops, with little roots....the very top is opening up to reveal new leaves!

When I took the most recent batch of explants, I went for ones that might develop like this one is now.

I have ordered special containers that allow better photography,

and rigged up a spot with a small tripod and light.

Today I also labelled the containers better.

Now they can be tracked through different photographs,

and will be easier to refer to by their ID....date, batch number, and letter.

This one, is from batch #2, planted in agar on April 6.....just recently got around to transferring it to a new jar.

Its reference ID would be something like this....K(kratom) 2(batch 2) A(plant 'A') 4/6 (planted 4/6) ...

Jar 6/4 (transferred to new jar on 6/4)

like this, written on the lids....

K(2) A 4/6---6/4 Jar

Edited June 19, 2013 by shonman

[planthelper]

I have ordered special containers that allow better photography,

and rigged up a spot with a small tripod and light.

get a cheap digital camera, and upgrade the focus and quality of your photos.

sorry mate, please get better pics, all well, lucky you, DL took care of you.

[Darklight]

Here is one I am also counting as a success, (so far)...

It has three branches and tops, with little roots....the very top is opening up to reveal new leaves!

When I took the most recent batch of explants, I went for ones that might develop like this one is now.

I have ordered special containers that allow better photography,

and rigged up a spot with a small tripod and light.

Today I also labelled the containers better.

Now they can be tracked through different photographs,

and will be easier to refer to by their ID....date, batch number, and letter.

This one, is from batch #2, planted in agar on April 6.....just recently got around to transferring it to a new jar.

Its reference ID would be something like this....K(kratom) 2(batch 2) A(plant 'A') 4/6 (planted 4/6) ...

Jar 6/4 (transferred to new jar on 6/4)

like this, written on the lids....

K(2) A 4/6---6/4 Jar

WOW! I am impressed! And happy for you! Thanks for keeping us up to date with this!

Photography and tissue culture are a hard mix. Half the time you can't get a clear picture because of condensation on the jars

You don't really need a bright light, just a steady camera. I just use my camera phone these days and make sure my hands are resting on something solid to help with the focus

Plants don't need to be suspended in agar, I usually only do that in media containing PPM for a couple of days straight after they have been sterilised. After that you can treat them like normal cuttings and only leave the parts of the stem below the agar that you want to have roots on

The first photo in post #73 has a dark spot at the front- it's not clear, is it fungus? That happens sometimes, comes in thru the top of the jar if they aren't properly sealed on. If it's fungus, get it out of there before it hurts your kratom! It will eat it in a few days!

The first photo in post #73 looks like your kratom is almost ready to be split into two!

Second pic: is that 3M tape over the hole? The mesh looks a bit large- it does need to be small to keep out the contam

I reckon you have more than one success there from all your pics. I hope I don't sound critical, because I'm really happy for you and grateful for your followups. Like your record keeping numbers too