Micropropagation, tropical trees, Mitragyna Speciosa

[Darklight]

I will start keeping better notes.

It is annoying to have one particular technique work out months later,

Then not remember what you did with that one!

It happens to all of us, I struggle to keep on top of it but it's worth doing, especially if you read them before you start your next one ( note to self )

A couple things I was wondering about......

1) how important is the dark cycle for the plants in jars when starting out?

Is it alot better than 24 hour?

Depends on the species and culture type. And I haven't done any controlled experiments on this, so I can't accurately say. Sometimes things have gone wrong with the light cycler and all I do is hope I found it early enough so the results I get have some sort of consistency

Dark is an important part of the growth cycle for a lot of species, 16/8 is optimal for most species in culture, but also a practical consideration inasmuch as once you have a controlled environment set up you try and work within it's parameters

Light quality and spectrum is important- remember to change your fluro tubes every 3 months or so as after 3 months they still throw light, but the amount of the Photosynthetically Active Radiation spectrum ( PAR ) is diminished and some species will definitely suffer as a result

2) are plants grown sterile in Tissue Culture/ jars..

Which almost by definition would be disease and pest free

Be easier to export or import between countries where of course, they are legal plants?

Plants in culture are *functionally* sterile, they may carry contanimants which are suppressed by plant growth metabolism ( cryptic contaminants ) but generally they are preferred to import because of the ease of detection of overt contaminants

However-

Plants can carry viruses of concern to the importing country. Virus free tissue culture is possible- many species have protocols for this. I'm not sure what the certification process is as I've never dealt with it

Many places have restrictions on importing cultures whose media contains additions which could mask contamination, such as antibiotics or PPM

[planthelper]

I try to answer but, I have no tissue culture experience, but I am sure dl, would say something if I get it wrong.

often people say, plants need time to sleep, but it's not true, but many plants have different relationships with light, but fore most this only plays a role when it comes to producing flowers.

one thing, I am sure and that is, that roots prefer it dark, but tissue culture roots are never in the dark...

in short, I would give them some night time, but only around 4h if you want to grow them fast, a longer photo period will mean more growth. this night time would give the plants as well some fluctuations which, they surely love.

if you work on root formation, less light might be better. etiolation, is a power full aid to achive root formation.

and yes, to all you said in question 2! only issue here is deflasking.

but I am sorry to say, that I find your avatar a bit creepy.

edit: dl did reply in the mean time.

Edited April 17, 2013 by planthelper

[shonman]

Thanks all...I guess that first avatar is a bit creepy, I thought about that too.
I was kind if at a loss for some cosmic avatar, so I just grabbed whatever was at hand and cropped it.

Edit-I have a New Avatar now .

Edited April 23, 2013 by shonman

[shonman]

sob, whimper......seven contaminated jars from my first attempt.

maybe ten or 12 left still not contaminated so far as i can tell... yet.

I suspect the contamination was on the plant parts themselves.

Here is a picture of an uncontaminated mitragyna speciosa I placed in agar,

and a Ps. Carthaginensis leaf, as of yet uncontaminated.....

Edited April 23, 2013 by shonman

[shonman]

Mitragyna Speciosa, first attempt at sterile culture

Edited April 23, 2013 by shonman

[shonman]

Psychotria Carthaginensis leaf, without mold...so far

Edited April 23, 2013 by shonman

[shonman]

Psychotria carthaginensis leaf culture, ... lots of mold....

[shonman]

Any suggestions?

I think these contaminants were on the plant pieces,

(especially on this Ps. Poepiggiana leaf, which was not in the best condition to begin with)

even though I sterilized them by cleaning briefly with alcohol and then 10% bleach, 10 minutes soaking time...

This first batch was expected to have setbacks......one was that I forgot to check the PH!

The agar was too hard.

I added different amounts of PPM, and no PPM to some jars.... those are probably contaminated.

[Darklight]

sob, whimper......seven contaminated jars from my first attempt.

maybe ten or 12 left still not contaminated so far as i can tell... yet.

I suspect the contamination was on the plant parts themselves.

Hey, fret ye not. Those numbers are entirely normal and are actually really good for plants taken from nursery stock- especially on your first try!

You have done *really* well- thanks for keeping us up to date with it

[Darklight]

Agar does look a little thick, never mind, you can change it next batch

Contam tends to show up 2-14 days after subculture, the sooner you take pics after it shows up the easier it is to diagnose. Once it runs all over the media it is harder if not impossible to pinpoint the origin/s

Are your fluro tubes fresh? They should be no more than 3 months old

I got the same % clean plants as you from a recent culture initiation. Really, you are fine

[shonman]

Thanks, Darklight!

Will make absolutely sure the florescent tubes are fresh.

There is also a Metal halide on a light mover that gives them light.

I wonder if that would be ok on its own...they seem far enough back.

A couple questions.....

1) can you recommend a good step by step book?

I realized how much more there is to know after trying to piece the procedure together from bits and pieces of info

2) do you wash re use your jars and lids?

3) what happens when you make the agar not dense enough?

I have about 16 jars like that, went too far to the other extreme with the agar.

Maybe it will be ok just to start out with, stick a few stems etc in there and see if they go.

4) I used 1/2 strength Woody plant medium, with one tablespoon sugar per liter.

6 grams agar...too little this time.....any suggestions? 8 grams agar?

5) Is the goal, at first, just to get a sterile plant culture that lives and not worry about rooting it?

And or propagate from stem and axillary bud sections after achieving the sterile culture?

Mitragyna seeds are notorious for not sprouting at all from many sources.....I can see the advantage of starting from seeds.

I have tried to sprout mitragyna seeds a couple of times, with ultimately no success in terms of getting a larger plant.

6) when you take cuttings from the mother plants, is it ok if they soak in filtered water overnight before sterilizing, etc?

7) when you do this kind of thing......if you were using little jars, do you punch a hole in the lid then cover with tinfoil?

And or wrap with clear plastic wrap?

Do you keep jars in a covered case, or out in the open?

I did this:

Used little pint jars from the store for canning

Punched a hole in the lid with a nail

Loosely put the lid on, covered the lid with tinfoil

Placed in the pressure cooker on high for 15 minutes.

Let it cool down, placed in a plastic bin with a lid on it,

Then placed it after cool into gallon ziplock bags to use later, bags inside the plastic bin.

After I place the plant pieces in the jars inside the clean box,

I wrap the jar with the tinfoil in Saran wrap.

I had a ten gallon aquarium in the clean box, and placed the jars in there.

It was laid on it's side, I covered the opening with clear acetate ( left over roll from old art projects)

Then took the whole thing out and put it on the shelf.

I was lazy and just left the flourescent on 24 hours,

But the metal halide and hps lights were on timers.

Both were used individually, at different times....

I had to work on one of them, and my other trees are nearby.

......I not sure the aquarium i placed the jars in is needed,

it just makes it harder for me to see what the plants are doing.....

All the other precautions...tinfoil, plastic wrap, then placing the jar in a clear sandwich ziplock bag,

Seemed like they might be sufficient...what do you think?

Thanks again, I am looking forward to getting this right!

Edited April 23, 2013 by shonman

[Darklight]

There is also a Metal halide on a light mover that gives them light.

I wonder if that would be ok on its own...they seem far enough back.

Ok- usually with plant tissue culture the inclusion of a carbon source ( in this case, sugar ) in the media means the plant only photosynthesises at 10%. Which is why we usually only need fluros.

The reduced light requirement and photosynthetic activity means IMO that we are trying to get the plant to metabolically respond to the media and the components therein, rather than work it out itself using light. One of the reasons deflasking can be slow is that the plant needs to remember how to photosynthysise and make it's own food after being hooked on sugar and hormones

In your case, where you are at the stage of establishing a clean culture, the extra light mightn't be an issue. But further down the track when you start adding hormones etc I'd stick to a couple of fluro tubes about 200-300mm above yout culture tubes

Or take good notes and keep track of your light sources. You could be onto something

A couple questions.....

1) can you recommend a good step by step book?

No, sorry, i don't know of any! Never heard of one! There are published protocols for a lot of species, and if you look around I think there are some step-by-step guides for African Violets and such. They're useful but probably not complete.

I think this lack could be because there are so many side-tracks that people need to know about ( hormone stock solution preparation and storage etc ) that it turns into chaos

Carol Stiff's Kitchen Culture Website may have some step by step guides

I realized how much more there is to know after trying to piece the procedure together from bits and pieces of info

Which is why I think it is totally cool that you have underaken your learning systematically and shared notes. We're all still learning

2) do you wash re use your jars and lids?

Yes I do. Triple rinse them with water after washing and air dry

3) what happens when you make the agar not dense enough?

Sometimes contamination can be hard to detect.

Sometimes nothing.

Agar and other gelling agents can affect what's called osmotic potential, which affects the plant's ability to uptake nutrients etc.

The other thing sloppy media can affect is gravitropism. If the media is so fine you can't orient your plant material in a conventional manner, some species will have to exhert extra energy ( which might otherwise be put into doing something you'd rather it do ) into growing upward. This change of priorities can come at a cost to your work. Callus regeneration and embryogenesis, for example, are protocols which are affected by the orientation of the plant material- you want thos little torpedo shaped clumps to be subcultured facing exactly the same way as they were on the last media so they can put all their energy into regeneration, not re-orientation. It ooes make a difference at that point

At this point I wouldn't worry about it as you're just trying to establish aseptic cultures, and you seem to be doing quite well Just put a standard amount in your next media

Maybe it will be ok just to start out with, stick a few stems etc in there and see if they go.

Yup! Keep notes!

4) I used 1/2 strength Woody plant medium, with one tablespoon sugar per liter.

6 grams agar...too little this time.....any suggestions? 8 grams agar?

OK, the 1/2 strength WPM is totally valid for induction. It may also be relevant for further stages.

One tblpsn- you will completely need to let me know what that is in grams. Please weigh your stuff rather than estimating, wherever you can. It means we can speak a common language

Low sucrose levels can be a good thing during culture establishment as it means that there is less for potential contaminants to feed off

6g could be too little agar. Try 8g/L

[Darklight]

 

5) Is the goal, at first, just to get a sterile plant culture that lives and not worry about rooting it?

 

Yes

And or propagate from stem and axillary bud sections after achieving the sterile culture?

That's one option and the one you should start with IMO

6) when you take cuttings from the mother plants, is it ok if they soak in filtered water overnight before sterilizing, etc?

I wouldn't. It allows things to breed and could weaken your plant. You need to wash contaminants away, not provide a breeding ground for them

It can be a good pretreatment to wash your TC cuttings in gently running water for a few hours before you sterilise them. Gentle agitation in running water which drains away can knock some of the surface contaminants off

7) when you do this kind of thing......if you were using little jars, do you punch a hole in the lid then cover with tinfoil?

And or wrap with clear plastic wrap?

What kind of thing? Actual culture for the plants? Or pretreating before sterilising them for culture?

I did this:...

... Seemed like they might be sufficient...what do you think?

OK, I am now completely lost. Are you saying you sterilised some plants then stuck them in a jar full of media with a nail hole in it? If that hole isn't covered up with something which completely excludes micro-organisms it could explain why you are having contam problems.

Wrapping the whole thing in plastic could be also interfering with the plant's access to light, I have no idea what wavelengths, if any, your plastic would filter out.

But I think you are wrapping them to stop contaminants getting in? In that case, next time grab some 3M micropore tape. You can get it from chemists.

http://mydesk.officemax.com.au/first-aid-medical/wound-management/3m-micropore-surgical-tape-rolls-1530-3-75mm.html

Put that over the hole in your lid before you autoclave your media and keep it there afterwards. You can replace it every few runs and it should be OK. You won't need to wrap your jars in anything further to keep contam out if you have properly sealed jars ( stretch electrical tape round the bottom of the lid where it meets the jar if you don't have proper seals. Ugly things can crawl up the threads on some jars and get into your cultures that way )

Gaseous exchange is a funny one. Given that the standard amount for media per jar is 1/3 media to 2/3 air, it often isn't an issue, even when cultures are fully sealed. However some species don't do well without it. Downside is if you leave your jars too long without subculturing the media can dry out. Whatever you do, make a note of it

The other thing to consider is the access to light your cultures have. If they are close to a white painted area they will get lots of reflected light from your fluros. If you're top lighting them in a non-reflected area and have opaque lids, they won't get as much. This is where a PAR light meter comes in handy. I wish I had one, they're exxy little buggers but very helpful so you can know and standardise the amount of light available to your cultures

In the absence of a PAR meter, your eyeballs are no substitute as they adapt to the amount of light available. I use the standard lighting system for tissue culture, use transparent lids and don't vary my lighting too much unless a protocol requires it. Basically with your light setup it will be suck it and see, I wish I could be more help but I can't. As long as it works for you, great

I am looking forward to getting this right!

Well, you know, you are already getting lots of it right! You are keeping good records and asking good questions. Really, that's half the battle with experiments

[shonman]

Please excuse any typos, I type too fast with two fingers, this is why I edit so much!

What I have been doing these first couple times, was punching a hole in the jar lid,

Covering that with tinfoil, just down the jar, then wrapping plastic wrap over that, and taping.

Then placing in a plastic baggie, maybe overkill.

I will order the tape you mentioned, and use electrical tape instead of blue no stick tape...

I have heard the doctors tape for bandages works well too....

I also got some 'tanglefoot' stick stuff that can be put on the tape,

but that could be a mess if I handled it wrong!

Thanks again for being my guide through the obscure, dark misty and miasmal swamps of beginning micropropagation!

I am enjoying working with it, even without having achieved final results so far.

This is a good sign that it is something I will stick with.

I love plants, and just want to start a nice little nursery with legal, unusual plants and Chinese medicinals.

My typo of tblpsn should have been tblspn..tablespoon.

I will measure from now on...

The seeds, (listed below) will be going into culture too,

- along with more plant matter, within a couple days.

Probably will do the plant matter today as I take cuttings.

I have started pasteurizing my cutting medium and sterilizing the plants, planting under a clean box, etc...

'filth culture' is something I thought I had mastered up to a point in the past.

But being indoors with carpeting has changed things.

...never have I lost so many cuttings to fungus and rot.

But that is changing!

Seeds I will be starting later this week:

(Thanks Jon at Bouncing Bear)

1 - Dream Herb

: One Seed Pack

1 - Kanna

: 20 Seeds

1 - Ma Huang

: One Seed Pack

1 - Mandrake Seeds

: One Seed Pack

1 - Peruvian Torch Cactus

: One See pack

Hmm,,,,,I will have to start using more scientific measurements, for sure.....

That way, I won't have to convert , for example '300 mm' to thumbs or grains of barley....(inches)

From Wikipedia

"In some other languages, the word for "inch" is similar to or the same as the word for "thumb"; for example, Catalan: polzada inch, polze thumb; French: pouce inch/thumb; Italian: pollice inch/thumb; Spanish: pulgada inch, pulgar thumb; Portuguese: polegada inch, polegar thumb; Swedish: tum inch, tumme thumb; Dutch: duim inch/thumb; Czech: palec inch/thumb; Slovak: palec inch/thumb; Hungarian: hüvelyk inch/thumb, Danish and Norwegian: tomme / tommer inch/inches and tommel thumb. Given the etymology of the word "inch", it would seem that the inch is a unit derived from the foot unit in Latin in Roman times."

And

"An Anglo-Saxon unit of length was the barleycorn. After 1066, 1 inch was equal to 3 barleycorn, which continued to be its legal definition for several centuries, with the barleycorn being the base unit.[9] One of the earliest such definitions is that of 1324, where the legal definition of the inch was set out in a statute of Edward II of England, defining it as "three grains of barley, dry and round, placed end to end, lengthwise".

Edited April 24, 2013 by shonman

[shonman]

Edit:

I am sticking to mostly micropropagtion in this thread, and some related info.

Horticulture and gardening under controlled conditions are another thread

Will post more there soon!

One thing that I will say though is:

CARPETING is not your friend in these arrangements!

It is a fungus and germ filled dishrag that can't be thrown away very easily.

Use a space you can clean and disinfect without any problem.

Edited April 24, 2013 by shonman

[migraineur]

Tell us more about your grow room. When I am free of living with others I want a complete room of my own to do this kind of stuff with. Sharing rooms sucks, especially when people bring in contaminates!!! *shakes fist*

[Darklight]

3M micropore tape allows gaseous exchange but it's pores are small enough to exclude most culture contaminants. Not sure about viruses tho

Previously I have advised against using it to close jars and I stand by that- over a long period of time it can become contaminated with fungus and/ or spores, which are released into the air when you take it off the jar and can contaminate other cultures

But I've seen it used in some labs as an air exchange system, like over a nail hole in a lid to allow gaseous exchange. As long as it's autoclvaved before each use, washed properly and replaced regularly it should be OK as it's not a large surface area

[Darklight]

Shonman, do you think it would help if you narrowed down your species range to one or two species to start with?

That's a lot of different cuttings and spp if I count correctly, it will be hard to get a consistant idea of what is working for you with so many variables

[shonman]

Ok, this next batch I will only try a few species, the main ones I am interested in right now.

At is point, I consider it successful in a beginner sort of way,

If any culture makes it through uncontaminated!

Edited April 25, 2013 by shonman

[shonman]

I have started another batch of attempted sterile culture of several plants, most notably, Mitragyna Speciosa.

This second batch, the agar was a bit too runny.

However, after a while, it would seem it solidifies more...over a week or so.

Perhaps some of the water is evaporating.

I decided to do a bit larger sections this time, about 2 inches or so, ( five cm)

Did about 17 jars, some were of other plants, like Psychotria Carthaginensis ( I think)

B. Caapi, Ma Huang seeds again, etc

But mostly Kratom.

Used 1/2 strength Woody Plant Media....

I took cuttings close to nodes, these had two nodes per cutting and a top.

Basicly, doing as I do when I take cuttings.

However, I have noticed, that often the emerging leaves on the top

Which are so great when taking cuttings the regular way...

Seem to often die back to the next node.

The cutting seems to live from there on down longer.

It seems that if there are small axillary buds present, they start to grow.

It seems in some ways, it is beneficial to entirely or almost entirely submerge the explants in the agar.

This time, I submerged almost all of the kratom cuttings/ explants,

Except the growing top...

Some have died back to the first node.

Others, less developed, seem to be doing a bit better.

Next time I take explants, I will focus on having two nodes, on on each end of the plant, or three...

With developing axillary buds being a priority.

I will take these from the section before the several top nodes, which I use to propagate the regular way.

Next batch of media is already mixed up, in jars, and sterilized.

Most of the plants from my first attempt at micropropagation have died.

Contamination mostly, but also some just turned brown and died,

Perhaps they needed more water, that batch of agar was too hard.

The right amount does seem to be about 8 gm/ liter.

Some seeds from the first batch have still not germinated.

I figured the first several times would be mostly practice.

I am learning, and understand that getting it right takes time.

All insights of those who have shed their light into this darkness

of micropropagation tech are very much appreciated!

Photos to follow soon!

This last batch is harder to see, and photograph

because I accidentally picked up canning jars with some #%~¥~texture on the sides.

........

MIcro prop mixture second batch.... notes

This batch labelled 5/6 or 05/06

Agar...6g/L at H 5.8 ...I used six, saw that somewhere online, but it wasnt hard enough

Edit: wrote 8g of agar , but then remembered, this was the runny batch I did before preparing the last jars, which are ready to work with on 'batch 3'...the next future batch

 

Took cuttings, used extra green and vigorous reddish tips.

sterilized, etc. in 10% bleach for 10 minutes

Cuttings were longer this time

also, more centered around nodes.

planted seeds all in one jar each kind, agar very liquidy .

ma hung, peruvian torch cactus, mandrake seeds.

Crammed some Ps. Viridis leaves into agar.

Also a viridis stem, and several caapi.

marority was kratom cuttings.

Stuck them into the agar as far as they would go,

mostly leaving just the growing tip sticking out of the agar, burying the rest.

It is possible the extra watery media will help the kratom cuttings.

Cuttings were mostly placed in pint canning jars, with a hole in the lid.

Sterilized with the agar in the pressure cooker beforehand.

Tinfoil was placed over the hole in the lid.

After plant cuttings were placed into jars,

they were labelled first by writing on the tinfoil in permanent marker.

saran wrap was placed over the tinfoil, taped tightly on the outsode with electrical tape around the lid to seal

then placed in small ziplock baggies to take out into the room.

Edited September 14, 2017 by shonman

[shonman]

This kratom ex-plant (I think its dead now, but not sure) was from the first batch....not enough water, agar too hard.

It seems to have dried out more than been contaminated.

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

 

Edited May 24, 2013 by shonman

[shonman]

Here is a closeup of batch two, watery media, Kratom axillary buds starting to grow

Edited May 23, 2013 by shonman

[shonman]

Here is another (batch 2, watery media) kratom explant, with axillary buds, still alive...

[shonman]

Here we (sort of) see browning on the stem of a kratom explant....batch 2, watery medium.....part alive, but dying back?

I apologize for the quality of some of these photos....My littlle digital camnera is great, but I need to learn to use it better.

Autofocus keeps getting the wrong things. i will figure out the closeup manual settings next time.

I have resized them etc so as not to take up too much space on this forum.

Edited May 24, 2013 by shonman

[shonman]

Another kratom explant, batch 2, watery medium, with axillary bud action.

I hope some of these make it!