I was wondering if anyone here has any experience with tissue cultures. I noticed that there is very little discussion of this on the Nexus so I wanted to get a thread going as this could be a very interesting route for growing ethnos and perhaps even manipulating alkaloid content.

I recently received supplies and will be having my go at this. To note I have no experience in any scientific field. Everything will be done in a kitchen environment and logged here, including recipes and methods.

Attached below is a paper listing alkaloid production in Cereus Peruvianus TC.

A quote from that paper: "The ratio of alkaloid concentration between mature plant and morphologically undifferentiated cells of callous tissue was 1∶1.7. A relationship between culture medium containing tyrosine and alkaloid production was also observed in the callous tissues of C. peruvianus".

You may find more discussion of TC on STS.

Presumably using a cactus species other than C. peruvianus could lead to the production of more interesting alkaloids than tyramine or hordenine.

Is formation of non-regenerative callous tissue a general function of higher plants, or are there some species where it's simply not worth bothering?

Thanks, I have seen the discussions on STS, but traffic is so low on that site and the Nexus has a very unique user base that I thought it would be good to bring the discussion here.


I believe so. Is it practical in all species? Probably not.

From wikipedia on callus: "Plant species representing all major land plant groups have been shown to be capable of producing callus in tissue culture".

Below are photos of T. Pachanoi and P. Alba leaves which showed contamination after 4 days. Seems to be fungal and originating from the explants and not the medium.

I realize now it is very important to choose explants which are young and very healthy. The explants I used all had some prior damage from being either torn (P. Alba Leaf), or even having some slight rot (T. Pachanoi). My sterile technique was also horrible and not worth mentioning. I suppose this was more a test of limitations... I am not surprised these contaminated.

Below P. Alba seeds, B. Caapi node, and two T. Pachanoi explants. All put in culture on 23/5.

100 ml 1/2 strength MS with 0.2g sucrose and 0.6g agar. PH was not adjusted as I do not have a reliable means yet to measure pH.

Hormones added: 22.5 uM (5mg/L) of BAP
5.4 uM (1mg/L) of NAA

This was poured into 3 different containers, equating to about 30ml per container.

For medium sterilization, I microwaved for less than 3 minutes as per advice I had received from a knowledgeable TC enthusiast. See attached paper on microwave sterilization of TC medium.

Containers were left to cool in a tub sprayed with bleach and anti-mold solution until lids could be snapped on.

Explants were washed under tap water for 1-2 minutes, then placed in a solution of 3% chlorhexidine gluconate and 3% cetrimide (pharmaceutical antiseptic) for 10-15 minutes, followed by a 3% bleach soak for 5 minutes, and finally rinsed x3 in distilled water.

Notes:
The microwave is definitely messing up the ratios of water:agar. I think the medium is too stiff. I am also skeptical of the short duration spent in the microwave, but only time will tell whether the medium gets contaminated.

Not having a proper pH meter may prove to be a big issue as it will change the stiffness of the agar and affect the plant's ability to take up nutrients.

IME TC is hardly necessary to propagate Psychotria. Most species of the genus that I have attempted readily propagate from leaf cuttings in plain old potting soil but preferably sphagnum moss. Leaf sections as small as a postage stamp will root and shoot as long as there is a vein in it somewhere. TC is just an unnecessary complication.

TC would be useful if attempting to grow thousands of clones from a limited amount of starting material, say if one were planning a plantation or selling plants on a commercial scale.