Tissue Culture / Micropropagation

I'm finishing up my degree in Horticultural Science in May, and my university actually didn't offer any classes strictly for micropropagation, I just did 2 semesters of independent research with a professor that did tissue culture work. Most universities won't have a horticulture specialization for micropropagation for a bachelor's degree, but some have master's programs with that specialization. University of Colorado in Fort Collins offers one, and they do alot of tissue culture research for the potato industry up there. I'm moving to Denver this summer, so I might end up going for my masters there.

I ended using TDZ and NAA for my hormones with Murashige and Skoog nutrients. The tissue cultures I started last week don't seem to be doing much, but they usually don't start to do anything for a week or two. I noticed that they soaked up a bunch of the bleach solution that I sanitized them with so it might not have been rinsed off all the way before I placed them on the media, so if they haven't done anything by next week I'm going to top my plant again and start some new ones.

I did the whole sanitizing procedure fairly quickly and rushed because I didn't want somebody walking into the lab and seeing me holding a flask full of what was obviously cannabis leaves and stems.

Next time I do some, I'm going to wait until later in the day when I know most people have gone home for the day.

 

I remember seeing that thread back when he was putting his grow box together, very well put together micro grow.

Hopefully I'll get a media mix together that will allow me to go around and collect genetics in Colorado when I move there this summer and put together a nice collection. The benefit of doing micropropagation is that I could gather 100s of strains and they wouldn't take up very much space at all, then I could just cycle out different strains in future crops.

So far my tissue cultures haven't really done much. The ones I started from auxiliary buds and shoot tips look like they might be starting to form new shoots growth, but I'm pretty sure the leaf cuttings aren't going to do anything. I haven't had much luck with leaf cultures in any of the plants I've micropropagated. I think this is because I've been focusing mainly on trying to induce shoot formation and avoid a lot of callus formation, but with leaf cuttings you need the dedifferentiation and callus formation before you induce shoot formation.

I'm going to try a slightly different approach tomorrow and start a few tissue cultures from shoot tips. I'm going to try and cut the shoot tip down to pretty much just the meristematic tissue, because those cells are the most embryonic and more readily divide and multiply. I'm also going to reduce the sanitizing solution from 20% bleach (which is what I'm used to using on seeds) to 10% Bleach. I think I might have overdone it a bit on the first samples and killed them with too much bleach.

Then next week I'm going to reformulate my media mix to induce callus formation and try some leaf cuttings again.

 

The media mixture contains a powdered nutrient salt mix dissolved in it. I'm pretty sure that the plant cells absorb the nutrients by diffusion directly across any surface that is touching the media and then the nutrients are distributed to the rest of the cells. So if the plant was suffering from a deficiency when you took the samples, then yes it might affect the initial growth of the tissue culture compared to samples taken from a completely healthy plant, but I think it will correct itself and recover quickly once they are in the media.

I started some more cultures on Monday. I filled the rest of the jars I had prepared from last time with the same media mix, and I also started some in a different media mix with BA and IAA as the hormones.

This is what my plant looked like before I cut off the two tops.



Most of the leaf samples that I started last time have started forming callus tissue. They first curled up, but stayed green, and then the cells started to swell up and form clumpy looking growth all over the place. Hopefully new shoots will start to develop once enough callus has formed. The shoot tip and auxiliary bud samples I used don't seem to be growing as fast, but they have formed some callus tissue also. I haven't had a single jar that was contaminated yet, so this has been my most successful tissue culture project so far.

Here is a picture of one of the jars with callus growth on the leaf samples.



Here is the lighted shelf I built this weekend to hold my culture jars. I just bought a $20 shelf and $12 two ft fluorescent light fixture from lowes and zip tied the light onto the bottom of the top shelf. Tissue cultures don't need alot of light so you can use T12 bulbs.

 

Hey guys sorry for the long wait on an update. I'm about to graduate from college so I've been really busy lately. Anywho.... so I've had some interesting results with my tissue cultures.

All of the jars that I started from leaf tissue have formed a lot of callus tissue but haven't formed any shoots, which I guess isn't too bad because at least they are still alive and I might be able to subdivide them into jars with increased cytokinin concentration later and induce them to put out some shoots.

Here's a few pictures of what I'm talking about.









I also tried one attempt at home and took a couple of shoot tips and cut them down to just the meristem cells and had some success in the same media with some shoots forming. They haven't grown much in the last couple of weeks so I will probably try moving them to some stronger media later, but I'm just going to let them sit there and see what they do for another month or so. I'm moving to Colorado this month so I'll be setting up a little home lab for experimenting with these more in a few weeks.

 

You could re-sterilize them if you wanted to, but if they aren't showing any contamination after 2-3 weeks then there isn't really a need to. The jar they are coming out of is sterile and if you autoclave the jar they are going into then it is sterile too, so the only way you'd get contamination is your tools not being completely sterile or not doing it in a sterile environment.

Hopefully in about 2-3 weeks I'll start a grow journal of my new grow I'll be starting once I get to Colorado and I'll eventually try and incorporate some of these tissue cultures into that grow. I just ordered a new Secret Jardin 4x4 grow tent and will be buying a 600watt HPS/MH light soon as well.

I've never grown in a grow tent before, do you think I could fit a 1000watt air cooled light in there or would a 600watt be better? My 400watt one I was using for my last few grows crapped out on me a few months ago so I am forced to upgrade which is good I guess.