Micropropagation techniques

[IceCube]

Here is another interesting journal article with pictures:

If anyone has access, I would like to request the following article please:

Vyskot, B.; Jara, Z. Clonal propagation of cacti through axillary buds in vitro.

J. Hortic. Sci. 59:449-452; 1984.

That reference describes the micropropagation of T. spach so it's obviously very interesting :D

Micropropagation of pitaya ( Hylocereus undatus Britton et rose).pdf

Micropropagation of pitaya ( Hylocereus undatus Britton et rose).pdf

Micropropagation of pitaya ( Hylocereus undatus Britton et rose).pdf

[tripsis]

An excellent book on the topic on a very advanced method of micropropagation, available as a PDF, is the following.

Photoautotrophic (sugar-free medium) Micropropagation as a New Micropropagation and Transplant Production System

Authors: Kozai, T.; Afreen, F.; Zobayed, S.M.A (Eds.)

Publishing: Springer

Published: 2005

The chlorophyllous in vitro cultures can grow vigorously in sugar-free medium by improving the in vitro environment to promote photosynthesis, transpiration and inorganic nutrient uptake of the cultures. Photoautotrophic micropropagation method using sugar-free medium, in which the growth of cultures or accumulation of carbohydrates in cultures is dependent upon the photosynthesis and inorganic nutrient uptake of cultures. This book is an excellent ambitious book that introduces the new propagation method for plant propagation and seeks to provide a comprehensive account of the latest advances in understanding sugar-free micropropagation for high quality transplant production. This is an ideal book that spans topics from physical state of culture environment to commercial application of photoautotrophic micropropagation. The issues discussed include definition, concept, principle, methodology, applications, advantages and disadvantages of photoautotrophic micropropagation. Outstanding features of this book are, 1) it is the first book on photoautotrophic micropropagation or micropropagation on sugar-free medium, 2) most of the authors are acknowledged as the experts in their designated research area; 3) this is a unique book which covers both biological and engineering aspects of photoautotrophic micropropagation and transplant production in closed system; 4) it also covers its concept, basics, applications and cost analysis; 5) it represents an integration of state-of-art, multidisciplinary technologies and knowledge. This book will be an important asset to the researchers and scientists in the field of plant development, plant tissue culture, biotechnology, horticulture, agriculture, forestry and environmental control.

Too large (and illegal) to upload unfortunately...

[tripsis]

Another on micropropagation.

Protocols for In Vitro Cultures and Secondary Metabolite Analysis of Aromatic and Medicinal Plants

Series: Methods in Molecular Biology, Vol. 547

Jain, S. Mohan; Saxena, Praveen K. (Eds.)

2009, XVIII, 350 p. 113 illus., Hardcover

ISBN: 978-1-60327-286-5

A Humana Press product

Given the vital and far-reaching applications of medicinal plant metabolites worldwide, the quality and consistency of the products as well as the very survival of various species are of the utmost importance. In Protocols for In Vitro Cultures and Secondary Metabolite Analysis of Aromatic and Medicinal Plants, expert researchers provide detailed, step-by-step protocols for the establishment of in vitro cultures of key medicinal plants, their mass multiplication in a controlled environment, and step-wise secondary metabolite analysis, genetic transformation, large-scale metabolite production in a bioreactor, and molecular markers. In addition, many of these protocols will provide a basis for much needed efforts of in vitro germplasm conservation or cryopreservation of medicinal plant species at the brink of extinction as well as efforts to protect them from the adverse impact of rapid climatic changes. As a volume in the Methods in Molecular Biology™ series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Comprehensive and authoritative, Protocols for In Vitro Cultures and Secondary Metabolite Analysis of Aromatic and Medicinal Plants is an ideal resource for scientists endeavoring to continue the research on this exciting natural branch of medicine.

both these book are well damned advanced, probably far more technical than the average person could handle, but I still think they could be useful, especially to those who have done micropropagation before.

Once again, too large too upload (and illegal)...

[Darklight]

pH I vote for pics to stay too

I have a few of these papers but no scanner at present and some of them are in books so it's no simple job. It will be a while before I can get round to this

Seriously, while you're starting they are more information than you will need and might confuse or give unrealistic expectations as to what is practicable rather than theoretical. What works in one lab often still requires more pfaffing around to get to work in another than is reasonable. It's the industry secret you only read about in 1% of reputable publications but it's a fact at bench level

Start by practicing the practical basics outlined in this thread. Ask questions based on your work- most people find they have them as they proceed.

Work out your basic techniques first, what you need to proceed for your first experiment. Don't clutter your mind during this, there is a kind of zen to it. Once you have that OK then go the research route.

Lots of ppl here are now experienced in myco tech, which isn't far different to plant tissue culture techniques re sterility theory and handling

Not sure what you have or can get easily and cheaply- but to start you'll need minimum hardware

An accurate balance of some kind for weighing stuff, pref to 0.01 g minimum accuracy. Some small balances will read this but be accurate to +/- 0.01 either side- unacceptable

1ml/10ml syringes without needles for measuring liquid solutions

pH tape of reasonable accuracy ( pH 4.0-7.0 ) or a pH meter to at least 0.1 accuracy

pressure cooker ( microwave sterility has been practiced but I've never done it )pH I vote for pics to stay too

containers for culture media ( some people have used chinese food containers but I find they don't stay sterile real well in varying temps)

Some way to seal your culture containers- nescofilm. NOT 3M tape

Some kind of sterile chamber- a perspex tunnel, glove box or flow hood

Somewhere to grow your cultures at a constant temperature + give regular light supply such as a timed 2 tube fluro light setup

Alcohol lamp for flaming instruments

Sterile petries- glass or gamma irradiated plastic

Forceps and disposable scalpels or disposable blades and good handle

Spray bottles for 70% methylated spirits

Fridge space to hold your perishables

To start learning your tech I'd recommend getting:

10 @ 1L Murashige and Skoog Basal medium with vitamins ( or share a 10L purchase amongst friends )

Agar or other naturally-derived gelling medium ( we'll skip the synthetics for now )

Hormones- BAP, IAA, IBA and Kinetin. You can buy these already made up in solution, they last three months in solution correctly stored so note the date you received them on the label

Coconut water, 100ml should be more than enough

Hope I haven't left anything out. If so I'll edit to backfill. Some of this gear is also relevant to myco work so I'm hoping you don't have to spend big bucks for an experiment

The above chems will give you enough leeway to experiment endlessly with basic theory and practise til you have a greater understanding of the process and the possibilities as opposed to working with extravagant theory and no practical base

[planthelper]

i can't add much to this topic unfortunately but, i would be very interressted in all sorts of DIY glove boxes, tunnels and flow hoods.

i remeber stories of people using cheapish bought air filtering systems (the airfilter was designed for another application) and using those without problems.

i'm a building stuff in the shed person, and would like to manufactor a glass, or perspex, stainless steel or corian glove box. perspex scratches easely, and those scratches might harbour contams after a while, so my thoughs ar to make it out of as scratch proof materials as possible.

i have researched this a bit on the net already, but i you know of inspirational pic's of DIY glove boxes, maybe post them here or give a link.

[Sola]

I'd love to give this a go with some of my plants. This is a fantastic thread, great information.

I'm going to start working on collecting the required material so hopefully I can experiment with this technique.

And as was mentioned some of the materials can be used when doing mycology as well so worth the purchase.

Has anyone had success with Agave tequilana? I hope to be able to contribute more ITF and ask some questions as I'm sure they'll come up.

[gecko]

Has anyone had success with Agave tequilana?

 

No, but I intend to give it a go when i manage to get hold of some.

A.tequilana cultivars that is.... A.tequilana 'Azul', the official one

as well as older cultivars 'Seguin' 'pes-mulae' or whatever I can get really.

also, I'd like to increase the numbers via micropropagation,

of some of my other mezcal Agaves.

A.duragnensis

A.deserti

A.sobria

A.potatorum

A.murpheyi

[tripsis]

Have you done much plant tissue culture work gecko?

I'm still very keen to get into this, but my girlfriend is dead set against me using some dangerous chemicals in the house, plus I'm pretty damned broke at the moment. It will happen at some point in the not too distant future though, especially once I have a laminar flow cabinet, which I'm determined to have before the year is out. So worry not Darklight, our discussions weren't for nothing, I promise!

[Darklight]

So worry not Darklight, our discussions weren't for nothing, I promise!

I never thought for a minute that they were You asked some cool questions and did it logically, I don't mind answering those at all

I'm still very keen to get into this, but my girlfriend is dead set against me using some dangerous chemicals in the house, plus I'm pretty damned broke at the moment.

Order or otherwise acquire the ones she's worried about in a 1mg/ml solution and store them in a sealed tupperware container in the fridge- dispense with syringe. Only downside is you'll need to make more up every 3 months as working strength solutions go off in that time

I'm thinking it's mostly the hormones she's worried about, like 2-4D? Look, seriously, if you even get to the point where you're using 2-4D you can pat yourself on the back because you've come a long way.

99.9% of people stay at the " I'm going to do this one day" phase. Some make it through the first stages of disinfesting their plant material and keeping it sterile in media for two weeks. Very, very few people stick with this long enough to produce five successive generations of well designed experimental media for their species of interest. If you get to the stage where you need 2-4D I'm sure some in solution will find it's way to you

It will happen at some point in the not too distant future though, especially once I have a laminar flow cabinet, which I'm determined to have before the year is out.

Please have another think about this. Getting a laminar flow cabinet is a big mistake if you haven't demonstrated to yourself that you will stick with this and have or can access the skills and consumables you will require to continue your work. Laminar flow cabinets are expensive and most are difficult to move around. If you even get a standard width one you may have to pull some doors off to get one in the house ( expect to deal with unhappy flatmates and partners while shifting them around and placate the hell out of them because you'll need to borrow them to help )

Start with a perspex tunnel or similar. Or borrow time in someone else's flow hood and see how you run experiments. After that if you believe you can honestly justify the hassle and expense of purchasing a laminar flow cabinet, and know you'll use it in a continuance of your new interest instead of leaving it to rot in a shed outside when you fall in love with your next hobby/ life phase- *then* buy a laminar flow cabinet

Oh, and while I'm in bossy mode ( sorry, don't mean to sound bossy )- don't even think about using cheap scales for measuring things like plant hormones.

If you can't source and calibrate a good analytical balance ( 0.001g maximum ) 2nd hand, then get a shotgun reloading beam scale. They're pretty bloody accurate to about 6-7mg and the grains/grams conversion charts are straightforward. Put a standard weighboat at the end, not the copper dish thingy, which does react with some things

Pic at http://www.shopping.com/RCBS-RCBS-Model-5-0-5-Balance-Beam-Powder-Scale/info

[tripsis]

Glad to know you didn't mind answering all my questions.

I'll look into diluted solutions. It is hormones like 2-4D my girlfriend is worried about. Anything potentially carcinogenic, etc. She'll come round to it eventually...

99.9% of people stay at the " I'm going to do this one day" phase. Some make it through the first stages of disinfesting their plant material and keeping it sterile in media for two weeks. Very, very few people stick with this long enough to produce five successive generations of well designed experimental media for their species of interest. If you get to the stage where you need 2-4D I'm sure some in solution will find it's way to you

Well, I study biology, so have a very keen interest in all this to start with. I expect plant tissue culture is more difficult than fungus tissue culture, but I've got experience with the latter and enjoy the work. Every time I read up on micropropagation I have a strong urge to do it. I doubt I'll give up before I've given it a damned good go.

Please have another think about this. Getting a laminar flow cabinet is a big mistake if you haven't demonstrated to yourself that you will stick with this and have or can access the skills and consumables you will require to continue your work. Laminar flow cabinets are expensive and most are difficult to move around. If you even get a standard width one you may have to pull some doors off to get one in the house ( expect to deal with unhappy flatmates and partners while shifting them around and placate the hell out of them because you'll need to borrow them to help )

Start with a perspex tunnel or similar. Or borrow time in someone else's flow hood and see how you run experiments. After that if you believe you can honestly justify the hassle and expense of purchasing a laminar flow cabinet, and know you'll use it in a continuance of your new interest instead of leaving it to rot in a shed outside when you fall in love with your next hobby/ life phase- *then* buy a laminar flow cabinet

Ah, but it's not only for micropropagation I want it for. Remember that I'm a keen grower of edible and medicinal fungi too, which of course requires a sterile workspace for much of the work. I wouldn't mind getting to the level where I could sell some at the farmer's markets, but that will never happen if I'm using a glovebox. As it so happens, I was lucky enough to walk past a laminar flow cabinet being taken from my uni a few days back. I inquired about it and found out there's another one available for $500, so will be arranging to pick it up this week. Already taken all the measurements and worked out where it will go. Won't be a problem getting it into the house, except for the fact it weighs ~150kg. A laminar flow cabinet has been a long term dream of mine and while I don't really want to have to own a massive piece of equipment, my life would be far easier with it, so the time (and opportunity) has come to get one.

Good to know about the scales. I'll keep my eyes out for one on Grays Online. Or better, will ask at uni tomorrow!

[Darklight]

Sounds like you'll get good use out of it

[Nezahualcoyotl]

I can post? Hooray!

Edit:wait.. this isn't right... damn it.

Edited March 3, 2012 by Nezahualcoyotl

[zero]

Has anyone out there done any tissue culture with pines or worked with hard wood?

sorry I know this is off topic.

[Peter_out]

I have a hint for all the budding Micropropagators watching this topic. All the info you need re techniques, materials & machinery is out there in super abundance. You just have to search.

I made my first home made laminar flow cabinet in the 1970s & it served for 20 years. Then I bought a huge 2.4m long double filter job which you could lay a sterile human in (overkill) !

But the thing I wanted to offer is the most effective chemical I found for inducing somatic embryos from undifferentiated callus & other organs: Paclobutrazol it is a chemical used in mainstream agriculture for manipulation of crops in various ways. It is very powerful, quite toxic so some say but very effective. It is apparently a Gibberellin antagonist as Cytokinins are Auxin antagonists & vica versa. It has a remarkable effect on tissue in vitro in very very low concentrations in the presence of rooting & shooting 'hormones' & on its own. Any one who is really serious about propagation in vitro should get some & try it in combination with other growth factors & alone. But remember in very tiny amounts.

Cheers

[Darklight]

I have a hint for all the budding Micropropagators watching this topic. All the info you need re techniques, materials & machinery is out there in

super abundance. You just have to search

 

You are so right. It's not rocket surgery, it takes dedication and good observation and note taking skills. Which many of you already have

Very much closer to having the design stats for the perspex tunnel thingy, a cheapo substitute for laminar flow cabinets. Excellent for those starting out and I'd recommend getting one before deciding whether to spend the $$ on laminar flow. I started out using one

Paclobutrazol it is a chemical used in mainstream agriculture for manipulation of crops in various ways. It is very powerful, quite toxic so some say but very effective. It is apparently a Gibberellin antagonist as Cytokinins are Auxin antagonists & vica versa. It has a remarkable effect on tissue in vitro in very very low concentrations in the presence of rooting & shooting 'hormones' & on its own. Any one who is really serious about propagation in vitro should get some & try it in combination with other growth factors & alone. But remember in very tiny amounts.

 

Ooh thanks! Do you know I'd totally forgotten about this one, never seen it used in any lab but there is heaps of documentation from ppl who do

I'll book it into my schedule for those species which have proved recalcitrant to 2-4D, of which there are a few by now

[Bigred]

can anyone recomend a good book for a beginer on this topic

[chronic]

I have this book. Plants from Test Tubes: An Introduction to Micropropagation

http://www.amazon.com/Plants-Test-Tubes-Introduction-Micropropagation/dp/0881923613/ref=sr_1_1?ie=UTF8&qid=1345560837&sr=8-1&keywords=Plants+from+Test+Tubes%3A+An+Introduction+to+Micropropagation

[Change]

I really miss seeing Tripsis post

[onyeka]

On 1/13/2011 at 11:39 PM, planthelper said:

i can't add much to this topic unfortunately but, i would be very interressted in all sorts of DIY glove boxes, tunnels and flow hoods.

i remeber stories of people using cheapish bought air filtering systems (the airfilter was designed for another application) and using those without problems.

i'm a building stuff in the shed person, and would like to manufactor a glass, or perspex, stainless steel or corian glove box. perspex scratches easely, and those scratches might harbour contams after a while, so my thoughs ar to make it out of as scratch proof materials as possible.

i have researched this a bit on the net already, but i you know of inspirational pic's of DIY glove boxes, maybe post them here or give a link.

Hi planthelper, have you had any experience in using microptopagation/tissue culture techniques with any of your catha?

Onyeka

[migraineur]

On 18/07/2017 at 9:43 PM, onyeka said:

Hi planthelper, have you had any experience in using microptopagation/tissue culture techniques with any of your catha?

Onyeka

 

Planthelper rage quit this forum in 2014, mate. 

[Solipsis]

Hey! I must admit I haven't read the thread yet but will after this.

 

I just had my first Loph seed germinated on MS medium! Next I would like to use tissue explants of rare cacti and do multiplication, I guess using areole activation? TC is amazing

 

Is anyone else doing micropropagation?

Edited October 4, 2019 by Solipsis