Tissue Culture Cloning

[PigSquishy]

Okay the Magenta Vessel can be put into an Autoclave, and they are said to be a "common choice" to put the explants from the cuttings to grow in and I was going to link you to one of the first sites I found for it in a Google Search, but in the end that experience just proved the very issue of why I am here asking about this very subject. So I looked up the container the one book I have on the Tissue Culture brings up and you asked about, found a site that sell the Kits, individual its for this process, etc... Even says to email the company if you don't find something you need, so all in all it sounds really good. I then did a search on "How To Do Leaf Tissue Culture" and found a .org address for how to do this process, which in turn links me back to the first place where I found that magenta vessel and other things for sale, says the lady has a Ph.D and teaches workshops on the subject and everything else... Really sounds top notch, but the more you dig around the site, it's poorly worded and written and the one link they give to their .org site comes up as saying its not a valid link, but if you do a search on that link you come up with their company which sells you stuff as a .com link... So right there I already feel like I am getting mislead and lied to before I even learned the first thing or bought the first thing from there...

I keep coming up with a lot of these "DIY Home Kits" for Tissue Culture and the issue I have with them is I do not want them just putting Agar into a bottle and calling it "a medium" so you can't run right out and replace what you need, but have to rebuy the kit because they don't tell you what you the name of the product are actually working with to prevent you from cutting them the middle man out of it. I have surfed around and browsed a few of those DIY Home Kits and they are all the same "almost everything you need" but hey they tell you that when you buy an engine rebuild kit too, but it doesn't mean you can go home and rebuild your car's engine with just that either. I found another site, which honestly only further confuses me...

http://www.studyread.com/plant-tissue-culture-techniques

1. Tissue or cell of an interesting plant is selected and sterilized (disinfected) by mercuric chloride or alcohol.

Sterilization of cells: The cells taken for tissue culture are to be surface sterilized. This helps the cell wall and tissue surfaces to be free from any bacterial or fungal infections. Care should be taken during their handling, transfer etc. to keep them free from infection.

2. Then tissue is placed in media and incubated with proper oxygen supply and right temperature.

Oxygen supply: Since tissue has no direct mechanism to take up oxygen, oxygen supply has to be provided. The gas should be free from contamination and also aseptic. The rate and pressure of flow of gas into the chamber of tissue culture should be optimal.

Perhaps you can see the issue I am having here... they are supposedly telling you everything "step by step" but seriously I was looking for quite a bit more details than what they are listing here and did i really miss something I need to supply oxygen?

Oh here is another DIY Home Kit listed parts that come included: (They Call It- Not Me)
EVERYTHING Almost Kit with CD + DVD

• KCK Manual, DVD, MSDS CD, and Comprehensive KCK CD
• MS Medium with Vitamins - 1 liter packet (10)
• Agar, Powdered - 9 gram packet (10)
• 3 - 100 mg powdered hormone (Choose 3: BAP, Kinetin, 2iP, NAA, IBA, Other?)
• 3 - 100 ml 0.1 M KOH for dissolving hormones
• Forceps - 8 inches
• pH papers
• Measuring spoons, smidgen spoons, 1 ml syringe
• 1 ml transfer pipettes
• Free samples (varies with availability)
• 150 baby food jar caps (Magenta bcaps)
• 3 GA7 Magenta vessels
• 1 liter microwavable measuring cup
• 30 ml PPM
• Optional Frank Tromble Micropropagation DVD
• Priority Shipping via USPS. Refrigerate on Arrival.

I honestly read that and felt lost, I am suppose to choose "3-of the powdered hormones" and I have no clue what any of them are or on what bases I would want to choose any of them. You can again see they list the "3 GA7 Magenta vessels"... I even can't get over "MSDS CD," is that really saying they don't send you an MDSD Sheet, but have it on CD for you to listen too... I am just stunned and shocked.

Magenta GA-7 Vessel:
http://www.carolina.com/lab-specimen...ker5?question=

Oh and I was reading some place else they wanted you to put the Tissue Cultures into another piece of equipment to grow it... All in all, at the end of the day I am still trying to find a good step by step instruction set to figure out everything I need to do step by step, including all the equipment I need. Thank You for adding to the list though, I did copy it all down... Perhaps I should start over with just asking you step one where do I begin and go from there...

[DingusKhan]

Agar is widely available from Amazon
https://smile.amazon.com/s/ref=nb_sb...-keywords=agar, or scientific or lab distributors.

If that isn't an option, a college science department, health clinic or veterinarian might be willing to order it for you, or if they're in a good mood, might give you a big scoop of it for a couple bucks. Might even come in little petri dishes.

I also watched a few of those NPK university videos, VERY detailed. I forget which one it was, but the guy did go over hormones, their roles and different available products like NAA, oligosaccharides, auxin, etc. I think he was talking about cloning, but still, I would assume the same hormones are still triggering the same response from the meristemic cells. If there are any other hormones, I'm sure you can replenish all the supplies a lot more cheeply than buying another kit.

[growmore]

That's why I said that kit was not very good as they don't tell you what the different components in their media is and unless you know that, you simply cannot replace what you have used.

[PigSquishy]

Oh yeah I know the video's over there very well, Harley loves educating people about hydroponics and plants... I have spoken to him a few times in email and sat in on the classes, which after the video ends, then he says "Does anyone here have any questions". He takes all the time he has to answer everyone's questions before closing the My Meeting window where people sit in on his classes for Free. I have also asked him once about these kits and that is when he told me the book he is reading as he himself as he is planning on putting together a class on the subject of Plant Leaf Tissue Culture, but then I heard he is involved in a study again right now so that work is on the back burner. But I will keep waiting for him to come out with a class on it.

Last I knew these two videos were not on NPK University but they are on YouTube and helped me a lot:

Full Plant Nutrient Rundown by Harley Smith (Part 1 of 2)

https://www.youtube.com/watch?v=BleWpBYweHE

Harley Smith, Plant Nutrient Research Developer for RAW Nutrients breaks down and talks about each individual plant nutrient and how to utilize them in your garden.
06:44 Nitrogen
12:55 Phosphorus
20:50 Potassium
23:48 Kelp/Seaweed Extract
27:00 Humic Acid
30:30 Fulvic Acid
36:40 Silica
41:10 Yucca Extract
48:25 B-Vitamins

Full Plant Nutrient Rundown by Harley Smith (Part 2 of 2)

https://www.youtube.com/watch?v=zdkwTo79NXE

0:25 Calcium/Magnesium
02:40 Amino Acids
13:20 Cane Molasses
20:30 Questions/Answers

[growmore]

Hi Greenthumb

Ok checked up your link to find out about Magenta vessels. That is just a brand name for a polycarbonate container available from any lab supply company. The other kit although more confusing is a lot better kit than the first one you mentioned. at least when you have used the media and hormones you can replace them.

The bit about needing an oxygen supply is totally wrong and is very dangerous. If you see a website suggesting this then I would leave the site and disregard everything they said. A typical way of keeping an aseptic work area is by the use of a naked flame inside your laminar air flow cabinet and every time you move from working from one culture to another is to dip your scalpel or forceps into 80% ethanol and then place over a flame to resterilise them. Open flames and oxygen are extremely dangerous, so unless you are using 2 separate work areas you run the chance of blowing yourself and the lab up.

You mentioned that you were confused about the reference to hormones. I had assumed that you had a general knowledge about what they were and how and why they are used. I hope you don't expect to just buy a kit and start growing. Buying what you need is a simple matter but knowing how and why you use them, is the complicated part. What one type of plant requires to grow successfully in vitro may be completely different for another plant. Even using different starting material ( leaf, Meristem pieces ) from the same plant will need different types and concentrations of hormones to grow.

I suggest you look at phytotechlab.com and look at their technical page as a good source of information. They are a very good company and what you read on that site is reliable and correct. It would be impossible for me to give you, step by step instructions without knowing exactly what type of plants and explant material you want to work with. So my first suggestion is to work out exactly what you want to do.

[PigSquishy]

I am very familiar with my compressed gases and their safety protocols, that is why when I saw that I was quoting it exactly... Sorry but as any space buff knows the Apollo 1 astronauts burned up alive on the ground because their capsule was filled with compressed oxygen, and it changed the future of spaceflight forever because of it. Seriously above all else I am #1 when it comes to doing it right and doing it safe, and I know better than to ever use oxygen inside, and if those who need oxygen for medical purposes were smart they would hang the "Oxygen In Use" sign on their front door.

Yes I know my basics of the hormones from cloning and basic biology, but as you so clearly pointed out different ones are needed for different things, and as I continue to read you can clone an entire plant from pretty much any part of the entire plant including its pollen. Which is exactly why I came to this point of wanting to be able to learn to do this, and I realize that this is going to be a lot more researching and reading to learn what I am going to need to know. Thank You for that website, that is exactly the kind of place I was looking to buy my things from where you can get everything you need and it is exact and precise in what you are buying especially when it comes to a medium or something like hormones. I do know enough to know there are different mixes and grades of things and those little tiny differences really can make a serious difference. Now what I really need is to find me a book or detailed website that teaches me the individual and proper steps...

Here is my plan of how I'm planning at going about this entire process... first I have been curious about this subject I've done a few months of researching on it, reading, asking questions, and even found a book to buy on the subject to do even more detailed reading on it., I've also watched the step by step video's by the company who makes the first kit so I got the basics of what I get if I buy their kit. I noted like you saw "medium" they don't say its Agar, they don't specify a specific anything for reference so if there is different grades of blends of it you still won't know what to buy and can't replace anything other than the entire kit, because it contains everything you need. The 2nd kit gives you a bit more details and lets you reorder some things, but again it seems like they are keeping you over the barrel by not telling you everything. Like you pointed out I may need different hormones and stuff for different parts of a plant, a different species of a plant... what are these kits specifically set up to do? Do all the kits grow all plant material? Lots of questions here... I read you can use pollen to Tissue Culture from, does it take a lot of pollen, different hormones? Right now I am at the point I am filled with questions... hence why I have come looking for others who like yourself have done this before, as I am hoping you can tell me websites to get my materials from, perhaps a book to read, a video to watch, a long article I need to go read... I am seriously willing to go put in the work, I am not asking anyone to come hold my hand and hold on while I go do Step #33... and come back now what?

You pointed out I needed a 15-lb pressure cooker, I assume that being it is in the "lab area" it will be being heated by a hot plate to carefully be able to control the heat more perfectly? Oh and that was another question, the bunsen burner, isn't it just going to be being used to sterilize and so my question then comes can I use an alcohol burner instead? I have also been looking more into those DIY laminar airflow cabinets and had some questions if they really are designed like the commercial one's you buy? I mean I am all for DIY and using what you have already, unless it comes at the cost of not doing it right.

At the end of the day before I start buying kits or anything else towards this end goal, I want an entire parts list compiled complete with prices, a step by step set of instructions to follow from A-Z so I can double check my parts list, I want to have at least one good book read on the subject and feel I have a firm grasp of everything needed of me. Then I will make good and sure I feel confident enough to guarantee success, as I will make sure I have resources in place to turn to encase things don't work out so I can figure out what I did wrong and fix it. Then and only then will I start spending the money towards this goal of being able to do it, because otherwise it's a waste of time, money and energy if I can't buy all of this full well knowing I am going to be guaranteed I will reap the rewards in the end for all of this hard work and effort.

[growmore]

Pressure cookers come in 2 different kinds. One has built in heating and are not suitable for tissue culture and the other variety needs placing on a hotplate and their pressure is controlled by a weight preventing the steam escaping, which governs their working pressure. Yes you could use a metho burner. You are interested in doing pollen culture, which is a very advanced area of to be working in. For something that technical you would need access to a university grade research lab and even then, being a novice your chances of success would be almost zero. Why are you interested to pollen culture and what do you hope to achieve? If you look closely at the website I mentioned you will also find that their is also a lot of information about what you need and how to set up a lab.

[PigSquishy]

Yep I have the type in which goes on the stove top/hot plate and then has the weights to set it for 5-10-15 lbs of pressure. My interest in pollen culture is simply the fact that it can be done, that is the problem with where I am at during this stage... I read and read and hear "you can do Tissue Culture with pollen, roots, stem, leaf material etc..." but they don't say anything beyond "you can do it". The problem is you can go to a store right now and buy a transmission rebuild kit for your car and I know plenty of people who have tried only to have to take the mountain of parts into the shop to have someone who knows what they are doing put it back together correctly because the directions only take you so far and don't tell you half of the steps of the things you already need to know. Hence why I am so very glad you are taking the time to help me, because I am only reading "you can do this..." it isn't telling me like you are that I would need a dedicated research lab to accomplish the work at hand... Hence why I am at the drawing board trying to look at everything in detail to get this all figured out on paper before I move forward.

[growmore]

We are talking about 2 completely different areas of tissue culture. Basic culture is quite straight forward and simple and most people would be able to achieve reasonable results with practice. Pollen culture on the other hand is very complicated and requires a lot more equipment and knowledge.
The question you need to ask yourself is not what can be done but what you want to do. Unless you have a clear idea on exactly what plants you want to culture and why you are setting yourself up for failure. I am not trying to discourage you but being realistic and offering my oppion. I cannot stress enough the fack that you will need to know exactly what you want to do.

[PigSquishy]

I know you and I are on the same point... but I don't think you are seeing that... I don't know enough yet to know that pollen culture and tissue culture are different, or that you need more equipment to do it... All I know is the little that said "you can use Tissue Culture to do...". I am still in the stage of wanting to understand and know what all can I do with this? How practical is it, what equipment is needed to do it, how hard is it to do... I am seriously loaded full of tons of questions about everything from "What is Plant TIssue Culture and What All Can You Do With It?". I don't care if it can be done with pollen, if you need a research lab to do it... I would at best have a small lab of my own where I would grow explants to keep in stasis. What kinds of plants? I don't know, what plants can I grow here for food and medicine that I can't or don't have in abundance, what plants could I sell and turn this knowledge into a cash turn around? Would this help me to get my greenhouse up and going to start my business up so I can one day grow big enough to be a nursery for plants and trees? Thus far I know only that the basics of what I am reading regarding this topic, I don't know a lot about it yet... that is why I keep trying to get you to see and understand that is why I came here to ask questions of where do I start and if anyone else has ever done it? It seems really cool, but I am the first to say "I DO NOT KNOW ENOUGH ABOUT THIS TOPIC AND SUBJECT YET" to be able to do it, know completely what it is, etc...

To try to give you a comparison "It would be like someone saying they know hydroponics means to grow plants in water"... that is a far cry from knowing how to run a hydroponic system to grow plants successfully and know about the nutrients, lighting, etc... I only know the basics of what this is, however if this was the topic of hydroponics I'd just do a search find sites like this one and I'd have people crawling over one another to help me learn everything you need to know about hydroponics. However when it comes to the subject of Tissue Culture it jumps into all kinds of subjects, cell division and not just those relating to plant tissues either, and there is no step by step beginner's guide. I am not even sure if this book I am waiting on is going to put any of this into perspective, I know it sounds cool... I know they sell the DIY Home Kits for it, and I know that it takes equipment and more knowledge than I currently have... stuff I am trying to learn and figure out what I can do with what... so how can I know exactly what I want to do with it, when I don't even know what all I can really do with it because I can't seem to find a site anywhere that outlines it for me. Believe me I keep trying to find a site to outline it, I keep trying new keywords for my search terms, and I keep responding trying to tell you this.

[growmore]

To tell you the truth I don,t know what else I can tell you.or how I can help. My only suggestion would be to go to a local university and access their library and talk to their biology department.

[bobsakamoto]

i don't know if this will help or not but as a former employee of 4 different universities and as a former graduate student i can say w/100% confidence that if you can just find the right person or people who are *SO SO* INTO this they will talk your ear off about it if you want them to. i know that's funny but i don't mean it to be. i promise it's true. so along those lines i'm just gonna throw this out there.... why don't you search for "expert author professor tissue culture cloning" or some combination thereof. alternatively, insert the word conference in there and see what you get. if you can find the tiny little group of academics for whom this topic is pretty much their *entire* life (grad students especially) then you could just write to them directly and ask how to get started, book recs, whatever you like. again, i don't know if you've done this already but just thought i'd put it out there...

[PigSquishy]

Which is a great idea... but that is why I am asking about it here... because I have been trying to find these so called "experts" but like yourself there is so many diversions of this... it would be like saying you have a Ph.D and thus you are a Doctor... a doctor of what, medicine? If so what kind of medicine... because this field is so diverse, and again like you stated earlier yourself, what plants am I going to be working with? Again it gets so diverse... so hey how about I go to a Cannabis Forum, ask if anyone knows anything about this topic, learn all I can from there.. learn how to do it on cannabis where others have tried and maybe done it and have some answers... learn all I did about it and by doing it just like I did with all the different types of cloning over the years... then find out what more I can do with it as I get better at it with time and practice. So I came here and ask what I assumed to be a basic question trying to get to the bottom of what I assumed was fairly straight forward answer...

If I bought a DIY Home Kit, do I just cut up a leaf inside the laminar flow hood keeping everything sterile, find out if I then grow the explants in there, another place, etc... and get a ballpark idea as to the cost of this and what all it is good for. I've heard mother plants can be kept small as explants for years, then grown to full sized mothers if you want to regrow that strain at a later point and keep it in stasis if you don't need it. How hard is it to keep it this way? I came asking with the assumption it couldn't be that hard, and hoping to find out if the different mediums were a preference like strain types, different folks just like working with one over another for their own reasonings. Didn't think this was over the top learning... I keep coming back to a sentence in the book:

The Cannabis Breeders Bible by Greg Green, Chapter 8 Tissue Culture, pg 92, paragraph-1 "TISSUE CULTURE IS A RELATIVELY NEW CLONING METHOD involving a certain degree of lab work. However this is twenty-first century and this kind of lab equipment can cost as little as $40. What follows is a basic rundown of what tissue culture is and how it works."

Now I know how to work in sterile conditions and how to work with medical tools and my basics of lab, I am very skilled with my hands and very knowledgeable, I have the dedication and the willpower to spend hours learning, studying and mastering this skill set; thus I came in search of answers. If I go accordingly to the first kit I researched over at GrowersHouse, a place I know I can trust because they won't carry a product unless the company gives them one for free and then picks the best staff member to give it a try and see if it works and is something they feel comfortable selling customers knowing it will have a high success rate. I know the common household method involves something to do with working within steam to keep the air around the tiny work area sterile. I know what it means to do this kind of work and I know full well to skip that and buy or DIY a laminar flow hood. So yeah I have the basics figured out and want to make sure I increase my success rate by getting set up correctly and understanding if this is really something worth all the time, money, energy of mastering this.