Breeding Program for a Super Cannabis

ShirkGoldbrick

ShirkGoldbrick

Active Member
First off, I'd like to say that this is entirely hypothesis on my part and the viability of the program, if it even works, in the creation of a super cannabis would be dependent upon random luck. This is much like the "random luck" encountered in planting seeds until you find a "keeper". However, if a keeper is found it may well blow away any other strain of cannabis on this earth in terms of quality due to increased genetic potential. None of these techniques are my own and I take no credit for the processes potentially used to these ends. I have not cited my sources because, well hey, this is a freaking weed forum and I imagine anyone bright enough may do the research themselves and perhaps find more means or information that would be a valuable contribution to this thread.

Outline

1) Background information on polyploids

2) Determining success in creating polyploids

3) Obtaining seeds

4) Induce tetraploids- polyploidization by colchicine

5) Breed triploids(potentially)


1) Background on Polyploids

Cannabis normally has 20 chromosomes. This is to say that when you plant a seed that it has inherited 10 chromosomes from its mother and 10 from its father. This means that cannabis is a diploid plant as it has 2N chromosomes naturally with N being 10 and 2N=20. To my knowledge there are no normal Tetraploid(4N)=40 or Triploid(3N)=30 naturally occurring variants of this plant.

Why Are Tetraploids desirable?

A) Genetic Variation:The plants contain double the genetic material as their diploid counterparts. This excess in genetic material may allow them to be more resistant to pests, diseases and environmental stress. They also may grow more vigorously and produce larger flowers than is possible by the diploid plant. One example is the increased size observed in commercial strawberries.

B) Breeding: as these plants contain twice as much genetic material as their diploid counter parts they will have twice as much influence on the determination of the genetics of the resulting seeds. As a result desirable traits of plants may be stabilized much more quickly through breeding. This breeding may create new species more quickly than is possible by breeding diploid plants. This is a technique extensively used with orchids.

Why Are Triploids Desirable?

By breeding a tetraploid and a diploid together a triploid may result. Any odd numbered polyploid will either produce small seeds, sterile seeds, or no seeds. An example would be the commercially grown banana, which contains no seeds. It is possible then that by breeding the desireable traits into a triploid one may create a defacto sinsemilla crop incapable of producing undesirable seeds in flower under any circumstance.


Variance in success of polyploidization

Through polyploidization may be induced through various means it should be noted that it is difficult to determine whether or not you have actually successfully induced polyploidy and in which tissues it has been induced.

A) Tissues and Chimeras

The plant consists of three primary tissues which of which the apical meristem is composed.

1) The outermost layer, or epidermis, polyploid induction here by itself is fairly worthless for our intents

2) The second layer, or the pith cells, polyploid induction here by itself will result in some polyploid offspring

3) The innermost layer, or root structure.

In the production of a polyploid none, one, some, or all of these tissues may be induced.

If all of the tissues are not induced then even if you produce a tetraploid through breeding the resulting generations may not not also be tetraploid. Also, over time the plant itself may convert completely back to diploid.


2) Determining Success in Diploid Creation

In order to determine whether or not you have successfully induced a diploid plant there are a number of measures which may be used. I will present some of the simplest here as it will be more practical for growers without access to advanced lab equipment used in flow cytometry.

For the simplest observations you should look for: Variegated foliage, difference in leaf shape/size, difference in growth patterns, difference in water and or nutrient consumption. All of these things would indicate at least partial polyploidization.

Other measures which may be undertaken include comparing stomata size, stomata density, and pollen size. These may be viewed under a microscope for determination. Multiple samples should be taken and averaged for both the control (known diploid) and test (induced polyploid).

Finally, a root tip squash may be performed.

A) Root Tip Squash

Note: Some of the chemicals used during this process are hazardous. It is recommended that chemical resistant gloves, goggles, and a long sleeve shirt be worn during the preparation.

Materials:
-Microscope
- Microscope slide and cover slip
-Water at 60 Celcius
-1M HCl acid
- Ethanol (95%)
-Glacial ethanoic acid
-Petri dish
-2 100mL beakers
- Sterile razor blade
- Toluidine blue stain
- Coffee filter
-Sterile sewing needle
-new wood pencil or a similarly sized wooden dowel

Methods:

1. Prepare Ethanoic acid by mixing 3:1 ethanol to glacial ethanoic acid.

2. Cut 3 cm of the plant root tip and place in the petri dish with the ethanoic acid for 10 minutes.

-At the same time heat the 1M HCl to 60 degrees celcius by placing a container containing the acid into a bath of water of the same temperature.

4. Wash the root tips in cold RO water for 5 minutes and place on a coffee filter.

5. Using the sewing needle, transfer the root tips to the hot HCl acid and leave for 5 minutes.

6. Repeat step 4.

7. Use the sewing needle to transfer the root tip onto a clean microscope slide.

8. Cut off and keep the newest 2mm of growth from the root, trash the remaining portion of the root.

9. Add a drop of toluidine blue, it is best to wait several hours at this point to allow the cells to take up the stain and harden which will reduce the chances of them bursting after squashing.

10. Break up the tissue with the sewing needle.

11. Place the cover slip over the roots and "tap" the slip into the roots "squashing" them by dropping the pencil or wooden dowel onto the cover slip from a height of approximately 2 inches approximately 20 times.

12. View the root tips under 400x magnification and look for the chromosomes. If cells are overlapping squash again. Count the chromosomes, if you were successful there should be 40 pairs.
 
ShirkGoldbrick

ShirkGoldbrick

Active Member
3) Obtaining Seeds

Clearly, if one wants to have the chances of the best success with this method then the seeds should already exhibit as many desirable traits as possible. It would be incredibly expensive to buy a large number of higher quality seeds from breeders, but that is one option. If you're going to go that route you might as well get unfeminized seeds and attempt to polyploid both a male and a female for breeding.

I have two main ideas to quickly enough obtain a high quality specimen to produce seeds. However, this may scare some people off as they may see it as breeding for hermaphroditism.

1) Obtain a known high quality strain, perhaps potentially a more legendary clone only strain

2) Obtain two such strains and graft the root stock of one onto the other. Observe the resulting branches the plant produces and label them. Some branches may show traits of one strain, other branches of the other, and some may show mixed traits. It may be necessary to take clones off multiple branches and flower the plant out to make the distinctions more clear. This in and of itself may be used to quickly propagate desired traits from one plant into another. This is similar to what was done to create the broom laburnum (which is infertile, but I doubt that would be the case with two cannabis strains).

A) Pollination

Next you need to self pollinate the plant to obtain seeds. The easiest way to do this is 2-3 weeks into flower to roll the hairs of a callyx between your thumb and forefinger until they separate. This will trick the plant into producing pollen sacks from which the pollen can be collected. Use this pollen to impregnate some of the lower branches and wait for maturity to collect the seeds


Induce Tetraploids

Note: Colchicine is a very dangerous and toxic chemical, extreme care should be taken when handling.

1. Germinate seeds utilizing the "paper towel" method in complete darkness.

2. Once the tap root has emerged several seedlings may be be carefully placed parallel onto a thin piece of cotton cloth that has been wetted with RO water. The tap root should be on the cloth and the stem end should be off the cloth.

2. Prepare a colchicine solution. Obtain 95% pure Colchicine from a chemical supplier. A few of them (as of 1964) are:

-Inland Alkaloid Company, Tipton, Indiana
-Mallinckrodt Chemical Works,New York City
-Eimer & Amend, New York City
-Merck & Co., Inc., Rahway ,New Jersey
-S. B. Penick & Company, 132 Nassau Street, New York City
-Gane and Ingram, Inc., 43 W. 16th Street, New York City
-Bios Laboratories, 607 West 43rd Street, New York 18, New York

To prepare your solution dissolve 22mg of colchicine per 1L of water. This will give you a 2% w/v solution.

Here's the trick: You can then place the wrapped seedlings in a vial filled with this solution immersing the stem end but keeping the tap root just in water. The wet cotton wrapped around the tap root acts as a plug. This method has a much lower mortality rate than simply immersing the seedlings in the solution.

4. 3 to 24 hours later remove the seedlings and plant into a rapid rooter placed into an aerponic cloner. As many groups as possible should be done to determine an optimum time for contact with the solution to successfully induce them into being tetraploids without killing them. The strength of the solution may be another variable that could be varied with additional groups.

After a few weeks the plants that live(many wont) should have developed a root structure which could be analyzed by doing a root tip squash. This is the reason for using the aeroponic cloner is for ease of access to clean roots.

If the plants have successfully turned tetraploid they should be further grown out and observed for exhibiting desirable traits. It may be necessary to take labeled clones off of these plants in order to flower them out and run a side by side with the diploid to compare flowering times and yields.

While I don't have any scientific data on the fact, many discussing this topic in other cannabis forms suggest not smoking the flowers from these first generation plants because of potential colchicine residue. Hypothetically though let us say that you had placed 10mL of your 2% into a vial for only one seedling and it absorbed all of the colchicine (which it wouldn't) that would be 0.22mg of colchicine. The half life of the residual would be 26.6-31.2 hours. So if you vegged the seedling out for a month and then flowered for 2 months and cured for 2 weeks that's (assuming 30 day months) 2496 hours. That's 80-93 half lives. The residual colchicine would be
0.0000000000000000000000000000222-0.0000000000000000000000000227 milligrams
remaining in the entirety of the plant. You could dry not just the flowers but also all of the other remaining plant material and flower weight/total mass and multiply it by the residual colchicine and that could possibly be the maximum residual left in all of the flowers. This could then be compared to the toxicity of colchicine.

However, quite likely, while colchicine can have a systemic effect I don't believe it would even stay around in the plant as it's miscible (dissolves in water infinitely) and would likely be transpired out of the plant over its growth.

How does it work, if it doesn't stay around? It just affects the cells that are currently in the seedling, then those cells (having doubled their genetic material) continue to maintain that level of doubled genetic material every time they divide as long as all three tissues were induced. If it stayed around you wouldn't get a tetraploid..you'd get a much larger amount of chromosomes. Take my advice at your own risk, this is a weed forum anyway.


Breeding

Tetraploids can breed with tetraploids. You could self pollinate a single plant or pollinate them with each other to produce seeds.

You could also breed them with their diploid predecessors to create triploids and eventually you would likely end up with a weed crop that would be clone only, have all the traits you wanted, and be incapable of producing seeds. If you get a triploid that produces seeds but that has all your desirable traits then keep breeding with other triploids until it no longer produces seeds(It wont be able to breed with diploids or tetraploids).

The whole point of all this is that because of the increased genetic material you can now breed the plant to have traits it never was capable of having in its diploid form. This doesn't mean the first one you create will be a super plant (although it might be) but it means that as a breeder you may be able to create and isolate traits that don't exist in the diploid form.
 
nomofatum

nomofatum

Well-Known Member
ShirkGoldbrick said:

2) Obtain two such strains and graft the root stock of one onto the other. Observe the resulting branches the plant produces and label them. Some branches may show traits of one strain, other branches of the other, and some may show mixed traits. It may be necessary to take clones off multiple branches and flower the plant out to make the distinctions more clear. This in and of itself may be used to quickly propagate desired traits from one plant into another. This is similar to what was done to create the broom laburnum (which is infertile, but I doubt that would be the case with two cannabis strains).
Click to expand...
This part is bullshit. Genetics don't move through the plant. New growth is from cell division of neighboring cells, the roots aren't neighboring the branches...
 
ShirkGoldbrick

ShirkGoldbrick

Active Member
nomofatum said:
This part is bullshit. Genetics don't move through the plant. New growth is from cell division of neighboring cells, the roots aren't neighboring the branches...
Click to expand...
They don't? How do you explain graft chimaeras? They can move through the plant.

This is the same case with the colchicine, if you turn enough of the cells of the seedling tetraploid (of the three different tissues) and they can spread throughout the plant and dominate it. This does not always happen, but it can, and that is what we're trying to do here.

Edit to add: Look up "Bizzaria of Florence"
 
Last edited:
nomofatum

nomofatum

Well-Known Member
ShirkGoldbrick said:
They don't? How do you explain graft chimaeras? They can move through the plant.

This is the same case with the colchicine, if you turn enough of the cells of the seedling tetraploid (of the three different tissues) and they can spread throughout the plant and dominate it. This does not always happen, but it can, and that is what we're trying to do here.

Edit to add: Look up "Bizzaria of Florence"
Click to expand...
I took a look, and still no reference of trans-locating of cells from one part to another. Instead I'm finding that chimeras are made when the graft sight is a growth node and it's comprised of a mix of both plants' cells. From that point up they are intertwined, but no DNA is flowing around, the trunk at least has to be a mix, one of the two sets of DNA can win out a branch or just a portion of a branch. The DNA never mixes, results of breeding would be the results of one of the two sets of DNA plus breeding partner, or mix of the two without a outside donor..
 
nomofatum

nomofatum

Well-Known Member
Bud Tipps said:
Have you tried this out?
Click to expand...
The majority of what he posted is proven and has been used professionally in the 70's-90's and even earlier. Only that one section had false info from what I can see.

polyploids by colchicine seed treatment, seed works best because you only have 1 cell that needs to mutate, no competition between different mutations or mutated vs non-mutated cells. They also do this in tissue culture on as small a sample of cells as possible surviving (again to minimize the competition between varying cells.)
 
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