Various Proposed Experiments to Induce Mutagenisis in Cannabis

[EnvyForJ]

Mutagenesis is the process of inducing mutations in the genetic code. This can be done using many different methods but is a tool the average breeder has no access to.
Breeders could want to use methods of inducing mutagenesis for various purposes such as creating new breeds of plants: such as more stealthy plants, or plants with a higher yield, novel phenotypes, higher potency, atypical growth, or a variety of other reasons.
Typical forms of mutagenesis are inducing polyploidity with colchicine; using high energy - small wavelength radiation such as gamma rays, x-rays, and Ultra-Violet light; using different compounds such as benzene, touline, or polyethylene glycol.

EnvyForJ here and im a student at a local college in colorado studying botany and biology, and me and a couple friends want to participate in Colorado Overgrow. We want to use Ducksfoot to accomplish this to raise the chance of establishing populations, but we have no access to any ducksfoot genetics so we have set up a couple experiments to try to modify the appearance of pot plants for use in this OP. We decided to use autoflower plants due to their quick successive generations and to simplify the experiments by removing light variables.

Right now we are waiting for Seeds for a bank at the moments as well as the equipment from amazon, but this will be our grow log.

We have done guerilla farming in the local mountains in a few rural communities in the past and never take pics but we will be using a safe camera to document and catalogue our journey of experimental breeding.
Cheers

 

[EnvyForJ]

[HASHTAG]#1[/HASHTAG] Use UV Light To Induce Mutagenisis In Cannabis Pollen

Use UV Led lights with a wavelength of 380nm - 405 nm and determine whether they could be used to induce mutations in Cannabis Pollen

Materials:
4 Petri Dishes, 100mm x 15mm
Mylar Reflective Sheeting
Ultraviolet LED Flashlight, 380nm-405nm or lower
(Im using a Opoway 51 Ultraviolet LED Flashlight)
Feminized, Autoflowering Cannabis Pollen from a Stable Line
(we will have to grow out and self fertilize a generation or two to determine if the autoflowering plants
are genetically stable)
A Good Grow Sight With Room To Accommodate At Least 16-23 Sea Of Green Style plants
(That includes all the necessary lighting systems, pots, water, and so on)
Flowering, Autoflower Cannabis Of The Same Line As The Pollen
Small Transparent Plastic Bags
Large Transparent Plastic Bags
Twine
Gibberellic Acid To Feminize The F1 and F2 Generations

Hypothesis:
Larger wavelengths of U.V. light can be used to induce mutations in cannabis polled based off of the
famous experiments of Lewis Stadler. Because of the inability o obtain any light with a wavelength of
360nm or less, the duration of exposure shall be lengthened.

Procedure:
There shall be four groups; A, B, C, & D; These groups shall be exposed to varying periods of ultraviolet light with group D being the control.

Create four simple enclosures by wrapping petri dishes in mylar without the lid. Place pollen into the enclosures equally and place the lid on top. On one side of the lid label the enclosures A, B, C, & D. Place D off to the side. Wrap the lids of A, B, & C with mylar while leaving an exposed portion of the lid the size of your flashlight head.

A, B, & C shall be exposed to 30 mins, 60 mins, and 90 mins of UV exposure respectively by placing the UV flashlight on the hole of the mylar wrapping.

Label four small plastic bags A, B, C, & D and place the exposed pollen into its appropriate bag.

On the cannabis plant locate four equally sized budsites. With care not to disturb the pollen in the bags, wrap the identified budsites with the pollen containing bags and seal with twine. Leave the bags on until the seeds have fully matured.

The generation that donated the original pollen as well as the plant that accepted the pollen are hee after refered to as the P1 generation. The seeds from the procedure above are to be refered to as F1: A, B, C, & D.

Record the number of seeds within the individual groups.

Incubate/Germinate all of the seeds, and record the number of seeds that sprout. Deformities or variations caused by mutagenisis should not have occured in the F1 generation, but record any discrepancies. Carve out an equally proportional population population in groups A, B, C, & D culling the rest.

Put the plants into flower as soon as possible (if they arent already auto flowering plants) and once they are a week into flower spray them with a diluted solution of Gibberellic acid 3 times a day in order to induce the growth of pollen sacks.

After 2-3 days of treatment with Gibberellic acid solution cover plants with large transparent bags and tie shut at the base in order to self pollinate.

Allow plants to self pollinate and create seeds. Once seeds mature, remove bag and harvest seeds.

The seeds harvested from the F1 generation will be referred to as the F2 generation, and should be the first plants to show any evidence of mutagenisis due to the assumption that most genetic mutations are either recessive mutations, deletion of genetic code which would be invisible in the F1 generation given stable genetics, or duplications which would either not show themselves at all or drastically alter the genetic code but still be either recessive or covered by redundancy rendering it invisible in the F1 generation.

Take seeds from the F2 generation proportionatly from each of the F1 plants in multiples of 4 so as to increase the chances of finding any mutations. You only need 4 seeds from group D.

Grow out all of the selected seeds. Catalogue and record any observable mutations as well as the Frequency of mutation in each group.

Once the plants are in flower all them to grow for a month or two in order to record any before unrevealed mutations. Then use the same feminization process from the F1 generation.

This point is the end of the procedure. The seeds from the F2 generation can be germinated and either used for their mutations in breeding or you could take the experiment further into the F2, F3, & F4 generations.


Q & A:

Why use pollen instead of seeds as the target for mutation?

Pollen is what was used in the original research done by Lewis Stadler. It is also more susceptible to mutation and large amounts of pollen allow for a larger chance of viable pollen remaining after UV exposure.

Why use autoflowering & feminized seeds?

While this experiment can be done on photoperiod and male cannabis having these traits eliminates various variables and simplify the experiment.



Conclusions:

By analyzing the data recorder from the quantity of seeds produced by each group you can determine if and how much exposure to large wavelength UV light kills viable pollen.

Any mutations observed in the F1 groups should be evidence of rare, dominant mutations, unless observed within the control group D: In which case the mutations may be a sign of instability in the line used.

Mutations observed in the F2 generation and their frequency will tell us how much exposure to UV light is needed to cause mutations. The frequency of mutations in the group should be around %25 for 1 mutation per F1 seed line. If their are %50 or more mutations in frequency in any of the groups it is evidence of multiple mutations per F1 seed line.
_____________________________________________________________________________________
Stadler, L.J; G.F. Sprague
(1936) "Genetic Effects of Ultra-Violet Radiation in Maize I. Unfiltered Radiation"

 

[EnvyForJ]

This is reserved Too

 

[EnvyForJ]

Reserved 3

 

[EnvyForJ]

Reserved4

 

[EnvyForJ]

Alright ive gotten some of my materials in and done some additional research

And while im waiting for the rest of my supplies to arrive ive started a half serious attempt to clone a plant to pass the time

 

[iampepe]

Welcome to AFN! Best of luck to you guys in your experiment.

 

[jingo]

Looks interesting think I'll sub up sounds like a long ride.

I thought gibberellic acid was used to increase flowers, what is it that causes male pollen? Frequency perhaps? Or repetition? Maybe some other variable?

 

[lost360]

Cool

Sent from my LGMS631 using Tapatalk

 

[tobe]

[HASHTAG]#1[/HASHTAG] Use UV Light To Induce Mutagenisis In Cannabis Pollen

Use UV Led lights with a wavelength of 380nm - 405 nm and determine whether they could be used to induce mutations in Cannabis Pollen

Materials:
4 Petri Dishes, 100mm x 15mm
Mylar Reflective Sheeting
Ultraviolet LED Flashlight, 380nm-405nm or lower
(Im using a Opoway 51 Ultraviolet LED Flashlight)
Feminized, Autoflowering Cannabis Pollen from a Stable Line
(we will have to grow out and self fertilize a generation or two to determine if the autoflowering plants
are genetically stable)
A Good Grow Sight With Room To Accommodate At Least 16-23 Sea Of Green Style plants
(That includes all the necessary lighting systems, pots, water, and so on)
Flowering, Autoflower Cannabis Of The Same Line As The Pollen
Small Transparent Plastic Bags
Large Transparent Plastic Bags
Twine
Gibberellic Acid To Feminize The F1 and F2 Generations

Hypothesis:
Larger wavelengths of U.V. light can be used to induce mutations in cannabis polled based off of the
famous experiments of Lewis Stadler. Because of the inability o obtain any light with a wavelength of
360nm or less, the duration of exposure shall be lengthened.

Procedure:
There shall be four groups; A, B, C, & D; These groups shall be exposed to varying periods of ultraviolet light with group D being the control.

Create four simple enclosures by wrapping petri dishes in mylar without the lid. Place pollen into the enclosures equally and place the lid on top. On one side of the lid label the enclosures A, B, C, & D. Place D off to the side. Wrap the lids of A, B, & C with mylar while leaving an exposed portion of the lid the size of your flashlight head.

A, B, & C shall be exposed to 30 mins, 60 mins, and 90 mins of UV exposure respectively by placing the UV flashlight on the hole of the mylar wrapping.

Label four small plastic bags A, B, C, & D and place the exposed pollen into its appropriate bag.

On the cannabis plant locate four equally sized budsites. With care not to disturb the pollen in the bags, wrap the identified budsites with the pollen containing bags and seal with twine. Leave the bags on until the seeds have fully matured.

The generation that donated the original pollen as well as the plant that accepted the pollen are hee after refered to as the P1 generation. The seeds from the procedure above are to be refered to as F1: A, B, C, & D.

Record the number of seeds within the individual groups.

Incubate/Germinate all of the seeds, and record the number of seeds that sprout. Deformities or variations caused by mutagenisis should not have occured in the F1 generation, but record any discrepancies. Carve out an equally proportional population population in groups A, B, C, & D culling the rest.

Put the plants into flower as soon as possible (if they arent already auto flowering plants) and once they are a week into flower spray them with a diluted solution of Gibberellic acid 3 times a day in order to induce the growth of pollen sacks.

After 2-3 days of treatment with Gibberellic acid solution cover plants with large transparent bags and tie shut at the base in order to self pollinate.

Allow plants to self pollinate and create seeds. Once seeds mature, remove bag and harvest seeds.

The seeds harvested from the F1 generation will be referred to as the F2 generation, and should be the first plants to show any evidence of mutagenisis due to the assumption that most genetic mutations are either recessive mutations, deletion of genetic code which would be invisible in the F1 generation given stable genetics, or duplications which would either not show themselves at all or drastically alter the genetic code but still be either recessive or covered by redundancy rendering it invisible in the F1 generation.

Take seeds from the F2 generation proportionatly from each of the F1 plants in multiples of 4 so as to increase the chances of finding any mutations. You only need 4 seeds from group D.

Grow out all of the selected seeds. Catalogue and record any observable mutations as well as the Frequency of mutation in each group.

Once the plants are in flower all them to grow for a month or two in order to record any before unrevealed mutations. Then use the same feminization process from the F1 generation.

This point is the end of the procedure. The seeds from the F2 generation can be germinated and either used for their mutations in breeding or you could take the experiment further into the F2, F3, & F4 generations.


Q & A:

Why use pollen instead of seeds as the target for mutation?

Pollen is what was used in the original research done by Lewis Stadler. It is also more susceptible to mutation and large amounts of pollen allow for a larger chance of viable pollen remaining after UV exposure.

Why use autoflowering & feminized seeds?

While this experiment can be done on photoperiod and male cannabis having these traits eliminates various variables and simplify the experiment.



Conclusions:

By analyzing the data recorder from the quantity of seeds produced by each group you can determine if and how much exposure to large wavelength UV light kills viable pollen.

Any mutations observed in the F1 groups should be evidence of rare, dominant mutations, unless observed within the control group D: In which case the mutations may be a sign of instability in the line used.

Mutations observed in the F2 generation and their frequency will tell us how much exposure to UV light is needed to cause mutations. The frequency of mutations in the group should be around %25 for 1 mutation per F1 seed line. If their are %50 or more mutations in frequency in any of the groups it is evidence of multiple mutations per F1 seed line.
_____________________________________________________________________________________
Stadler, L.J; G.F. Sprague
(1936) "Genetic Effects of Ultra-Violet Radiation in Maize I. Unfiltered Radiation"

Hi
that is a interesting project. It would be great to read the resaults here at afn.
One question why you want to use fem seeds why not work with regular seeds. You could get hermi problems.
Do you use the seeds to study or do you want to sell your mutants.

I will tag my friend @bonobo a duckfoot breeder who create a autoflower duckfoot.

cu tobe