Preservation of an Afghani Landrace from 25 year old seeds

[TheHatman]

Thy for likes, your interesting and comments

There’s no need to pick at the young seedling and risk damaging it. I’m speaking of when dealing with really old and weak seeds that need all the help they can get.

Thx for telling us your method. In the regular case it´s sufficient to moisture the shell - and I do it the same way like you do. But there are individuals -e.g. 25yo seeds that are for a main part necrotic- that didn´t make it this way. In these cases, moistening is not enough and you have to remove the shell like I wrote in my chapter. Such seedlings also need light on intact tissue as soon as possible.

 

[resin_lung]

Thx for telling us your method. In the regular case it´s sufficient to moisture the shell - and I do it the same way like you do. But there are individuals -e.g. 25yo seeds that are for a main part necrotic- that didn´t make it this way. In these cases, moistening is not enough and you have to remove the shell like I wrote in my chapter. Such seedlings also need light on intact tissue as soon as possible.

Yes, I agree that the “drop of water” method will not be enough to help seeds “that are for a main part necrotic”. That is to say that when most of the cells in a plant embryo(The plant embryo, sometimes called the seed embryo, is the part of a seed that contains the earliest forms of a plant's roots, stem and leaves.)are dead.

But its not that it isn’t the best way to remove a stuck shell and or the thin skin under the shell that contains the endosperm(the part of a seed which acts as a food store for the developing plant embryo, usually containing starch with protein and other nutrients.)

The drop of water method works equally well for removing the dry skin casing as it does for dry shells.

The reason it wont work is because when most of the cells in the embryo are dead...... they are dead, and no amount of sunlight or nutrient or time will change that.

So the seed never germinates and thus will never break ground.

This is because if there are still any living cells within the embryo, they will likely be closer to the center than they are to the outer portions of the embryo.

This means that the nearly dead seed will absorb more water than it would if it were a healthy seed because its not transpiring and other reasons, possibly too complex to discuss here.

Too much water and not enough oxygen is often disastrous when seeds are fresh. When they’re nearly dead, its always disasterous.

These are those ungermed seeds you’ll find filled with white mush when they're dug up out of the soil, or between your paper towels, after enough time has passed and all hope has been lost.

The only way someone should reasonably expect to get a living plant from a mostly necrotic embryo is to... macerate?(Been a while) the nearly dead embryo in order to allow those few viable cells hidden within the embryo to come in direct contact with the culture gell they are going to be grown on.

The hope is that they’ll devide and form callus(clumps of undifferentiated, totipotent
stem cells).

These clumps of stem cells can be coaxed to form whole plantlets. Theoretically anyways.

Thoretically, because every plant is different enough that for one reason or another, one plant may respond well and in a predictable manner to a commonly used protocol and it’s full sibling may not. Also...it seems that there arent enough people with the the knowledge, equipment or discipline to put in the work required to find a protocol that will work.

Even if most of the cells in the embryo are fine, it could just as eaisily be that the edosperm itself that has gone bad. So although the embryo may be totally healthy and viable, it doesnt have access to the necessary nutrients(proteins, starch etc etc.) needed to survive.

In this situation, an endosperm transplant can theoretically be preformed, replacing the old/bad with new/good.

Fresh endosperm can be carefully collected from a plant specific donor. In this case, hemp seed endosperm is perfect. I have a ton of finola hemp seed(kilos) so thats what I used but anything in the cannabis family should be fine, if not perfect. So use what you got.

This type of unviable seed(bad endosperm) are easier to identify than the mostly necrotic seeds mentioned earlier.

They are those old seeds we’ve all tried to pop that seem to resond well to the paper towel germination method, at first anyway, only to sputter and stall out after only growing a tiny little tail about an 1/8th of an inch or so long.

The barley seed tea soak tries to basically accomplish the same thing as an endosperm transplant but in a much simplified way. I dont believe the soak would work on seeds with bad endosperms but may work just enough on seeds with partially spoiled endosperm.

Once the embryo grows enough root to get at the nutrient in the soil, all thats needed is some light. But light wont do a seedling, embryo or plant anygood without a food source.

Light isnt plant food. Plants make their own food by combining carbon dioxide, which they get from the air, with water, containing nutrients, which they get from the soil.

To power the whole process, the plant uses the energy it gets from light/sunlight. Its the green pigment in the leaves, chlorophyll, that traps the energy to be used to combine all those raw materials into food(photosynthesis).

So stripping the shell off of a mostly dead seedling or a seed with a viable embryo but a spoiled food supply, so that it can get some light doesnt make a whole lot of sense to me. Especially concidering that if its planted in soil, it will never grow enough to get above ground anyway.

Stripping the shell is totally necassary for anykind of embryo rescue or endosperm transplant, but not because its keeping it from getting at the light.

Its completely necessary when a heathy seedling is above ground and wasn't able to strip it off on its own. In that case, when the seedling has everything but the energy it needs to make food, the lack of energy it would normally get from light, will surely be its doom.

But fresh seeds and vigorous seedlings aren’t whats being discussed here.

I hope I’m not comming off as jerk or anything. My intentions weren't to hijack your thread.

I enjoyed what i read before I decided to post a comment and I’m looking forward to what I’ll read after this post.lol

 

[TheHatman]

To everyone here:
Please take care of yourself and stay healthy!

Greetz,
hatman

 

[TheHatman]

Sorry guys, I´ll write the next chapter soon! Corona changed my work the last weeks/month so I hadn´t time for my thread. It takes many time to translate into english. But I should start next chapter soon.



So thanx 4 waiting and stay tuned,


Greetz,
hatman

 

[JustSumTomatoes]

Thank you Hatman! I have really enoyed this thread.

 

[techattack]

Oh man i love fatty leafs. Will grab a chair on this thread.

 

[OrgAeroMan]

Good luck, Hatman, maybe tissue culture would be the most feasible. I hope you can figure these little buggers out, the world could always use some more great Afghan bud/hash! Take care

 

[TheHatman]

3rd Chapter: Leap into Life (Part III) - From living Dead to living Beings
[2022-04-15]


INTRODUCTORY WORDS
I am glad that the preservation project has been completed and that I am “only” reporting it retrospectively here now. I don't know if I would have had the calm and, above all, the time to do the report properly. Because I still remember the great stress at that time: „Is there a chance for a few to survive? And then will they become at least male AND female? Or is an old line dying out here?”

Like a lion I stared at the prey, photographed and measured the few specimens twice a day to see if they were making any progress - notes everywhere around me and my laptop was filling up with mass of development data. In addition to that thriller, craftsmen and technicians went in and out of the house continuously for more than 2 months. Folks, it wasn't a funny time, but I got through it with optimism and a joyful drive.

My girl couldn´t catch me for a simple crap talk during this time. She only could give herself my grower shit non-stop between breakfast, lunch and coffee: but only verbal of course

But it's done and she has understood how important my project is and is looking forward to the next breeding steps , for which she want to support me. But until you get the future stuff of oldskool affies, a few thousand more pages will follow here ;-)

Let's go.. and finally end 3rd chapter with part III:


DEALING WITH NECROTIC COTYLEDONES
Which grower doesn't know them? Healthy cotyledons, that are recognizable by a fresh appearance with a yellow to light green colour, which, grew up at exposed light, and quickly acquire a lush green hue.
In comparisation prehistoric dino seeds can quickly scare you with their vampire-like appearance. Some specimens were only missing these big eyes to pass as an alien strain

Because such cotyledons are largely white, greyish and often transparent. They feel lifeless, stiff and waxy - and it stays that way, because realize: They are just dead! Miracle healers, witch spells and something like that don´t help. On the contrary it only rob your important time and can even promote dangerous fungus cultures under certain circumstances.

! Insertion: If you already have experience with supporting agents or chemical additives and if you can rule out dangers or unfavorable hormonal changes, risk it. For me experiments were too dangerous and because I already have experience with bringing such aliens back into life with clean working methods, I have avoided various methods, solutions or chemicals/hormones (methods e.g. with hydrogen peroxide; gibberellic acid) to be on the safe side. Although gibberellins in the correct dosage can support the germination process, tests show an increased incidence of leaf and growth anomalies.

Between the necrotic cotyledons, the first and second true pair of leaves are already created from birth in the seed. If the organic decay in the embryo is already far advanced, then these first pairs of leaves are also sometimes severely affected. The lesions are shown by white-grey to transparent, rigid necrosis, which of course doesn´t get living, while other parts of the leaves slowly begin to develop. Sometimes the pairs of leaves are even completely dead - but if there is still intact tissue deep inside, one or two main sprouts will develop from the rudimentary nodes after a long time and assuming root growth.

These conditions usually lead to crippling of the leaves or even the overall growth. Such cripples aren´t to be confused with genetic mutation, because they are caused by environmental conditions and not genetically. Therefore, these mutants can also become correctly growing specimens that can be used for genetic save, because they develop healthy seeds.

On the left we see one of my Skunk variations from seeds that are as old as Afghanica. In this case, the shoot tip was necrotic after the first pair of leaves, which is why several shoots grew



The breaking up of necrotic cotyledons
Light is needed for tissue that is still intact to form chlorophyll and start photosynthesis. Because the cotyledons are often solidified like wax, the sprout can´t get out. Now it is important to help by carefully breaking off. The necrotic areas break and you can open up the former germ layers and expose living tissue. This allows the important light to shine on these spots and allows the plant to focus on the essential task of survival. The fact that the necrotic parts of the cotyledons break off is not a disadvantage at this point. Who can do much with a dead part of a body?

>>> First examine the cotyledons for remaining endosperm residues and, if present, remove them with a needle (see 3rd Chap, part II) so as not to scalp the seedling accidentally.

>>> Then open with a sewing needle or break with your fingers. In doing so, care must be taken not to damage healthy tissue, because every intact part is a motor for cell division and a factor for growth.

Left: An accident while breaking, healthy tissue has also been damaged here!
Right: If you take a closer look, you can see the necrotic-white first pair of leaves lying on the cotyledon



On the hunt for chlorophyll
After the interior has been exposed, it is particularly important to provide the seedlings with a light source suitable for the vegetative phase. It is also important to note that the seedlings are sensitive and that strong light sources such as an metal halide lamp must initially be placed up to twice as far away from the young plants. With luck, there is still intact tissue in the sprout system, which after a while begins to produce chlorophyll.

In some cases there is only a spark of green life deep inside. It should be clear that the development of this compared to healthy, fresh seedlings takes much longer - so patience is needed here again. The more chlorophyll there is, i.e. the more green there is, the closer the lamp can get to the plant up to the minimum distance.
The other environmental conditions (soil, humidity, temperature..) should be in a suitable range for the growth of cannabis seedlings.

Here you can see the comparison: on the left a real necrotic goth vs seedling from a fresh seed.


From now on, the plant has to do it on its own: In the steps before, we removed the nut shells, endosperms and necrotic cotyledons, which the seedling was no longer able to do itself due to dead tissue and lack of nutrient reserves. Such weak plants would certainly not be the first choice in a conventional grow. Because such specimens in particular often grow only with difficulty or not at all.

In addition, the demand for such hardships cannot be a fat, sticky pinup girl. However, this is solely due to the storage time and the resulting conditions - but from a genetic point of view, every specimen that manages to flower and develop healthy seeds is to be regarded as healthy. Their offspring will again produce exemplary plants that grow quickly and at best become hot pinup girls at best.

Under a microscope you can see that chlorophyll is formed in the lower part of the first pair of leaves.



The following photos show examples of growth:

Done: having escaped from the darkness of the beyond, the fighters start their way into life (The seedling on the right has something animalistic, doesn't it?)

Lucky: The first true pair of leaves is half Necrotic, it could have been dead.

Here separately and exclusively for you: the growth of the main actress in this report: „A4“ !




<see next post for finishing 3d chapter pt III>

 

[TheHatman]

<<continuation of chapter 3, pt. III>>


THE HORROR CABINET

As the final part of this chapter, a little horror show.
Here we see a few specimens with distinctive, faulty growth behavior that are due to the times.

This first pair of leaves of that affie is only a skeleton, the leaf mass itself is dead only the leaf veins are intact.

The photo shows necrotic spots.
Left: Only parts of a S.E. are affected (one of my brazilian genetics)
Right: The cotyledons of an Afghani are completely dead.

Here are a few more little crabs whose further growth will be delayed - selected examples of different varieties:
Above: there is stunted growth
Bottom left: the first pair of leaves is probably dead
Bottom middle: partially necrotic pair of leaves, which is cripple
Lower right: A leaf has stunted due to necrosis




I haven't continued here for a long time. Covid and the whole situation put me off for a while. Nonetheless, I hope you're back on board and continue to diligently post inspirational comments. This makes the report come alive and I really enjoy it! In any case, thank you for your interest and reading! And.. sorry if I'm partially incomprehensible. i translate my chapters from german and sometimes things can go wrong. Thank you for your understanding

The following 5th chapter (coming soon) will be about how the individuals survived the growth phase and got ready for flowering.

See you soon and spring greetings..

hatman



---------------------------------------------------------------------------------
>> Continue with 4tg Chapter: Tour de Cannabis - Race of the Growing Oldskool Afghanicas (coming soon..)
<< Back to 3nd Chapter Part II: Your Support Methods
<< Back to OVERVIEW

 

[exploziv]

The level of detail in here is crazy! I will come and read all thread!

 

[TheHatman]

The level of detail in here is crazy! I will come and read all thread!

Thanks! kool that you are here. Enjoy it!
btw I really like the new forum software for editing (yeah). But unfortunately I couldn't format some older chapters because my posts contain too many characters (over 10000 characters :-D). is there a way?


Hey guys,
hello to everyone who's back!! Finally I uploaded a chapter after more than 2 years. I hope you like the new post, which I had to split into 2 posts due to the limited caracters.


I´ve created and edited the table of contents in the star post for easy use:

INDEX
[Last Update: 2020-03-06]

>> Prolog
>> 1st Chapter: The Purity - An old Afghan Landrace
>> 2nd Chapter: There´s life in the dog! - The Germination Process
>> 3rd Chapter: Leap into the Life (Part I) - The Beginning of the Growth
>> 3rd Chapter: Leap into Life (Part II) - Your Support Methods
>> 3rd Chapter: Leap into Life (Part III) - From living Dead to living Beings
>> 4th Chapter: Tour de Cannabis - Race of the Growing Oldskool Afghanicas (coming soon)

See you soon, keep breeding and oh yes: save the landraces!
hatman

 

[Sam_Skunkman]

I did not read every post just to long for me. My question for you is how many plants Males & Females do you think need to be used with each reproduction cycle to preserve all the genes found in the original landrace? Most Cannabis is Dioecious, all is Heterozygous, and an Obligate Outcrosser, wind pollinated, man can create a Homozygous Cannabis variety but they are not found in nature. Any Monoecious Cannabis made by man must be maintained by man, in the wild they will soon revert to Dioecious with many intersex individuals.
Because of this unlike tomatoes which are not Dioecious or Heterozygous it is not easy to just breed with a few plants to maintain a Cannabis landrace, genes will be lost every reproduction. Tomatoes are easy to maintain a variety unless they are modern F1 hybrids.

 

[TheHatman]

We’ll fix it.

Thx, that would be very nice




@Sam_Skunkman First, thanks for your interest in my project and your thoughts. I am very happy that a legend like you is part of the thread.

My question for you is how many plants Males & Females do you think need to be used

(..) genes will be lost every reproduction (..)

My afghani genetics are quite old and I've managed to germinate and reproduce a handful of specimens. So I´m glad it worked at all. I read in a study that you need a population of N=30 to get all traits in a variety (i would have to search to find out which study it was).

So I could save a few individuals, so I was only able to include only these few in my project (I tried to popp 800+ beans....). In a second step, I did an open pollination with the offspring (N=50; m/w) in order to fluff up rarer traits in the gene pool. As a third step, I plan to make an incross with this fluffed up generation and one of my other older afghani lines to maximize the gene pool of both. Of course i want to get both lines parallel as well as the incross line. But I will describe everything in detail here in my threat.
Certainly, traits normally get lost with every breeding step. But i try zu minimize that effect as much as possible with my "controlled" randomization method. I will report on this later in my thread. I´ll be happy to tag you then in this future post..

greets and happy easter ,
Hatman

 

[Sam_Skunkman]

Here are several papers that show the number of plants required to conserve a landrace of Cannabis, basicly because Cannabis is a Dioecious, Heterozygous, wind pollinated, and an Obligate Outcrosser, you need a minimum of 1000 Males and 1000 females all freely pollinating at the same time in the same location, otherwise you will lose genes every reproduction, this is what is required for conservation, breeding is different, requiring more or less plants, dependant on the goals.
I also included several other papers about seeds and storage and use.

-SamS


Not Cannabis specific
A Program for Estimating the Optimum Sample Size for Germplasm Conservation.
Hernandez, C. M., & Crossa, J.
Journal of Heredity, 84(1), 85–86.(1993).
doi:10.1093/oxfordjournals.jhered.a111284


Not Cannabis specific
Methodologies for estimating the sample size required for genetic conservation of outbreeding crops*
J. Crossa
Theor Appl Genet (1989) 77:153-161
doi: 10.1007/BF00266180.


Cannabis, the multibillion dollar plant that no genebank wanted
Davoud Torkamaneh and Andrew Maxwell Phineas Jones
Genome Volume 65, Number 1, January 2022
https://cdnsciencepub.com/doi/10.1139/gen-2021-0016


Not Cannabis specific
Determinants of Genetic Diversity
Hans Ellegren and Nicolas Galtier
Nature Reviews Genetics volume17, pages422–433 (2016)
doi:10.1038/nrg.2016.58


Development and Standardization of Rapid and Efficient Seed Germination Protocol for Cannabis sativa
Aleksei Sorokin, Narendra Singh Yadav, Daniel Gaudet, Igor Kovalchuk
BIO-PROTOCOL 11(1) January 2021
DOI: 10.21769/BioProtoc.3875
https://www.researchgate.net/publica...annabis_sativa


Not Cannabis specific
Preventing seed quality loss during storage. The role of oxygen is underestimated
Steven P.C. Groot
https://www.researchgate.net/publica...underestimated
DOI: 10.13140/2.1.5013.3447


Not Cannabis specific
Prolonging the longevity of ex situ conserved seeds by storage under anoxia.
Groot, S. P. C., de Groot, L., Kodde, J., & van Treuren, R.
Plant Genetic Resources, 13(01), 18–26.(2014).
doi:10.1017/s1479262114000586


Not Cannabis specific
Seed storage at elevated partial pressure of oxygen, a fast method for analysing seed ageing under dry conditions.
Groot, S. P. C., Surki, A. A., de Vos, R. C. H., & Kodde, J. Annals of Botany, 110(6), 1149–1159.(2012).
doi:10.1093/aob/mcs198


Cannabis Domestication, Breeding History, Present-day Genetic Diversity, and Future Prospects
Robert Connell Clarke, Mark D Merlin
Critical Reviews in Plant Sciences 35(5-6):293-327 November 2016
DOI: 10.1080/07352689.2016.1267498
https://www.researchgate.net/public...nt-day_Genetic_Diversity_and_Future_Prospects



Large-scale whole-genome resequencing unravels the domestication history of Cannabis sativa
GUANGPENG REN, XU ZHANG, YING LI, KATE RIDOUT, MARTHA L. SERRANO-SERRANO, XYONGZHI YANG, AI LIUGUDASALAMANI RAVIKANTH, MUHAMMAD ALI NAWAZ, LUCA FUMAGALL
Science Advances
DOI: 10.1126/sciadv.abg2286
https://www.science.org/doi/10.1126/sciadv.abg2286

The Conservation of Cultivated Plants​

Bruce Benz (2012) The Conservation of Cultivated Plants. ​

Nature Education Knowledge 3(10):4

The Conservation of Cultivated Plants

How can we guarantee the human population will be able to feed itself as it swells from 7 billion in 2012 to 9 billion by 2044?

www.nature.com

 

[exploziv]

My friend sci-hub can help you unlock those research papers as well! For any need that may be to read any of that.

 

[Sam_Skunkman]

For looking up DOI's

sci-hub: https://sci-hub.se

 

[Calaika]

Gotcha!


Sweets!