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InvisibleBuddha420
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Cloning vs print culturing
    #13383752 - 10/25/10 04:14 AM (13 years, 4 months ago)

Let me try to phrase my question correctly. First I will explain what I understand so far about the proccess, correct me if I'm wrong.

My colonization chamber is kept at 85F, please don't start on the temps with me =) The gt strain I'm working with, loves the high temp.

I sprinkle the spores onto an agar mix, that becomes Transfer #0 (T0). A few days later it grows in, I pick out the best-looking patches of rhizomorphic myc., even though sometimes there is none. I usually make 2-4 transfers, depending on dish availablity. Those become T1(A through D, as per dish respectivly). In about 10 days the dishes will be about 90% colonized. I pick the best overall looking dish and cut it up into 4 pieces. Then I place the cuts into 4 jars of rye. Mix them untill I have the kind of placement that I want. In about 6-7 days they will be about half way colonized, at this point I shake the jars up to spread the myc. and increase origins of colonizatin. After this step, it takes another few days to fully colonize. I label these jars as JT1 - Jar Transfer 1. I use all 4 to inoculate 4 sets of sterilized jars. Thats 28 jars, 7 jars per set. My pc cooks 14 jar at a time, so thats two sets of 14. These 28 become JT2. From here I take 4 for next 4 sets, and 24 go towards cake making. I usually use 4 jars per cake, so thats 6 cakes. I case each cake with a 1 inch layer of 50/50+ casing mix (pete, verm, gypsum, and hydrated lime). I also add gypsum to the rye. Then I incubate the cakes for exactly 7 days, after I put them out to fruit. Fantastic results. Here is a post I made to show off some of my cool grows:

http://www.shroomery.org/forums/showflat.php/Number/13383636


Now, finally to my question. Using the references above: with transfer, both petri and grain to grain, when the numbers go up, T1,2,3,4...JT1,2,3,4...at around JT6 the cakes are already fruiting skinny, flimsy fruits. At JT8 they dont fruit at all. Now if you're an experienced cultivator, you would make prints and start the life cycle over to RESET (keyword in my question) the genetics. Recently I've discovered the cloning method. Apparantly I didn't have to make a print, and through the whole ordeal, I simply picked the best looking fruit, took a tiny piece from the inside at the part where the stem joins the cap. Then I follow the same exact steps as before... T0 (except now its CT0)...so my question is, does this technique provide the same kind of genetic "RESET" that the spore culturing provide?

What if I make a print out of a JT7 fruit..sometimes they come out huge, even at that large transfer #. Will the JT7 information be carried over to the print? The fact that it was taken from a cake that has old genetics, or is it a complete reset?

Same question as above, except with cloning. Does it matter if you clone a JT7 fruit or a JT3 fruit?

Does it matter which flush you collect the print or clone from? 1st flush best, 3rd flush worst? Or it doesn't matter? Because in some cases the cake will have too many pins for the 1st flush, so they come out small but packed like hair, no good prints there, because caps are small. Then the 2nd or 3rd flushes can produce insane monsters that make 3 inch wide prints (I'm not kidding, I'll take a pic when I decide to open it again). Fruits are 5-6g alone...question is, does it matter if I took the print off a huge monster with an enormous cap, or from an average looking fruit from the masses of the 1st flush?


Thank you so much for reading this long post with a million questions...when in the beggining I implied that I only had one question....sorry... =P

RR, you are an inspiration. If not for your grain preparation technique, I would be nowhere!! The face you make on that DVD after you take jars out of the PC is priceless. Before I did it your way, I had no 1st flush...3/4 all my grows for a long time produced short aborts for the 1st flush. 2nd and 3rd flushes were normal, even awesome...just no 1st. Thanks again, your DVD, and most of all your posts, have shed much light onto my hobby.


--------------------
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InvisibleDoc_T
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Re: Cloning vs print culturing [Re: Buddha420]
    #13383806 - 10/25/10 04:41 AM (13 years, 4 months ago)

Cloning doesn't reset the genetics like starting from spores does.
A spore print carries genetic potential, like eggs and sperm.
A dikaryonic mycelium is a complete organism like a fertilized egg is.


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InvisibleBuddha420
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Re: Cloning vs print culturing [Re: Doc_T]
    #13383872 - 10/25/10 05:14 AM (13 years, 4 months ago)

Quote:

Doc_T said:
Cloning doesn't reset the genetics like starting from spores does.
A spore print carries genetic potential, like eggs and sperm.
A dikaryonic mycelium is a complete organism like a fertilized egg is.




Can you please see if you can answer some of the other more specific questions I asked at the end of the post?? I explain the "JT7" reference in the 1st paragraph. Sorry if its too much to read...


Also..when I made my 1st clones, it actually shaved a whole 3 days off the fruitig cycle, it now fruited on 17th day, as opposed to 20th. Then when I started using RR's grain preparation technique, I shaved another day and sometimes even 2!! A little over 2 weeks from chamber to full 1st flush.

So I cannot keep taking pieces of mature fruits (or pins), putting them into petri dishes, and then using that to "RESET"?? Will doing that produce the same kind of degerative effect that over-transferring grain to grain does?


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Re: Cloning vs print culturing [Re: Buddha420]
    #13383877 - 10/25/10 05:19 AM (13 years, 4 months ago)

I can't make heads or tails out of that- what is it you want to know?
Are you asking whether spores carry the genetics of the parent caps? Yes.


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OfflineFeelers
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Re: Cloning vs print culturing [Re: Buddha420]
    #13383887 - 10/25/10 05:25 AM (13 years, 4 months ago)

Quote:

Will the JT7 information be carried over to the print? The fact that it was taken from a cake that has old genetics, or is it a complete reset?




It's not quite a complete reset (in a print), it's akin to your parents having another child; it's more likely to look like you than your 3rd uncles' children, but it could look completely different to you also. This is where "strain" (or more accurately race) stabilization comes from. You keep selecting for the children you want, and eventually you will lower the variation in the gene pool down to the traits your searching for. Which often comes with side effects mind you.

If you have a true mono culture, then all your mushrooms should be genetically identical, and cloning from the myc would be as good as the mushroom (in terms of genes involved). 3rd flushes in my opinion are the mushrooms' desperate last attempt to make spores, and the extreme lack of nutrients/metabolite buildup/dessication/cellular exhaustion probably cause all the whacked out diversity that is often seen. Your wife wont look the same after 3 kids either. :grin:

Commercial operations have been fighting the "cloning effect" since forever and it's referred to as strain degradation. They try as hard as possible to create each new batch from as far up the chain as possible, because each transfer step down equals a loss in yield. I don't know how much this would change things for a hobbyist, although I hear that strains do degrade pretty quickly from sucessive cloning, I'm sure I've read somewhere that a spore can only grow so far a distance from it's initial germination(ignoring and including transfers) say like 40 cms before it starts having issues. At a genetic level this all comes down to cells replicating and loosing information in the process. Have a look at Telomeres for one reason why this happens, it's also a reason why we get old. :blush:


NOW - the real thing your after, is cloning from a multispore. It's like a mega random lottery and the big winners in this one are the ones you should look to clone from. The idea would be to start with a multispore and set up your long life culture cloned from the best of the fruits. Remember though that like all life it might be great at one thing but poor at another, in a way that might not be immediately obvious. For example it could be huge in size but low in actives. Also throw into the mix that a clone from a multispore is often not made of just one strain, and it could have 3 or more strains that need to be sectored out on a plate before you get a true "isolated strain" (isolate).

Heavy. :ooo:

I hope that helps, I think I've been accurate. RR is the commercial grower who has a lot of expreience in this department. :thumbup:

Edited by Feelers (10/25/10 05:34 AM)

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InvisibleBuddha420
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Re: Cloning vs print culturing [Re: Doc_T]
    #13383890 - 10/25/10 05:28 AM (13 years, 4 months ago)

Quote:

Doc_T said:
I can't make heads or tails out of that- what is it you want to know?
Are you asking whether spores carry the genetics of the parent caps? Yes.




Look, all I was saying was...grain to grain transfers I labeled as JT1 - Jar Transfer (being 1st jar with the piece of agar in it) then every following grain transfer I labeled JT2..etc. The higher the # the less vigor in the myc. and then it becomes less produtive.

Does cloning from fruit to petri to begin my cycle again create the same kind effect that over-transferring does? Because so far, I've been getting great results, but I've only went through 3 full generations, and I see no degradation yet. So maybe culturing a tiny fiber from a fruit in a petri dish, in some way resets something? Or will I eventually fail with this proccess?


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Re: Cloning vs print culturing [Re: Feelers]
    #13383895 - 10/25/10 05:30 AM (13 years, 4 months ago)

^^^ good stuff.

Cloning does not reset the genetics like starting from spores.
Cloning old tissue results in old tissue.


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InvisibleBuddha420
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Re: Cloning vs print culturing [Re: Feelers]
    #13383900 - 10/25/10 05:36 AM (13 years, 4 months ago)

Quote:

Feelers said:
For example it could be huge in size but low in actives.





Absolutely...I'm working with a sub-strain of gt i made, the fruits are monsterous, caps are 3 inches in diameter...but they're not nearly as potent at the smaller stuff, its very noticable too...

Thanks for the prompt reply...best piece of understanable information to my exact questions. Cloning has actually proven to have good effects...the proccess itself, not the strain I created...because I used diff sub-strains, as soon as I would make the 1st clone culture from a print culture, the clone culture will fruit 3 days sonner than the traditional print culture. I have a million picks with many grows where I've experimented...but I'm too lazy to actually read up on it..whenever I try, the informatio is too hard and inaccessible to me. I'm not that edumucated. =P

Thanks again.



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InvisibleBuddha420
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Re: Cloning vs print culturing [Re: Doc_T]
    #13383901 - 10/25/10 05:38 AM (13 years, 4 months ago)

Quote:

Doc_T said:
^^^ good stuff.

Cloning does not reset the genetics like starting from spores.
Cloning old tissue results in old tissue.




thats MAY not be true...I have a grow in progress I've taken from an old tissue, so far I've put the cakes out to fruit, in 16 days we'll have our answer...if they'll have a monster flush, I'll post it here to show =)


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Re: Cloning vs print culturing [Re: Buddha420]
    #13383906 - 10/25/10 05:42 AM (13 years, 4 months ago)

No, it's true. Read that telomere link, and read about the Hayflick limit.
You may be getting satisfactory results, but that does not change the cellular mechanics involved.


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OfflineRogerRabbitM
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Re: Cloning vs print culturing [Re: Buddha420]
    #13384435 - 10/25/10 10:29 AM (13 years, 4 months ago)

Quote:

Buddha420 said:
Will the JT7 information be carried over to the print? The fact that it was taken from a cake that has old genetics, or is it a complete reset?



Genetics yes, senescence no.

Quote:

Buddha420 said:
Same question as above, except with cloning. Does it matter if you clone a JT7 fruit or a JT3 fruit?



Cloning keeps the same cell lines alive, so always clone from early flushes to keep the youngest mycelium.

Quote:

Buddha420 said:
Does it matter which flush you collect the print or clone from?



As said above, clone from early flushes.  Prints can be taken from any flush.
RR


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Re: Cloning vs print culturing [Re: Doc_T]
    #13384503 - 10/25/10 10:44 AM (13 years, 4 months ago)

hello,

Reading your post it seems to me that you have the basic idea of transfers and traditional transfer technique down pat, It seems to me that your basic question revolves around strain viability and preserving the vigor of youth in your strains for the purpose of ongoing spawn run efforts.

I was surprised to see that you are experiencing a decline around gen 6, from my own experience GT that I have worked with do not experience this decline until well past the 50th generation. ( I will admit to having pushed so far in my early days of cultivating but did not push it so far all by myself ) I would then wonder if there was some sort of environmental condition contributing to this decline.



Regarding your question about genetics, it has been discussed here in the past inside of recent memory the question of strain viability and the question of sub-strains. Every time you take your strain to spore and then restart new genetics from spores, on agar you have created a new individual descended from a common genetic base. Although this strain has a common genetic ancestry, it is as unique as you are from your cousins. It may have similar features, and act in a similar manner, but is a unique recombination of the genetic potential that is inherent in the gene pool present in that strain. It has been discussed here regarding the question of custom engineering strains, and I would say that it seemed the opinion of most members that after 7 cycles of re-origin through spores that you have the potential for separating out a distinct sub-strain that is notably different from the originating parent. This conversation was in context of the originating genetics being wild, and depending on where and how your genetics came to you it may be that this has already occurred, possibly a further explanation for experiencing senescence at gen 6

Having experiencing senescence with the very mushroom you describe I can tell you soon after I became very interested in the process of preservation and I will share my take on the subject.

When I receive new genetics in the form of a spore print, I will sample a small portion with an inoculating loop and transfer this material into a test tube 2/3 full of sterile water. I let this tube sit in my lab occasionally picking it up and shaking the hell out of it, observing the clumps in the bottom to get a feel for if they are breaking up and re hydrating. Many spore prints have dried out and I feel as it takes a while in immersion for them to want to come out to play. This tube is ignored until I get around to pouring a special run of non peroxided agar, so it may be several days to a week before they are let out and worked with further. I use 60mm plates because they are cheap and I pour no less than 20. If a strain is valuable I would do any where from 20 to 40 plates.

After I am satisfied that the 10 or so ML solution in the tube is saturated with many invisible to the naked eye spores in solution, I prepare a 500 ml beaker with about 300 ML sterile water solution, and combine the contents of the tube with the water int the beaker that is then placed on a stir table and I then proceed to whip the hell out of it to ensure maximum dispersion. When I think that there is adequate homogeneous solution, while stirring, I use a sterile syringe and draw 10 ml at random. I then put 1 drop on each plate and roll the drop on the plate until the entire surface is wetted. The spores are going to tend to stick to the agar, and arrive fully hydrated, thereby providing their best chance for germination.

Getting a sense of proper dilution is the key to seeing individual points of origin, most folks tend to put their spores too much in concentration, causing so many points of origin on top of each other that they then become difficult to separate out into non sectored strains. When properly diluted the spores find each other on the agar surface, form dicaryotic myc and then go from there. Then the playing field is level and you can properly see that all strains are not created equal and then you may pick your genetics.

If I do 40 plates for something that is very valuable to me, I may take 5 to ten subcultures on 60 mm plates, each labeled F1 to show generation, but also labeled "A,B,C" etc. to show unique points of origin. After growing out and showing no signs of sectoring,  I usually have a 100% success rate in achieving an isolate in 1 transfer. I may subculture onto peroxidated Agar to ensure that there are no hidden contams hitch hiking on the myc, mold is obvious, low levels of bacteria isn't always so visible.

At this point I may have 10 candidates for cultivation. Many times I have prepared 20 or so PF tec style jars, inoculating them using a LC/fragmentation technique, and then injecting each jar. In this example each candidate might get two jars that are labeled "A,B,C" etc these jars are then incubated together and set into fruiting conditions side  by side, while maintaining their identity markers,

Proof being in the pudding,, I make summary judgments of the mini trial, some strains looking great on agar proving poor providers of fruit, other strains refusing to fruit at all (rare) and other strains emerging as the obvious winners.


At this point you are ready to preserve your strains in culture slants. Many times I have inoculated slants from the F1 generation that were sub cultured from the multi-spore germination (MSG) These slants are then labeled as F2 with a date and which MSG sub culture provided their origin. I usually only keep 2 or 3 winners from this process, making at least 2 slants for each sub strain, just in case. Typically one slant is the designated mother and the other left undisturbed. When I make one plate from one of these slants, then I find that I can usually subculture that plate over many generations, usually no longer than a year of spawn generation, depending on how much I run a particular strain, I generally make no more than 10 transfers from the slant before returning back to said slant to start a new path of descent. Accounting for the potential for making sister plates from a single mother, this gives you unlimited potential for spawn origins while maintaining genetic integrity, viability and isolation of any given source material.

A clone is a copy of the parent mushie, and is be genetically identical to the individual myc that gave that fruit rise. If you clone a mushroom that is already experiencing strain senescence, there is a temporary re-invigoration of that material and it's ability to provide fruit, that soon drops out after 3 or 4 generations of transfers.

Cloneing is an powerful tool, one that I mainly use on wild mushrooms, or mushrooms that are hard collect spores from.  Every wild clone that I have collected had demonstrated a vigorous willingness to fruit. Wild genetics are powerful because nature maintains multi-strain genetics in any given patch, that patch then transports via wind or water many genetically compatible spores from different individual parents, that then recombine to from unique strains from the common gene pool. These mushrooms are so strong in culture that I find little use for spores, other than back up and extremely long term storage, and preservation efforts.


Also important to note, you indicate an interest in an earlier post showing an In-vitro mushroom that had dropped a print inside a dish lid, I rigged my plate incubator with a 15w daylight compact fluorescent bulb for the purpose (primarily) of providing a low out output heat source that is regulated by a thermostat. Intermittent exposure to daylight stimulates some plates to fruit in vitro, giving me clues as to the willingness of certain sub-strains to fruit over others the pic you found was a part of an experiment to find out if precursors in substrate affected potency, the particular dish in question was fruiting stimulated by having placed one grain of sterile reed canary grass seed next to the inoculation point in the dish, combined with exposuere to day light,VIOLA!!



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Re: Cloning vs print culturing [Re: RogerRabbit]
    #13384607 - 10/25/10 11:16 AM (13 years, 4 months ago)

Hi; not only are you dealing with the older cellar life cycle clock that a tissue clones would yield. The spores derived from the fruit bodies of multi generational monoculture clones have a tendency to be weaker growers over time. This is caused by both the lack of genetic diversity within a mono culture and the genetic adaption to the near sterile in vitro growing conditions that we grow in.

There are a number of us that have been around long enough to have seen this in years past. Back at the Alamo when growing with Jim Bowie, no not really so let me digress. Back in the early 80’s when there were few parent strains. Over time you would see this declining vigor and ability to stay off contaminations with each subsequent grow. Then someone would find some old spore prints that were still viable and the cycle would start over again. That’s the beauty of people like Mushroom John and other hobbyist that is introducing new strains and fresh strains from the field is a fresh gene pool.

What I learned personally from this experience is that multi spore is better over the long run than a mono culture.  That when you do attain a new or fresh strain of spores that grows out with desirable characteristics (fast colonization, contamination resistant’s and superior fruit crops with multiple flushing) is to get as many spore prints from the second flush of the second grow as you can get and store them as long term insurance.

But this is just my option and options are like ass holes we all have one; CheeWizz

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OfflinePrimalSoup
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Re: Cloning vs print culturing [Re: Buddha420]
    #13387223 - 10/25/10 08:53 PM (13 years, 4 months ago)

Good info ^^^^^ :bow:

Note with care what Mycoelf said about how the genetics came to you.  The further from the wild a strain is, the more selected it is for certain characteristics, the less genetic variability remains.  If the strain you're fruiting is distant that way from the wild it may have a much shorter time to senescence (the G6 performance) than might another one that hasn't been stepped on so often.

The way "strains" are marketed doesn't help this situation any, and I suspect the way "strains" are passed around has something of the same effect - it's been said before but how many people in their own cultivation (and dissemination) are careful to keep the "strain" close to its supposed phenotype?

Anyway, that's why wild prints are of interest to me, allowing minimal selection for the qualities I'm looking for, without having to inherit selection I may not care about (color, ability to fruit upside down, massive massive fruitbodies, technicolor spores, whatever).

Other than that :popcorn::nothingtoadd::lol:

Peace
-PS


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Re: Cloning vs print culturing [Re: PrimalSoup]
    #13387360 - 10/25/10 09:16 PM (13 years, 4 months ago)

Quote:

PrimalSoup said:
Good info ^^^^^ :bow:

Note with care what Mycoelf said about how the genetics came to you.  The further from the wild a strain is, the more selected it is for certain characteristics, the less genetic variability remains.  If the strain you're fruiting is distant that way from the wild it may have a much shorter time to senescence (the G6 performance) than might another one that hasn't been stepped on so often.

The way "strains" are marketed doesn't help this situation any, and I suspect the way "strains" are passed around has something of the same effect - it's been said before but how many people in their own cultivation (and dissemination) are careful to keep the "strain" close to its supposed phenotype?

Anyway, that's why wild prints are of interest to me, allowing minimal selection for the qualities I'm looking for, without having to inherit selection I may not care about (color, ability to fruit upside down, massive massive fruitbodies, technicolor spores, whatever).

Other than that :popcorn::nothingtoadd::lol:

Peace
-PS





Starting over from a wild print is an enormous amount of work...I remember 5 years ago when I got my 1st print from the internet....it looks nothing like the stuff I got going now, and its a variant of that original print from 5 years ago. So that's 5 years of continuous  selective breeding. The fact that I have a monoculture now is for sure...

Question: What exactly is the result of making a single cake from jars of various sub-strains?

In one of my experiments I got fruits that look like neither of the sub-strains used to make the cake. But have characteristics of both. Does that make any sense?


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Re: Cloning vs print culturing [Re: Buddha420]
    #13387493 - 10/25/10 09:42 PM (13 years, 4 months ago)

:popcorn:

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Re: Cloning vs print culturing [Re: total]
    #13387663 - 10/25/10 10:19 PM (13 years, 4 months ago)

:popcorn:


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Re: Cloning vs print culturing [Re: nexus1946]
    #13388394 - 10/26/10 01:03 AM (13 years, 4 months ago)

Quote:

total said:
:popcorn:



Quote:

nexus1946 said:
:popcorn:




:hehehe:




Alright...lets slow down a bit...can someone give an easy, comprehensible explanation of what a monoculture is...then whats a multi-spore culture? Are we still talking about the same sub-strain? You see, I lack the knowledge of basic genetics..what I possess is observational experience. If I make so many xfers...it degenerates...if I start from print, it resets...those were my basic guidelines and I never bothered to go deeper into it. Untill I opened Stament's book again and came across cloning...which looked soooo simple, and saved time too, compared to culturing from a print. So hell, I started making clones. Before you know it, by 8th generation of clones, they got really skinny with small caps...at 1st i thought i had FAE issues, but no....identical fruiting environment, different genetics, and bam! Great results.


So, inspired by the great information in this thread, I have found an old print from 06, I think its the oldest I have..the great big problem here is that I never cataloged stuff up until recently when I started tracking the genetic makeup of my cultures. By tracking I mean, give sub-strains names and preserve as much information about transfers, amount of transfers, etc...this print I have found has nothing but the date on it. BUT at the time I made the print, I did not know about cloning, and was only using the print culture method, which is the right way I guess...well, maybe not right, but different for sure =)

I actually thought the bigger the better...I guess I found out the hard way...when most of my yield was like this:

1-3 fruit per flush....BUT WHAT FRUITS!!! this was 1/4 oz alone







i took a print from it:









here is another example, but here the monster is from a different sub-strain...this is the cake that was made from 2 jars foreign online sub-strain and 2 jars of my cloned sub-strain...








now the rest of my stuff looks nothing like those....here is a pic of an outstanding healthy cake, this is the 3rd flush of a 3rd generation clone.










this one here is the 1st flush of a 4th generation clone...still looks great. This cake here was made from 5 1qt jars, and ended up giving up 4 great flushes.





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Re: Cloning vs print culturing [Re: Buddha420]
    #13388905 - 10/26/10 06:22 AM (13 years, 4 months ago)

Quote:

You see, I lack the knowledge of basic genetics




It's a big topic. Start at the beginning, and work your way into it.
Spores combine genetics like eggs and sperm do, so an understanding of human genetics is applicable.


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Re: Cloning vs print culturing [Re: Buddha420]
    #13390660 - 10/26/10 02:37 PM (13 years, 4 months ago)

Quote:

Starting over from a wild print is an enormous amount of work...




Maybe so. :lol:

Quote:

Question: What exactly is the result of making a single cake from jars of various sub-strains?




It's whatever you get - I only do clones...

Quote:

.it looks nothing like the stuff I got going now, and its a variant of that original print from 5 years ago. So that's 5 years of continuous  selective breeding. The fact that I have a monoculture now is for sure...




This is what's going senescent on you now?  No wonder. :shrug:  Again, you don't propagate from clone to clone to clone - you want to make an isolate (clone) that performs well and preserve it at the earliest point in time you can.  Then you return to that preserved culture for new grows.

Peace,
-PS

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