Cloning Plants By Tissue Culture

pharmacoping said:

whoaaa, you a smarty pants guile ! thanks man, good to see a serious mind here.
I buy it already diluted, because I never learned how to drive a calculator

Click to expand...

I buy mine in nearly pure/powdered form so I don't have to deal with unknowns.
I didn't go digging for a calculator either (if I messed up that's why) all the math here was a factor of 10, all I had to do is move the decimal point (the kind of thing you feel comfortable doing in your head when you are stoned). Its partly why i didnt want to get into figuring out everything's specific mass to use volume and density to sort it out... Especially since it felt like I was going to have to do all the looking to get the figures I needed... and it would have required that I payed attention to my math....

but with water having a specific gravity of "1" (meaning a liter weighs a kilogram) it just made all the math simple (a factor of 10, so I only had to move the decimal point). The only other consideration is that ethanol has a specific gravity of about .780 (meaning a liter of it weighs about 780 grams).. Its my understanding that alcohol (drinking alcohol) is not flammable at room temperature in concentrations under 50% (100 proof).. meaning that the base solvent couldn't be more than half ethanol. Half the difference in specific gravity between water and ethanol is 11% so your margin of error dealing with the unknown base solvent would be less than that (10.something at the most).. this would translate to similar accuracy in your final solution.. Besides you are only accurate as you can prove, anything beyond that is strictly speculation...

nitro20% said:

yep should have went with a less concentrate. newbie's!!! think that we can do cheaper/better.no you are right with that one. Thats how i was thinkin it was broke down.. but listen to this one: NxE=JUSTBUYTHEKIT with a remainder of PRESURECOOKER!!!ha aha ha haha. Newbie x Experience= always a realization.but back to work (with pictures i know)i think i have to re-size them cause that firewall thing..connection..etc. would let this old ass Dell through.lights closet,hepa running now,bleach n clean. to ace for plastic(mini lab)powered gloves no good. to my buddy's ,oshit things to do, cant rush this.LATER!!!oh goin with @1000ml-1 to 1.5 ppm iba, which will make ppm naa @ .5 lower,,perfect!!!

Click to expand...

If you haven't figured that one out yet its just 3 drops per Liter

The concentrations you describe seem low to me, unless the cells you want to influence are in like constant direct contact with it... Are you putting this stuff into your culturing gel? or using it in an aeronautic or DWC type cloner?

It seems that mimicking the culturing gels they use here (to promote rooting) in a liquid solution (simply omit the gelling agent) should make about the best cloning machine solution ever..

Guile said:

If you haven't figured that one out yet its just 0.75ml/L, apx: 3ml per gallon or Tablespoon per 5gal jug..

The concentrations you describe seem low to me, unless the cells you want to influence are in like constant direct contact with it... Are you putting this stuff into your culturing gel? or using it in an aeronautic or DWC type cloner?

It seems that mimicking the culturing gels they use here (to promote rooting) in a liquid solution (simply omit the gelling agent) should make about the best cloning machine solution ever..

Click to expand...

yeah i know they seem low,,but for all i read on tissue cultureing for the harder woods like/and mj(theres alot of studies on tissue culturing on mj) they all seemed to use a .5 to a 2.0 ppm of iba for the first to weeks or so, and naa run about .5 ppm less. NOW,, im with that other guy, every thing i say is a lie QUOTE.ive had no/to nill guide (except for u guys) so what ever i made up is the truth so far,,with no results...but its easier then that.....DDDDUUUUUU,,,1 part dip n grow to 19 parts water=50 ppm!!!!so easy its hard!@#!@!#$ SSSOOO @ 1ml (concentrate) per 1200ml water= 1.5625 ppm. YYYYEEEEEAAAA i did it.im confident with that! to my buddys house,, theres some sick plants to cure!

nitro20% said:

yeah i know they seem low,,but for all i read on tissue cultureing for the harder woods like/and mj(theres alot of studies on tissue culturing on mj) they all seemed to use a .5 to a 2.0 ppm of iba for the first to weeks or so, and naa run about .5 ppm less. NOW,, im with that other guy, every thing i say is a lie QUOTE.ive had no/to nill guide (except for u guys) so what ever i made up is the truth so far,,with no results...but its easier then that.....DDDDUUUUUU,,,1 part dip n grow to 19 parts water=50 ppm!!!!so easy its hard!@#!@!#$ SSSOOO @ 1ml (concentrate) per 1200ml water= 1.5625 ppm. YYYYEEEEEAAAA i did it.im confident with that! to my buddys house,, theres some sick plants to cure!

Click to expand...

One of us is off by atleast decimal point (I'm willing to accept if its me, but please point out where).. I get 20:1 making 500ppm and 1ml to 1200ml water to get 8.3ppm

1ml to 6.5L (about 7 quarts, or 1 and 3/4 gallons) should give you about 1.5ppm (IBA and 0.5ppm NAA) if my math is right..

If there are 20,000 drops in a liter (20 props per ml) , and your concentrate is 10,000ppm then you will want to use 3 drops per liter to achieve 1.5ppm

Brings to mind how they say to only use one drop per gallon super-thrive, most everything I read seems to suggest the ideal concentration of vitamin B to be quite low sometimes around 0.5ppm.
The concentration of "B vitamins" in "Superthrive" must be similar to the hormone concentrations in "Dip n Grow"

thats funny cause last night right at bed time 3:00 am i came up with that same answer. and this morning with the final confident answer it was about 3-4 drops in a liter @ 1.5 ppm iba.which is what im doing right now!!! (WITH PICS I KNOW) gettin my mini plastic lubor-u-tory' ready. cooked the tools and got king kush, diesel, blue dream, head band,grand daddy purps,and my own afi-gui-snow roturallas. told u it was a universal language. i did it probably wrong but got the same result,,,thats scary!!!this is what i did, just took and made the dip n grow scale into ml per parts water. then stared with 1ml concentrate @ 199ml. parts water still equals 50 ppm.then to the calculator divided by 2 a few times,, 1ml concentrate to 398 parts water = 25ppm iba. 1ml to 796= 12.5ppms iba.etc etc...?yeah?no? the reciepe is 3 drops of concentrate to 1l , hopefully reach 1.5 or so.cant be to picky,i picked this cat.

nitro20% said:

thats funny cause last night right at bed time 3:00 am i came up with that same answer. and this morning with the final confident answer it was about 3-4 drops in a liter @ 1.5 ppm iba.which is what im doing right now!!! (WITH PICS I KNOW) gettin my mini plastic lubor-u-tory' ready. cooked the tools and got king kush, diesel, blue dream, head band,grand daddy purps,and my own afi-gui-snow roturallas. told u it was a universal language. i did it probably wrong but got the same result,,,thats scary!!!this is what i did, just took and made the dip n grow scale into ml per parts water. then stared with 1ml concentrate @ 199ml. parts water still equals 50 ppm.then to the calculator divided by 2 a few times,, 1ml concentrate to 398 parts water = 25ppm iba. 1ml to 796= 12.5ppms iba.etc etc...?yeah?no? the reciepe is 3 drops of concentrate to 1l , hopefully reach 1.5 or so.cant be to picky,i picked this cat.

Click to expand...

Go with what is recommended by the manufacturer. my margin for accuracy can't match theirs (they know whats in it).

Though if you had those figures yesterday we might have gotten here sooner..

If you give me the mixing instructions from the label (or package insert) there should be enough information to figure most any concentration out..

its that web site. ppm of dip n grow. thats what i went off of.its say hhhh nothing, no box, but it did come with a 1 to 1 dilution cup. @ tsp per. concentrate line IS 1tsp. im trying not to 2nd guess myself but man if my little break it down by 2s equation seemed pretty accurate. 1 ml per 1200=1.5625ppm.every time.and thats 15 to 20 drops per1200ml.broke that down 6 times so 9 to 14 drops per l.but the thing is,, ratio of 3 drops per L, will not hurt the first 10 day stage, to weak.i will have some time to "get it together" so what do u think. well i guess thats what u been doing.

i take it , the chart on their website wont help you none nitro ?

i dont speak tissue culture, but i will .... gotta LOT of reading to do.


can you guys lay out some real simple info for me ?

are these pieces of plant sitting in a hormone or solution , in an airtight environment ? or is this stuff open in a sterile "lab" (or box)





soil

I just found this PDF file... PARTS-PER-MILLION MATRIX - Dip'N Grow (I can't believe you got me to do your homework) This has all the info you need on it and would have made it pretty simple to figure out any concentration you would need based on whats listed.

Considering that the undiluted strength is 10,000ppm I have a feeling we figured things out pretty well yesterday..

3 to 17 gives you 1500ppm (thats using 3 parts concentrate out of 20 parts total) given that it is 1000 times as strong as you want you would apply a 1000 to 1 ratio making 20,000 parts only 3 of which are concentrate. 3 parts out of 20,000 with 20,000 drops in a liter means 3 drops per liter..

If you use 1:199 (1 out of 200) = 50ppm which is 33.3333 times as strong as you want. so you multiply the water used accordingly (to get the correct disillusion) you end up with 1:6633 (6.63L assuming you used 1ml concentrate) otherwise 1 drop to 331.65ml (332 plus 1 equals 333ml or 1/3 of a liter, or 3 drops per liter)

If I'm not mistaken my math from yesterday showed that 1ml to 7 quarts or 6.62L (10ml, less than a tablespoon of water from the above formula) would give you "about" 1.5ppm
6620ml divided by 20 (the number of drops in a ml) equals 331ml per drop or 3 drops per liter (give or take a drop or 2 of water).

I would be inclined to use the figures biased on 1:199 as it is the closest to the scale you are trying to obtain (smaller margin of accuracy). Or my figures from yesterday, they seem to be more accurate than I had hoped.


About the 1:1200 thing...
Starting with 1 drop @ 10,000ppm, adding 1000 (999) drops of water will get you to 10ppm (this is way more than 20% from where you want to be) adding the other 200 drops (for a total of 1200) will give you 8.3ppm (like 5 and a half times as high as you want to be)

This is a very interesting read! Just read the beginning and I'm intrigued! Gonna roll up a blunt and read up a lil more... Quick question does this also apply to weed plants? I don't see why not but you never know..

Boyz N Da Hood said:

Quick question does this also apply to weed plants? I don't see why not but you never know..

Click to expand...

yes , any plant. i think. im sure some are a lot easier then others. i dont even speak the language yet , but its very interesting.





soil

The way I have it figured you will probably need a pressure cooker (the kind they use for canning meat and low acid foods) and some wide mouth canning jars (1 cup jobie's if you can find them, I have some 12oz ones and they seem a little tall).

You can probably make your incubator out of a couple plastic (Sterilite) totes nested together to make a warm water bath using chlorinated water and fish tank heaters for temperature control. I would probably cover the rim of the upper bin with weather strip (make as air tight as possible) and mount a UV light inside the lid and just leave it running because the metal lids to your jars will keep most the light off your culture so it should be relatively unaffected however it might cut down on contamination in the bin (I have an ozone generator idea too that could maintain darkness but it might be a little harsh).

Other than that alot of sterilization (in your pressure cooker or using alcohol). I have seen a few recopies for making the gel, just add that mixture to your jars before pressure cooking them. Allow jars to cool to room temperature before adding the plant material you prepared with sterile tools/instruments, and procedures (if you wanted to introduce a virus you would do so at this point). Wash your hands, tools, jars, and working surface with alcohol before each opening/or use of the tool, incubator, or jar. (lint free alcohol wipes might be handy).

It seems doable... I have a feeling the "don't open the door until its done cooking" rule might apply.. I would probably prepare several jars checking each with a diminishing degree of frequency. check the same one every day, another every other, the following every 4th, so on.. As fungus and other contaminates claim victims just move the others up in queue. The odds are the air in your room is contaminated, so it might be a struggle (though I guess you could make a "clean room" out of plastic sheathing, wear a respirator/mask, and build a bigger ozone generator, but seems like too much hassle for some clones).

This 1.5ppm IBA 0.5ppm NAA ratio we worked out getting from Dip'n Grow should work for rooting (I personally considered using it in a DWC cloner), I assume you would also add some nutrients (I've even seen sugar) and of curse the gelling agent before pouring into your Mason jars and pressure cooking (per instructions for canning meat). This should work on a pretty small growing tip (like micro propagation might imply, it would just be a very small cutting).
If you want to create a new growing tip from cells harvested elsewhere it seems like it could be a bit more involved..

no fish tank heaters, or chlorine baths necessary. absolutely NO UV lights,or ozone, other than that, pretty close.

I naturally check my baby's often, as transfers happen daily sometimes. a small 100 watt 4 lite T5 fixture hangs over the totes on a timer. storing/growing a clone is exactly the same protocol as culturing a new growing tip , cept for the recipe. dont open any containers unless doing another sterile transfer. a closet works great. mites are your enemy dust mites love the recipe, they carry mold. keep the area sprayed with lysol, and no traffic. really is no big deal. you'll see. sugar is a must in culture, they are not photosynthesizing to make carbs, so sugar is tube fed. pressure cooker, aluminum foil, paper towels, scalpel, tweezer, baby food jars/lids, bunson burner, denatured alcohol. no respirator, tyvek suits, or sterilized rooms here. I use a simple grow tent, spray lysol upon entry/exit, inside and outside the tent. your mold will come from your explants, and sterilizing those pieces is really the only difficult part. next to figuring out recipes

small bits of plant material are either suspended or laying on, or partially submerged in an in vitro specialized mix gelled, and then sterilized in a pressure cooker, while being sealed within their incubator, a baby food jar/lid is awesome for rootballs. the collection of jars is kept in a still, clean area with some light. transfers of these cultures to new vessels, or dividing them to make multiple "clones" is done in a tipped rubbermaid, wiped with alcohol, and quickly done. one hand will open the jar while the other is tweezing bits into it, and closed quickly. the jars are not "canned" with tight fitting snapped lids after a transfer, but will preserve the gel for a long time if sealed and dark. temp should be 70-80 f, lite on 14-16 hrs a day. roots get none. hormones(cytokins and auxins) sugar,agar,water,correct basal salts,plant preservative are in the mix at varying rates.
I stock hundreds of rootballs of specified strains, until needed to sprout. they are transferred to a sprouting/shooting mix, covered, and then go to the veg room in cycle. roots are divided daily to replenish stock.

hope this helps

yep you are 100% right....i came up with that number (2-3 drops per l.) before i found that site. sorry man i thought i put the page up once(about the ppm).and my math was all messed up in the last entries ,,the funny thing is,,,is rhat i 2nd quessed our stoner math and change it...not goin to say what i did,,but i swear i saw the tiny little plantet pieces go from a kinda limp to straight up, kinda like when the wind blows up the hole of my pants,,,booing! so either it will work or not.if not it was good practice for sanitation. its hareder then u think. at the last bit of mix i started to notice hairs and things.i thought i was keepin everthing covered well.i wonder if there is such contaminates,, if the pressure cooking takes care of that to.i have agar comeing,, the health stores prices are 11$ for 6 tbls.holy shit..so if this turns into hot jars,,which is very likeley,,ill do it again @ 2-3 drops per 1000l.

im a first batch, heard about tissue culture decided it was cool 3 day ago PROFESSIONAL,,, beeeiiiiiitttcchhhheesss!!! JUST KIDDIN, K this is what i know,,no results,,,but the first cutting, the actual top from the mother,,goes in to a mostly glucose based and a form of plant preservative material (PPM)or(nadcc)and gel agent with VERY WEAK!!! iba & naa.these planlets are like premature babies basically with no nerve stucture,,just a tissue with parts or a cell to start from,,from here the goal is to reach CULLIS state. which is when the plant cells know exactly what to do and were to go..by then if all things are a go, the plantlet u started 40 days ago can be split into 3 to 10 plantlets a jar.some for more micro-production and the ones for soil treat like barely rooted clones for 7 days,,till strong enough to GO FOR IT!! fuck,,,its fun though..SUGGESTION: do not buy or get any thing till u are SERIOUS BOUT THIS,,,CLEAN ROOM,PRESSURE COOKER, A PLACE WITH NONE TO NO MOVEMENT,HEPA FILTERS,E.T.C.THIS IS NOT ANYTHING LIKE CLONNING OR ROOTING A PLANT,THIS IS A WAY TO WEEN OUT A PLANTS DEFECTS DUE TO AGE,PEST,E.T.C. OR IF YOU REALLY HAVE NO LIFE TO LIFE AND NEED SOMETHING TO DO (ME)THE KITS ONLY GIVE PART OF THE THINGS NEEDED FOR 90 % RESULTS AT BEST (I THINK)I BOUGHT EVERY THING I NEEDED AND IM IN IT FOR 300 +$(no kit). I HAD AQUARIUMS,HEPA FILTER MACHINE (200.00), LIGHTS, VENTING, ITS GOES ON AND ON.. THATS JUST THE BASICIS,,I THINK..



I might be really high, but this is my experience,,please for the overly confident kit makers,,no hard feelings,,but every ones sanitary view might not be enough for the home they live in.cats,dogs,birds e.t.c. In the 3 years ive lived in this house,,my shoes have NEVER been past the sweep of my doors. my dog gets bath once a week, i am to say the least CLEAN.
yep,,you are right, i have NO life. not by choice though,,lets just say,,if u are deciding to take up a meth hobby,,,use clean needles!! it might come bite u right in the LIVER 12 yrs. later.....

oh and to make 1,000,000,000 plants in 1 year from 1 planlet, that Is the main goal in micro-reprodution by tissue culture..
damn dip and grow could turn into dipnGO GO GO GONE!!! move a bond from here to there and you can get some drastic effects, especially if proteins can break apart the rings at the changed points and the change of the bond location in the pyrrol ring worries me a bit). The long butryic acid chain seems like something that could be a candidate for replacement to produce illegal tryptamines or their analogs. My question is, "Given that tryptamine is banned in this paranoid age, have we found a new widely available precursor chemical?"quote to a study

oh and sorry to ALL of us that had to listen/read that illiterate excuse for math i was 100% responsible for.note: when breaking down ppms by 2s, must take that new volume and double, but not the parts per million in question.please feel free to pass warning,,i deserve it.

pharmacoping said:

no fish tank heaters, or chlorine baths necessary. absolutely NO UV lights,or ozone, other than that, pretty close.

I naturally check my baby's often, as transfers happen daily sometimes. a small 100 watt 4 lite T5 fixture hangs over the totes on a timer. storing/growing a clone is exactly the same protocol as culturing a new growing tip , cept for the recipe. dont open any containers unless doing another sterile transfer. a closet works great. mites are your enemy dust mites love the recipe, they carry mold. keep the area sprayed with lysol, and no traffic. really is no big deal. you'll see. sugar is a must in culture, they are not photosynthesizing to make carbs, so sugar is tube fed. pressure cooker, aluminum foil, paper towels, scalpel, tweezer, baby food jars/lids, bunson burner, denatured alcohol. no respirator, tyvek suits, or sterilized rooms here. I use a simple grow tent, spray lysol upon entry/exit, inside and outside the tent. your mold will come from your explants, and sterilizing those pieces is really the only difficult part. next to figuring out recipes

Click to expand...

you make it sound simple, man i was having a rough time thinkin i was not being clean enough.like i said there was hair in the mix at the end,but it got in there as i filled the jars, probably. have a 30 gal aquarium that i turn on end with 2 layers plastic,1st is THE COVER. then there is the one that i put [+ +][ +] holes in the two openings.so is it ok to take the pressure cooked med. and put them in my tank (91% alcohol & gloves every step of the way) to cool and so i might cook more or cuttins ready? how long is SAFE for an unsealed lid protected from the particles of death that fall @ 1 ft. per min.??ha ha. man, they have been cultured for about 9 hours, 1st look, i must be high as fuuuukkkk, could they be,,, should i say it?? are they GROWING? could it be that fast??????if i "HOT JARED THEM" (ppm) would i see signs of the to?

I leave the jars in the pressure cooker overnight to cool, while hot they draw air into themselves, ...........unless transferring or hardening off, the lid should stay affixed to the tops of your containers. some tape or wrap the containers. I've considered keeping the containers inside zip locks for more protection, but has not been necessary in yrs since I moved into the tent.

yep, you see growth really fast. i didnt cut arm holes in my latest sterile tub transfer area, no issues . arms,gloves,outside of jars, are all sprayed with alcohol/lysol, seems good here

pharmacoping said:

no fish tank heaters, or chlorine baths necessary. absolutely NO UV lights,or ozone, other than that, pretty close.

Click to expand...

I find that my cuttings seem to respond well to warmth.. I keep my place pretty cool in the winter months 60-65F (many basement "project arias" never go much above that without adding heat somehow) I have noticed an improvement in rooting times when I bring cuttings up in temperature to around 70-75F (actually I have gone as far as 85F without running into problems).

I know this might sound silly but I believe a plants bimetallism is pretty closely related to heat..
The only reason I said to use chlorine in the water bath is for when you take out the second (upper) bin (that you keep your jars in) an set it on something else to top off the water bath you don't transfer contaminant.
I chose that method for temperature control because I always have fish tank heaters around (I use them to culture other things in water) and I find heating mats/pads to be less accurate..

I haven't used UV light or Ozone around small cuttings before so I have no idea how harsh you can be before damaging your plants, I figure UV defuses pretty quickly over distance so keeping it near the lid (where the air will get in) and with the metal lids on the jars it might be a good way to manage that micro environment.
I have used homemade ozone generators to "sterilize" my grow room. A healthy dose of ozone after lights out can help keep large/dense buds from rotting as they mature (you just put it on a timer like you would your lights or pumps). A large enough dosage will even take care of a mite problem (though you would have to let it go for hours).

I just figured biased on the amount of failure I have heard about that keeping the cleanest (and most regulated) environment should yield the best results..

I agree, cleanliness is key, but I wouldnt keep any water anywhere near the growing cultures, even bleach water.
Ozone will steal your terpine profile in a grow room, not a good idea except for maybe exhaust, if you dont have a sealed grow.

the t5 lamp inside the tent keeps temps around 70-80, esily maintained in a tent, in a closet, in my lab room, which is climate controlled, and also where traditional cloning was taking place.

lowering humidity and increasing air flow is what will stop bud rot in its tracks, very effectively.

My opinion, 99% of my mold failures in vitro are due to infected explant material, not the pressure cooker, or room, or tools. everything is flamed off before coming in contact with another specimen. the jars, once taped up, have been impenetrable thusfar to any mites or mold, here, since moving inside tent a year ago. more failures were seen when the grow chamber was on a shelf in a room, but not one jar has gone moldy (in the tent) if they've made it for 5 days without. dirty explants cause me mold within the first few days of transfer, occasionally. and I have killed explants with bleach, alcohol trying to achieve this issue, until last year when I got good at it. I used to transfer inside a sterile zip lock, with the vessel and tools inside ! that worked very well for me, as my head could see the view from the top, very helpful with timy bits.

I like the "mote" with the two bins, but believe its overkill. 10,000 ppm(bottle of C02) for a couple hours will kill every living bug in your room, and please your plants. repeated with the breeding cycle of mites(or any pest) will rid the garden for good....then, dont take in stranger clones, unless tissue cultured, of course !!