Tissue Culture Information

[theberry]

Hi Everyone

Here is a little information on Tissue Culturing let me know what you think and if you have any questions



As long as you can keep a clean area you can tissue culture. TC (tissue culturing) helps regenerate the plants cells by multiplying them. When you clone with traditional methods you harm the plant and over time the plant is no longer able to produce fruit as well as it use to. With tissue culturing it rebuilds the plants cell structures to make it stronger and healthier to produce a better fruit. Compared to a mother plant they are mini versions of adult plants and grow to it environment. Unlike cloning you use less water, less light, and less maintenance. It does take a little more time to do the actual TC but there is hardly any maintenance once you are done. In the end the plants yield more and are fuller greener and healthier.

The hardest part of tissue culturing is keeping it clean

All Hormones listed are needed throughout tissue culture developments. Strangely enough sugar is a primary base, it provides carbohydrates to the plant, without carbohydrates, the plant can't grow. (For example: when you cut flowers, sugar is added to the water as a food source.)

BA is a hormone used for the grouping of tissue cells.

NAA is an organic hormone used for stem, root, callus, and leaf growth. NAA can also be utilized after plants are put into the ground.

PPM is a plant preservative added to increase plant life during the In-vitro stage. There has been no scientific proof that plants need PPM. (In-Vitro/artificially grown in medium)

TDZ and IBA and INA and HCI are used to assist in the rapid tissue growth of all cells.

A cannabis plant may have between 5 to 30 chromosomes (and average of 1,500 genes for each chromosome). There are 20-30 types of cannabinoids (which are glands and secretions from glands in the cannabis plant). Cannabinoids have about 30,000 to store the information it needs to make the proper enzymes, structural proteins and hormones are necessary for the organism's life and cell structure.

 

[DrWeedly]

Very nicely done! I've just received my PhD in molecular biology (winegrapes, some tissue culture for transgenics) and finally have the time to get back to my favorite plant. Your explanation of tissue culture is well done, I'm glad to see someone with knowledge is willing to break it down correctly in this forum. Two weeks ago I initiated my first callus cultures from 1cm X 1 cm sections of leaf tissues from several strains. Just starting to see the beginnings of a callus forming on one of the leaf squares. I'll be back when I have some data, sure I'll have some questions too.
love you man

 

[thegreengrowe]

Some hard data and procedures from someone actually doing it. Nice! Looking forward to your postings, success or set backs. I will be experimenting with TC, as soon as spring planting is completed. Thanks and good clonong,
May your growing dreams be made real!
thegreengrower

 

[theberry]

Thank you for your comments. I am looking forward to posting my results with you. And hearing your results. Good luck growing and may the bud god's bless you this harvest. You can check out some video's on youtube also if you like. you can find me on the BillBerry Farms Channel.

 

[Wild Heart]

I am so gonna follow this I've been looking into tc a lot lately

 

[PotChimp]

I was thinking of trying tissue culturing myself. It seems like it would be a good idea if I were to want to condition a plant to be a parent for seeds (especially females). Cull out the weak (if there are any) and breed.

 

[420Troy420]

can you kind and genius folks explain in a lamen variety of descriptive words detailing some instructions on how this is done ? thanks

 

[man0os]

Here are some basics:

Preparing Media

When ordering media we find a baffling number of options in the catalogs. One of the most complete media is in the Sigma catalog: Murashige and Skoog shoot multiplication medium B (MSMB) (Sigma Catalog No. M7149). At the appropriate time, order a pretransplant medium (Murashige syngonium stage III Pretransplant Medium with sucrose: Sigma M 8650) You will also need a gelling agent, preferably a blend of agar and agar substitute, such as Agargel.
Purchase sterile distilled water from a local grocery store. For 1 liter of medium use a 2 liter container (because the medium boils up). Add the powdered medium to the water and stir. (Don't add the agar (gelling agent) at this stage because it gums things up when adjusting the pH).

Adjust the pH to pH 5.7 using 1N NaOH or 1N HCl (carefully, by the drop) and pH indicator paper (3.5 - 6.8), or a pH meter. (If you don't have a pH meter, the chemistry teacher might.) Now add the agar. Use about 5 grams per liter of medium . Heat and stir until the the medium is clear. (The clarity tells you that the agar has melted.) Dispense into test tubes.


Sterilizing the Medium

Pour about one and a half inches of water into the pressure cooker. Place the tubes upright in the cooker. To hold them upright place them in a wide mouth jar, make a wire or wooden rack, or tie them with string in bundles of ten. They must not fall over. Process at 15 pounds for 15 minutes according to the instructions which come with your cooker.


Preparing Explants

An explant is the part of a plant that you put in culture. The example used here is a strawberry runner tip. Select a young runner where the bud on the end has not yet opened. Cut it off the plant one inch or so from the end. Transport the tips to your classroom in a plastic bag in which you have placed a wet, but not dripping wet, paper towel. If the runner tips are not going to be processed immediately, they can be held in a refrigerator for a day or two.

Fill a pint jar half full of sterile water (boiled, pressure cooked tap water or the store-bought bottled distilled water. Add 2 or 3 drops of liquid dish washing detergent (or Tween 20, a wetting agent). Place the runner tips in the jar and replace the screw-on lid. Vigorously shake the jar by hand for one minute. Pour off the water, rinse two or three times with fresh sterile water. Repeat this operation (or dip in 70% alcohol for a few seconds and rinse. In another container add 30 ml of household bleach to 270 ml of sterile water(10% bleach). To this add 2 drops of detergent. Add the explants and shake intermittently for 10 minutes. Quickly drain and add sterile water, cover and shake. The explants are now ready to take to the transfer chamber.


Starting the Explants

The transfer chamber should be ready with the walls and workspace wiped or sprayed with 10% bleach/sterile water solution or 70% isopropyl alcohol (not if using a Bunsen burner). There should be a container of 10% bleach/sterile water and a container of 1% bleach/sterile water to sterilize and rinse the instruments and gloved hands of the operator.

Immerse the forceps and knife for 30 seconds or more in the 10% bleach then rinse in the 1% bleach and rest them on a sterile holder or paper towel to dry for a few seconds. With the forceps place a sterile paper towel on the workspace. With the forceps place a runner tip on the towel. Place the forceps in the other hand to hold the tip while the first hand uses the knife or scalpel to cut off 1 cm of the stem. Place the knife in the 1/10 bleach, move the forceps to that hand. Grab a sterile test tube of medium with the other hand, hold it by the base. With the little finger of the hand holding the forceps, remove the cap and cradle it there while you use the forceps to firmly lay the bud on the medium. Recap the test tube and seal with a piece of Scotch tape (or Parafilm).


Growing

Place the test tubes in the planter tray (or other appropriate holder ) and place the tray on a shelf under fluorescent light which is 8-10 inches above the top of the tubes. Room temperature is o.k. Continuous light is acceptable, but 16 hours light/ 8 hours darkness is standard.

Check daily for contaminants. If any are found, sterilize tube and contents before discarding the contents.

Transfer the explant every two weeks or so until it is actively growing. In one to two months you should be able to divide the culture into two pieces, each of which is about 0.5 cm in diameter. Continue to divide and transfer until you have enough plantlets. The plantlets should be singulated as you transfer to prerooting medium which has no hormones (or only IAA).


Transplanting

When the plantlets begin to root, perhaps two to four weeks, transplant them to a light artificial soil mix, such as peat/pearlite, in a seedling tray. Cover with clear plastic and place on a lighted shelf or in a shaded greenhouse. After two or three weeks begin leaving the plastic off for a period of time each day. The time the plantlets are left uncovered should get longer each day, until after about a week, the cover can be left off completely. (Tissue cultured plantlets are more delicate than seedlings as the stomates remain open until they slowly adjust to normal humidity and light.


Conclusion

After having successfully tried plant tissue culture, you will wonder about nearly every plant you see, asking yourself, "I wonder how that plant will respond in tissue culture?"

Source: Plant TC.protocol.php

 

[420pharms]

Im impressed T.C. on 420mag thanx, been looking into it for a while is Phytotech a good supplier? I have had a DYI kit in storage that requires I make agar I would rather by it R.T.U. My late uncle was the driving force to go T.C. he had alot of experience with it professionally that he and I were going to use together, but he died and without his resources I felt too overwhelmed to attempt to go in-vitro . I feel more inspired now that I see others willing to do the same. Same plant f1 vigor thousands in a baby food jar awesome.

 

[TorturedSoul]

Agar? Ask a high school biology teacher (if you know one) what they use in their petri dishes. It is doubtful that they create their own by adding a mix of nutrients to the agar; they probably order the stuff through a place such as Edmond Scientific (I'm told you can no longer BS them into selling you white phosphorous, lofl - used to ship it under kerosine so it wouldn't self-ignite... But I digress into old high school memories.) or the like.

Culture Medium Nutrient Agar | Edmund Scientific
Culture Medium Nutrient Agar - $24.95 for a ¼-pound (dry) jar.

Remember to follow proper sterilization procedures.

 

[420pharms]

Thanx for the Edmond tip, I called and spoke with Bill he has it for sale pre charged for different stages, I am going to get started soon with bill as my supplier. and your right that agar, sugar gel is available R.T.U. Im not sure why my kit wanted to make it the inst. dvd spends alot of time showing how to make it we thought we might do it for kicks but felt that it was a waste of time.

 

[TorturedSoul]

Im not sure why my kit wanted to make it the inst. dvd spends alot of time showing how to make it we thought we might do it for kicks but felt that it was a waste of time.

Possibly because the RTU stuff is a general-purpose nutritive agar, while the recipe on your DVD is more of a special-purpose plant one? Might be the case, or maybe the author didn't spend his formative years paging through scientific catalogs in hopes of discovering a handy-dandy "Things That Explode in Creative Ways" section (in addition to the usual sex/party/sex type stuff)

Good luck and I'd be interested in learning of any progress that you make.

 

[lofryhash]

I will 'second' the fact that it requires a 'clean room' - and that does mean CLEAN.
You should also include a stereoscopic multi-lit microscope with camera input for sure. AND... unless you are learned in cellular biology and professional lab technique - get trained first. And don't forget a good autoclave- screw the bleach!

 

[s1ingblade]

Would sure like to have an opportunity to give tissue culture a shot. Gotta save up and either buy a used flow hood or build one