|
Sterilize Your Cuttings |
 |
Now your ready to get started. The first thing you need to do is make sure you can get the sterile water in the pint jar opened. Spray the outside of the jar with alcohol because it will create a suction when you break the seal. Unfortunately this also makes the jar slick so opening it is that much harder. All you want to do is get the lid to turn enough to know it will open when the time comes. If you can't get it open nuke it for a few seconds to heat the lid a bit. Then it should open some, leave it a little loose and place back in the sterile area. Let it cool back down to room temperature if need be.
Now you need to collect your cuttings. Collect them the same way you would to use them for propagation any other way. I put them on a plate to transfer them around. Do not take too many at a time until you get more experience under your belt. You can chop up an entire plant to use if you want, but I wouldn't until you are familiar with tissue culture.
Let's use a sundew leaf for our example. Cut it free from the plant as close to the body of the mother plant as you can without harming the plant. You do not want to touch the cutting with your hand, nor do you want to drop it. That is why I use a plate to tote it around. Use good sharp scissors to take it with and hold it with tweezers or forceps. Do not use the same anything you are using for tissue culture. Your tissue culture tools should be used in the sterile area only, always. No exceptions!
Now that you have your cuttings on your plate take them to your sterile area. Take the cap off the small bottle of 90% alcohol you prepared to dip cuttings and soak tools in. Loosen the cap on the bleach so you can remove it with one hand.| 3) Place your forceps in the jar of alcohol you dipped the cutting in, let them soak while the cutting is in bleach. If your going to do any cutting place your razor blade/scalpel in alcohol to soak at this time too. |  |
| You want to place and remove the cutting as fast as you can without opening the lids anymore than you have too. Do not breath on or in anything, keep your head outside of the sterile area. Only open the sterile water jar when your placing or removing tissue. It is imperative you get all of the bleach rinsed off. If you have to sneeze or cough get out of the room. Wash up good before returning. |  |
| 11) Push the cap down tight on the media and seal with florists or medical tape. 12) Label the media jar. You want the type of plant and date. |
 |
Make sure to dip your utensils into the 90% alcohol before you touch sterile tissue each time. Gently shake off the excess, a little left on them isn't going to hurt anything.
It is best if you use one cutting per jar. The more you put in the better the chance for contamination. I put several in one jar but I have been at this awhile.
In 4 - 12 weeks you should start to see plantlets emerge. The time and amount depends on the plant and media type. If you use BAP your going to get a lot, several hundred probably. Usually after a couple of weeks you will notice the tissue starting to swell. If you did a good job all will go well. If you didn't it will crash, usually within the first week or just after shoot growth starts.
If you are using BAP or any other shoot growth regulators you need to transfer them to plain media to allow root formation before you transfer them to soil. I generally take them out of the growth regulator media after three - five weeks and place them in plain media. This can be just a fast easy transfer in a sterile environment. They will still divide and multiply quit well but they will also develop roots. Its easier than trying to separate them all later and keep them sterile to get back into culture. Should you decide to divide them before putting them in plain media soak each plantlet in 4% PPM for 10 minutes before placing in the plain media.
You can soak cuttings in 2% - 4% PPM after the rinse and before placing in the media for a few minutes. This will ensure the tissue didn't get contaminated during the rinse, but this is usually not an issue. If you have plenty of PPM or your working on something that must work this time I would use it. Instructions for making it can be found in the sterilization section on the tissue culture page.
Here are a couple tips after you get the hang of things. Do not try any of this until you have succeeded a few times. Sometimes when you remove the cuttings from bleach the cut end will turn white. You can wipe down a plate with alcohol and lay the cutting on it after the rinsing is completed. Take the blade out of alcohol and let the majority of alcohol run off. Then cut the white tip off. Hold it with your forceps and make one good clean straight cut.
Use the same technique to cut larger leaves into smaller pieces. You want to do this after the rinse instead of before sterilization to prevent as much bleach damage as possible. You can also cut the margin (outside edge) off of the leaves to encourage more shoot growth.
Anytime you are taking tissue out of a sterile environment and putting it back into a sterile environment use a 4% PPM soak. You can take cuttings from the plants to start new cultures with. This works extremely well because you are using sterilized tissue. The PPM soak ensures it stays that way. See the sterilization page for 4% PPM details.