Sciencemadness Discussion Board

Tissue cultures

GreenD - 23-2-2012 at 12:06

I'm not sure the popularity of botanists or horticulturists on this site (already spoke to bot0nist), but I'm having trouble with tissue cultures.

Three main components to tissue cultures;
1.Sterilization technique
2.Plant material
3.Nutrient/Gel medium

I am not having problems with #1 as I am having no infections or blooming. However, my plants simply do not grow.

I have in the past tried many vines; passion flower, piper sp., and some others as well as orchids. Orchids are tricky, but I've taken the appropriate part of the plant (stems before flower blooms).

Here is where my problem most likely lays:
I disinfect my plant material from 10-30 minutes in strong or diluted bleach. I then dip them in IPA, and drop them onto the gel medium, pressing very lightly to get some stability.

The bleach (first trial) was diluted 1:10 with h2o and was allowed for 15 minutes. Some infection found, but zero growth in any medium.
Second trial - Bleach was undiluted and mixed with small amount of tween-20 <1%, and allowed to sit for 15 minutes. 1 Infection (out of 8) but zero growth in any containers.
Third trial - Strong bleach, tween-20 <1%, 30 minutes. No infections except for one which cap broke, no growth.

I dipped each specimen in IPA enough to fully cover surface, shook off IPA in container, transferred to medium.

The nutrient medium I use is ~4% sugar + the 'appropriate' amount of Murishige & Scooge mix (indicated on the bottle). I also have orchid medium for orchids.

But nothing grows. If anyone has experience with this, please help me out here. I've read papers, but they do not hint at what I'm doing wrong.
I will try again with strong bleach, 10, 15, 20 minutes, tween <1%. And perhaps a sterile paper towel to remove the liquid before placing on medium.


[Edited on 23-2-2012 by GreenD]

aaparatuss - 23-2-2012 at 17:23

it seems you may be over doing the steri portion. try some with little or none to get a control.

i think success also comes in numbers, like you may have to do 50 cultures to get one to go.

also are you useing hormones? MS medium is just nutrients correct?

i have ordered stuff from
http://www.phytotechlab.com/Default.aspx they are quite happy to service you and you get a nice news letter as well....

one hormones for callus formation and then others added for root growth later on..... its an art and tricky, so don't get frustrated . hope you find a technique that works!

warteo - 23-2-2012 at 18:51

I agree about the overdoing of the sterilizing. I would especially look at replacing the IPA dip with washes in sterile water. PLANT CELL CULTURE by Collin & Edwards has this to say:
Quote:

1. The plant material is washed to remove any large particulate matter such as soil, then dipped in 70-100% ethanol to facilitate wetting of the hydrophobic plant surfaces.
2. The plant material is surface sterilized in sodium hypochlorite solution (5-20%) for a suitable period of time (5-10 min) then washed in at least three changes of sterile distilled water.
3. If the explant is a section of stem or petiole, any cut surfaces that have been in contact with the sterilant should be removed before transferring to sterile medium. This is necessary because the sterilant will kill exposed plant cells which may then form a barrier to the uptake of the nutrient medium.
4. The explant is sectioned and transferred to callus-inducing medium. Normally, 10 mm stem sections or 10 mm diameter leaf portions are used.


Some further thoughts I had are:
What temperature are you keeping the cultures at? 17 - 25C is considered a good range, depends somewhat on plant species.
How long have you waited? Depending on plant it can take 2 - 8 weeks for callus to initiate.


[Edited on 24-2-2012 by warteo]

GreenD - 24-2-2012 at 08:17

Thanks guys.

I will replace the IPA was with DI water, and do a pre-wash in ethanol. And reduce my bleach time to half.

aaparatus - Phyto is who I go through, they are very helpful.

The temperature is good, perhaps on the high side (I put the jars on a heat blanket set on low) And I have waited quite some time. The first & second species turned brown and were obviously dead after about 3 weeks.

I also included some IAA - which I believe is cell elongation, or perhaps it is cell splitting. But apparently did not help.

Doing 50 samples is out of my abilities, but I think perhaps 10 would be adequate to get a good handle on things.

Also I am taking plants from greenhouses back to my house (with permission). This can be anywhere from 10 minutes to 45 minutes of the plant in a small baggie, do you think this is enough to cause death of the plant cells? When I get home, the majority of the plant is still quite green and fresh.

In fact... OFF TO THE GREENHOUSES!
[Edited on 24-2-2012 by GreenD]

[Edited on 24-2-2012 by GreenD]

GreenD - 27-2-2012 at 07:10

I have obtained about 30 samples of various plants.

I introduced an ethanol pre-wash to the plants. The ethanol was combined with ~1% tween for good submersibility.
I pressurized sink water to 15 psi @ ~100°C for 15 minutes, meanwhile the plants were soaked in dilute bleach - the bleach had been opened for a week (stupid room mates) so the content was very low I am sure.
The dead edges of contact in the plant were chopped off by flame-sterilized scissors, and placed in a agar medium with MS medium, a small amount of orchid medium, and a few drops of IAA. [amounts were approximately that labelled on the appropriate bottles).
So far, no infection.

The sections used were about 200% larger than previously done, which allows for more chance to growth, but also greater chance of infection. Multiple different plants were put in same jars, which may cause decreased chance of success, but I was limited on jars.

If this doesn't work I will need to gain more jars, construct a sterile box and buy some new bleach :mad:

GreenD - 5-3-2012 at 08:08

Update;

Not pleased.

Although I only have 2 infections out of 10 (to be expected without a HEPA flow hood), 6 of the remaining 8 samples turned incredibly black (I believe this is complete death). I've never seen this before - the tissue usually does not discolor this quickly, it happened over night. The orchid sample I collected turned black almost immediately on contact with the agar medium.

There approximately 3 tissue samples that remained green, and still have a chance to survive.

But the discoloration is incredible. I have heard, though, that under stress such as this orchids will excrete lots of indoles and auxins, staining the agar medium black. However, this was so prevelant and so quickly, I don't think this is just some "stress" - I believe the plants are dead.

Will update with any new results.