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Pataskala,OH

A few years ago someone on this message board (I think Matt Debacco) was trying to culture tissue from the plants that produced the largest pumpkins. The vine I got mailed to me that year never sprouted side vines so I couldn't use it, but it was interesting. Did the bugs ever get worked out with that? Did anyone ever get a normal looking plant? Has anyone grown anything big off a cloned vine yet? I was just wondering because I am taking a plant physiology class and we were talking about plant tissue culture recently.

9/20/2016 1:48:39 PM

Austin MN

I have the some of the best tissue culturist in the world right next to me at the U of M and iv been tissue culturing for 3 plus years so I'd be happy to take cuttings from kins this year to grow out!

9/20/2016 2:34:17 PM

Austin MN

Not sure on Matt's culturing but I was able to grow out culture from Chris Stevens 2328 he grew and I had the 1625 that grew Josiah's 1965. I could get great growth off them but the biggest issue was disease would really take over

9/20/2016 2:35:58 PM

Austin MN

If not a clean sample.

9/20/2016 2:36:15 PM

Springfield, Missouri

I did tissue culture for a few years. It was very easy to produce callus tissue, but difficult to form plantlets from callus. Callus tissue is a mass of undifferentiated cells (stem cells). Other people I talked to had the same problem. Bryson Ensign might still be working on it?

My thoughts are that tissue culture is a viable tool for more advanced biotec such as androgenesis, but not worth it for simple cloning. I have kept pumpkin clones alive for 3+ years via cuttings. Not to mention callus tissue is prone to mutation and genetic degradation if maintained for long periods of time.

9/20/2016 3:54:29 PM

Midway, UT

Joe Scherber has gotten a few different "clone" plants from Matt. The most interesting was the 1725 plant that grew Ron Wallace's 2009. Unfortunately the only cross that took was a cross with my plant that produced a poor pumpkin.

9/20/2016 3:58:50 PM

Connecticut

I was able to give tissue culture a valiant attempt and there were some limitations with it.

First off this is the best way to produce thousands of identical plants the difficult part is timing. It essentially takes longer than September to May to create the volume of mature plants that could be produced. Once we as growers determine plan to be valuable it is typically around this time of year. What makes this difficult is the plants are "dirty" and when you bring them into a sterile environment it is hard to keep them "clean".

One advantage is that the plants do grow fast and the hope was to be able to transition them quickly to essentially out run potential disease/contamination.

I determined that form a growers stand point clones were a better option as this was a way to get material to growers to use, but this has its own set of potential issues as well.

So, I settled on growing a winter pumpkin as this allowed me to send seeds to growers that were essentially a form that all growers were accustom to which made the handling process much better. The challenge from my end was timing and the enormous amount of work during the winter season. One the bright side my 220 DeBacco combined the idea of cloning a plant and winter growing and proved that this could produce very powerful genetics. The "little" 220 pound pumpkin ended up producing two pumpkins in the top-15 weighed the same year the 220 was grown.

9/21/2016 6:32:10 PM

Connecticut

Now to answer your actual questions...

1.) Did the problems ever get worked out? = Essentially, the issue of contamination is still the main issue and even taking steps to prevent this the problem is the end of the season plant material is internally contaminated.

2.) did anyone ever get a normal looking plant? = Yes, but it seems the plants needed more water and were not very heat tolerant for the entire growing season. I am not sure why this was the case but it was certainly a common pattern that growers reported to me.

3.) Has anyone grown something big off of a clone? The 850 Adams is the biggest pumpkin grown from the clones that I distributed. However, many growers had planned on specifically using them as pollinators.

850 Adams
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=173182

832 A. Berard ’11
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=173183

767 J. Post ’11
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=173184

650.5 N. Petti ’11
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=173185
*Yes a clone produced a Howard Dill Winner!

616 C. Horn ’11
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=173186

9/21/2016 6:32:19 PM

Connecticut

If there is any interest in seeing the actual process I used for propagation here are some links to my 2010 diary...

Growth Chambers were used
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126630

Stock Plant harvesting
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126645

Sterilizing
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126646

Balance of cleaning but not killing the plant material
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126647

In the hood
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126648

Hurry up and wait
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126649

Keeping the tools sterile
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126650

Final cut
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126651
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126652

Agar jars
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126653

Growth chamber
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126654
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126655

4 Days after placing in agar
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126656

10 days later
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126657

Transplanting
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126658

Repeated trials
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126659

Trial #2 more nodes in less space
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126660

Test Tube Pumpkin plant
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126662

Labeling is an important step...
http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=126663

9/21/2016 6:40:09 PM

Austin MN

Matt did you ever try heat treatment to clean up the tissue. I was able to heat treat and suppress the disease and bacteria than regrow out the the tissue to a plant that I could grow out.

9/21/2016 7:55:45 PM

Connecticut

The contamination was determined to be internal, and what made the pumpkin plant material a challenge is the overall size of tissue present. The amount of heat needed would increase the odds of tissue damage so chemical treatments were selected to offer the greatest benefit with least chance for plant tissue damage. This process also allowed for a timed soaking to allow for more complete infiltration of the plant tissue. It was effective just not to the degree needed for the required length of time to get the plant to grow and then develop enough to be able to survive the shock of coming out of the "easy" tissue culture environment.

9/21/2016 11:16:48 PM

Springfield, Missouri

I believe Matt and I are talking about two different types of cloning, each with their own merits. As I said in my previous post, I believe that cloning plants via cuttings is suitable for our goals. This type of cloning is know as vegetative propagation. It appears that Matt is conducting vegetative propagation by the photos he has provided. This method utilizes nodes, and can be done in soil or agar, with or without plant growth regulators. Pluripotent cells aka callus are not used in this method.

Tissue culture is an alternative cloning method. Tissue culture requires agar or broth, sterilize meristematic explant, plant growth regulators, and nutrient supplementation. Tissue culture relies on the formation of callus (stem cells). Callus can be divided thousands to millions of times, thus millions of clones can be produced in a matter of weeks from a single callus.

I will try to provide photos for both methods within the next day.

9/22/2016 4:07:32 PM