Showing of my cloning attempt...

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NHguy03276
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Showing of my cloning attempt...

Post by NHguy03276 » Fri Jan 04, 2019 12:26 am

So this is the most successful I've been with cloning. First attempt, the ex-plants browned out after 3 weeks, second attempt browned out after 5 days (waited too long to take the stem), and then this.

These are Phal. Tying Shin 'Baby Smile' at 52 days.

Image

I know, old hat for many of you, but I'm super excited...

link to the Imgur album with more pics: https://imgur.com/a/Lg1qWPS

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Re: Showing of my cloning attempt...

Post by Ben Belton » Fri Jan 04, 2019 5:32 am

Are you cloning or are you making stem props?
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Re: Showing of my cloning attempt...

Post by WaltonInlet » Fri Jan 04, 2019 5:43 am

If I may stick my neck out on a limb, sir, there is no such thing as "old hat" when it comes to stems, except for a small handful of people with a ton of experience. Even the most experienced slice/dice/culture folk occasionally have disappointments -- usually with their favorite plant (Murphy's Law does apply to orchids...).

Congrats on your stem props. Keep us up to date on when you are ready to go commercial and charge for your services! It seems that most people are always in search of someone patient enough to do stems for customers/friends. As others have noted here, sometimes doing stems for customers is just too much of a pain: impatient customers want immediate results (and they are not going to get it), there is too little profit, if any, and sometimes things just do not work out.

Do you plan to replate on a rooting media, or does your first media allow for that already when the stems get older?

Again -- many sincere congratulations: myself, I've been growing for nearly 50 years and, outside of a commercial lab at an orchid company I used to work for, I have *never* successfully propped anything. If you enjoy distilled spirits, pour yourself a fine glass of wine and celebrate!!!

P.S. I just saw the note from Ben Belton and will second his question here; looks like stemming, but not cloning from multiple protocorms, at least in the photo. Still, looks great and congratulations are in order, in my opinion.

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Re: Showing of my cloning attempt...

Post by NHguy03276 » Fri Jan 04, 2019 12:33 pm

Thank you for the words of encouragement.
WaltonInlet wrote:looks like stemming, but not cloning from multiple protocorms, at least in the photo. Still, looks great and congratulations are in order, in my opinion.
Ben Belton wrote:Are you cloning or are you making stem props?
Ok, first, yes, they are Stem Props. I consider Stem Prop a type of cloning, since there is no sexual transfer of genetic material, and the new plant, as I understand it should be genetically identical (unless something goes wrong in the process) to the parent plant. I also consider divisions and other cuttings "cloning" for the same reasons. If this is incorrect terminology, I apologize for any confusion.

I'm also considering, since I have 4 plantlets in vitro, using one once it gets bigger for both root tip and thin leaf sectioning... I've attempted both and had some initial results that looked like PLBs were beginning to form, but they all browned out within a couple weeks. However they were using adult plants. I'm not sure I really want to do that just yet, but it is something I'm considering.

As for media, currently I'm using P793 propagation media and this is the third replate at the moment. I plan on keeping it on P793 until I see root formation of significance, then moving to P668 until they get big enough to harden off... I haven't made it that far yet to figure that step out...
WaltonInlet wrote: impatient customers want immediate results

Yeah, I this alone will likely prevent me from going commercial for anyone who is not a seasoned grower. Plus even under ideal conditions, there is no guarantee of success. I ran my own business making/selling art pieces for a number of years, I took every commission I could get as they paid the bills, and hated every commission because impatient people.
WaltonInlet wrote:Again -- many sincere congratulations: myself, I've been growing for nearly 50 years and, outside of a commercial lab at an orchid company I used to work for, I have *never* successfully propped anything.
I was extremely lucky, in that I work for a contract manufacturer of medical devices. As a contract manufacturer, contracts/product lines come in, and go out for many reasons. When a new contract requires a special piece of equipment, it is built into the contract, and when the contract ends, even though the customer paid for it, the equipment is often left with us. Management then decides if we keep or gets rid of the equipment, and often unneeded equipment gets offered up for an internal auction by the employees.

I was able to pick up a Captair Flowair 700 Laminar flow hood for $50.00, No one else there (including management) even knew what a Laminar flow hood even was. It definitely makes a huge difference. I tried both steam column and glove box with some success before getting the hood, but they were both a pain.

But even without a Laminar cabinet, I've been seeing more people report success of seed staring and stem props in a couple other forums. It is actually quite nice to see more people making the attempt.

Again, thank you. I'm hoping to be able to report more successes in the upcoming future.

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Re: Showing of my cloning attempt...

Post by Ben Belton » Fri Jan 04, 2019 8:40 pm

Hey,

I thought I'd make a couple comments. Looks like you are doing great, so I'm not being insulting. I just hope to hit on something that might help you out possibly if you hadn't heard it.
NHguy03276 wrote:Ok, first, yes, they are Stem Props. I consider Stem Prop a type of cloning, since there is no sexual transfer of genetic material, and the new plant, as I understand it should be genetically identical (unless something goes wrong in the process) to the parent plant. I also consider divisions and other cuttings "cloning" for the same reasons. If this is incorrect terminology, I apologize for any confusion.
Yeah, technically you are correct in that by your definition this is cloning. However from a jargon point of view, and if you ever had the opportunity to sell your stems to a hybridizer you want to use, stem prop. In orchid reproduction when someone refers to cloning, they mean what you describe below with the taking of tissue from a root, growing it in hormones, cutting it into chunks, growing the chunks, cutting them up... etc, etc until you have 10,000 plants. All the exposure to multiplication hormones increases the odds there will be mutation. A serious hobbyist or especially a hybridizer does not want to find the plant they spent $$$ on has a mutation. Remember mutations can't always be seen. Hybridizers want stem props that have only been minimally exposed to the hormones, the chance for mutation is greatly reduced, and the genetics of the mother plant has been preserved. (Wow I can't type tonight) So yeah, you are technically correct, but serious hobbyist and especially hybridizers will not buy a plant they want to breed with if they think it is a "clone."

To be even more confusing sometimes people use the word clone interchangeably with cultivar. Again, you will find a hybridizer or serious hobbyist will quickly interrupt you and ask which you mean. I have asked multiple times myself. They will ask if you mean clone or stem prop, because they are not going to buy a clone.
NHguy03276 wrote:As for media, currently I'm using P793 propagation media and this is the third replate at the moment. I plan on keeping it on P793 until I see root formation of significance, then moving to P668 until they get big enough to harden off... I haven't made it that far yet to figure that step out...
The hormone in the P793 that causes the multiplication (BAP) unfortunately has the side effect of inhibiting root formation. You aren't going to get roots unless you leave them there for months until the BAP degrades. Your babies are a perfect size. You are good to go ahead and move them over to a grow out medium. They will pop out roots and take off in no time.

As a side comment.... P793 has 2mg/L of BAP which is the hormone that causes multiplication. Other people who do this and I buy the hormone and add more to the mix. I had 3-4 additional mg/L for a total of 5-6mg/L and have had good luck with that. I know someone that adds for a total of 10mg/L. In at least one of your cultures below you should have gotten multiple plants. A little extra BAP will help you. I have gotten 40 plants from one culture. Usually I only get 2-5 though. Don't get over excited and add too much or you increase your chances of mutation. Once you do start to get multiples, you'll need larger jars. I like baby food jars.

One comment on the risk of mutation. Someone I know who has made 100's of stem props told me they have never had a mutation. At this point, I've made 100's of stem props and never gotten a mutation. While the risk is there, stem props aren't exposed to the hormones over and over and over like in the cloning process, so the actual incidence seems to be tiny.
NHguy03276 wrote:currently I'm using P793 propagation media and this is the third replate at the moment.
You may have had reason for doing this, but I put my stems into P793 and don't move them until they are ready for final flask. Every time you move them you risk contamination. If you are careful, the risk is minimal, but if you are stemming a plant you have that is rare, it is looking unhappy, and this might be the only chance you have to not lose it, why risk it? Also it takes a lot of time. Yeah the media turns all black, but it will be fine.
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Re: Showing of my cloning attempt...

Post by NHguy03276 » Sat Jan 05, 2019 12:25 am

Ben Belton wrote: I thought I'd make a couple comments. Looks like you are doing great, so I'm not being insulting. I just hope to hit on something that might help you out possibly if you hadn't heard it.
Thank you for the info, and no insult taken. I am just getting started, and effective communication is more important than being technically correct. When I speak with one of my co-workers, who runs my machines on the day shift, we use a machinist shorthand that is technically incorrect, but because we have worked together for many years, running the same part, were can quickly convey a lot of information even if 'technically incorrect'.

Ben Belton wrote: You may have had reason for doing this, but I put my stems into P793 and don't move them until they are ready for final flask. Every time you move them you risk contamination. If you are careful, the risk is minimal, but if you are stemming a plant you have that is rare, it is looking unhappy, and this might be the only chance you have to not lose it, why risk it? Also it takes a lot of time. Yeah the media turns all black, but it will be fine.


Ok, Good to know. I really don't like replating if I don't have to. Yeah, every time I crack open a flask, I get nervous. The actual time to do the replate of 4 stems took less than 10 minutes, but it was almost 1 hr of prep time, wiping surfaces down, making sure flasks were wiped, and then rewiped, etc. etc. anything to reduce the risk of contamination... But I'd rather not if I don't really have to. The reason I have been replating is I was given a warning from someone who has a bit more experience than I do that phenolic exudate buildup would inhibit growth and possibly kill the plantlet. If this is not the case, I'll gladly leave them alone.

As for grow out media, is P668 acceptable for this, and would you use it full strength or half strength? Much of what I've see suggest half strength for Phals. Also, thanks for the info that BAP inhibits root development. I did not know that, and would probably have been wondering why no roots were forming...

Curiosity, how often do you find Stem Props failing. I've had a couple attempts fail after promising starts, and wondering if it is just my inexperience or if they just have a measurable fail rate...

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Re: Showing of my cloning attempt...

Post by raybark » Sat Jan 05, 2019 5:56 am

Everyone commenting so far is lightyears beyond my knowledge and experience in this realm, but isn't activated carbon added to media to absorb the phenols?

The proper use of nomenclature is important, both for scientific clarity, but for the economic angle, as well. You're going to get a lot more for a stem prop of a desirable plant than you'll ever get for a clone.

Lastly, once you get those puppies out of flask, you're definitely going to want to give them some Concentric Ag Garden Solution. Many folks dip the plants in a fungicide or disinfectant, but that is of no value, as the plants are sterile to start with, and fungicides generally don't impart protection against future infection. Garden Solution (I do wish they'd complete the name change to "Synergro") inoculates the plant with beneficial bacteria and fungi that stimulates growth, transfers water and nutrients directly into the plant, gleaned from the entire volume of medium, rather than what's in close proximity to the roots, and exude antibiotics into the medium and plant to combat pathogens. I'd say that the majority of my sales of the product are to breeders these days, as they experience much greater yields when using it.
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Re: Showing of my cloning attempt...

Post by Ben Belton » Sat Jan 05, 2019 9:23 am

NHguy03276 wrote: Yeah, every time I crack open a flask, I get nervous. The actual time to do the replate of 4 stems took less than 10 minutes, but it was almost 1 hr of prep time, wiping surfaces down, making sure flasks were wiped, and then rewiped, etc. etc. anything to reduce the risk of contamination...
Yeah, welcome to lab work. People think it is fun and easy. Many people have wanted me to do some for them in the past. I just can't. The actual bit in the hood doing the actual work IS fun and easy. However, as you say, it takes hours of time making flasks and then the prep time to get everything cleaned. For every 2 hour session in the hood doing the actual lab work, I probably spend 4 or more hours outside the hood making flasks and cleaning.

NHguy03276 wrote: The reason I have been replating is I was given a warning from someone who has a bit more experience than I do that phenolic exudate buildup would inhibit growth and possibly kill the plantlet. If this is not the case, I'll gladly leave them alone.
I'm not going to say this is completely untrue. My comment would be that in my experience it isn't a huge issue. I have cultures die all the time. I always feel I may have over sterilized. I could list several reasons I think they died (contamination not included). I am busy. I have a regular job. I sometimes don't get lab work done for years, that should have been months after started. And, the stem props are fine. Yours may do better than mine because you replated into fresh media. Mine are going to sit forever. Been doing this a long time now. I have a few pictures below.
NHguy03276 wrote:Curiosity, how often do you find Stem Props failing. I've had a couple attempts fail after promising starts, and wondering if it is just my inexperience or if they just have a measurable fail rate...
Contamination aside, I usually have a few stems that just up and die with no obvious reason. I don't know if I sterilized them too much, there wasn't enough tissue to start with to sustain them, they were out of media too long and air got up into the tissue. I have no idea. Sometimes it's crushing how many just spontaneously die and sometimes it's only a couple. Sometimes they die within a few weeks, and I've had them up and die after 6 months of great growth.

Pictures Yay!

In this picture you can see that the tissue just died. No phenols to blame. It just died. Why? No idea
Image

Here you can see there are lots of phenols and everything is doing fine. I also got 2 plants. If I had moved them to fresh media would they have done better or grown faster? Maybe/Probably But I'm lazy and busy. If I had time, I could go ahead, cut them apart, and move these into final media. Maybe this will get done in 2019... if they are lucky. This was done in July 2018 and I took this picture today for you, so they have been there 6 months. I'm not saying this is the right way or the best way or the most efficient. I'm offering perspective on moving stuff multiple times and the affects of phenols etc etc
Image

Here are some old pictures I had. Both are of stem props. Lots of plants. Lots of phenols on the left. Not so much on the right. Everything looks good. I should have cut these apart months before this pictures was taken. I'm sure I was behind like always. They do better the larger they are anyway. Note that despite the size of the plants, there are no roots. Also back to something I mentioned yesterday, if you start getting multiple plants, those tubes aren't going to work. These are baby food jars.
Image

More stem props. You can cut these apart at this size. It can be tricky though. If you accidentally butcher it, you can plant the nubs and often they will do fine anyway. A couple things about this one. It is rare that I get this many plants. Yes, you can push it with more hormones, but you always increase your risk of mutation. I usually get 2-5. Secondly when you cut the large babies off, you often find that you have a knot of tissue underneath that is still green and has a few tiny bumps on it. Those are potentially plants that grow into more plants. In that case, I take the knot of tissue left and put it onto final media. Sometimes a year later, after already once harvesting all the plants on it, you get something similar to the picture below. One culture yields 2 crops of stems. This sounds awesome but from a business point of view, the Phal orchid world is a small place. You often don't have buyers for 30 plants of the same cultivar.
Image

This picture is a little different. This is actually a stem prop before I cut everything apart. What had happened is that it was growing multiples like one of the ones above, but it was not growing well. Maybe from phenols. It was too small or the baby plants too dense to cut apart without butchering it. So I moved it to final media where everything could continue to grow. I can cut them apart now without killing everything. Ironically they are harder to cut apart at this stage. Roots are going everywhere and they are hard to avoid. Plants are bigger and it is harder to get my blade between them. However, even if I slice up a few things, the plants are big and they recover in no time. These will be cut apart and placed individually into final media to grow out. I don't worry about slicing roots. I try to avoid it, but not a big issue.
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Re: Showing of my cloning attempt...

Post by Ben Belton » Sat Jan 05, 2019 9:26 am

raybark wrote: isn't activated carbon added to media to absorb the phenols?
Not in this case. You could add charcoal, but it's not there. If it were, the media would be black like in my last picture above. As I think about it, it might not be a good idea to add charcoal. Charcoal is added to absorb organic components like phenols. I am guessing it would also absorb the multiplication hormone, and that wouldn't be good.
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Re: Showing of my cloning attempt...

Post by Ben Belton » Sat Jan 05, 2019 9:52 am

Just a side note, I am about to go on vacation. Glad to answer anyone's questions. Please feel free to post. However my ability to reply is going to be limited started tomorrow. I have already pointed Dewey in this direction and if people get interested we'll get Peter to pull in Robert and Rob. They will do 100x better than me. I learned most of this from them.

To anyone interested in this topic, but who don't have a laminar flow hood, Dewey only has a glove box. I say "only" but he has turned out flaskings of orchid seedlings from multiple genera and Phal stem props with a lower contamination rate than many people with laminar flow hoods. So if someone wants to insist you need a hood, I call BS and use Dewey as an example.
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Re: Showing of my cloning attempt...

Post by kyle » Sat Jan 05, 2019 11:23 am

Great discussion!

What are your protocols for sterilizing the stems?

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Re: Showing of my cloning attempt...

Post by NHguy03276 » Sat Jan 05, 2019 11:39 am

Ben Belton wrote:. So if someone wants to insist you need a hood, I call BS .

I absolutely agree... Before getting lucky and getting a hood, I used a unsealed glove box, and while I didn't have much success getting plants to grow, I did have solid success at maintaining sterility (which at that point was just as much a success as getting plantlets to grow). I think I had 1 flask out of 8 contaminate. On another forum, there is a small group of us that are all learning, reporting our results, trying different things, and one of the more successful people uses only a steam column to work in. I personally dislike the steam column, but they had bad luck with glove box and prefer it. Having a Laminar is like having a Cadillac, you can live without one, but it is fun to have one.
Ben Belton wrote:
raybark wrote: isn't activated carbon added to media to absorb the phenols?
Not in this case. You could add charcoal, but it's not there. If it were, the media would be black like in my last picture above. As I think about it, it might not be a good idea to add charcoal. Charcoal is added to absorb organic components like phenols. I am guessing it would also absorb the multiplication hormone, and that wouldn't be good.
It is my understanding that is indeed the case that charcoal will absorb the BAP, and it is why it is not added to P793 mix. I have heard of some people adding a little bit to their own recipes, but with mixed results... Personally at this point, I'm more interested in repeatably and consistency, that I don't want to tinker too much with the formulas until I gain a bit more understanding.
raybark wrote:Lastly, once you get those puppies out of flask, you're definitely going to want to give them some Concentric Ag Garden Solution.
Thanks, I hadn't even heard of this before, but it will definitely go on the list now. looks like it will significantly help with the hardening off process.


Thanks for all the great information... Sometimes asking a few questions from experienced people can give more information than months of reading and experimentation. And Ben, Have a Great Vacation.

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Re: Showing of my cloning attempt...

Post by NHguy03276 » Sat Jan 05, 2019 11:53 am

kyle wrote:Great discussion!

What are your protocols for sterilizing the stems?

Kyle

Once I cut the stem into the sections, I remove the bract covering the node and then soak in a .5% sodium hypochlorite solution for about 10-15 minutes. Then soak in sterile DI water for about 5 -10 minutes, and then rinse in a second sterile DI water bath. So far this has worked pretty good for me.

There are some that recommend a second 10 minute soak at 1% sodium hypochlorite, but I haven't done this.

As for the %'s a lot of directions say a 10% bleach solution, but there from when commercial bleaches were 5.25% sodium hypoclorite as a standard... (meaning the 10 parts water 1 part bleach would be just about .5%) but with today's concentrated bleaches, you need to adjust for the % of hypochlorite to get the .5% or 1% depending on what you are shooting for. Also, a number of bleaches have additive that are a pain... I try to find a simplest form of Clorox without scents and such, and make sure the % sodium hypochlorite is printed on the container.

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Re: Showing of my cloning attempt...

Post by NHguy03276 » Sat Jan 12, 2019 4:06 am

Ben Belton wrote: A serious hobbyist or especially a hybridizer does not want to find the plant they spent $$$ on has a mutation. Remember mutations can't always be seen. Hybridizers want stem props that have only been minimally exposed to the hormones, the chance for mutation is greatly reduced, and the genetics of the mother plant has been preserved.
I think I understand this better today than I did when I first posted this thread. I had two plants bloom with very disappointing blooms, because when I bought them online, I didn't really get the difference between clone/stem prop. This also explains the different prices I sometimes see on the same plant (P. LD Bear Queen, Sometimes $20, other times $250)

So yeah, I bought these:
ImageImage

and I got these:

ImageImage

sorry for the potato quality in my photos, but I was too disappointed to try for a better pic. I guess I got what I paid for... Of course buying sight unseen is always a gamble, sometimes you win, sometimes you lose...

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Re: Showing of my cloning attempt...

Post by Ben Belton » Tue Jan 22, 2019 8:55 am

Well the difference in the first one seems pretty extreme for a cloning mutation. It can happen, but usually it is more subtle. Are you sure you received the correct thing?

Did you move your stem props to regular media yet? Usually they take off pretty quick. I'm curious how they are doing.
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