tissue culture versus peat substrate

[dudo klasovity]

Hi there!

Many times growers have asked the question, how much faster is the propagation of seedlings in vitro than when sowed on a regular substrate. Well, I got my hands on some high quality seeds (thanx Iggy;-)) recently, so I made a little experiment which would give us a hint about the answer to this question (at least as far as some drosera species are involved). For I was very curious about the outcome myself, I picked 3 species for this experiment and compared the germination ratio, survival ratio and also, most importantly, the size and eagerness of the plants to grow.

Today, 2 months after start, I am capable of submiting a few pictures and can bother you with some figures:-)

Conditions: - the species I picked were:

drosera admirabilis

drosera sp. South Africa (a.k.a drosera sp. Pretty Rosette)

drosera ascendens 'red form'

- the plants grew in environmentally more-less identical conditions (humidity, light levels, temperatures,...etc)

- in vitro cultures contained no hormones, the cultures were not replated

- experiment took 2 months, the all seeds were sown on substrate/medium the very same day

RESULTS:

drosera admirabilis:

germination (in vitro): after 12 days, (ex vitro): after 20 days

germination ratio (in vitro): very high,>95%, (ex vitro): high, apx. 70%

survival ratio (in vitro): 100%, (ex vitro): 10%

size (in vitro): almost uniform size of all plants, median of 10mm, (ex vitro): strong variation in size, median of 4mm

viability (in vitro): >90%, (ex vitro): low, 30%

PICTURES:

drosera admirabilis on peat/sand

drosera admirabilis, acclimatised seedlings from in vitro:

drosera sp. 'Pretty Rosette':

germination (in vitro): after 13 days, (ex vitro): after 13 days

germination ratio (in vitro): very high,>95%, (ex vitro): very high, apx. 80%

survival ratio (in vitro): 100%, (ex vitro): 60%

size (in vitro): uniform size of all plants, diameter 12mm, (ex vitro): almost uniform in size, median of 3mm

viability (in vitro): >95%, (ex vitro): 80%

PICTURES:

drosera sp.'South Africa' on peat/sand

drosera sp. 'South Africa' aclimatised from in vitro

drosera ascendens 'red form'

germination (in vitro): after 10 days, (ex vitro): after 15 days

germination ratio (in vitro): very high,>95%, (ex vitro): very high, apx. 70%

survival ratio (in vitro): 90%, (ex vitro): 80%

size (in vitro): almost uniform size of all plants, median of 15mm,(ex vitro): almost uniform in size, median of 3mm

viability (in vitro): >85%, (ex vitro): 50%

PICTURES:

drosera ascendens 'red form' on peat/sand

drosera ascendens 'red form' from in vitro

Seeing this, I would say, that the benefit of in vitro propagation of drosera via seeds, is not as much in a provision of high germination rate (it does not vary from the regular ones, when fresh seeds are used), as much as in the fact, that in vitro growth is substantial for high survival rate and rapid growth.

The final answer, which was, how much faster does the plant grow in vitro than on soil, I am not able to provide. For this, I would need to measure the time the plant reaches maturity (which can be many months). Unfortunatelly, I cannot provide the numbers since my test period was only 2 months, and the data cannot be reached by mere extrapolation of known figures, simply because the growth is not linear, but exponential function of time. Moreover, exponentiality is influenced by many factors, mainly feeding in the early stages.

Nevertheless, I think the pics give us a pretty good idea about the benefit of TC. I must add, that growing plants this way, also preserves the original DNA spam of the plants since they are not being cloned;-)

[Petr D.]

Really interesting experiment, I must say. And the in-vitro pictures are really amazing. All lined up in rows, looking healthy ;-) Thats evey drosera holic's dream I guess You have definitely found a way how to do this right Dudo.

I don't have any experience with in-vitro, but lately I have been sowing alot of seeds too. The germination rate is rly not an issue as long as you start with fresh seeds. But it gets harder to grow them to adult size. I usualy end up with batch of seedling differing in sizes. If they are too crowded, they stop growing just after germinatiion and you have repot, wait, repot wait ;-) Some seedlings grow fast, some refuse to grow at all.

Looking at your in-vitro pics, it looks more than just efficient way to propagate, but I have no doubts there is alot of hard work and research until you get such nice results.

Edited February 11, 2010 by Petr D.

[faunista]

Very interesting comparison!

... but the most interesting thing is that now you have a few D. ascendens red for me!

[jimscott]

Just a wee bit of contrast!

[FlytrapCare]

Nicely done! Thanks for sharing this experiment.

[Daniel O.]

Hi Dusan,

really good results you have there.

In vitro i´ve not tried till now, but it seems so as if i have to try it one day, nethertheless i´ve had not so good experiances with in vitro plants i´ve received in the past.

A lot of plants haven´t be able to produce seed at the beginning nethertheless i´ve pollinated them.

D. spec. Duida (probably D. yutajensis) for example has flowered in several collections of different growers (my plants have also flowered) but nobody was able to collect any viable seed till now, perhaps because of the in vitro propagation.

But all in all this method seems to be really very effective, congratulation.

Best regards,

Dani

Edited February 12, 2010 by Daniel O.

[droseraman]

Nice work!!! You have so many seedlings- your transplanting looks fantastic!

Just out of curiosity, I have a few questions since I now feel tempted to try TC thanks to your wonderful report :)

1. Could you have left the plants in tc longer, or had they used up all the nutrients?

2. What recipe did you use for these particular batches (or a link to where you have said this before)

3. Did you use gibberellic acid? I'm guessing not, but thought I'd check...

I greatly appreciate your help!

Hi there!

Many times growers have asked the question, how much faster is the propagation of seedlings in vitro than when sowed on a regular substrate. Well, I got my hands on some high quality seeds (thanx Iggy;-)) recently, so I made a little experiment which would give us a hint about the answer to this question (at least as far as some drosera species are involved). For I was very curious about the outcome myself, I picked 3 species for this experiment and compared the germination ratio, survival ratio and also, most importantly, the size and eagerness of the plants to grow.

Today, 2 months after start, I am capable of submiting a few pictures and can bother you with some figures:-)

Conditions: - the species I picked were:

drosera admirabilis

drosera sp. South Africa (a.k.a drosera sp. Pretty Rosette)

drosera ascendens 'red form'

- the plants grew in environmentally more-less identical conditions (humidity, light levels, temperatures,...etc)

- in vitro cultures contained no hormones, the cultures were not replated

- experiment took 2 months, the all seeds were sown on substrate/medium the very same day

RESULTS:

drosera admirabilis:

germination (in vitro): after 12 days, (ex vitro): after 20 days

germination ratio (in vitro): very high,>95%, (ex vitro): high, apx. 70%

survival ratio (in vitro): 100%, (ex vitro): 10%

size (in vitro): almost uniform size of all plants, median of 10mm, (ex vitro): strong variation in size, median of 4mm

viability (in vitro): >90%, (ex vitro): low, 30%

PICTURES:

drosera admirabilis on peat/sand

drosera admirabilis, acclimatised seedlings from in vitro:

drosera sp. 'Pretty Rosette':

germination (in vitro): after 13 days, (ex vitro): after 13 days

germination ratio (in vitro): very high,>95%, (ex vitro): very high, apx. 80%

survival ratio (in vitro): 100%, (ex vitro): 60%

size (in vitro): uniform size of all plants, diameter 12mm, (ex vitro): almost uniform in size, median of 3mm

viability (in vitro): >95%, (ex vitro): 80%

PICTURES:

drosera sp.'South Africa' on peat/sand

drosera sp. 'South Africa' aclimatised from in vitro

drosera ascendens 'red form'

germination (in vitro): after 10 days, (ex vitro): after 15 days

germination ratio (in vitro): very high,>95%, (ex vitro): very high, apx. 70%

survival ratio (in vitro): 90%, (ex vitro): 80%

size (in vitro): almost uniform size of all plants, median of 15mm,(ex vitro): almost uniform in size, median of 3mm

viability (in vitro): >85%, (ex vitro): 50%

PICTURES:

drosera ascendens 'red form' on peat/sand

drosera ascendens 'red form' from in vitro

Seeing this, I would say, that the benefit of in vitro propagation of drosera via seeds, is not as much in a provision of high germination rate (it does not vary from the regular ones, when fresh seeds are used), as much as in the fact, that in vitro growth is substantial for high survival rate and rapid growth.

The final answer, which was, how much faster does the plant grow in vitro than on soil, I am not able to provide. For this, I would need to measure the time the plant reaches maturity (which can be many months). Unfortunatelly, I cannot provide the numbers since my test period was only 2 months, and the data cannot be reached by mere extrapolation of known figures, simply because the growth is not linear, but exponential function of time. Moreover, exponentiality is influenced by many factors, mainly feeding in the early stages.

Nevertheless, I think the pics give us a pretty good idea about the benefit of TC. I must add, that growing plants this way, also preserves the original DNA spam of the plants since they are not being cloned;-)

[dudo klasovity]

Thanx for nice comments everybody!

Peter: You hit the nail exactly on head, in the second paragraph you described what I am trying to avoid by using TC :-) Tedious work with tiny seedlings, endless waiting and slowly bringing the plantlets up, often a Sisyphean task I must say. And you are right, the results didnt come easy, it took some research and losses in the beginning.

Droseraman:

1. Yes, I could have left the plants on medium longer, even for up to 8 months in a fridge if needed. When you mix a good formula it is possible to grow plants at normal temperatures for 2-5 months before deflasking or replating on fresh media.

2. The recipe I used was worked up by trial-error, altough I started with something resembling diluted MS medium, but since I have access to chemicals, I rather mix my own medium, because it is easier to modify the amount of some components. But often, just 30%MS works just fine. South american species from my experience are quite sensitive to ammonium nitrate though and need better dilution.

3. No hormones, no GA3 (it is hormone too). For non-dormant seeds you do not need any pretreatment, just as if you sowed them on regular substrate. In fact, when I tried (just out of curiosity) to use hormones (cytokinins and/or auxins), the results were poor. The seedlings often died soon after germination, or were etiolated and unhealthy looking, with feeble growth rate. The seeds contain neccessary hormones in them already, so generally the use of hormones is not recommended.

Edited February 12, 2010 by dudo klasovity

[naryn]

I find it interesting reading about TC on the forum but i have absolutely no idea how to start doing in vitro work. Is there some guide/website that you can suggest for reading up on the subject and how to get started with it?

tx

naryn

[dudo klasovity]

Hi Naryn,

learning TCing is pretty much in most cases a self-educating process (unless you can take some university biochemistry/biology tutorial on this subject).

I recommend reading any book about TC, just type in keywords of what you are looking for on google books or ebay books or other search engine. I do not think you will find a book specialised purely on TC of carnivorous plants, but luckily we have the internet and forums such as:

http://www.flytrapcare.com/phpBB3/viewforu...a0b3f4385a2a71d

or http://www.labflytrap.com/

or http://www.world-of-carnivores.com/tissue_culture.html

..and so on and so forth.......when you do your research you can start experimenting. Getting neccessary skills and experience is possible via experimentation only, but the reading provides many useful information. So far, not too much research have been done in this particular area, so do not expect miracles! ;-) GOOD LUCK!

[naryn]

Very useful links, thankyou, i will take my time reading through the information.

[mobile]

Is there some guide/website that you can suggest for reading up on the subject and how to get started with it?

Try here: http://www.world-of-carnivores.com/tissue_culture.html

Carl

Edited February 12, 2010 by mobile

[droseraman]

Thanks for the response!

Unfortunately this leads to even more questions.

I'm doing some research now, but I think I'll have a few more questions for you...:)

[UtricSeb]

Hi Dudo,

This is very good information and a great success on your part with TC.

Can you please share how are you sterilize the seed? I and a friend have just started with TC this last December, only some of the seed have germinated but are growing very slow. Our first try was with D.capensis because we don't want to risk valuable seed while learning.

I have doubts about our seed sterilization process, maybe some seed got killed and those germinating can't grow well. We are using MS with vitamins at 50% strength.

Thanks for your help.

Sebastian.

[dudo klasovity]

Hi Sebastian!

Using the seeds for TC has many advantages, amongst which the biggest one is that they are relatively hard to kill during the sterilization sprocess in comparison with e.g. leaf tissue. I use a very simple sterilization protocol, using 1,5% NaOCl solution (30% solution of bleach, which is in fact about 5%NaOCl solution) and one rinse with sterilised water. Thats it. The time period neccessary to sterilize the seeds varies from 2 to 5 minutes (it depends on the species, now I am talking about drosera seeds). Pinguicula seeds need less and nepenthes seeds more time in average.

Sometimes, when your sterilization is too strong, you bleach the seeds too much, the germination rate is very low and the seedlings dont grow well, or die early. It can also mean, of course, that there is a toxic element contaminating the medium, which can come from low grade chemicals or agar impurities. Most of drosera dont mind low grade agar (used for food), but some do and die. It is a matter of experience to find out details and particularities of propagation of each species.

[UtricSeb]

Hi Dudo,

Thanks for your reply. It will surely help us.

[aflubenov]

Hello Dudo!

Some questions, may be you can help me.

I see that your tissue cultured droseras are not in Agar.... it seems like it are in sustrate... Is that right?

So, what you did was sterilyse sustrate, seeds and recipients and then, without using hormones, put the seeds and cover the recipient?

I am asking it because I am new at this and it is hard to get Agar in my town, so if there is any alternative using sustrate, it will be great to me!

How do you sterilized the sustrate? It is enought putting in the microwave?

Thanks in advance!

[dudo klasovity]

Aflubenov: the pictures of drosera from tissue culture were taken one week after deflasking (deflasking is taking the plants from agar sterile agar medium and planting them in regular substrate, which is NOT sterilised).

If you wonder what the sundew looked like while in vitro, here are some pics of the same plants on agar:

drosera admirabilis

drosera sp. 'South Africa'

\

drosera ascendens 'red form' (germinating on medium)

[droseraman]

I am asking it because I am new at this and it is hard to get Agar in my town, so if there is any alternative using sustrate, it will be great to me!

How do you sterilized the sustrate? It is enought putting in the microwave?

Most people aren't able to buy agar locally. You will have to order it from a chemical company online, or a place like amazon.com. While amazon might not carry micropropagation grade agar, they for sure carry other brands that dudo had mentioned may or may not work depending on the species.

If you do some searching, I'm guessing you'll be able to order something in unless the postal system in your town is different than ours (which I could have overlooked).

[MFS]

A good source of agar is Asian food stores, at least here in Tasmania.

However I've had the best success with Gelcarin or Gelrite, which are purified carageenans. These gel to a clear (rather than off-white) gel, and seem to grow plants less susceptible to hyperhydricity.

Dudo: Have you tried PMM?

Like you I just used bleach (and ethanol for vegetative explants) and sterile water rinse, but I know of people having had great success with it on CPs.

Finally, what system do you use to sterilise the seeds? Syringes? Tea bags?

I used a tea-bag system in the past, but it was a lot of work and very fiddly.

Miguel.

[dudo klasovity]

To get hands on agar is not a problem (at least not here in Czech Republic), you can get it in packets by 10g in any good store with healthy food line and it is very cheap. The problem is, that since agar is not commercially synthesized, but exploited from natural souces, there are no 2 identical packets of agar. Each agar differs in quality. That means that agar works just fine for your plants but one time you buy new packet which is of low quality or contains toxic elements and it might harm your culture (especially the more sensitive ones). That is why it is a good idea to buy pure, TC grade agar. I had really great results with Sigma-Aldrich agar.

I never used Gelrite nor Gelcarin since SA Agar never disappointed me. Media with agar really are hazy, in comparison with sheer see-through Gelrite media, but for me this is not a disadvantage.

MFS: You mean PPM? Yes, I use PPM sometimes for really expensive seeds, just to doublecheck on thwarting the contamination, at least in early stages of the growth. I use it incorporated in medium. For disinfection I dont use it. I have heard some people use alcohol, PPM and antibiotic treatment for really stubborn tissues like Nepenthes for example. I mostly work with drosera and pinguicula tissues, and nepenthes seeds which are significantly easier to sterilise.

So for me it is just bleach and sterilised water, I try to keep it simple if possible.

For sterilising seeds, I use simply folded filtration paper used in any laboratories. It can withstand the corrosiveness of bleach for prolonged time, so it is really easy to work with and the cheapest thing in the world:-) Tea bags sound too fragile for this , no?

Edited February 17, 2010 by dudo klasovity