It’s been a while since I posted anything – its just been real busy lately. I figured I had to put up a quick little update on my tissue culture experiments with C. nurii.
Pictures are worth a thousand words…
Although I’ve always been a little interested in micropropagation, my recent obession with Cryptocoryne is what pushed me into actually trying it out. Cryptocoryne are not exactly fast growing plants. They’re not easy to flower, let alone artifically pollinate to try and get seeds. Additionally, their natural environments are increasingly under threat by development. Sounds like a great candidate for micropropagation!
Over the last year or so, I’ve been experimenting with Micropropagation of Cryptocoryne and Lagenandra. Although a lab would be ideal, it’s quite possible to do this at home. In the next part of this article, I will document techniques and procedures of how to propagate these aroids.
Cryptocoryne/Lagenandra Micropropagation Protocol
Each species of plant will have it’s own specific conditions that it’ll do best in. What I’m going to describe below is based off the Cryptocoryne wendtii micropropagation protocol that Dr. Micheal Kane of the Univerity of Florida has published. Some research papers I’ve read indicate different levels of BAP are sometimes used with different species of crypts. Feel free to experiment, but always use controls and repeat your experiments in a scientific manner to ensure that the results are accurate before publishing them.
Explants
For explants, new runners seem to work really well, a 2 cm piece is all you need. Since runners are growing rapidly, this section is cleaner that the rest of the plant to begin with. Additionally, the cells at the runner tip are rapidly dividing and growing, exactly what we need.
You can also use the crown, using a plant, take off all but the newest leaf by peeling the older ones off the rhizome. Use material from emersed grown plants only. Submersed plants are too bacteria laden, you’ll have a very hard time cleaning them without killing them.
Sterilization
Wash the explant under running water that’s cool, not cold. Don’t use warm water. The best way I’ve found to do this is to put the plant segment in a jar and cover the mouth with some mesh and a rubber band to hold it in place. The just put it under the faucet for 10 minutes.
Then a 5 second dip in 70% alcohol, followed by 15 minutes in a 10% bleach solution. If your growing conditions are not exactly clean, you can increase this time to 20 minutes. Rinse off in sterile water for 5 minutes twice.
This is the most difficult part of tissue culture. Too much sterilization, and you kill the explant. Too little, and you end up with mold / fungal growth.
Cut off 1/2 cm off the ‘cut’ end since it’s probably dead from the bleach treatment and in a sterile environment, place the explant in Initiation media.
If you were successful, you should start seeing new growth in about a week.
If you end up with something looking like this – the explant was not sterile.
Initiation Media
The media you’ll first establish the explant on consists of the following:
1 liter of distilled water
Murashige & Skoog (MS) Basal Salt Mixture
6.5 grams of Agar
1 ml of PPM (Antimicrobial agent)
1 ml of 6-BENZYLAMINOPURINE SOLUTION(Also known as BAP or BA) (1.0 mg/mL)
30 grams of table sugar or lab grade sucrose
2-3 drops of a food color to make it easier to tell different media apart.
pH adjusted to 5.7 using baking soda / vinegar
Once the explant is established and shows growth, its transfered to a different media, one that’s designed to help that single shoot to multiply like crazy! This is known as Stage 2.
Over the next few weeks, the plants multiply, using up the nutrients in the media. Every six weeks or so, the plants are transferred to fresh media. If enough growth has occurred, the culture is divided in several jars.
Multiplication Media
The media for multiplication is the same as the one used for Initiation, except you’ll use
4.5 ml of 6-BENZYLAMINOPURINE SOLUTION instead of 1 ml.
Every 6 weeks, transfer the explant to fresh media. Cultures that are dividing well, can be separated into several portions and transfered to multiple jars. This cycle can continue every 6 weeks until you have enough material to take to the next stage.
Once enough plants have been produced, plantlets are transferred to a media that allows / promotes rooting. In a lot of plants, rooting may occur without the use of any plant growth regulators.
Rooting
The media for rooting is the same as the one used for Initiation, except that 6-BENZYLAMINOPURINE will not be used. Within a few weeks the effects of BAP used in Stage 2 will wear off and the plantlet should start developing roots.
This is the last stage of our micropropagation where rooted plantlets are moved out of the sterile environment and planted in a traditional soil less mix.
Acclimation
Rooted plantlets are removed from the culture and the media is washed off. A quick dip in an anti-fungal and the plantlet can be planted in a high humidity in either rockwool, Metro Mix 390 or other soilless growing media. The humidity levels are slowly decreased and the plants harden off. Often, they’re still affected by residual plant growth regulators and exhibit lush growth.
Some links that may be of interest:
Dr. Carol Stiff’s Home Tissue Culture website – Best place to get your supplies
http://www.hometissueculture.org/
Frank’s YouTube Channel – One of the best resources online
http://www.youtube.com/user/fbt2007
Home Tissue Culture Yahoo! Group
http://tech.groups.yahoo.com/group/hometissueculture/
Back in October of 09, a friend of mine, Kai Terukuni, went collecting in Lingga island in Indonesia. Among some of the crypts he found and collected was one he labelled Cryptocoryne sp. Desa Musai Timur, Pulau LINGGA[LNG-7].
A few pictures of the habitat by Kai.
Additionally, here’s a link to some video of the habitat.
A fairly shaded shallow stream with a sandy bottom.
I was lucky to receive a specimen from this locality in November.
This plant flowered around the end of December 09 and then again in early January.
Cryptocoryne scurrilis? I’ll send the pictures to Jan to be identified.
I spent a couple of hours this week catching up on the recent discussions on the Home Tissue Culture Yahoo Group. The Yahoo group, albeit hard to navigate through, is chock full of really good information and was started by Carol Stiff who’s known for the Kitchen Tissue Culture website. As you peruse through the posting, you cannot help but notice a LOT of discussion on sterilization methods and chemicals used, and why shouldn’t you? Sterilization, is after all, the most important step in micro-propagation.
Recently, a hobbyist, Gregorio J. Placeres, came up with a new sterilization process. Made from household chemicals, AAHPPAA for short (fer real?!), is a ‘new’ oxidizer which is proving to be an effective and more efficient alternative to using bleach in the sterilization process.
To make it the instructions are as follows:
In a two cup Measuring cup, place 100 ml of White Vinegar (5% acetic acid).
Place the cup in the microwave for 1 min.
Add 400 ml of Hydrogen Peroxide (3%)
Transfer the solution to a Dark Bottle and place in the fridge for one hour.
After that you can use this desinfect the surface of your explant.
Since this is still pretty new, experimentation is currently underway with determining the exact protocol. However, generally, this liquid will sterilize your explant in a mere 2.5 minutes. Additionally, unlike the traditional bleach method, no rinse off is needed on tough plants. Tender plants might need a quick rinse.
If this works, this would be a real time saver! Cheap and easy to make too!
I’m going to try it out this weekend. Stay tuned!
Recently, I was copied on an email from Claus Kettner in which he describes adding a water pump to his crypt setup.
Google translates the email as follows:
Dear Friends,
We are all constantly on the lookout for new techniques useful in the keeping, care and propagation of our water plants.
We are careful to admit in our plants Standwasser enough, but not too much fertilizer on. I have resolved to fertilize my plants so to about 400 uS. With the
Rainwater that I use, already contributes about 20 uS. In addition, my water is kept constantly by a small pump to move (from the belief that moving water is better than standing and also) the risk of algae (Kamhaut the water is prevented). The
Beaker culture creates in an occasional change of water also help. For these reasons, I have little in all my pools circulators.
I now noticed after a few months ago that in the basin with the most water spray (leaves) always dampened the growth of plants is stronger than in the rest of the basin. The growth (size) of C.pygmea foremost plant, is second from right, much stronger than before this change. Certainly the repot showed while its effects.
With a few plants, the rest C.alba everything that I have received from Niels, Alfred and Peter.It gives me a major concern of the stock of C.alba keep us stable in the culture.
Best regards
Claus
I did have pumps in my setup for water movement, but I removed them since they kept getting clogged. Kettner appears to have circumvented this issue by having a pre filter. Maybe its time to add them back in.
Additionally, he mentions that he fertilizes his plants with 400 uS of fertilizer. While here in the US, we normally measure fertilizer by tsp/gallon, in Europe, most crypt growers use a more scientific approach to concentration. From all appearances, it looked like MiracleGro (the blue is tell-tale) that everyone used. Mix enough in some RO/Rainwater to give you EC of 400 uS and you’re set. I also believe Kai Witte mentioned that most people do complete water changes on their crypt setups to ensure that adequate fertilizer levels are maintained.
Kettner’s setup looks so clean compared to mine! I need to clean up my setup.
I’m starting to play around with YouTube videos on my blog. Here’s the first one showing me taking a clump of Lagenandra meeboldii out of culture before transfering it into my aquarium.
Due to popular demand, I’ve written up a series of articles on Micropropagation of Cryptocoryne.
This is the first of a series of articles to come.
Introduction
Micropropagation is a technique used to propagate plants using small fragments of the growing tip, stem, leaves, flowers and sometimes even just individual cells of a parent plant. It is often used to rapidly propagate plants for commercial use, especially in the case of plants that do not reproduce rapidly by regular means. Walk into a Garden Center today, and chances are, the plants you see there for sale were likely produced via this technique.
Micropropagation is also used for conservation of rare plant species and that’s specifically why I got interested in it.
Micropropagation differs from other propagation techniques in that it requires the plants be grown in aseptic conditions. There are for major stages of Micropropagation:
Initiation
The first stage of Micropopagation, is also, in my opinion, the most difficult. This is where a segment of the plant, you’re trying to propagate is cut from the plant. This peice is referred to as the explant. Even though you cannot see this, the explant is covered in bacteria and fungus/mold spores. It is then a tricky game of trying to disinfect the plant to kill off all the surface contaminants without killing the plant itself. Once decontaminated, the segment is placed in a sterile nutrient media under sterile conditions.
Multiplication
The explant is placed in media that contains certain hormones, called cytokinins. These hormones exist naturally in the plant, usually the highest concentration of which is at the growing tip. Cytokinins stimulate cell division, shoot initiation/bud formation and growth of lateral buds. By adding Cytokinins to the media in certain quantities, we can promote the rapid generation of shoots from plant cells. The goal of this stage is to maximize shoot production. After a certain interval, the shoots are seperated and placed into their own containers to multiply further. One shoot turns into 5, which after the are divided, turn into 25, then 125, then 625, 2135, 15625, 78125, 390625, 1953125 – almost 2 million plants.
Often multiplication factors are increased in lab conditions due to less contamination issues and ideal conditions. Its often normal to have a 7-9 fold increase in plant mass every 6 weeks in the lab. At home, I’ve found my numbers very close to this as well.
Rooting
Once enough material has been produced, plants can be induced into producing roots by placing them in a different media. This time, hormones called Auxins are used. For some plants, this step can be skipped and the plants can be placed in hormone-free media, which is usually enough to get the plant to start producing roots.
Acclimatization
At this stage, shoots are removed from the sterile environment and placed in soil in a high humidity environment. Humidity is the gradually reduced to harden off the plant. Usually plants still have some residual hormones in them often exhibit strong lush growth.
I’ve gotten enough L. meeboldi going invitro to start taking some out. This is right out of Stage 2. The plants had already started to develop small roots since the jar was over 8 weeks old, so instead of dividing it further, these two jars were ‘harvested’.
Pics w/ camera phone. 
Sorry.
This is approx 25+ individual plants / crowns in the clumps shown above! I have 40 more jars of meeboldii like this.
