Cryptocoryne tissue culture / micropropagation Part 2

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.

Stage 1 – Initiation
Stage 1 consists of selecting the explant, disinfecting it and placing it in initiation media.

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.

meristem_selection

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.

sterilization

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.

initation

If you end up with something looking like this – the explant was not sterile.

fungus

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

Stage 2 – Multiplication

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.

multiplication

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_3

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.

Stage 3 – Rooting

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

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.

Stage 4 – Acclimation

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/