Introducing Darlingtonia californica to Tissue Culture

Table of Contents

Overview: Carnivorous Plants

Carnivorous plants are a unique category of plants in the plant kingdom that feed on living animals and insects or other small organisms to obtain nutrients for their growth and development.

These plants have a special mechanism to catch their prey and digest them with their secretory enzymes. The mechanism in these plants is developed to their natural habitat condition, which consists of low-nitrogen or nutrient-deficient soil.

To fulfill their nutrient needs these plants developed beautiful and attractive structures, flowers, nectars, and scents to attract their prey towards them and then capture and absorb the required nutrients from their body.

The carnivorous plant group is composed of around 600 species. The plants are mostly distributed to the 9 families and 19 genera. It has a wide range of values including, economical, medicinal, and ecological/diversity values. And, because of this, they are a common attraction to growers, culturists, farmers, and biologists worldwide.

Some carnivorous species are on the verge of extinction and some have already been extinct due to their overexploitation.

Though these plants can be grown through seeds or cutting, these conventional approaches are not suitable to conserve the plant species or grow them on a large scale to fulfill their market demands.

In such a case, tissue culture can be an efficient and effective approach for the propagation of carnivorous plants.

But, how do we do it?

In this article, we will learn about the procedure to the tissue culture of one of the carnivorous species, Darlingtonia californica.

So, learn and shoot us your questions at the end of the article at anjali@plantcelltechnology or info@plantcelltechnology.com.

Micropropagation on Darlingtonia californica

Darlingtonia californica is a carnivorous plant, commonly known by the names of Cobra Lily, Cobra plant, or California pitcher plant. It belongs to the family of Sarraceniaceae. The leaves of the plants are tubular, forked, and range in colors from yellow to purple green. The plant has large, rambling root systems. However, they are very delicate towards the temperature change.

Unlike other carnivorous plants, Darlingtonia californica does not collect rainwater in its pitcher. However, it controls the water level inside the pitcher through its root. It absorbs water from its roots whenever required and releases it back into the soil when there is an excess of it or there’s no need.

The plant hides the main exit (a tiny hole) from its prey by curling it inside and displays many false exits to the prey. So, when they are tired of trying, they fall into its trap. Additionally, to attract prey the plant has lubricating secretions and downward-pointing hairs, like other carnivorous plants.

Figure: An image of Darlingtonia californica.

Explant sterilization of Darlingtonia californica

Here’s a general protocol to sterilize the explant of Darlingtonia californica. So, this needs to be tweaked based on the size of the tissue—especially the exposure time of tissues to the chemicals.

• Collect the cleanest and disease-free tissue from the mother plant.
• Rinse the tissue under warm running tap water for 20-30 minutes.
• Keep the explant in soapy water for around 10-15 minutes.
• Wash the explant thoroughly under running tap water.
• Dip the tissue in alcohol. However, if the explant is too small (like taken from a leaf) avoid the step. It can kill the tissues.
• Transfer the explant to a 3% H2O2 solution and sterilize the plant with continuous stirring. If the explant is small, keep the exposure time for about 3-5 minutes. However, if it's bigger like a flower stalk, then keep the explant for about 6-8 minutes.
• Transfer the explant to 10%-20% bleach with 5-7 drops of Tween 20. The exposure time of the explant in the chemical will be based on its size. However, it generally ranges from 6-12 minutes.
• Rinse the explant 2-3 times for 3-5 minutes in sterile water.

Media Preparation for Darlingtonia californica

• Prepare the culture media for Darlingtonia californica with: ⅓ MS media, 1ml/L BA, 0.1ml/L NAA, 1 ml/L PPM, 25 grams sucrose, and 8 g/L agar or 3g/L gellan gum.
• Take a 1-liter container and fill it 1/3rd using sterile water.
• Then, add all the chemicals mentioned above with continuous stirring.
• Adjust the pH of the media to 5.6-5.8 using HCL (to decrease the pH if it’s high in the media or NaOH to increase the pH of the media if it’s too low).
• When all the components are mixed, make up the volume to 1 liter with continuous stirring.
• Cap the container and place the culture media in a pressure cooker or autoclave for sterilization at 121 °C for 20 minutes.
• Once the media is sterilized, let it cool down.
• Dispense 15-20 ml media (this varies based on the size of your jars) in each sterilized tissue culture jar.

NOTE: You can also dispense the culture media in each jar before sterilization.

• After the media is solidified, transfer the sterilized explant to the culture vessel using a sterilized forceps.

NOTE: You can keep your forceps in alcohol for sterilization. However, before use burn the tip of the forceps in an alcohol lamp and then rinse it with sterile water. Or, you can also use the sterilized media to cool down the tip of the forceps.

• Incubate the cultures at a temperature between 22 °C to 28 °C.
• For subculturing the plant after a few weeks, use the same media as used to establish the culture.

Rooting of Plant

• Prepare a rooting media with ½ MS media with 2g/L activated charcoal, 30 g/L sugar, and 8 g/L agar, and 0.5 ml/L kinetin.
• Autoclave the media at 121 °C for 20 minutes.
• Divide the shoots and transfer them to the rooting media.

Acclimatization of Darlingtonia californica

Follow the steps for acclimation of your cultured plants:

• Take out your rooted cultures and remove the plant from the media using forceps.
• Wash the plantlet under running tap water to wash off any media sticking to the plant root.
• Divide the plantlet into small groups and transfer them to a mix of organic peat moss with perlite or sphagnum moss.
• Acclimate the plants in a suitable greenhouse condition with a humidity dome.
• For the first week open the slider in the dome to allow well-exchange of air for 15-20 minutes and then close it. After the first week, you can gradually start keeping the lid hole open.
• Final step is to keep increasing the intensity of the sunlight to match the outer environment.

Get The Best Tissue Culture Experience With PCT Products and Equipment

Plant Cell Technology is helping tissue culturists worldwide by providing unique and world-class products and services that smoothen their process. The PCT Store has MS media, agar, gellan gum, Plant Preservative Mixture (PPM), culture vessels, Biocoupler (TM), and masks in its store to facilitate your processes.

And, that’s not it! Plant Cell Technology also offers consultation services to culturists of all sizes that help to get instant solutions to your tissue culture problems.

You can either book a one-on-one consultation call or a physical visit to your lab. We help you at every step of the tissue culture process, ranging from establishing a tissue culture lab to preventing contamination problems or any specific challenges in your process.

So, visit plantcelltechnology.com today and learn more about our products and services and how they help you excel in your tissue culture processes.

Happy Culturing!