28 Mar 2023

Sterilizing Carnivorous Plant Seeds Using PPM™

Anjali Singh

Table of Contents

It won’t startle you if someone talks about animals eating plants, but what if I tell you that some plants turn the tables!

Yes, carnivorous plants are predatory flowering plants that attract, capture and absorb nutrients from animals. It’s their adaptation to survive in nutrient-poor conditions, especially to obtain an adequate amount of nitrogen. They consume insects, protozoans, crustaceans, small water and soil-living invertebrates, mice, and some arthropods.

These plants capture their prey by using special mechanisms like pitfall traps, flypaper traps, snap traps, suction traps, and Lobster-pot traps. You can read more about these traps in the article Subculturing a Year-old Cephalotus Culture Using PPM™.

The carnivorous plants have evolved independently in many plant lineages, which has created families like Droseraceae, Drosophyllaceae, Biblidaceae, Nepenthaceae, Cephalotaceae, Sarraceniaceae, Dioncophyllaceae, Roridulaceae, Bromeliaceae, and Lentibulariaceae.

In the article, you will learn about two carnivorous plants: Nepenthes truncata and Sarracenia waccamaw,and the procedure of sterilizing their seeds using PPM™ (as shown in the video (above).

Nepenthes truncata

Source:https://www.deviantart.com/mokele/art/Nepenthes-truncata-48019842

Order: Caryophyllales

Family: Nepenthaceae

Genus:Nepenthes

Species:Nepenthes truncata

Nepenthes, commonly known as pitcher plants, can be found in the diverse habitat of Madagascar, India, Northern Australia, New Caledonia, and the Philippines. Different species can be found in different habitats, like on beaches, cold wind swept ridge tops, sand, acid bogs, or alkaline volcanic soil.

The species N. truncata is endemic to the Philippines and grows at the height of 0-1500 m above sea level. Additionally, the plant uses the pitfall trap mechanism to capture its prey.

Sarracenia waccamaw

Order: Ericales

Family: Sarraceniaceae

Genus:Sarracenia

Species:Sarracenia waccamaw

Source: https://www.botany.org/Carnivorous_Plants/

Sarracenia is commonly known as trumpet pitchers, and is one of the largest genera of pitcher plants. The genus is indigenous to the eastern seaboard of the United States, Texas, the Great Lakes area, and southeastern Canada.

The genus includes herbaceous perennial plants whose leaves are modified into a funnel/trumpet or pitcher shape to capture its prey. The extrafloral nectaries present on the rim/lip of the pitcher attract insects and make them slip into the pitchers where they get digested by the plants using proteases and other digestive enzymes.

Figure: Pitcher of the species of Sarracenia.

The nectar is present in the peristome zone of the pitcher that lures insect prey. And the operculum helps prevent the plant’s pitcher from getting filled with rainwater, which can result in the loss of prey and dilution of digestive juices.

Source: https://en.wikipedia.org/wiki/Sarracenia

Procedure to sterilize the seeds of the carnivorous plants

Carnivorous plants are threatened in their natural habitats because of uncontrolled exploitation for trade and extensive human activities. In nature, the growth of these plants through seed takes about 223 days with a low percentage of germination.

So, tissue culture practices have been very efficient in the mass production of these plants, to conserve their genes, and maintain their genetic diversity. Tissue culture propagation using cuttings of plants is one potential method, but it will lead to loss of genetic diversity in the plants. So, in-vitro seed germination is a method of propagation often used to preserve genetic diversity, as seeds are heterozygous (containing different forms of a single gene), and increase the percentage of germination.

Here’s a stepwise protocol to sterilize carnivorous plant seeds using Plant Cell Technology’s trademark product, PPM™.

Material Required

Nepenthes truncata and Sarracenia waccamaw seeds, Eppendorf, 5% PPM™, 95% distilled water (autoclaved), 4X MS slats (1X=4.54 grams), micropipette, surfactant, bead sterilized, forceps, culture containers, and parafilm or saran wrap.

Procedure

1. Label and number the tubes in which you will be sterilizing the seeds and record it in your notebook as well.

2. To sterilize the seeds, you need to prepare 10 ml of sterilizing solution that consists of 0.5 ml PPM™, 9.5 ml water, and 0.1816 gram MS salts. Below are the calculations for different volumes of the sterilizing solution.

Table: Calculation of different volumes of sterilizing solutions

3. To prepare the solution, take a 10 ml tube and add 9.5 ml of autoclaved distilled water using a micropipette. Then, change the tip of the micropipette and add 0.5 ml PPM™. Lastly, add 0.1816 grams of MS salts.

4. Add a drop of Tween 20 or dish detergent to work as a surfactant (it reduces the surface tension of the solution and makes it more effective).

5. Then, add 4-5 Nepenthes seeds in four separate tubes. The sterilization time will be different for all four tubes.Tube-1: 2 hours, Tube-2: 4 hours, Tube-3: 6 hours, and Tube-4: 8 hours.

6. Repeat step 5 for Sarracenia waccamaw seeds.

7. Add 0.5 ml sterilizing solution to all the tubes using a micropipette.

8. Put seeds in the media using sterilized forceps by uniformly spreading them in the culture plate/box.

9. Make sure to label your containers with the plant name, sterilization treatment, and date.

10. Seal the container using parafilm or saran wrap.

11. Place containers in 16 hours of artificial light.

12. First seeds should start germinating after 2-3 weeks, however, some seeds might take a longer time (especially Nepenthes seeds).

At Plant Cell Technology, we are committed to helping our customers with their culturing processes as much as we can. The purpose of the video above is to educate our customers on the sterilizing process of carnivorous plant seeds using PPM™.

PCT (Plant Cell Technology) has its own tissue culture lab setups where it tests the efficiency of its products, and designs protocols for culturing different plants in a laboratory environment.

Along with providing tissue culture services and equipment, PCT is also taking steps toward creating informative videos to help and educate its customers. So, don’t forget to check out our Plant Cell Technology website for more “how-to” videos.

Also, make sure to check out our other videos on our Youtube channel “PlantCellTechnology”, and don’t forget to subscribe to the channel so you don't miss any future videos from PCT. Do share your thoughts on the video and content with anjali@plantcelltechnology.com. And, if there is any specific tissue culture topic/subject you want us to make a video about, let us know at the given email. We will do our best to bring you what you want!