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Subculturing a Year-old Cephalotus Culture Using PPM™

Posted by Anjali Singh on 9th Sep 2020

Subculturing a Year-old Cephalotus Culture Using PPM™


Insectivorous / Carnivorous Plants

Insectivorous plants, or Carnivorous plants, have a unique adaptation for living in soil with low or poor nutrient conditions, especially nitrogen. They fulfill their nutrient needs by trapping insects, arthropods, and occasionally small frogs or mammals for their food.

Carnivorous plants belong to five different orders that include a dozens of genera and more than 500 species. The largest family of carnivorous plants is Lentibulariaceae of order Lamiales. These plants absorb nitrogen by trapping prey in their modified leaves (or traps).

Carnivorous plants follow different mechanisms to capture or trap their prey. These mechanisms are considered active or passive traps, depending on whether there is any movement involved in capturing the prey. The different trap mechanisms include:

  1. Pitfall trap: This is the most common type of trap found in pitcher plants. In this trap, the leaves are folded into a hollow, deep, slippery pool that is filled with digestive enzymes.
  2. Flypaper trap: This trap can be observed in sundews and butterworts. Stalked glands cover the leaves and secrete a sticky, mucilaginous substance on the leaf surface to capture the prey.
  3. Snap trap: This trap is found in plants like waterwheel and Venus flytrap. It involves rapid leaf movement (snap shut) when trigger hairs are touched by insects.
  4. Suction trap: This trap mechanism is found in bladderworts. Leaves are modified in a bladder shape with a hinged door lined by trigger hairs. The plants that use this mechanism trap their prey by generating an internal vacuum.
  5. Lobster-pot trap: This mechanism is observed in corkscrew plants, in which inward-pointing hairs are present that force prey to move deeper into the digestive organ.

Description of Cephalotus

Classification

Kingdom: Plantae

Division: Angiosperms

Clade: Eudicots

Clade: Rosids

Order: Oxalidales

Family: Cephalotaceae

Genus: Cephalotus


Cephalotus plants also bear ordinary, simple lanceolate leaves, other than the modified pitchers. The ordinary leaves are observed in the spring and fulfill the plant’s carbon requirements by the process of photosynthesis. After reaching peak production, the pitcher leaves start appearing. The flowers have six ovate–elliptic sepals, 12 filaments with mauve stamens, and six pale green flask-like ovaries.Cephalotusfollicularis is the only species of the genus Cephalotus. It is a small perennial herb that is endemic to the Southwest part of Australia, where it is abundantly present on the margins of freshwater wetlands, ditches, and slow streams. It is a slow growing plant that takes many years to reach the flowering stage.

These plants have the pitfall trap mechanism to capture their prey. The mouth of the pitcher is red and purple in color to attract the insect. And, the smooth ridges on the mouth direct insects inside the pitcher.

Figure: Left hand side: An illustration of the inside of Cephalotus flower Right hand side: A close-up image of Cephalotus mouth.

In-vitro Propagation of Cephalotus

The propagation of Cephalotus through seed is a slow process and the method of vegetative propagation is also not expeditious to produce a large number of plants. So, the tissue culture technique is an efficient method to culture the rare or endangered species of plants, like Cephalotus.

To culture the Cephalotus in labs, LS (Linsmeier and Skoog) media is used which contains the same components as MS media. The only variation is the addition of 100 mg/L of myoinositol and 0.4 mg/L thiamine HCL. The in-vitro tissue culture technique has the potential to increase the production by 10 fold (compared to conventional methods) in just 6 months.

The video (above) shows the process of subculturing of Cephalotus. The steps followed are discussed in the section below.


Subculturing of a Year-old Cephalotus Culture Using PPM™

PPM™ (Plant Preservative mixture) is a featured product of PlantCellTechnology (PCT). It is effective in fighting any kind of contamination (airborne, waterborne or endophytic) in cultures. To test and show the efficiency of the product and to educate the customers for its use, PCT uses PPM™ to avoid contamination in all the cultures of it’s labs.

Material Required: A year-old culture of Cephalotus, PPM™ (Plant Preservative Mixture) from Plant Cell Technology, media, autoclaved distilled water, micropipette, glass bead sterilizer (or you can use any sterilizing technique available to you), culture containers, forceps, and a laminar flow hood.

Procedure:

  1. Take a year-old culture of Cephalotus (a culture this old would be out of media, a few plantlets would have died, and the culture would be contaminated).
  2. Prepare 1% PPM™ solution in a 250 ml jar or container. Using micropipette add 2 ml of PPM to 200 ml of water and mix it well.
  3. Soak the old Cephalotus culture in the prepared solution for 1 hour.
  4. After one hour, pour off/discard the PPM solution and transfer the plant from the jar to a sterilized plate.
  5. Sterilize the forcep using glass bead sterilizer at 290 ℃.
  6. Remove the big chunks of dead plants using the sterilized forceps.
  7. Then again place the forceps back in the bead sterilizer for 10-15 seconds.
  8. Cool the forceps and then divide the plants in small sections and remove the dead tissue as much as possible.
  9. Transfer the small section of the plants to culture containers containing fresh media.
  10. Subculture as many plates as you want or until you run out of the culture.


NOTE: If you don’t have a laminar flow hood, you must wear a mask and work as fast as you can.

At Plant Cell Technology, we are committed to helping our customers with their culturing processes as much as we can. This video was an initiative to educate our customers on the process of subculturing Cephalotus using PPM™.

PCT (Plant Cell Technology) has its own tissue culture lab setups where it tests the efficiency of the products and designs the 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 views 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!


References

  1. Ko, C., Lin, T., Ho, C., & Shaw, J. (2010). In Vitro Regeneration of Cephalotus follicularis, HortScience horts, 45(2), 260-264. Retrieved Sep 7, 2020, from https://journals.ashs.org/hortsci/view/journals/hortsci/45/2/article-p260.xml
  2. https://botany.org/Carnivorous_Plants/
  3. https://www.britannica.com/plant/pitcher-plant

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