this slide is about the basics of protoplast culture. isolation method, staining, and culture

1. Protoplast culture

3. 1. The protoplast in culture can be regenerated into a whole plant.
2. Hybrids can be developed from protoplast fusion.
3. It is easy to perform single cell cloning with protoplasts.
4. Genetic transformations can be achieved through genetic
engineering of protoplast DNA.
5. Protoplasts are excellent materials for ultra-structural studies.
6. Isolation of cell organelles and chromosomes is easy from
protoplasts.
7. Protoplasts are useful for membrane studies (transport and
uptake processes).
8. Isolation of mutants from protoplast cultures is easy.
Importance of protoplast culture

18. FDA Staining Test:
• FDA is cell-membrane permeable and accumulates inside of
viable cells as fluorescein.
• FDA: fluorescein diacetate
• Since fluorescein is less hydrophilic, the leakage of fluorescein
from cells is rather high. FDA is also utilized for flow cytometry.
1. 0.5 mg/ml FDA stock solution with DMSO. Dilute 10 ul of the stock
solution with 5 ml PBS(-).
2. Prepare a cell suspension and wash cells with PBS(-). Prepare
1×105-1×106 cells/ml cell suspension
3. Add 15 ul FDA solution to 30 ul cell suspension, and incubate at
37ºC for 15-30 min.
4. Put 10 ul stained cell suspension on a glass slide and cover with a
cover glass.
5. Observe the cells under a fluorescence microscope with 488 nm
excitation and 530 nm emission filters.
Viability test

20. Evan’s Blue Test
Evans blue is a nonfluorescent dye used to test the
viability of plant protoplasts. It's used to check the
integrity of the cell membrane.
Tetrazolium reduction test
The assay relies on oxidoreductases to reduce tetrazolium into
purple formazan crystals. The formazan crystals are then
solubilized, and the absorbance reflects viability.

24. • There are different methods of protoplast culture such
as:
• Liquid culture
• Agar culture
• Droplet culture
• Co-culture
• Hanging droplet culture
• Immobilised/bead culture
• Feed layer technique

25. Liquid culture
• This method is generally preferred in most cases during early
developmental stages of protoplasts, because it allows easy
dilution and transfer
• Protoplasts easily get divide in liquid media, osmotic pressure
of the medium can be regulated and can be effectively reduced
during further growth of protoplasts.
• The disadvantage of this method is that it does not permit the
isolation of single colonies derived from one parent cell.

26. Agar culture
• The protoplasts are cultured on the plate using
Bergmann’s cell plating technique.
• Here, the protoplasts are fixed in a position for cell division
and further growth.
• The agar technique is efficient to prevent the formation of
clumps in the cultures.

27. Droplet Culture:
Suspending protoplasts in liquid culture media are placed
on Petri dishes in the form of droplet, the cultured
protoplasts clump together at the center of droplets. The
liquid medium can be changed at regular interval.
Co-culture:
Sometimes to induce division the newly isolated protoplast
suspension is mixed with a reliable fast growing protoplast
suspension and mixed protoplasts are plated. Some growth
factors help to induce the proper growth and development
of the isolated protoplasts.

28. Hanging droplet culture

29. Immobilized bead Culture:
The protoplasts suspension can be mixed with any kind of
polymer like alginate, carrageenan, etc. and then small beads
are made by dripping into the liquid medium and then
cultured into liquid medium with slow shaking condition.
Micro-Drop Culture
In this technique, protoplasts are cultured in a special dish,
known as Cuprak dishes. The dish has outer and inner
chambers. The inner chamber has several walls, in which
individual protoplast droplets in nutrient media can be added.
The outer chamber contains water to maintain the humidity
for the growth of the cultures.

30. Feed Layer Technique
In this technique protoplast suspension is exposed to X-rays
and then plated on agar plates. It’s used to culture
protoplasts at low density.