Protoplast culture isolation and purification , application , method of Protoplast culture , factors affecting of Protoplast culture.

1. PROTOPLAST
CULTURE ISOLATION
BY
SNEH SAHU
SANT GAHIRA GURU UNIVERSITY AMBIKAPUR CHHATTISGARH

2. INTRODUCTION
• Is a naked Cell surrounded by a plasma membrane it can regenerate cell wall grow and
divide.
• Is fragile but can be cultured and grow in to a whole plant.
• is used by biotechnolists microbiologists , pathologists.
• In 1960 , E.C. cocking contributed to the enzymatic islation and culture of protoplast.
A typical cell wall
• The plant cell wall is multilayered structure.
1. The middle lamella – pectin compounds cellulose.
2. The primary wall – cellulose and hemicellulose
3. Secondary wall - many cells produce an additional layer.

3. PROTOPLAST ISOLATION
Refer to the separation of protoplast from plant tissue important to isolate viable and unijored Protoplast as gently And
as Quickly as possible . Imolues two methods –
Mechamical method
Tissue is immersed in 1.0M sucrose until protoplasm Shrunk away from their enclosing cell wall
• plasmolysed tissue is cut with a sharp knife at such a thickness that only cell wall are cut.
• undameged Protoplast incstrips are released by osmotic swelling when placed in a low concentration of sucrose
solution.
• problem encountered – some cells release anut complete Protoplast while the rest produces broken dead Protoplasts.
Enzymatic method
• Refers to the use of enzymatic to dissolve the cell wall for releasing Protoplasts.
Advantages
• used variety of tissues and organs such as fruits , roots, petiols , leaves

4. CONTI….
• Osmotic shrinkage is minimum.
• cells remain intact and not infured.
• protoplast readily obtained.
Involves two mathods
1. Direct mothod
• Incubation of leaf segments overnight in enzymes solution
• mixture is filtered and centrifuged
• protoplast forms pellet
• Then washed with sorbitol and re-centrifuged.
• clean Protoplasts float
• they are pipetted out.

5. CONTI…
2. Sequential method
• two enzyme mixtures are used one after the other
• leaf segments with a are vacuumed infyctrated for 5 min transferred to a water bath at 25°C
and subjected to slow shaking.
• The enzyme mixture the then replaced by fresh enzyme mixture A and leaf segments are
incubated for another hour
• The mixture is filtered using nylon meth and centrifuged for 1min.
• washed 3 times withv13% mannitol
• cells are then incubated with , enzyme mixture B for above 90min. At 30°C
• the mixture is centrifuged for 1 min. So that protoplast form a pallet and clean 3 times with
sorbitol.

6. PURIFICATION OF PROTOPLAST
• Protoplast are purified by remaining undigesred Material( debris )
bursts Protoplast enzymes.
• debris – are removed by Filtering the preparation through a nylon
mesh .
• Enzymes are removed by centrifugation uvereby the Protoplast
settle to the bottom of the tube and the Protoplast settle to the
bottom of the tube and the supermanant removed with the help of a
pipette
• Intact Protoplast are seperated from broken Protoplast through
centrifugation and removed by a pipette as they are collected at the

7. PROTOPLAST CULTURE
• Isolated protoplast cen be cultured in an appropriate medium to refarm cell wall
and generate callus.
Optimal culture conditions.
1. Optimal density to the culture.
2. Optimal auxin to cytokinin ratio , glucose and sucrose.
3. maintain osmoprostectant in the medium.
4. Temperature- 20-28°C PH- 5.5- 5.9 , 0.25% casein hydrolysate . BAP and NAA.

8. CULTURE OF PROTOPLAST
• protoplast culture in suitable nutrient media first generate a new cell wall .
• The formation of a complete all with a wall is followed by an increase and induction of
cell division.
• The first cell division may occur Within 2 to 7 day of culture. Resulting in small clamps
of cell , also known micro colony within 1 to 3 weeks.
• from such clumps , there are two routes to generate a complete plant.
1. Plants are regenerated through organogenesis From callus masses.
2. The micro calli can be made to develop into somatic embryos , which are then
converted into whole plant through germination.

9. IMPORTANCE OF PROTOPLAST CULTURE
• study of osmotic behaviour
• study of IAA action .
• study of plasmalemma
• study of cell wall formation
• organelle isolation.
• study of morphogenesis
• virus uptake and replication.
• study of photosynthesis
• Gene transfer.

10. FACTOR AFFECTING PROTOPLAST CULTURE
1. Plant species and varieties – small Genetic difference leads to Verifying Protoplast
responses to culture conditions.
2. Plant age and organ – age of donor plant and it’s developmental stage. Stage are
germinating embryos , plantlets leaves.
3. Pre- culture conditions- Protoplast culture are different influenced by climatic
factors and different culture of seedings yields Protoplast having different responses
when cultured.
4. pre- treatment to the tissue, before isolating Protoplast – cold treatment
plusnolysis and hormone increase the chance of recovery of uiable protoplastes and
their plating efficiency.
5. Density – gucial as it influences plating efficiency and surviving of Protoplast. At
higher density , Protoplasts complete with one another while at lower density losses
of metabolites from Protoplasts is more.

11. APPLICATION OF PROTOPLAST
Protoplasts can be used to study
• Metabiotic Studies including photosynthesis.
• for DNA transformation.
• for somatic hybridization
• Ingesting foreign material into the cytoplasm.
• single cell systems.
• cell wall synthesis.

12. CONCLUSION
In a nutshell , Protoplast culture has formed it’s many uses and
applications in Field’s such as genetic engineering and crop breeding.
Genetic transformation of trant gene. DNA somatic hybridization by
Protoplast fution of species or subspecies resistant to traditional.
Cross breeding and isolation of subcellular organelles ore all
examples of how the development of Protoplast systems haS lid To
on increase in the versatiling of plant.

13. Thank you