The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
INTRODUCTION
2. HISTORY
3. BASIC COMPONENT OF MEDIA
1. Inorganic nutrient
2. organic supplements
3. Carbon and energy source
4. Growth Regulators
5. Solidifying Agent
6. PH
4. TYPES OF MEDIA
5. MS MEDIA
6. IMPORTANCE
7. CONCLUSION
8. REFERANCE
An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell
A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.
Until two decades ago the genetic resources were getting depleted owing to the
It was imperative therefore that many of the elite, economically important and endangered species are preserved to make them available when needed.
The conventional methods of storage failed to prevent losses caused due to various reasons.
A new methodology had to be devised for long term preservation of material.
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
The presentation gives overview of production of secondary metabolites using callus culture as well as tissue culture techniques. Various batch and continuous culturing process are described on the basis of secondary metabolite to be synthesised.
1.What is plant tissue culture?
2.Production of virus free plants.
3.History.
4.Virus elimination by heat treatment.
5.Virus elimination by Meristem Tip culture.
6.Factor affecting virus eradication by Meristem Tip culture.
7.Chemotherapy.
8.Virus elimination through in vitro shoot-tip Grafting.
9.Virus Indexing.
10.Conclusion .
11.References .
WHAT IS ARTIFICIAL SEED..?
Artificial seed can be defined as artificial encapsulation of somatic embryos, shoot bud or aggregates of cell of any tissues which has the ability to form a plant in in-vitro or ex-vivo condition.
Artificial seed have also been often referred to as synthetic seed.
HISTORY
Artificial seeds were first introduced in 1970’s as a novel analogue to the plant seeds.
The production of artificial seeds is useful for plants which do not produce viable seeds. It represents a method to propagate these plants.
Artificial seeds are small sized and these provides further advantages in storage, handling and shipping.
The term, “EMBLING” is used for the plants originated from synthetic seed.
• The use of synthetic varieties for commercial cultivation was first suggested in Maize (Hays & Garber, 1919).
Suspension Culture and Single Cell Cultures, Culturing methods, maintenance a...Ananya Sinha
Suspension Culture and Single Cell Cultures, Culturing methods, maintenance and application
Generally, suspension culture is a one stop technology to produce secondary metabolites on a large scale in-vitro, irrespective of the climatic condition or nutrient availability (as required in field plants).
In this presentation, we will see the importance of suspension culture, culturing methods and it's application (mostly with respect to plants) and also focus on what exactly is a single cell culture.
INTRODUCTION
2. HISTORY
3. BASIC COMPONENT OF MEDIA
1. Inorganic nutrient
2. organic supplements
3. Carbon and energy source
4. Growth Regulators
5. Solidifying Agent
6. PH
4. TYPES OF MEDIA
5. MS MEDIA
6. IMPORTANCE
7. CONCLUSION
8. REFERANCE
An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell
A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.
Until two decades ago the genetic resources were getting depleted owing to the
It was imperative therefore that many of the elite, economically important and endangered species are preserved to make them available when needed.
The conventional methods of storage failed to prevent losses caused due to various reasons.
A new methodology had to be devised for long term preservation of material.
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
The presentation gives overview of production of secondary metabolites using callus culture as well as tissue culture techniques. Various batch and continuous culturing process are described on the basis of secondary metabolite to be synthesised.
1.What is plant tissue culture?
2.Production of virus free plants.
3.History.
4.Virus elimination by heat treatment.
5.Virus elimination by Meristem Tip culture.
6.Factor affecting virus eradication by Meristem Tip culture.
7.Chemotherapy.
8.Virus elimination through in vitro shoot-tip Grafting.
9.Virus Indexing.
10.Conclusion .
11.References .
WHAT IS ARTIFICIAL SEED..?
Artificial seed can be defined as artificial encapsulation of somatic embryos, shoot bud or aggregates of cell of any tissues which has the ability to form a plant in in-vitro or ex-vivo condition.
Artificial seed have also been often referred to as synthetic seed.
HISTORY
Artificial seeds were first introduced in 1970’s as a novel analogue to the plant seeds.
The production of artificial seeds is useful for plants which do not produce viable seeds. It represents a method to propagate these plants.
Artificial seeds are small sized and these provides further advantages in storage, handling and shipping.
The term, “EMBLING” is used for the plants originated from synthetic seed.
• The use of synthetic varieties for commercial cultivation was first suggested in Maize (Hays & Garber, 1919).
Suspension Culture and Single Cell Cultures, Culturing methods, maintenance a...Ananya Sinha
Suspension Culture and Single Cell Cultures, Culturing methods, maintenance and application
Generally, suspension culture is a one stop technology to produce secondary metabolites on a large scale in-vitro, irrespective of the climatic condition or nutrient availability (as required in field plants).
In this presentation, we will see the importance of suspension culture, culturing methods and it's application (mostly with respect to plants) and also focus on what exactly is a single cell culture.
Defination,growth curve, types and kinetics of growth curve, applications and advantages and disadvantages . Environmental factors affecting the cell growth.
Induction and maintenance of callus and
cell suspension cultures.Callus Culture :
● Callus represents an unorganized or undifferentiated mass of cells.
● They are generally composed of parenchymatous cells and
usually undergo division.
● When an explant is cultured in a medium supplemented with
sufficient amount of auxins, it starts producing mass of cells from
the surface of the explant.
● The concentration of auxins required for each type of explant will
be different and is mainly dependent on the physiological state of
the explant tissue.
● Callus cultures can be maintained for a very long time by
intermittent sub-culturing to a fresh medium.
● The callus cultures can be manipulated for different purposes by
changing the hormone concentrations in the media
Usage of callus Culture :
● Rregeneration of plantlets.
● Preparation of single cells
● Suspension cultures.
● Protoplasts preparation
● Genetic transformation studies.
In some instances, it is necessary to go through a callus phase prior to
regeneration via somatic embryogenesis or organogenesis.
● Callus cultures are suitable for the generation of useful somaclonal variants.
● Callus cultures can be used for in vitro selection of cells and tissue variants
Induction of callus Culture :
● Induction of cell division in the permanent tissue is highly
dependent on the high auxin content (e.g., 2, 4-D) in the
medium.
● The hormone requirement for callus induction may be auxin
alone, cytokinin alone or auxin and cytokinin in different ratios.
● The type of growth regulator requirement and its concentration
in the medium depends strongly on the genotype and
endogenous hormone content of an explant
Transformation of any kind of tissue explant into proliferating
callus mass depends highly on the plant genotype, the source
of origin of the explant and the physiological state of the tissue
at culture.
● For the initiation of callus cultures tissues from young seedling
or from juvenile part of the mature plant either grown in vivo or
in vitro are generally taken, either steri lised (when grown in
vivo) or cut aseptically (when grown in vitro) and inoculated
aseptically on a nutrient medium provided with different
combinations of exogenous growth substances
Maintenance of Callus Culture:
● After callus induction, the callus is grown further on a new
medium, which is referred to as sub-culturing.
● During subculturing the callus tissue shows sigmoid growth
pattern on a particular media.
● The time interval for sub-culturing is calculated according to
the growth pattern of the particular tissue type and genotype
of the plant.
● When the callus growth reaches to the stationary phase of
growth then it needs to be cultured on to a fresh medium.
○Maintenance of Callus Culture:
● After callus induction, the callus is grown further on a new
medium, which is referred to as sub-culturing.
● During subculturing the callus tissue shows sigmoid growth
pattern on a particular media.
● The time interval for sub-cultur
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Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
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3. • CALLUS CULTURE:
• Callus is an amorphous aggregate of loosely arranged
parenchyma cells, which proliferate from mother cells.
• Cultivation of callus usually on a solidified nutrient
medium under aseptic conditions is known as callus
culture.
• These culture need to be sub-cultured every 3-5
weeks.
• Risk free, easy to handle.
4.
5.
6. INITIATION OF CALLUS CULTURE:
1. Selection and preparation of explant
2. Selection of culture medium
3. Transfer of explant
4. Incubation of culture
MAINTENANCE OF CALLUS CULTURE:
1. Depletion of nutrients in medium
2. Gradual loss of water
3. Accumulation of toxic waste
7. • SUSPENSION CULTURES:
• Tissues and cells cultured in liquid medium is
known as Suspension culture.
• They often produce suspension of single cells and
clumps.
• Liquid cultures are constantly agitated, generally by a
gyratory shaker @ 100-250ppm.
• This enhances the aeration and dissociation of cell
clumps into smaller pieces.
• This suspension cultures are used for large scale
production.
• Need to be sub-cultured every week.
8. 1. INITIATION OF SUSPENSION CULTURE:
1. Isolation of cells:
a) From callus culture
b) From plant organ- by Mechanical and enzymatic
method
2. Growth Pattern of suspension culture
3.Maintenance of suspension culture:
1. Batch cultures
2. Continuous cultures
9.
10. • Batch cultures:
• A technique used to grow microorganisms or cells.
• A limited supply of nutrients for growth is
provided; when these are used up, or some other
factor becomes limiting, the culture declines.
• Cells, or products that the organisms have made, can
then be harvested from the culture.
• The cell number or biomass of batch culture exhibits
a typical sigmoidal growth curve.
11. • LAG Phase: during this phase cell number or
biomass is unchanged.
• LOG Phase: during this phase the cell number
rapidly increases in geometrical progression.
• Stationary Phase: In this phase end phase where
cell number does not change.
12. • Batch cultures are maintained by sub-culturing.
• These cultures are mostly used in initiation of
cell suspension i.e. single cell culture.
• These are most convenient to use than
continuous cultures hence mostly used.
13. • Continuous culture:
• These cultures are maintained in a steady state for a
long period by draining out the used (or) spent
medium and adding the fresh medium.
• such subculture systems are either closed (or) open
type.
• Closed:
• The cells separated from used medium taken out for
replacement and added back to the suspension culture.
• So that the cell biomass keeps on increasing.
14. • Open:
• Both cells and the used medium are taken out from
open continuously cultures and replaced by equal
volume of fresh medium.
• The replacement volume is so adjusted that cultures
remain at sub-maximal growth indefinitely.
