Germplasm conservation

1. In-situ conservation: In this conservation of germplasm is done in their natural environment such as national parks, gene sanctuaries, etc. It provides the natural habitat for the preservation of land plants with genetic diversity.

1. a) Environmental hazards may lead to loss of germplasm
2. b) The maintenance and cost is very high
3. Ex-situ conservation: in this method seeds, or in-vitro cultures of plant cells, tissues, or organs can be used to preserve genetic material as gene banks for long-term storage. This is the most convenient method compare to in-situ conservation.

1. Seeds are the most common and conventional material to conserve plant germplasm.
2. The majority of plants are propagated through seeds.
3. Seeds occupy a small place and are convenient for transport to various places.
4. Seed viability is reduced over some time
5. Seeds are prone to pathogenic infection and may lead to their destruction
6. Vegetatively propagated plants have no use for this seed conservation e.g. for potato, Ipomoea etc
7. Clones cannot be maintained by seed conservation.

In-vitro methods for germplasm conservation

1. Cryopreservation (freeze-drying)
2. Cold storage
3. Low pressure and low Oxygen Storage

1. Cryopreservation (freeze-drying): In this method, the storage of plant tissues and cells is done in a frozen state. Plant cells or tissues are preserved for the long term at a very low temperature. The principle of cryopreservation is the use of Cryoprotectants to reduce temperature to bring plant cells and tissue culture to a non-dividing state or zero metabolism. Cryoprotectants such as glycerol, mannitol, dimethylsulfoxide (DMSO), and proline are used to store plant tissues at low temperatures. These Cryoprotectants prevent damage caused to cells by freezing or thawing.

1. Over solid carbon dioxide (at -79⁰ C)
2. Low-temperature deep freezers (at -80⁰ C)
3. In vapor phase nitrogen (at -150⁰ C)
4. In liquid nitrogen (at- 196⁰ C)

2. Cold storage: in this method germplasm is conserved at low and 1-9⁰ C non-freezing temperatures. This technique allows us to slow down the growth of plant material, in contrast, to completely stop cryopreservation. Hence the method is also known as slow germplasm conservation. Cryogenic injuries are avoided by this method. This method is simple, cost-effective and the yield and survival rate of germplasm is good.

3. Low-pressure storage (LPS) and low oxygen storage (LOS): in LPS the atmospheric pressure around the plant material is reduced, which partially decreases the pressure exerted by the gases around the germplasm. This lowered partial pressure declines the growth of plants in-vitro. This LPS system is used for short-term and long-term storage of plant material. In LOS the atmospheric pressure i.e. 260 mm Hg is maintained by the addition of inert gases especially nitrogen while the oxygen concentration is reduced. The partial pressure of Oxygen below 50 mm Hg declines plant tissue growth. This is due to the reduced availability of O₂, which decreases CO₂ This phenomenon results in reduced photosynthetic activity and thereby inhibiting plant tissue growth and dimension. Long term conservation of plant material by LOS inhibits plant growth after certain dimensions.

Applications of Germplasm storage

1. Plant materials like cells and tissue cultures of several species can be cryopreserved and maintained for a long time
2. Cell cultures used for the production of secondary metabolites can be conserved for a long time by cryopreservation
3. Plant material free from pathogens can be stored and can be propagated whenever needed.
4. Somaclonal variations in culture can be conserved by this cryopreservation.
5. Conservation of plant material from endangered species can be done
6. Germplasm developed from genetic manipulations can be conserved.
7. Globally information can be exchanged by the establishment of germplasm banks.

Limitations

1. It requires expensive instruments for conservation
2. Trained personnel is required for these techniques.

Multiple-choice Questions

1. b
2. d
3. d
4. d
5. a
6. b
7. a
8. d
9. d
10. b

1. U. Satyanarayana, biotechnology, 1st e.d. Kolkata, books & allied (p) ltd publishers, 2005  page: 565-571

The post Germplasm Conservation and Its Applications: MCQ for DBT BET, GPAT, GATE, & CSIR NET appeared first on Gpatindia: Pharmacy Jobs, Admissions, Scholarships, Conference,Grants, Exam Alerts.

Advantages for production of secondary metabolites

1. Production of compounds can be done in controlled conditions as per market demand.
2. Environmental factors, seasonal variations, pest, and microbial diseases, and geographical constraints can be avoided by the in-vitro cultural system
3. Product formation can be facilitated by control over cell growth.
4. The consistency of quality products can be achieved as they are produced from a specific cell line.
5. Product recovery is easy
6. In-vitro cultures are important when plants are expensive or difficult to grow in fields.
7. Mutant cell lines can be developed for the production of novel compounds.
8. Plant tissue culture techniques can minimize time and labor cost

Disadvantages

1. In compared to intact plants, production of secondary metabolites is lower in Invitro culture
2. There are chances of cell damage during culture due to vigorous stirring
3. Strict aseptic conditions have to be maintained, otherwise, any infection to the culture can affect production.

Examples of secondary metabolites used as drugs

Multiple-choice questions

1. a
2. b
3. d
4. a
5. a
6. c
7. b
8. b
9. c
10. b

Reference

1. U. Satyanarayana, biotechnology, 1st e.d. Kolkata, books & allied (p) ltd publishers, 2005  page:507-516

The post Production of Plant Secondary Metabolites: MCQ for DBT BET, GPAT, GATE, & CSIR NET appeared first on Gpatindia: Pharmacy Jobs, Admissions, Scholarships, Conference,Grants, Exam Alerts.

Protoplast is the plant cell without a cell wall. This protoplast possesses other cellular components and a plasma membrane. Hence, this protoplast is a functional plant cell without a barrier i.e. cell wall.

Steps involved in protoplast culture

1. Sources of protoplast
2. Isolation of protoplast
3. Purification of protoplast
4. Viability check of protoplasts
5. Culture of protoplasts

1. Source of protoplast: Tissues and organs like leaves, roots, shoot apices, fruits, embryos and microspores are widely used as sources for protoplast. Callus and suspension cultures are also good sources for protoplast isolation.

2. Isolation of protoplast: It can be done by two methods

a) Mechanical method: In this method, the cells of the epidermis are subjected to plasmolysis, by which protoplast causes to shrink away from the cell wall. Then, the protoplast is released by tissue dissection. The main disadvantage of this method that, the yield and viability of protoplast is very low. This process is tedious and laborious.

b) Enzymatic method: This method has an advantage over the mechanical method, which has a high yield of protoplasts with viable cells. Hence, it is a widely used method. Enzymes like cellulases, hemicellulases, pectinases, are used to degrade cellulose, hemicellulose, and pectin in the plant cell wall.

One of the two methods is used to isolate protoplast by enzymatic method.

1. Two-step method: in this method the tissue is treated with pectinase to degrade middle lamella, to separate cells from tissue. These free cells are treated with cellulose to remove the cell wall, which results in the release of protoplast.
2. One-step method: in this method the tissue is treated simultaneously with cellulose and pectinase. Hence it is the most preferred method.

3. Purification of protoplast: It is done by filtration to remove cell clumps and undigested tissues, followed by centrifugation and washing of protoplast.

4. Viability test of protoplast: The following are the methods used to test the viability of protoplast

a) Testing photosynthetic activity of protoplast

b) Measurements of cell wall formation can be observed using Calcofluor white (CFW) stain, which bind to the newly formed cell wall and emits fluorescence.

c) Measurement of oxygen uptake by protoplast by using oxygen electrode

d) Phenosafranine stain: dead protoplast uptake this stain and turns red, whereas viable protoplast remains unstained.

e) Fluorescein diacetate (FDA) staining: viable cells get stained by the FDA and can be detected by fluorescence microscopy.

5. Culture of Protoplast 

Cell wall development around the protoplast membrane is the prior step of protoplast culture. The development of the cell wall is followed by cell division to form small colonies. Protoplast is generally culture in semi-solid Agar medium or Liquid medium.

Advantages of liquid culture over Solid culture

1. It is easy to transfer
2. Dilutions can be easily done in liquid culture
3. Cell density can be easily manipulated
4. Osmotic pressure can be easily changed in a liquid medium

Nutritional Requirements of Culture media

It is similar to those used in callus culture EXCEPT:

1. The culture medium should not contain ammonium
2. Very fewer quantities of iron and zinc are used
3. The Calcium concentration is 2 to 4 times higher than the ordinary cell culture.
4. Glucose is the preferred carbon source for protoplast culture
5. A high Auxin/kinetin ratio is preferred to induce cell division
6. A high kinetin/Auxin ratio is preferred for regeneration

Protoplast culture methods

2. Feeder layer technique: in this technique the protoplast cell suspensions are exposed to x-rays, which inhibits cell division and metabolic activity followed by platting them on agar plates.
3. Co-culture of protoplast: this technique is proffered when morphologically distinct protoplasts are used. So, generally, protoplasts of two different plant species can be co-cultured.
4. Micro drop culture: In this technique, cuprak dishes, specially designed with the inner and outer chamber are used. The inner chamber contains a droplet of protoplast in the nutrition medium is added to wells. Whereas, the outer chamber is filled with water to maintain humidity. This technique has an advantage when there are few protoplasts in the droplet of the medium.

Regeneration of protoplast

Protoplast development can be done in two steps

1. Cell wall formation: generally the process takes two to several days to develop cell wall in cultured protoplasts. The protoplast loses its spherical shape as the cell wall develops.
2. Callus or Whole plant development: generally after cell wall formation, cell size increases, and cell division is initiated within 2-7 days. This cell division results in the formation of small colonies and visible colonies are formed by the third week. These colonies are developed in an osmotic free medium to form a callus. This callus may undergo several manipulations and undergo embryogenic or organogenic differentiation which results in the development of the whole plant.

Applications of protoplast culture and protoplast

1. The whole plant can be generated by a culture of protoplast.
2. Two protoplasts can be fused to form hybrids known as somatic hybridization
3. Protoplast can be used for Single-cell cloning
4. Chromosomes and cell organelles can be easily isolated from protoplast
5. Transport and uptake studies or membrane studies can be easily done by protoplast
6. Protoplast is used in genetic engineering for genetic transformations
7. Ultrastructural studies can be done using protoplast

The millions of single cells are formed by protoplast isolation and protoplast culture, which can be used for wide varieties of studies

Multiple Choice Questions

1. Protoplasts are__________

a) Cell without a nucleus

b) Cell without a cell wall

c) Cell without plasma membrane

d) Cell without genetic material

2.__________ is the first step for protoplast culture

a) Selection of Explant

b) Viability testing for protoplast

c) Isolation of protoplast

d) Preparation of culture media

3. ___________ is the advantage of mechanical method for isolation of protoplast

a) The process is very tedious

b) Very less viable protoplast are isolated

c) Both

d) Only B

4. Cellulase enzyme in isolation of protoplast is used ________

a) To degrade proteins

b) To degrade cellulose

c) To degrade pectin

d) To degrade hemicellulose

5. ____________ is used to check viability of protoplast

a) Phenosafranine staining

b) Fluorescein diacetate (FDA) staining

c) Both

d) Only A

6. The following are the advantages of liquid protoplast culture EXCEPT:

a) It is easy to transfer

b) Dilutions can be easily done in liquid culture

c) Cell density can be easily manipulated

d) Less time consuming

7. Co-culture of protoplast is appropriate for ___________

a) Protoplast from two different organs of the same plant

b) Protoplast from the same organ of different plant

c) Protoplast from two different plant species

d) Protoplast from two same plant species

8. ___________media is used for protoplast culture

a) Synthetic media

b) Natural media

c) Nutritional media

d) None of the above

9. High Auxin/kinetin ratio in nutritional media for protoplast culture is preferred ______

a) To induce cell regeneration

b) To induce cell growth

c) To induce cell division

d) All

10. Advantage of Micro-drop protoplast culture technique is _____________

a) It requires Large numbers of protoplasts

b) It requires a Small number of protoplasts

c) It requires a large amount of culture media

d) It requires less amount of water

Answer Key

1. b
2. a
3. c
4. b
5. c
6. d
7. c
8. c
9. c
10. b

Reference

1. Satyanarayana, biotechnology, 1st e.d. Kolkata, books & allied (p) ltd publishers, 2005  page:523-528

The post Protoplast Culture and its Applications, MCQ for GPAT, GATE, DBT BET & CSIR NET appeared first on Gpatindia: Pharmacy Jobs, Admissions, Scholarships, Conference,Grants, Exam Alerts.

In-plant genetic engineering, plant genes can be modified for required characteristics. Desired plant genes can be transferred from one species to another, or introducing new genes can be done by genetic transformation technology. The transferred genes are known as a transgene, where the process is called transgenesis. This results in new genetically transformed new plants, also known as transgenic plants. These transgenic plants are developed on the basics of the Totipotency of the plant cell, where desired or genetic traits are introduced to the new plants for successive generations.

Gene transfer methods

Gene transfer technique is the primary requirement for genetic transformation technology. It avails transfer for genes to plant cells by various gene transfer methods.

Gene Transfer methods are classified as:

1. Vector mediated gene transfer method

2. Direct transfer or vector less gene transfer method

a) Physical methods

I. Electroporation

II. Microinjectile

III. Liposome fusion

IV) Silicon carbide fibers

b) Chemical methods

I) Polyethylene glycol (PEG) mediated

II) Diethylaminoethyl (DEAE) Dextran mediated

III) Calcium phosphate precipitation

1.Vector mediated gene transfer method

Gene transfer is generally done by Agrobacterium-mediated transformation or the use of Plant viruses as vectors.

a) Agrobacterium-mediated gene transfer: a soil-borne, gram-negative bacterium Agrobacterium tumefaciens is used for gene transfer. It is a motile and a rod-shaped bacterium belongs to the bacteria family of Rhizobiaceae. It is a phytopathogen and hence, treated as nature’s effective plant genetic engineer. In this method, the agrobacterium carrying vector with the desired gene is developed. Then explant is co-cultured with Agrobacterium. Followed by, killing of agrobacterium with the use of antibiotics without harming explant cells. Finally, transformed cells are selected and regenerated into the whole plant. It is a natural method of gene transfer because Agrobacterium can infect any explant. Large fragments of DNA can be transferred by this method. The stability of the transferred DNA is good and hence whole plants can be regenerated effectively.

b) Virus mediated gene transfer: viruses are used as natural vectors for genetic engineering because they infect the plants and multiply the transferred genes by viral genome replication. Hence they are efficient for gene transfer methods. Three groups of viruses are used for gene transfer in plants e.g. Caulimoviruses, geminiviruses, and RNA viruses. By the use of virus vectors the, transmit of the desired genes to generations is not possible.

2. Direct transfer

a) Physical methods

I) Electroporation: In this technique uptake of DNA is done by, incubating plant material in a buffer solution containing DNA. This mixture solution is subjected to high voltage electrical impulses, which causes pores in the cell membrane. Reversible Uptake of DNA is facilitated by the pores on the cell membrane, this DNA then integrated into the host cell genome. This technique is simple and rapid to use. The physiological state of transformed cells is not altered. The efficiency of Transformation can be improved by electrical field strength optimization. Under normal conditions, DNA transformation in cells is very low. Plant regeneration is not easy by this method, especially protoplast is used.

II) Microinjection: In this method, mechanical transfer of gene to target cell is done by using a pipette of 0.5 to 10µm tip into the cytoplasm of a plant cell. Recipient cells are immobilized in Agarose embedding and then held by suction holding pipette. Later, this genetically modified cell is then cultured and grown into a transgenic plant. This process is very slow and is only performed by skilled or trained personnel.

III) Liposome mediated gene transfer: In this method liposomes carrying desired genes are fused with protoplasts and transfer the genes. Liposomes are first attached to the target protoplast, then these liposomes fuse with the protoplast at the site of attachment and release plasmid inside the cell. DNA is protected from the environment by encapsulation, and hence DNA is stable, can be stored prior to transfer. This method is used for a wide range of plants. Regeneration of plants from protoplasts is difficult using this method.

IV) Silicon carbide fibers mediated transformation: in this method silicon carbide fiber of 0.3 to 0.6 µm diameter and 10-100 µm length. The DNA coated silicon fibers are vortexed with suspension culture of plant material. These fibers penetrate the cell wall and plasma membrane hence, DNA adhering fibers enter the cell and get integrated with the host genome. This process is simple and cost-saving. These fibers are carcinogenic hence should be carefully handled. This technique is not applicable to embryonic plant cells, as they are hard and compact, so resistant to SCF penetration.

b) Chemical mediated gene transfer:

I) Polyethylene glycol mediated transformation: In this procedure plasmid, DNA is added to the protoplast suspension followed by slow addition of 40% PEG-4000 w/v dissolved in mannitol and calcium. This mixture is then incubated. These protoplasts get transformed, by destabilization of the plasma membrane by PEG in presence of divalent calcium ions. By this method, a large number of protoplasts can be transformed easily. There are chances of DNA degradation and rearrangement.

II) Dextran mediated transfer (DEAE): High molecular weight polymer diethyl aminoethyl (DEAE) dextran is complexed with desired DNA to be transferred. This method does not yield stable transformations.

III) Calcium Phosphate Co-Precipitation mediated transfer: The calcium chloride solution and isotonic phosphate buffer is mixed with DNA. This mixture allows precipitating DNA-Calcium phosphate precipitate. This precipitate is then exposed to the culture medium of actively dividing cells, by which the DNA is transformed into the cells. This method required a large amount of DNA samples.

Multiple Choice Questions

1. Plant gene transfer method is used in genetic engineering to __________

a) Transfer modified gene

b) Introduce new gene

c) Both

d) Only A

2. In Genetic engineering the a modified or new gene transfer is done to ___________

a) Make diseases resistant plant

b) To introduce new traits

c) To increase the yield of secondary metabolites

d) All

3. Which of the following is used in vector-mediated gene transfer?

a) Agrobacterium tumefaciens

b) Agrobacterium lufifeciens

c) Agrobacterium mutafaciens

d) All

4. Agrobacterium tumefaciens is _________

a) Fungi

b) Virus

c) Protozoa

d) None

5. ______________is the physical method for direct transfer of genes

a) PEG mediated

b) Dextran mediated

c) Calcium mediated

d) Liposome fusion

6. Large fragments of DNA can be transferred by ___________

a) Virus mediated transfer

b) Agrobacterium tumefaciens transfer

c) Both

d) None

7. __________ method uses high voltage electrical impulses for gene transfer

a) Liposome fusion

b) Microinjectile

c) Electroporation

d) Silicon carbide fibers

8. Silicon carbide fibers used in the direct method of gene transfer are carcinogenic

a) True

b) False

9. _____________method of gene transfer requires skilled personnel

a) Liposome fusion

b) Microinjectile

c) Electroporation

d) Silicon carbide fibers

10. In liposome-mediated direct gene transfer method genes are __________

a) Gene is stable

b) Gene is protected

c) Both a) and b)

d) Nor protected nor stable

Answer Key

1. c
2. d
3. a
4. d
5. d
6. b
7. c
8. a
9. b
10. c

Reference

1. Satyanarayana, biotechnology, 1st e.d. Kolkata, books & allied (p) ltd publishers, 2005  page:573-588

The post Gene Transfer Methods in Plant Genetic Engineering: MCQ for GPAT, GATE, & CSIR NET appeared first on Gpatindia: Pharmacy Jobs, Admissions, Scholarships, Conference,Grants, Exam Alerts.