AR-7867

Integrated UG/PG IV Sem. Biotechnology

Examination, 2013

Plant and Animal Tissue culture: Application and Techniques

Section-A

1. The somatic embryo were first observed in -------------------- cell suspensions.

(b) Daucus carota

2. Most commonly used hormone for somatic embryogenesis is:-

(d) 2,4 D

3. EDTA is a chelating agent. Fe-EDTA is used in plant tissue culture medium. In this state iron

is gradually released into the culture medium as it is utilized by the living cells.

4. Mercuric chloride, Sodium hypochlorite--- These agents may disrupt the cell membrane of

microorganisms and protect the explants from microbial contaminations.

5. Serum free medium regulates:

C. Regulates differentiation

6. Which one of the following is commercially available serum free medium

C. MCDB-131

7. Callus tissue means an unorganised proliferative mass of cells produced from isolated plant

cells, tissues or organs when grown aseptically on artificial nutrient medium

under controlled experimental conditions.

8. Cell suspension culture is a type of culture in which single cells or small aggregates of cells

multiply while suspended in agitated liquid medium.

9. The cell fusions can be achieved using:-

b. Polyethylene glycol

10. In Hybridoma technology, the myeloma cells lack hypoxanthine guanine phosphoribosyl

transferase (HGPRT) enzyme.

Section B

1. Give an outline of a plant tissue culture laboratory.

Ans.:- Plant tissue culture laboratory:- an ideal tissue culture laboratory should

have at least two big rooms and a small room. One big room is for general laboratory

work such as preparation of media, autoclaving, and distillation of water etc. the other

big broom room is for keeping cultures under controlled light, temperature and

humidity. The small room is for aseptic work and for keeping autoclaved articles.

The general laboratory for tissue culture should be provided with the following

articles and arrangements:-

A washing area:- This is a very important for a tissue culture laboratory. It should be

provided with a large sink, running hot and cold tap water, brushes of various sizes,

detergents and bucket of single distilled water for final rinse of the washed glass

goods. Another separate bucket with lid also required for disposing off the used or

infected media before cleaning.

Hot air oven:-It is necessary for drying the washed glass goods.

Refrigerator:- It is essential for storing various thermolabile chemical like vitamins,

hormones, amino acids, casein hydrolysate, yeast extract etc. Stock solutions of salts

are also kept to prevent contaminations.

Distilled plant :- A single distillation and double distillation water plants are

indispensible.

Weighing Balance:- It is required for weighing chemicals, sugars, agar and other.

pH meter:- It is necessary for measurement and adjustment of pH of nutrient

medium.

Vacuums pump:-It is required for filtering liquid media, sugar solution etc.

Autoclave:- It is very important for sterilization of nutrient media , glass goods

instruments etc.

Working tables:- These are necessary for preparation of medium.

Heater:- It is needed for heating or warming the medium to dissolve agar or to melt

the agarified medium.

Microscope:-It is required for the observation of morphological changes in cells.

Microtome:- It is needed for the sectioning of cultured tissue.

Wooden racks:- These are required for keeping the various chemicals.

Laboratory for aseptic inoculation:- This room should be without any window or

ventilation to make room dust free. The doors should have an automatic door closer.

Laminar air flow cabinet is the most suitable, convenient and reliable instruments for

aseptic work.

Culture Room:-the culture room means the room for keeping or inoculating the

culture under controlled temperature, light and humidity. The culture room is also

fitted with double doors to make it dust free and to maintain constant room

temperature. To maintain the temperature around 25+20C inside the culture room air

cooler are used. This room is also provided with specialized designed shelves for

keeping flasks jars bottles etc. cultures can be grown in light or in dark. For light

arrangement each culture racks provided with fluorescent lamps which are

photoperiodically controlled by an automatic timer. A thermometer and hygrometer

are fixed on the wall at the safety corner of the room to check temperature and

relative humidity respectively. The culture room should have a shaker for suspension

culture.

Glass goods and instruments:-different glass goods are used to culture plant tissues.

Scalpel, surgical scalpel, forceps, needle scissors and spatula are also needed in plant

tissue culture laboratory.

2. What is culture medium? State the basic composition of a general plant tissue culture

medium.

Ans. :- Excised plant tissues and organs will only grow in vitro on a suitable artificially

prepared nutrient medium which is known as culture medium.

A culture medium is composed of inorganic salts, an iron source, vitamins, amino acids,

growth substances and a carbohydrate supply.

Inorganic salts are supplied in two groups –as macro nutrients and micronutrients.

Macro Nutrients:-The salts needed in higher amount called macro nutrients. These include

nitrogen, phosphorus, sulphur, magnesium, calcium, potassium. Nitrogen is mostly provided in

two forms as nitrates and as ammonium compounds. It is a constituent of amino acids, proteins,

certain hormone and chlorophyll. Phosphorus is vital for cell division as well as in storage and

transfer of energy in plants. Potassium is necessary for normal cell division, for synthesis of

proteins chlorophyll and for nitrate reduction. Sulphur is present in some proteins. Calcium as

calcium pectate is an integral part of the walls of plant cells and helps maintain integrity of the

membrane. Magnesium is a component of chlorophyll and a co-factor for many enzyme

reactions.

Micro nutrients are required in very trace quantities and may get carried into medium as

impurities in other ingredients. It includes Ni, Mn, Zn, Cu, Na, K, Al etc. all these are needed in

very little amount but absent of these plants get different types of deficiencies.

Fe-EDTA is used as iron source.

Vitamines to achieve the best growth of the tissues it is often essential to supplement the

medium with one or more vitamins. Myoinositol , Nicotinic acids, Pyridoxine HCl, Thiamine

HCl, Biotine may used in plant tissue culture medium.

Amino Acids Glycine, Cystein may use.

Undefined Supplements Numerous complex nutritive mixtures of undefined composition, like

casein hydrolysates, coconut milk, corn milk, malt extract also used to promote the growth of

certain organs.

Carbon source glucose, sucrose fructose can be used in plant tissue culture medium.

Growth hormones: auxin used for cell division and root differentiation. The auxins commonly

used in tissues culture are IAA,IBA,2,4-D etc. Cytokinins it is concerned with cell division and

shoot differentiation. Most commonly used cytokinins are BAP, kinetin, zeatin etc. Gibberallin

generally GA3 is used.

Gelling agents: agar, agarose gelrite may used as gelling agents in plant tissues culture medium.

pH: The pH of the medium is usually adjusted between 5.0 and 6.0

3. Give the preparative methods of aseptic plants.

Ans. Surface sterilization of plant tissue may cause some deleterious effects because

most of the sterilants are toxic chemicals. Seed can more or less resist such

deleterious effects due to the presence of its seed coat. So to avoid the surface

sterilization of plant tissue, seeds are surface sterilized and are cultured on simple

basal nutrient medium. Seeds in culture germinate and give rise to an aseptic

seedling. Explants from such seedlings grown under aseptic and controlled conditions

are the most suitable material for culture and need no further surface sterilization.

Procedure:-

1. Wash the dry seeds thoroughly with tap water

2. Dip the seeds in 5% teepol solution for 10-15 minutes. Decant the teepol solution

and wash the seeds again with tap water and finally with distilled water.

3. Rinse the seeds with 70% ethyl alcohol for 1 minute.

4. In Laminar Air flow, seeds will transfer into the autoclaved bottle and pour 0.1%

HgCl2 solution. Leave for 10-15 minutes stir the bottle frequently.

5. Decant the sterilant and wished 3-4 times with autoclaved distilled water.

6. Transfer the seeds from bottle to autoclaved Petri-dish with the help of sterile

forceps.

7. Transfer the seeds to culture medium.

8. Incubate the seeds in continuous dark either at room temperature or at 25-280C

Fig: The preparation of aseptic plants from seeds.

4. Surface sterilization of plant tissue material.

Ans. Plant materials which to be cultured, should be surface sterilized to remove the

surface borne microorganisms. This procedure is done in front of laminar air flow or

inside the inoculation chamber before the plant material is inoculated onto the culture

medium.

Procedure of surface sterilization of plant materials.

Plant materials or explants washed with tap water

Immersed in liquid detergent for 10-15 minutes, then washed with tap water and

finally in distilled water.

Dip the explants in 70% ethyl alcohol for 60 seconds

Immediately transfer the material into an autoclaved bottle and pour 0.1% HgCl2 or 5-10%

sodium hypochlorite solution and keep them for 10-15 minute in stirring conditions.

Decant the sterilant and wash the explants thoroughly with autoclaved distilled water. Then the

explants are ready for culture.

Fig: The procedure for surface sterilization of plant material and inoculation of

explant for culture.

5. Define monolayer and Feeder layer.

Mono Layer:- Monolayer that given the opportunity, the cells will attach to the

substrate and that normally the cells will be propagated. Many types of cells can

adhere to and grow on glass or on specially treated plastics with negatively charged

groups on the surface. The cultured cells secrete collagens and other matrix

components these bind to the culture surface and function as bridge between it and

the cells. cells cultured from the single cells on a glass or a plastic dish form visible

colonies in 10-14 days.

Surface materials:-glass and disposal plastic, palladium and metallic surface.

Fig: Stage in the subculture of monolayer cells

Feeder Layers:- while matrix coating may also help attachment, growth and

differentiation some cells do not clone well is related to their inability to survive at

low cell densities. One way to maintain cells at clonogenic densities but, at the same

time, to mimic high cell densities, is to clone the cells onto a growth arrested feeder

layer. The feeder cells may provide nutrients, growth factors, and matrix constituents

that enable the cloned cells to survive more readily. These feeder layers may consist

of mouse embryo, fibroblasts, normal fetal intestine, glial cells etc.

Fig: Feeder Layer

6. Write a note on development and maintenance of cell lines.

Ans. Cell Lines:-After the first subculture, or passage the primary culture becomes known as a

cell line and may be propagated and sub cultured several times. With each successive

subculture, the component of the population with the ability to proliferate most

rapidly will gradually predominate, and non proliferating or slowly proliferating cells

will be diluted out.

MAINTENANCE of CELL LINES:-

Once a culture is initiated, whether it is a primary culture or a subculture of a cell line, it will

need a periodic medium change, or ‘‘feeding,’’ followed eventually by subculture if the cells are

proliferating. In non proliferating cultures, the medium will still need to be changed periodically,

as the cells will still metabolize and some constituents of the medium will become exhausted or

will degrade spontaneously. Intervals between medium changes and between subcultures vary

from one cell line to another, depending on the rate of growth and metabolism; rapidly growing

transformed cell lines, such as HeLa, are usually sub cultured once per week, and the medium

should be changed four days later. More slowly growing, particularly non transformed, cell lines

may need to be sub cultured only every two, three, or even four weeks, and the medium should

be changed weekly between subcultures.

Significance of Cell Morphology:-

Whatever procedure is undertaken, it is vital that the culture be examined carefully to confirm the

absence of contamination. The cells should also be checked for any signs of deterioration, such

as granularity around the nucleus, cytoplasmic vacuolation, and rounding up of the cells with

detachment from the substrate. Such signs may imply that the culture requires a medium change,

or may indicate a more serious problem, e.g., inadequate or toxic medium or serum, microbial

contamination, or senescence of the cell line. Medium deficiencies can also initiate apoptosis.

During routine maintenance, the medium change or subculture frequency should aim to prevent

such deterioration, as it is often difficult to reverse.

Replacement of Medium:-

Four factors indicate the need for the replacement of culture medium:

(1) A Drop in pH:-The rate of fall and absolute level should be considered. Most cells stop

growing as the pH falls from pH 7.0 to pH 6.5 and start to lose viability between pH 6.5 and pH

6.0, so if the medium goes from red through orange to yellow, the medium should be changed.

Try to estimate the rate of fall; a culture at pH 7.0 that falls 0.1 pH units in one day will not come

to harm if left a day or two longer before feeding, but a culture that falls 0.4 pH units in one day

will need to be fed within 24–48 h and cannot be left over a weekend without feeding.

(2) Cell Concentration:- Cultures at a high cell concentration exhaust the medium faster than

those at a low concentration. This factor is usually evident in the rate of change of pH, but not

always.

(3) Cell Type:- Normal cells (e.g., diploid fibroblasts) usually stop dividing at a high cell density

because of cell crowding, growth factor depletion, and other reasons. The cells block in the G1

phase of the cell cycle and deteriorate very little, even if left for two to three weeks or longer.

Transformed cells, continuous cell lines, and some embryonic cells, however, deteriorate rapidly

at high cell densities unless the medium is changed daily or they are sub cultured.

(4) Morphological Deterioration:- This factor must be anticipated by regular examination and

familiarity with the cell line. If deterioration is allowed to progress too far, it will be irreversible,

as the cells will tend to enter apoptosis.

SUBCULTURE

When a cell line is sub cultured the regrowth of the cells to a point ready for the next

subculture usually follows a standard pattern. A lag period after seeding is followed by a

period of exponential growth, called the log phase. When the cell density (cells/cm2 substrate)

reaches a level such that all of the available substrate is occupied, or when the cell

concentration (cells/mL medium) exceeds the capacity of the medium, growth ceases or is

greatly reduced. Then either the medium must be changed more frequently or the culture must

be divided. For an adherent cell line, dividing a culture, or subculture as it is called, usually

involves removal of the medium and dissociation of the cells in the monolayer with trypsin,

although some loosely adherent cells may be sub cultured by shaking the bottle, collecting the

cells in the medium, and diluting as appropriate in fresh medium in new bottles.

Standardization of Culture Conditions:-

Standardization of culture conditions is essential for maintaining phenotypic stability.

Although some conditions may alter because of the demands of experimentation, development,

and production, routine maintenance should adhere to standard, defined conditions.

Medium:- The type of medium used will influence the selection of different cell types and

regulate their phenotypic expression.

Serum:- The best method of eliminating serum variation is to convert to a serum-free medium

unfortunately, serum-free formulations are not yet available for all cell types, and the

conversion may be costly and time consuming.

Plastics:- Most of the leading brands of culture flasks and dishes will give similar results, but

there may be minor variations due to the treatment of the plastic for tissue culture. Hence it is

preferable to adhere to one type of flask or dish and supplier.

Cell line maintenance:- Cell lines may alter their characteristics if maintained differently from

a standard regime. The maintenance regime should be optimized and then remain consistent

throughout the handling of the cell line. Cultures should also be replaced from frozen stocks at

regular intervals.

7. Describe the process of preparing cell suspension. What are the benefits of using

aqueous medium over solid medium?

Suspension culture is a type of culture in which single cells or small aggregates of cells

multiply while suspended in agitate liquid medium.

Callus proliferates as an unorganized mass of cells. So it is very difficult to follow many

cellular events during its growth and developmental phase. To overcome such limitations of

callus culture, the cultivation of free cells as well as small cell aggregates in a chemically defined

liquid medium as a suspension was initiated to study the morphological and biochemical changes

during their growth and developmental phases. To achieve an ideal cell suspension, most

commonly a friable callus is transferred into the agitated liquid medium where it breaks up and

readily dispersed. After elimination of large callus pieces only single cells and small cell

aggregates are again transferred to fresh medium and after two or three weeks a suspension of

actively growing cells is produced. This suspension can then be propagated by regular sub-

culture of an aliquot to fresh medium. Ideally suspension callus should consist of only single

cells which are physiologically and biochemically uniform.

Fig: Methods of cell suspension culture and regeneration of plant through

embryogenesis.

The suspension culture eliminates many of the disadvantages in comparison to solid

medium. There are several benefits in suspension culture.-

1. Movements of cells in relation to nutrient medium facilitates gaseous exchange,

remove any polarity of the cells due to gravity and eliminates the nutrient

gradients within the medium and at the surface of the cells.

2. It contributes many informations about cell physiology, biochemistry, metabolic

events at the level of individual cells and small cell aggregates.

3. It is also important to build up an understanding of an organ formation or

embryoid formation starting from single cell or small cell aggregates.

4. Suspension culture derived from medically important plant can be studied for the

production of secondary metabolites such as alkaloids.