Biotechnology

Lecture 1

Applications on Plants

Tissue culture or In vitro culture

Plant tissue culture:

tissue culture means the culturing of pieces , tissues or cells of a plant in glass or plastic vessels containing specific nutritional medium (consists of macro and micro nutrients, hormones, energy source, ..).

All growing materials (vessels, media, plant tissues, …etc.) must be sterilized, then the culture is then placed under suitable light and temperature for the tissues to grow and differentiate.

Components of tissue culture lab:

1- Preparation room: The first room where the plant materials, media and vessels are prepared, washed and sanitized.

Instruments: Autoclave, Balance, water distiller, Fridge, hot plate stirrer, pH meter, glassware (Petri dishes, pipettes, flasks, …etc.)

Components of tissue culture lab:

2- Transfer room: It is a sanitized room where the plants are planted on a sanitized media.

Instruments: Tissue culture hood, Bunsen flame or electric heater for sanitizing blades and forceps, microscope.

3- Culture room: a room equipped with suitable temperature (25 oC) and light (16 hrs light and 8 hrs darkness) used for growing the tissues, contains plastic or aluminum racks where the vessels are placed.

1- Autoclave 2- sinks 3- Hood 4- Racks 5- Benches 6- Fridge 7- water distiller 8- 9- 10 – pH meter, hot plate stirrer , balance

Components of growing media

1- Macro and micro nutrients:

Non organic matter, important for the life and survival of all plants

Macro nut.: N, P, K, Ca, Mg, S, Na. needed in quantities more then 0.5 mil mol / lit.

Micro nut.: Mn, Ni, Al, Co, Mo, B, Fe, I, Cu, needed in quantities less then 0.5 mil mol / lit.

2- Source of carbohydrates and sugars:

Organic matter needed as a source of energy, the most important carbohydrate used is the sucrose (Glucose + Fructose).

Sucrose is added to the media in a conc. Of 2 – 6%

3- Vitamins:

Added to the media in a very small quantities. It works as a catalyst, regulator or as a cofactor in the enzymatic reactions and the metabolic pathways.

Ex: vit. B, C, D, E

Components of growing media

4- Hormones and growth regulators:

They are added to the growing media in a very small quantities and they are added to regulate the growth and differentiation of the tissues, they are categorized in 3 groups:

1- Auxins: a group of compounds responsible for the elongation of cells, plant apical dominance and promote root formation. Ex: (IAA, IBA, NAA, 2,4-D)

2- Cytokinin: a group of compounds responsible for the cell division and promote the formation of buds and lateral shoots Ex: Kinetin, Zeatin , BA, TDZ.

3- Gibberellins: growth regulators used in breaking the dormancy of buds and seeds and cell elongation and growth Ex: GA3

5- Gelling agents:

It is used to give a solid texture (Gelatin texture) to the growing media. Ex Agar, Agarose, Gelrite.

5-9 gms/lit. of the Gelling agent is added to the media.

If the gelling agent is not added to the media, the media becomes liquid and it must be placed on a shaker after planting and is called in this case (liquid media).

Tissues are placed first on a medium containing cytokinins (at conc. 0.5-10 mg/lit) for the production of shoots then the formed shoots are then transferred to a new medium containing Auxins (at a conc. of 1-20 mg/lit) for the production of roots.

If both Auxins and cytokinins are added to the medium, the tissues will grow to undifferentiated cells called callus , the callus differentiate to shoots or roots after that if placed on a medium containing the suitable growth regulator.

The explants used in tissue culture:

1- Meristem culture: The cells at the growing tip of the plant are used, and it is used if a free virus plant is needed to be obtained.

2- Shoot tip culture: The cells at the growing tip of the plant are used + the leaf primordia at the growing tip.

3- Organ culture: if a while organ from a plant is used Ex: leaf culture, root culture, stem culture, bud culture …etc.

4- Embryo culture: used to grow the mature or immature embryos, it is used with tiny seeds or if the seed doesn’t contain enough endosperm to feed the embryo during seed germination Ex: Orchids

5- Pollen and anther culture: if 1n plant is needed for the heredity studies or genetics program.

6- Ovary culture: if 1n plant is needed for the heredity studies or genetics program.

7- Protoplast culture: which is a cell without a cell wall, used in protoplast fusion and in plant transformation programs.

8- Single cell culture: One cell only is placed on a medium, but because of the difficulty of one cell to grow by its self and differentiate a nursing cells must be used.

Factors affecting the growth and differentiation in in-vitro culture:

A- External factors:

1- Chemical factors: - The Auxin to cytokinin concentration A:C if the A>C (Root formation), A<C (Shoot formation), A=C (Callus).

- Sugar concentration and type: example fructose sugar if used instead of sucrose will cause the production of secondary products (Taxol), also if the concentration of sucrose is increased in the medium this will lead to the production of antioxidants and phenolic compounds.

- 2,4-D if added in increased conc will lead to the formation of mutations in the growing tissues

2- Physical factors: - Light: Total darkness and very high light intensity cause the stopping of differentiation and tissues form callus only.

It was found that light at wave length of 400-450 nanometer enhance the formation of flowers, blue wave length enhance antioxidant formation while the red light enhance the cytokinin therefore increase the bud and lateral shoot formation.

- Temperature: The optimum temperature for the tissues to grow and differentiate is 25 oC.

-pH: The best pH for the tissues to grow is between 5-6 any fluctuation will cause the change in membrane permeability and the absorption of nutrients.

B- Internal Factors:

-Species of stock or mother plant: plants are different in their rate of growth and differentiation on the media for example Nicotiana plant and family Solanaceae grow and differentiate very fast on the growing media while woody plants are slower in their rate of growth.

- Physiological age of the explants: as the explant ages its rate of growth and differentiation decreases, also the possibility of genetic distortion increases.

- Cell to cell or Tissue to tissue: it was found that the relationship between cells in the same tissue or the tissues in the same organ had a great influence on the growth and differentiation of the tissue

Sanitation of utilities, glasses, media and explants:

-All utilities (papers, Aluminum foil, blades, forceps…) glasses (Petri dishes, flasks, beakers….) are sanitized in autoclave for 30 min. at 121 oC and pressure 15 pound/inch2.

-Media are autoclaved for 20 min. at the same temp. and pressure.

- Explants are sanitized using Clorox bleach at 10% for 10 min. , 2 drops of tween 20 are added to reduce the water surface tension.

- Tissue culture hood and hands are sanitized by alcohol 75%.

Practical uses of tissue culture:

1- Protoplast fusion:

Protoplast fusion is used in the cases of sexual incompatibility between parents or in the cases of male sterility.

Cell wall of cells are removed and 2 parental cells are fused together, after fusion the cell wall is formed once again. The cell wall is removed by the use of cellulase enzyme for 4-12 hrs at 25-30oC and pH 5-6.

The osmotic of the solution used pressure is very important because if the osmotic pressure increases it might cause plasmolisis to the protoplast leading to the death of the cells. The osmotic pressure is stabilized by the use of Ca or K chloride or Mg sulfate.

In 1978 was the first successful trial to make a fusion between 2 cells from different species which is known as somatic hybrid plants this fusion was done between tomato cells and potato cells forming a new plant called pomato or potomato.

The fusion is done by two ways:

1- Chemical fusion

Depends on the presence of Ca++ ions and PEG in the solution. The PEG causes the coagulation and gathering of cells together.

This way is not widely used because the PEG is toxic substance and if not washed quickly it might cause the death of the cells.

2- Electro fusion

The protoplasts are placed in an electric circuit, the –ve charges on the cell membrane causes the cells to migrate to the +ve pole and the coagulation of the cells together.

Selection of the cells fused successfully is done by the use of a microscope and a micro pipette.

The 2 parental cells must have 2 different colors or shapes so that the selection of the fused cells become easier .

After selecting the successfully fused cells, the cells must be transferred to a suitable growing media to form callus then the plant.

Benefits of protoplast fusion:

-Transferring characteristics (drought tolerance, salinity tolerance, …) from one species to another

- making a hybrid of 2 parents even if they haven't reach their maturity stages (orchids).

-Overcoming the male sterility in some species

Problems of protoplast fusion:

-Difficult to select the successfully fused cells.

- most hybrids are sterile and have deformations in their genome due to the lost or addition of organelles.

- the successfully fused cells might not differentiate to a full plant.

Practical uses of tissue culture:2- Micropropagation: Means the production of numerous number pf plants from a single tissue, this is done by placing the tissue on a medium containing cytokinins in order to enhance the formation of lateral shoots, then the shoots are removed and placed once again on medium containing cytokinin to enhance more shoot formation (Sub-culturing). After a large number of shoots are formed, the shoots are transferred to rooting medium (contains auxins) .After root formation the small plants are transferred to trays containing peat moss + fungicides and covered for 15 days , the covered is then removed gradually and the plants are transferred to the green house.

2- Production of secondary products: Benefits 1- Saving the land for food crops.1- Saving the land for food crops. 2- The production of secondary products in small areas in its final form.2- The production of secondary products in small areas in its final form.3- Stress can be induced properly without the interfere of external factors.4- No contamination and no pesticides are used.4- No contamination and no pesticides are used.5- Production at any time of the year (no seasonal variation).5- Production at any time of the year (no seasonal variation).6- Producing constant amount that can meet with market demand (Bioreactors).

Practical uses of tissue culture:3- Saving endangered plants:Wild and endangered plants can be propagated by the use of tissue culture in large numbers and replanted once again in the wild or its place of origin. 4- genetic improvement:-Somaclonal variation or the use of chemicals causing mutation might lead to the formation of a plant that caries a desired characteristics which might enter after that in a genetics program. 5- production of virus free plants:-Using meristem culture leads to the production of a virus free plants and it is very important in fruits and vegetable production

6- production of roots or tubers:- By the use of tissue culture potato tubers or tuberous roots can be formed directly from the explant without the need of a whole plant for the production of roots