Agricultural Biotechnology

How can we obtain better Crops ?

1. selection

2. breeding

3. hybridization

4. cloning

5. grafting

6. radiation mutagenesis

7. chemical mutagenesis

8. gene splicing

9. genomics/gene expression

10. tissue culture

Plant cell & tissue culture

• In 1905, first attempt was made to culture monocot cells on artificial medium, but did not succeed in producing plant from cultured cells

• During 1930s, roots and shoots were successfully cultured from cells on artificial nutrient medium… thus the beginning of “organ culture”

• Since then, remarkable achievements are made in culturing plants from tissues, cells, anthers, pollens, protoplasts and embryos.

• During 1980s, emergence of genetic engineering technology has further paved the way of crop improvement by transferring genes from unrelated species to plant cells.. Leading to transgenic crops or genetically modified plant species.

Basic terms

Cell culture

Culturing whole plants cells, tissues and organs on a suitable nutrient medium.

Organ Culture

In vitro culture of whole plants from roots tips, shoot apices, and embryos etc. is called organ culture.

Plasticity– Ability to be shaped or formed. – Ability of differentiated cells to undergo trans-differentiation– Many of the processes involved in plant growth and

development adapt to environment. – Allows plants to alter their metabolism, growth and

development to best suit their environment. – Abilities to initiate cell division from almost any tissue of plant

and to regenerate lost organs or undergo different developmental pathways in response to particular stimuli.

• Ability of a single cell to divide and produce all the differentiated cells in an organism.

• Totipotent cells formed during sexual and asexual reproduction include spores and zygotes.

– For example, a plant cutting or callus can be used to grow an entire plant.

• When plant cells and tissues are cultured in vitro they generally exhibit a very high degree of plasticity, which allows one type of tissue or organ to be initiated from another type.

• This regeneration of whole organisms depends upon the concept that all plant cells can, given the correct stimuli, express the total genetic potential of the parent plant.

Totipotency

Explant

Any excised portion of the plant such as leaf, stem, root etc. that is used for culturing a whole plant.

Callus

Undifferentiated and unorganized mass of cells.

Protoplasts

These are naked plant cells from which the cell wall has been removed by treatment with an enzyme cellulase.

Regeneration

It is a process in which cells are made to divide and differentiate to gave rise to a whole plant.

Basic Technique

1. Collection of explants (such as seedlings, buds, stems, leaf, immature embryos etc.)

2. Sterilization of the material by dilute solution of surface – sterilants, e.g. sodium hypochlorite, hydrogen peroxide, alcohol etc.

3. Removal of surface – sterilants by washing with sterile distilled water.

4. Transferring the material to sterile glass tube containing autoclaved nutrient medium. This is called inoculation.

5. Culture to be incubated in a tissue culture room where temperature is maintained at 56° ± 5° for 3 – 4 weeks in low light (relative humidity 60%). Calli will be developed.

6. Transfer the callus into a liquid medium.

7. Regeneration of plants from cell suspension culture.

8. Regenerated plants are transferred to pots and kept in glass house for acclimatization.

This is called hardening effect in natural conditions.

Plant tissue culture

Nutrient Media

Culture media used for in vitro cultivation of plants cells are composed of three basic components:1. Essential elements, or mineral ions, supplied as a

complex mixture of salts2. An organic supplement supplying vitamins and / or

amino acids3. A source of fixed carbon; usually supplied as the

sugar sucrose

• Macroelements

– Carbon, nitrogen, phosphorus, potassium,

magnesium, calcium and sulphur.

• Microelements

– Manganese, iodine, copper, cobalt, boron etc.

• Iron source

• Organic Supplements

– Vitamins, amino acids

Plant growth regulators

1. Auxin– Promotes cell division and growth– Promotes root formation

2. Cytokinins– Promote cell division (purine derivatives)– Promotes shoot formation

3. Gibberelins– Regulate cell elongation– Important in determining plant height and fruit-set

4. Abscisic acid– Inhibits cell division – Used in tissue culture to promote distinct developmental

pathways such as somatic embryogenesis

5. Ethylene– Most commonly associated with fruit ripening

Ratio of auxin and cytokinin determines the type of regeneration

• Intermediate ratio

– Callus formation

• Low auxin to cytokinin ratio

– Shoot formation

• High auxin to cytokinin ratio

– Root formation

• Production of somaclonal variations

– Variation seen in plants that have been produced by

plant tissue culture.

– Chromosomal rearrangements are an important source

of this variation.

– Results are

• Aneulpoids

• Sterile plants

• Morphological variants

– All resulting in crop improvement

Applications of cell and tissue culture

• Embryogenesis

• Somatic embryogenesis is an asexual form of plant propagation in nature that mimics many of the events of sexual reproduction.

• Plants may be produced artificially by the manipulation of tissues and cells in vitro.

• In vitro somatic embryogenesis is an important prerequisite for the use of many biotechnological tools for

– genetic improvement

– mass propagation

– Production of haploids

– Production of triploids

Transgenic plants and crop improvement

What is a transgenic?

Concept Based on the Term Transgene

Transgene – the genetically engineered gene added to a species

Transgenic – an organism containing a transgene introduced by technological (not breeding) methods

Transgenics are a Biotechnology Product

• Transgenic plants possess a gene or genes that have been transferred from a different species.

• The term "transgenic plants" refers to plants created in a laboratory using recombinant DNA technology.

• The aim is to design plants with specific characteristics by artificial insertion of genes from other species or sometimes entirely different kingdoms.

Transgenic plants with beneficial traits

• Stress tolerance– Withstand biotic and abiotic stress

• Biotic stress– Attacks by insect pests, viruses, bacteria, fungi etc.

• Abiotic stress– Caused by non-living environmental factors

• Drought• Extreme temperatures• Soil conditions• High winds

Why are transgenics important?

• We can develop organisms that express a “novel” trait not normally found in the species

Extended shelf-life tomato (Flavr-Savr)

Herbicide resistant soybean (Roundup Ready)

Thanks