Biotechnology:

Principles, Applications,
and Social Implications

The techniques used by the biotechnology industry to improve productivity and

to modify genes and introduce them into transgenic organisms

Dr. Dinesh D. Khedkar

Shri Shivaji Science College,
Amravati

What is Biotechnology?
How about some definitions
General Definition
The application of technology to improve a biological
organism
Detailed Definition
The application of the technology to modify the biological
function of an organism by adding genes from another
organism

U. S. National Science Foundation

The controlled use of biological agents such as
microorganisms or cellular components for biological use.

European Federation of Biotechnology

The integrated use of Biochemistry, Microbiology and
Engineering Sciences in order to achieve technological
application of the capabilities of microorganisms, cultured
tissues / cells and parts thereof.
NDSU

Extension

It is concern with the exploitation of biological agents or their

components for generating useful products / services
Agriculture
Medical

Industrial
Veterinary
Environment
NDSU

Extension

These definitions imply biotechnology

is needed because:
Nature has a rich source of variation
Here we see bean has many
seedcoat colors and patterns
in nature

But we know nature does not

have all of the traits we need

But nature does not contain all the

genetic variation man desires

Fruits with vaccines

Grains with improved nutrition

What controls this natural variation?

Allelic differences at genes control a specific trait
Definitions are needed for this statement:
Gene - a piece of DNA that controls the
expression of a trait

Allele - the alternate forms of a gene

What is the difference between

genes and alleles for Mendels Traits?
Mendels Genes
Plant height

Seed shape

Smooth Wrinkled
Allele

Tall

Short
Allele

In General, Plant Biotechnology Techniques

Fall Into Two Classes
Gene Manipulation
Identify a gene from another species which controls
a trait of interest
Or modify an existing gene (create a new allele)
Gene Introduction
Introduces that gene into an organism
Technique called transformation
Forms transgenic organisms

Genes Are Cloned Based On:

Similarity to known genes
Homology cloning (mouse clone used to obtain human gene)
Protein sequence
Complementary genetics (predicting gene sequence
from protein)
Chromosomal location
Map-based cloning (using genetic approach)

Homology Cloning
Clones transferred
to filter

Human clone
library

Mouse probe
added to filter

Hot-spots are human

homologs to mouse gene

Complementary Genetics
1. Protein sequence is related to gene sequence
NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COOATG GAT-GCT
TGG-AGT-AAA
C
C
C
G
A
TCT
G
C
A
G

2. The genetic code information is used to design PCR primers

Forward primer: 5-ATGGAT/CGCN-3
Reverse primer: 5-T/CTTNC/GT/ACCA-3
Notes: T/C = a mixture of T and C at this position;
N = a mixture of all four nucleotides
Reverse primer is the reverse complement of the gene sequence

Gene Manipulation
It is now routine to isolate genes
But the target gene must be carefully chosen
Target gene is chosen based on desired phenotype
Function:
Glyphosate (RoundUp) resistance
EPSP synthase enzyme
Increased Vitamin A content
Vitamin A biosynthetic pathway enzymes

The RoundUp Ready Story

Glyphosate is a broad-spectrum herbicide
Active ingredient in RoundUp herbicide
Kills all plants it come in contact with
Inhibits a key enzyme (EPSP synthase) in an amino acid pathway

Plants die because they lack the key amino acids

A resistant EPSP synthase gene allows crops
to survive spraying

RoundUp Sensitive Plants

Shikimic acid + Phosphoenol pyruvate

+ Glyphosate
Plant
EPSP synthase

3-Enolpyruvyl shikimic acid-5-phosphate

(EPSP)
Without amino acids,
plant dies

Aromatic
amino acids

RoundUp Resistant Plants

Shikimic acid + Phosphoenol pyruvate

+ Glyphosate
Bacterial
EPSP synthase

RoundUp has no effect;

enzyme is resistant to herbicide

3-enolpyruvyl shikimic acid-5-phosphate

(EPSP)
With amino acids,
plant lives

Aromatic
amino acids

The Golden Rice Story

Vitamin A deficiency is a major health problem
Causes blindness
Influences severity of diarrhea, measles

>100 million children suffer from the problem

For many countries, the infrastructure doesnt exist

to deliver vitamin pills
Improved vitamin A content in widely consumed crops
an attractive alternative

-Carotene Pathway in Plants

IPP
Geranylgeranyl diphosphate
Phytoene synthase

Phytoene

Problem:
Rice lacks
these enzymes

Phytoene desaturase

-carotene desaturase

Lycopene
Lycopene-beta-cyclase
Normal
Vitamin A
Deficient
Rice

-carotene
(vitamin A precursor)

The Golden Rice Solution

-Carotene Pathway Genes Added
IPP
Geranylgeranyl diphosphate
Daffodil gene

Phytoene synthase

Phytoene

Vitamin A
Pathway
is complete
and functional

Phytoene desaturase
Single bacterial gene;
performs both functions

-carotene desaturase

Lycopene
Daffodil gene
Golden
Rice

Lycopene-beta-cyclase

-carotene
(vitamin A precursor)

Metabolic Pathways are Complex

and Interrelated
Understanding pathways
is critical to developing
new products

Modifying Pathway Components

Can Produce New Products
Turn On Vitamin Genes =
Relieve Deficiency
Modified Lipids =
New Industrial Oils

Increase amino acids =

Improved Nutrition

Trait/Gene Examples
Gene

Trait
RoundUp Ready

Bacterial EPSP

Golden Rice

Complete Pathway

Plant Virus Resistance

Viral Coat Protein

Male Sterility

Barnase

Plant Bacterial Resistance

p35

Salt tolerance

AtNHX1

Introducing the Gene or

Developing Transgenics
Steps
1. Create transformation cassette
2. Introduce and select for transformants

Resistant Transgenics
Insect Resistance

Cold Tolerance

Transgene=
Bt-toxin protein

Transgene=
CBF transcription factors

More Modern Examples

Salt Tolerant

Mercury Resistance

Transgene=
Glyoxylase I

Transgene=
Mercuric ion reductase

The Next Test Is The Field

Herbicide Resistance

Non-transgenics

Transgenics

Final Test
Consumer Acceptance
RoundUp Ready Corn

Before

After

The Public Controversy

Should we develop transgenics?
Should we release transgenics?

Are transgenics safe?

Are transgenics a threat to non-transgenic
production systems?
Are transgenics a threat to natural
eco-systems?