Biotechnology:

Principles, Applications,
and Social Implications
From Protein to Product
The techniques used by the biotechnology industry
to modify genes and introduce them into transgenic organisms

Phil McClean
Department of Plant Science
North Dakota State University

NDSU

Extension

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
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

NDSU

But we know nature does not have

all of the traits we need

Extension

But nature does not contain all the

genetic variation man desires

Fruits with vaccines

Grains with improved nutrition

NDSU

Extension

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
NDSU

Extension

What is the difference between

genes and alleles for Mendels Traits?
Mendels Genes
Plant height

Seed shape

Smooth Wrinkled
Allele

Tall

NDSU

Short
Allele

Extension

This Implies a
Genetic Continuum
A direct relationship exists between the gene, its alleles,
and the phenotypes (different forms ) of the trait
Alleles must be:
similar enough to control the same trait
but different enough to create different phenotypes

NDSU

Extension

Allelic Differences for Mendels Genes

Plant Height Gene

NDSU

Gene: gibberellin 3--hydroxylase

Function: adds hydoxyl group to GA20 to make GA1
Role of GA1: regulates cell division and elongation
Mutation in short allele: a single nucleotide converts
an alanine to threonine in final protein
Effect of mutation: mutant protein is 1/20 as active

Extension

Allelic Differences for Mendels

Seed Shape Gene

Gene: strach branching enzyme (SBE) isoform 1

Function: adds branch chains to starch
Mutation in short allele: transposon insertion
Effect of mutation: no SBE activity; less starch, more
sucrose, more water; during maturation seed looses
more water and wrinkles
NDSU

Extension

Central Dogma of Molecular Genetics

(The guiding principle that controls trait expression)

Protein

Trait
(or phenotype)
Translation
Seed shape

DNA
(gene)

Transcription

RNA
Plant height

NDSU

Extension

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)

NDSU

Gene Introduction
Introduces that gene into an organism
Technique called transformation
Forms transgenic organisms

Extension

Gene Manipulation Starts

At the DNA Level

The nucleus

contains DNA

NDSU

Source: Access Excellence

Extension

DNA Is Packaged
Double-stranded
DNA

is condensed
into

Chromosomes
NDSU

Source: Access Excellence

Extension

Chromosomes Contain Genes

Chromosome

Gene

Source: Access Excellence

NDSU

Extension

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)
NDSU

Extension

Homology Cloning
Clones transferred
to filter

Human clone
library

Mouse probe
added to filter

Hot-spots are human

homologs to mouse gene

NDSU

Extension

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

NDSU

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

Extension

Complementary Genetics
(cont.)
3. Use PCR to amplify gene fragment
a. template DNA is melted (94C)
3
5

5
3

b. primers anneal to complementary site in melted DNA (55C)

3

c. two copies of the template DNA made (72C)

5

NDSU

Extension

PCR Animation

NDSU

Denaturation: DNA melts

Annealing: Primers bind
Extension: DNA is replicated

Extension

PCR Again

NDSU

Extension

Complementary Genetics
(cont.)
4. Gene fragment used to screen library
Clones transferred
to filter

Human clone
library

NDSU

Hot-spots are human gene

of interest

PCR fragment
probe added to filter

Extension

Map-based Cloning
1. Use genetic techniques to
find marker near gene
2. Find cosegregating marker
3. Discover overlapping clones
(or contig) that contains the marker

4. Find ORFs on contig

Gene Marker

Gene/Marker

Gene/Marker

Gene/Marker

NDSU

5. Prove one ORF is the gene by

Mutant + ORF = Wild type?
transformation or mutant analysis Yes? ORF = Gene

Extension

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
NDSU

Extension

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
NDSU

Extension

RoundUp Sensitive Plants

Shikimic acid + Phosphoenol pyruvate
+ Glyphosate

Plant
EPSP synthase

3-Enolpyruvyl shikimic acid-5-phosphate

(EPSP)

Without amino acids,

plant dies

NDSU

Aromatic
amino acids

Extension

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

NDSU

Aromatic
amino acids

Extension

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
NDSU

Extension

-Carotene Pathway in Plants

IPP
Geranylgeranyl diphosphate
Phytoene synthase

Phytoene

Problem:
Rice lacks
these enzymes

Phytoene desaturase
-carotene desaturase

Lycopene
Lycopene-beta-cyclase

-carotene
(vitamin A precursor)

NDSU

Normal
Vitamin A
Deficient
Rice

Extension

The Golden Rice Solution

-Carotene Pathway Genes Added
IPP
Geranylgeranyl diphosphate
Daffodil gene

Vitamin A
Pathway
is complete
and functional

Phytoene synthase

Phytoene
Single bacterial gene;
performs both functions

Phytoene desaturase
-carotene desaturase

Lycopene
Daffodil gene

-carotene
(vitamin A precursor)

NDSU

Golden
Rice

Lycopene-beta-cyclase

Extension

Metabolic Pathways are Complex

and Interrelated
Understanding pathways
is critical to developing
new products

NDSU

Extension

Modifying Pathway Components

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

Increase amino acids =

Improved Nutrition

NDSU

Extension

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
NDSU

Extension

Introducing the Gene or

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

NDSU

Extension

Transformation Cassettes
Contains
1. Gene of interest
The coding region and its controlling elements
2. Selectable marker
Distinguishes transformed/untransformed plants
3. Insertion sequences
Aids Agrobacterium insertion
NDSU

Extension

Gene of Interest
Promoter

TP

Coding Region

Promoter Region
Controls when, where and how much the gene is expressed
ex.: CaMV35S (constitutive; on always)
Glutelin 1 (only in rice endosperm during seed development)

Transit Peptide
Targets protein to correct organelle
ex.: RbCS (RUBISCO small subunit; choloroplast target

Coding Region
Encodes protein product
NDSU

ex.: EPSP
-carotene genes

Extension

Selectable Marker
Promoter

Coding Region

Promoter Region
Normally constitutive
ex.: CaMV35s (Cauliflower Mosaic Virus 35S RNA promoter

Coding Region
Gene that breaks down a toxic compound;
non-transgenic plants die
ex.: nptII [kanamycin (bacterial antibiotic) resistance]
aphIV [hygromycin (bacterial antibiotic) resistance]
Bar [glufosinate (herbicide) resistance]

NDSU

Extension

Effect of Selectable Marker

Non-transgenic = Lacks Kan or Bar Gene
Plant dies in presence
of selective compound

X
Transgenic = Has Kan or Bar Gene
Plant grows in presence
of selective compound

NDSU

Extension

Insertion Sequences
TL

TR

Required for proper gene insertions

Used for Agrobacterium-transformation
ex.: Right and Left borders of T-DNA

NDSU

Extension

Lets Build A Complex Cassette

pB19hpc (Golden Rice Cassette)

TL

aphIV

35S Gt1

psy

35S rbcS

crtl

TR

T-DNA
Border

Hygromycin
Resistance

Phytoene
Synthase

Phytoene
Desaturase

T-DNA
Border

Insertion
Sequence

Selectable
Marker

Gene of
Interest

Gene of
Interest

Insertion
Sequence

NDSU

Extension

Delivering the Gene

to the Plant
Transformation cassettes are developed in the lab
They are then introduced into a plant
Two major delivery methods
Agrobacterium
Gene Gun

NDSU

Tissue culture
required to generate
transgenic plants

Extension

Plant Tissue Culture

A Requirement for Transgenic Development

A plant part
Is cultured

Callus
grows
Shoots
develop

Shoots are rooted;

plant grows to maturity

NDSU

Extension

Agrobacterium
A natural DNA delivery system
A plant pathogen found in nature
Infects many plant species
Delivers DNA that encodes for plant hormones
DNA incorporates into plant chromosome
Hormone genes expressed and galls form at infection site
Gall on
stem

NDSU

Gall on
leaf

Extension

The Galls Can Be Huge

NDSU

Extension

Natural Infection Process Is Complex

NDSU

Extension

But Natures Agrobacterium

Has Problems
Infected tissues cannot be regenerated (via tissue culture)
into new plants
Why?
Phytohormone balance incorrect regeneration
Solution? Transferred DNA (T-DNA) modified by
Removing phytohormone genes
Retaining essential transfer sequences
Adding cloning site for gene of interest

NDSU

Extension

The Gene Gun

DNA vector is coated onto gold or tungsten particles
Particles are accelerated at high speeds by the gun
Particles enter plant tissue
DNA enters the nucleus and
incorporates into chromosome
Integration process unknown
NDSU

Extension

Transformation Steps
Prepare tissue for transformation
Tissue must be capable of developing into normal plants
Leaf, germinating seed, immature embryos

Introduce DNA
Agrobacterium or gene gun

Culture plant tissue

Develop shoots
Root the shoots

Field test the plants

Multiple sites, multiple years

NDSU

Extension

The Lab Steps

NDSU

Extension

Lab Testing The Transgenics

Insect Resistance

Cold Tolerance

Transgene=
Bt-toxin protein

Transgene=
CBF transcription factors

NDSU

Extension

More Modern Examples

Salt Tolerant

Mercury Resistance

Transgene=
Glyoxylase I

Transgene=
Mercuric ion reductase

NDSU

Extension

The Next Test Is The Field

Herbicide Resistance
Non-transgenics

Transgenics

NDSU

Extension

Final Test
Consumer Acceptance
RoundUp Ready Corn

After

NDSU

Before

Extension

The Public Controversy

Should we develop transgenics?
Should we release transgenics?
Are transgenics safe?
Are transgenics a threat to non-transgenic
production systems?

NDSU

Are transgenics a threat to natural

eco-systems?

Extension