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

But we know nature does not have

all of the traits we need

NDSU
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 Mendel’s Traits?
Mendel’s Genes
Plant height Seed shape

Smooth Wrinkled
Allele

Tall Short
Allele

NDSU
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 Mendel’s Genes
Plant Height Gene

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

NDSU
Extension
 Allelic Differences for Mendel’s
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 RNA
Transcription
(gene)
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)

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

NDSU
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
Mouse probe
library
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-COO-
ATG 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;

NDSU
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’

3’ 5’

5’ 3’

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

3’ 5’

5’ 3’

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

3’ 5’

NDSU
5’ 3’

Extension
 PCR Animation

Denaturation: DNA melts

Annealing: Primers bind

NDSU
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 PCR fragment
probe added to filter

Hot-spots are human gene

NDSU
of interest
Extension
 Map-based Cloning
Gene Marker
1. Use genetic techniques to
find marker near gene

Gene/Marker
2. Find cosegregating marker

3. Discover overlapping clones

(or contig) that contains the marker Gene/Marker

Gene/Marker
4. Find ORFs on contig

5. Prove one ORF is the gene by Mutant + ORF = Wild type?

NDSU
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

X
Plant
EPSP synthase

Without amino acids,

X
3-Enolpyruvyl shikimic acid-5-phosphate
(EPSP)
plant dies

X
X
Aromatic

NDSU
amino acids
Extension
 RoundUp Resistant Plants
Shikimic acid + Phosphoenol pyruvate
+ Glyphosate
RoundUp has no effect;
Bacterial enzyme is resistant to herbicide
EPSP synthase

3-enolpyruvyl shikimic acid-5-phosphate

(EPSP)
With amino acids,
plant lives

Aromatic

NDSU
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 doesn’t 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: Phytoene desaturase
Rice lacks
these enzymes ξ-carotene desaturase

Lycopene
Lycopene-beta-cyclase
Normal
 -carotene

NDSU
Vitamin A
“Deficient” (vitamin A precursor)
Rice Extension
 The Golden Rice Solution
-Carotene Pathway Genes Added

IPP

Geranylgeranyl diphosphate

Daffodil gene Phytoene synthase

Phytoene
Vitamin A
Phytoene desaturase
Pathway Single bacterial gene;
is complete performs both functions
and functional ξ-carotene desaturase

Lycopene
Daffodil gene Lycopene-beta-cyclase

 -carotene

NDSU
Golden
Rice (vitamin A precursor)
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
Trait Gene

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
ex.: EPSP
-carotene genes

NDSU
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
 Let’s Build A Complex Cassette
pB19hpc (Golden Rice Cassette)

TL aphIV 35S Gt1 psy 35S rbcS crtl TR

T-DNA Hygromycin Phytoene Phytoene T-DNA

Border Resistance Synthase Desaturase Border

Insertion Selectable Gene of Gene of Insertion

Sequence Marker Interest Interest 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

Tissue culture
• Gene Gun required to generate
transgenic plants

NDSU
Extension
 Plant Tissue Culture
A Requirement for Transgenic Development

Callus
grows
A plant part Shoots
Is cultured 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 Nature’s 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= Transgene=
Bt-toxin protein CBF transcription factors

NDSU
Extension
 More Modern Examples
Salt Tolerant Mercury Resistance

Transgene= Transgene=
Glyoxylase I 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

NDSU
Before After
Extension
 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?

NDSU
Extension