Understanding Biotechnology - Forest Science Labs

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Understanding
Biotechnology


Steve Strauss, Professor, OSU

Forest Science, Genetics, Molecular and
Cellular Biology

Director, Outreach in Biotechnology

http://wwwdata.forestry.oregonstate.edu/orb/


Steve.Strauss@OregonState.Edu



Outreach website

http://wwwdata.forestry.oregonstate.edu/orb/

Educational activities

Food for Thought Lecture Series / 2005
-
2008


Streaming video
-

OPAN/OPB usage


The plan


What is biotechnology


GMOs


State of usage in the world


How it works


The general concerns surrounding them


Non
-
GMO biotechnologies (Dave Harry)


Genomics and DNA markers


Break
-
outs for grass seed specifics


Commercialization issues, GMO testing, grass
industry biotechnologies

What is biotechnology?

Amer. Heritage Dictionary (2000)


1. The use of
microorganisms

or biological
substances such as
enzymes
, to perform
industrial processes.


2a.

The application of the principles of
engineering and technology to the life
sciences; bioengineering.

A more crop oriented definition
of biotechnology


Use of technologies that affect physiology,
genetics, management, or propagation


Most common uses


Microorganisms for fermentation of plant
products


Plant tissue culture for propagation


DNA sequencing and indexing for
identification (DNA fingerprinting)


Gene isolation, modification, and insertion
(genetic engineering, “modern biotechnology”)


GE, GEO or GM, GMO

Why emphasize GE forms of
biotechnology?



GE crops have been taken up rapidly
by farmers when available, have had
large benefits, and have great
economic and humanitarian potential


Exploding science of genomics fuels
rapid discovery, innovation

Rapid rise of GE crops in

developed and developing world

http://www.isaaa.org

Many social issues with major
impacts on use / acceptance


Few GMO crop types in production


Maize, soy, cotton, canola


Insect, herbicide tolerance traits


Small amounts of viral resistance (squash,
papaya)


Benefits of reduced tillage, reduced pesticide
use, improved yields, reduced costs


But other traits and crops mostly on hold


Substantial
social

resistance and obstacles to
their use

Defining GMOs


GEO / GMO = creation of a

“recombinant DNA modified organism”


It’s the method,
can use native or foreign genes


DNA isolated, changed/joined in

a test tube, and re
-
inserted asexually


Vs. making crosses or random mutations in
conventional breeding


Powerful breeding tool but can generally handle
one to a few genes at a time


Simple traits can be
designed
, but without constraints
from native gene pools


That’s why its called genetic
engineering
, though we
are modifying, not building, a new organism

Assembling a gene


Controls level of
expression, and


Where and when
expressed


Provides stability to
messenger RNA, and


Guides processing into
protein

Coding sequence

Promoter

Terminator

Can mix and match parts & can
change sequences to improve
properties

Protein

Promoter

(controls expression)

Gene

(encodes protein)

Examples of promoter : gene

combinations produced via
recombinant DNA methods

Phenolic pathway enzyme

(bacteria)

35
S
-
CAMV (plant virus)


RNA degrading enzyme

(bacteria)

Pollen sac (tobacco)

Herbicide tolerant

Male
-
sterile

FMV (plant virus)

Insect toxin protein (bacteria)

Insect

resistance

Oilseed (canola)

Insulin (human)

Improved

nutrition

Recombinant DNA modification
of native plant genes

How are GE plants produced?


Step 1

Getting whole plants back from cultured
cells =
cloning

Differentiation of new plant organs
from single cells

Leaf
-
discs

First step is de
-
differentiation
into “callus”
after treatment
with the plant
hormone auxin

Shoots, roots, or embryos
produced from callus cells using
plant hormones

Step 2

Getting DNA into plant cells

Main methods

-

Agrobacterium tumefaciens

-

Biolistics [gene gun]



Agrobacterium

is a natural plant
genetic engineer

Agrobacterium gene insertion


Gene of interest

Agrobacterium tumefaciens

Engineered

plant cell

T
-
DNA

Ti
Plasmid

Only a few cells get modified so
need to identify and enrich for the
engineered cells

Not all cells are engineered, or engineered the
same. Thus need to recover plants from that
one

cell so the new plant is not
chimeric

(i.e.,
not genetically variable within the organism)

Hormones in plant tissue culture

stimulate division from plant cells

Antibiotics in plant tissue culture

limit growth to engineered cells

Other kinds of genes can also be used to favor transgenic cells

(e.g., sugar uptake, herbicide resistance)

Transformation of bentgrass

(Wang and Ge 2006)

Glyphosate
-
tolerant Fescue

Conventionally
-
bred Patented Varieties

GE traits under development in
forage and turfgrasses

Wang and Ge, In Vitro Cell Develop. Biol. 42, 1
-
18 (2006)



Nutritional quality


Lignin reduction, increase of sulfur
-
rich proteins


Abiotic stress tolerance


Drought, frost, salt


Disease/pest management


Fungal, viral, herbicide tolerance


Growth and nutrient use


Flowering time, phosporus uptake


Hypoallergenic pollen


Bioethanol processability


Problems and obstacles to wider
use of GE crops


Regulations complex, uncertain, changing, and
very costly


Three agencies can be involved


Environmental and food/feed acceptability criteria
complex, stringent compared to all other forms of
breeding


Unresolved legal issues of

gene spread, safety assessment, liability,

marketing, and trade

restrictions

Legal actions


USDA sued over process for granting field
trial permit for GE bentgrass and GE
biopharma crops


USDA sued over deregulated Roundup
-

resistant alfalfa


First time an authorized crop forced to be
removed from market


USDA required to do EIS for alfalfa, one
was already underway for bentgrass


Scotts fined $ 500K over Roundup Ready
bentgrass field trial

Strong and well funded political
and legal resistance

Intellectual property issues


New, costly, overlapping “utility patents” issued
for genes and crops since 1980


Patent “anticommons”


Major costs, uncertainties for use of best technologies
and usually need several licenses for an improved
crop


Major litigations ongoing for years to decades


Basic
Agrobacterium

gene transfer method


Bt insect resistance gene innovations


Regulatory risks make large companies very
reluctant to license to small companies,
academics


Public sector, small companies find it very hard
to cope with the costs, obstacles


Strong polarization on benefits vs. risks


A highly vocal, concerned minority (~20%)


A majority whose level of acceptance
varies widely among applications
depending on benefits and ethical views


Strong resistance to animal applications, and
to impacts that appear to harm biological
diversity


Very low knowledge of the science,
technology

Varied public approval

Rutgers survey data
-

USA (2005)

http://www.foodpolicyinstitute.org/resultpub.php


http://www.foodpolicyinstitute.org/docs/reports/NationalStudy2003.pdf



Seven in ten (70%) don't believe it is possible
to transfer animal genes into plants


Six in ten (60%) don't realize that ordinary
tomatoes contain genes


More than half (58%) believe that tomatoes
modified with genes from a catfish would
probably taste fishy


Fewer than half (45%) understand that eating a
genetically modified fruit would not cause their
own genes to become modified


Education needs: Gullibility


"People seem to have a great number of
misconceptions about the technology. As a
result, they seem to be willing to believe
just about anything they hear about GM
foods.“


Very few universities take an active role in
outreach, education


University of California system an exception

Summary


GE is a method, not a product


GE crops a major presence and with major
science and technology push forward


GE method highly regulated, causing great
costs and uncertainties both for field
research and commercial development


Social/legal obstacles slowing or blocking
investment outside of the major crops and
large corporations

Understanding
Biotechnology



Part 2:

Genomics and DNA Markers

David Harry

Department of Forest Science

Assoc. Director, Outreach in Biotechnology

http://wwwdata.forestry.oregonstate.edu/orb/


david.harry@oregonstate.edu



DNA
-
based

Biotechnologies


Genetic engineering (GE, GMO)


direct intervention and manipulation


gene manipulation and insertion through an
asexual process


Genomics & DNA markers


are generally descriptive, examining the
structure and function of genes and genomes


manipulating genes and genomes is indirect,
through selection and breeding

Some definitions


Genes


a piece of DNA (usually 100’s to 1000’s of bases long)


collected together along chromosomes


serves as a structural blueprint or a regulatory switch


Genome


an entire complement of genetic material in the nucleus of an
individual (excluding mitochondria and chloroplasts)


genes, regulatory elements, non
-
coding regions, etc


tools for describing genomes include
maps

and
sequence


DNA marker


some type of discernable DNA variant (variation, or
polymorphism) that can be tracked


tracking the +/
-

of markers offers powerful tools for managing
breeding populations and, increasingly, for predicting offspring
growth performance


For today:


Basics of DNA markers


DNA markers & fingerprints


are fixed for the life of an individual


can be used to identify individuals


Marker inheritance (parent to offspring)


nuclear markers


parentage verification


genome mapping


Associating markers and traits


maps and associations


marker breeding (MAS/MAB)

Genes are located on
packaging platforms called
chromosomes

Genomes, genes, and DNA

DNA markers reveal subtle
differences in DNA sequence

A

A

A

T

T

C

G

G

A

A

A

T

T

C

C

G

T

C

C

A

C

G

T

G

T

C

C

A

C

G

T

T

G<>C

G<>T

Marker “1”

Marker “2”

Age

A DNA fingerprint is fixed
throughout an individual’s life

MCW
-
305

MCW
-
184

MCW
-
087

DNA Fingerprints to Verify Identities

22 Paired Samples Collected at Different Times

S D

Progeny

Pedigree errors: “non
-
parental”
marker types

Genetic Map:

Perennial Ryegrass

Gill et al. 2006

X

X

Then,

evaluate genetic
makeup early to select
young birds

First,

associate performance and
genetic makeup

X

X

How might genetic markers
accelerate breeding?

a

b


c

d

e

f

g

h

i

j

k

l

m

Hypothetical genes (QTLs)
affecting economic traits

Linkage Map

1

2

3

4

Trait 2

Trait 1

Mapping loci affecting quantitative
traits (QTL) in chickens

Distance along chromosome Gga 3 (cM)

Genes in the circled
region appear to affect
breast
-
meat yield

High
-
throughput Genotyping

Illumina
-

BeadStation500G
-
BeadLab

~150,000 data points per week at UCDavis Genome Center

Marker Assisted Breeding


in Conifers


Quantitative Trait Locus (QTL) Mapping


Association Mapping

Pinus taeda


(loblolly pine)

Pseudotsuga menziesii


(Douglas
-
fir)

Pinus elliottii

(slash pine)

Genomics & DNA Markers:
Summary


DNA markers can be used as fingerprints to
distinguish individuals, and


cultivars, varieties, etc


increasingly used to protect intellectual property
(utility patents, PVP)


Marker inheritance allows parentage to be
verified, facilitating pedigree control


DNA markers can be associated with phenotypic
traits


Once marker
-
trait associations have been
established, marker data can augment
phenotypic observations to accelerate breeding