Plant Biotechnology:

Principles and Techniques

Instructor: Eric BONCOMPAGNI

Nice Sophia Antipolis University - France

Part 4

sites.unice.fr/EB

Genetic engineering of plant for fundamental

research purpose

Learn about gene expression level

Spatio-temporal expression = where and when a gene is expressed

Reporter gene method: gene encoding a protein that will be easily

noticeable in the plant

Should not be present in plant before transformation

Chimera construct: promoter from gene of interest + reporter gene

β-glucuronidase (uidA gene)

From Escherichia coli bacteria

Structure et activité: enzyme with β-

glucuronidase activity

Quantitative detection : fluorometer, photometer

Qualitative detection : histochimical analyzes

Blue precipitate will form after X-Gluc (5-bromo-

4-chloro-3-indolyl-β-D-glucuronide) is clived

Luciferase (Luc gene)

From Photinus pyralis firefly

Activity: luciferine degradation

Quantitative detection : luminometer

Qualitative detection : light emission

Tabacco with luc

gene expression

H2O2-induced luciferase activity surrounding

an area of hypersensitive cell death in a

gst1::luciferase transgenic Arabidopsis line

The chemical reaction catalyzed by firefly luciferase takes place in

two steps:

•luciferin + ATP → luciferyl adenylate + PPi

•luciferyl adenylate + O2 → oxyluciferin + AMP + light

Green Fluorescent Protein (GFP)

From Aequorea victoria jellyfish

Activity: fluorescence (absorption: 395 nm; emission: 508 nm)

Quantitative detection : luminometer

Qualitative detection : fluorescent microscope, confocal

microscope

- Resistance to diseases and pathogens (bacteria, fungi, viruses,

insects…)

- Resistance to novel herbicides

- Protection against abiotic stress: salinity, drought, frost…

- Improved nutritional value in food products (ex: provitamin A and

Tomato; see exercice on Thursday 26 December)

- Improved aroma, taste and structure of agricultural products

- Improved fiber quality (ex: cotton)

Genetic engineering of plant for agricultural

purpose

First generation of agriculture transgenics on the market

Insect resistant cotton

Bt toxin kills the cotton boll worm

transgene = Bacillus thuregiensis Bt protein

Insect resistant corn

Bt toxin kills the European corn borer

transgene = Bt protein

Normal Transgenic

Herbicide resistant crops

soybean, corn, canola, sugarbeet, lettuce, strawberry,

alfalfa, potato, wheat

transgene =

modified EPSP synthase

or

phosphinothricin-N-acetyltransferase

Herbicide Resistance: 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

Non-transgenics

Transgenics

spraying

=> plant dies

=> plant lives

Herbicide Resistance: Roundup Sensitive Plants

=> Non-transgenic

Shikimic acid + Phosphoenol pyruvate

5-Enolpyruvyl shikimic acid-3-phosphate

(EPSP)

Plant

EPSP synthase

Aromatic

amino acids

X

X

X=> Without amino

acids, the plant diesX

+ Glyphosate

Transgene: Bacterial

EPSP synthase

Shikimic acid + Phosphoenol pyruvate

5-enolpyruvyl shikimic acid-3-phosphate

(EPSP)

Aromatic

amino acids

Herbicide Resistance: Roundup Resistant Plants

=> Transgenic

With amino acids, the plant

lives

RoundUp has no effect

bacterial enzyme is resistant to

herbicide

+ GlyphosateXPlant

EPSP synthase

Golden Rice – increased Vitamin A content

… but not without controversy

transgene = three pathway enzymes

Next generation of agriculture transgenics on the market

• Vitamin A deficiency is a major health problem: causes blindness;

Influences severity of diarrhea…

• For many countries, the infrastructure doesn’t exist to deliver vitamin

pills

=> Improved vitamin A content in widely consumed crops

an attractive alternative

• >100 million children suffer from the problem

-Carotene Pathway Problem in Plants

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene

(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Problem:

Rice lacks

these enzymes

Classical

Vitamin A

“Deficient”

Rice

The transgenic Golden Rice

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene

(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Daffodil gene

Single bacterial gene;

performs both functions

Daffodil gene

-Carotene Pathway Genes Added

Vitamin A

Pathway

is complete

and functional

Golden

Rice

Transgenic plants could be used as edible vaccines

to serve human health needs…

Phytoremediation

- cleanup contaminated sites

- uses plants designed to take up the soil pollution

(heavy metals)

Environmental applications of transgenic plants

• Works like any vaccine

• A transgenic plant with a pathogen protein gene is developed

• Potato, banana, and tomato are targets

• Humans will eat the plant

• The body produces antibodies against pathogen protein

• Humans are “immunized” against the pathogen

The most cultivated GMO species (2003)

GMO field

species

Area of field

used for GMO

(%)

soybean 55

oilseed rape 16

cotton 21

corn 11

Disadvantages, risks with GMO

• Pollination

- transfer of pollen and genes by insects away from the field with GMO

But usually inside the species; rare between relative species; very rare transfer between different species.

• Toxical properties for nontarget organisms

- insects, birds, plants, animals, people eating plants..

Political strategy in European countries

• Consideration of EU Directives EU -

different possibilities for different types of

agriculture: classic, ecological, GMO…

• Consumer must have possibility to

choose between GMO and others;

declarations on food articles are obligated

Biotechnology definition

The application of the technology to improve the biological function of an

organism by adding genes from another organisms

Conclusions – Take home message

Important Plant Improvement Methods

• Breeding

Crossing two individuals from the same (or close)

species; produces a new and improved variety.

• Transformation

Adding a gene from another species; the

essential biotechnology procedure to produce

transgenic plants.

Transgenic – an organism containing a transgene introduced by

biotechnological (not breeding) methods => develop organisms that express

a “new” trait not normally found in the species

Isolating genes

Modifying genes so they function better

Preparing genes to be inserted into a new species

Developing transgenes

Genetic engineering is the basic tool set of biotechnology

Transformation Cassettes

1. Gene of interest

• The coding region and its controlling elements

2. Selectable marker

• Distinguishes transformed/untransformed plants

3. Insertion sequences

• Aids Agrobacterium insertion

Plant Biotechnology:

Principles and Techniques

Instructor: Eric Boncompagni

Nice-Sophia Antipolis University - France

Part 5: Exercices

Transcription factors are key regulators of the modulation of gene expression thatcontrol developmental processes and the response to environmental stresses. Inorder to characterize the signaling pathway (s) involving the transcription factorAtMYB44, a team of researchers proposes to study the phenotype of transgenicplants exhibiting a modification of AtMYB44 gene expression.

Five independent and homozygous Arabidopsis thaliana 35S :: AtMYB44 lines,named T-10, T-14, T-17, T-18 and T-21, are selected and analyzed. The constructionused during the transformation is presented in Figure 1.

EXERCISE

Figure 1: Diagram of the constructionused for the transformation of A.thaliana. The coding sequence of thegene MYB44 from A. thaliana(AtMYB44) is placed downstream ofpromoter of the 35S gene of theColeopoly mosaic virus. flower (P 35S)and upstream of the 3 'sequence ofthe gene of the nopaline synthaseBam (T). OUR LB / RB: Left and rightedges of T-DNA; NPTII: gene encodingneomycin phosphotransferase type II.

The selected transgenic lines are analyzed by Southern blot. The resultobtained is presented in Figure 2.

Figure 2: Southern blot analysis of the 5transgenic lines and the wild line (WT).The genomic DNA of the plants isdigested by XbaI (X) or EcoRI (E). Thecoding sequence of the NPTII genelabeled radioactively is used as a probe.

The researchers continue the analysis of 35S :: AtMYB44 transgenic linesand of a AtMYB44 KO mutant line (noted atmyb44) using hybridizationapproaches.

Figure 3: (A) Northern-blot analysis of AtMYB44gene transcripts. 15 μg of total RNA extractedfrom the wild-type (WT) lines, the 5 different 35S:: AtMYB44 transgenic lines and the atmyb44mutant are deposited on gel and stained withethidium bromide (Total RNA, bottom panel).After transfer on a nylon membrane, the RNAs arehybridized with a probe corresponding to thecoding sequence of the gene AtMYB44 (AtMYB44,top panel). (B) Western-Blot Analysis of theAtMYB44 Protein Level in the wild lines (WT), 35S:: AtMYB44 (T-18, T-21) and mutant atmyb44 atusing polyclonal antibodies specifically raisedagainst the protein AtMYB44. *: bandcorresponding to the protein AtMYB44. bandscorresponding to 2 unknown proteins interactingwith the raised antibody against AtMYB44.

Random insertion of the T-DNAposition effect

Identify a

gene

of interest

Isolate the

gene

Cloning in a binary

vector

Transfom Agrobacteria

Transgenesis

Selection

Regenerate

EvaluationIntrogression

Take home message – the steps for

making a GM crop

Plant Biotechnology:

Principles and Techniques

Instructor: Eric Boncompagni

Nice-Sophia Antipolis University - France

Part 6: Research article analysis

BP04:Introduction to

Biotechnology

TUTORIALS – Article analysis

Eric BONCOMPAGNI

Nice Sophia Antipolis University

France

Basic research needed to improve food

affordability and quality

Basic research contributes to food affordability

• Higher yields

• Less loss to drought, flooding,

temperature extremes

• Less loss to pests and

pathogens

• Improved shelf life

• Better understanding of plant

biochemistry, nutrient uptake,

photosynthesis, growth and

development

• Improved tools for breeding and

genomics

Virus-resistant tomatoesFlooding-tolerant rice

Broccoli that stays

fresh longer

Reprinted by permission from Macmillan Publishers Ltd. (NATURE) Xu, K., Xu, X., Fukao, T., Canlas, P., Maghirang-Rodriguez, R., Heuer, S., Ismail, A.M., Bailey-Serres, J., Ronald, P.C., and Mackill, D.J. (2006).

Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice. Nature 442: 705-708. ; Broccoli photos courtesy Jocelyn Eason, Plant and Food, New Zealand; Anderson, L.K., Lai, A., Stack,

S.M., Rizzon, C. and Gaut, B.S. (2006). Uneven distribution of expressed sequence tag loci on maize pachytene chromosomes. Genome Research. 16: 115-122; William M. Brown Jr., Bugwood.org

• Improve nutritionnal quality

* enrichment in vitamine A (golden rice)

* enrichment in iron

* enrichment in essentials amino acids (Met)

• Improve digestibility

• Modify fathy acids content to prevent cardivascular disease

• Reduce amount of allergens

•…

Basic research contributes to food quality

vitamine A

Vitamine A (retinol)

Humans cannot produce vitamine A:

- 50% de pro-Vitamine A (β carotene = a carotenoide) from plants converted in

vitamine A in the intestin

- Ingestion of vitamine A of animal origin

b.usuhs.mil/biochem/nutrition/NOTES/

Vitamin A deficiency is a leading cause of

blindness

100 million children

are Vitamin A

deficient

Up to 500,000

children become

blind every year and

half of these die

within 12 months of

losing their sight

Improving dietary

intake of carotenes

can reduce child

mortality by 25 %

WHO data

Occurrence of

Vitamin A

deficiency (VAD)

Phytonutrients: carotenoids

Reprinted from Cazzonelli, C.I. and Pogson, B.J. (2010). Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci. 15: 266-274 with permission from Elsevier. Corn photo by Doug Wilson.

Carotenoids include β-carotene,

the precursor to vitamin A, but

also lutein, zeaxanthin and

lycopene, which have health-

promoting benefits

β-carotene is

converted to

vitamin A after

ingestion

lycopene

lutein

Carotenoids are more

than just vitamin A

precursors

Carotenoids may help prevent age-related

macular degeneration

Age-Related Eye Disease Study Research Group. (2007). The Relationship of Dietary Carotenoid and Vitamin A, E, and C Intake With Age-Related Macular Degeneration in a Case-Control

Study: AREDS Report No. 22. Arch Ophthalmol. 125: 1225-1232. http://www.nei.nih.gov/photo/eyedis/images/EDA17_72.jpgl Image courtesy CDC and Mary Anne Fenley (number 13053)

Age-related macular

degeneration is a leading

cause of sight loss

Diets high in lutein,

zeaxanthin, zinc and

vitamins C, and E may

protect eyes

Lutein and zeaxanthin accumulate

in the macula, giving it an orange

color. They are thought to absorb

high energy light and protect the

retina from damage

Some phytonutrients show good evidence for a

protective function

Epigallocatechin gallate

from green tea that may help

eliminate immortality from

some cancer cells

Isothiocyanates from

broccoli and other

cruciferous vegetables

may detoxify carcinogens

Lycopene, a carotenoid

abundant in tomatoes, has

been shown in some studies

to protect against several

forms of cancer

Genistein an isoflavonoid

from soy beans may

protect against cancer

Caroténoïdes precursor is:

geranylgeranyl pyrophosphate

(GGPP)

-carotène synthesis requires 4

enzymes:

phytoene synthase, phytoene

desaturase,ζ-carotene desaturase,

lycopene cyclase

Biosynthesis occurs in plastids

Pro-vitamin A biosynthesis

Produce rice with more vitamine A

Production of the ‘Golden Rice’

Geranyl geranyl pyrrophosphate (GGPP)

Phytoène

Lycopène

ß-carotène

Phytoene

synthase

Phytoène

desaturase

z -carotène

desaturase

lycopène

ß-cyclase

z -carotène

Erwinia

uredovora

double

desaturase

crtI

Plante Erwinia

Which construction was used?

How where the plants transformed?

How were the transformants selected?

Which validations should be performed on the selected lines?

Expression of bacterial CrtI in tomato

Which construction was used?

How where the plants transformed?

How were the transformants selected?

Which validations was performed on the selected lines?

Expression of bacterial CrtI in tomato

RNA interference (RNAi) in plant

P35S-GUS

CHS DICER

RISC

dsRNA

RNA-induced silencing complex

RNAse III

sRNA

GENE XP GENE XRDR6

Transgene trigger Endogenous

gene

The silencing occurred with

transgenes having homology

to the silencing locus

could be induced by stable or transient expression of transgenes

1990, Napoli, Lemieux & Jorgensen

Bacterial crtI has only 35%

Homology with tomato gene:

NO SILENCING!

Transformation with purified plasmid can

be done with either electroporation or a

simple freeze/thaw transformation method.

Alternatively, a mobilizable plasmid can be

placed into Agrobacterium using the

triparental mating method.

Requirements

A helper strain, Carrying a conjugative

plasmid (such as the F-plasmid) that codes

for genes required for conjugation and

DNA transfer.

A donor strain, Carrying a mobilizable

plasmid that can utilize the transfer

functions of the conjugative plasmid.

A recipient strain, you wish to introduce the

mobilizable plasmid into.

BP13:Introduction to Biotechnology

TUTORIAL – Practicals preparation

Eric BONCOMPAGNI

Nice Sophia Antipolis University

France

Practicals

BP13:Introduction to

Biotechnology

Group 1 Group 2

Practical– Protoplasts isolation and fusion

Somatic fusion, also called protoplast fusion, is a type of genetic

modification in plants by which two distinct species of plants are fused

together to form a new hybrid plant with the characteristics of both, a somatic

hybrid.

Protoplasts may also be used for genetic transformation.

However, the poor efficiency of regeneration from these simplified

systems makes this application limited to some plant species.

Production of somatic hybrids

The plant cell wall is made of cellulose fibers forming with

hemicelluloses a network which is embedded in the pectin

matrix. The adjacent cells are linked by the middle

lamella mainly composed of pectin (Figure 1).

The cell wall

+

Protoplasts-Spherical

-Unable to divide

-FragilesCellulases and pectinases

Protoplasts result from cell wall digestion

Protect the cells (resistance of protoplasts to turgor pressor)

. Cell adhésion. Cell morphogenesis. Regulate cemm growth. Cell division.

Isotonic

mediumHypertonic

medium

Hypotonic

medium

Osmosis

Spontaneous net movement of solvent molecules through a

selectively permeable membrane into a region of higher

solute concentration

When making protoplsts you should use a isotonic medium

• Get rid of cell walls and debris:

Filter should be of the appropriated size 10 to 30 µm depending on

cell/plant type

• Washings: Stop enzymatic lysis, isotonic medium

• Controls:

verify loss of cell wall (Calcofluor White),

check viability (Fluorescein Diacetate) => should be around 80 %

Protoplasts purification

Need to open plasmic membrane: Spontaneous fusion

(soybean, maize), Ca2+ - high pH, PEG or electrical choc.

Hybridization by protoplast fusion

Protocole

Isolation of protoplasts and fusion

MES (MW =195.2): 50 mL, 20 mM

TRIS highly basic salt (MW= 121.14): 50 ml, 25 mM

Buffer solution:

mix the 2 solutions TRIS and MES until getting pH 5.5 then add 0.9M of mannitol (MW

=182.17)

Washing buffer at pH 7.5: what element will change?

Digestion solution: add enzymes in 50 mL of buffer solution:

Solution 1: Macerozyme (0.1%) and Cellulase (1%)

Solution 2: Pectolyase (0.1%) and Caylase (1%)

PEG-fusion solution:

Solution 3 (20 mL): PEG (0.4%) by heating in a water bath.

Solution 4 (20 mL): glucose (5.4%) + CaNO3 4H20 (0.75%)

Mix the solutions 3 and 4, adjust at pH 7 (KOH 0.1N) and complete to 50 mL.

Evans’s blue: 1% in H2O

Exercise

Protocol

Protocol

Protocol

Protocol

Use of Malassez cell.

Determination of protoplast number

1 rectangular unit (in blue)

Volume = 0,01µL

Protocol

Whole plants can be regenerated via cell and tissue culture

Protoplast purification

Protoplast fusion for

hybridization and

protoplast selection

Whole plant regeneration

=> Hybrids with new traits

Examples of protoplast

regeneration in different

plant species

Agricultural valorization