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BP04:BP04:

Introduction toIntroduction to

BiotechnologyBiotechnology

NicolasNicolas PaulyPauly

nicolas.pauly@unice.fr

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Universit de Nice Sophia Antipolis

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Institut Sophia Agrobiotech

From genes to ecosystems

Institut Sophia Agrobiotech

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Research Area: Biotic Interactions and Plant Health

Research for Sustainable Agriculture and the Environment

Reduction of fertilizer use:

Molecular bases of plant-pathogen interactions

Molecular bases of plant- symbiotic bacteria interactions

Plant Breeding for resistance

Stimulate defence mechanisms

Inhibit pathogen development

Reduction of pesticide use:

Optimize the symbiosis

Reduce the negative impact of stress

http://www.paca.inra.fr/

Rhizobium Medicago truncatulasymbiosis (D. Hrouart & A. Puppo)

Legumes

Legumes represent a crop with nutritional properties particularly valuable for foodand feed (20 to 40% proteins in seeds, production of health-promoting secondarycompounds, blood cholesterol-reducing effect ).

P. vulgaris V. faba P. sativum

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Sinorhizobium meliloti / Medicago sp. Interactions

MedicagoMedicago truncatulatruncatula

SinorhizobiumSinorhizobium melilotimeliloti

Fixed nitrogen(ammonia)

RootRoot nodulesnodules

N2

Rhizobium

Fixed carbon(malate, sucrose)

Medicago

Rhizobium Medicago truncatulasymbiosis

1. Redox signalling: From symbiose establishment to nodule functionning

2. Understanding senescence mechanism in the root nodule

Nitric Oxide

GlutathioneHydrogen

peroxide

Redox

Signalling

- Spatiotemporal dynamics

- Cross-talks

- Production systems

- molecular targets

Rhizobium Medicago truncatulasymbiosis

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Plant biotechnology

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Plant Biotechnology

Haplodiplodisation

Culture of meristems

Micropropagation

Save of embryo

Protoplast fusion

Induction of variability

Plant biotechnologies:

in vitroculture

Molecular biology

Agronomy :

New CultivarsClonal micropapagation

Cultivar identification

Fundamentalresearch

Industry:

Synthesis of natural products or proteins

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Plant totipotency

Overview

Definition

Birth and development of in vitroculture

Application et limitation of totipotency

Mecanisms underlying totipotency

Biological significance of plant cell totipotency

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Some definitions

Specific to plants

In plants, the totipotency can be defined as the property of some cellsthat may regenerate a plant when they are placed under appropriateconditions (possibly via a stage of dedifferentiation)

Exemple : micropropagation of Saint Paulia

2 monthslater

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Historical aspects

Totipotency as the theme ofplant biotechnology birth

Historical of tissue culture and plant organs

Scientific context in the beginning of XXth century

Cellular theory (Schleiden et Schwann)

Microbiology and biochimistry

How to study the behavior of single cells?

Growth in axenic cultures Characterisation of growth substances

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G. Haberlandt : the concept of totipotency ofthe plant cell

Historical aspects

Two main important ideas:

culture of isolated cells constitute a potentialresearch model

keep alive isolated cells

No cellular multiplication

We can potentially regenerate a whole plant from a single cell totipotency

Failure (bad choice of explants, ignorance of growth substances)

Emergence of culture techniques

Haberlandt (1902) : concept of totipotency

White (1934) : in vitroculture of tomato root

Gautheret (1935) : use of auxin to grow willow cambium

1939 : First callus culture of carrot

Tissue culture is possible using growth substances and / ormeristematic tissues

https://www2.carolina.com

Historical aspects

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Emergence of culture techniques

Braun (1941) : work on crown gall

Miller (1955) : cytokinins

Murashige and Skoog : development ofeffective culture media containing cytokinins andauxins

In vitro tumor growth withouthormon supply!!

Historical aspects

Validation of Haberlandt hypothesis

1956 (Muir) cell suspensioncultures

1958 (Reinart et Stewart) Carrotsomatic embryogenesis

Historical aspects

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Validation of Haberlandt hypothesis

1965 (Vasil et Hilderbrandt) Regeneration of a tobaccoplant from a single cell

1971 (Nagata et Takabe) Regenerating a whole plantfrom a protoplast

Historical aspects

Developement:agronomic tools

1965 (Morel) in vitro propagation oforchids

1972 (Sharp) : tomato plants fromhaploid pollen

1973 : hybrid from a protoplastfusion

Historical aspects

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Development: production ofsecondary metabolites

1977 : culture of tobacco cells in a reactor of 20000 liters

1983 (Mitsui Petrochemical) : industrial production ofsecondary metabolites

Historical aspects

Development : GMO

Van Montaigu (1983) : kanamycinresistant tobacco plants

1994 : Flavr Savr (Calgene)

1996 : GM maize in the United States

Historical aspects

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Sussex, Plant Cell 2008

Conclusions

Initial problem :

Search for a isolated cell model

Validation of plant cell totipotency

Identification of the role of growth substances

Development of many techniques

Transgenesis

Historical aspects

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Totipotency: applications andlimitations

Meristem

SAM

Node

Culture ofmeristem

rootingPlantules

Leafy stem

Indirectmorphogenesis

Callogenesis

callus

Cellsuspension

Indirectcaulogenesis

Indirect somaticembryogenesis

Direct

morphogenesis

Caulogenesis

Direct somaticembryogenesis Artificial

seeds

Main methods of micropropagation

From Lindsey et Jones 1989

Explants (root,stem, leaves, )

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Cells more or less totipotent

Meristems:

a pool of totipotent cells

Other cell types: totipotency more or less easy to express

Use of exogenous growth substances

direct regeneration

Regeneration through a callus stage

interspecificvariability

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Limitations of totipotency :technical impossibility

Differentiation irreversible xylem (!)

Depending on the method of preparation of protoplastsrecalcitrance to regeneration

For many species of agronomic interest, theprotoplasts are not totipotent (recalcitrant) ex: Vitis

vinifera

Limitations of totipotency: Somaclonal variation: regenerated plants often

have problems

Loss of characters (chimeras)

Chromosomal deletions

Changing the character juvenile

Impact on fertility

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Mechanisms underlyingtotipotency

In plant cells

Why do plant cells are totipotent:common arguments

Small number of cell types

Only 3 or 4 basic types of organs (root, stem, leaves)whose flowers, tendrils, thorns, fruits and tubers are derivatives

Large genomic plasticity

growth may remain nearly normal despite profoundchromosomal rearrangements

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Steps related to totipotency

Step 1 :

Differentiated cells: need of dedifferentiation

Meristematic stem cells

Step 2 : initiation of mitotic activity use of growthsubstances

Step 3 : autonomous tissue growth with respect to

exogenous growth substances

Current scientific issues:- Understanding the mechanisms of dedifferentiation- Understanding the nature of stem cells

Understanding the mechanisms ofdedifferentiation

Modulation de lexpression gntique via desmcanismes pigntiques

Reconformation de la chromatine

Modification des histones

Mthylation de lADN

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Understanding the nature of stemcells

Stem cell concept in plant biology

Concept of stem cell niche in plant biology

Basic techniques of vitroculture

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Equipment and media are steri lizedby autoclaving

Manipulation and transfer of the explant are madein a laminar flow hood

The explant was cultured in steriletubes, petri dishes or bottles

Concept of axenic conditions

Technical aspects

Laboratory organisationMedia

preparation and

sterilization

Subculturing

under sterile

conditions

Growth

chambersLaundry

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Sterilization of explants

Ethanol

Sodium or calcium hypochlorite

Hydrogen peroxide

Silver nitrate

Sterilization protocol of explants

Selecting explant

1/ Ethanol 10 s

2/ Ca(OCl)2 7%, 10 mn

3/ 3 washes in sterile H2O

Division and

cultivation of the

explant

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Culture medium

Water

Gelling agents :

agar phytagel :

polymer of bacterial origin (glucuronic acid, rhamnose and glucose)

translucent

!! Interference with kanamycin!!

Growth conditions

Culture medium

Carbon supply?

Most of in vitro plant cultures are htrotrophs

Choice of carbon source

Sucrose

Glucose

Maltose

Case by case basis:

growth substances

genotype

Growth conditions

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Culture medium

Minerals

Macroelements : N,P,K, Ca, Mg, S

ex : NO3- / NH4

+ ratio

Microelements : others trace elements

Use of silver nitrate as ethylene antagonist

Growth conditions

Plant growth substances

Auxins (from tryptophan)

Cytokinins (from adenine)

AuxinCytokinins Gibberellins

Abscisic Acid

Ethylene

BrassinosteroidsSalicyla

tes

JasmonatesStrigolactones

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Plant growth substances

Auxins

Growth conditions

Natural:

Indole Acetic Acid = IAAN

CH2

CO2

H

HCH2CO2H

Synthetic:

Naphthalene Acetic Acid NAA

2,4-D (dichlorophenoxyacetic acid )

OCH2

CO2

H

Cl

Cl

At cellular level: they stimulate elongation

At tissular level: they stimulate root growth at low

concentration (except 2,4-D)

Roles of auxins

They inhibit stem elongation and auxiliary bud

Growth conditions

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Cytokinins

Naturals

Isoprenod CK (ex: zeatin)

Aromatic CK (ex: BA)

SyntheticsKinetinBenzyl AdenoPurine (BAP)

Plant growth substances

Growth conditions

At cellular level: they stimulate cell division

At tissular level: in general, they inhibit RAM growth

Activation of the SAM

Role of cytokinins

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Gibberellins (gibberellic acid)

ent-Kaurene (from geranyl-geranyl PP)

Plant growth substances

Growth conditions

Gibberellins (gibberellic acid)

Complex effects:

Stimulation of internodal growth, leaf expansion

Inhibition of root growth in the light Instead promotes root growth in the dark

Growth conditions

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Culture medium

Various organic compounds

Amino acids (glycine, cysteine)

Vitamins (thiamin: B1, nicotinic acid: PP, folic acid: B9)

Complex organic mixtures

Antioxidants

Growth conditions

Organogenesis and callogenesis

Undifferentiated cells

Differentiated cells (organ cultures)

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Callogenesis

Callus of undifferentiated cells from organs(leaves, roots ...)

Subculturing on fresh medium regularly, infinitegrowth

Genetic drift

Used to make cell suspensions or regeneration

How to obtain callus and undifferenciated cells?

undifferentiated cells

Cut

callogenesis

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callogenesis

callogenesis

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OrganogenesisSkoog and Miller (1957)

Auxin

Cytokinin

organogenesis

auxins cytokinins

rooting

callus

buds

Axilary buds

R=1: callus only

R1: roots on callus

FLEURS

organogenesis

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organogenesis

organogenesis

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Solid medium

Callus

Cell suspension

Direct way!

Obtaining of cell suspension cultures

callogenesis

callogenesis

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callogenesis