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BP04:BP04:
Introduction toIntroduction to
BiotechnologyBiotechnology
NicolasNicolas PaulyPauly
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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