Vocabulaire - مفردات - Vocabulary
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Cultures in vitro | زراعات زجاجية | in vitro cultures |
Culture de protoplastes | زراعة البروتوبلاست | Protoplast culture |
HISTORY OF THE POMATO (POMATE).
The 'pomato' is a somatic hybrid plant obtained by fusion of protoplasts between the potato and the tomato, two species of the genus Solanum (family Solanaceae) whose incompatible genomes prohibit sexual hybridization. This hybrid was first created in 1978 by a German biologist Georg Melchers at the Max Planck Institute for Biology in Tübingen.
What is protoplast ?
Protoplast is a plant cell without a cell wall. It consists of plasmalemma containing all the other cellular components in it. Protoplast culture is used to regenerate a whole plant using suitable artificial medium and environmental conditions...
بالإمكان خلق هجين السيتوبلاسم (cybrid) بتقنية زراعة البروتوبلاست. البروتوبلاست هي خلايا نباتية بدون جدار خلوي. وهي تتكون من غشاء بلازمي يحتوي على جميع المكونات الخلوية الأخرى التي يحويها. يستخدم استزراع البروتوبلاست لانتاج نبات كامل باستخدام وسط اصطناعي وظروف زراعية مناسبة. |
in vitroLes protoplastes sont des cellules végétales sans paroi cellulaire. Ils sont constitués de cytoplasmes contenant tous les autres composants cellulaires. La culture de protoplastes est utilisée pour régénérer une plante entière en utilisant un milieu artificiel et des conditions de culture appropriés. |
Interests of the protoplast technique
Protoplast cultures are important in plant breeding programs:
--- Fusions of intraspecific or interspecific protoplasts that do not fuse normally (different species and genera).
--- Modifications of the genetic heritage: Genetic transformation of plant species: creation of new varieties:
------- By Introductions of cellular organelles of different species.
Example: introduction of chloroplasts, mitochondria…: cytoplasmic genetic material (eg chloroplasts).
------- By direct introduction of genes: the technique can easily introduce exogenous genes into the protoplasts: because they can absorb nucleic acids.
Examples:
- Introduction of a disease resistance gene.
- Introduction of a gene responsible for nitrogen fixation….
- Increased yields
- Fruit quality
- Resistance to abiotic stresses….
The protoplast culture is the basis of the methods of genetic transformation of plants: creation of transgenic plants (GMOs).
Techniques for obtaining a protoplast
Plant material used:
In tissue culture, protoplasts are first isolated using mechanical and enzymatic methods and then cultured on a liquid or semisolid agar medium. usually leaf fragments are used.
Mechanical method:
The mechanical method is old and applies to tissues with large cells such as onion cells.
In this method, a small piece of the plant epidermis is taken and subjected to plasmolysis, causing protoplasts to shrink away from the cell wall. Then, the tissue is dissected for the release of the protoplasts.
Enzymatic method:
It’s the most widely used technique of protoplast isolation.
The plant cell wall is composed of cellulose, hemicellulose, and pectin, thus, the enzymes used for protoplast isolation include cellulase, hemicellulase, and pectinase.
The duration of application and the concentrations of the enzymes used must be checked.
First, pectinase separates cells from middle lamella, and then cellulase separates the protoplast from the rest of the cell wall.
Protoplast culture
• Liquid culture media.
• Rich mineral media (Murshige and Skoog), B5 (Gamborg) ..
• sugars with low osmotic potential:
--- Glucose 0,35 M.
--- Glucose + mannitol.
• High doses of phytohormones
--- strong auxins (2,4-D + ANA) + cytokinins: reconstruction of the wall.
Culture conditions:
• High density of protoplasts (50 000 – 500 000/ ml)
• First days in the dark to avoid tissue oxidation.
Two methods of creating varieties by protoplasts:
1- Protoplast fusion
2- Introduction of foreign genetic material.
1. Protoplast fusion:
• Spontaneous fusion possible (soya, corn).
• addition of Ca2+ or NaN03 ions (charge attraction).
• work at basic pH.
• add osmolysants: examplefor PEG.
• Electrical shock.
Handling example
How is genetic material distributed?
- Cybrid may be defined as 'plant or cell which is a cytoplasmic hybrid produced by fusion of a protoplast and cytoplast (fluid inside a cell but outside the cell's nucleus)'.
- Cybrid has nuclear genes from only one parent and cytoplasm from both the parents.
The phenomenon of formation of cybrid is regarded as cybridization. Regenerants from a cybrid will have plastomes (genomes of plastids) from both parental species. However, the functional genome results from only one species, due to chromosome elimination.
The plants derived from such regenerants will, therefore, genetically be hybrids only for cytoplasmic traits.
Techniques for verifying the formation of somatic hybrids:
--- Cytogenetics (number of chromosomes, cytometry…)
--- Vitroplant morphology.
--- Biochemical markers (fluorescent substances): microscopy-fluoremetry
--- Molecular studies (DNA).
2. Introduction of genetic material:
Work steps for a protoplast culture with introduction of new genetic material:
Stage 1: Placing the cells in a concentrated solution of mannitol or sucrose: plasmolysis of the cells and detachment of the protoplasts from the cell walls.
Step 2: Enzymatic digestion of the walls by enzymes: pectinases, celulases, hemicellulases… at specific times and concentrations.
Preparation of protoplast suspension (example of density 104 to 105 cells/ml):
During this stage, it is possible to introduce exogenous genetic material.
Step 3: Culture on regeneration media rich in sugars and containing phytohormones, in particular auxins, for the reconstitution of cell walls.
Then there is formation of microcals.
then somatic embryos (or buds)
Step 4: Culture on suitable media (reduction of auxin content for example) to obtain vitroplants (possibly modified/original plant).
- Examples:
---- Long shelf life tomato: change in ethylene metabolism.
---- Production of rice capable of producing vitamin E: Golden rice.
---- Transformation of a Medicinal Plant: Datura innoxia Mill: for production of secondary metabolites
---- Plants that produce insecticides….
---- Herbicide resistant plants
SEE TRANSGENESIS course.
Quiz 1
QUESTION 1. Les
protoplastes peuvent être obtenus par un traitement avec:-
cocher la réponse juste -
(!Protéases)(!Cellulases)(!Pectinases)(Cellulases et pectinases)
QUESTION 2. Choisir l'information incorrecte concernant les protoplastes - cocher la réponse juste - (!Ils peuvent être obtenus chez les plantes et les bactéries)(!Ils peuvent servir pour transférer les gènes contenus dans les mitochondries et chloroplastes - cytoplasme-)(!Ils doivent être conservés dans un milieu hypertonique pour ne pas les éclater par turgescence)(Les sucres alcools comme le mannitol et le sorbitol, inhibent leur plasmolyse)
QUESTION 3. cybrid is a hybrid carrying(!Cytoplasms of two different plants.)(!Genomes and cytoplasms of two different plants.)(Cytoplasms of two different plants and the genome of one plant.)(!Genomes of two different plants.)(!Cytoplasm of several plants and genomes of two plants.)
QUESTION 4. Choisir l'information FAUSSE concernant un cybride:(!Il est produit par la fusion d'un protoplaste et d'un cytoplaste -cytoplasme sans noyau-)(!Il a des gènes nucléaires d'un seul parent et un cytoplasme des deux parents)(!Il a des plastomes -génomes de palastides- des deux espèces parentales)(Il a le génome fonctionnel résultant de deux espèces)
QUESTION 5. Le nombre de cybrides pouvant résulter de la fusion de deux protoplastes est:(!1)(2)(!3)(!4)
Quiz 2 (QUESTION A CASES VIDES) AU SUJET DES PROTOPLASTES.
Remplir les cases vides par des mots ou nombres convenables (écriture en MAJUSCULES sans espace, sinon, copier et coller les mots proposés.
Pour la vérification des réponses,
remplir toutes les cases, d'abord.
Après clic sur les réponses, les cases à réponses justes restent pleines, les cases à réponses fausses (encore vides) doivent être remplies dans les tentatives suivantes. Le score est comptabilisé comme le nombre de tentatives pour avoir tout juste
Comme les cellules du poireau sont de couleur verte grâce aux chloroplastes
et les cellules du chou rouges sont rouges grâce aux anthocyanes des vacuoles, j'ai opté pour tenter une fusion des protoplastes, bien distingués par leurs couleurs. Ainsi, j'ai commencé à traiter les tssus des deux plantes par CELLULASE + PECTINASE (choix: CELLULASE, PECTINASE, CELLULASE + PECTINASE, PEPTIDASE, GLUCOSIDASE). Les cellules obtenues perdent leurs PAROIS (choix: MEMBRANES PLASMIQUES, GLYCOCALYX, PAROIS, PROTEINES MEMBRANAIRES).
Ces protoplastes doivent être maintenus en PLASMOLYSE
(choix: PLASMOLYSE, TURGESCENCE, ACTIVITE, INACTIVITE)
dans un milieu de culture contenant des SUCRES ALCOOL
(choix: ACIDES AMINES, SUCRES ALCOOL, HORMONES, VITAMINES, NaCl).
La fusion des protoplastes est activée par le PEG
(choix: MANNITOL, SORBITOL, PEG, VITAMINES, GLUCOSE).
Obtenue sous microscope, l'image suivante montre probablement
une fusion de CYTOPLASMES (choix: NOYAUX, CYTOPLASMES, NOYAUX + CYTOPLASMES) des deux espèces de plantes.
LIENS UTILES
Somatic embryogenesis and artificial seeds التطوير الجنيني الجسدي و البذور الاصطناعية
-
Micropropagation et culture in vitro d'espèces végétales
الإكثار الدقيق و الزراعة الزجاجية للأصناف النباتية
- Conformité génétique, variations somaclonales et contraintes
الزراعات الزجاجية. التطابق الوراثي، التغيرات الجسدمستنسخة
Genetic conformity (true-to-type), somaclonal variations and constraints
- Palmier dattier (Phoenix dactylifera L.). Micropropagation in vitro
الزراعة الزجاجية لنخيل التمر
- Vitro-plants. Modifications - تغيرات نواتج الزراعات الزجاجية
- Biotechnologies. QCM
البيوتكنولوجيات. اختبار للمعرفة
- Embryogenèse. Cours
- Vitro-plants du palmier dattier (Phoenix dactylifera L.) obtenus par culture des tissus in vitro
زراعة الأنسجة عند نخيل التمر
- Embryogenèse
somatique che le palmier dattier
- Marquage en vitro-culture