Androgenesis. In vitro culture of haploid plants - الزراعات الزجاجية. النشوء الذكري

Vocabulaire - مفردات - Vocabulary

French	Arabic	English
Cultures in vitro	زراعات زجاجية	in vitro cultures
Culture de protoplastes	زراعة البروتوبلاست	Protoplast culture
Organogenèse	تطوير عضوي	Organogenesis
Embryogenèse somatique	تطوير جنيني جسدي	Embryogenesis
Explant avec bourgeon	مزدرع ب برعم	Explant with bud
Androgenèse	النشوء الذكري	Androgenesis

ANDROGENESIS (Motherless plants).

Androgenesis is the regeneration of whole plants from the culture of male sex cells (immature pollen grains (= microspores)),
- either by culture of isolated pollen,
- either by culture of whole anthers.

The Objective of androgenesis:

The objective of androgenesis and gynogenesis is to obtain doubled haploid plants, (after spontaneous or artificial doubling by colchicine). Thus pure lines are produced in a few months instead of 8 to 10 years by the classic technique of self-fertilization.
Obtaining inbred lines is an almost always necessary step in plant breeding programs. Indeed, homozygosity confers stability and phenotypic uniformity, and thus makes the agronomic behavior of lines predictable. A high homozygosity is required for the registration of new cultivars, and it allows, at the same time, to reduce the frequency of undesirable alleles during the selection phase.

الهدف من النشوء الذكري هو إنتاج نباتات أحادية الصيغة الصبغية، يتم تثنيتها لتعود ثنائية الصيغة. وبالتالي يتم إنتاج السلالات النقية في غضون بضعة أشهر بدلاً من 8 إلى 10 سنوات بواسطة التقنية الكلاسيكية للتخصيب الذاتي.

L'objectif de l'androgenèse est l'btention de plantes haploïdes doublées. Ainsi des lignées pures sont produites en quelques mois au lieu de 8 à 10 ans par technique classique d'autofécondations.

Advantages of the androgenesis:

- Expression of recessive genes that can be masked in the diploid plant (dominance). - Expression and easy identification of mutants.
- Use of androgenesis in plant selection & genetic improvement programs. Thus, different characters can be researched for improvement. For examples:
--- Fruit shape.
--- Early flowering.
--- Resistance to diseases or abiotic stresses (drought, salinity, cold, etc.).
--- Yield.
--- Richness in secondary metabolism compounds.

Disadvantages of Androgenesis by anther Culture:

- Production of genetically different/heterogeneous plants.
- Production of a heterogeneous population at different levels of ploidization.
- The asynchronous development of pollen will lead to the suppression of younger grains by older grains due to the release of toxic substances.
- Plants from anther culture would not be of purely gametophytic origin.

Advantages of androgenesis by pollen cultures over anther cultures

- The pollen culture is a haploid and unicellular system.
- Pollen grains bearing an androgenic response can be isolated using the density gradient centrifugation method.
- Production of a homogeneous population.
- Production of genetically identical plants.
- Pollen grains can be easily modified by exposing them to mutagens or genetic engineering.
- Pollen culture is 60% more efficient than anther culture in terms of embryo production.

Haploid plants are sterile and stunted: they must undergo diploidization (= doubling of the number of chromosomes). Diploid and completely homozygous plants are obtained.

How to double the chromosomes?

- By colchicine:

colchicine

At low concentrations colchicine applied (eg on the terminal bud) makes it possible to double the chromosomes in metaphase.

- By culture of the calluses: spontaneous diploidization after a few weeks, eg in tobacco.

Stages of an androgenic culture

Two scenarios:
-- Formation of calluses, then embryos (indirect embryogenesis), e.g. in rice. -- direct formation of embryos (direct embryogenesis): rapid acquisition of bipolarity: ex. in tobacco.
Note: after passing through callus, there is a possibility of the appearance of chromosomal aberrations.
Importance of anther maturation stage.
- Need for a very precise stage of anther development at the time of culturing: e.g. --- uninucleate microspore stage in grasses.
--- stage of pollen mitosis in tobacco.
To find these stages, one must look for a relationship between the stage of microspore maturation and the morphology of the flower.
The best stages are always before the flower buds open. - Ex: petal length in tobacco.
- Position of the ear in the sheath in wheat (size of the ear at 2/3 of the sheath of the leaf ex wheat).
Possibility of verification under the microscope: staining of the cells with carmine.

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Stages of an androgenic culture (continued)

Importance of genotypes
The androgenesis capacities of each species depend on the genotypes used (barley, pepper, wheat, etc.).
Action of External Factors.
-- Anther Pre-Treatment
----- by cold: Ex in Nicotiana tabacum
----- By an osmotic shock: Ex of barley anthers:
pre-treatment with different concentrations of mannitol, sorbitol and sucrose or PEG (poly ethylene glycol).
-- Culture media. In general, culture media rich in auxins are required.
----- Use of 2.4 D at a rate of 0.5 to 2 mg/l, generally in association with a cytokinin (in particular BAP and Kinetin).
----- The media must be rich in sugars: eg 80 to 120 g/l (= osmotic shock which favors the development of haploid tissues compared to diploid tissues).
----- Additives: Activated carbon (to prevent oxidation of phenolic compounds), L glutamine…
Preparation and Culture of microspores isolated in suspension (on liquid media)

Stages of culture of isolated microspores.
- Collection of flower buds and pre-treatment in cold temperatures at 4°C for 3 to 5 days.
- Sampling of the anthers and culturing them for one week (preculture of the anthers) on agar medium.
- Grinding of the anthers.
- Filtration and centrifugation of the ground material to isolate the microspores.
- Culture of microspores in a liquid medium enriched with glutamine; calluses then androgenetic embryos.

QUIZ

QUESTION 1. Which one of the following is used for the in vitro development of haploid plants?(!Embryogenesis)(Androgenesis)(!Organogenesis)(!Microcutting)

QUESTION 2. En culture in vitro des plantes, la création rapide de lignées pures homozygotes est obtenue par:(!Embryogenèse somatique)(Androgenèse)(!Organogenèse)(!Microbouturage)(!Culture de cellules isolées)

QUESTION 3. Choisir un avantage de l'androgenèse parmi les propositions suivantes:(!Embryogenesis)(Expression des gènes récessifs masqués par la dominance)(!Multiplication conforme)(!Assainissement des plantes)(!Formation de graines artificielles)

QUESTION 4. Parmi les propositions suivantes, Choisir celle représentant un avantage de l'androgenèse via la culture de microspores par comparaison à la culture d'anthères :(!Production d'une population hétérogène de plantes à différents niveaux de ploïdisation.)(Production d'une population homogène de plantes au même niveaux de ploïdisation.)(!Les plantes issues de cette androgenèse ne seraient pas d'origine purement gamétophytique.)(!Méthode de culture engageant uniquement la formation d'embryons via une callogenèse)

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