• Embryo culture is also known as embryogenesis/ embryo rescue.
• Growth of a species during its embryonic stage in artificial culture medium (in-vitro) with aseptic conditions.
• First in 18^th century by Charles bonnet in Phaseolus and Fagopyrum
• First successful embryo culture inside laboratory performed in 1904, by Hanning
• The fusion of anther(pollen) and ovary (ovule).
• Formation of a zygotic embryo.
• The zygotic embryo germinates and leads to the formation of the whole plant.
• The two embryonic leaves are known ad
• Cotyledons are attached to the embryonal axis.
• The shoot of an embryo is called plumule.
• The root of an embryo is called the radicle
• The axis part above radical is known as hypocotyl.
• The part present below plumule is called epicotyl.
• The embryo grows asymmetrically (asymmetric division).

Anther (pollen)                 *                  ovary (ovule)

Zygote (zygotic embryo)

Stages/methodology:

1. Explant/mother plant collection:

• for the culture of embryo’s, the best explant taken is the seed.
• Take a seed àimbibition (soaked in water)à the seed became swell à perform the surface sterilization
• For sterilization, there are several methods that can be used such as laminar airflow, fungicides, detergents, heat sterilization.

2. Establishment:

In the process of Establishment:

• Media preparation
• Sterilization of the media
• Inoculate the zygotic embryo (cytokinin > auxin)

3. On culture medium:

• In this process, we have to prepare two types of culture medium for:
• Shoot induction
• Callus formation
• for culture media most commonly used medium is:
• Modified MS medium (Murashige and Skoog, 1962)
• Additional coco water, yeast extract, tomato juice.

Nutrient requirement:

• Carbohydrates: the best carbohydrate, the carbon source is sucrose.
• Vitamins
• Mineral salts: used in MS medium such as MgSO4, CaCl2.
• Growth regulators: auxins/cytokinin’s
• pH 5-7.5
• Temperature 25-30 degree for 14-30 days.

Cell division

Two cell stage:

• Zygotic embryo divides asymmetrically.
• It divides into two cells

Terminal cell and basal cell.

• Terminal cell/apical cell contains most of the cytoplasm
• Terminal cells give rise to Hypocotyl, shoot apical meristem, and cotyledons.

Basal cell

• Basal cell is large contain large vacuole
• Basal cell give rise to Hypophysis and suspensor

Eight cell stage –> sixteen cell stage –> globular stage

Globular stage:

• spherical or globular shaped.
• Give rise to the embryo axis.

Heart stage:

Known as the cotyledon stage.

These rise to torpedo stage embryo

Heart and torpedo stage of seed germination form apical meristem, radical, cotyledons, and hypocotyl.

Mature stage:

• Fully formed embryo, separate cotyledons.
• Suspensor and basal cells start disappearing.

4. Shoot multiplication:

For shoot multiplication, the amount of cytokinin should be > auxin

5. Rooting:

The use of Auxin leads to rooting.

6. Transplant:

After keeping a fully developed embryo, it needs to be transplanted into the field in favorable conditions.

Advantages:

• Recovery of Endangered plant species.
• Propagation of rare hybrids e.g., lilies
• Some seeds are infertile, we can use those in embryo culture (dormancy overcome).
• It reduces the breeding cycle for plants.
• Study of embryo and embryo development.
• Mass production of plants.
• To study seed dormancy and germination.

Disadvantages:

• Difficulty in genetic modification