EXERCISE 9

SOMATIC EMBRYOGENESIS

Somatic embryogenesis (SE) is a means by which plants can regenerate bipolar structures from a somatic
cell. During the process of cell differentiation, the explant responds to endogenous stimuli, which trigger
the induction of a signaling response and, consequently, modify the gene program of the cell.

The SE process also occurs in nature. For example, under certain environmental conditions such as heat
and drought, the plant Kalanchoe produces, around their leaves, small bipolar structures, which develop
later into plantlets. Fig. 1

Figure 1. Kalanchoe leaves with the plantlets.

Once the somatic cells are induced to generate cells with embryogenic capacity, the new cells can form
structures capable of regenerating a complete plant. In the case of Zygote embryogenesis (ZE) an
asymmetric cell division occurs, whereas in SE this is often not observed. Once the somatic embryos reach
the cotyledonary stage, they initiate a shoot meristem, and seedling growth begins.

Observe somatic embryogenesis on the leaves of a succulent species for about 3 weeks. Draw the
regeneration of a plant at the base of the leaves and label the parts.
Plant Tissue Culture

Tissue culture is the in vitro aseptic culture of cells, tissues, organs or whole plant under controlled
nutritional and environmental conditions often to produce the clones of plants. The resultant clones are
true-to type of the selected genotype. The controlled conditions provide the culture an environment
conducive for their growth and multiplication. These conditions include proper supply of nutrients, pH
medium, adequate temperature and proper gaseous and liquid environment.

Plant tissue culture technology is being widely used for large scale plant multiplication. Apart from their
use as a tool of research, plant tissue culture techniques have in recent years, become of major industrial
importance in the area of plant propagation, disease elimination, plant improvement and production of
secondary metabolites. Small pieces of tissue (named explants) can be used to produce hundreds and
thousands of plants in a continuous process. A single explant can be multiplied into several thousand
plants in relatively short time period and space under controlled conditions, irrespective of the season
and weather on a year round basis. Endangered, threatened and rare species have successfully been
grown and conserved by micropropagation because of high coefficient of multiplication and small
demands on number of initial plants and space.
Watch the video on the basics of plant tissue culture.
https://www.youtube.com/watch?v=OFHipYNDVGE

Micropropagation
Micropropagation is the tissue culture technique used for rapid vegetative multiplication of
ornamental plants and fruit trees. It starts with the selection of plant tissues (explant) from a healthy,
vigorous mother plant. Any part of the plant (leaf, apical meristem, bud and root) can be used as explant.
The whole process can be summarized into the following stages as shown in Figure 2.

Figure 2. Flow chart summarizing tissue culture experiments.

Stages in Micropropagation
Stage 0: Preparation of donor plant
Any plant tissue can be introduced in vitro. To enhance the probability of success, the mother plant should
be ex vitro cultivated under optimal conditions to minimize contamination in the in vitro culture [16].

Stage I: Initiation stage

In this stage an explant is surface sterilized and transferred into nutrient medium. Generally, the combined
application of bactericide and fungicide products is suggested. The selection of products depends on the
type of explant to be introduced. The surface sterilization of explant in chemical solutions is an important
step to remove contaminants with minimal damage to plant cells. The most commonly used disinfectants
are sodium hypochlorite, calcium hypochlorite, ethanol and mercuric chloride (HgCl2). The cultures are
incubated in growth chamber either under light or dark conditions according to the method of
propagation.

Stage II: Multiplication stage

The aim of this phase is to increase the number of propagules. The number of propagules is multiplied by
repeated subcultures until the desired (or planned) number of plants is attained.

Stage III: Rooting stage

The rooting stage may occur simultaneously in the same culture media used for multiplication of the
explants. However, in some cases it is necessary to change media, including nutritional modification and
growth regulator composition to induce rooting and the development of strong root growth.

Stage IV: Acclimatization Stage

At this stage, the in vitro plants are weaned and hardened. Hardening is done gradually from high to low
humidity and from low light intensity to high light intensity. The plants are then transferred to an
appropriate substrate (sand, peat, compost etc.) and gradually hardened under greenhouse.

GROUP OUTPUT:

Read the article:

Micropropagation from Inflorescence Nodal Segments of Phalaenopsis and Acclimatization of Plantlets

Using Different Substrates https://www.mdpi.com/2311-7524/8/4/340

Create a schematic diagram of the micropropagation of Phalaenopsis to show the crucial stage of the
process.
Answer the following questions:

1. What are some of the plants that we might use for tissue culture?
Plants that are used for tissue culture can include but are not limited to plantains like banana, yam, sweet
potato, pineapple, sugar cane, grapes among others. These are crops that are typically of high agricultural
value especially in developing regions such as Africa and Southeast Asia (Hasnain et al., 2022).

2. Why is tissue culture used for propagation of some plants rather than just planting seeds?
Tissue culture is a preferred method for propagation for some plants due to its aseptic techniques in
growing as well as cultivating sterile environments for growth. These types of plant propagation prevent
the infection of pathogens like viruses, bacteria, and fungi that can hinder the development of healthy
plant individuals especially in seed growth. Tissue culture plants are also characterized by the product’s
uniformity such that of the Cavendish banana where its production results in a consistent appearance
which is appealing to commercial growers and distributors (Gulzar, et al., 2020).

3. What is a sterile environment? Why is a sterile environment important in tissue culture?

A sterile environment is an environment with the complete absence of any living organisms including
bacteria, fungi, yeast, and their spores (Walleser, 2020). Maintaining a sterile environment in tissue
culture is important because any contamination would lead to errors, inconsistencies to cell lines or
cultures, and may interfere with the overall result of the experiment.