A Primer on Plant Cell & Tissue Culture

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Review this Website for basic Information on “Plant Tissue Culture:

http://perso.wanadoo.fr/technivit/in%20vitro%20plant%20tissue%20culture.htm

1] The material used to inoculate a culture is called the “Explant”. It could be a single cell, a chunk of tissue, an embryo, a seed, a shoot tip or a root tip.

2] A Single Cell Culture is the culture of a single cell.

3] A Cell Suspension Culture is composed of cells and cell clusters usually suspended in a liquid medium.

4] A Tissue (Callus) Culture is an undifferentiated, proliferating mass of parenchyma cells usually on agar or another “solid” substrate.

5] An Organ Culture is the growth of an Embryo, Shoot Tip, Root Tip or other organ in an organized manner. In other words, a root culture would involve taking a root tip and having it grow into a long root in vitro. A shoot tip would produce a shoot and an embryo would produce a whole plant.

The choice of the Explant is the most critical part of any attempt to get cells to grow in vitro.

The smaller and less organized an Explant becomes, the more difficult it is to establish in culture (in vitro).

The culture of single cells and their growth into organized structures is extremely hard to do today!

The ability of Explants to grow and regenerate in vitro is partly Genetic in that species from certain families (Apiaceae & Solanaceae) have been easy to culture while others, especially many monocots, have been difficult (recalcitrant).

The Developmental Status of the Explant is also very important. Explants that contain actively dividing (Meristematic) cells are the easiest to culture.

Embryos and Shoot Apical Meristems are good explants if you want to regenerate whole plants or shoots that can be rooted. Root Apical Meristem cultures produce roots but the roots usually can’t produce shoots.

Storage Tissues are good explants if you want to form a callus culture. A Callus is a mass of proliferating cells that are largely parenchymatous and do not resemble any differentiated plant organ like a root, a leaf or an embryo.

Callus cultures can sometimes be stimulated to form organs or embryos, however.

Callus cultures are often used to create liquid suspension cultures. The latter can regenerate tissues, organs & embryos under certain conditions.

Suspension Cultures are often used to make Protoplasts.

Protoplasts are cultured plant cells which have had their cell walls enzymatically removed. Protoplasts are often used for genetic engineering because it is relatively easy to introduce foreign DNA into them. Some protoplasts can regenerate complete plants.

Differentiation a la Salvador Webi

The Benchmark for Undifferentiated Plant Cells is the Meristematic Cell. The Zygote is one example. Cells in Shoot and Root Apical Meristems are very good examples.

Meristematic cells have thin cell walls, are Isodiametric, Densely Cytoplasmic (microvacuolate), with a large Nuclear:Cytoplasm Ratio. Plastids are in the form of minute Proplastids. They actively synthesize DNA and divide frequently.

It should be easy to generate Callus Cultures from Meristematic Tissues.

Isolated Shoot Apical Meristems and Root Apical Meristems have been cultured. Root tips were used to make the first organ cultures.

Shoot Tips and Shoot Apical Meristems have been used to propagate disease-free plants. The principal goal of this work was to avoid callus formation and generate a normal Shoot that could be rooted later.

Cell Differentiation involves changes in the extent and type of Cell Wall formation that occurs, cell shape, vacuolation and plastid development.

Epidermis & Ground Tissues

Parenchyma cells have thin primary cell walls, large vacuoles and well developed plastids (Chloroplasts, Chromoplasts or Amyloplasts). They have a variety of shapes and do not divide once they mature BUT they retain the ability to divide in response to a stimulus like wounding or the application of plant growth regulators (PGRs) like Auxin & Cytokinin.

Parenchyma Tissues are used to establish callus and suspension cultures. This involves wounding and the application of PGRs in the culture medium.

Epidermal cells have many traits in common with Parenchyma. However, they do not have well developed plastids and secrete a cuticle. Guard cells do have well developed plastids and they have elaborate cell wall elaborations which have functional significance.

Epidermal Tissues are not routinely used to establish plant cell or tissue cultures BUT they have been used for this purpose occasionally. Trichomes are highly differentiated epidermal cells. It would be difficult to start cultures using these as explants. Guard cells would also be unlikely to produce cultures.

Collenchyma cells have unevenly thick, unlignified cell walls. Their plastids are not prominent but they have a large central vacuole. They are typically elongate and narrow. They do not divide after they mature. It is unclear as whether or not they can divide like parenchyma.

Sclerenchyma cells have thick, lignified secondary walls and poorly developed plastids. They can have many shapes. Fibers are highly elongate. Sclerenchyma cells are not dead at maturity BUT are probably unlikely to divide in vivo or in vitro.

Parenchyma, Collenchyma and Sclerenchyma are typically called Ground Tissues. Parenchyma and Sclerenchyma can be found in the Vascular Tissues (Phloem & Xylem).

Vascular Tissues

Sieve Tube Elements, Sieve Cells, Tracheids and Vessel Members are enucleate and are unlikely to divide after maturation. Vessel Members are DEAD!

Vascular Tissue is not likely to produce cell or tissue cultures BUT both contain Parenchyma cells which might divide in vivo or in vitro.

Plant Regeneration in vitro

Complete plants can form via Organogenesis or Embryogenesis.

Organogenesis typically involves the production of shoots which are later induced to form roots.

Embryogenesis involves the production of embryos that resemble zygotic embryos. These are induced to form seedling-like organisms which produce complete plants.

Embryogenesis is the preferred process for many reasons!