Cultura de Células e Tecidos

2018/2019

Módulo 3. Cultura de Células e Tecidos Vegetais

Plant Cell and Tissue Culture

Overview

1. Key concepts
2. Types of plant tissue culture
3. Pathways of plant regeneration
4. Culture media and plant growth regulators

Bibliography:
George et al. (eds) 2008 Plant Propagation by Tissue Culture. 3rd ed. Springer
Smith R.H. 2013 Plant Tissue Culture, Techniques and Experiments. 3rd ed. Elsevier
Plant Cell and Tissue Culture

Range of techniques that allows the growth of plant parts (cells,

tissues, organs) in vitro under conditions of asepsis in a medium
with nutrients and phytohormones and control of abiotic factors
such as light, temperature, oxygen and carbon dioxide
Plant Cell and Tissue Culture

1756 - First observation of callus formation, by Henri-Louis Duhumel du

Monceau

1838-1839 - Cell theory (Schleiden, 1838 and Schwann, 1839) holding

that the cell is the unit of structure and function in an organism
and therefore capable of autonomy

1878 – Vöchting showed the limits of divisibility of plant segments and

formation of callus

1902 - Haberlandt proposed the concept of in vitro cell culture

Objectives and applications
• Mass propagation of uniform plant material
• Production of disease-resistant plants
• Production of virus-free plants
• Embryo-rescue
• Safe transfer of germplasm
• Production of haploid plants
• Plant morphogenesis
• Secondary products
• Plant genetic engineering
 Production of virus-free plants

• Virus infection can result in lack of

plant vigour, loss in yields, necrosis
of leaf tissue, undesirable streaks in
flower petals, and twisted or
contorted leaves.

• The shoot apex or meristem tissue of

rapidly growing plants usually does
not contain the virus.

• Virus-free tissues in the shoot

meristem of a plant can be cultured
in vitro to develop into virus-free
plants.
 Embryo-rescue

• Plant breeding and development

programmes make use of plant
crosses, or hybrids.

• When crosses are wide it may be

that various incompatibility
problems will cause the developing
embryo to begin to die (abort) if it is
left on the plant.

• Using plant cell culture techniques

the embryo can be rescued from the
plant and cultured on a nutrient
medium in vitro. Often this hybrid
embryo will continue its
development in vitro into a plant.
Safe transfer of germplasm

• A concern with the transportation of

whole plants across international borders
is that the plants or soil adhering to the
root system will contain insects, bacteria
or fungi that will cause disease problems
at the new location of the plant.

• An alternative to transporting whole

plants is the use of plant cell cultures.
These can be transported relatively easily
and can bypass lengthy quarantine
procedures for moving plant material.
 Production of haploid plants
• Many breeding improvement programmes
require homozygous diploid plant
material. To develop this plant material
normally requires many years of
backcrossing.

• Using cell culture, haploid and

homozygous diploid plants can be derived
in only a few months.

• Anther culture is used in plant breeding

improvement programmes, particularly in
cereal breeding programmes, to obtain
haploid plants.
Plant morphogenesis
• Plant cell culture techniques has been used extensively to better our
understanding of plant morphogenesis – the study of inception and
development of plant form from cell to plant structure.

• Current studies focus on defining the medium and growth conditions to

regenerate whole plants from callus or cell cultures.
 Secondary products
• Many plant secondary
metabolites have
widespread interest for
production of
pharmaceuticals, valuable
metabolites, flavouring
compounds and natural
products.

• In many cases cultivation

is difficult or the plant
material is unreliable.

• Plant tissue culture can

provide controlled year-
round production of the
compound.
 Plant genetic engineering
• Insert foreign DNA into plant cells
and regenerate whole plants that
have the foreign DNA integrated
into the plant genomic DNA.

• This new DNA will then be

expressed and inherited by the
progeny from that plant.

• Genetically altered crop plants

contain foreign DNA or genes for
insect or herbicide resistance.
 Micropropagation
• Plant tissue culture technique used for
producing plantlets

• Implies the culture of aseptic small sections of

tissues and organs in vessels with defined
culture medium and under controlled
environmental conditions.

• Important tool for both fundamental research

and commercial applications.

• Almost all plant biotechnology methodologies

ultimately require the successful culture of
plants cells, tissues or organs.

• Many examples: ferns, woody trees, vines,

shrubs, conifers, vegetables, grasses, date and
oil palms, and bananas.
Micropropagation: advantages

• Clonal propagation
• The plants are often more uniform
• May be the only way to propagate vegetatively
• Plant growth is generally fast
• Plants may mature faster than those propagated by seeds
• Speed of propagation: high productivity
• Axenic: provided that the original explant is free of contaminants, the
resulting plants will all be uncontaminated
• Programming
• Large quantities in a small space
Micropropagation: disadvantages

• Requires a clean environment

• Requires equipments and facilities for intensive operation and well-trained
personnel
• Technical expertise in management positions
• Protocols not optimized for all species
• Propagules may be expensive
• The regeneration may not be possible; especially for adult hardwood
trees; obtaining roots is more problematic than shoots
• Plants may not have an uniform growth and instead have different growth
rates and maturation in vitro
The micropropagation laboratory
Micropropagation equipment

• Dionizer
• Balance (analytical or other)
• pH meter
• Filter-sterilizer
• Autoclave
• Laminar flow chamber
• Incubator
• Plant growth chamber
Key factors for sucessful PTC

Growth medium
Minerals, growth factors, carbon source, hormones

Environmental factors
Light, temperature, photoperiod, sterility

Explant Source
Different species show differences in amenability to tissue culture.
In many cases, different genotypes within a species will have
variable responses to tissue culture.
 Growth medium

Shoot tip: phytohormones

An explant, such as an axillary bud, is (auxins and gibberellins)
removed from the sources of many
chemicals and these have to re-supplied
to allow growth

Roots: water, vitamins, mineral salts

and phytohormones (cytokinins)
Growth medium

• Inorganic salt formulations

• Source of carbohydrates
• Vitamins
• Water
• Plant hormones: auxins, gibberelins, cytokinins
• Solidifying agents (agar)
• Undefined supplements
Environmental factors

In vitro plant growth conditions:

Stoppered vessels
Culture room:
Low light intensity (±30-50 µmol/m2/s)
Temperature: 22-24 ºC
Photoperiod: 8:16 h day/night
Axenic
 Explant

Fragment of plant tissue obtained from a

plant and will be propagated to obtain a
new plant.

The explants best suited for

micropropagation naturally contain
meristmatic tissue cells.

Leaves are also used (the younger, the

better), as well as root-tips.
 Sources of explants
• Shoot tips, axillary buds, seeds, hypocotyl
(from germinated seed), leaves

• Desirable properties of an explant:

• Easily disinfected
• Juvenile
• Responsive to culture
Axillary bud

• The choice of explant conditions determines

the degree of success in micropropagation
and their source should be carefully chosen

• The explants should be from young adult

plants, preferably in areas of active growth
Cellular differentiation
Cellular dedifferentiation
Dedifferentiation
Differentiation andand differentiation
dedifferentiation
 Totipotency

The potential or inherent capacity of a plant cell to develop into

an entire plant if suitably stimulated
Basic steps in Plant Tissue Culture

Preparation Disinfection Manipulation of

media of the explant tissues

Storage Growth of tissues

Cryo-preservation

Regeneration of
Transfer to soil
Plants
 Basic steps in Plant Tissue Culture

1. Selection/disinfection of explant
2. Initiation stage
3. Multiplication stage
4. Rooting
5. Acclimatization
 Basic steps in Plant Tissue Culture

In vitro

1. Selection/disinfection of explant
2. Initiation stage
3. Multiplication stage
4. Rooting Not necessary in SE

5. Acclimatization

Ex vitro
Key concepts

Asepsis Without infection or contaminating microorganisms.

Aseptic Technique Procedures used to prevent the introduction of fungi, bacteria,

viruses, mycoplasma, or other microorganisms into cell, tissue, and organ
cultures. Although these procedures are used to prevent microbial contamination
of cultures, they also prevent cross contamination of cell cultures.

Axenic Culture A culture without foreign or undesired life forms. An axenic

culture may include the purposeful cocultivation of different types of cells,
tissues, or organisms.

Callus An unorganized, proliferative mass of differentiated plant cells; a wound

response.

Cell Culture Maintenance or cultivation of cells in vitro, including culture of single

cells. In cell cultures, the cells are no longer organized into tissues.
Key concepts

Cell Line The product of the first successful subculture of a primary culture.
Cultures from a cell line consist of lineages of cells originally present in the
primary culture.

Cell Strain Strain derived from either a primary culture or a cell line by the
selection or cloning of cells having specific properties or markers.

Clonal Propagation Asexual reproduction of plants, the results of which are

considered to be genetically uniform and originated from a single individual or
explant.

Clone In plant culture terminology, the term may refer to a group of plants
propagated by only vegetative and asexual means, all members of which have
been derived by repeated propagation from a single individual.

Cryopreservation Ultralow temperature storage of cells, tissues, embryos, or

seeds. This storage is usually carried out using temperatures below −100°C.
Key concepts

Differentiated Cells that maintain, in culture, all or much of the specialized

structure and function typical of the cell type in vivo.

Embryogenesis The process of embryo initiation and development.

Explant Tissue taken from its original site and transferred to an artificial medium
for growth or maintenance.

In Vitro Propagation Propagation of plants in a controlled, artificial environment,

using plastic or glass culture vessels, aseptic techniques, and a defined growing
medium.

Meristem Culture In vitro culture of a generally shiny, dome-like structure

measuring less than 0.1 mm in length when excised, most often excised from the
shoot apex.

Micropropagation In vitro clonal propagation of plants from shoot tips or nodal

explants, usually with an accelerated proliferation of shoots during subcultures.
Key concepts

Morphogenesis (1) The evolution of a structure from an undifferentiated to a

differentiated state. (2) The growth and development of differentiated structures

Organogenesis In plant tissue culture, a process of differentiation by which plant

organs are formed de novo or from preexisting structures. In developmental
biology, this term refers to differentiation of an organ system from stem or
precursor cells.

Regeneration In plant cultures, a morphogenetic response to a stimulus that

results in the production of organs, embryos, or entire plants.

Somatic Embryogenesis In plant culture, the process of embryo initiation and

development from vegetative or nongametic cells.
Key concepts

Type I Callus A type of adventive embryogenesis found with gramineous

monocotyledons, induced on an explant where the somatic embryos are arrested
at the coleptilar or scutellar stage of embryogeny. The embryos are often fused
together, especially at the coleorhizal end of the embryo axis. The tissue can be
subcultured and maintains this morphology.

Type II Callus A type of adventive embryogenesis found with gramineous

monocotyledons, induced on an explant where the somatic embryos are arrested
at the globular stage of embryogeny. The globular embryos often arise
individually from a common base. The tissue can be subcultured and maintains
this morphology.

Undifferentiated With plant cells, existing in a state of cell development

characterized by isodiametric cell shape, very little or no vacuole, and a large
nucleus and exemplified by the cells in an apical meristem or embryo.

Vegetative Propagation Reproduction or plants by a nonsexual process involving

the culture of plant parts, such as stem and leaf cuttings.