Plant regeneration pathways

Plant regeneration by micropropagation of plants can be divided into three

types that differ depending on the starting material and the type of response
obtained:

Existing meristems
Microcutting or nodal sectioning: uses pre-existing meristematic cells to
regenerate the whole plant (‘Cultura de segmentos nodais’)

Organogenesis
Relies on the production of organs either directly from an explant or callus
structure (de novo)

Somatic embryogenesis
Embryo-like structures which can develop into whole plants are formed from
somatic cells, in a way similar to how zygotic embryos are formed
 Organogenesis in vitro
• Process during which organs (buds, roots and shoots)
can be produced de novo from explants under certain
physical and chemical conditions

• Vascular system is in connection with the tissues of

origin

• Unlike microcutting, the meristems are induced de

novo

• Applications:
– plant modification and improvement
– clonal propagation
– germplasm storage
– study of regulatory mechanisms of plant development
 Organogenesis in vitro
Plant regeneration via organogenesis involves the differentiation of
adventitious meristems (meristemoids) into organs by altering the
concentration of plant growth hormones in nutrient medium.

Indirect organogenesis

Direct organogenesis
Embryogenesis
• In flowering plants, the process of double fertilization involves a haploid
sperm fertilizing a haploid egg cell to form a diploid zygote
• The zygote undergoes a series of morphological, biochemical, and
molecular events to develop into an embryo
Zygotic versus somatic embryogenesis
Zygotic Somatic

Main diferences:
• Absence of a suspensor
• Absence of seed coat
• Vascular independence from initial explant
 Somatic embryogenesis (SE)
• Process by which somatic cells or tissues develop into differentiated
somatic embryos (bipolar structure, resembling a zygotic embryo)

• These somatic embryos can develop into whole plants without undergoing
the process of sexual fertilization as done by zygotic embryos

• in vitro method of plant regeneration widely used as an important

biotechnological tool for sustained clonal propagation

Bipolar structure
 Somatic embryogenesis (SE)

• The plant can be derived from a single somatic cell or a group of somatic
cells.

• All the plantlets produced have the same genetic makeup.

• Combined with genetic engineering, micropropagation through somatic

embryogenesis provides an efficient means of producing a large number of
elite or transgenic plants
Stages of somatic embryogenesis
SE is a complex process comprising three main stages:
a) induction: tissues acquire (direct or indirectly) embryogenic
competence Auxins/cytokinins
b) expression: competent cells develop into somatic embryo structures:
Proliferation
Histo-differentiation No plant growth regulators
Maturation
Germination/conversion

c) acclimatization: transfer to ex vitro conditions

 Direct vs indirect somatic embryogenesis
Direct SE: production of somatic embryos from the
explant cells called pre-embryogenic determined
cells

Preembryogenic determined cells are already

destined for embryogenic development prior to
explanting, requiring only growth regulators or
favorable conditions to enter cell division.

Indirect SE: production of somatic embryos from

Indirect
Direct

induced embryogenic determined cells in

unorganized callus
Direct somatic embryogenesis
 Desinfection Induction Expression Aclimatization
Light

Elongation
Inoculation
Induction
MS + 3 mg/l NAA (Dark)

Medium without growth

regulators (MSWH)

Globular Embryo Repetetive SE

Aclimatization
NAA
SE PROTOCOL STANDARD

Cotyledonar Embryo
Expression

Germination

Light

Plants
Application of SE
 Cryopreservation
• Long-term storage of somatic embryos through cryopreservation (-196 °C) while
being field tested.
• Flexibility to rapidly deploy suitable clones, under changing breeding goals and/or
environmental conditions

• Development of clonal
somatic seedlings

• Amenability to
process scale up-
bioreactor
 Basic steps in Plant Tissue Culture

In vitro

1. Selection/disinfection of explant
2. Initiation stage
3. Multiplication stage
4. Rooting
5. Acclimatization

Ex vitro
Step I: selection and disinfection

• Selection of the plant tissue (explant)

from a healthy vigorous ‘mother plant’
- this is often the apical bud, but can
be other tissue.

• This tissue must be disinfected to

remove microbial contaminants.
 Step I: selection and disinfection
• Bacteria and fungi will overgrow the explant on the medium unless they
are removed.

• Pre-treatments to clean up the explant

Transfer plants to a greenhouse to reduce endemic contaminants
Force outgrowth of axillary buds
Remove endemic surface contaminants by washing

• Example of desinfection protocol:

Etanol 70% (v/v) 30 s
NaOCl 20% (v/v) + TEEPOL 0.01% 15 min
Sterilized water
Culture medium

• Important factors:
Concentration of the desinfection agent
Time of desinfection
Species-dependent
Step II: Initiation

• Establishment of the explant in a

culture medium

• The medium sustains the plant cells

and encourages cell division

• Culture medium: solid or liquid

• Each plant species/genotype has

particular medium requirements that
must be established by trial and error
Step II: Multiplication
• The explant gives rise to a callus
which is manipulated by varying:
- sugar concentrations
- auxin: cytokinin ratios

• The callus may be subdivided a

number of times

• Light and temperature are critical

 Step II: Rooting

• The shoots develop roots in the

multiplication medium

• The shoots are transferred to a

growth medium with relatively
high auxin concentration
Step II: Acclimatization
• The transfer of the plantlets from
the in vitro to the ex vitro
conditions

• Tissue culture plants moved to a

nursery and potted up
 Culture Media
• In vitro plant cells, tissues and organ cultures are not fully autotrophic

• Need carbohydrates for:

- maintaining the osmotic potential
- obtaining energy and carbon sources for developmental processes
(shoot proliferation, root induction and emission, embryogenesis
and organogenesis) which are highly energy demanding
developmental processes in plant biology
- the plant tissue culture media should contain the nutrients required
by the whole plant

• Culture media are largely responsible for the success/insuccess of in vitro

growth and morphogenesis of plant tissues
 Culture Media: components
• Plant tissue culture media should generally contain some or Shoot tip - Auxins
all of the following components: and Gibberellins
Macronutrients (> 0.5 mM)
Micronutrients (< 0.5 mM)
Vitamins
Amino acids or nitrogen supplements Leaves -
Source(s) of carbon
sugars, GAs
Plant Growth Regulators (phytohormones)
Solidifying agents

• The optimum concentration of each nutrient for achieving Roots - water,

maximum growth rates varies between species vitamins
mineral salts and
cytokinins
• Sucrose is most widely used transport-sugar
in the phloem sap of many plants.
Sterilization (by autoclaving) of the
medium, hydrolyses sucrose to glucose and
fructose
 Culture Media

The composition of the culture media is primarily

dependent on:
• the plant species
• type of material used for culture i.e. cells,
tissues, organs, protoplasts

Medium
White´s The earliest
MS Formulated for organogenesis, the
most widely used
B5 Cell suspension/callus
Nitsh´s Anthers
Culture Media: types
MS: Murashige and Skoog
G5: Gamborg et al.
W: White
LM: Lloyd and McCown
VW: Vacin and Went
Km: Kudson modified
M: Mitra et al.
NN: Nitsch and Nitsch
Culture Media: composition
Culture Media: composition
Culture Media: composition
Culture Media: composition
Plant Growth Regulators
• ‘Plant hormones’ or ‘phytohormones’

• Numerous substances that profoundly

influence the growth and differentiation of
plant cells, tissues and organs

• Also function as chemical messengers for

intercellular communication
Plant Growth Regulators
Plant Growth Regulators
Auxins

IAA is the only natural auxin

NAA (2,4-D naphthalene-acetic acid)

Cytokinins

BAP (6-benzyloaminopurine)

2iP (6dimethylaminopurine)

Kinetin (N-2-furanylmethyl-1H-purine-
6-amine)

Zeatin (6-4hydroxy-3-methyl-trans-2-
butenylaminopurine).

Zeatin and 2iP are naturally occurring

cytokinins
Plant Growth Regulators
Plant Growth Regulators
Physical factors
Physical factors
 Plant uniformity: somaclonal variation
• Somaclonal variation: variation seen in plants that have been produced by PTC,
particularly common in plants regenerated from callus

• Variations can be genotypic or phenotypic, which in the latter case can be either
genetic or epigenetic in origin

• Typical genetic alterations:

- changes in chromosome numbers (polyploidy and aneuploidy)
- chromosome structure (translocations, deletions, insertions, duplications)
- DNA sequence (base mutations)

• It is a valuable tool in plant breeding - variation in plants regenerated from

somatic cells can be used in the development of crops with novel traits

SOMACLONAL VARIATION OF CHRYSANTHEMUM

PROPAGATED in vitro FROM DIFFERENT EXPLANTS TYPES
Genetic stability

Regeneration pathways can be ranked in order from high to low in

terms of genetic stability:
1. micropropagation by preformed structures (nodal explants)
2. adventitiously derived shoots (direct organogenesis)
3. somatic embryogenesis
4. organogenesis from callus, cell and protoplast cultures
Summary: What is needed for PTC?

• Appropriate tissue (some tissues are better than others)

• A suitable growth medium
• Aseptic conditions
• Growth regulators
• Frequent subculturing
Main applications
As an emerging technology, the plant tissue culture has a great impact on both
agriculture and industry, through providing plants needed to meet the ever increasing
world demand.

It has made significant contributions to the advancement of agricultural sciences in

recent times and today they constitute an indispensable tool in modern agriculture.

• Production of improved crop varieties

• Production of disease-free plants (virus)
• Genetic transformation
• Production of secondary metabolites
• Production of varieties tolerant to salinity, drought and heat stresses

Alternatives to production of medicinal compounds from plants, biotechnological

approaches, PTC have potential as a supplement to traditional agriculture in the
industrial production of bioactive plant metabolites.

A number of medicinally important alkaloids, anticancer drugs, recombinant proteins

and food additives are produced in various cultures of plant cell and tissues.