PLANT BIOTECHNOLOGY

2016-2017 (UC 42314)

Glória Catarina Pinto: gpinto@ua.pt
MOD 4_ 26/10/16
 Micropropagation

Initial explants

Type of final response MOD 4

Propagation of existing meristems Somatic embryogenesis

MOD 2/3
Organogenesis
MOD 3
Embryogenesis
 In flowering plants, the process of double fertilization involves
a haploid sperm fertilizing a haploid egg cell to form a diploid
zygote.
 Subsequently, the zygote undergoes a series of morphological,
biochemical, and molecular events to develop into an embryo.
SOMATIC EMBRYOGENESIS

 Somatic embryogenesis has been defined as a non-sexual developmental

process that produces a bipolar embryo (presenting both shoot and root
meristems) from somatic tissue (Merkle et al. 1995, Dodeman et al.
1997).

 Somatic embryogenesis is used currently as a powerful tool in

biotechnology. It is also used to study the development of the embryo.

 This process was reported as the best example of totipotency in plants

(Thorpe 2000). Developmental stages similar to zygotic embryogenesis
occur and yield an embryo with no vascular connection to the parent
tissue (von Arnold et al. 2002).
Somatic embryogenesis is a natural
phenomenon that was moved from nature to APOMIXIS
the laboratory • Apomixis (asexual seed formation) is the result
of a plant gaining the ability to bypass the most
fundamental aspects of sexual reproduction.
• Development of seeds from maternal tissue
inside ovule.
• Produce plants that are genetically identical as
maternal plants
• If apomixis is engineered into sexual crops in a
controlled manner, its impact on agriculture will
be broad and profound. Will allow clonal seed
production and thus enable efficient and
consistent yields of high-quality seeds, fruits,
At the edges of the leaves of several and vegetables at lower costs.
species of Kalanchoë appear small
bipolar structures.
Applications
 It can be used to produce plants commercially, or to carry out basic studies of cell
differentiation, gene expression, molecular genetics, and many others.

 Long-term storage of somatic embryos through cryopreservation while being field

tested.

 Flexibility to rapidly deploy suitable clones given changing breeding goals and/or
environmental conditions, ability to manage genetic diversity and genetic gain.

 Development of clonal somatic seedlings

 Amenability to process scale up-Bioreactors

 Allow mass production of selected clones from relatively small quantities of control
pollinated seed from controlled crosses where outstanding parents are difficult to
flower and/or only a small quantity of seed is produced.

 Embryogenesis may assist in the application of genetic transformation

Zygotic versus Somatic embryogenesis
Somatic and zygotic embryos have similar developmental stages:

Dicots: globular, heart, torpedo, and cotyledonary

Conifers: globular, early cotyledonary and late cotyledonary embryos stages.

 In vitro plant cells can undergo dedifferentiation or re-differentiation to enter a
new biological program that gives rise to somatic embryos.

Main diferences:
• Absence of a suspensor
• Seed coat
• Endosperm
• Unicelular or multicelular origin

Genetic transformation Quimeras

History
 The history of the study of somatic embryogenesis is plenty of
discoveries of very different natures: from the role of growth
regulators, mainly auxins, to the function of the components of the
media of culture.

 This was first observed in suspension cultures of carrot (Daccus

carota) by Steward, Mapes and Mears (1958) and in carrot callus grown
on na agar medium by Reinert (1959).
Main steps of somatic embryogenesis
Induction and expression
SE is a complex process that has been traditionally divided in
two main stages:

a) INDUCTION, where tissues acquire (direct or

indirectly) embryogenic competence
Signals
b) EXPRESSION, where competent cells develop
into somatic embryo structures.
Direct vesrus indirect SE

Different patterns of the in vitro origin of somatic

embryos have been distinguished.

Direct production of somatic embryos from the

explant cells called pre-embryogenic determined
cells,

Indirect production of somatic embryos from

induced embryogenic determined cells in
unorganized callus.

Pre-embryogenic determined cells are already

destined for embryogenic development prior to
explanting, requiring only growth regulators or
favourable conditions to enter cell division.
 In PEDCs the embryonic pathway is predetermined and cells appear to only
wait for a signal to start independent mitotic divisions to express their
potential.
 IN IEDCs is requires dedifferentiation and re-determination to the
embryogenic state by exposed to specific PGRs (2,4-D). Dedifferentiation in
callus.

 Once the embryogenic state has been reached both cell types proliferate and
development in a similar manner.
Regardless of the type of inducing embryogenic cells usually present a number of cytological and
ultrastructural characteristics:
I. small and isodiametrical cells,
II. having thin primary walls with a dense little vacuolated cytoplasm,
III. high nucleus / cytoplasm ratio and prominent nucleus.

Fig. 2. Histology of somatic embryogenesis in coriander. (A)

Embryonic cells at different stages (1, single cell; 2, diad; 3,
triad; 4, tetrad stages, bar = 20 m m). (B) Globular to early
heart stage embryos (bar = 20 m m). (C) Early cotyledonary
embryo, and (D) cotyledonary embryo (bar = 20 m m).
 Somatic embryogenesis in coriander.

(A) Embryogenic callus on MS medium

supplemented with 4.52 m M 2,4-D
and 3% (w/v) sucrose.

(B) Cluster of cotyledonary-staged

somatic embryos developed on the
callus.

Direct SE
 Induction
 Factors: Genotype and type of explant, culture medium
composition and culture conditions
Hormonal requirement
Formation of somatic embryos usually requires the presence of an
auxin or stress conditions. The most commonly used auxin It is
2,4-D, but others such as NAA, IBA, IAA dicamba or picloram can
also be successfully applied. The type of auxin used, as well as
concentrations applied vary widely from species to species.

carbohydrates
The presence of high concentrations of carbohydrates, in particular
sucrose, has proved, in some species, potentiates the embryogenic
response. (OSMOTIC POTENTIAL)
Others
Source of Nitrogen
This expression stage is usually divided in

Development (Histo-differentiation)

Academic point of view

Maturation

Germination/conversion to plants

Applied research

Emblings

Acclimatization
Development and Histo-differentiation
 Auxin must be removed or decreased for embryo development
 Continued used of auxin inhibits embryogenesis
 Histo-differentiation
 Maturation
 Require complete maturation with shoot and root apical meristem and
cotyledons;
 Storage of crucial reserves: Proteins, lipids, Carbohydrates No dormancy
No dissecation
 Often requires ABA for complete maturation

AVOID
Precocious germination

Germination and convertion of somatic embryos in plants.

Emblings

 MAY requires GA3 and Cytokinins

Aclimatization
 MS + 3 NAA MSWR MSWR MSWR MSWR
3 weeks 4 weeks 8 weeks 12 weeks 16 weeks

induction expression

Somatic embryos formed were whitish, compact, and mostly at the globular stage, although
other advanced stages could be found, showing some assynchronism of the process.

Germination and conversion were observed independently of the medium,

although MSWH continued to give the highest number of cotyledonar embryos
and of plantlets.

Primary SE
 Example: effect of genotype and yearly variation on SE

Seeds from 13 OP families, produced by parents were used over three consecutive years beginning
in 2002. However, due to a lack of seed, no observations were made in 2004 for CB02 and CB08.
These parents are a part of Celbi’s breeding program.

● SE induction varies across E.

globulus families and over the
Initiation of SE by OP families years of seed production tested.
35.0

all years

● Somatic embryogenesis was

30.0
2002

25.0
2003 initiated on explants from 84%
of the OP families tested in 2002
Percent initiation

2004

20.0 and 100% of the families

tested in 2003 and 2004.
15.0

10.0
● The year 2003 gave best results
for percentage of induction and
5.0 total number of somatic embryos
produced.
0.0
CB09 CB13 CB04 CB06 CB12 CB08 CB03 CB07 CB02 CB05 CB01 CB11 CB10

OP families
Current bottlenecks to the application of somatic embryogenesis technology for large scale production

• Low induction rates

• Low number of somatic embryos per explant
• Low conversion of somatic embryos to somatic seedlings

Repetitive somatic embryogenesis protocol

In contrast to primary somatic embryogenesis induced from explant cells, secondary

somatic embryogenesis is the phenomenon whereby new somatic embryos are induced
through existing somatic embryos.

In woody plants, secondary somatic embryogenesis can maintain the embryogenic

competence of cultures for many years and thus provide useful research material.
Multiplication

EC and NEC EC NEC

Fig. 3. Histological preparation showing globular- and heart-

staged secondary somatic embryos developed on primary
embryo (bar = 20 m m). pe, primary
Repetitive Somatic embryogenesis in Eucalyptus globulus

zygotic embryos
Induction medium

primary somatic embryos Dark

(isolation)
MSWR

MS + NAA

Secondary somatic embryos

This strategy led to successive cycles of new embryo production via secondary somatic embryogenesis
maintained in our laboratory (Dbio/ UA) for years
 Secondary SE

Germination and
Somatic embryo
B
conversion
E

A C D
B

SE

SE
ZE

E F G
 Desinfection Induction Aclimatization
Light
Inoculation

Elongation
MS + 3 mg/l NAA (Dark)
Indução

MSWH

MSSH
Globular Embryo Repetetive SE dark

Aclimatization
NAA
SE PROTOCOL STANDARD

Cotyledonar Embryo
Expressão

Germination Multiplication

Light

Plants
Quercus suber

EC and NEC EC NEC

Synthetic seeds
The concept of synthetic seeds was first mentioned by Murashige (1977). The synthetic or
artificial seed was defined as an encapsulated single somatic embryo inside a matrix
covering. Later, synthetic seeds of alfalfa were successfully produced by encapsulating
somatic embryos in alginate hydrogel (Redenbaugh et al., 1984).
Synthetic seed technology

The synthetic seeds are defined as an alternative to seeds consisting of

somatic embryos surrounded by artificial coats which is most equivalent to
an immature zygotic embryo.

The encapsulation technology provides the somatic embryo with protection

from mechanical damage and a supply of nutrients for the growing
embryo.

Synthetic seeds could be easily handled for storage, transport, and sowing,
the same as a zygotic seed. Hydrated and desiccated forms of
encapsulation technology were employed in synthetic seeds production.

Synthetic seed technology is one of the most important applications of

plant tissue culture, as it combines the advantages of clonal propagation
with those of seed propagation (i.e., storability, easy handling and
transport, use of sowing equipment, protection against diseases and
Artificial Seeds of Cactus (Echinocereus sp.)
pests). Through Biotechnological Methods
Factors affecting success of synthetic seed technology

The genotype of the somatic embryo

the encapsulating agent used

matrix

Evidence also suggests that the conversion rate may decrease

with increased storage times and storage temperatures

NOTE: only suitable for some specious whose somatic embryos

are tolerant of desiccation
Most efforts involve using different coating agents to
encapsulate the propagules. These agents include:
• sodium alginate,
• potassium alginate,
• sodium pectate, among others,

Carbon sources, plant growth regulators, and antimicrobial

agents were also added to the hydrogel to facilitate growth and
increase the survival rate of the encapsulated propagules.

Finally, the encapsulated propagules were cultured on media or

in the field for plantlet conversion.
Synthetic seed technology is today an important tool, available to breeders
and scientists, for the in vitro propagation and conservation of plants.

The synthetic seeds are used

today in advanced procedures of
cryopreservation (such as the
“encapsulation-dehydration”
method) with very promising
results for the long-term
preservation of plant germplasm.
“Encapsulation-dehydration” method

Pre-culture in high
sucrose medium

regrowth Thawing
Somatic embryogenesis limitations:
• Absense of synchronization in the development of embryos;
• The presence of non embryogenic cells and embryogenic in the same population with predominance of the
latter;
• Potential embryogenic expression only during certain periods the time;
• Weak knowledge of the mechanism of action of auxin and relative to embryogenesis induction or with respect
to other its effects.