INTRODUCTION

• Totipotency is the ability of a single cell to divide and produce all of

the differentiated cells in an organism.
• In other words, totipotency is the genetic potential of a plant cell to
produce the entire plant.
• Isolated cells from differentiated tissue are generally non dividing and
quiescent; to express totipotency they undergo dedifferentiation and
then redifferentiation.
• In Latin, totus means "entirely“ and "potens" means "having power".
• The possibility of regenerating an entire plant from a single or few
non-zygotic cells was proposed by Gottleib Haberlandt (1854-1945) in
1902.
• Haberlandt is now popularly called the Father of Tissue Culture.
Adult plant cells are totipotent, meaning they have the ability to give rise to a fully differentiated plant. Because
of this, it is possible to collect cells from a mature plant and use those cells to produce clones of that plant.
In the 1950s Steward and his co-workers succeeded in growing carrot
plants from isolated phloem cells.

Fig 1: Expression of Totipotency in Culture

 High ratio of cytokinin to auxin = shoot development (caulogenesis).
Low ratio of cytokinin to auxin= roots development (rhizogenesis).

Fig 2 : Differentiation of totipotent plant cell

 Some factors that affect cellular totipotency are :

I. Source of explant : Organ that served as tissue source,

physiological and ontogenic age of organ, season in
which explant is obtained, size, overall quality of explant.
II. Nutrient media and constituents : Inorganic macro- and
micronutrients, carbon/energy source, reduced nitrogen
source, plant growth regulator.
III. Culture environment (Physical form of medium) :
Presence/absence of agar, pH of medium, light quality and
quantity, temperature, relative humidity and gaseous
atmosphere in the vessels.
 Embryo Culture
• Sterile isolation & growth of an immature or
mature embryo in vitro, with the goal of
obtaining a viable plant.
• The first attempt to grow the embryos of
angiosperms was made by Hannig in 1904
from two Crucifers Cochleria & Raphanus.
 Types of Embryo culture
• Mature Embryo Culture

• Immature Embryo Culture/ Embryo rescue

 Mature Embryo Culture
• It is the culture of mature embryos derived
from ripe seeds.
• This type of culture is done when embryos do
not survive in vivo or become dormant for
long periods of time.
• This culture is done to eliminate the inhibition
of seed germination.
 Immature Embryo Culture
• It is the culture of immature embryos to rescue
the embryos of wide crosses.
• This type of culture is mainly used to avoid
embryo abortion with the purpose of
producing a viable plant.
 Embryo Rescue
• In immature embryo culture, wide hybrids
often suffer from early spontaneous abortion
due to several barriers which operate at pre- &
post- fertilization levels.
• Pre- fertilization barriers include all factors
that hinder effective fertilization .
(due to inhibition of pollen tube growth)
 Embryo Rescue
• Post fertilization barriers retard the
development of the zygote after fertilization
and normal development of the seed.
(due to failure of endosperm)
• This is known as embryo-endosperm
incompatibility where the endosperm produces
toxins that kills the embryo.
 Media Requirement
• For Mature embryos-
Basal salt medium with a carbon energy
source.
• For Immature embryos-
In Addition require different vitamins, amino
acids & growth regulators are required.
 Culture-medium factors

• mineral salts – K, Ca, N most important

• carbohydrate and osmotic pressure
- 2% sucrose works well for mature embryos
- 8-12% for immature embryos
- transfer to progressively lower levels as
embryo grows
 Culture-medium factors

• Amino acids
- reduced N is often helpful
- up to 10 amino acids can be added to
replace N salts, incl. glutamine, alanine,
arginine, aspartic acid, etc.
 Culture-medium factors
• natural plant extracts
- Coconut milk (liquid endosperm of
coconut)
- enhanced growth attributed to undefined
hormonal factors and/or organic
compounds
- others – extracts of dates, bananas, milk,
tomato juice
 Culture-medium factors

• Plant Growth Regulators

- globular embryos – require low conc. of
auxin and cytokinin
- heart-stage and later – moderate level of
auxin & low level of cytokinin
- GA and ABA regulate "precocious
germination"
EMBRYO CULTURE
OF
MAIZE
 SCIENTIFIC CLASSIFICATION OF
MAIZE
Kingdom - Plantae
Order - Poales
Family - Poaceae
Genus - Zea
Species - mays
 The Kernels of Maize has a
pericarp of fruit fused with
the seed coat and the entire
Kernel is reffered as seed

 The Cob is close to a multiple

fruit in structure, except that
the individual fruits (Kernels)
never fused to a single mass
 Procedure
Immature cob is taken from the healthy field
↓
Its Immature seeds (Kernels) were extracted out
↓
Surface sterilize the immature seeds by treating them with
0.1% mercuric chloride solution for 7 minutes
↓
Give three washings with autoclaved distilled water
↓
Dissect out the embryo from the seed under totally aseptic
conditions
↓
Culture the embryos in the jars containing MS media
↓
Incubate the jars at temperature 28 ̊-30 ̊C
EMBRYO CULTURE OF MAIZE
Applications of Embryo Culture
• Prevention of embryo abortion in wide crosses
• Production of haploids
• Overcoming Seed dormancy
• Shortening of breeding cycle
• Prevention of embryo abortion with early
ripening stone fruits
 Prevention of embryo abortion
• Embryo rescue from a cross between an early
Japanese cv.- Sunago wase & a Chinese cv.-
Yuhualu obtained “Zaoxialu” an extra early
maturing peach cultivar (It matures in 55days).
 Wide crosses in embryo culture
Crossing species • Resistance traits
1. Solanum tuberosum x S. • Potato leaf roll virus
etuberosum

2. Brassica oleracea x B. • Triazine resistance

napus

3.Oryza sativa x O. minuta • Bacterial blight

 Production of haploids

• Production of monoploids
- useful for obtaining "haploids" of barley,
wheat, other cereals.
-
 Production of haploids

• The barley system uses Hordeum bulbosum as a pollen parent.

• Hordeum vulgare is the seed parent.

• Zygote develops into an embryo with elimination of HB

chromosomes.

• Eventually, only HV chromosomes are left.

• Embryo is "rescued reulting in monoploids of female parent H

vulgare.
 Overcoming Seed dormancy

• Embryo rescue technique is applied to break

seed dormancy.
• Production of seedlings from seed of naturally
vegetatively propagated plants such as
Banana & Colocasia, whose seeds do not
germinate in nature.
 Shortening of breeding cycle

• Hollies (Ilex) are important plants for Christmas

decorations.
• Under proper germination conditions, 3 years
are required for seedsof I. opaca from mature
berries to complete their embryonic
development.
• The excised embryo goes through all the
developmental sequences in vitro & plants can
be obtained in 2-3 weeks time.
 Prevention of embryo abortion in stone
fruits
• Some species produce sterile seeds that will
not germinate under appropriate conditions
e.g.- Peach, Nectarine, Plum.
• Embryo culture has been practised as a general
method in horticultured crops including Peach,
Nectarine & Plum.
• Embryos are excellent materials for in vitro clonal
propagation. This is especially true for conifers and members
of Graminae family.

• Germination of seeds of obligatory parasites without the host

is impossible in vivo, but is achievable with embryo culture,