13. 1. Mechanical Method
• Used for vacuolated cells like onion bulb scale, radish and beet root tissues
• Low yield of protoplast
• Laborious and tedious process
• Low protoplast viability
(Thomar, 2010)
15. Enzymatic Method
• Used for variety of tissues and organs including leaves, petioles,
fruits, roots, coleoptiles, hypocotyls, stem, shoot apices, embryo
microspores
• Mesophyll tissue - most suitable source
• High yield of protoplast
• Easy to perform
• More protoplast viability
16. Enzymatic Method
Leaf sterlization, removal of
epidermis
Plasmolysed
cells
Plasmolysed
cells
Pectinase +cellulase Pectinase
Protoplasm released
Release of
isolated cells
cellulase
Protoplasm
released
Isolated
Protoplasm
31. • The “thin alginate layer’’ (TAL) technique by Golds et al. (1992), in which
protoplasts are enmeshed in an alginate medium and placed in liquid
culture medium.
• The new, improved technique is termed extra thin alginate film (ETAF)
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43. Embryo Culture
• 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 (1904) from two Crucifers Cochleria &
Raphanus.
45. 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 invivo or become dormant
for long periods of time.
• This culture is done to eliminate the
inhibition of seed germination.
49. 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.
50. 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)
51. 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.
52. Embryo-rescue process.
• ( A ) Emasculated inflorescence;
• ( B ) inflorescence bagged after
pollination;
• ( C ) ovules inoculated onto embryo
formation medium;
• ( D ) embryo inoculated onto
germination medium;
• ( E ) plant subculture on rooting
medium;
• ( F ) plant in pot covered with plastic
cup;
• ( G ) hardened plants in greenhouse;
• ( H ) surviving plants growing in soil.
53.
54.
55.
56.
57. Media Requirement
• For Mature embryos-
Basal salt medium with a carbon energy source.
• For Immature embryos-
Different vitamins, amino acids & growth regulators are
required.
58. 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
59. 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.
- requires filter-sterilizing a portion of the
medium
60. 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
61. 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"
62.
63.
64.
65.
66.
67.
68. 7. Zygotic embryo rescue to recover individuals from crosses between seedless varieties.
70. 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
71. 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
73. Applications of Embryo Culture
• Production of haploids
• Prevention of embryo abortion in wide crosses
• Overcoming Seed dormancy
• Shortening of breeding cycle
• Prevention of embryo abortion with early ripening stone fruits
74. Production of haploids
• Production of monoploids
- useful for obtaining "haploids" of barley,
wheat, other cereals.
- the barley system uses Hordeum bulbosum
as a pollen parent.
75. Production of haploids
• 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" by culturing 10 PP to avoid abortion.
76. 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.
77. Wide crosses in embryo culture
Crossing species
1. Solanum tuberosum x S.
etuberosum
2. Brassica oleracea x B.
napus
3.Oryza sativa x O. minuta
• Resistance traits
• Potato leaf roll virus
• Triazine resistance
• Bacterial blight
78. 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.
79. Shortening of breeding cycle
• Under proper germination conditions, 3 years are required for
seeds 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.
80. 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.
81. - ANTHER AND MICROSPORE
CULTURE
• Haploid plants are derived from microspores (pollen) cultured
individually or in anthers
Processes Leading to Production of Haploid Plants:
- Androgenesis: haploid plant derived from male gamete, most
common method in vitro
- Parthenogenesis: from unfertilized egg
• Chromosome elimination:
chromosome elimination in somatic cells, most common method used
with plant breeding
82. Production of haploid
• In vitro methods:
– Anther/microspores culture
(androgenesis) - production of haploid plants from
microspores
• Anther culture for production of haploids reported
in about 250 species
• Solanaceae, Cruciferae, Gramineae,
Ranunculaceae most common
– Ovule culture (gynogenesis) - production of haploid plants from
unfertilized egg cell
83.
84. Androgenic methods
From the male gametophyte of an angiosperm plant,
i.e. microspore (immature pollen)
- The underlying principle is to stop the development of
pollen cell → direct development in a plant (no gamete phasis)
- Anther techniques are simple, quick and efficient:
- immature anthers sterilized
- acetocarmine test for pollen development
- solid media, pollen callus → shoots
- disadvantages: plants may originate from various parts of
the anther → plants with various ploidy levels
85. Diagrammatic illustration showing various modes of androgenesis and haploid plant
formation by anther and isolated pollen culture. The homozygous plants are obtained
by treating haploids with colchichine. (Bajaj et al. 1983
88. Gynogenic methods
Megaspores or female gametophytes of angiosperms can be
triggered in vitro to sporophytic development.
• Culture of unpollinated ovaries and ovules represents an
alternative for the production of haploid plants in species for which
anther culture has failed (e.g. albino plants).
• Not used as much as androgenic method.
• Problems in dissection of unfertilized ovules/ovaries.
• Promising for gymnosperms.
• In addition to unpollinated ovaries, pollinated can be also used in
some cases.
89. - NUCELLUS CULTURE
• Nucellus culture has been utilized to study factors responsible
for the formation of adventives embryos (the embryos arise
adventitiously from cells of nucellus or integuments, e.g. citrus,
mango)
• The adventives embryos are of considerable importance to the
horticulturists. Genetically uniform reproduce the
characteristic of the maternal parent, they are disease-free
clones retaining growth vigor and fruiting characteristics
90. - ENDOSPERM CULTURE
• The induction of organogenesis has always been a challenging problem.
• Applications: production of triploid plants (from triploid endosperm)
e.g. in Citrus, banana, apple, tea, mulberry).
• Endosperm culture can also be used as a nurse tissue for raising hybrid
embryos.
