Toxicokinetics studies.. (toxicokinetics evaluation in preclinical studies)
embryo-culture plant tissues culture.ppt
1.
2. 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.
4. 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.
5. 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.
6. Media Requirement
For Mature embryos-
Basal salt medium with a carbon energy
source.
For Immature embryos-
Different vitamins, amino acids & growth
regulators are required.
7. 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
8. 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
9. 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
10. 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"
11. 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)
12. 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.
15. 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
16. 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
18. 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
19. 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.
20. 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.
21. 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.
22. 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
23. 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.
24. 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.
25. 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.
