Plant Tissue Culture - In-vitro Pollination and Fertilization in Plants

1. Culture of In-vitro Pollination
and Fertilization
A BIODICTION (A UNIT OF DR. DIVYA SHARMA)
DR. DIVYA SHARMA
ASSISTANT PROFESSOR

2. In-vitro Pollination
Vitro ------ Glass or Glassy substance
In-vitro ----- Glass or Glass tube
 In-vitro technique ------ Cultivation of plant tissue or other organs on artificial
media in a test tube or conical flask.
 The process of seed germination following stigmatic pollination of cultured pistil
has been referred to as in-vitro pollination.

3. Types of in-vitro Pollination
 Ovular pollination:
[Application of pollen to excised ovule]
 Ovarian pollination:
[Application of pollen to excised ovary]
 Placental pollination:
[Application of pollen to ovule attached to
the placenta]
 Stigmatic pollination:
[Application of pollen to stigma ovule]

4. Method of in-vitro Pollination
 In-vitro pollination can be accomplished by procedure by following appropriate
sterilization procedure, suitable medium, and selection of suitable explant
 In nature, intraspecific hybridization occurs less frequently. This is due to hindering
factor:
 Factors: barrier to the growth of the pollen tube on the stigma or style
 Solution: in such cases the style or part of it can be excised and pollen grains
either placed on the cut size of ovary
Eg: Pavaver sommiferum

5. Requirements of in-vitro fertilization in
crop improvement
 In certain occasions pollen fails to germinate on the stigma
 In certain genotypes the growth of the pollen tube in the style partially or
completely stagnates
 Fertilization not takes places after self or cross pollination is refereed as self or
cross incompatibility
 In such situations ovule fertilization is done by bringing the pollen artificially to
contact with the ovules is called in-vitro fertilization (IVF)

6. In-vitro Fertilization
 The process of seed formation following
stigmatic pollination of cultured pistil has
referred to as in-vitro pollination and
development of seed through in-vitro
fertilization.
History
 Kranz et al., 1990, 1991
 Faure et al., 1993
 Kranz and Lorz, 1993

7. Types of in-vitro Fertilization
 Stigma fertilization
 Placental fertilization
 Fertilization of an isolated ovule
without a placenta

8. Stigma fertilization
FEMALE MALE
ADVANTAGESFERTILIZATION
Nicotiana rustica, Nicotiana tabacum, Petunia violacea, Antirrhium majus, Pisium sativum,
Lathyrus odoratus, Zea mays and Glycine species
 Anther is placed on the stigma
 Useful when ovaries fall off the
plant prematurely, resulting in
lack of progeny
 Pollen from a ripe anther is
collected and has been
extremely sterilized
 Emasculated flower is
sterilized and then isolated in-
vitro

9. Placental fertilization
FEMALE MALE
ADVANTAGESFERTILIZATION
Caryophyllaceae, Gossypium and Zea mays
 Useful to determine whether the
pollen grains germinate, if they
penetrate the embryo sac and
whether fertilization follows
 Mature closed anthers at a stage
of ready to open in-vitro are
externally sterilized
 Anthers are opened under sterile
conditions and the pollen grains
placed near the ovules
 Flower is extremely sterilized and
placenta explants with unfertilized
ovules are dissected and
inoculated onto a nutrient
medium

10. Fertilization of an isolated ovule without a
placenta
FEMALE MALE
CHALLENGEFERTILIZATION
 Little success with this method
since it is extremely difficult to
induce embryo formation in in-
vitro fertilized
 Mature closed anthers at a stage
of ready to open in-vitro are
externally sterilized
 Anthers are opened under sterile
conditions and the pollen grains
placed near the ovules
 Flower is externally sterilized and
ovules are isolated in-vitro

11. Use of in-vitro fertilization
 Placental pollination is sometimes possible when the plants are completely self-
incompatible in-vivo.
Eg.: Petunia axilaris, Petunia hybrids
 Cross fertilization may be possible.
Eg.: Nicotiana alata X N. tabacum, intergeneric crosses with different members of the
Caryophyllaceae
 Production of haploids by parthenogenesis
 The abscission of a flower or ovary is sometimes unavoidable. In such a cases
stigma fertilization may be effective.
 To study the physiology of the fertilization

12. Techniques : Isolation of pollen and egg
cells
Pollen
 Isolated from pollen grains by osmotic
shock in 540 mos mol/kg in water
mannitol solution.
Egg cell
 Isolated by microdissection from the
ovules incubated at 40ºC for 60 minutes.
 Enzyme solution containing pH7
o Pectinase (0.75%)
o Pectolyse 1/23 (0.23%)
o Hemicellulose (05%)
o Cellulose onozuka RS (0.5%)

13. Techniques
Methodology of gametes there in fusion and culture has been standardized for maize plant
Isolation of sperm from pollen grains is executed by osmotic shock in mannitol solution
Isolation of ovules by microdissection of ovule incubated to 40-60 minutes
Micro droplet mannitol solution placed on UV sterilized cover slip with the aid of micro-pillaries
connected to a computed controlled dispenser to isolate and select the egg cell
The coverslips the over layed with 300 µL autoclaved mineral oil

14. Methods of in-
vitro fertilization
Isolation --- fusion ----
fertilization ---- culturing -----
subculturing

15. Methods of in-vitro fertilization
By using micro pump isolated sperm is transferred into micro-droplet containing egg
After fixing, egg is fertilized by electrofusion due to supplying maximum of B negative DC pulses
Fertilize then cultured on a semipermeable transparent membrane of a millicell-CM dish filled with
0.1 mL of nutrient solution
In 50 light intensity 26± 0.2 temperature the dish is then inserted in the middle of 3cm petriplate
where 1.5 of a feeder cell suspension layer of another maize line remains
A high frequency of sperm-egg cell fusion was replaced
Fertilized egg divide to form embryo from which regenerated full plants within 86 days

16. Points to be considered for in-vitro
fertilization in plants
1. The pollen grains and the ovules must be in the correct physiological and
morphological state
2. The choice of nutrient medium is extremely important
3. Stigma should not be in sterilizing agent for too long to stop the stigma exudates
dissolve in sterilitants
4. It is better not to remove the sepals from the flowers, since they enpromote ovary
growth
5. If placenta fertilization is failed try stigma fertilization
6. Temperature may be a decisive factor

17. Applications
 Micropropagation for ornamental plant increased by bud proliferation and
multiple shoot formation
 Artificial synthetic seeds are produced by somatic embryos
 Plant tissue culture a plant cell potato and tomato were brought together there
protoplasmic fusion and hybrid cell was made to develop into a pomato plant
 Useful for mutation breeding, triploidy through endosperm culture for inducing
parthenocarpic fruits

18. A Biodiction (A Unit of Dr. Divya Sharma)