Ovary culture and

Cryopreservation
VINAYKUMAR S N
OVARY CULTURE
Invitro culture of unpollinated ovaries and ovules represents an alternative for
the production of haploid plant, it is called as Gynogenesis.
It is the formation of saprophyte form the female gametophyte on artificial
nutrient medium.
First successful report on induction of gynogenic haploid was in barley by San
Noeum in 1976.
Later works have been done in Wheat, Rice, Maize, Tobacco etc.
In most cases optimum stage for ovary culture is nearly mature embryosac but
in rice free nuclear embryosac stage is most responsive.
 Invitro gynogenesis is used as a alternative technique in species where anther or
pollen culture is unsucessfull.
 The gynogenic plants may arise through direct embryogenesis or the gametic
cells may form a callus followed by plant regeneration.
 Both ovary slice culture and ovule culture can be carried out simultaneously for
achieving invitro gynogenesis.
Two methods
Ovary culture.
Ovule culture.
Ovary culture
•It is a technique of culture of ovaries isolated
either from pollinated or unpollinated flowers.
•Developed by Nitsch in 1951.
•Ovary is a ovule bearing region of pistil.
•Medium containing mineral salts, sucrose,
Vitamin B, IAA and coconut milk.
•In some species like tomato and gherkins
excised ovaries grown in culture forms fruits
that ripen and produce viable seeds.
 Ovary culture from fertilized ovary, provided the flowers have been fertilized
two or more days before excision.
 Ovaries of unpollinated flowers do not grow on simple nutrient medium.
 Use of some synthetic auxins such as 2,4 – D, 2,4,5- T (2,4,5-
trichlorophenoxyacetic acid), NOA (Napthoxyacetic acid) in nutrient medium
induces the development of ovaries of unpollinated flowers.
Procedure
oCollection of pollinated or unpollinated flower from healthy plant.
oWash them in tap water, dip in 5% Teepol solution for 10 mins and again wash
to remove traces of Teepol.
oTransfer flowers to LAF, surface sterilize them by immersing in 5% sodium
hypochlorite solution for 5-7 mins. Wash them with sterile distilled water.
oIn a sterile petridish, using flamed forceps and a surgical scalpel, dissect out the
calyx, petals, anther filaments etc. of the flower to isolate the ovary.
othe ovary can be cultured either through induction or regeneration process. In
induction, the ovaries float over the liquid medium. In contrast, the ovaries grow
on the solid nutrient medium in the regeneration process.
o Incubate the cultures for 16 hours at 250 C. For ovary regeneration, keep the culture
plates in a daylight regime by using a fluorescent lamp. Place the culture tubes in the
dark for the induction process.
Ovule culture
Ovules are aseptically isolated from the ovary
and are grown aseptically on chemically
defined nutrient medium under controlled
conditions.
Ovule is a mega sporangium covered by
integuments.
Ovules are attached with placenta inside ovary
by means of its funiculus.
Invitro ovule culture helps to understand the
factors that regulate the development of zygote
through organized stages to a mature embryo.
Procedure
Collect the open flower. If fertilized ovules are desired, collect the open flowers
where anthers are dehisced and pollination has taken place. To ensure the
fertilization, collect the flower after 48 hrs. of anther dehiscence.
Remove sepals, petals, androecium, etc. from the ovaries containing either
fertilized or unfertilized ovules.
Soak the ovaries in 6% NaOCL solution.
Rinse the ovaries 3-4 times with sterile distilled water.
Using sterile technique, ovules are gently prodded with the help of spoon
shaped spatula by breaking the funiculus at its junction placental tissue.
 The spatula with ovules is gently
lowered into the sterile solid or liquid
medium as the culture vial is slanted
about 45 0C.
 Damaged or unorganized ovules are
rejected when possible during transfer.
 Incubate the culture in either dark or
light at (16 hrs. 3000 lux) 25 0C.
 Selection of healthy parent plant

Check ovary/ovule development stage by histology

Select appropriate flower bud and surface sterilized

Inoculation of ovary/ovule in suitable medium

Incubation of ovary/ovule culture

Regeneration of callus/embryo

Haploid plant recovery

Triggering factors
i. Pre-treatment
Cold/heat shock treatments
cold pre-treatment of the inflorescence/ flower enhances gynogenesis
eg: 24-28 hrs. at 40 C in sunflower.
24 hrs. at 70 C in rice.
ii. Medium
MS medium, N6, B5 etc.
sucrose; 3-5 %
solid or liquid medium
phytohormones; auxins, cytokinins, 2,4-D
Advantages
•In normal process some hybrids fail to develop due to early embryo abortion and
premature abscission of fruits. Thus ovule culture can be used to rescue them.
•Useful to study the early development of embryo, fruit, different aspects of fruit
physiology including respiration, maturation and disease.
•Effect of phytohormones on parthenocarpic fruit development can be studied
from the culture of un-pollinated pistil.
•Successful in inducing polyembryony.
Disadvantages
The frequency of haploids production is very low in ovary culture as only 1-5%
of the responding ovaries are there.
This technique requires high technical skills and management. 
Gynogenesis can be employed only in limited species, for example, wheat, rice,
maize, sugar beet etc.
CRYOPRESERVATION
oCryo is Greek word (Krayos - frost)
oIt literally means preservation in “frozen state”.
oPrinciple – To bring plant cells or tissue to a zero metabolism and non dividing
state by reducing the temperature in the presence of cryoprotectant.
oBiological material remains genetically stable and metabolically inert, while
minimizing ice crystal formation.
oAny biological activity including the biochemical reactions that would cause
cell death are effectively stopped.
It can be done:

 Over solid carbon dioxide (at - 790 C)

 Low temperature deep freezer (at - 800 C)

 In vapor phase nitrogen (at - 1500 C)

 In liquid nitrogen (at - 1960 C), most widely used cryopreservation.

Procedures
A. Equilibrium (slow programmable) freezing.
• Expose the cell to cryoprotectant in a gradual step-wise fashion to slowly allow
equilibrium of cell with the cryoprotectant while releasing the water. Once the cells
have been cleared of the majority of cellular water, they are placed in a plastic or
glass container.
• Volume of liquid surrounding the cells for slow freezing is typically less than a
teaspoon and may only be a few drops. The pre-labeled container is filled, sealed
and put away in a programmable freezer, which slowly decreases the temperature of
the container over a period of minutes or hours to very low temperatures.
• When container reaches a temperature between -300 C and -850 C, the container
holding cells can be directly plunged into liquid nitrogen to complete the cooling to
-1960 C.
B. Non-equilibrium or Ultra freezing (Vitrification)
• This process uses higher concentration of cryoprotectant coupled with an almost
instantaneous freezing rate achieved by plunging cells directly into liquid
nitrogen.
• Vitrification bypasses the ice-crystal formation phase and moves the water
directly into a glass-like phase.
• For vitrification, cells are usually placed on the tip of a straw and excess
cryoprotectant is removed, leaving just enough so that the cell clings to the
container by surface tension, prior to plunging in liquid nitrogen.
• Because of rapid freezing, the duration of exposure to cryoprotectant is much less.
• Warming of the cell to return it to normal metabolic functioning must also be
incredibly rapid.
Cryoprotectants
Substances used to protect biological tissue from freezing damage.
They operate simply by increasing the solute concentration in cells.
To be biologically viable they must
• Easily penetrate cells.
• Not be toxic to cells.
Types of cryoprotectants
Based on their ability to diffuse across the cell membrane, there 2 types.
1. Penetrating cryoprotectant
 They can penetrate the cell membrane and enter cell cytoplasm.
 Form hydrogen bond with water to prevent ice crystallization.
 Act by replacing water and therefore controlling cell size changes as well as
preventing intercellular ice formation and prevent excessive dehydration during cell
cryopreservation.
 DMSO (Dimethyl sulfoxide), glycerol, ethylene glycol.
2. Non-penetrating cryoprotectants
• They do not penetrate the cell membrane.
• Large molecules usually polymers such as PEG or saccharides such as
sucrose.
• Act by dehydrating the cells before freezing, thereby reducing the amount of
water that the cell needs to lose to remain close to osmotic equilibrium during
freezing.
• Inhibit ice growth by the same mechanism as penetrating types but without
entering the cell.
• They help to prevent damage to cells during recovery from cryopreservation
by preventing solutes, particularly larger protoplasmic elements, from
escaping the cell too rapidly.
 Risks involved in cryopreservation
techniques
i. Solution effect: as ice crystals grow in freezing water solutes are excluded,
causing them to become concentrated in the remaining liquid water. High
concentration of some solutes can be very damaging.
ii. Extracellular ice formation: can cause mechanical damage to cell membrane
due to crushing.
iii. Dehydration: direct damage caused by associated stress on cells.
iv. Intracellular ice formation: while some organisms and tissues can tolerate
some extracellular ice, any appreciable intracellular ice is almost always fatal
to cells.
Applications of cryopreservation
Embryo storage for research
Fertility preservation
Efficiency of assisted reproduction
Reduce the implantation of multiple embryos
Biodiversity conservation
Germplasm conservation
Maintaining the disease free stock.
Thank
you…