- In a general procedure for pollen culture anther are collected from sterilized flower buds in a beaker containing basal media.
- The pollen are then squeezed out of the anther by them against the side of beaker with the glass rod.
- Another tissue are removed by filtering the suspension through a nylon sieve having a pore diameter which is slightly wider than the diameter of pollen.
- Smaller microspores don’t regenerate does layer good and viable microscopes can lie concentration by filtering the microscope suspension through nylon sieves.
- These pollen suspension is then centrifuged at low speed 150 x g for 5 minutes.
- The microspore was obtained are then mixed with an appropriate culture medium at a density of 103 2104 microspore 1 ml.
- The final suspension is then pipetted 8 into small Petri dishes.
- The responsive microscope from embryos or Calli and its subsequent development to plant formation can be achieved by transferring to suitable media.
- Success in anther culture is predominantly dependent on the genotype of the anther donor material and it is also known that anther culture ability is genetically controlled.
Physiological status of the donor plants
- The physiological status of the plants at the time of anther excision influences the sporophytic efficiency of microscopes.
- In order to allow for the in vitro development of pollen into an adult plant it is very important to start with healthy pollen cells.
- Therefore it is good to culture anther from plants grown under the best environmental condition.
Stage of pollen:
- It has been established that selection of anther of an appropriate stage of pollen development is most critical.
- Anther with microspore ranging from Detroit to the binucleate stage are responsive.
- But as soon as starch deposition has begin in the microscope no sporophytic development and subsequently no microscopic structure formation occur.
- Data has established that uninucleate microspores are more prove to experimental treatment for culture before or during first mitosis.
Pre treatment of anthers
- The underlying principle of androgenesis is to stop the development of the pollen cell whose fate is normally to become a gamete and to force its development directly into a plant.
- In general cold treatment between 3 degree Celsius and 6 degree Celsius for 32 15 days give good response message respond better to a temperature of 4 degree Celsius.
- Floral birds or anther plants in some species when subjected to 30 degree Celsius for 24 hours for 40 degree Celsius for 1 hour stimulus embryogenesis.
Various chemical are known to induce pathogenesis 2 chloro methyl phosphoric acid has a pronounced effect in increasing the haploid production in various species.
- Sucrose is essential for androgenesis.
- Sugar are in dispensable in the basal medium as they are not only the sucrose of carbon but are also involved in of regulation nitrogen metabolism is quite and important feature.
- The presence of nitrate ammonium salt as well as a.
- Appeared to play very special role at different stages of development process.
- Haploid can be diploidized to produce homozygous plant by following methods:-
- it has been extensively used as a spindle inhibitor to induce chromosome duplication.
- The plant leaves when still attached to the anther are treated for 24 to 42 hours with 0.5% call kissing solution washed thoroughly and replanted.
- Haploid cell are in general unstable in culture and have a tendency to undergo endomitosis to form diploid cell.
- This propertyof cell culture has been exploited in some spaces for obtaining homozygote or diploidization.
- A small segment of system is an auxin or cytokinin medium to induce callus formation.
- Young flowers buds with immature anther which the microspore are confined with in the anther set at the appropriate stage of pollen development are surface sterilized and rinsed with sterile water.
- The calyx form the flower buds is removed by slammed group the Corolla is slit open and stamens are removed and placed in a sterile petri dish.
- One of the anther is accused in acetone carmine to test the stage of pollen development.
- It is found to be correct stage each other is gently separated from the filament and the intact anther are inoculated horizontally on nutrient media.
- Injured anther may be discarded because wounded often stimulate callusing of the anther wall tissue.
- In case of serials spikes are harvested at the uninucleate stage of microspore development and surface sterilized.
- Anther can be planted and solid agar media in petri dishes.
- Normally 10 to 20 anthers are plated in a 6cm Petri dish.
- Anther can be cultured on a liquid media and 50 anthers can be cultured in 10 ml of liquid medium.
- In responsive anther the world tissue gradually turn brown and within 3 to 8 weeks they open due to the pressure extended by the growing pollen callus or pollen plants.
- After they have attend a height of about 3-5 CM the individual plantlet emerging from the colours are separated and transferred to a medium that would support for the development.
- It is very quick for practical purpose.