2. ANTHER CULTURE
It is a method of Androgenesis, i.e. production of
haploid plantlets by the in-vitro condition, in which the
anther is separated from the bud aseptically and cultured
on the nutrient media to give rise to the haploid plantlets.
Its primary objective is to produce haploid androgenic
plants by using the regeneration capacity or
the totipotency of the anther.
3. Methods
Anther culture is a plant tissue culture method, which can be done by either of
the two methods, namely direct or indirect method.
1. The direct method of an anther culture involves Embryogenesis. In this method,
the anther behaves as a zygote and forms embryoid that eventually develops
haploid plantlets.
2. The indirect method of an anther culture involves Organogenesis. In this
method, the anther undergoes cell division repeatedly to form the callus tissue,
which later gives rise to the haploid plantlets.
4. 1. First, select the unopened buds of size about 17-22mm in length. Reject the buds that are
beginning to open or already open. While selecting buds, the size of the sepal must be equal to
the size of a petal, which is considered ideal for the anther culture.
2. Transfer the buds into sterilized airtight plastic bags.
3. Then, transfer the selected buds to the laminar airflow chamber.
4. After that surface sterilization of buds is carried out in the chamber to maintain the aseptic
conditions. Firstly, the buds are surface sterilized by 70% ethanol for 10 seconds and then 20% of
sodium hypochlorite for 10 minutes.
5. Then, wash the buds three times by the distilled water.
6. After washing, transfer the buds to the sterilized Petri plate.
7. Then, separate the stamen from the bud through a scalpel.
8. Remove the filaments from an anther.
9. After that, transfer an anther onto the solid or liquid nutrient medium and incubate it for 3-4
weeks at 24-28 ֯C in the dark.
10. Then either by embryogenesis and organogenesis, haploid plantlets would appear from the anther
culture within 3-4 weeks. During this stage, incubate the culture at 24-28 ֯C for 12-18 hours in light
and 6-12 hours in the dark.
11. At last, about 50mm tall plantlets will appear that are then transferred into the pot containing bio
compost followed by washing. Then a sterilized glass beaker is used to cover the pot and remove
the glass beaker after some week and transfer the plant to the large pot.
5. Factors Influencing Anther Culture
Some factors that influence the anther culture technique can be categorized into
intrinsic and extrinsic factors.
Intrinsic Factors
Anther wall: It provides nourishment during the developmental stages of the
anther.
Stage of Anther: For anther culture pre-mitotic, mitotic and post-mitotic stages of
an anther is preferred. Pre-mitotic is the stage, in which the first meiotic division
occurs in the anther and the pollens are at an immature stage. In the mitotic stage
of an anther, there is the division of the pollen. The post-mitotic stage is the bi-
cellular stage, where the development in pollen grains form embryoids.
6. Bud size: Its preferable size is up to the length of 17-22 mm.
Age of plant: The anther culture prefers the buds from the younger plants.
Extrinsic Factors
Culture medium: In the culture medium sucrose, iron, vitamins, coconut milk,
hormones and growth regulators (auxins or cytokinins) play an essential role in
the induction of haploid plant.
Temperature: It varies with the different plant species. Example: In Datura
stramonium, the optimum temperature is 20 ֯C for the formation of embryoids.
In Nicotiana tabacum, the optimum temperature is 25 ֯C for the formation of
embryoids. In Brassica campentris, the optimum temperature is much higher, i.e.
35 ֯C for the formation of embryoids.
