2.  Androgenesis

In androgenesis, the male gametophyte (microspore or immature pollen) produces haploid plants. The basic principle is to stop the development of pollen into a gamete (sex cell) and force it to develop into a haploid plant or sporophyte. The remarkable discovery that haploid embryos and plants can be produced by in vitro culture of anthers of Datura (Guha and Maheshwari 1964,1966) brought renewed interest to haploidy. This method of androgenic haploid production was quickly attempted in many species to hasten the breeding programme in several economically important plants. Haploid production through anther/microspore culture scores higher over other methods due to the fact that anthers harbour large numbers of haploid microspores per anther and is a potentially efficient means to generate homozygous true-breeding progeny lines in plant breeding programs.

 

2.1. Methodologies

In androgenesis, immature pollen grains are induced to follow the sporophytic mode of development by the application of various physical and chemical stimuli. There are two methods for in vitro production of androgenic haploids –

(A) Anther culture, and (B) Isolated pollen (microspore) culture.

2.1.A. Anther culture

(a)	Select the flower buds from an elite plant and determine the stage of microspores by acetocarmine squashes of anthers or by staining them with fluorescent dye DAPI (4,6-diamidino-2-phenylindole).
(b)	Surface sterilize the selected size of buds (ca 2mm size flower bud) to initiate in vitro anther cultures.
(c)	Dissect the buds under a stereo-microscope, using pre-sterilized Petriplates, forceps and fine needles. Discard the damaged anthers, if any, and remove the filament gently.
(d)	Inoculate the anthers, bearing early-to-late uninucleate stage of microspores, in a nutrient medium and maintain the cultures in defined conditions.
(e)	As the anthers proliferate, they produce embryos/callus.
(f)	The callus/embryos formed can be transferred to a suitable medium to finally produce haploid plants and then diploidize them by colchicine to produce homozygous diploids.