2.1.B. Microspore (Pollen) culture

Haploid plants can also be produced from isolated immature pollens or microspores (male gametophytic cells):

• (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 to initiate in vitro anther cultures.

(c)  Extract the microspores by pressing and squeezing the buds with a glass rod against the sides of a beaker.

(d)  Filter the pollen suspension to remove anther tissue debris.

(e)  Wash and collect the viable and large pollen (smaller pollen do not regenerate) by filtration.

(f)  Culture these microspores on a solid or liquid medium.

(g)  As the microspores undergo multiple divisions, they produce multicellular and multinuclear structure.

(h)  The callus/ embryos formed can be transferred to a suitable medium to finally produce a haploid plants and then diploid plants by colchicine treatment (Figure 9.2).

Figure 9.2. Androgenic haploid production pathways