More often, anthers rather than microspores are cultured, since methods for extraction and culture of microspores differ and have been successful only in a few species (Datura inoxia, Nicotiana Sylvestris, N. tabacum, Oryza sativa, etc.). The following parameters have been recognized as particularly important for successful anther and microspore culture; (i) conditions of growth of donor plant; (ii) genotype of donor plant; (iii) the pretreatment; (iv) the developmental stage of anther/microspore; (v) the culture medium and the conditions during culture growth. The different steps involved in anther and microspore cultures are shown respectively. In either case, flower buds are brought to a laminar flow chamber and sterilized using appropriate chemical treatment before their dissection. While removing anthers from the flower buds, care is exercised to avoid injury, because injury leads to development of callus, giving a mixture of diploids, haploids and aneuploids.

As shown the anthers generally cultured on solid agar medium, where they may directly give rise to embryoids or may lead to callus formation, before differentiation. The embryoids develop into haploids plantlets, which are colchicines treated to get doubled haploid (DH) homozygous plants to be field tested for selection.

Microspore culture may be preferred over anther culture, even though the degree of success is low in this case. Microspores are collected first using the following steps: (i) anthers, dissected as above, are taken in a sterile beaker containing liquid medium, and are pressed with a  glass rod or syringe piston to allow the microspores squeeze out; (ii) the suspension with anther and microspores is filtered through a nylon sieve, which allows only the microspores to pass through; (iii) the filtrate is centrifuged thrice, for 5 minutes each, at 500-800 rpm (the pellet resuspended in a fresh medium each time); (iv) the microspores are inoculated on a solid or in a liquid medium maintained at 25˚C and 16/8 hr photoperiod.