Production of haploids of neem (Azadirachta indica A. Juss.) by anther culture

Androgenic haploids of the neem tree (Azadirachta indica A. Juss.) were produced by anther culture at the early- to late-uninucleate stage of pollen. Haploid formation occurred via callusing. The best medium for inducing callusing in the anther cultures was Murashige and Skoog's basal medium (MS) (9% sucrose) supplemented with 1 μM 2,4-D, 1 μM NAA and 5 μM BAP, while anther callus multiplied best on MS medium supplemented with 1 μM 2,4-D and 10 μM Kn. These calli differentiated shoots when transferred to a medium containing BAP; 5 μM BAP was optimum for young calli (75% cultures differentiated shoots), but older calli showed the best regeneration with 7.5 μM BAP. Shoots elongated at a lower concentration of BAP—0.5 μM. These shoots were multiplied by forced axillary branching and rooted in vitro. The plants were subsequently established in soil. Of the plants that regenerated from anther callus 60% were haploid, 20% were diploid and 20% were aneuploid.