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 muM 2,4-D, 1 muM NAA and 5 muM BAP, while anther callus multiplied best on MS medium supplemented with 1 muM 2,4-D and 10 muM Kn. These calli differentiated shoots when transferred to a medium containing BAP; 5 W BAP was optimum for young calli (75% cultures differentiated shoots), but older calli showed the best regeneration with 7.5 muM BAP. Shoots elongated at a lower concentration of BAP - 0.5 muM. 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.