Maternal haploid induction in African marigold (Tagetes erecta L.) through in vitro culture of un-fertilized ovules

Abstract

A protocol for production of haploids from two heterozygous male sterile lines of Tagetes erecta using un-pollinated ovules as explant was standardized. Various factors affecting the in vitro gynogenic response viz., growth regulators, ovule developmental stage, basal media, sucrose concentration, photoperiod and cold shock duration were assessed in Tagetes erecta. Direct induction of parthenogenic embryos occurred in cultivar ‘Local Orange’ when the un-pollinated ovules were cultured in EMS (MS medium enriched with coconut water, AgNO3, PVP etc.) medium supplemented with 2 mg l−1 BAP and 0.5 mg l−1 NAA or 2 mg l−1 BAP along with 2 mg l−1 2,4-D. The developmental stage of the flower buds was vital in embryo induction from the excised ovules. The ovules collected from half-open flower buds were more responsive to gynogenesis as compared to ovules collected from un-open and fully open flower buds. EMS basal medium was found best for gynogenesis over the other commercially available basal media. Cold pre-treatment of flower buds at 4 oC had no stimulatory effect and negatively affected the gynogenesis. The dark culture condition was imperative for direct parthenogenic embryo induction than 16 h light duration. The ploidy levels of 17 regenerants were determined by cytological studies, revealed 47.06% as diploids, 41.18% as haploids and 11.76% as mixoploids. These results were reconfirmed with flow cytometry analysis. The determination of ploidy level by counting the number of chloroplasts in stomatal guard cells of marigold was also established for rapid screening of haploids. The resultant haploids were successfully diploidised and are being utilized in the hybrid breeding programme at our institute. The standardized protocol for doubled haploids (DHs) production by un-pollinated ovule culture paved the way for Tagetes F1 hybrid breeding. This is the first successful report of in vitro gynogenesis in Tagetes spp for production of doubled haploids (DHs). The protocol for obtaining high frequency parthenogenic embryo induction from unpollinated ovules was standardized and validated for rapid production of homozygous lines.