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.

标准化了使用未授粉的胚珠作为外植体从两个万寿菊杂合子雄性不育系生产单倍体的方案。影响体外雌激素反应的各种因素。，在直立塔吉木中评估生长调节剂，胚珠发育阶段，基础培养基，蔗糖浓度，光周期和冷休克持续时间。当未授粉的胚珠在补充有2 mg l ^-1 BAP和0.5 mg l的EMS（富含椰子水，AgNO ₃，PVP等的MS培养基）中培养时，单性生殖胚发生在品种'Local Orange'中。^-1 NAA或2 mg l ^-1 BAP以及2 mg l ^-12,4-D。花芽的发育阶段对于从切下的胚珠诱导胚胎至关重要。与从未开放和完全开放的花芽中收集的胚珠相比，从半开放的花芽中收集的胚珠对雌核发育更敏感。与其他市售基础培养基相比，发现EMS基础培养基最适合女性生殖。在4ºC下对花蕾进行冷预处理没有刺激作用，并且对雌核发育产生了负面影响。暗培养条件对于直接孤雌性胚胎诱导比16 h光照持续时间势在必行。通过细胞学研究确定了17个再生体的倍性水平，其中二倍体为47.06％，单倍体为41.18％，混合倍体为11.76％。通过流式细胞仪分析再次证实了这些结果。还建立了通过计算万寿菊气孔保卫细胞中叶绿体数量来确定倍性水平的方法，用于快速筛选单倍体。产生的单倍体已成功地二倍体化，并已在我们研究所的杂交育种计划中得到利用。非授粉胚珠培养产生双倍单倍体（DHs）的标准化方案为万寿菊F ₁杂交育种。