Standardization of microspore developmental stages and plant growth regulators for induction of haploid through anther culture in Chrysanthemum genotypes
摘要
Haploids and doubled haploids are essential for creating new varieties that alter resistance, productivity, and various features in ornamental plants like Chrysanthemum morifolium Ramat. The current study is aimed at standardizing the observable morphological markers for each flower bud stage in Chrysanthemum varieties, viz., Pusa Guldasta and Akhite, and different combinations of plant growth regulators to improve the efficacy of androgenesis. Anthers of average length ranged from 0.92 to 1.22 mm in the case of Pusa Guldasta and 1.01 mm to 1.32 mm in the case of Akhite, carrying 70–90% microspores at vacuolated late uni-nucleate and early bi-nucleate stage. To improve the visualization of nuclei and reduce the wall autofluorescence, various concentrations of FeCl₃ were added as a mordant to Carnoy’s fixative, followed by DAPI staining. The effect of Triton X-100 was also examined by incorporating it in DAPI stain to enhance membrane permeabilization, which gave uniform staining and acceptable contrast. Among the various combinations of growth regulators, anthers cultured in media containing 1.0 mg L−1 TDZ along with 0.2 mg L−1 NAA showed a significantly high androgenic response. By flow cytometric analysis, we characterized 14.28% of plants as tri-haploids, 57.14% as hexaploids (same ploidy level as the parent), and the remaining 28.57% were mixoploids (higher ploidy level). The study has demonstrated the most reliable method for accurately visualizing the stages of microspores along with combinations of plant growth regulators, which is necessary for a successful anther culture in Chrysanthemum. Besides, this study will be crucial for incorporating molecular and genetic studies, and advanced biotechnologies for genetic improvement in Chrysanthemum.