ABSTRACT
Objective: To observe the morphology, measure the activity of pollen of Senna obtusifolia, determine the optimum conditions for pollen germination, induce the formation of callus from its anthers as well as identify its ploidy levels, and thus to lay a basis for its haploid breeding. Methods: Microscopic observation of the anther squashed method was used to reveal the morphology of pollen; The pollen activity was investigated using I2-KI staining and the size of the flower buds with the highest pollen activity was determined; Liquid culture and microscopic observation were performed to determine the optimum culture medium, pH value, and temperature for pollen germination; Microscopic observation of the acetocarmine-stained pollen was performed to determine microspore development stages; The optimum conditions for callus formation were studied by in vitro anther culture; The phenol fuchsin staining was improved for the identification of callus ploidy levels. Results: Pollen of S. obtusifolia is ellipsoid with three germinal furrows, and mostly two germination holes showed germination; The highest pollen activity was found in buds with vertical diameters about 1.0-1.1 cm; The optimum culture medium for pollen germination was determined to be 10% sucrose + 1% boric acid, with maximum germination cultured for 1-3 h at 25 ℃; The uni-nucleate microspore stage was in buds with vertical diameters about 0.9-1.1 cm; Calluses were successfully obtained by culturing the uni-nucleate stage anthers in an inducing medium MS + 1.0 mg/L 6-BA + 2.0 mg/L NAA + 3% sucrose + 0.6% agar and callus enrichment culture was done in MS + 1.0 mg/L 6-BA + 1.0 mg/L IAA + 1.0 mg/L 2,4-D + 1.0 mg/LGA3 + 3% sucrose; There was ploidy separation in callus, with the haploid and diploid cells coexisted. Conclusion: The optimum culture medium for pollen germination is 10% sucrose + 1% boric acid; The optimum anthers for in vitro culture is within flower buds with a vertical diameter of 0.9-1.1 cm; The chimeric calluses obtained from anthers of S. obtusifolia lay a solid foundation for the further induction of haploid plants from its pollen for breeding.