目的 观察钝叶决明Senna obtusifolia花粉粒形态,测定花粉活力,确定花粉萌发最适条件和花药诱导培养产生诱导愈伤组织的培养条件及其倍性鉴定,为决明单倍体育种奠定基础。方法 采用小孢子压片显微观察花粉粒形态;I₂-KI染色法观测花粉活力并确定活力最大的花蕾大小;液体培养结合显微观察确定花粉萌发的最适培养液组成、温度和萌发时间;醋酸洋红染色法确定小孢子发育时期;花药离体培养确定产生愈伤组织的条件;改良苯酚品红染色法鉴定愈伤组织倍性。结果 钝叶决明花粉粒呈椭球形并具有3条萌发沟,多为2孔萌发;花蕾纵径为1.0~1.1 cm时,花粉的活力最高;花粉萌发的最适培养液是10%蔗糖+1%硼酸,以25℃培养1~3 h为最佳萌发期;小孢子处于单核靠边期时花蕾纵径为0.9~1.1 cm;愈伤组织诱导培养基组成为MS+1.0 mg/L 6-BA+2.0 mg/L NAA+3%蔗糖+0.6%琼脂;愈伤组织增殖培养基组成为MS+1.0 mg/L 6-BA+1.0 mg/L IAA+1.0 mg/L 2,4-D+1.0 mg/L GA₃+3%蔗糖;愈伤组织存在倍性分离现象,具有单倍体和二倍体细胞。结论 钝叶决明花粉萌发的最适培养液是10%蔗糖+1%硼酸;花药离体培养最佳时期为花蕾纵径为0.9~1.1 cm,获得的嵌合体花药愈伤组织为进一步进行决明单倍体诱导和育种奠定了基础。

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.

作物种质资源利用与创新基地建设(104510-205001);国家大学生创新实验训练项目(201310635024)