玉米花粉与唐菖蒲花粉原生质体中肌动蛋白微丝的荧光显微观察

FLUORESCENCE MICROSCOPIC OBSERVATIONS ON ACTIN FILAMENT DISTRIBUTION IN CORN POLLEN AND GLADIOLUS POLLEN PROTOPLASTS

用异硫氰酸荧光素-鬼笔碱(FITC-phalloidin)作为显示肌动蛋白的荧光显微探针,观察了玉米花粉和唐菖蒲花粉原生质体中微丝分布格局的变化。玉米花粉水合后微丝呈不规则网络状。萌发前转变为极性排列状态,由与萌发孔相对一极向萌发孔极辐射,并汇集于萌发孔处。在部分花粉中观察到与萌发孔相对一极有一个微丝聚集的中心。唐菖蒲花粉原生质体刚分离后有不规则的微丝细网。培养4—7小时,在部分原生质体中观察到微丝分布与再生新壁的位置吻合。培养17—18小时,部分原生质体萌发正常的花粉管,此时原生质体中微丝呈极性分布并向花粉管汇集;花粉管中微丝呈纵行排列,其格局与由花粉粒萌发的花粉管相似。当原生质体表现不正常时,微丝呈现各种异常的分布。

Actin filament(AF)distribution in Zea mays pollen and Gladiolus gandavensis pollen protoplasts was localized by FITC conjugated phalloidin fluorescence microprobe.The pollen was incubated in Brewbaker and Kwack(BK)medium,and the pollen protoplasts were isolat- ed enzymatically and cultured in K3 medium containing various supplements by a previously reported method.Samples were fixed for 30 min with 1.5% paraformaldehyde dissolved in 0.1 tool/l phosphate buffer(pH 7),half strength of BK elements,1 mol/l EGTA and sucrose, stained for 30—60 main with 1 μg/ml FITC-phailoidin in the buffer solution,and observed by a fluorescence microscopy.In hydrated corn pollen grains,the AFs constituted an irregular network.Prior to germination a part of the pollen grains showed polarized pattern of AFs. At the opposite pole to the germ pore,there was a center from which AF bundles radiated and converged toward the pore,often making a spndle-shaped configuration.In just isolated gladiolus pollen protoplasts,the AFs appeared as irregular fine network.After 4—7 h of cul- ture,the AF distribution coincided in some cases with the unevenly regenerated new wall area as exhibited by FITC-phalloidin and Calcofluor White ST double staining,indicating a pos- sible involvement of AF in wall synthesis.After 17—18 h of culture,a part of the pollen protoplasts went on germination.The AFs became polarized in such protoplasts and converged into the tubes produced,and ran longitudinally along the tubes just like in the tubes germinated from pollen grains.However,in ungerminated pollen protoplasts,the AFs behaved abnormal- ly,showing various irregular arrangements.When protoplasts bursted,the actin aggregates of- ten located at the protrusion site from which the protoplasts would burst,and were discharg- ed into the medium.In neither corn pollen nor gladiolus pollen protoplasts AFs were observed within the generative or sperm cells.