Thin-Layer Lactose Agar for Pollen-Tube Culture ofTradescantiaTo Enhance Planar Distribution of Chromosomes

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Abstract
Mature pollen grains of Tradescantia paludosa were sown on a thin layer of lactose-agar medium at 38-39 C. After 16 hr incubation, slides were fixed in aceto-alcohol (1:3; for 1-3 hr, hydrolyzed in 1 N HC1 at 60 C, and treated with water at 65 C. Delamination of the upper layer of medium was accomplished in cold water. The delaminated upper layer of solidified medium containing most of the ungerminated pollen and underdeveloped pollen tubes was flushed off with running water. The remaining single layer of pollen tubes was flattened and firmly affixed to the slide by pressing under a coverglass on a hot surface at 80 C. The quick-freeze technique was used to remove the coverglass prior to staining, dehydration and permanent mounting. Preparations made according to this procedure gave a planar chromosome distribution and approximately 160 analyzable metaphase figures per slide, thus facilitating aberration analysis and autoradiography. Comparative studies on the effectiveness of colchicine, Colcemid, and acenapthene in arresting mitotic nuclei at metaphase indicated that Colcemid was more effective than the others, but that it caused deformities of the metaphase chromosomes and induced chromatid breaks at a concentration of 0.2 mg/ml or higher. Colchicine is a more favorable chemical than the other two.