Study On Pollen Mother Cell Meiosis And Male Gametophyte Development In Autotetraploid And Diploid Broccoli

On the bases of studies on techniques in chromosome preparation, meiotic behaviours of pollen mother cells (PMCs) and development of male gametophyte were observed on autotetraploid and diploid broccoli, which laid bases for genetic breeding of polyploid broccoli.1. Techniques in chromosome preparationTechniques of meiotic chromosome preparation for diploid and autotetraploid broccoli were discussed, among which different sampling materals were required, suitable fixation with Karnuo's solution for 24 hours could improve slide-making quality and stained in decolored base fuchsin and warmed on flame could improve the slide resolution.Clearing with methyl salicylate after staining in Ehrlich's hematoxylin for 24 to 36 hours could display the dynamic process of twice miotic division of nuclear clearly, which was the best technique for the observation of male gametophyte development in broccoli.The roots of germinating seeds were used to study the effects of the root length, pretreating methods and macerating time with 1N HCl on the slide-preparation of the mitotic chromosomes in broccoli. The results revealed that the root long about 0.6 to 1.2cm with good condition have the highest mitotic index of cells. Pretreated with 8-hydroxyquinoline for three hours and macerated with IN HCl under 60℃for 8～10 minutes showed the best effect.2. Meiosis of pollen mother cells Cytological studies were conducted on pollen mother cells (PMCs) meiosis of broccoli using chromosome preparation technique. Results showed that the PMCs meiosis of autotetraploid was accompanied by simultaneous cytokinesis and the tetrads were tetrahedral or decussate type. The PMCs meiosis of autotetraploid was similar to the diploid with its particularity: In metaphaseⅠ, multivalent, quadrivalent, trivalent, bivalent and univalent were observed. Some of the chromosomes were found not to rank at metaphase plate in metaphaseⅠand metaphaseⅡcells. In anaphaseⅠand anaphaseⅡcells, there were lagged chromosomes, chromosome bridge and fragment. During anaphaseⅡand telophaseⅡchromosome segregation was not synchronous and equal. At tetrad stage, dyad, triad, tetrad with micronucleus and polyad appeared. The emergence frequency of abnormal phenomena in each stage of meiosis had been counted, it was 31.5% in autotetraploid and only 10.2% in diploid, which indicated that the genetic stability of autotetraploid broccoli was much lower than that of diploid. Experimental results of artificial germination of pollens showed that the pollen germination rate was only 46.2%, which indicated that abnormal meiosis of PMCs was the cytogenetic reason of fertility reduction of autotetraploid broccoli pollen.3. Development of male gametophyteThe development of male gametophyte in autotetraploid broccoli was observed using Ehrlich's hematoxylin staining-methyl salicylate clearing technique. Results showed that the male gametophyte in autotetraploid broccoli shared the similar development process with diploid broccoli, through which one vegetative cell and one generative cell were produced by one miotic division, and the generative cell would divide into two sperms by the other miotic division and in the end 3-celled mature pollen with four or three apertures was formed. Abnormality appeared mainly in uninuclete stage of male garnetophyte development with an average frequency of 12.14%, which was the important reason for fertility reduction of autotetraploid broccoli pollen.