Application Of Sporogenous Cells In Chromosome Of Male Mulberry Resources And FISH Analyses

Mulberry is a perennial woody plant belonging to Morus L.As an important ecological and economic tree,mulberry has a long planting history in China.The cytological study of mulberry began at the beginning of the last century mainly focused on the number of the chromosome instead of the karyotype analyses.As a carrier of genetic material,the chromosomes play the important roles in transmission of genetic information.The karyotype analyses of mulberry can gain the cytological datas which are useful for the classification and the breeding of mulberry,but also have important significance for studying the origin,phylogeny and phylogenetic relationship of mulberry.The mulberry chromosomes of are not only small in shape but also large in number.The results of the traditional method of chromosome identification had not been good enough,which made the study of mulberry cytology greatly restricted.The application of wall removal and hypotonic method facilitated the study on the cytology of mulberry.The materials commonly used in mulberry chromosome ploidy research are callus,young leaves,shoots,root tips and shoot tips.The characteristic of low cell division index of chromosomes in mulberry vegetative organs is responsible for less chromosome splitting phase in the metaphase using experimental materials mentioned above,and thus hinders the futher study.However,in the plant sporulation period,archesporial cells form a lot of cells that are vigorous to split through several mitosis.These cells are sporogenous cells.In these cells,the mitotic phase of the metaphase chromosome is easily obtained after being treated by 8-hydroxyquinoline.Clearer split phase can be observed.Since of these unique characteristics,sporogenous cells are one of the optional materials of mulberry chromosome research.In this study,young leaves and sporogenous cells in male flowers were used as experimental materials to prepare metaphase chromosomes.The number and ploidy analyses of various mulberry germplasm resources were verified.Fluorescence in situ hybridization was carried out in seven selected tetraploid mulberry germplasm resources.25 S rDNA was used as probes.The number of 25 S rDNA loci were used to presume whether 25 S rDNA could act as the criterion or not to differentiate the autotetraploid or allotetraploid of mulberry.The main results are as follows: 1.The chromosome number and ploidy analyses of mulberry young leavesThe most vigorous young leaves were taken as materials,and the samples were prepared by method of enzymolysis wall removal and hypotonic treatment.The leaves were pretreated with 8-hydroxyquinoline at room temperature for 3 hours in dark and then digested by the mixture of 5% cellulase and 5% pectinase in a 1:1 volume ratio for 3 hours to get the best samples.The number of chromosomes in 23 mulberry germplasm resources were analyzed.It was found that mulberry germplasm resources have 4 kinds of the number of chromosomes with 28,35,42,84 in the somatic cells,but mulberry germplasm resources with 28 chromosomes in the somatic cells were the most abundant.2.The chromosome number and ploidy analyses of mulberry sporogenous cells in male flowersThe young flowers in the budding period were as the materials,and the chromosome samples were prepared by the method mentioned above.The pretreatment was carried out in the same was described above,but the enzymolysis time was extended to 6 hours to get better samples.The chromosome numbers of 11 mulberry male germplasm resources were analyzed.The results indicated that 9 mulberry germplasm resources contained 28 chromosomes,and a mulberry germplasm resource had 42 chromosomes.In addition,mulberry germplasm resource with 126 chromosomes was found for the first time.3.FISH analyses of 25 SrDNA sequence in mulberry chromosomesThe metaphase chromosome splitting phase samples were prepared using the sporogenous cells from Yunnan wild male mulberry germplasm resources(No 3,No 4,No 5,No 12,and No 14),Nongsang 14 and Wupisang were aslo used.The cytological studies of these materials were carried out using the 25 S rDNA repeat sequences as probes for the fluorescence in situ hybridization.It was found that the 25 S rDNA signals in these mulberry resources were 2,suggesting that the number of hybridization signals of 25 r DNA may not be able to be used as a criterion to estimate the homology or heterogeneity of mulberry tetraploid materials.