Tetraploid Induction And Karyotype Analysis Of Populus Deltoides × Populus Ussuriensis

The fresh leaves,petioles and stems of P.deltoides × P.ussuriensis were used as explants,colchicine was used as mutagen,and the polyploid of P.deltoides × P.ussuriensis was induced by mixed culture method and immersion method combined with tissue culture technology.The mutation system suitable for mixed culture and immersion induction of polyploid of P.deltoides × P.ussuriensis was established.Meanwhile,the changes of growth,morphology,cytology and physiological indexes of the mutant plants were studied with untreated diploid as control,and the chromosome preparation of P.deltoides × P.ussuriensis was optimized.The main results are as follows:1.Different mutagenic methods and explants had different effects on polyploid induction.In the solid mixed culture method,stems were used as explants,placed on the medium containing 50 mg/L colchicine for 2 d,the highest polyploid induction rate was 16.67%;in the liquid mixed culture method,leaves and stems were used as explants,placed on the medium containing 30 mg/L colchicine for 2 d and 1 d,the highest polyploid induction rate was 13.33%.The stems were used as explant and soaked in colchicine solution of 0.4%and 0.6%for 6 h and 12 h,the highest polyploid induction rate was 6.67%.Among them,the order of total induction rate of different explants from large to small was stem,leaf and petiole;the order of total induction rate of different induction methods from large to small was solid mixed culture method,liquid mixed culture method and immersion method.2 Flow cytometry showed that the DNA content of single cell in the leaves of the mutant plants was twice that of diploid plants,which proved that the mutant plants were tetraploid.the root system of tissue culture seedlings was more developed and grew faster;the length,width and area of leaves became larger,the ratio of length to width decreased,the veins and petioles became thicker,the surface of leaves became rough;the stomata and guard cells became larger,the stomatal density became smaller,the number of chloroplasts increased;the chlorophyll content of leaves increased.3.The effect of chromosome preparation of P.deltoides × P.ussuriensis was greatly affected by root length,pretreatment method,dissociation time and temperature.When the root length was 1.5 cm,more metaphases were obtained and more metaphase cells were suitable for karyotype analysis.The chromosomes were pretreated with p-dichlorobenzene saturated solution at 0℃ for 2 h,which have clear morphology,good dispersion and suitable length.The dissociation was performed with 1mol/L hydrochloric acid at 60℃ for 10 min or at 25℃ for 18 min,which was easier to press and obtained better chromosome morphology and structure.The karyotype analysis showed that the diploid chromosome number was 38,the karyotype formula was 2n=2x=38=32m+6sm,the nuclear asymmetry coefficient was 56.61%;the tetraploid chromosome number was 76,the karyotype formula was 2n=4x=76=64M+12sm,the nuclear asymmetry coefficient was 57.43%;both of them found satellites on the short arm of the third group of chromosomes,the karyotype was 1 B.