Techniques Of Triploid And Teraploid Induction In Populus Adenopoda Maxim

Populus adenopoda Maxim, is a native tree species that is often used as timber sources to the mountainous regions of southern China. Polyploid breeding has been proven to be a valuable approach in populus improvement. In the present paper, P. adenopoda was employed as polyploid breeding materials, in order to provide new populus varieties of puplpwood plantation for those regions. The occurrence of spontaneous2n pollen and its cytological mechanisms, chromosome doubling of female gametes and zygote of P. adenopoda were studied by combining the cytogenetics and techniques of polyploid breeding. And these investigations provide a platform for polyploid breeding of P. adenopoda. The main results are described as follows:1. The production of spontaneous2n pollen in P. adenopoda was firstly investigated in China, and its cytological mechanisms were revealed. Nine clones of P. adenopoda collected from four provinces could produce2n pollen at different frequency, and varying from0.49%to12.56%. The meiosis analysis of anthers by squashed technique and indirect immunofluorescence showed that the non-disjunction of sister chromatid resulted from lack of spindles microtubule in anaphase Ⅱ, and the partial failure of cytokinesis caused by the absence of phragmoplasts in telophase Ⅱ, were the main cytological mechanism for2n pollen formation in P. adenopoda.2. Based on the relationship between flower bud morphological characteristics and the female meiotic stage of P. adenopoda, the female bud at different morphological characteristics were treated with heat and colchicine to induce megaspore chromosome doubling. In the resulting progeny,19triploids were obtained. The optimal periods and treatment intensity with high temperature and colchicine in P. adenopoda were determined respectively. When approximately one fourth of the catkins protruded from the bract scales, cytological observation revealed that most megaspore mother cells were at the period from diplotene to diakinesis, was the optimal period for megaspore chromosome doubling induced by high temperature. The treatments with38-41℃for4-6h were efficient, and the highest efficiency of triploid production was40.0%. When the catkins emerged from bract scales slightly, cytological observation showed that major cells were in pachytene stage, which was the optimal period for megaspore chromosome doubling induced by colchicine. The appropriate treatment condition was immersed the flower buds with0.3%colchicine solution for24h.3. On the basis of the optimal pollinated time was determined in P. adenopoda, the pollinated female catkins during embryo sac development were treated with high temperature and colchicine to induce embryo sac chromosome doubling. In the offspring seedlings,381triploids were detected. The optimal treatment periods and treatment conditions for embryo sac chromosome doubling by high temperature and colchicine in P. adenopoda were investigated respectively. When high temperature was employed to induce embryo sac chromosome doubling, the treatments with38℃for4-6h or with41℃for2-4h during18-24h after pollination were both effective, and the highest frequency of triploid production was83.33%. Furthermore, correlation analyses combined with cytological observation showed that the percentage of triploid production was positively correlated with the rate of uni-, two-and four-nucleate embryo sacs, revealed that three mitotic divisions in embryo sac development provide chances for embryo sac chromosome doubling induced by high temperature. When the colchicine solution was used, the optimal treatment combination was immersed the female catkins during12-18h after pollination with0.3%colchicine solution for24h. Further correlation analyses combined with cytological observation showed that the percentage of triploid production was positively correlated with the rate of uni-and two-nucleate embryo sacs.4. The combination between pollination time of female catkins and developmental changes of the cottonlike fiber in ovaries were established to mark the zygote division stage of P. adenopoda, and43tetraploids were produced by inhibiting the first zygote division with high temperature exposure and colchicine. When treated by high temperature, the cottonlike fibers in the ovary start to enclose the funiculus was the efficient state for zygote chromosome doubling, and the highest rate of tetraploid production was14.12%by treating the catkins with44℃for2h. When treated with colchicine, the female catkin5d after pollination and the cottonlike fiber in the ovary did not occur was optimal stage for zygote chromosome doubling, and soaking the pollinated catkins with0.3%colchicine solution for30h was effective.5. The comparative study on the stomatal features, leaf characteristic, photosynthetic index and endogenous hormone between triploids and diploids of P. adenopoda was conducted. The results showed that the stomatal size, leaf size, photosynthetic rate and the content of stimulated hormones in triploid offsprings were higher than that of the diploids in average. However, the concentration of intercellular CO2and the content of inhibited hormones in triploids were lower than that of diploid controls.6. The investigation of stem height and basal diameter of two-year-old diploids and triploids of P. adenopoda revealed that triploids had significant advantages in growth compared with diploid plants. Cross combination between Guiyang and Hunan, the average values of stem height and basal diameter in triploids exceeded the diploids by17%and38%, respectively. In Guiyang cross combination, the average values of stem height and basal diameter in triploids exceeded the diploids by44%and47%, respectively.