| Hybridization is one of the most important methods to create genetic variations and develop new varieties.Currently,most of the improved poplar varieties used in forest production are developed by crossing two parents and genetic testing in China.However,when the number of parents is large,the traditional hybridization is challenged by heavy workload and low selection efficiency.In the present study,based on observation of the pollen morphology,we mixed pollen derived from 10 plus trees of Populus tomentosa by equal volume,equal weight and equal numbers,respectively.Subsequently,three F1 offspring populations was obtained by crossing mixed pollen with Populus alba x Populus glandulosa.Then the male parent would be identified by SSR molecular marker for the three F1 hybrids.To study the possibility of mixed-pollen pollination in P.tomentosa,experiments of pollen germination in stigma and in vivo was conduced to determine the differences in pollination competition ability among different clones.The main findings were as follows:(1)There were significant differences in pollen diameter among 10 different clones of P.tomentosa.The average pollen diameter of clone 'lumao 50' was the largest(33.7±3.9?m).However,The smallest average pollen diameter was seen in clone 4123(27.2±3.4 ?n).In this study,there existed low frequency of natural 2n pollen among the 10 clones.The frequency of natural 2n pollen in clone 5017 were the highest(4.00±1.00%).the lowest frequency of natural 2n pollen were found in clone 1340(0.33±0.58%).Moreover,the frequency of natural 2n pollen in clone 5017 were significantly higher than that of clone 1340,5025 and 5042.(2)After pollen was mixed by equal volume,equal weight,equal pollen grain number,respectively,hybrdization was conduced by crossing with Populus alba x Populus glandulosa.The total of seeds derived from the three cross combination was 7685.Subsequently,1423 seedlings were obtained after sowing.The ploidy level of all the survival seedlings was detected by flow cytometer and all the seedlings were found to be diploids,suggesting that natural 2n pollen did not take part in pollination.(3)The male parent of 718 seedlings was identified by using 15 SSR primer pairs with well polymorphism and the ratio of identification was 84.1%.Among three offspring populations,there was significant differences in frequency of seedlings with different male parents.The highest frequency of seedlings derived from clone 'Lumao 50' was observed in regard of mixed-pollen ways,and The frequency of seedlings derived from clone 'Lumao 50' was significantly higher than that of other male clones.The frequency of seedlings derived from clone 1340,6305 and 5025 followed as clone 'Lumao 50'.However,the frequency of seedlings derived from clone 5017 was the lowest.The results suggested that there was heavy pollen competition among the sampled clones in P.tomentosa.(4)2-8h after pollination,pollen grains derived from 10 clones could germinate on the stigma of Populus alba x Populus glandulosa,respectively.With time passing by,the pollen germination rate and the length of pollen tube gradually increased in the same cross combination.Analysis of Variance of the pollen germination rate and the length of pollen tube revealed that clone had significant effect on pollen germination rates and the length of pollen tube.The average pollen germination rate and the length of pollen tube of the clone 'Lumao 50',6305 and 1340 was the highest.However,the lowest average pollen germination rate and the length of pollen tube was found in the clone 5017.This suggested that the pollen collected from the clone 'Lumao 50',6305 and 1340 had advantage over other clones during pollination.(5)Germinating pollen of 10 clones in P.tomentosa in vitro were conducted on solid medium,respectively.5h after being cultured,some germinated pollen grains were seen for all the clones,and there were significant differences in pollen germination rate and the length of pollen tube.Pollen germination rates and length of pollen tube of clone 'lumao 50',1340 and 6305 were significantly larger than that of other clones,suggesting that the numbers of offspring derived from clone 'lumao 50',1340 and 6305 would be more than that of other clones.Therefore,to achieve the desired objective,it was necessary that experiments of pollen germination was conducted to screen the clones with the similar pollen germination rates and length of pollen tube before hybridization of mixed-pollen pollination was done.indicating the key to performance mixed-pollen pollination. |
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