Induction Of 2n Pollen And Creation Of Triploid Germplasm Of Longan And Its Intergeneric Hybrids

Longan（Dimocarpus longan Lour.）is an evergreen tree of the Dimocarpus Genus,which originated in the tropical area of South China.It is an important economic fruit tree in South China,and has rich nutritional value.But the factors of small fruit,low edible rate and lack of seedless varieties in the market restrict the development of longan to a great extent,so it is of great significance to cultivate seedless longan through triploid breeding.In this study,we used the main longan cultivar‘Shixia’and the longan and litchi intergeneric hybrid‘Shizi 19’created by our research group as materials,and for the first time,we created stable triploid by inducing 2n pollens to cross sexually.The main research contents are as follows:（1）In order to determine the best induction period,we studied the relationship between the microspore mother cells meiosis and the diameter of male flower bud.The results showed that when the diameter of male flower bud was between 1.5 mm and 1.8mm,the majority of microspore mother cells were in the stage from pachytene to diakinesis.At this time,the induction effect is the best.（2）In the best induction period,colchicine cotton dip,colchicine injection and high temperature induction were used to treat male flower buds,in order to find the best 2n pollen induction combination.The results showed that among the three methods,the damage of male flower bud induced by high temperature induction was small and the 2n pollen rate of the pollen obtained was the highest,which was 7.95%,it was 6.74 times that of the control.（3）According to the nature that 2n pollen volume is 1.28 times of normal pollen,by adjusting threshold and PMT value of each parameter,flow cytometry was used to establish an experimental scheme for rapid detection of 2n pollen rate in pollen,The results showed that when the threshold value was 1038,the PMT values of GFP,SSC and FSC were 40.05,28.04 and 2.5 respectively,the particle signal cluster was obvious and stable.Compared with the results of diameter measurement by microscope and abnormal proportion statistics of tetrad stage,the results showed that there was no significant difference in 2n pollen rate obtained by the three methods,indicating that the method of flow cytometry is not only convenient and fast,but also reliable.（4）An experimental method of sorting 2n pollens of longan was established by flow cytometry.The results showed that the sorting rate was fast,the average diameter of the sorted pollens was 1.28 times that of the unselected pollens,the 2n pollens rate was at least5 times higher than that before screening,and 29.25%of the pollens retained the ability of germination.（5）Using the selected 2n pollen to artificially pollinate the longan female buds with a brush during the blooming of female flowers,884‘Shixia’artificial inbred offspring and2778‘Shizi 19’artificial inbred offspring were obtained.A preliminary ploidy identification of the artificial inbred offspring was performed using a root-tip chromosome counting method,and 4‘Shixia’inbred triploid offspring and 3‘Shizi 19’inbred triploid offspring were found.（6）The ploidy of 6 triploid longan and its intergeneric hybrids was determined by flow cytometry.The results showed that the fluorescence mean value of the peak of G₀+G₁was about 1.5 times of that of diploid longan and its intergeneric hybrids.The results showed that the 6 triploid of longan and its intergeneric hybrid were true triploid.For the first time,the triploid of longan and its intergeneric hybrid was created by inducing 2n pollen and combining with flow cytometry,which is of guiding significance for longan to create polyploid.