Production Of Citrus Triploid Plants By Interploid Crossing With Twelve Tetraploids As Pollen Parents

Seedless citrus fruit with high quality is one of the major goals of citrus breeding.Due to abnormal meiosis,citrus triploids can't form a normal gamete type and are usually infertility.Ploidy hybridization is one of the effective ways to obtain triploid seedless citrus.This study mainly used some seedy diploid varieties as female parents to cross with allotetraploid somatic hybrids and double diploid,aiming to generate triploid progenies with three different genomes through embryo rescue techniques.The main experimental results are as follows:1.Using monoembryonic diploid varieties as female parent to produce triploids by 2X × 4X.Eleven interploidy crosses were carried by using six monoembryonic diploid cultivars as female parents,and seven tetraploids as pollen parents.4526 seeds were obtained from 299 pollinated fruits and 362 regenerated plants were obtained after embryo rescue.285 triploids and 2 tetraploid were verified by flow cytometry analysis,incidences of triploids and tetraploid were respectively 78.73% and 0.55%.At present,all of the survived polyploid plants have been transplanted to the greenhouse,and most of the plants have been grafted to the field.7 pairs of SSR primers with good polymorphism were selected to identify 36 regenerated plants of three combinations.The results showed that 16 diploid plants were non-hybrid offspring,20 triploid plants were sexual offspring of both parents.2.Using polyembryonic diploid varieties as female parent to produce triploids by 2X × 4X.Eleven hybrid combinations were conducted by using four polyembryonic diploid cultivars as seed parents and nine tetraploids as pollen parents.8181 immature seeds from 829 fruits were sowned in vitro.After ploidy identification,185 plants were proved to be polyploids from 2697 regenerated plants,including to 107 triplods and 78 tetraploids,incidences of triploids and tetraploids were respectively 57.84% and 42.16% among polyploids in 2015-2016.The survived polyploid plants have been transplanted to the greenhouse.2122 seeds of 223 young fruits were germinated using the hybrid combination of W.Murcott tangor as the female parent in 2017.However,most of embryoids are at the stages of inducing shooting and rooting induction,polyploids has not been obtained at present.During the Embryo rescue,embryogenic calluses were initiated from the hypocotyls of plants and young embryoids,and the calluses of different paternal types had different embryogenic ability.The embryogenicity of callus was gradually weakened in the process of cultivation on the MT solid medium.After ploidy identification,the calluses derived from twelve different plants were diploids and one line of callus was a mixture of diploid and triploid.In addition,genetic heterozygosity of callus needs to be identified subsequently.At present,these embryogenic calluses have been used as experimental materials for related studies in our research group.3 pairs of SSR primers with good polymorphism were selected to identify 30 regenerated plants of Bendizao tangerine × 4X Ponkan.The results showed that 29 triploid plants were sexual offspring of both parents,and 1 tetraploid plant was a double diploid from doubled parental nucellar cells.3.Production of citrus germplasms by 2X × 2X crossing.Two hybrid combinations were conducted by using W.Murcott tangor as seed parents and two diploid varieties as pollen parents.987 seeds from 476 young fruits were cultured by embryo rescue.Nine triploids and two tetraploids were obtained from 90 regenerated plants of W.Murcott tangor × Red tangerine,and the incidence rates were respectively 10.00% and 2.22%.Embryoids of W.Murcott tangor × Egan NO.1 Ponkan are still at the stage of shooting and rooting induction.276 seeds of Fortunella hindsii × Blood orange hybrid were sown in vitro,67 regenerated plants have been transplanted to the greenhouse.