Efficient Exploration Of Tetraploid Seedlings From 11 Local Citrus Cultivars And Production Of Triploid Plants

Based on the presence of spontaneous doubling of nucellar cells in citrus polyembryonic cultivars,using the morphological characteristics of polyploidy seedlings combined with rapid ploidy analysis of flow cytometry is an efficient approach to explore tetraploids of citrus.The interploid crosses between diploid and tetraploid combined with immature embryo rescue techniques and rapid determination of ploidy are the most classic strategy in citrus ploidy breeding.Based on the above methods,this study intended to efficiently explore tetraploid germplasm resources by using 11 local cultivars such as Wenling Gaocheng;in addition,nine interploid crosses using five monoembryonic diploids such as Huanglingmiao tangerine,and two polyembryonic diploids(W.Murott tangor and Bendizao mandarin)as the female parents and six tetraploids(five autotetraploids and one allotetraploid somatic hybrids)as male parents were used to produce triploid seedlings.The main results are as follows:1.Exploration of tetraploids from 11 citrus local cultivars and germplasm resourcesFollowing the germination and sowing of mature seed,through the primary identification of seedling morphology,4,7,10,88,7,24,8,10,11,36 and 20 putative tetraploid seedlings were screened out from the 72,373,303,1992,465,890,277,315,709,1259 and 1251 seedlings of 11 local cultivars including Wenling Gaocheng,Xinhui sweet orange,Quzhou Xiangcheng,Sour orange,Shitougan,Changshan Huyou pummelo,Nova tangelo,Page tangelo,a hybrid of blood orange with Clementine,Bendizao tangerine × VM and a cybrid(Bonanza navel orange + Wampee).After further confirming the ploidy level of these putative tetraploids by flow cytometry,a total of 76 tetraploids,of that 2,4,6,24,1,14,6,2,3,12 and 2 tetraploids were obtained respectively.By analyzing the genetic origins of these tetraploids using simple sequence repeat(SSR)markers,it showed that the genetic profiles of 72 tetraploids seedlings were identical with that of their diploid counterparts,indicating all these 72 seedlings were originated from chromosome doubling of nucellar cells of their diploid parents.And the remaining two tetraploids from Changshan Huyou pummelo and two tetraploids from Bendizao tangerine × VM showed some bands different from their corresponding diploid,indicating they might be originated from sexual hybridization with unknown pollen parents.2.Production of citrus triploid new germplasm based on the nine 2X × 4X interploid crosses1)Using monoembryonic diploid varieties as the female,six 2X × 4X interploid crosses were used to produce triploids.Five monoembryonic diploids(Hongmeiren tangerine,Fallglo tangor,Huanglingmiao tangerine,Kiyomi tangor and Nules clementine)were used as the female parents and crossed with four tetraploids,including three autotetraploids(4X PK,4X Suc and 4X BDZ)and one allotetraploid somatic hybrids(SP).At 85 days after pollination(DAP),372 young fruits were collected and 5849 immature seeds were extracted.By culturing them in vitro using embryo rescue techniques,363 regenerated plants were obtained.Aftering determining their ploidy level using flow cytometry,it showed that a total of 233 triploid plants and 6 tetraploid plants were obtained,including two triploids from ‘Fallglo × 4X BDZ’,two triploids from ‘Nules clementine ×4X PK’ 180 triploids and two tetraploids from ‘Fallglo × 4X Suc’,six triploids and one tetraploid from ‘Huanglingmiao tangerine × SP’,43 triploids and one tetraploid from‘Kiyomi tangor × 4X PK’ and two tetraploids from ‘Hongmeiren tangerine × 4X PK’,respectively.Analyzing the genetic origins of 77 triploids and 2 tetraploids from ‘Fallglo ×4X Suc’ using simple sequence repeat(SSR)markers,it showed that they were all hybrids of their both parents.2)Using polyembryonic diploid varieties as female parent,three 2X × 4X interploid crosses were used to produce triploids.Two polyembryonic diploids(W.Murcott tangor and Bendizao tangerine)as the female parent and two autotetraploids(4X HJ and 4X RH)as male parents were used to produce triploid plants.A total of 1020 flowers were pollinated and 124 fruits were set.The average fruit setting rate was 13.44%.By conducting in vitro immature embryo rescue,totally 799 plants were regenerated from the 1078 immature seeds.Using flow cytometry to determine their ploidy level,13 seedlings were proven to be polyploids,including six triploids and five tetraploids from ‘W.Murcott tangor × 4X HJ’,one triploid from ‘W.Murcott tangor × 4X RH’ and one triploid from ‘Bendizao tangerine × 4X HJ’.