Induction And Molecular Identification Of Citrus Doubled Haploid Via Anther And Micropore Culture

With the rapid development of high-throughput sequencing technology,homozygous genotypes were much focused for its simplicity to analyze.Citrus is one of the most important fruit crops in the world.Although homozygous germplasm was obtained for a few genotypes,the populations are small and the genetic background are similar.Therefore,expanding the group of citrus homozygous genotypes remains to be challenged.Because of self-incompatibility,high heterozygosity of the genome and long duration of the generation cycle in most citrus species,the conventional inbreeding is not applicable to produce homozygous lines of Citrus.Anther culture as a commonly used technique for citrus homozygosis breeding is a better choice to expand citrus homozygous groups.Isolated microspore culture provides the possibility to observe the process of embryo regeneration intuitively,and effectively reduces the workload of subsequent screening,and has been applied to many species.The present study aims to expand the citrus homozygous groups through anther culture.Isolated microspore culture was conducted with various modification.Ploidy analysis was conducted for the existing homozygous lines of sweet orange.The results are as follows:1.85 regeneration embryoid were obtained by anther culture of nine citrus varieties,including 32 of trifoliate,24 of Mexican lime,23 of Early flowering lemon,three of‘ Early Gold' sweet orange,one each of ‘Anliu' sweet orange,‘Rhode Red' Valencia sweet orange and ‘Red Anliu' sweet orange,respectively.Of them,only 46 embryoids regenerated buds.Some of the buds were grafted on trifoliate by shoot-tip grafting and then transplanted into the greenhouse,others are still in subculture.DNA was extracted from some embryoids,to identify genotypes by SSR molecular marker.As a result,the embryoids of trifoliate,‘Red Anliu' sweet orange and ‘ Early Gold' sweet orange are all heterozygous,whereas those of Mexican lime and Early flowering lemon are homozygous.In addition,the regeneration plants are all diploids.By comparing the effects of bud size,flowering time and duration of low temperature pretreatment onregeneration rate of anther culture,we found that the regeneration rate of Early flowering lemon and Mexican lime,which flowered early in December and January,was significantly higher than trifoliate that flowered in March and the six other citrus varieties that flowered in April.The highest regeneration rate occurred in low temperature pretreatment at 7-8 days.2.Isolated microspore culture was used on 10 citrus varieties with culture conditions modified as follows:(1)using three different pretreatment liquids on the basis of the 0.4 mol/L mannitol;(2)using various modifications of MCM or BH3 basal medium;(3)setting four cell culture densities.It was found that 0.4mol/L mannitol containing 1% DMSO increases the possibility of microspore cells developing into dense cell mass as embryoid,whereas the other treatments showed insignificant effects.Dense cell mass can not continue developed.3.Clear chromosome images were obtained by chromosome section of seven homozygous callus lines of ‘ Early Gold' sweet orange.It was found that the double haploid callus lines are easier to doubling than haploid callus.The chromosome images of homozygous callus can be used for subsequent homozygous cytologic research.