Anther Culture Of Wild Diploid Potato S.verrucosum And Identifition Of Regenerated Plants

Due to high heterozygosity and tetrasomic inheritance,genetic studies and breeding of cultivated potato(Solanum tuberosum L.)is relatively slow.S.verrucosum(PI 275256)is a wild relative of cultivated potatoes characterized by its diploidy and self-compatibility.Using PI275256 as dornor plant,we studied the relationship between bud size and anther developmental stage and the different hormone combinations for regenerated plants induction.A total of 23 regenerates were obtained and subsequent analysis of ploidy and homozygosity was performed.The main results are as follows:1.The optimum bud developmental stage for anther culture was determined.The mononuclear stage of PI 275256,which is best for anther culture,is when its bud length and anther length reaches about 6.0mm and 4.0mm respectively,with the bud color being light green.2.An effective and stable anther culture system was established.We designed 11 media by adding 3 hormones: NAA,6-BA and KT to MSbase in different concentration combinations.The 2 mg/L NAA+1 mg/L6-BA+0.5 mg/L KT+MS,with a callus response rate of 11.88%,was the best medium found in this study.In total,23 regenerated plants were obtained by anther culture.3.The ploidy of regenerated plants was identified.Of the 23 plants obtained,6 were characterized as diploid and 17 were determined as monoploid through guard cell chloroplast counting and flow cytometry.4.The homozygosity of regenerated plants was studied.The homozygosity for regenerated plants was investigated with 5 polymorphic SSRs that located on different chromosomes.As expected,all monoploids were homozygous at 5 loci;surprisingly,6 diploid plants were also homozygous for 5 SSRs.We propose the chance that the 6 diploids being spontaneous doubling of monoploid during anther culture(in which case,the homozygosity is expected to be 100%)is 96.875%.