Development Of New Apple Germplasms By Anther Culture

Apple(Malus × domestica Brokh.)is one of the most important world-wide fruit with high degree of heterozygosity and self incompatibility.At present,the major apple cultivars derived from grafting varieties contains rich genetic resources that are ideal as parental materials.Haploid breeding through embryogenesis during anther culture is an efficient way to regenerate new homozygous plantlets in short period.The regenerated plantlets greatly enriched the present apple breeding parental germplasm and play important roles in identification of favorable traits related genetics as well as providing a solid basis for further apple new cultivars breeding.In this study,a few major apple cultivars were investigated including cultivar ‘Gala',‘Fuji' and ‘Danxia'.The uni-nucleate to early bi-nucleate phase anther(not open bud)were collected for subsequent experiments.Firstly,the effects of low temperature treatment on the expression of glucose transporter SWEET gene were examined.The expression levels of MdSWEET1(expressed in apple)were detected using quantitative PCR,at the time points of 10 and 30 days of low temperature treatment.Next,the apple anthers after 30 days of low temperature treatment were selected for subsequent in vitro anther culture.Genotypes of the regenerated plantlets were identified using apple SSR markers.Finally,the morphological characteristics of the regenerated germplasm were analyzed.The results are as follow:1.The Q-PCR analysis showed that expression level of MdSWEET1 was significantly decreased.The down-regulation of MdSWEET1 is known to affect development and differentiation of pollens,thus suggesting that it may be related to the induction of somatic embryogenesis.2.The pollens with 30 days low temperature treatment were subjected to in vitro anther culture.In total 74,200 anthers of ‘Gala' were inoculated.From the more than 50,000 uncontaminated cultures,386 embryos were induced(0.7% embryoid induction).During next differentiation culture,64 plantlets were obtained(regeneration rate 16.6%).With subsequent subculture,30 plantlets survived as new germplasm.Flow-cytometry analysis(FACS)showed the 30 regenerated plantlets consisting of 28 diploid,1 haploid and 1tetraploid.Under the same anther culture,9 and 7 plantlets derived from ‘Danxia' and ‘Fuji'cultivars were respectively regenarated.3.To identify the genotypes of the regenerated plantlets,130 SSR markers were selected from HIDRAS database SSR for PCR amplifications.The PCR products sizes were checked using gel electrophoresis(PAGE).For ‘Gala',20 SSR markers showed to be able to differentiate pollens from somatic cells.For ‘Danxia' and ‘Fuji',16 SSRs and 17 SSRs were identified respectively.4.Corresponding to each of apple 17 linkage groups,17 SSRs from above 20 SSRs were further selected for genotyping.Using capillary electrophoresis,genotypes of the 30 regenerated plantlets were examined,demonstrating these 17 SSRs can reliably distinguish individual plantlet.5.After 60 days subcultures,the morphological characteristics of Gala plantlets were analyzed.The observations showed that plant height,leaf length-width ratio were significantly different.Variance were also observed within the diploid plantlets: Gala05 line was relatively high with wider leaf base,while Gala07 line showed smaller and thicker leaves and Gala18 line smaller leaves and less amount of leaves.Moreover,diploid plantlets showed a trend of weaker growth than that of the parental “gala” but stronger than that of haploid and homozygous tetraploid.Our study demonstrates that haploid anther culture is a powerful approach to create new apple germplasm.The regenerated plantlets as new apple germplasm are very valuable materials for traits related genetics identifications,and more importantly to advance the present apple breeding.