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《Journal of Fruit Science》 2016-06
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A study on the influence factors of single-seeded formation in loquat

XU Fangjie;ZHANG Xueying;SHI Chunhui;WANG Xiaoqing;XI Xiaojun;JIANG Shuang;CHEN Rong;Pomology and Forestry Research Institute,Shanghai Academy of Agriculture Science·Shanghai Key Laboratory of Protected Horticultural Technology;Chongming Agricultural Technology Extending Service Centre,Shanghai;  
[Objective]Loquat(Eriobotrya japonica.Lindl.) is an commercially important fruit tree,which belongs to the Rosaceae family and is native to southern China.One of the major problems which restricts the development of the industry is its low flesh recovery due to seediness.Normal diploid loquat fruit usually has 4-5 seeds and a very thin flesh layer,leaving only a 50%-60% flesh recovery.Seedless variety is one of the important targets of loquat breeding.Our research group has obtained several single-seeded loquat lines and collected some basic data such as fruit set,fruit weight,and yield per plant as well as total soluble solids(TSS) and titratable acids(TA).We found that although the fruit set rate is very low,sometimes only 10-20 fruits per plant,all fruit are single-seeded,with a high flesh recovery of 75%-90%.TSS of the fruit is 10%-11%.The results suggested that the single-seeded loquat lines are a important germplasm resource.However,the mechanisms of the formation of the single-seed trait are still unclear.Seed degeneration or embryo abortion is generally observed during seed development in multi-seeded loquat cultivars,but it could not clearly explain why finally only one seed remained in each fruit.We believe that the single-seeded loquats might have some unique characters in seed formation.So we selected the single-seeded loquat as material to study the mechanisms and influence factors of single-seeded trait,with the purpose to provide theoretical evidence for breeding loquat cultivars with higher flesh recovery.[Methods]In this study,we used 3 single-seeded loquat lines ' 10-6','2-27' and ' 10-47' as materials,and normal diploid loquat variety 'Dawuxing' as the control.First,flow cytometry(FCM) was used for loquat ploidy identification,and then TTC staining and pollen germination in medium(agarose gel containing different concentrations of sucrose) were used to test pollen viability.Meanwhile,single-seeded loquat was used to observe the pollen morphology by scanning electron microscope(SEM).The reciprocal pollination experiment was carried out between each single-seeded loquat lines and 'Dawuxing',which was used to observe pollen germination in each pollination combinations.Pistils of single-seeded loquats were stained with aniline blue for observing pollen tube growth under the fluorescent microscope.Also,the meiosis of pollen mother cells of the 3 single-seeded loquats was observed with the method of ordinary tablet compressing and staining with aceto-carmine liquid.Finally,paraffin sections were employed to observe the anatomical structure of embryo and embryo sac in single-seeded loquats.[Results]The flow cytometry results showed that all of the 3 single-seeded loquat lines were triploid.There were no differences in shape or size among fresh,air-dried(AD) and fixed pollens.The fresh pollen grains of Dawuxing were spheroidal or subprolate in shape with smooth surface,the pollen exine sculptures were unexceptionally reticulate,most of which were 3-colporate.However,most of pollen grains of single-seeded loquat were paramorphia,including rough-surfaced,germinal furrow deformed,or even empty pollen grains.The morphological disorders of single-seeded loquat lines indicated that their pollen grains might be functionally sterile.In vitro germination tests showed that Dawuxing had a very high germination rate of up to 92%,while germination rate of ' 10-6',' 10-47' and '2-27' was only 21%,11% and 13%,respectively,in mediums supplied with 10%sucrose.Higher concentrations of sucrose might provide better germination condition,and the germination rate of 10-6,10-47 and 2-27 increased to 27%,17% and 22% in mediums containing 15% sucrose.The suspension of fresh pollen grains in 0.4% TTC solution was dropped on a glass slide and then inspected under microscope directly.High viability pollen grains would be dyed red.'Dawuxing' showed 90% TTC-staining rate,while '10-6' and '2-27' had only 8% and 27%TTC-staining rate,respectively,and ' 10-47' had totally unstained pollen grains,suggesting that the pollen viability of the 3 single-seeded loquat lines are very low.During the flower bud differentiation,we sampled loquat anthers and crashed them with tweezers,then did ordinary tablet compressing and stained with 0.2%aceto-carmine,and found that single-seeded loquat lines showed a large number of abnormal triads during the tetrad period of pollen mother cells(PMC) meiosis,indicating that abnormal meiosis of PMC might be one of the most important reasons for pollen abortion in single-seeded loquats.Aniline blue staining could clearly display pollen tube growth.In our experiment,we found that more than 5 pollen tubes reaching style bottom in self-pollinated pistils of 'Dawuxing' within 24 h,but in the crosspollination between single- seeded loquats and ' Dawuxing',we observed pollen tube growth retardation in single-seeded loquat styles.Some of pollen tubes in the style of '2-27' reached 1 000 μm,and some of pollen tubes in style of '10-47' and '10-6' were 100-200 μm within 24 h.Meanwhile,field test showed that self-pollination of single-seeded loquats bore no fruit,cross-pollination of ' 10-6' × ' 1-24'(a multi-seed seedling) produced 5 hybrid fruit,'2-27' × 'Ninghaibai' had only 3 hybrid fruit generated from 50 flowers.All the hybrid fruit were single-seeded,these results induced that single-seeded trait formation might not be related to male fertility.The results of paraffin sections showed that embryo sac degradation was very common in single-seeded loquats.Sometimes the embryo sac appeared in the form of cavity or residues,which might cause most of premature embryo sac to be degenerated at very early stage of fruit development.However,we also observed complete normal embryo sac development process in singleseeded loquat,which accord with the typical features of polygonum type development.It was possible that very few ovules survived and developed into full seed,which might have potential to produce single-seeded fruit finally.[Conclusion]All of the results indicate that the formation of single-seeded fruit might be related with the female gamete dysplasia due to the typical tripical genetic characteristics:non-synchronism of chromosome pairing during meiosis.The formation of very few fertile gametes from triploid meiosis might play the key role in single-seed formation.
【Fund】: 上海市自然基金青年项目(12ZR1449200);; 上海市农口青年人才成长计划项目(沪农青字(2016)第1-23号);; 上海市农科院青年科技基金项目(农青科技2012(019))
【CateGory Index】: S667.3
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