浙江地方特色品种温岭高橙无核潜力新种质创制及分子鉴定OA北大核心CSTPCD
Production and molecular identification of potentially seedless germ-plasms derived from Wenling-gaocheng,a citrus local cultivar in Zhejiang province
[目的]温岭高橙是浙江台州地方特色品种,栽培历史悠久,且具有一定的保健和药用价值,但果实有核限制了其在台州以外地区的发展,因此利用细胞工程高效育种技术对温岭高橙进行无核化改良.[方法]通过2条育种途径,一是以二倍体温岭高橙为母本与异源四倍体为父本倍性杂交,授粉后90d利用幼胚离体挽救培养和流式细胞仪倍性鉴定创制三倍体无核新种质;二是以雄性不育胞质杂种华柚2号为母本与温岭高橙有性杂交,通过成熟种子催芽播种,创制二倍体无核新种质.利用SSR分子标记对杂交后代进行分子鉴定.[结果]以温岭高橙为母本、异源四倍体NS和BDZNS为父本配置2个倍性杂交组合,授粉145朵花,坐果74个;胚抢救幼嫩种子1520粒,离体培养获得再生植株553株;用流式细胞仪对再生植株进行倍性鉴定,获得三倍体45株、四倍体8株;以华柚2号为母本与温岭高橙有性杂交,授粉69朵花,坐果20个,获得成熟种子312粒;通过催芽播种获得实生后代203株,倍性鉴定均为二倍体;用SSR分子标记对2个倍性杂交组合的21株三倍体和8株四倍体后代及华柚2号×温岭高橙随机选取的29株二倍体后代进行分子鉴定,显示所鉴定的三倍体及二倍体后代均含有父本特异条带,为其杂交双亲的有性后代,而倍性杂交获得的四倍体后代与母本条带完全一致,推测其为母本珠心细胞自然加倍形成的同源四倍体(双二倍体).[结论]研究结果为温岭高橙无核新品种培育奠定了宝贵的材料基础.
[Objective]Seedlessness is an important trait for citrus fresh fruits because consumers do not like fruits with seeds.Wenling-gaocheng,a natural hybrid of pommelo and orange,is an excellent local cultivar in Zhejiang Province because of its unique flavor and rich nutritional value.However,it failed to expand the market outside Taizhou city due to the abundance of seeds in the fruits.The fruits of the triploid plants are generally seedless.There are several ways to produce citrus triploid plants.Among them,using diploid varieties as female parents and allotetraploid as the male parents is the most efficient way.The triploid plants produced by interploidy hybridization with allotetraploid as the male parent generally possess abundant genetic variations,which is conducive to breed new cultivars with good fruit traits.Production of diploid progenies using cytoplasmic male sterile(CMS)cultivar Huayou No.2 as the female parent crossed with the seedy diploids is another useful way to breed seedless citrus germplasm because the CMS trait in citrus is female inherited,and the CMS trait can be inherited to all progenies.Therefore,the above two strategies were employed in this study to produce seedless proge-nies with the lineage of Wenling-gaocheng,to produce promising materials for selecting seedless culti-vars with the lineage of Wenling-gaocheng.[Methods]Two crossing strategies were employed in this study.The first one was to produce triploid progenies using Wenling-gaocheng as female parent and two alloteraploids as male parents.The pollination of the two interploidy crosses was conducted at the full-bloom stage and the immature fruits were collected on the 90th days after pollination(DAP).The immature seeds were removed from the fruits and cultured in vitro in the germination medium.When the seedlings regenerated,their ploidy level was determined by the flow cytometry.Another strategy was to produce diploid progenies with CMS traits using Huayou No.2 as the female parent and the Wenling-gaocheng as the male parent.The pollination of this cross was conducted at the full-bloom stage and the fruits were collected at the mature stage.After the seeds were extracted from the fruits and the episperms were peeled,they were placed in a controlled chamber to accelerate germination.The seeds were sown in pots after germination in a plant growth chamber.After the seedlings grew with three or more leaves,their ploidy level was determined by the flow cytometry.At last,the genetic origin of all seedlings from the three crosses were determined by SSR markers.[Results]In the two interploi-dy crosses,a total of 145 flowers were pollinated and 74 fruits were set,with an average fruit setting rate of 51.03%.1520 immature seeds were in vitro cultured via embryo rescue and 553 seedlings were obtained.Among them,239 plantlets were regenerated from the cross of Wenling-gaocheng×NS and 314 plantlets were originated from the cross of Wenling-gaocheng×BDZNS.After determining their ploidy level,a total of 45 triploids and 8 tetraploid seedlings were screened out.Three SSR markers were used to determine their genetic origin.All the analyzed triploids were the hybrids of their parents and all the tetraploids had the same bands with Wenling-gaocheng,indicating that all the tetraploids might be derived from the nucellar doubling of Wenling-gaocheng.For the cross of Huayou No.2×Wenling-gaocheng,a total of 69 flowers were pollinated.From the 20 fruits,312 mature seeds were ob-tained.After sowing and germination,203 seedlings were obtained,all of them were identified as dip-loid by flow cytometry.Four polymorphic SSR markers were selected to determine their genetic origin of 29 randomly selected progenies and it showed that all the diploid progenies were parental hybrids.[Conclusion]The diploid hybrids derived from the cross of Huayou No.2×Wenling-gaocheng and the triploid hybrids derived from the two interploidy crosses could be used for seedless breeding of new va-rieties with Wenling-gaocheng genetic background.The autotetraploid progenies of Wenling-gaocheng also would be valuable for crossing with diploid Wenling-gaocheng to produce autotriploids.
张成磊;师小舒;陈昊;谢善鹏;卢鑫;伍小萌;刘高平;郭文武;解凯东
台州科技职业学院·台州市园艺生物技术重点实验室,浙江台州 318020华中农业大学果蔬园艺作物种质创新与利用全国重点实验室,武汉 430070浙江省台州市黄岩区果树技术推广总站,浙江台州 318020
园艺学与植物营养学
柑橘无核育种倍性杂交华柚2号
CitrusSeedless breedingPloidy hybridizationHuayou No.2
《果树学报》 2024 (004)
590-597 / 8
台州市科技计划项目(22nyb06);浙江省黄岩区农业揭榜挂帅项目(JBGS2022-HYNY02);浙江省高校国内访问学者项目(FX2022164);台州市农业科技项目(23SNK05);台州科技职业学院乡村振兴研究重点课题(TKXZ2022001)
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