首页|四倍体胡柚与鸡尾葡萄柚有性杂交创制三倍体新种质

四倍体胡柚与鸡尾葡萄柚有性杂交创制三倍体新种质

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[目的]柑橘三倍体具有无核或少核的特点,通过4x×2x杂交方式创制三倍体种质,以弥补鸡尾葡萄柚种子多、抗寒性差的缺点。[方法]以四倍体(双二倍体)胡柚为母本与二倍体鸡尾葡萄柚杂交,果实成熟后对种子大小进行分类并进行组织培养,采用流式细胞仪、SSR分子标记鉴定再生子代的倍性及遗传组成。[结果]共授粉40朵花,坐果19个,坐果率为47。5%。获得的230粒种子中,大种子占56粒,再生49株,2株为三倍体(占大种子再生后代的4。08%);小种子117粒,再生69株,35株为三倍体(占小种子再生后代的50。72%);败育种子57粒,再生4株,2株为三倍体。经SSR分子标记鉴定,39株三倍体子代均为双亲杂交后代,同时鉴定出1株杂种四倍体后代。[结论]创制了一批异源三倍体和四倍体新种质,为选育抗寒、无核的杂种葡萄柚新品种奠定了种质基础。
Regeneration of triploid citrus germplasm by the crossing between tetra-ploid Huyou and Cocktail grapefruit
[Objective]Seed number has a crucial effect on the fruit quality for the fresh-fruit market and processing production in citrus.Triploid citrus can produce seedless fruits,and as polyploids,it usu-ally shows the superiority of vigorous growth,enlarged fruits and enhanced stress resistance.In citrus breeding,the 4x × 2x interploidy cross is an effective way to obtain triploids and to select new seedless species.Cocktail grapefruit(Citrus paradisi'Cocktail')has excellent fruit taste with high total soluble sugar and low titratable acid contents but has the defect of abundant seeds and poor tolerance to cold.So reducing the seed number to enhance the economic value has raised the awareness among citrus breeders.Changshan Huyou(Citrus aurantium'Changshanhuyou')is a native citrus species in Zheji-ang province and exhibits rich flavor and excellent cold resistance,and its seed is a mixed type of single and multiple embryos.A tetraploid Changshan Huyou was identified and bear seedy fruits for several years.Therefore,we attempted to produce triploid hybrids by interploidy hybridization between the tet-raploid Changshan Huyou and the diploid grapefruit.[Methods]The 4x × 2x interploidy cross was con-ducted using a tetraploid Changshan Huyou(4x)as the female parent and the diploid Cocktail grape-fruit(2x)as the pollen parent in this study.Pollen viability was determined by the result of Alexander staining and germination rate in vitro before artificial pollination.In the florescence stage of Cocktail grapefruit,the pollen was collected using a 2 mL centrifuge tube and stored in dry environment at 4 ℃ for a short period.Seeds were extracted from the ripened fruit of hybrids and divided into developed seeds of normal size,developed seeds of small size(1/3-1/6 of normal size)and undeveloped seeds.The normal size and small size seeds were sowed on Murashige and Tucker(MT)culture medium,and undeveloped seeds were sowed on MT+1.0 mg·L-1 GA3 culture medium.The seedlings were transplant-ed after the plant growing 4-5 functional leaves.The ploidy of progenies was then measured by flow cy-tometry using young leaves.After DNA extraction,the genetic origin of the offspring was analyzed us-ing 4 Simple Sequence Repeats(SSR)markers,which could display different profiles in Changshan Huyou and Cocktail grapefruit.The morphological differences were compared using one-year-old seed-lings from offsprings and parents.[Results]The pollen grains of Cocktail grapefruit had a staining ac-tivity of 89.40%and in vitro germination rate of 30.02%,indicating that the pollen of Cocktail grape-fruit had satisfactory vitality for further pollination.As a result,a total of 40 flowers of the tetraploid Changshan Huyou were used in pollination,and 19 hybrid fruits were harvested in November with a fruit-setting rate of 47.5%.A total of 56 developed seeds(average 3.0 seeds per fruit),117 small devel-oped seeds(6.2 seeds per fruit),and 57 undeveloped seeds(3.0 seeds per fruit)were obtained,and 49,69 and 4 seedlings were germinated in vitro with an average germination rate as 87.50%,58.97%and 7.02%for these seeds,respectively.Ploidy analysis showed that 39(31.96%)and 83 progenies were proven triploids and tetraploids,respectively.Among the offsprings of each group,a total of 2,35 and 2 triploid seedlings were identified from the developed seeds,small developed seeds and undeveloped seeds,respectively.The majority(89.74%)of triploids were originated from the small developed seeds.The hybrid nature of all the 39 triploids and 1 out of 22 tetraploids randomly determined were con-firmed by the SSR marker of F14,P72,MEST86 and CAG01,and were indicated as the characteristic stripes of both Changshan Huyou and Cocktail grapefruit.The remaining 21 tetraploids were confirmed to be derived from selfing or nucellus embryos due to the presence of the characteristic stripe of the fe-male parent.The leaf shape index of male parents,female parents,triploid F,seedlings(3x),and a tetra-ploid F1 seedling(4x)were 1.73,2.00,1.83 and 1.65,respectively.The triploid and tetraploid offsprings obtained from hybridization were closer to the tetraploid female parent in terms of leaf shape index,without statistical difference from the female parent but with significant difference(p<0.05)from the male parent.Leaf shape index of 3x hybrids was between 4x and 2x parents,however,the leaves of the 4x hybrid were wider and rounder than 4x female parent.The leaves of 4x hybrid offspring were signifi-cantly wider than the leaves of 3x hybrids.[Conclusion]The tetraploid Changshan Huyou was used as the maternal parent and hybridized with diploid pollen of Cocktail grapefruit in this study.After culture in vitro,ploidy identification and SSR molecular marker analysis,a total of thirty-nine heterozygous triploid offsprings were obtained,which mainly germinated from small developed seeds harvested from hybrid fruits in 4x × 2x interploidy cross,providing an effective strategy to ensure the seedless germ-plasms in citrus breeding by ploidy hybridization.

Cocktail grapefruitHuyouPloidy breedingTriploidTetraploidSSR markers

徐天予、张迟、王刚、叶潇玲、陈翔、叶生月、张小琴、张敏

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浙江农林大学·省部共建亚热带森林培育国家重点实验室,杭州 311300

浙江农林大学园艺科学学院·农业农村部亚热带果品蔬菜质量安全控制重点实验室,杭州 311300

常山县农业农村局,浙江常山 324200

桐庐县农业农村局,浙江桐庐 311500

浙江省农产品绿色发展中心,杭州 310003

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鸡尾葡萄柚 胡柚 倍性育种 三倍体 四倍体 SSR分子标记

2025

果树学报
中国农业科学院郑州果树研究所

果树学报

北大核心
影响因子:1.486
ISSN:1009-9980
年,卷(期):2025.42(1)