番木瓜均质胚性细胞悬浮系的建立和高效植株再生  

Development of embryogenic cell suspension cultures and efficient plant regeneration of Carica papaya L.

作　　者：魏岳荣[1] 周陈平 邝瑞彬[1] 杨护[1] 黄炳雄[1] 杨敏 WEI Yuerong;ZHOU Chenping;KUANG Ruibin;YANG Hu;HUANG Bingxiong;YANG Min(Institute of Fruit Tree Research,Guangdong Academy of Agricultural Sciences/Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization,Ministry of Agriculture and Rural Affairs/Guangdong Province Key Laboratory of Tropical and Subtropi-cal Fruit Tree Research,Guangzhou 510640,Guangdong,China)

机构地区：[1]广东省农业科学院果树研究所·农业农村部南亚热带果树生物学与遗传资源利用重点实验室·广东省热带亚热带果树研究重点实验室,广州510640

出　　处：《果树学报》2023年第2期376-385,共10页Journal of Fruit Science

基　　金：广东省省级乡村振兴战略专项资金种业振兴项目;佛山市财政专项资金—2022年高水平广东省农业科技示范市建设项目;广东省重点领域研发计划项目(2019B020214005);广东省基础与应用基础研究基金项目(2021A1515010739)。

摘　　要：【目的】提高番木瓜体细胞胚发生的同步性及其植株再生率,为番木瓜大量快繁、细胞工程和分子育种技术研发提供技术基础。【方法】以紫晖110~120 d果实的未成熟合子胚为外植体,经胚性愈伤组织诱导、液体悬浮培养和体细胞胚发生过程,建立均质胚性细胞悬浮系和高效植株再生技术体系,重点比较了不同质量浓度2,4-二氯苯氧乙酸(2,4-Dichlorophenoxyacetic acid,2,4-D)对胚性愈伤组织诱导、不同质量浓度6-苄基氨基嘌呤(6-Benzylaminopurine,6-BA)+萘乙酸(1-naphthlcetic acid,NAA)组合和活性炭(activated carbon,AC)对子叶期体细胞胚萌发与生根的影响。【结果】4 mg·L^(-1)2,4-D可诱导62.86%未成熟合子胚形成胚性愈伤组织。经5个继代周期的筛选培养,可建立由单细胞和小细胞团组成的均质胚性细胞悬浮系。采用液体培养方式能诱导大量球形胚的形成,被转移至含5 g·L^(-1)AC的半固定培养基成熟培养30 d后,可获得大量子叶期体细胞胚。在含0.4 mg·L^(-1)6-BA+0.02 mg·L^(-1)NAA的萌发培养基中体细胞胚萌发率为97.58%,胚根端愈伤化比率为95.48%,补充5 g·L^(-1)AC后可获得92.62%体细胞胚同步萌发和生根,并显著降低愈伤化比率至33.10%;在不添加任何植物生长调节剂、仅含5 g·L^(-1)AC的成熟培养基中,体细胞胚同步萌发和生根率为88.33%,愈伤化比率降低至11.67%。正常萌发生根的体细胞胚在含0.1 mg·L^(-1)6-BA+2 mg·L^(-1)吲哚丁酸(3-Indolebutyric acid,IBA)+10 g·L^(-1)AC的再生培养基中可获得81.36%的植株再生率。【结论】以紫晖110~120 d未成熟合子胚为外植体,最佳的胚性愈伤组织诱导培养基为1/2 MS培养基+4 mg·L^(-1)2,4-D+400 mg·L^(-1)谷氨酰胺+60 g·L^(-1)蔗糖。最优体细胞胚发生方案为胚性细胞悬浮系(embryogenic cell system,ECS)经液体培养方式诱导球形胚形成,在含1/2 MS盐+MS维生素+50 mg·L^(-1)肌醇+400 mg·L^(-1)谷氨酰胺+30 g·L^(-1)蔗糖+5 【Objective】Embryogenic callus from different papaya explants can be induced to generate somatic embryos,which develop into plants.However,there are some problems in this pathway,such as genotype dependence,limited number and asynchronous development of somatic embryos,poor rooting quality and low plant regeneration rate caused due to callus formation at the base of somatic embryos.This study was designed to overcome these obstacles,and to establish an efficient plant regeneration technology for large-scale rapid propagation of papaya seedlings,cell engineering and molecular breeding.【Methods】Hybrid Carica papaya L.‘Zihui’was used as the experimental materials.Immature zygotic embryos (IZE) excised from fruit 110 to 120 days post-anthesis were used as the explants to induce embryogenic callus on the medium containing half-strength MS (Murashige and Skoog,1962) basal salts and vitamins,400 mg·L^(-1)glutamine and 60 g·L^(-1)sucrose supplemented with 0-5.0 mg·L^(-1)2,4-dichlorophenoxyacetic acid (2,4-D) for 60 days.Light yellow and fragile embryogenic calluses were allowed to proliferate on the same medium at 28-30 days intervals for 3-4 months to get enough embryogenic calluses.In the process of liquid culture,900μm mesh sieves in the first subculture and 154μm mesh sieves subsequently were used to filter out large particle cultures in the medium,until homogeneous embryogenic cell suspension was obtained.Embryogenic suspension cells were transferred to100 m L conical flasks with 30 ml liquid somatic embryo induction medium (MSI) containing halfstrength MS basal salts and full-strength vitamins,50 mg·L^(-1)myo-inositol,400 mg·L^(-1)glutamine and30 g·L^(-1)sucrose,and agitated at 110 r·min-1in a gyratory shaker for 21 days.The induced somatic embryos were transferred to semi-solid somatic embryo mature medium[MSM:MSI+5 g·L^(-1)activated carbon (AC)+4.0 g·L^(-1)gel]allowed to further develop into cotyledonary somatic embryos,and then budded and rooted into plantlets on germinating medium.The effe

关 键 词：番木瓜 未成熟合子胚 胚性细胞悬浮系 体细胞胚发生 植株再生 

分 类 号：S667.9[农业科学—果树学]