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.

【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 g·L^(-1)AC的培养基中促进体细胞胚的成熟及后续的同步萌发和生根,可有效缓解胚根端愈伤化现象。

【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 effects of different concentrations of 6-benzyl amino purine (6-BA),naphthalene acetic acid (NAA) and AC on the germination and rooting of cotyledonary somatic embryos were investigated.【Results】Complete immature zygotic embryos can be easily and rapidly separated by extrusion method without the assistance of stereoscope.4 mg·L^(-1)was the optimum 2,4-D concentration for embryogenic callus induction of IZE from Zihui cultivar,and the maximum embryogenic callus induction rate was 62.86%.After 5 subculture cycles of sieving culture of embryogenic callus in liquid medium,homogeneous embryogenic cell suspensions with a large number of single cells and small cell groups were established.A large number of spherical embryos were induced by liquid culture for 21 days,and then developed into cotyledonary somatic embryos synchronously on MSM in 30-45 days.About 97.58% of the cotyledonary somatic embryos germinated on MS medium supplemented with 0.4 mg·L^(-1)6-BA and 0.02 mg·L^(-1)NAA,95.48%of which showed callus formation at the base of somatic embryos,the rooting rate was 18.18%,and the quality of roots was poor.The supplementation of 5 g·L^(-1)AC generated 92.62% cotyledonary somatic embryos with synchronous germination and rooting,and significantly reduced the callus rate to 33.10%.Synchronous germination and rooting were also achieved in MSM medium without any plant hormone,with a synchronous germination and rooting rate of 88.33%,and the callus rate was reduced to 11.67%.The plant regeneration rate was 81.36% when these plantlets were transferred onto the regeneration medium with 0.1 mg·L^(-1)6-BA,2 mg·L^(-1)IBA and 10 g·L^(-1)AC.Plants with well-developed shoots and roots were subsequently hardened to seedlings in a potting mixture with 2/3 peaty soil and 1/3 vermiculite.【Conclusion】The optimal embryogenic callus induction medium was 1/2 MS medium+4 mg·L^(-1)2,4-D+400mg·L^(-1)glutamine+60 g·L^(-1)sucrose with immature zygote embryos of Zihui at 110-120 days as explants.Spherical embryos can be efficiently induced from ECS with liquid culture,and subsequent synchronous germination and rooting of somatic embryos in the medium containing 1/2 MS salt+MS vitamin+50 mg·L^(-1)inositol+400 mg·L^(-1)glutamine+30 g·L^(-1)sucrose+5 g·L^(-1)AC.This regeneration protocol established in C.papaya L.‘Zihui’achieves a high frequency of somatic embryogenesis with good synchronization and plant regeneration.