【作者】 周秀梅；

【导师】 成仿云； 齐力旺；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2008， 博士

【摘要】 牡丹是中国特产的传统名花。但其繁殖系数低、育种周期长,一直是生产和育种发展面临的重大问题。体胚发生方式再生植株是植物再生的一个重要方式,在许多木本植物中已经取得成功,但牡丹的体胚发生研究很少。为建立稳定的牡丹体胚发生体系,本研究以紫斑牡丹(Paeonia.rockii)‘书生捧墨’、杨山牡丹(P.ostii)‘凤丹白’和滇牡丹(P.delavayi)的种胚为外植体,在离体培养条件下,首先通过试验确定了适于牡丹体胚发生的种源,并在此基础上对外植体发育时期、类型及其诱导培养基的组成进行了筛选;第二,探讨了蔗糖溶液预处理、光照条件和诱导时间对‘书生捧墨’体胚诱导与发生的影响;第三,采用最优混合设计研究了诱导培养基中BAP、蔗糖、CH和硝酸银浓度对‘书生捧墨’体胚诱导与发生的影响;第四,采用试验获得的体胚材料,进行了体胚增殖、分化能力保持和成熟、萌发与植株再生的初步研究;最后,以‘凤丹白’的绿色子叶为外植体进行了牡丹体胚间接发生的探索性研究,同时对体胚的直接发生和间接发生进行了细胞学的初步研究。主要结论如下:1.在供试的3类牡丹中,紫斑牡丹‘书生捧墨’最适于诱导体胚的直接发生;‘书生捧墨’花后90d的完整种胚是适于诱导牡丹体胚直接发生的外植体;诱导培养基中,基本培养基种类、BAP、NAA和蔗糖浓度对体胚诱导率的影响均极显著,它们的最佳组合为:MS或改良MS+蔗糖100.0 g/L+BAP 0.5 mg/L和NAA 0.0 mg/L,其诱导率最高为63.3%,比前人的结果(紫斑牡丹花后90d种胚外植体的体胚诱导率19%)提高了44.3%。2.对外植体进行蔗糖溶液预处理能促进体胚发生。经1.0M蔗糖溶液预处理1 d的‘书生捧墨’花后70 d的幼胚,在无激素培养基上起始诱导后再转到含有BAP的培养基上培养,22 w时体胚诱导率最高达到了84.6%,并能减少连体胚比率。蔗糖溶液预处理时间对体胚发生效果与诱导培养基间存在交互作用。在供试的3种诱导培养基上,1.0 M蔗糖溶液预处理‘书生捧墨’花后90 d的种胚2~3 d为好。3.光照的有无和诱导培养时间长短对体胚的发生有一定的影响。‘书生捧墨’花后85 d的完整种胚为外植体诱导体胚发生时,宜在25℃±2℃和全黑暗条件下诱导培养60 d后再转接到无激素培养基上培养较好。4.利用416-B最优混合设计,进一步剖析影响‘书生捧墨’体胚发生的主导因素为BAP、蔗糖、CH和硝酸银,并根据试验结果建立了它们对其体胚诱导率的多元二次回归方程:?=-1.5413-0.0824X1+1.0886X2-0.1532X3+0.0755X4+0.3720X12-0.1425X22+0.1377X32+0.4941X42+0.2816X1X2-0.1651X1X3-0.3375X1X4+0.2400X2X3(R2=0.9996),分析了主导因素的一级效应和互作效应,最后综合寻优求得最佳组合为:BAP 0.1 mg/L、蔗糖150.0 g/L、CH 600.0 mg/L、硝酸银10.0 mg/L。用改良MS培养基附加该组合,理论上体胚诱导率可达到97.5%。5.‘书生捧墨’的体胚可通过诱导次生体胚和体胚分化能力保持的方式反复发生从而实现增殖。次生体胚诱导培养基与初生体胚诱导培养基的组成相似,但BAP的浓度要降低;已分离过体胚的初始外植体或分离过次生体胚的初生体胚,能在胚性保持培养基上保持其体胚分化能力。6.‘书生捧墨’的体胚可以在无激素的MS或SH固体培养基中成熟,AC起促进作用;萌发培养基中少量添加BAP或IAA能使外形成熟的体胚正常萌发,IAA能促进胚根的伸长,但BAP(高于0.5 mg/L)易导致胚轴过度膨大,NAA使胚轴高度愈伤化,高浓度的GA3导致早熟萌发。体胚与种胚一样具有上胚轴休眠特性,必修经过一段时间的低温或GA3处理打破休眠,才能使胚芽伸长。7.剥取滇牡丹、‘书生捧墨’和‘凤丹白’的种胚时,发现都存在双胚和畸形胚现象。双胚比率在1.5%左右,双胚均能成苗,或仅有一个能成苗;畸形胚主要有单子叶、3子叶或多子叶、胚轴合生、胚根合生、子叶合生等。8.‘凤丹白’的绿色子叶为外植体诱导愈伤组织时,以SH为基本培养基,2,4-D和NAA配合使用较好,愈伤组织诱导率高达100%;有2,4-D存在时,诱导80 d可获得球形胚。9.‘书生捧墨’体胚的直接发生中,体胚起源于表皮的突起之上;间接发生中体胚起源于愈伤组织的边缘或内部细胞,添加ABA能促进胚性愈伤组织中体胚的分化。更多还原

【Abstract】 Tree peony was famous in the world and its wild species were originated in China. But its low propagation coeffidient and long breeding period have always been the big problems for the production and breeding .Somatic embryogenesis is the important approach for plant regeneration and it has been used successfully for many plants.But the research reports on tree peony were rare.So this research was carried out to set up a steady regeneration system and to provide a platform for the propagation in vitro or transgene breeding. Firstly, the suitable explant donator were filtrated from Paeonia rockii‘Shu Sheng Peng Mo’、P.ostii‘Feng Dan Bai’and P.delavayi in vitro. Based on this,the developing stage and type of the explant to somatic embryogenesis were filtrated and decided, and the basal medium type and the concentration of BAP, NAA and Sucrose in the induction media on somatic embryogenesis were also explored. Secondly, the effect on induction rate of pretreating the explants with 1.0 M sucrose solution, as well as the light conditions and induction periods were experimentalized to improve the somatic embryos. Based on 416-B Optimized Mixed Design, the best suitable concentrations of BAP, Sucrose, CH and AgNO3 in induction medium were decided by DPS, so the somatic embryogenesis system was optimized.Thirdly, the secondary somatic embryogenesis from the abnormal and semitransparent somatic embryos, and the maintainability of continuance differentiation capability of somatic embryo from the original explants which had produced somatic embryos or the original somatic embryos which had produced secondary somatic embryo were experimented to get repetitive somatic embryogenesis. Fourthly, maturation and germination of the somatic embryos and the transformation from somatic embryo to plantlets were studied to explore the ways for more plants.At last, the research on indirect somatic embryogenesis from the green cotyledon of‘Feng Dan Bai’was done for the first time. At the same time cytology researches on direct or indirect somatic embryogenesis were done.The main results were as follows: Among Pa rocki‘iShu Sheng Peng Mo’,P. osti‘iFeng Dan Bai’and P. delavayi, P. rockii‘Shu Sheng Peng Mo’was the most suitable one for inducing direct somatic embryogenesis, the better developing stage of embryo explants was 90 days after flowering; for the explants type, the whole embryo was best. The basal media and the concentration of BAP, NAA and Sucrose had the most significant effects on the induction rate of somatic embryos. The most suitable combination of the four factors was: MS or Revised MS medium+Sucrose 100.0g/L+BAP 0.5 mg/L+NAA0.0 mg/L, and the highest induction rate was up to 63.3%, which is 44.3% higher than the result 19% gained by the former researcher.Somatic embryogenesis in tree peony was promoted and the amount of fused somatic embryos was reduced by pretreating explants with 1.0 M sucrose solution. It was pretreating the young embryos 70 d after flowering with 1 M sucrose solution for 1d firstly and culturing them on the auxin free inducing medium, then subculturing them on the medium with BAP that had better effect on direct somatic embryogenesis in‘Shu Sheng Peng Mo’. After 22 w cultured, the induction rate was up to 84.6%. There was an interaction between the pretreatment period and the induction medium for somatic embryogenesis. For the 3 induction media used in the experiment, it was better that pretreating the seed embryo explants 90d after flowering with 1.0 M sucrose solution for 2 or 3 d.Induction period and light had some effect on the somatic embryogenesis in tree peony. For the embryos 85 d after flowering of‘Shu Sheng Peng Mo’, it was better to be cultured for 60 days in dark and 25±2℃and then transferred to the hormone free medium.Based on 416-B Optimized Mixed Design,The main factors to affect the somatic embryogenesis of 85d embryo explants in‘Shu Sheng Peng Mo’were BAP, Sucrose, CH, and AgNO3 And for the somatic embryo induction rate,the multiple quadratic regression equaton was:?=-1.5413-0.0824X1+1.0886X2-0.1532X3+0.0755X4+0.3720X12-0.1425X22+0.1377X32+0.4941X42+0.2816X1X2-0.1651X1X3-0.3375X1X4+0.2400X2X3(R2=0.9996).The main and interaction effects were analyzed. And the best levels of the four factors from the 500 combinations was gained by DPS ,they were BAP 0.1 mg/L, Sucrose 150.0g/L, CH600.0 mg/L and AgNO3 10.0 mg/L.For the embryo explants 85d after flowering in‘Shu Sheng Peng Mo’, the highest theoretical inducing ratio can up to 97.5% with Revised MS with this combination。Somatic embryos of‘Shu Sheng Peng Mo’were proliferated vir secondary somatic embryogenesis. For somatic embryogenesis and secondary somatic embryogenesis, the inducing media in composing were similar, but the latter was with BAP in low concentration. The capability to differentiate somatic embryos continuously of donators including the initiated explants and the somatic embryos which had developed somatic ones were maintained in the media for preserving the capability.The somatic embryos of tree peony maturated on MS or SH solid hormone free medium, which was promoted by AC. When the somatic embryos developed to the cotyledon stage, it was necessary to separate them from the donators in time and culture to make them germinate, otherwise, they geminated poorly or even not. The medium with lower BAP or IAA concentration made the mature somatic embryos in shape germinate well but the media with higher BAP concentration (>0.5 mg/L) made the hypocotyls swelle strongly.While the medium with lower IAA concentration made the radicles elongated. It was not suitable to add NAA and GA3 in higher concentration to the germination medium, because NAA in high concentration made the hypocotyls callus highly and GA3 in high concentration made the somatic embryos germinate precociously. Just like the seed embryos, somatic embryos of tree peony have dormancy epicotyls, so it is required to treat the dormancy epicotyls with lower temperature or GA3 in order to make them elongate.The phenomenon of double embryos in the tree kinds of tree peonies used by this research was approved, and the ratio of double embryos was about 1.5%. Moreover, the development of the two embryos was synchronous or not, and both or one of them two grew up to seedling. And the abnormality in the embryos of tree peony was found firstly.Calli were induced from the green cotyledon pieces of‘Feng Dan Bai’on SH basal media with 2,4-D and NAA in different concentration, and higher induction rate (100%) was gained.The globule embryos were found on the media with 2, 4-D after 80d induction.For direct somatic embryogenesis, somatic embryos were found originating on the protuberances from the superficial of the cotyledons or the hypocotyls, but for indirect one, they were originated from the superficial and inner cell of the calli.The somatic embryogenesis indirectly were improved by adding ABA in the media.更多还原