牡丹组织培养技术研究进展

Research Advances in Tissue Culture of Tree Peony

牡丹是我国特有的观赏、药用和油用植物,高效的组织培养技术是加速其育种和繁殖的重要基础。牡丹组织培养技术历经约半个世纪的研究,主要进展包括以下3个方面:1)器官组织培养再生:牡丹器官组织培养再生主要包括器官发生、体细胞胚发生和无菌短枝扦插这3条途径。(1)牡丹器官发生途径包括愈伤组织和分生结节培养,牡丹的各种繁殖器官和营养器官皆可作为外植体诱导得到愈伤组织,但愈伤组织进一步分化器官难度较大,且尚未得到完整再生植株;牡丹分生结节培养以黄化嫩茎和叶柄薄层为外植体,前人已初步建立了紫斑牡丹和牡丹芍药组间杂种——伊藤的分生结节发生体系,并实现了不定芽和叶状体的分化,但分化率极低,且未能获得再生植株,还发现牡丹分生结节中含有丰富的芍药苷和丹皮酚,这证实了其在药用成分生产中的应用潜力。(2)牡丹体细胞胚发生途径包括直接发生和间接发生,前人通过筛选基因型、培养基和发育时期等实现了多个牡丹品种的体细胞胚直接发生,但体细胞胚萌发率低,且再生成苗困难;间接发生途径以叶柄为外植体,已诱导得到愈伤组织并获得体细胞胚,但体细胞胚发生率极低。(3)牡丹无菌短枝扦插技术历经约30年的研究,完善的启动、增殖、生根和驯化移栽技术体系已经建立,但该技术仍无法实现产业化应用,主要原因在于试管苗的顶芽休眠、两步生根成本高以及移栽成活率低。2)胚珠和胚离体培养成苗:分别以完成器官分化的胚珠(花后60天)和成熟胚(花后90天)为外植体,前人通过筛选植物生长调节剂建立了紫斑牡丹和杨山牡丹的胚珠和胚离体培养体系,并获得了组培苗,但组培苗在驯化移栽阶段死亡。3)花药(花粉)离体培养成苗:以处于第1次分裂期的花粉为外植体可诱导得到花粉胚,培养过程中不同发育状态的花粉胚表现出不同的器官分化能力,其中聚集成簇的花粉胚无法分化出芽和根,而独立生长的花粉胚可分化出根系,但无法分化出芽。综上,牡丹组织培养研究目前仍处于应用基础研究的初级阶段,后期研究一方面需要针对褐化、器官间接发生困难以及试管苗的生根和移栽问题进行技术优化,另一方面亟需从生理甚至是分子的层面对组织培养中的褐化、器官发生和顶芽休眠机理等展开研究,从而为组织培养技术的优化提供理论支撑。

The tree peony（ Paeonia Sect. Moutan ）, native to China, is famous for its ornamental value, medical use and edible oil production; an effective tissue culture system is an urgent requirement for its breeding and propagation. Tissue culture of tree peony has been extensively studied during the past half-century, and the main advances include the following 3 aspects： 1） Organ and tissue regeneration culture： there are 3 ways for regeneration including organogenesis, somatic embryogenesis and in vitro shoot proliferation. （1） Tree peony organogenesis includes callus culture and meristematic nodules culture. Vegetative and reproductive organs can both be used as explants to induce callus, but it is very difficult to induce shoots from callus, and a complete plant is still not obtained. With the yellowing stem segments and thin-layer petiole as explants, effective meristematic nodules culture systems have been developed for P . rockii and P . itoh by selecting plant growth regulators and improving culture medium. Adventitious buds and thallus were induced from the meristematic nodules; however, a complete plant is not obtained. In addition, the meristematic nodules of tree peony have been found to contain abundant peoniflorin and paeonol which indicate its value in pharmaceutical ingredients production. （2） Somatic embryogenesis： tree peony somatic embryogenesis includes direct and indirect ways. Somatic embryos have been induced in many tree peony cultivars by selecting genotype, culture medium and embryo development stage; however, the somatic embryo has been found difficult for germination and plant regeneration. Tree peony petioles have been used as explants to induce callus though indirect organogenesis, and the somatic embryo have been induced from callus with however extremely low induction rate. （3） In vitro shoot proliferation： in vitro shoot proliferation of tree peony has been studied for nearly 30 years. Effective initiation, multiplication, rooting and acclimatization system have been established; however, the protocol is unviable for commercial use due to the dormancy of the rooted shoots, the high cost of the two-step rooting protocol and the poor survival rate during acclimatization. 2） Ovule and embryo culture： Using mature ovule （60 days after flowering） and mature embryo （90 days after flowering） as explants, ovule and embryo culture systems have been developed for P . rockii and P . ostii by selecting plant growth regulators,and complete plants have been obtained which however died during acclimatization. 3） Anther （pollen） culture： Pollen at the first stage of division is the best explant for anther （pollen） culture. In tissue culture, the pollen embryos in different development states vary in organ differentiating capacity. The clustered pollen embryo can not produce shoots or roots, while the pollen embryo developing independently can produce roots, however without shoots produced. In conclusion, tissue culture of tree peony is still in the primary stage of applied basic research, the future technology research should focus on browning, the difficulties in indirect organogenesis, and the rooting and acclimatization problems to optimize the protocol. Meanwhile studies on the physiological and the molecular mechanism of the browning, organ differentiation, apical shoot dormancy, etc. is essential, in order to provide a theoretical basis for the establishment of tree peony tissue culture system.