【作者】 秦磊；

【导师】 成仿云；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2012， 博士

【摘要】 牡丹(Paeonia sect. Moutan)是我国的传统名花,但传统的繁殖方法效率低下,操作过程繁琐,严重阻碍了产业发展的步伐。组织培养技术是解决牡丹繁殖难题的有效方法。经过半个世纪的研究,仍然没有形成能够在生产中应用的技术。分生结节培养是与体胚培养具有同等重要价值的体外再生方式,在大量难于离体培养的木本植物中实现了植株再生,是有望解决牡丹繁殖困难的新途径。本研究以叶柄薄层为外植体,从品种、植物生长调节剂(PGRs)等多种因素对愈伤组织诱导、分生结节诱导、增殖和分化的影响进行研究,同时对分生结节诱导、发生与发育过程进行了形态学和细胞组织学观察,初步实现了分生结节到不定芽和叶状体的分化,对最终在牡丹中建立高效的植株再生技术体系具有重要的参考价值。主要结论如下：1.愈伤组织是分生结节产生的前提。本研究表明愈伤组织的诱导受品种、外植体发育阶段和取材位置、PGRs浓度及其组合的显著影响。在供试的7个品种中,以’Golden Era’愈伤组织诱导率高、体积大且质地紧实；取材于开花期到花谢1周的外植体,经培养可以获得诱导率高,且褐化程度低的愈伤组织；近叶片和近基部的叶柄薄层外植体愈伤组织诱导率高,且很少发生褐化现象,而中部叶柄薄层外植体在培养过程中褐化现象非常严重；PGRs以1 mg/L的2,4二氯苯氧基乙酸(2,4-D)为参考,浓度过高会发生玻璃化和褐化现象,同时噻苯隆(TDZ)对愈伤组织形成具有促进作用,在’Golden Era’中经1 mg/L 2,4-D+1 mg/L TDZ处理,可得到诱导率高、体积大且质地紧实的愈伤组织。2.牡丹愈伤组织发生和发育的过程在外部形态上可划分为0-4五个级别,内部细胞组织学特征表现为经历了启动、分裂和形成三个时期。在启动期,外植体形成层细胞最先被活化,启动分裂过程；继而进入细胞旺盛增殖的分裂期,先后于外植体形成层和皮层薄壁细胞处产生了可见的愈伤组织,愈伤细胞体积小且细胞质浓；然后进入形成期,愈伤组织增殖速度减缓,只有接触培养基的周缘愈伤组织保持增殖。3.牡丹分生结节是从愈伤组织内部分化形成的。分生结节存在两种结构,一种是以维管单元为中心,外层由核大、细胞质浓的薄壁细胞构成,另一种是以特化的薄壁细胞为中心,外层由数层薄壁细胞包围形成。牡丹分生结节的形成受品种与取材位置、PGRs以及培养基中氮素存在形式和蔗糖浓度等多因素共同调节。本研究在’Golden Era’、’Bartzella’和’High Noon’品种中获得了分生结节,其中取材于中部叶柄薄层的诱导率略高于近叶片和近基部;经SH附加0.2 mg/L 2,4-D和6 mg/L 6-芐氮基腺嘌呤(BA)培养,’Golden Era’和’Bartzella’分别在培养基中含有N03-/NH4+比值为2.4和50 g/L蔗糖和N03-/NH4+比值为9.5和30g/L蔗糖时,分别获得了78.0%和55.6%分生结节诱导率。4.增殖是通过分生结节进行繁殖的重要环节。本研究表明荼乙酸(NAA)和TDZ对牡丹分生结节的增殖作用因品种不同。’Golden Era’培养于含有0.5mg/LNAA和1 mg/L TDZ,’Bartzella’于8 mg/L BA和5 mg/L TDZ的SH培养基上,分别获得83.3%和91.7%的分生结节增殖率。在液体悬浮培养条件下,分生结节主要以数量增殖为主,’Golden Era’的最佳液体培养周期为30天(d)。5.分化是通过分生结节进行繁殖的技术关键。本研究在’Golden Era’中通过两步培养法,初步实现了不定芽和叶状体的分化。第一步是在添加PGRs组合为0.25 mg/LNAA+0.5 mg/L TDZ或0.5 mg/L TDZ的SH培养基上,光照条件下培养45d后;第二步是转接到添加25g/L蔗糖+2g/L活性炭(AC)的SH培养基上培养,最终获得16%的不定芽和叶状体分化率。在此过程中,降低PGRs浓度和光照培养是影响分化的主要因素,此外,继代次数少、培养周期长的分生结节更容易分化。细胞组织学观察表明不定芽起源于分生结节表而,并与分生结节之间存在维管连接。更多还原

【Abstract】 Tree peonies (Paeonia sect. Moutan) are Chinese traditional flowers. However, low propagation rate and time-consuming operation by traditional propagation methods are the main obstacles hindering the development of tree peonies. And that is why tissue culture is becoming an issue in breeding and propagation of this species. Since about half a century when the in vitro culture of tree peonies was firstly reported, there is still a long way to form a protocol in the production. Meristematic nodules (MN) represent a morphogenetic pathway of importance equal to that of embryogenesis. And MN culture leads many recalcitrant woody plants amenable to in vitro regeneration. Therefore, it is reasonable to say MN culture as the most valuable and promising way to solve the challenges of tree peonies with low propagation rate. In this study, thin cell layers (TCLs) from petiole were used as materials to examine the influence of cultivars, plant growth regulators (PGRs) and other factors on the pre-induction callus formation, MN induction, multiplication and differentiation. Meanwhile, histological analysis was used to examine the induction, genesis and development of MN and determine their anatomical characteristics. The process is realized basically from MN to adventitious buds or leaflets. The induction conditions were defined and can be used as reference for the in vitro protocol for tree peonies propagation. Main results were as follows:1. Callus formation was the premise of MN formation. The result suggested that cultivar, developmental stage and position of explant significantly affected callus formation. Among the seven tested cultivars,’Golden Era’performed best and produced the higher induction rate and amount of compact callus. Explants taken between flowering and 1 week after blossom fall showed higher callus induction rate and negligible browning. Petiole layers explants taken from the basal or top petioles showed lower browning rate and higher callus induction rate. While, the explants taken from middle petioles showed serious browning problem. The optimum formulation for callus formation was SH medium containing 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Higher concentration of 2,4-D leaded to vitrification or browning. Meanwhile thidiazuron (TDZ) promoted callus formation, and the effect was more noticeable when TDZ was combined with 2,4-D.1 mg/L 2,4-D and 1 mg/L TDZ produced the higher formation rate and good quality callus of’Golden Era’.2. The genesis and development of tree peonies callus were divided into 0-4 five grades. The cytohistological observation suggested that callus formation underwent three stages, namely activation, division and formation. During activation, fascicular cambium cells were most notable and started the division process. Cells kept division during the second stage division and led to the formation of callus in cambial area firstly and then in the region of cortex parenchyma cells. These callus tissues formed a compact cell cluster and the callus cells were characterized by dense cytoplasm, nucleus in center and small in volume. However, callus proliferated at a slower rate during formation stage, only the peripheral callus cells which facing the medium kept division.3. Tree peonies MN differentiated from the callus internally. There were two kinds of structures in the center of tree peonies MN. Some of tracheids organized as vascular centers and some specialized parenchyma cells could also act as centers, and then these centers were surrounded by loosely arranged, plastid-dense cell layers. This way, MN formed. The formation of tree peonies MN were regulated by cultivar, pre-formation of position of explants, PGRs, nitrogen form and sucrose concentration. Callus of’Golden Era’,’Bartzella’and’High Noon’proliferated into nodule structures and the explants from the central part of petiole produced higher induction rate of MN than from the basal or top petioles. The optimum formulation for MN was SH medium containing 0.2 mg/L2,4-D and 6 mg/L N6-benzyl adenine (BA) for both of’Golden Era’and’Bartzella’. While the ratio of NO3-/NH4+ was 2.4 and sucrose concentration was 50 g/L in the medium,’Golden Era’produced highes MN induction rate (78.0%). And when the ratio of NO3-/NH4+ was 9.5 and sucrose concentration was 30 g/L in the medium, ’Bartzella’produced its highes MN induction rate (55.6%).4. Proliferation was an important part of the MN protocol, a-naphthalene acetic acid (NAA) and TDZ influenced the MN multiplication according to cultivar. Maximum MN multiplication rate was gained in 0.5 mg/LNAA and 1 mg/L TDZ for’Golden Era’(83.3%) and 8 mg/L BA and 5 mg/L TDZ for’Bartzella’(91.7%). The MN mainly increased their number when cultured in liquid medium. And the best liquid culture period was 30 days for’Golden Era’.5. Differentiation was the key of the MN protocol. The results suggested that the differentiation of ’Golden Era’MN has experienced two steps. The first step related to transfer the MN to SH medium containing 0.25 mg/L NAA+0.5 mg/L TDZ or 0.5 mg/L TDZ under light condition for 45 days. The second step, the SH medium containing 25 g/L sucrose+2 g/L activated carbon were useful. Then 16% rate of adventitious buds and leaflets were observed. Among these factors, lower concentration of PGRs and light condition are major factors for differentiation. In addition, fewer subculture time and long subculture cycle were more easily differentiated. Epidermic cell divisions preceded shoot formation of MN and the communication-vascular bundles between buds and the MN was observed.更多还原