【作者】 安佰义；

【导师】 李玉花；

【作者基本信息】 东北林业大学 ， 园林植物与观赏园艺， 2005， 硕士

【摘要】 本文以牡丹(Paeonia suffruticosa Andr.)单瓣品种‘凤丹白’为主要试材,初步建立了牡丹组培离体再生系统。 实验测定了不同花型品种叶片间及‘凤丹白’5种不同类型外植体(幼叶、成龄叶、幼茎、暗处理叶片、子叶)间总酚含量和PPO活性,分析了总酚含量、PPO活性与外植体褐化的关系;通过不同的消毒、预处理、培养基类型和培养条件的选择,对牡丹幼叶初始培养的褐变进行了研究;以幼叶、未木质化叶柄、子叶为外植体,研究了培养基类型、激素种类及浓度配比、碳源、培养条件等因素对牡丹组培离体再生系统建立的影响;研究了不同培养基组分对成熟胚直接诱导丛生芽“玻璃化”程度的影响;以休眠侧芽为外植体比较了60个牡丹品种的扩繁能力。 研究结果如下: (1) 确定了牡丹初始培养的较佳外植体类型。不同品种间或同一品种的不同类型外植体间,总酚含量、PPO活性、初始外植体褐化率差异显著。子叶和幼茎的总酚含量、PPO活性较其它类型外植体低,是初始培养最佳的外植体。 (2) 筛选了有效降低外植体褐变的技术措施。外植体经过浓度为10%的次氯酸钠(有效氯1%)消毒10min后,用无菌的抗氧化剂预处理液(MS+150mg/L柠檬酸+100mg/L抗坏血酸,pH4.5)对外植体进行30min预处理,以WPM作为基本培养基,辅加500mg/L PVP或15mg/L Vc,在弱光、较低的温度(18～20℃)培养,可有效减轻外植体的褐变。 (3) 筛选出牡丹离体培养的培养基类型。诱导愈伤组织的最佳培养基为WPM培养基,添加2,4-D 0.5mg/L+TDZ 0.5mg/L或2,4-D 1mg/L+TDZ 1mg/L的激素组合。利用来源于子叶的愈伤组织进行不定芽分化的培养基为WPM+0.5mg/L TDZ。以成熟胚为外植体直接诱导丛生芽的最佳培养基为WPM+6-BA2.5mg/L+2,4-D 0.1mg/L。生根最佳培养基为1/2 WPM+2mg/L IBA。在低温、暗培养条件下处理10天,在生根前进行5周壮苗处理,有利于提高无根苗的生根率。移苗前打开瓶盖炼苗3～5天,培养室内保持温度严格控制在25℃±1℃,湿度大于80%,可使幼苗移栽成活率达到80%以上。 (4) 解决了牡丹丛生芽诱导后的“玻璃化”现象。在WPM培养基中添加NAA0.1mg/L+6-BA1mg/L,选用7g/L琼脂可有效改善丛生芽的“玻璃化”现象。 (5) 牡丹基因型的差异是影响离体扩繁的主要原因。以休眠侧芽为外植体,通过对60个牡丹品种间的扩繁能力比较,发现扩繁能力的大小取决于基因型。不同品种间扩繁能力差异显著,不同品种间繁殖系数介于2～5之间。总的来看,在连续继代培养48周后(约1年),扩增率下降,只有‘大胡红’、‘紫金绫’、‘丛中笑’保持较高扩增率。更多还原

【Abstract】 Micropropagation of tree peony (Paeonia suffruticosa) was studied and in vitro regeneration system was established on single form cultivar ’Feng Dan Bai’.The content of total phenols and activity of PPO was analysed among cultivars with different flower form and 5 different explants of ’Feng Dan Bai’. The relationship was analyzed between total phenols content, activity of PPO and browning of explants. We investigated the effects of sterilization, pre-treatment, medium composition and culture condition on the browning during culture of young leaves used as explants. With young leaves, petioles and cotyledon as explants, the influence of medium composition, the kind and concentration of hormones, carbon source and culture condition were studied to establish regeneration system of tree peony. The influences of medium composition on the vitrification of cluster-buds regeneration by direct induction from mature embryos as explants were studied. With dormant axillary buds as explants, the reproductive capabilities were compared among 60 cultivars of tree peony.The results are as follows.(1) The aptitude explants for incipient micropropagation of tree peony were selected. Odds were significant in the content of total phenols, activity of PPO and browned degree among cultivars or kinds of explants. With lower content of total phenols and lower activity of PPO, cotyledons and young stems were seclected as explants for in vitro incipient culture.(2) Effective technique to reduce browning of explants was experimented. Following operation were : sterilizing with 10%NaOCl for 10 min, pre-treatment with antioxidant solution (MS + citric acid 150mg/L+ Vc100mg/L) for 30min, use of WPM as the basic medium, adding PVP500mg/L or Vc15mg/L to the medium, and culture explants at low temperatures(18~20℃) and dim light.(3) The aptitude mediums for in vitro culture of tree peony were selected. WPM medium with 2, 4-D 0.5mg/L+TDZ 0.5mg/L or 2,4-D 1mg/L+TDZ 1mg/L, was suitable for the induction of callus. WPM medium with TDZ 0.5mg/L was better for the bud redifferentiation of callus originated from cotyledon. With mature embryos as the explants, WPM medium with 6-BA2.5mg/L + 2,4-D 0.1mg/L is the aptitude medium for the direct-induce of cluster-buds. As for rooting, 1/2WPM medium with 2mg/L IBA showed a better result. Pre-treatment at low temperatures and dark conditions for 10 days and 5 weeks culture in WPM medium without hormones could improve a rooting rate. In the process of seedlings acclimatization, removing container cap for 3 to 5 days before transplanting, controlling temperature strictly at 25℃ ± 1 ℃ and humidity at 80% could improve a surviving rate of seedlings to 80% after transplanted.(4) Vitrification on cluster buds was controlled effectively by modified WPM medium with addition of NAA0.1mg/L + 6-BA1mg/L and agar 7g/L.(5) Genotype plays a important role in the micropropagation of tree peony. With dormantaxillary buds as explant, an obvious odd is showed in reproductive capabilities of 60 cultivars of tree peony with its coefficient ranging from 2 to 5. Reproductive capability of axillary buds was relative to genotype significantly. In addition, reproductive rates decreased for most cultivars after 48 weeks subculture except for three cultivars — ’Da Hu Hong’, ’Zi Jin Ling’ and ’Cong Zhong Xiao’.更多还原