【作者】 谭钺；

【导师】 高东升；

【作者基本信息】 山东农业大学 ， 果树学， 2013， 博士

【摘要】 自然休眠是落叶果树为抵御冬季寒冷环境发展出的一种生物学特性，其解除需要一定时间的低温积累。低温积累不足会导致芽萌发及后续枝叶生长异常，严重影响树体生产能力。由于这一特性，自然休眠严重限制了设施果树生产及温暖地区的落叶果树的栽培生产。目前对休眠解除机制的研究已涉及生理、生态、生化、分子生物学等多个领域，研究还发现除低温外许多处理也具有快速打破自然休眠的作用。为探讨自然休眠快速解除的机制及关键调控因子，为安全、高效快速破眠技术的开发提供参考，本研究选择三种不同类型的破眠处理，50℃高温（物理类）、单氰胺（化学类）和TDZ（生长调节剂类），对其破眠过程进行了研究。许多研究表明呼吸代谢、H₂O₂代谢和Ca²⁺在休眠解除过程中都发生标志性变化，而Ca²⁺又是细胞分裂重要的调控因子，因此，本研究也围绕这三个因子在休眠解除中的地位和相互关系展开。试验于2010年²013年在山东农业大学园艺学院进行，以‘曙光’油桃（Prunus persica var. nectariana cv. Shuguang）为试验材料。试验分析了休眠芽的细胞周期特点，研究了三种破眠处理的破眠作用及对H₂O₂代谢、呼吸代谢和激素含量的影响，并检测了破眠剂单氰胺、TDZ及信号物质H₂O₂对芽休眠组织Ca²⁺转运的影响。初步总结了休眠快速解除过程中呼吸代谢、H₂O₂代谢和Ca²⁺的关系。主要结果如下：1.芽分生组织在萌芽期同时存在二倍体细胞群（G1期）和四倍体细胞群（G2期），而在自然休眠期没有明显的四倍体（G2期）细胞群，染色体复制受到抑制。2.三种破眠处理中50℃高温和单氰胺破眠效果较好，而TDZ破眠效果较差。对于花芽，单氰胺处理后芽萌发最早、萌芽率最高，50℃高温次之，TDZ最差；而对于叶芽，50℃高温破眠效果最好，单氰胺次之，TDZ最差。单氰胺对枝条和芽具有显著的毒害作用，主要表现为枝条颜色改变、枝梢干枯、芽及周围组织坏死、脱落等。3.自然休眠期，芽分生组织细胞内Ca²⁺大量分布于细胞核和细胞质基质区域，质膜钙通道活跃，组织对Ca²⁺呈现吸收状态。低浓度的单氰胺和TDZ对休眠芽分生组织Ca²⁺转运影响较小，但在有效破眠浓度下，单氰胺能够显著降低组织对Ca²⁺的吸收速率，TDZ则显著提高组织对Ca²⁺的吸收速率。4.50℃高温和单氰胺都能抑制休眠芽CAT表达和CAT活性，并引起H₂O₂积累，但对POD活性影响不大。50℃高温引起的CAT活性抑制和H₂O₂积累发生较早，持续时间较短，单氰胺引发的CAT抑制和H₂O₂积累发生较晚，持续时间也较长。TDZ对芽内H₂O₂含量、CAT活性、POD活性均未表现出显著影响。5.在深休眠期对休眠枝条外施H₂O₂后，没有显著的破眠作用。但外源H₂O₂改变休眠芽Ca²⁺转运状态。低浓度H₂O₂降低组织Ca²⁺吸收速率；高浓度H₂O₂使组织对Ca²⁺的转运由吸收转变为释放，这种变化与休眠解除前后Ca²⁺区域分布的变化一致。高浓度H₂O₂还诱导组织对K+离子的释放速率升高，对Na+离子的吸收速率降低，对Mg²⁺的吸收速率升高。6.50℃高温、单氰胺和TDZ对休眠芽呼吸代谢影响不同。在总呼吸速率上，50℃高温处理后芽呼吸速率数小时内都维持在低水平；单氰胺能够直接抑制休眠芽的呼吸作用，抑制程度与浓度呈正比，但处理后1d内芽呼吸速率又显著升高；TDZ对休眠芽呼吸速率没有显著影响。在呼吸代谢电子传递途径上，50℃高温和单氰胺都直接抑制电子传递的CYT途径，对ALT途径没有规律性影响；在底物途径上，高温和单氰胺在处理后1⁴d活化PPP途径，TCA途径在萌芽前活化。TDZ对呼吸途径变化影响较小，仅在处理后第12d表现出对PPP的小幅活化。7.50℃高温和单氰胺处理后，芽内激素（GA、ZR、IAA、ABA）含量均出现下降。高温处理后，芽内4种激素含量均在第4d大幅下降，但萌芽时GA、ZR、IAA含量又升高，ABA含量则仍低于对照；单氰胺处理后，除ZR外，芽内其他3种激素含量也出现下降，但幅度小于高温处理。更多还原

【Abstract】 For deciduous fruit trees, endodormancy is a biological characteristic developed to resistthe cold conditions in winter. The release of endodormancy requires accumulation of chillingtemperatures. Chilling deficiency leads to aberrant budburst and tree growth, which reduceproduction capability of fruit trees seriously. For this reason, endodormancy became one ofthe important limiting factors of protected fruit production and fruit production in warmregions. Studies on dormancy release mechanim now involved various fields such asphysiology, ecology, biochemistry and molecular biology. Experiments also showed thatmany treatments have the effect of breaking dormancy besides chilling temperature. In orderto explore mechanism of short time required endodormancy release, find out the key controlfactors and provide basis for the research of efficiency dormancy-breaking technology,dormancy release processes induced by50℃high temperature（physical treatment）, hydrogencyanamide （chemical treatment） and TDZ （plant growth regulator treatment） were studied inthe experiment.Our study was focused on positions and mutual relationships of respiration, H₂O₂metabolism and Ca²⁺for the reason that they all changed significantly during endodormancyrelease and Ca²⁺was an important regulatory factor of cell division. The experiments wereconducted in College of Horticulture Science and Engneering, Shandong AgriculturalUniversity during2010²013, using one-year-old shoots of ‘shuguang’ nectarine （Prunuspersica var. nectariana cv. Shuguang）. Cell cycle characters of endodormant buds wereanalyzed and effects of the three dormancy-breaking treatments on budburst, respiration,H₂O₂metabolism and hormone contents were studied. Effects of hydrogen cyanamide, TDZand H₂O₂on Ca²⁺transports of dormant bud tissues were also measured. And a preliminarysummary on relations among respiration, H₂O₂metabolism and Ca²⁺were also made. Themain results were as follows:1. Diploid cell group （G1phase） and tetraploid cell group （G2phase） exsited in budmeristem during budburst. But no tetraploid cell group （G2phase） exsited in bud meristemduring endodormancy, which indicated the inhibition of chromosome replication.2.50℃high temperatureand hydrogen cyanamide were more effective in breaking budendodormancy and TDZ showed poorly effects. For floral buds, hydrogen cyanamide resulted in the earliest budburst and highest budburst rate and was the most effective in breakingendodormancy.50℃high temperature was less efficiency and TDZ was the worst. But forvegetative buds,50℃high temperature was the most effective and TDZ had the lowestefficiency. Hydrogen cyanamide also showed toxic effects, which performed as bark colourchanging, shoot tip drying and bud necrosis.3. In endodormant bud meristem cells, high level of Ca²⁺existed in the region of nucleusand cytosol. The Ca²⁺channel was active and meristems were at the state of absorbing Ca²⁺.Hydrogen cyanamide and TDZ at low concentration showed little effects of Ca²⁺transport ofendodormant bud meristem. However, at the effective concentration for dormancy-breaking,hydrogen cyanamide reduced and TDZ raised the absorbing rate of Ca²⁺.4. On H₂O₂metabolism, both50℃high temperature and hydrogen cyanamide inhibitedCAT expression and CAT activity in buds and induced H₂O₂accumulation. POD activitychanged little. The CAT inhibition and H₂O₂accumulation induced by50℃high temperatureoccurred earlier and lasted shorter than that in hydrogen cyanamide treatment. TDZ showedno significant effects on H₂O₂content and activity of CAT and POD.5. Exogenous H₂O₂showed little dormancy breaking effects after application. However,exogenous H₂O₂affected the Ca²⁺tansport of dormant primordiums. H₂O₂reduced theabsorption rate of Ca²⁺at lower concentration and led to transfer direction altering fromabsorption to release at higher concentration. Exogenous H₂O₂also induced the increase of K+release and Mg²⁺absorption, and decrease Na+absoption.6. Respiration of dormant buds was influenced differently by the treatments. Both50℃high temperature and hydrogen cyanamide reduce respiration rates of dormant buds whileTDZ showed no significant effect. Bud respiration rate remained low several hours after50℃treatment. Inhibitory effects of hydrogen cyanamide were in direct proportion to itsconcentration. Interestingly, hydrogen cyanamide also showed promoting effect on respiration1d after treatment. For respiratory electron transfer pathways, both50℃treatment andhydrogen cyanamide inhibited the CYT pathway but show no regularly on the ALT pathway.For the substrate pathway, the PPP pathway was activated in the1st to the4th day after50℃and hydrogen cyanamide treatment, and the TCA pathway was activated before budburst. Nosignificant effects of TDZ on respiratory pathways were detected except the activation of PPPpathway12d after treatment.7.50℃high temperature and hydrogen cyanamide also affect content of hormones suchas GA, ZR, IAA and ABA. In50℃treatment, contents of the4hormones decreasedsignificantly at the4th day. At the time of budburst, contents of GA, ZR and IAA rised and ABA content were still less than control. In hydrogen cyanamide treatment, contents of GA,IAA and ABA also decreased but the range was smaller than that in with50℃treatment.更多还原