【作者】 冯金玲；

【导师】 陈辉；

【作者基本信息】 福建农林大学 ， 森林培育， 2011， 博士

【摘要】 芽苗砧嫁接是植物嫁接繁殖中的一种新方法。我国对芽苗砧嫁接技术的研究时间不长,处在初期阶段,而且仅应用于油茶、银杏、板栗、核桃等少数植物上,尚未大规模推广应用。科学研究上,芽苗砧嫁接仅在嫁接法、接穗的选择、嫁接时间、栽培基质等方面进行初步的探讨。本论文以油茶芽苗砧嫁接体为材料,研究芽苗砧嫁接口愈合过程的机理。采用石蜡切片法对油茶芽苗砧嫁接口的愈合进行解剖学研究。观察结果表明,在嫁接后的第4天,嫁接口产生隔离层；在嫁接后的第8天,嫁接口的砧木产生愈伤组织；在嫁接后的第16天,嫁接口的接穗产生愈伤组织；在嫁接后的第22天,嫁接口的砧木与接穗连接；在嫁接后的第29天,嫁接口的形成层形成；在嫁接后的第35天,嫁接口的愈伤组织维管束形成,接穗连接成功。通过油茶芽苗砧嫁接,对嫁接口发育中的植株死亡率、可溶性糖含量、相对电导率、POD、SOD及CAT酶活性进行研究,探讨芽苗砧嫁接亲和性生理机理。结果表明,上述指标的总体变化趋势一致,表现为先增后降的趋势。各个指标的高峰值出现的具体时间虽各不同,但都集中在嫁接后18天左右出现。相关分析得出植株死亡率与POD呈极显著正相关,与相对电导率呈显著正相关,与SOD相关不显著。为探讨油茶芽苗砧嫁接口和作为对照的愈伤口的发育差异,在处理后的2-55 d对嫁接口和愈伤口间隔取样,进行苯丙烷代谢若干生理指标检测。结果表明：嫁接口的苯丙氨酸解氨酶(PAL)活性极显著低于愈伤口；过氧化物酶(POD)活性在嫁接后的第14-35天极显著高于愈伤口；苗木重量、纤维素含量、绿原酸含量和多酚氧化酶(PPO)活性与愈伤口无显著差异；嫁接口各生理指标变化的拐点数量及所处时间与愈伤口不同。相关分析表明：嫁接口的PPO活性与苗木重量,绿原酸与纤维素含量,绿原酸与苗木重量之间的相关性与愈伤口相当；绿原酸含量与PPO活性,绿原酸含量与POD活性,纤维素含量与苗木重量之间的相关性明显高于愈伤口；而PPO活性与POD活性,PPO活性与纤维素含量之间的相关性却低于愈伤口。采用酶联免疫吸附法(ELISA)测定油茶芽苗砧嫁接口和对照愈伤口的内源激素生长素(IAA)、赤霉素(GA3)、细胞分裂素(ZR)和脱落酸(ABA)含量的动态变化,研究嫁接口愈合过程中内源激素含量的变化规律,及其和愈伤口变化的异同点。研究结果表明：内源激素IAA含量、ZR含量和GA含量的变化与嫁接口和愈伤口的发育阶段相吻合,ABA含量变化更复杂。嫁接口内源激素含量变化比愈伤口更激烈,说明嫁接口发育比愈伤口更复杂。嫁接口的IAA含量和GA含量整体上低于愈伤口,ZR的含量和ABA含量总体上与愈伤口相当,说明内源激素IAA和GA对嫁接口的愈合更重要。利用AFLP对不同发育阶段的油茶芽苗砧嫁接口,油茶芽苗砧愈伤口以及接穗母树、砧木和接穗萌发芽进行分析。从64对引物组合中筛选出2对,2对引物共扩增出82个位点,多态性位点数16,多态性比例0.13-0.23。结果得出：油茶芽苗砧嫁接口发育过程中,条带数变化相对比较大,特别是在嫁接后的第4、16、29、35天。油茶芽苗砧愈伤口发育过程中,条带数几乎没有变化。接穗萌发芽的条带数比接穗母树和砧木的多,在E1M1组合引物中,有3条带是在接穗母株无,而在接穗萌发芽和砧木存在；有3条带是在接穗母株和砧木中都为无,但在接穗的萌发芽中却为有；有1条带是在接穗母株和砧木中都为有,而在接穗的萌发芽中却为无。由上分析,油茶芽苗砧嫁接口愈合过程中条带数的变化,其原因需进一步深入研究。以油茶芽苗砧嫁接口为研究对象,运用蛋白质组学方法和技术比较油茶芽苗砧嫁接口愈合过程中不同发育阶段的蛋白质组变化。应用双向电泳对嫁接口蛋白进行分离,从2-D凝胶上检测到分离的蛋白质点约1000个,用Image Master TM 2D Platinum software7.0凝胶图谱进行分析,获得40个表达量差异在2倍以上的差异蛋白。MALDI-TOF-TOF/MS串联质谱分析和数据库检索鉴定了其中34个差异表达蛋白,鉴定率为85%。鉴定得到的蛋白中,与油茶芽苗砧嫁接口愈合关系密切的植物光合作用相关蛋白,呼吸作用相关蛋白,能量代谢相关蛋白,转录和翻译相关蛋白,次生代谢相关蛋白,细胞生长和分化相关蛋白,调控相关蛋白以及抗性相关蛋白的生物学功能都得到了讨论。这些蛋白在油茶芽苗砧嫁接口愈合过程中差异表达,说明油茶芽苗砧嫁接口愈合的过程中发生了一系列生理生化代谢变化。更多还原

【Abstract】 The nurse seed grafting is a new plant propagation of grafting technique. The time of study on this technique which only apply to some plants, for example Camellia oleifera, Ginkg o biloba, Castanea mollissima, Carya cathayensis, is not long in china, which is in its infancy. The nurse seed grafting technique is not on a large scale applied yet. On scientific research, it was carried out preliminary exploration on the grafting method, selection scion, grafting time, cultivated media. The paper adopted the nurse seed grafting union as the experiment material, studied the mechanism of healing process of the nurse seed grafting union.Conventional paraffin sectioning was adopted to conduct the anatomical examination of the nurse seed grafting union during the development. The results showed that at day 4 after grafting the nurse seed grafting union come to being the isolation layer; at day 8 after grafting, come forth the callus in the root stock; at day 16 after grafting, the callus was found in the scion; at day 22 after grafting, the root stock connected the scion; at day 29 after grafting, the nurse seed grafting union formed the vascular cambium; at day 35 after grafting, the vascular bundles was formed, which indicated that the nurse seed grafting union in Camellia oleifera survived.The death rate, soluble sugar content, the relative conductivity, SOD, CAT and POD activity in camellia oleifera grafted chimera through nurse seed grafting were studied for inoculating the mechanism of physiological affinity. The results show that the content and activities of these index increase gradually in early period after grafting and decrease gradually in later period after grafting. The times on the peak value of these index are different, but they appear centrally at 18 days approximately after grafting. The death rate is extreme- significantly and significantly positive correlation with the POD activity and the relative conductivity respectively, but has no significant correlation with the SOD activity.To discuss on the difference of development between the nurse seed grafting union in Camellia oleifera and the healing wound (the contrasts), the test materials were interval sampled during 2-55 days after treatment and some physiological phenylpropanoid metabolic indices were measured. The results showed that the phenylalanine ammonialyase (PAL) activities of the grafting union were highly significant lower than the healing wound; that during 14-35 days after treatment the peroxidase (POD) activities of the grafting union were highly significant higher; The weight of seedling, the content of cellulose, the content of chlorogenic acid and the polyphenoloxidase (PPO) activities of the grafting union had no significant difference; The number and the time of break point in the grafting union physiological indices differentiated from the healing wound. By correlation analysis, the correlations of the PPO activities and the weight of seedling of the grafting union, the content of chlorogenic acid and the content of cellulose, the content of chlorogenic acid and the weight of seedling were equal to the healing wound; The correlations of the content of chlorogenic acid and the PPO activities, the content of chlorogenic acid and the POD activities, the content of cellulose and the weight of seedling were obviously higher than that of the healing wound; But the correlations of the PPO activities and the POD activities, the PPO activities and the content of cellulose were lower than that of the healing wound.In order to find out variation rule in the development of the nurse seed grafting union and different from the nurse seed grafting union and the healing wound, the contents of endogenous hormones IAA, GA3, ZR and ABA from both of them were determined by ELISA. The results showed that for the contents of IAA, ZR and GA, the nurse seed grafting union and the healing wound conform to the stage of development of their, the change in the contents of ABA became more complicated. The endogenous hormone change in the nurse seed grafting union was greater than in the healing wound, which showed that the development of the nurse seed grafting union was more complicated than the healing wound. The contents of IAA and GA3 from the nurse seed grafting union as a whole was below the healing wound, the contents of ZR and ABA approximately was the same to the healing wound, which showed that endogenous hormones IAA and GA3 were very importance to the development of the nurse seed grafting union.The difference of development between the nurse seed grafting union in Camellia oleifera and the healing wound, the mother tree of the scion, the root stock and the germinate buds from the scion were examined by AFLP(Amplified Fragment Length Polymorphism) technology.2 primers pairs were selected from 64 primer pairs to amplify the genetic DNA. Eighty-two loci were obtained, which sixteen were exhibited polymorphism, the average percentage of polymorphic loci was from 13% to 23%. The results showed that the number of bands changed relatively large during the development of the nurse seed grafting union, especially at day 4,16,29 and 35 after grafting; the changes in the number of bands during the development of the healing wound were small; the number of bands in the germinate buds from the scion were more than the mother tree of the scion and the root stock; from E1Ml primer pair, there were three bands not in the mother tree of the scion but in the root stock and the germinate buds from the scion; there were three bands not in the mother tree of the scion and the root stock but in the germinate buds from the scion; there was one bands not in the germinate buds from the scion but in the mother tree of the scion and the root stock. According to the above analysis, the gene changes during the development of the nurse seed grafting union in Camellia oleifera would be left behind to further research.Proteomics method was used to compare and identify the differential proteins of the development of the nurse seed grafting union in Camellia oleifera. Over 1000 protein spots were detected on 2 DE gels, mage Master TM 2D Platinum software7.0 was applied on gel pattern analysis,40 differentially expressed proteins were detected.34 differential proteins were identified by MALDI-TOF-TOF/MS and protein database searching, the identification rate was 85%. Biological function analysis showed these identificated proteins related with photosynthesis, respiration, energy metabolism, transcription and translation, secondary metabolism, the cells growth and differentiation, regulation and stress tolerance. The differential expression of these proteins showed that the healing of the nurse seed grafting union in Camellia oleifera changed a series of physical and chemical metabolism.更多还原