| 其他摘要 | 喀斯特地区地质环境脆弱，碳酸盐岩的造壤能力低，营养元素缺乏，土壤贫瘠。喀斯特地区土壤还存空间分布的不均匀性。土壤中的氮是喀斯特土壤肥力的重要组成部分，土壤中的氮含量可以作为反映喀斯特土壤肥力的重要指标。而由于喀斯特土壤的不均匀分布，必然导致了土壤氮含量存在较大的差异性。此外，土壤中的无机氮一般呈现低铵多硝的赋存特征。脆弱的地质环境还导致了脆弱的植被系统，喀斯特地区通常的生物多样性缺乏，植被结构简单，生态问题非常严重。氮素的同化是植物生命活动最重要的生理过程之一，氮还是植物生长发育过程所必需的营养元素，是蛋白质、核酸、酶和叶绿素等重要组成成分。因此，研究不同氮环境下植物（组培苗）的无机碳氮的同化情况及同位素响应情况，将为喀斯特地区植物的氮管理提供指导依据。同时要恢复喀斯特地区脆弱的生态系统，必定需要大量的喀斯特适生植物的苗木。而植物组织培养技术为供应大量的喀斯特适生苗木提供了技术支持。此外，在研究组培苗的无机氮利用机制时，同时研究不同氮环境对组培苗自养能力的影响，这将为获取优质的适生植物组培苗提供有效的氮管理。本论文选择喀斯特适生植物诸葛菜组培苗和非适生植物甘蓝型油菜组培苗作为研究材料。为两种组培苗提供不同的氮环境（硝态氮作为单独氮源；硝态氮浓度是铵态氮浓度2倍组成的混合氮源；恒定浓度的铵态氮变化浓度的硝态氮组成的混合氮源；恒定浓度的硝态氮与变化浓度的铵态氮组成的混合氮源），两种稳定氮同位素值差距较大的硝酸盐作为本研究中的硝态氮，本研究中所用的铵态氮的稳定氮同位素值与两种硝态盐的稳定氮同位素值的差值均较大。测定组培苗叶片的碳氮元素含量来探讨了不同氮环境下组培苗的碳氮同化情况；测定组培苗叶片的稳定碳同位素值来探讨了不同氮环境对组培苗自养能力的影响；根据硝态氮作为单独氮源下组培苗叶片的稳定氮同位素分馏值来探讨了硝酸盐同化能力与硝酸盐供应的关系，并比较了适生组培苗和非适生组培苗的硝酸盐同化能力；利用双向稳定氮同位素标记技术量化了不同氮环境下组培苗硝态氮和铵态氮的利用情况；以上研究结果为揭示喀斯特地区植物的无机氮利用策略提供了新的依据。本研究的主要结论如下：（1）硝酸盐作为唯一氮源时对组培苗碳氮同化及同位素影响的研究。通过研究不同硝酸盐浓度下组培苗生长和生理指标，我们发现诸葛菜组培苗的生长对硝酸盐浓度需求较小，而甘蓝型油菜组培苗的生长对硝酸盐浓度的需求较高。在合适的硝酸盐浓度下，两种组培苗的自养能力均较高。增加硝酸盐的供应量能显著促进两种组培苗叶片氮含量的增加，而叶片碳含量则呈现缓慢下降的趋势。两种组培苗在各硝酸盐浓度下均发生了稳定氮同位素分馏，但发生分馏的程度不一样。两种组培苗的叶片稳定氮同位素分馏值均与其叶片氮含量呈现显著的负相关性。（2）无机氮浓度对组培苗碳氮同化及同位素的影响。我们发现增加无机氮的浓度能显著促进诸葛菜组培苗和甘蓝型油菜组培苗叶片的氮含量的增加，而叶片碳含量则呈现缓慢下降的趋势。虽然在无机氮浓度处理中，硝态氮的浓度都是铵态氮浓度的2倍，但是两种组培苗叶片的氮含量主要来自铵态氮，铵态氮是两种组培苗优先利用的氮源，且是高效利用。诸葛菜组培苗在各无机氮浓度下叶片稳定碳同位素值无显著变化，而甘蓝型油菜组培苗的叶片稳定碳同位素值则随着无机氮浓度的增加而显著偏负。诸葛菜组培苗在最低的无机氮浓度下长势较好，增加无机氮的浓度对诸葛菜组培苗的生长无显著促进作用，而甘蓝型油菜组培苗的生长量则随着无机氮浓度的增加而增加。诸葛菜组培苗对无机氮营养的较小需求可能是其作为喀斯特适生植物的特征之一。（3）铵态氮浓度恒定下，硝态氮浓度对组培苗碳氮同化及同位素的影响。在铵态氮浓度都为20mM的情况下，增加硝态氮的浓度导致诸葛菜组培苗和甘蓝型油菜组培苗叶片氮含量增加的幅度并不大，但两种组培苗叶片的氮含量均高达5%以上（叶片干重的百分比）。然而，组培苗叶片的碳含量就明显受到硝态氮浓度影响，在硝态氮浓度为5mM时，两种组培苗的叶片碳含量都是最低的。通过双向稳定氮同位素标记技术，我们发现在所有处理下，铵态氮都是两种组培苗的主要吸收利用的氮源，即使在硝态氮浓度为40mM,铵态氮对两种组培苗叶片氮含量的贡献仍然超过了65%。硝态氮的浓度对两种组培苗生长量和叶片稳定碳同位素值影响非常显著，在5mM硝态氮浓度下，两种组培苗的生长量均最小，且有最偏正的稳定碳同位素值。（4）硝态氮浓度恒定下，铵态氮浓度对组培苗碳氮同化及同位素的影响。在硝态氮浓度都为20mM的情况下，增加铵态氮的浓度能显著促进诸葛菜组培苗和甘蓝型油菜组培苗叶片氮含量的增加，然而，在最高铵态氮浓度（40mM）下,两种组培苗的叶片碳含量均出现了显著下降，而其它铵态氮浓度下两种组培苗叶片碳含量均无明显变化。通过双向稳定氮同位素标记技术，我们发现很低浓度的铵态氮（0.5mM和1mM）都能被两种组培苗吸收利用。随着铵态氮浓度的增加，两种组培苗铵态氮的利用份额都逐渐增加。此外，两种组培苗的生长量对铵态氮浓度的响应也明显不同。高浓度的铵态氮（40Mm）还抑制了两种组培苗叶绿素的合成，从而导致了两种组培苗很偏正的稳定碳同位素值。 关键词：喀斯特，无机氮，稳定同位素，氮同位素分馏。; The geological environment is fragile in karst region,where carbonate rocks had low capacity for building soils. Lack of nutrient elements resulted in poor soil. The distribution of soils was uneven in karst region. The nitrogen in soils was the vital components of soil fertility and the nitrogen content in soils could be as an important indicator for reflecting the soil fertility in karst region. The uneven distribution of soils resulted in variable soil nitrogen content in karst region. Morever, compared to the ammonium concentration，the nitrate concentration was rich in karst region. The fragile geological environment was be accompanied by vegetation system. The biodiversity was poor and the vegetation structure is simple in karst region. The ecological problem is very serious. The assimilation of nitrogen is one of the most important physiological processes of plant life activities. Nitrogen is essential for plant growth and development process. It is a vital component of proteins, nucleic acids, enzymes, and chlorophyll. Hence, the study of the carbon and nitrogen assimilation and isotope response of plants (plantlets in vitro) will contribute to effective nitrogen management for the plants in karst region. Meanwhile, vast seedlings which are adapted to grow in karst region are necessary for recovering the fragile ecosystems in karst region. The plant tissue culture technology makes it possible to supply vast seedlings which are adapted to grow in karst region. Moreover, studying the effect of variable nitrogen condition on the photosynthetic capacity of plantlets in vitro will contribute to effective nitrogen management for culturing high quality plantlets in vitro. The Orychophragmus violaceus (Ov) and Brassica napus (Bn) plantlets were selected in this study. The Ov plantlets are adapted to grow in karst regions, while the Bn plantlets are not. The Ov and Bn plantlets were grown in variable nitrogen condition (the nitrate as the sole nitrogen; the inorganic nitrogen treatment which the nitrate concentration was twice of the ammonium concentration; the nitrogen treatment which contained constant ammonium concentration and variable nitrate concentration; the nitrogen treatment which contained constant nitrate concentration and variable ammonium concentration.). The nitrate with highδ15N（natural nitrogen isotope composition）values and lowδ15N values was selected for this study, the ammonium, whose theδ15N values are obviously different with theδ15N values of the nitrate, was also selected for this study. Foliar carbon and nitrogen content of plantlets was measured to study the carbon and nitrogen assimilation status of plantlets. Foliar δ13C (natural carbon isotope composition) values of plantlets was also measured to study the effect of variable nitrogen conditions on the photosynthetic capacity of the plantlets. When the nitrate was the sole nitrogen source, the foliar stable nitrogen isotope fractionation of plantlets was employed to study the relationship between the nitrate assimilation ability and nitrate concentrations. Meanwhile, the nitrate assimilation ability of Ov and Bn plantlets was compared. The amount of nitrate and ammonium which were utilized by plantlets was quantified via the bi-directional stable nitrogen isotope labeling technique. The above results provided new basis for revealing the inorganic nitrogen use strategy of the plants in karst region.The main conclusions of this study are as follows:(1) The study of carbon and nitrogen assimilation and isotope response of plantlets under different nitrate concentrations: The biomass and physiological index of plantlets which were cultured under different nitrate concentrations were measured. We found that the nitrate demand of Ov plantlets was small，while the nitrate demand of Bn plantlets was relatively high. The Ov and Bn plantlets had better photosynthetic capacity under suitable nitrate concentrations. The foliar nitrogen content of Ov and Bn plantlets increased with increasing nitrate concentrations, while the foliar carbon content showed a slow decline with increasing nitrate concentrations. Nitrogen isotope discrimination occurred in both Ov and Bn plantlets cultured under different nitrate concentration. However, the nitrogen isotope fractionation value was different for both Ov and Bn plantlets cultured under different nitrate concentration. An negative correlation between foliar nitrogen isotope fractionation value and foliar nitrogen content was observed for both Ov and Bn plantlets.(2) The effects of inorganic nitrogen concentration on carbon and nitrogen assimilation and isotope of plantlets. We found that increased inorganic nitrogen concentration could significantly promote the increase of foliar nitrogen content for both Ov and Bn plantlets, while the foliar carbon content decreased slowly. Although the nitrate concentration was twice of the ammonium concentration in this treatment, the foliar nitrogen content of both Ov and Bn plantlets was mainly consisted of the ammonium. The ammonium was prior to utilize for both Ov and Bn plantlets, and utilization efficiency of the ammonium was high. The foliarδ13C values of the Ov plantlets had no significantly changes under all inorganic treatments, while the foliarδ13C values of the Bn plantlets decreased with increasing inorganic nitrogen concentration. The growth of Ov plantlets was good under the lowest inorganic nitrogen concentration. The increased inorganic nitrogen concentration did not promote the growth of the Ov plantlets. However, the biomass of the Bn plantlets increased with increasing inorganic nitrogen concentration. The small demand of inorganic nitrogen of the Ov plantlets could make it to adapted to grow in karst region. (3) When the ammonium concentration was constant, the effect of nitrate concentrations on the carbon and nitrogen assimilation and isotope of plantlets: When the ammonium concentration was 20 mM, the foliar nitrogen content of both Ov and Bn plantlets did not show a big increment. whilethe foliar nitrogen content of both Ov and Bn plantlets was still beyond 5%(expressed as a percent of dry weight).However, the foliar carbon content of plantlets was significantly affected by nitrate concentrations. When the nitrate concentration was 5mM，the foliar carbon content of both Ov and Bn plantlets was minimum. Through the bi-directional stable nitrogen isotope labeling technique, we found that the ammonium was the main nitrogen form which was assimilated for both Ov and Bn plantlets under all treatments. Even if the the nitrate concentration was 40mM，65 percent of foliar nitrogen of both Ov and Bn plantlets derive from the ammonium. The biomass and δ13C values were significantly affected by nitrate concentrations. When the nitrate concentration was 5mM，the biomass of both Ov and Bn plantlets was minimum, and theδ13C values of both Ov and Bn plantlets was maximum.(4) When the nitrate concentration was constant, the effect of ammonium concentrations on the carbon and nitrogen assimilation and isotope of plantlets: When the nitrate concentration was 20 mM, the foliar nitrogen content of both Ov and Bn plantlets increased with increasing ammonium concentrations. However, when the ammonium concentration was 40mM, the foliar carbon content of both Ov and Bn plantlets declined significantly . The foliar carbon content of both Ov and Bn plantlets under other ammonium concentration. Through the bi-directional stable nitrogen isotope labeling technique, we found that low ammonium concentrations (0.5 mM and 1 mM) could be absorbed and assimilated by both both Ov and Bn plantlets. The share of the ammonium which was utilized by both both Ov and Bn plantlets increased with increasing ammonium concentrations. In addition, the effect of ammonium concentrations on the biomass was different for both Ov and Bn plantlets. The synthesis of chlorophyll was inhibited by high ammonium concentrations (40Mm). Consequently，theδ13C values of both Ov and Bn plantlets was maximum. Key Words: Karst, Inorganic Nitrogen, Stable Isotope, Nitrogen Isotope Fractionation, Element Content |
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