【作者】 徐稳定；

【导师】 党志；

【作者基本信息】 华南理工大学 ， 环境工程， 2014， 博士

【摘要】 近年来，镉米事件被多次报道，土壤镉(Cd)污染已危及到人们的日常生活。在治理Cd污染土壤的各种方法中，植物修复以其成本低、不破坏土壤结构、易为社会接受等优点日渐受到重视。同时在修复植物的选择上，玉米因其种质资源丰富，易栽培，生长快，生物量大，适应性强且籽粒中含量低等特点，更是受到国内外不少研究者的关注。根据超甜38(CT38)玉米的营养生长和生殖生长特性，研究其生长周期中的两个关键阶段——幼苗过渡期和开花授粉期的Cd耐受机理和整株富集、分布状况，及授粉过程对整株玉米的Cd富集量和其在不同器官中的富集比例状况，最后，将实验室的研究成果在Cd污染土壤的实地修复中加以验证。获得的主要研究结果如下：1.探索了玉米CT38幼苗在过渡期对Cd的耐受机制。在CK(空白，背景值0.13mg/kg，下同)、1.00、5.00、20.00、50.00、100.00、200.00mg/kg土壤Cd含量的盆栽土中直接播种玉米CT38，测定其发芽状况，观察幼苗从利用胚乳生长到光合作用自养的过渡期内的生长情况，收集、测定其吐水水滴中重金属Cd含量，研究表明：玉米CT38种子的发芽率除Cd含量200mg/kg处理之外，其余处理与空白对照没有显著性下降(n=3,p=0.05，下同)。除根部截留之外，植物通过吐水排Cd也是玉米苗期重要的耐受机制。在5mg/kg以下的含Cd土壤中，植物能完全耐受重金属Cd的逆境胁迫，所吐水滴中Cd浓度低于检测限(<0.1ug/L,石墨炉原子吸收)；在20~100mg/kg之间的含Cd土壤中，玉米植株可通过植物吐水排出多余的Cd而能正常的生长，吐水水滴中最高Cd浓度为10.4mg/L；当达到200mg/kg时，植物遭受重金属胁迫而受损伤，植物吐水排Cd能力减弱，最高Cd浓度降低为8.8mg/L，生长遭受严重的抑制，地上部和地下部干重仅为对照的25.4%和48.3%，并出现幼苗死亡现象。玉米秧苗吐Cd之前，随着土壤中Cd含量不断增加，转运系数(S/R)不断降低；而秧苗吐Cd现象出现以后，S/R则维持在0.5左右。另外，根部和土壤中Cd含量在植物吐Cd以后也呈显著线性相关，植物吐Cd之后，根部Cd含量是土壤中中的11.2倍左右。玉米秧苗吐水排Cd对耐受、富集Cd有重要的意义。2.研究了土壤中不同浓度的Cd胁迫对玉米CT38生长和生理影响。在Cd浓度分别为CK、1、5、20、50、100、200mg/kg的盆栽土壤中种植玉米CT38，授粉后分别测定玉米CT38植株的株高、叶面积、叶绿素含量、叶片中超氧化物歧化酶(SOD)，过氧化氢酶(CAT)和过氧化物酶(POD)的活性以及丙二醛(MDA)的含量，并在扫描电镜下观察玉米的花粉状况。结果表明：灌浆期玉米CT38植株的高度和叶面积均随着土壤中重金属Cd含量的升高先增大后减小，5mg/kg以下低浓度Cd引起的升高并未达到显著水平，50mg/kg以上高浓度Cd的抑制效果显著。灌浆期玉米叶绿素含量随着土壤中重金属Cd含量的增加呈现先升高后降低的现象，5mg/kg处理下最高；而叶绿素a/b则并未表现出明显下降趋势。随土壤中重金属Cd含量升高，灌浆期玉米叶片SOD活性先升高后降低，20mg/kg处理时最高，CAT和POD活性则一直升高；玉米植株体内MDA含量则一直上升，且在20mg/kg浓度之后，上升较为明显。籍此，我们结合苗期的状况认为，玉米生长后期抵抗Cd胁迫的耐性不及苗期明显，苗期100mg/kg处理仍能正常生长，拔节后50mg/kg处理即表现出严重的矮化症状。另外，随着土壤中重金属含量的升高，玉米的生殖系统受到威胁，花粉的萌发孔发生明显的变异。在50mg/kg Cd胁迫下，玉米的花粉萌发孔明显凹陷，100mg/kg Cd胁迫下无花粉产生。3.研究了整株玉米对Cd的富集和分配。对完成生长周期的玉米CT38植株，分别测定其营养生长器官(根、茎、叶)和生殖生长器官(雄花和雌穗，雌穗包括苞叶、穗柄、穗轴、花丝和籽粒)的生物量和重金属含量，并计算出各器官对Cd的富集量和富集比例。结果显示：虽然生物量干重在低浓度Cd胁迫下增加，高浓度Cd胁迫下降低，但玉米植株生殖器官的干重占整株的比例与对照相比没有明显的差异。虽然玉米各器官重金属Cd含量随土壤Cd含量升高而增加，但营养器官和生殖器官富集重金属的比例却没有明显变化，玉米营养器官(根、茎、叶)富集的重金属占整株的78.9~88.5%，对重金属Cd有截留作用；而玉米生殖器官雄花和雌穗对重金属Cd的富集作用相对较弱,在雌穗内，苞叶、穗轴、穗柄等非食用部位对籽粒富集有分流作用，所富集的重金属占雌穗的50%以上；重金属进入籽粒之前要先经过营养器官的截留和生殖器官非食用部位的分流，最后到达籽粒中Cd的量占整株的6%以下。4.研究了授粉及籽粒生长对整株玉米CT38富集Cd的影响。对干凈盆栽土上生长的玉米CT38，在拔节期进行一次CdCl2溶液汚灌处理，汚灌后盆栽土中Cd的含量分别达5、50、100mg/kg水平，玉米完成自然生长周期后，测量重金属含量，对比授粉与不授粉植株对Cd的富集量和各器官的富集比例，结果发现：尽管不授粉玉米整株的生物量干重比授粉的低16.4~22.4%，但不授粉的玉米植株比授粉玉米植株富集的Cd多46.2%；整株玉米营养器官和生殖器官之间的分配并未随授粉控制而发生明显变化，可能与整株玉米的生理结构有关。5.对广州市白云区4个自然村蔬菜基地土壤中的5种重金属镍(Ni)、铜(Cu)、锌(Zn)、镉(Cd)、铅(Pb)的含量进行测定与分析，并运用单因子污染指数以及多因子污染指数法中的算术平均法和内梅罗污染指数法对土壤中重金属的污染状况进行评价。结果表明，所调查的四个行政村的蔬菜基地土壤主要是重金属Cd污染，Ni、Cu、Zn、Pb四种金属并未超标。从污染程度来看，横沥村＞南浦村＞方石村＞大岭村。根据土壤重金属污染评价结果，我们认为选人和镇横沥村为修复示范工程地址最为合适。6.在实地修复工程中进一步检验玉米CT38对Cd的富集能力，并结合本课题组前期工作，对氨三乙酸(NTA)的添加量和添加时间进行探索，根据整株Cd富集量的大小确定最佳添加措施。另外，采用袋封花丝方式阻止玉米授粉，与正常授粉玉米对比修复前后土壤中的Cd含量，用地学统计软件(GS+软件)做图，评价整体修复效果。结果表明：在实地修复中，玉米CT38比土著玉米华珍(HZ)在整株上能富集更多的Cd。在无NTA添加的条件下，CT38比HZ多25.9%；而在授粉后添加25mmol NTA/株玉米，CT38富集的Cd比HZ多39.3%；授粉后添加25mmol NTA/株玉米，虽能显著增加玉米CT38对重金属的富集量，但各器官富集比例并未发生明显的变化。而授粉前添加NTA，能改变各器官中重金属的比例，且随着NTA添加量的增加茎中富集比例下降，叶中富集比例上升。通过实地修复工程，进一步验证了不授粉玉米植株的修复效果比授粉玉米植株高。不授粉的修复措施下，土壤中重金属Cd含量从修复前的0.4~0.5mg/kg降低至修复后的0.24mg/kg以下；而授粉的修复措施下，重金属Cd含量从修复前的0.4~0.5mg/kg降低至0.24~0.28mg/kg以内。同时，实地修复的玉米籽粒中重金属含量为0.03~0.062mg/kg，符合《食品安全国家标准食品中污染物限量》(GB2072-2012)规定的低于0.1mg/kg要求，证实了“边修复边生产”的方案是可行的。更多还原

【Abstract】 In recent years, there have been many reports about contaminated rice in China whichtells us that Cd pollution has been endangering the people’s daily life. Among all the methodsfor cleaning up Cd-contaminated soil, phytoremediation is particularly attractive because ofits special characteristics. Compared with other plants that could be used forphytoremediation purposes, maize is highly regarded by researchers around the world becauseof its excellent characteristics that include availability of many genotypes, ease of cultivation,fast growth, high biomass yield, versatile adaptation and low concentration of pollutant thatends up in seeds.The life cycle of maize includes two key phases: the vegetative phase and thereproductive phase; and two key periods: seedling time and pollination time. Therefore, westudied the mechanism of cadmium tolerance on maize seedlings and the effect of pollinationon the accumulation of Cd in maturing plants. Also, by covering the ear silk in order toprevent the pollination process, we studied the effect of pollination on the Cd accumulationand distribution in every organ of the plant. Finally, we tested our lab results by using thetechnology to clean up contaminated soil in a trail field. The primary results from the presentstudy are presented below:1. We have studied the mechanism by which maize seedlings tolerate Cd in soil duringthe seedling phase. Maize seeds, genotype CT38, were grown in pots containing variousamounts of Cd: CK(Blank) with a background level0.13mg Cd/kg soil and1.00,5.00,20.00,50.00,100.00and200.00mg/kg of soil. We determined the germination rate andinvestigated the growth conditions of maize seedlings during the transition period from theendosperm stage to living solely by photosynthesis. We analyzed the Cd content in theguttation. The germination success of the maize CT38seeds were not statistically affected bythe Cd, except at the highest level of the200mg Cd/kg soil (n=3, p=0.05). Besides theintercept in the root, the tolerance mechanism was also related to the maize’s ability toexclude Cd by guttation. When the Cd content in soil was below5mg/kg, the maize couldendure the pollution without Cd appearing in guttation. When the Cd content in soil was from20mg/kg to100mg/kg, the maize could still endure the pollution while excluding Cd fromguttation，in which the highest Cd concentration was no more than10.4mg/L. However,when the Cd content in the soil was200mg/kg，the capability of the maize to exclude Cdweakened, Cd concentration in the guttation was only8.8mg/L, and the plant could notendure the high Cd pollution any more. The dry mass weight of shoots and roots of maize plants in200mg Cd/kg soil were only25.4%and48.3%, respectively, of that in the blankcontrol. What’s more，there were some dead plants among those grown in the200mg Cd/kgtreated soil. If the maize seedlings had not excluded Cd from guttation, the Transfer Factor(TF) would have become smaller with the increasing of Cd content in soil. Once the maizeplant exclude Cd from guttation, the TF was about equal to0.5and did not change withincreasing Cd content in the soil. Furthermore, there was a strong liner relationship betweenthe Cd content in the maize root and the Cd content in soil after the Cd appeared in the maizeseedling’s guttation. The Cd concentration in the root is11.2times higher than that in the soil.In short, the phenomenon of excluding Cd from guttation is an important feature of the maizeseedlings’ ability to endure and accumulate Cd in soil.2. We investigated the effect of different Cd concentrations in soil on the growth andphysiological features of maize plant. Maize seeds were planted in a series pots with differentlevels of Cd; i.e.,0,1,5,20,50,100mg Cd (as CdCl2/kg of soil). After pollination in thepustulation period, we measured the height and leaf area of the plants and determined thechlorophyll content. In addition, we recorded changes in the activity of three antioxidantenzymes (i.e., SOD, CAT and POD) and the content of malonaldehyde (MDA). We alsoexamined the condition of the plants’ pollen by the Scanning Electron Microscopy (SEM).The results showed that the height and leaf area of maize plant during the postulation perioddid not significantly increase from the blank to the5mg/kg level, but decreased significantlyfrom50to100mg/kg. The chlorophyll content of the maize during the postulation periodincreased at first, reaching a maximum in the5mg/kg treatment plant and then decreased.The Cd content in the soil did not affect the concentration of chlorophyll a/b in the plants. Asthe concentration of Cd in the soil increased, the activity of SOD initially increased and thendecreased with increasing Cd, but the activity of CAT and POD increased in all treatmentgroups. The content of MDA in the plants also increased in all treatments, especially at thelevels of20-100mg Cd/kg soil. Comparing this behavior with the response of the seedlings,we concluded that the maize plant after the elongation phase cannot tolerate the Cd stress aswell as the seedlings did. In the seedling phase, maize could endure100mg Cd/kg soilwithout any obvious ill effects, but after entering the pollination phase after elongation, themaize plants exhibited the serious shorten symptom. What’s more, the reproductive organs ofmaize plant have been affected, and the germ pore of the maize pollen changed with theincreasing of Cd content in the soil. The operculum of pollen germ hole became smaller anddeeper at50mg Cd/kg soil treatments and, more seriously, there was no pollen among plantssubjected to the100mg Cd/mg soil treatments. 3. We have studied the accumulation and distribution of Cd in maize plants. For thematuring maize plant, we determined the dry mass and Cd concentration in every organ,including vegetative organs and reproductive organs. The vegetative organs are the root, stemand leaf, while the reproductive organs include the flower and ear that is divided into bract,ear stalk, ear cob, silk and seeds. From the original data on the dry mass weight andconcentration, we calculated the amount of Cd accumulated and the percentage in every organ.The results showed that low Cd treatments can increase the dry mass weight slightly, whilehigh Cd concentrations in the soil can cause the dry mass weight to decrease significantly.However, the percentage of dry mass weight found in the reproductive organs is almost thesame under all treatment conditions. Further, even though the Cd concentration in every organincreased with increasing amounts of Cd in soil, the distribution of Cd in vegetative andreproductive organs is nearly the same under all of the treatment levels. Specifically, thepercentage of Cd accumulated in vegetative organs was about78.9~88.5%, which shows thevegetative organs are the major sink for absorbed Cd. Only a much lower percentage Cdentered the flower and the ear. In the maize ear the non-edible organs, including bracket, coband ear stalk, absorbed more than50%of Cd that accumulated in the reproductive organs.We conclude that the presence of the non-edible organs can prevent the Cd from entering theseeds, which are the most important and worthy organs of the whole maize plant. In summary,after considering all of the sinks for Cd in the maize plant, we find that the percentage of Cdin the seeds is not more than6%of the Cd accumulated by the plant.4. We studied the effect of pollination on the Cd accumulation in maize. For the maizegrowing on the clean soil, we irrigated them in a solution of CdCl2once before pollination inorder to make the Cd content in pot soil5,50,100mg Cd/kg soil. When the maize plantsfinished their life period, we determined the Cd concentration in every organ and comparedthe results between the plants that were allowed to pollinate and those that were preventedfrom being pollinated. The comparison showed that even though the dry mass weight ofun-pollinated plants was16.4~22.4%lower than that of the pollinated plants, the Cdaccumulation in un-pollinated plant is more46.2%higher than in the pollinated plant.However, the distribution between vegetative organs and reproductive organs did not changeunder different pollination conditions. This result shows that the physiology of the maizeduring pollination plays an important role in the accumulation of Cd by the plants.5. We field-tested our lab results on vegetable fields in four villages in the BaiyunDistrict of Guangzhou City where the soil is heavily polluted by heavy metal. The soils wereanalyzed for five heavy metals; i.e., Ni, Cu, Zn, Cd and Pb. The heavy metal conditions were assessed using single pollution indices for each contaminant and two kinds of integratedpollution indices, including the arithmetic mean and the Nemerow pollution index. The resultsshowed that the Cd pollution is the most serious condition in the investigated soils and thatthe levels of the other metals (i.e., Ni, Cu, Zn and Pb) in the soil are safe. The Cdcontamination in the fields of the four villages decreased in the following order: Hengli＞Nanpu＞Fangshi＞Daling. Based on these results, we chose the vegetable fields in Henglivillage as the location for phytoremediation.6. The phytoaccumulation ability of maize CT38was tested in the field trail. Building onour results obtained in our laboratory, we studied the optimal amount and time for addingnitrilotriaceticacid (NTA) that would results in the maximum Cd accumulation in the wholemaize plant. Furthermore, we prevented pollination by covering the silk with a plastic bag inorder to maximize the Cd accumulation that we saw. The Cd concentrations before and afterphytoremediation of the fields were used to generate maps (drawn by Geological Statisticssoftware (GS+, Version5.0)) to visualize the overall effect of phytoremediation. The resultsshowed that the maize CT38accumulated more Cd than did the native maize species, HZ.Without the addition of NTA, CT38accumulated25.9%more Cd than HZ did. Further, afterthe addition of25mmol of NTA, CT38accumulated39.3%more Cd than did HZ. AddingNTA after pollination, although the amount of Cd accumulated increased, the relativedistribution of Cd within the organs of the plant did not change. While adding NTA beforepollination cannot increase the Cd accumulation but can change the percentage of every organ.The percentage of Cd in the leaf increased and the percentage in the stem decreased when theNTA was added. By using phytoremediation engineering techinques in the field, we haveverified that the un-pollinated maize can accumulate more Cd than pollinated maize. Throughun-pollinated phytoremediation, the Cd content in field decreased from0.4~0.5mg Cd/kg soilto0.24mg/kg or less, while the decrease with pollinated phytoremediation was from0.4~0.5mg Cd/kg of soil to0.24~0.28mg/kg. Most importantly, the Cd concentration in seedsin the field phytoremediation study was in the range of0.03~0.062mg Cd/kg of seeds, whichwas below the Chinese government’s permitted concentration (0.1mg Cd/kg seeds) in coarsecereals (GB2072-2012). Therefore, this study shows that it is possible to conduct maizephytoremediation of Cd-contaminated soil while, at the same time, harvesting a crop, forsubsequent consumption by animals or humans.更多还原