A route-directed hybrid genetic approach for the vehicle routing problem with time windows
环境政策分析,英文版(练习翻译)

123 Array 4567891011111121314151617181920111212223242526272829301113132333435363738394011171should not automatically bar adoption of measures to prohibit or otherwise regulate the activity, and, in stronger versions, further asserts that uncertainty provides an affirmative justification for regulating an activity or regulating it more stringently than in the absence of uncertainty. Strong versions of PP hold that regulators should adopt “worst case” presumptions regarding the harms of activities posing an uncertain potential for significant harm; should prohibit such activities or require them to adopt best available technology measures;that regulatory costs should be disregarded or downplayed in such decisions;and that the proponents of such activities should bear the burden of establishing their safety in order to avoid such regulatory controls. The article also considers the relevance for regulatory decisions of uncertainties regarding the costs of regulating an activity as well as uncertainties regarding harms. It further considers the implications of the circumstance that regulatory decisions about a given environmental issue may be made sequentially over time and benefit from additional information developed in the interim between earlier and later decisions. The essay concludes that, while preventive regulation of uncertain risks is often appropriate and should incorporate precautionary elements where warranted by consideration of risk aversion or information acquisition, strong versions of PP do not provide a conceptually sound or socially desirable prescription for regulation.I. INTRODUCTION: ENVIRONMENTAL DECISION MAKING UNDER UNCERTAINTY AND THE PRECAUTIONARY PRINCIPLE The following are examples of regulatory decisions involving uncertain risks. In each case, consider whether a regulator should permit the potentially harmful activity to commence or continue, or, alternatively, to prohibit or otherwise regulate it, and the implications of sequential regulatory decision making and the opportunity to develop additional information to reduce uncertainties regarding the risks of harms posed by an activity and/or the costs of regulation.•Whether to prohibit the sale of meat products from cattle that have received bovine growth hormone injections.•Whether to prohibit the construction of an astronomic observatory atop Mt.G raham, in New Mexico, the only known habitat of the Mt. G raham Red Squirrel, a subspecies of the western red squirrel located only on Mt. Graham;other subspecies of the red squirrel are abundant in the Western United States.72RICHARD B. STEWART12345678910111111213141516171819201112122232425262728293011131323334353637383940111•Whether to prohibit field releases of crop plants that have been genetically modified using DNA technologies.•Whether to adopt a National Ambient Air Quality Standard (NAAQS) to limit short-term (10-minute) exposures to elevated levels of sulfur dioxide (SO 2). Several laboratory studies indicate that asthmatics exposed to higher short-term SO 2exposures experience temporary airway resistance that makes breathing more difficult.•Whether to prohibit introduction of sucralose, an artificial beverage sweetener that has been touted as safer than saccharin or aspartame, the artificial sweeteners currently in use.•Whether to prohibit the dumping of wastes of any sort at sea.•Whether to develop defenses against collisions with the earth by asteroids and other near-earth objects.•Whether to eliminate the use of chlorine to treat drinking water.•Whether to prohibit the use of glyphosate (“Roundup”), a broad-spectrum non-selective herbicide that is harmless to animals.•Whether to prohibit or tightly regulate the conversion of rainforest to agricultural or other uses.•Whether to immediately initiate strict limits on greenhouse gas emissions in order to limit the potential adverse effects of climate changes attributable to such emissions.In evaluating these and other environmental regulatory decision-making issues,the extent of available knowledge regarding environmental harm that may be caused by the activity in question can be conceptually classified in three ideal type categories:Type 1. The harm that the activity will cause is known and determinate. If,for example, the Mt. Graham observatory is built, the Mt. Graham red squirrel population will be wiped out within 20 years.Type 2. The harm is probabilistic in character but its probability distribution is known. For example, if the observatory is built, there is a 40% probability that the squirrel population will be wiped out within 20 years and a 60% probability that it will survive another 1000 years, at which point it will become extinct from natural “background” causes. In this situation we deal with a risk of harm, but the risk (comprising both the probability of an adverse effect occurring and the magnitude of the adverse effect if it occurs) is determinate.Type 3. There is a risk of harm that is uncertain. Thus, the probability of harm occurring, and/or the magnitude of the harm if it occurs, is not determi-nate and is subject to substantial uncertainty. To take the Mt. Graham example,Environment al Regulat ory Decision Making Under Uncert aint y 737312345678910111111213141516171819201112122232425262728293011131323334353637383940111it may be uncertain, based on current knowledge, whether any adverse effect on the squirrels will occur. If any adverse effect does occur, its magnitude is uncertain. Thus, it may be uncertain what percentage of the population may be lost, whether any given level of loss will result in extinction of the subspecies,and when possible losses or extinction may occur. There may also be cases where the type of harm, if any, that may occur is not known.The difference between Type 2 cases and Type 3 cases is obviously one of degree, but the distinction between the two ideal types is very useful for purposes of analysis. Very many environmental problems are Type 3 cases,characterized by uncertainty regarding risks of harm, although the nature and degree of uncertainty varies widely from case to case. These uncertainties have many potential causes, including lack of data, limitations in scientific understanding of causal relationships, medical and ecosystem complexity, and “trans scientific” gaps in the capacities of science.1There may also be substantial uncertainties regarding the costs of prohibiting or otherwise regulating the activity in question. In many situations, such uncertainties can be reduced by the development of additional information and knowledge as discussed further below.The common law traditionally awards damages only ex post for harm that has occurred and has been shown to have been caused by another’s activity. It grants injunctive relief ex ant e only if an activity poses an imminent and substantial likelihood of serious irreparable harm. Many environmental risks of types 2 and 3 would not qualify for an award of damages or prophylactic relief under this standard. In theory, ex post liability for harm caused could provide the requisite incentives for actors to manage their activities so as to prevent excessive risks of harm appropriately. In practice, however, these incentives have for a variety of reasons proven inadequate to prevent excessive environ-mental harm from occurring.2Accordingly, administrative programs of preventive ex an t e regulation have been widely adopted in the United States and other countries to regulate activities that pose substantial risks of environmental harm, even in cases where it is not certain that harm will actually occur. International agreements, such as the Vienna Convention for the Protection of the Ozone Layer and the Framework Convention on Climate Change, have also been adopted to address such risks.Preventive regulatory programs have been adopted not only in cases where activities have been shown to cause harm, but also in cases involving risks of harm, including cases of substantial uncertainty in the risk of harm.3Quantitative risk analysis and cost-benefit analysis are increasingly being used in connection with the preventive approach to regulation.4Under many preventive regulatory74RICHARD B. STEWART12345678910111111213141516171819201112122232425262728293011131323334353637383940111programs, regulators have the burden of establishing a significant risk of harm before imposing regulatory controls,5although under some programs, such as U.S. FDA new drug and food additive approvals and EPA registration of pesticides, the applicant bears the burden of showing product safety.In recent years, environmental advocates and many environmental law scholars, particularly in the field of international environmental law, have argued that environmental regulatory decisions and policies should follow a precautionary principle (PP).6The focus of PP is on appropriate regulatory policy in Type 3 cases where the risks of harm posed by an activity are characterized by substantial uncertainty. PP advocates argue for a precautionary approach to regulation in the face of such uncertainty. They often criticize prevailing preventive approaches to regulation on the grounds that they place the burden on regulators to show that an activity will cause serious harm or poses a high probability of serious harm before regulatory controls may be adopted. They argue that, given the lack of scientific capacities to predict which activities will cause serious or irreversible harms, this approach results in seriously inadequate environmental protection. They often also contend that existing preventive regulatory approaches give undue weight to costs in establishing controls.7Various versions of PP, mostly weak ones, have been incorporated or invoked in a number of recent international environmental declarations and conventions,including the Framework Convention on Climate Change 8and the EU Maastricht treaty.9These documents and the writings of PP advocates and of academics provide widely varying formulations of PP. It has been claimed that PP is already, or is becoming established as a binding principle of customary international law.10PP skeptics and critics, however, have contended that the heterogeneity of PP formulations, many of which are quite vague and indeterminate, demonstrates that that there is no single or determinate PP.11Thus, they have concluded that the precautionary principle is a “composite of several value-laden notions and loose, qualitative descriptions” and that accordingly its “operational usefulness ... is doubtful.”12They also deny that PP has been established as customary international law.13Criticisms of PP as indeterminate and conceptually fuzzy have merit. With a very few exceptions, there is a remarkable lack of analytic care or rigor regarding the substance of, and justification for, various versions of PP by those who advocate or favor their adoption. One can, however, identify four different PP conceptions that have emerged in legal instruments, international and national governmental declarations, advocacy statements, and the academic liter-ature that can serve as a useful basis for analysis and evaluation. These four versions of PP are as follows:Environment al Regulat ory Decision Making Under Uncert aint y 757512345678910111111213141516171819201112122232425262728293011131323334353637383940111PP1. Scientific uncertainty should not automatically preclude regulation of activities that pose a potential risk of significant harm (Non-Preclusion PP).PP2. Regulatory controls should incorporate a margin of safety; activities should be limited below the level at which no adverse effect has been observed or predicted (Margin of Safety PP).PP3. Activities that present an uncertain potential for significant harm should be subject to best technology available requirements to minimize the risk of harm unless the proponent of the activity shows that they present no appreciable risk of harm (BAT PP).14PP4. Activities that present an uncertain potential for significant harm should be prohibited unless the proponent of the activity shows that it presents no appreciable risk of harm (Prohibitory PP).What unites these different formulations is a focus on uncertainty regarding risks as the key factor guiding regulatory decisions. Some discussions of the PP blur the distinction between known (Type 2) and uncertain risks (Type 3),but the most careful commentators make clear that the precautionary principle is addressed to uncertain risks (Type 3) as such.15PP1 and PP2 are weak versions of precautionary approaches. Unlike the strong versions, PP3 and PP4, they do not mandate regulatory action and do not make uncertainty regarding risks an affirmative justification for such regulation.Thus, PP1 is negative in character; it states that uncertainty should not preclude regulation but does not provide affirmative guidance as to when regulatory controls should be adopted or what form they should take. This is the approach that is most widely invoked in international treaties and declarations. While the exact wording may vary, this principle of non-preclusion always sets up a threshold, e.g. an uncertain risk of serious damage,and then makes the negative prescription that, once that threshold has been triggered, regulators cannot rely on this fact alone to deny regulation. For example, the Bergen Ministerial Declaration states: “Where there are threats of serious or irreversible damage, lack of full scientific certainty should not be used as a reason for postponing measures to prevent environmental degradation.”16The Cartagena Protocol goes further by clarifying that uncertainty can not, in and of itself, justify the decision not to regulate, nor,presumably, the alternative decision to impose regulation:Lack of scientific certainty due to insufficient relevant scientific information and knowledge regarding the extent of the potential adverse effects of a living modified organism shall not prevent [a] Party from taking a decision, as appropriate. Lack of scientific knowledge or scientific consensus should not necessarily be interpreted as indicating a particular level ofrisk, an absence of risk, or an acceptable risk.1776RICHARD B. STEWART12345678910111111213141516171819201112122232425262728293011131323334353637383940111This principle of non-preclusion rejects the common law position that harm must be shown to have occurred or be imminent before legal liabilities or controls may be imposed. It also rejects the position, often asserted by industry,that significant uncertainty about risks should preclude imposition of preven-tive regulatory controls. Of all the formulations of the PP, this approach is the most often invoked and is most likely to be recognized as a part of customary international law; it is already widely accepted that a preventive approach, under which regulatory controls are adopted to prevent or reduce risks of harm even though the magnitude or even the occurrence of harm is uncertain, is justified in at least some circumstances.18Yet, the very generality and lack of specific prescriptions of PP1 may preclude it from being recognized as a binding norm.19PP2 likewise fails to specify when or what form of regulation should be adopted, but instructs that, whenever regulation is adopted, it should incorporate a margin of safety. Unlike PP1, PP2 is operative only after regulators have made the determination to regulate. Once this decision is made,regulators must first determine the maximum “safe” level of an activity, and then only allow the activity at some degree lower than that level (the “margin of safety”). This is a common approach in U.S. environmental law. An example is the Sustainable Fisheries Act of 1996, in which the optimum allowable yield from a fishery “is prescribed on the basis of the maximum sustainable yield from the fishery, as reduced by” relevant factors including “ecological” factors.20PP2 is consistent with (although it does not necessarily mandate) many commen-tators’ views that PP requires that regulators allow “large margins for error” in risk assessments.21It represents one formulation of the PP premise that: “Given scientific ignorance, prudent pessimism should be favoured over hazardous optimism.”22PP2 is not explicitly set forth in any international agreements and declarations, but its approach is implicit in some international agreements that require or provide for the adoption of precautionary measures.23The weak versions of the PP are fully compatible with and are often reflected in many well-established preventative regulatory programs that have been adopted at the domestic level by many countries and by international agreement over the past 30 years. These programs often authorize prophylactic regulation of uncertain risks in appropriate circumstances even in the absence of a showing that harm will actually occur. In many cases, they explicitly require a margin of safety in setting regulatory standards.24Thus the weak versions of PP do not represent or justify any basic change in the preventive approach to regulation that has generally prevailed over the past 30 years. They accordingly provide no basis for arguing that existing preventive regulatory programs are not sufficiently “precautionary” and need to be fundamentally changed in order to reflect precautionary principles.Environment al Regulat ory Decision Making Under Uncert aint y 777712345678910111111213141516171819201112122232425262728293011131323334353637383940111There are, however, important differences between established programs of preventive regulation and the strong versions of PP. Weak precautionary programs generally do not make the existence of uncertainty regarding risks as such a mandatory or distinct basis for imposing regulatory controls. PP3 and PP4, on the other hand, require regulators to regulate, or regulate more stringently, activities that pose risks that are more uncertain relative to risks that are less uncertain, and thus represent a significant change in regulatory concept and result.Under PP3, when regulators determine that there is a serious but uncertain risk, they must impose BAT measures. For example, the Second International Conference on the Protection of the North Sea calls for parties to:[R]educ[e] polluting emissions of substances that are persistent, toxic and liable to bio-accumulate at source by the use of the best available technology and other appropriate measures. This applies especially when there is reason to assume that certain damage or harmful effects on the living resources of the sea are likely to be caused by such substances,even where there is no scientific evidence to prove a causal link between emissions and effects (“the principle of precautionary action”).25Such a prescription does not appear to allow regulators to decide what sort of regulation is required, including no regulation: if there is an uncertain risk of serious harm, BAT measures should be imposed. However, some flexibility may remain under PP3 since the intensity of BAT controls may vary depending on the magnitude of the potential risk relative to the costs of controls, in accor-dance with a principle of proportionality.26PP4 imposes an even more stringent prescription upon regulators. Under this formulation, if there is an uncertain but serious risk of harm, the activity in question should not be undertaken at all until it is proven to be safe by the proponent of the activity. Thus, the Final Declaration of the First European “Seas at Risk” Conference provides that:The “burden of proof” is shifted from the regulator to the person or persons responsible for the potentially harmful activity, who will now have to demonstrate that their actions are not/will not cause harm to the environment. If the “worst case scenario” for a certain activity is serious enough, then even a small amount of doubt as to safety of that activity is sufficient to stop it taking place.27While this version of the PP presumably allows regulators some latitude to determine how serious an uncertain risk must be to invite regulation, it requires prohibition of the activity once the relevant risk threshold is met.The strong versions of PP, PP3 and PP4, are the focus of this essay.Accordingly, unqualified references to PP in the following discussion should be understood as referring to the strong versions of PP. Unlike the weak versions 78RICHARD B. STEWART12345678910111111213141516171819201112122232425262728293011131323334353637383940111of PP and the preventive approach to regulation generally, they make the existence of uncertain risks of significant harm both a sufficient and mandatory basis for imposing regulatory controls. We may term this the “uncertainty-based potential for harm” prescription for regulation. Different PP formulations incorporating this precept vary in the criteria for determining the potential for harm threshold that triggers the requirement of regulation, including how great the probability of harm must be, its character, and its magnitude. Some formulations, for example, stress that the probability of harm must be substan-tial and the harm that may eventuate must be “serious and irreversible.”28Other formulations enunciate less demanding criteria.29In some strong PP formula-tions, once the applicable risk threshold is met, regulation is mandatory;regulatory compliance costs, including the social costs involved in forgoing the benefits of activities subject to regulatory prohibition or restriction, are not included as a factor to be considered in the regulatory decision.30Some formu-lations explicitly allow for consideration of costs, but relegate them to a distinctly secondary role, while others introduce the principle of proportion-ality, tailoring the extent and character of the regulatory response adopted to the gravity of the risk in question.31Under PP3, for example, the costs of BAT controls might be taken into account in determining whether a given technology is “available.” It might be concluded that very costly technology controls are not as a practical matter “available.” Under PP4, in cases where potential risks are judged less serious or where the social benefits of the activity are high,prohibitory controls might be adopted for only a limited initial period subject to “sunset” provisions or reconsideration, or field trials may be permitted.32Strong versions of the PP also often hold that the burden of resolving uncertainty should be borne by the proponent of an activity rather than by regulators or opponents of the activity.33Accordingly, in order to avoid or lift regulatory prohibitions or BAT requirements, the proponent of an activity bears the burden of demonstrating that it does not present a potential for significant harm. Proponents of regulation, however, bear some initial threshold burden of production and persuasion. They must establish that an activity poses risks (albeit uncertain) of harm, including a potential for significant harm. Once that threshold burden is satisfied, however, the burden shifts to the activity proponent to resolve the uncertainty and show that that it does not have a potential for significant harm.34The normative core of the strong versions of PP, which distinguishes PP-based regulation from preventive regulation generally, is the principle that uncertainty regarding risks is an affirmative justification for adopting regulatory controls or adopting more stringent controls than would be appropriate in the case of activities posing more determinate risks. In the face of uncertainties regardingEnvironment al Regulat ory Decision Making Under Uncert aint y 797912345678910111111213141516171819201112122232425262728293011131323334353637383940111risk, PP holds that decision makers should err on the side of precaution and envi-ronmental protection and, in effect, make “worst case” presumptions about the probability and magnitude of harm that an activity poses; precisely how “worst case” is defined (“reasonable worst case,” etc.) varies in different PP formula-tions.35The justifications advanced by PP proponents for adopting its prescrip-tions center around limitations in our ability to predict which activities will cause serious, irreversible environmental harms.36The predictive capacity of science is limited. For example, science has often been unable to predict, in a sufficiently timely fashion to support effective preventive action, the occurrence of serious environmental harms such as asbestosis, stratospheric ozone depletion, or the ecological harms caused by DDT. Thus, a regulatory policy that requires regulators to demonstrate that an activity causes harm or even a significant risk of harm before imposing controls will result in the occurrence of serious environmental harms. Some of these harms, such as biodiversity loss or highly disruptive changes in natural systems resulting from rapid global warming, may be irreversible and seriously harm future generations. Accordingly, decision makers should err on the side of pre-caution and protection of the planet by adopting PP-based regulatory controls on activities involving uncertain risks that pose a potential for significant harm.The PP literature provides little in the way of helpful guidance on what regulators must show in order to establish a potential for harm that triggers PP.37While some PP proponents appear to assume that nature is inherently vulnerable and precarious rather than resilient, such a general presumption is not sufficient to show that a given activity triggers PP. The bovine growth hormone dispute suggests that a showing that a substance similar in chemical structure to the substance in question can cause harm may be sufficient.38The BtCorn-Monarch butterfly controversy suggests that a report of a single experimental study, albeit one quite unrepresentative of field conditions, can be enough to trigger PP controls if it is sufficiently widely publicized.39Under strong versions of PP, once the risk posed by an activity satisfies the threshold that triggers a worst case presumption, regulators must then follow a set of relatively stringent regulatory prescriptions. They must prohibit or impose BAT requirements on the activity; shift the burden to the activity proponent to show that the activity is “safe” in order to avoid or lift these regulatory require-ments; and disregard or downplay regulatory costs in implementing regulatory requirements. Thus, PP can be analyzed as containing two basic components:First, a worst case presumption for uncertain risks that meet a triggering threshold. Second, a set of regulatory decision rules that are mandatory once the presumption is triggered. These components can be analyzed separately.80RICHARD B. STEWART12345678910111111213141516171819201112122232425262728293011131323334353637383940111。
定向突变 生物合成

定向突变生物合成英文回答:Directed mutagenesis is a technique used in molecular biology to introduce specific changes or mutations into a DNA sequence. This technique allows scientists to study the effects of these mutations on gene function and protein structure. There are several methods available for directed mutagenesis, including site-directed mutagenesis and random mutagenesis.Site-directed mutagenesis is a targeted approach where specific nucleotides in the DNA sequence are changed to create a desired mutation. This can be achieved using techniques such as PCR-based mutagenesis oroligonucleotide-directed mutagenesis. For example, if I want to introduce a point mutation in a gene to study its effect on protein function, I can design a pair of primers that contain the desired mutation and use them in a PCR reaction to amplify the gene with the mutation.Random mutagenesis, on the other hand, introduces random mutations throughout the DNA sequence. This can be done using techniques such as error-prone PCR or chemical mutagenesis. Random mutagenesis is useful when studying the effects of a large number of mutations or when trying to create a library of variants with different properties.Once the mutations are introduced, the mutated DNA sequences can be cloned into an expression vector and used to produce mutant proteins. The mutant proteins can then be purified and characterized to determine their functional and structural properties. For example, if I introduce a mutation in a protein involved in enzyme catalysis, I can compare the catalytic activity of the mutant protein to the wild-type protein to understand the role of the mutated residue in the reaction.Directed mutagenesis has a wide range of applicationsin biotechnology and biomedical research. It can be used to study the function of specific genes and proteins, identify key amino acid residues involved in protein-proteininteractions, and engineer proteins with improved properties. For example, directed mutagenesis has been used to create enzymes with enhanced catalytic activity, antibodies with improved binding affinity, and receptors with altered ligand specificity.中文回答:定向突变是分子生物学中一种用于在DNA序列中引入特定改变或突变的技术。
临床检验中的分子诊断技术进展考核试卷

C. Western blot
D. Southern blot
11. 哪种技术可以用于检测单个细胞中的基因表达?( )
A. RNA-Seq
B. Single-cell PCR
C. qPCR
D. Northern blot
12. 以下哪种技术不是基于DNA芯片的检测方法?( )
A. Microarray
B. SNP array
C. CGH array
D. Western blot
13. 在分子诊断中,以下哪种技术用于检测基因重排?( )
A. PCR
B. FISH
C. Southern blot
D. Northern blot
14. 哪种技术可用于检测病原微生物的快速诊断?( )
D. PCR
8. 哪种技术被称为下一代测序技术?( )
A. Sanger sequencing
B. NGS
C. AFLP
D. RFLP
9. 以下哪个不是NGS技术的优点?( )
A. 高通量
B. 成本低
C. 准确性高
D. 速度快
10. 在分子诊断中,以下哪种技术用于检测DNA甲基化?( )
A. PCR
C. Western blot
D. Northern blot
19. 哪种技术可以用于检测miRNA的表达?( )
A. PCR
B. Northern blot
C. RNA-Seq
D. Western blot
20. 在分子诊断中,以下哪种技术用于检测病毒载量?( )
A. PCR
B. ELISA
C. Western blot
清华大学硕士论文博士论文格式及范文详解

Key words: molecular excited state, rydberg state, predissoc iation, Na2, CaCl
目录
摘 要 ......................................................................................................... I ABSTRACT(英文摘要).......................................................................... I 目 录 ....................................................................................................... I 符号对照表ﻩﻩI 第一章 引 言 ............................................................................................ﻩ1
清华大学硕士论文博士论文格 式及范文详解
———————————————————————————————— 作者: ———————————————————————————————— 日期:
名称
章标题 一级节标题
二级节标题 三级节标题 正文段落
表题与图题
文献
页眉 页码
清华大学硕士论文博士论文编辑排版建议采用的字体、字号
号 仿
的
宋 或
高华
温文
水仿
2024年6月四级英语试卷

2024年6月四级英语试卷Part I Writing (30 minutes)Directions: For this part, you are allowed 30 minutes to write an essay on the topic "The Importance of Cultural Heritage Protection". You should write at least 120 words but no more than 180 words.Part II Listening Comprehension (25 minutes)Section A.Directions: In this section, you will hear three news reports. At the end of each news report, you will hear two or three questions. Both the news report and the questions will be spoken only once. After you hear a question, you must choose the best answer from the four choices marked A), B), C) and D).News Report 1.Questions 1 and 2 will be based on the following news item.A new study shows that urban green spaces can significantly reduce the risk of certain diseases. The research, which was conducted over a five - year period in several major cities, found that people living near large parks or gardens had a lower incidence of respiratory and heart diseases compared to those in areas with little or no greenery.1. What did the new study mainly find?A) Urban green spaces are getting smaller.B) People prefer to live near large parks.C) Green spaces can reduce disease risks.D) Respiratory diseases are on the rise.2. How long was the research period?A) Three years.B) Four years.C) Five years.D) Six years.News Report 2.Questions 3 and 4 will be based on the following news item.A well - known tech company has announced a major breakthrough in battery technology. The new battery is said to be more efficient, longer - lasting, and safer than current models. This could have a huge impact on the development of electric vehicles and portable electronics.3. What is the achievement of the tech company?A) A new type of electric vehicle.B) A major breakthrough in battery technology.C) A new marketing strategy for electronics.D) An improvement in portable electronics.4. Which fields could be influenced by this achievement?A) Only electric vehicles.B) Only portable electronics.C) Both electric vehicles and portable electronics.D) Neither electric vehicles nor portable electronics.News Report 3.Questions 5 to 7 will be based on the following news item.A group of international scientists has discovered a new species of deep - sea fish. The fish was found at a depth of over 2,000 meters in the Pacific Ocean. It has unique physical characteristics that distinguish it from other known species.5. Where was the new species of fish discovered?A) In the Atlantic Ocean.B) In the Indian Ocean.C) In the Arctic Ocean.D) In the Pacific Ocean.6. At what depth was the fish found?A) Over 1,000 meters.B) Over 2,000 meters.C) Over 3,000 meters.D) Over 4,000 meters.7. What makes the new fish different from others?A) Its color.B) Its size.C) Its unique physical characteristics.D) Its living habits.Section B.Directions: In this section, you will hear two long conversations. At the end of each conversation, you will hear four questions. Both the conversation and the questions will be spoken only once. After you hear a question, you must choose the best answer from the four choices marked A), B), C) and D).Conversation 1.Questions 8 to 11 will be based on the following conversation.M: Hi, Lisa. How was your weekend?W: It was great. I went to a concert with some friends.M: That sounds fun. Who did you see?W: A local rock band. They were really good.M: I like rock music too. Do you often go to concerts?W: Not really. This was my first time in a while.8. What did Lisa do on the weekend?A) She went to a movie.B) She went to a concert.C) She went to a party.D) She stayed at home.9. What kind of band did Lisa see?A) A pop band.B) A jazz band.C) A rock band.D) A classical band.10. Does Lisa often go to concerts?A) Yes, she does.B) No, she doesn't.C) She used to.D) She never goes.11. What does the man like?A) Pop music.B) Jazz music.C) Rock music.D) Classical music.Conversation 2.Questions 12 to 15 will be based on the following conversation. W: John, I'm thinking about taking a Spanish course.M: That's a great idea. Why do you want to learn Spanish?W: Well, I'm planning a trip to Spain next year, and I think it will be useful.M: You're right. Spanish is also very useful in business these days.W: Do you know any good language schools?M: There's one near my office. I heard it has very good teachers.12. What is the woman thinking about?A) Taking a French course.B) Taking a Spanish course.C) Taking a German course.D) Taking an Italian course.13. Why does the woman want to learn Spanish?A) She is interested in Spanish culture.B) She has Spanish friends.C) She is planning a trip to Spain.D) She wants to work in Spain.14. What else is Spanish useful for according to the man?A) Only for travel.B) Only for culture.C) For business.D) For nothing else.15. What does the man know about a language school?A) It has cheap courses.B) It has good teachers.C) It has a convenient location.D) It has a lot of students.Section C.Directions: In this section, you will hear three passages. At the end of each passage, you will hear three or four questions. Both the passage and the questions will be spoken only once. After you hear a question, you must choose the best answer from the four choices marked A), B), C) and D).Passage 1.Questions 16 to 18 will be based on the following passage.The Internet has changed the way we communicate and access information. However, it also brings some problems, such as information overload and the spread of false information. To deal with these problems, we need to develop good information - literacy skills. This includes being able to evaluate the reliability of sources, distinguish between fact and opinion, and manage the amount of information we consume.16. What has the Internet changed?A) Only the way we communicate.B) Only the way we access information.C) Both the way we communicate and access information.D) Neither the way we communicate nor access information.17. What problems does the Internet bring?A) Only information overload.B) Only the spread of false information.C) Both information overload and the spread of false information.D) Neither information overload nor the spread of false information.18. What skills do we need to develop?A) Technical skills.B) Social skills.C) Information - literacy skills.D) Management skills.Passage 2.Questions 19 to 22 will be based on the following passage.In recent years, more and more people are choosing to live in small towns instead of big cities. There are several reasons for this. First, small towns usually have a lower cost of living. Second, they offer acloser - knit community where people can get to know their neighbors better. Third, the environment in small towns is often cleaner and more peaceful.19. What trend is mentioned in the passage?A) More people are moving to big cities.B) More people are living in small towns.C) People are moving from small towns to big cities.D) People are moving abroad.20. What is one advantage of small towns?A) High - paying jobs.B) A lot of entertainment options.C) A lower cost of living.D) Easy access to big cities.21. What can people do in small towns?A) They can only work.B) They can get to know their neighbors better.C) They can enjoy big - city life.D) They can avoid all problems.22. How is the environment in small towns?A) Dirty and noisy.B) Clean and peaceful.C) Polluted.D) Dangerous.Passage 3.Questions 23 to 25 will be based on the following passage.A new study has found that reading books can have a positive impact on our mental health. People who read regularly are less likely to suffer from depression and anxiety. Reading can also improve our cognitive abilities, such as memory and concentration.23. What has the new study found?A) Reading books is a waste of time.B) Reading books can harm our mental health.C) Reading books can improve our physical health.D) Reading books can have a positive impact on our mental health.24. Who are less likely to suffer from depression and anxiety?A) People who watch a lot of TV.B) People who play video games.C) People who read regularly.D) People who do no hobbies.25. What can reading also improve?A) Our physical strength.B) Our social skills.C) Our cognitive abilities.D) Our appearance.Part III Reading Comprehension (40 minutes)Section A.Directions: In this section, there is a passage with ten blanks. You are required to select one word for each blank from a list of choices given in a word bank following the passage. Read the passage through carefully before making your choices. Each choice in the word bank is identified by a letter. Please mark the corresponding letter for each item on Answer Sheet 2. You may not use any of the words in the word bank more than once.The concept of time is something that humans have been trying to understand for centuries. Time is a fundamental part of our lives, yet itis also one of the most difficult things to define _(26)_. We use time to organize our days, plan our activities, and measure the passage of our lives. But what exactly is time?Some scientists believe that time is a physical property of the universe, like space or matter. Others think that time is a human _(27)_, created by our minds to make sense of the world around us. There are also those who argue that time is an illusion, and that there is no such thing as a past, present, or future.Despite these different views, one thing is certain: time is a precious _(28)_. We all have a limited amount of time in our lives, and we need to make the most of it. This means using our time _(29)_ to achieve our goals, whether they are personal, professional, or academic.One way to make better use of our time is to learn how to manage it effectively. This involves setting _(30)_, prioritizing tasks, and avoiding distractions. Another important aspect of time management is learning to say no to things that are not important or that will take up too much of our time.In addition to managing our time, we can also try to _(31)_ our perception of time. This can be done by engaging in activities that weenjoy, which can make time seem to pass more quickly. On the other hand, when we are bored or waiting for something, time can seem to drag on _(32)_.Finally, it is important to remember that time is not just aboutgetting things done. We also need to take time to relax, enjoy life, and spend time with the people we love. After all, life is not just about work and achievement, but also about _(33)_ and relationships.Word Bank:A) accurately.B) construct.C) efficiently.D) endlessly.E) goals.F) illusion.G) limited.H) manage.I) precious.J) property.K) relaxation.L) resource.M) sense.26. A) accurately.27. M) sense.28. L) resource.29. C) efficiently.30. E) goals.31. B) construct.32. D) endlessly.33. K) relaxation.Section B.Directions: In this section, you are going to read a passage with ten statements attached to it. Each statement contains information given in one of the paragraphs. Identify the paragraph from which the information is derived. You may choose a paragraph more than once. Each paragraph is marked with a letter. Answer the questions by marking the corresponding letter on Answer Sheet 2.The Benefits of Bilingualism.A) Bilingualism, or the ability to speak two languages fluently, has been shown to have numerous benefits. For one thing, it can improve cognitive abilities such as problem - solving, memory, and concentration. Studies have found that bilingual individuals tend to perform better on tasks that require these skills than monolingual individuals.B) Another benefit of bilingualism is that it can enhance cultural understanding. People who are bilingual are often more exposed to different cultures, which can lead to a greater appreciation and understanding of cultural differences. This can be especially important in today'sglobalized world, where cross - cultural communication is becoming increasingly common.C) Bilingualism can also have a positive impact on career opportunities. In many industries, being bilingual is seen as an asset. For example, in international business, bilingual employees can communicate moreeffectively with clients and partners from different countries. In thefield of education, bilingual teachers are in high demand, especially in areas with large immigrant populations.D) In addition to these practical benefits, bilingualism can also havea positive effect on mental health. Research has shown that bilingual individuals are less likely to develop age - related cognitive decline,such as dementia. This may be because using two languages keeps the brain active and stimulated.E) However, learning a second language is not without its challenges. One of the main difficulties is grammar. Different languages have different grammar rules, and it can be difficult to master them all. For example, in some languages, the verb comes at the end of the sentence, while in others, it comes in the middle.F) Another challenge is vocabulary. Building a large vocabulary in a second language can take a long time. There are often many words that have no direct equivalent in one's native language, which can make it difficultto understand and use them correctly.G) Despite these challenges, the benefits of bilingualism far outweigh the difficulties. There are many ways to learn a second language, such as taking classes, using language - learning apps, or immersing oneself in a language - speaking environment.H) One effective way to learn a second language is through immersion. This involves living in a country where the language is spoken or spendinga significant amount of time in such an environment. Immersion can help learners pick up the language more quickly and develop a more natural accent.I) Another way to learn a second language is through language exchange. This involves finding a partner who speaks the language you want to learn and who wants to learn your native language. You can then practice speaking with each other, which can be a fun and effective way to improve yourskills.J) In conclusion, bilingualism is a valuable skill that can bring many benefits. Whether it is for cognitive, cultural, career, or mental health reasons, learning a second language is well worth the effort.34. Bilingual individuals may perform better on tasks requiring problem - solving skills. (A)35. Bilingualism can help people understand cultural differences better.(B)36. In international business, bilingual employees have an advantage.(C)37. Bilingualism can reduce the risk of age - related cognitive decline.(D)38. Grammar is one of the main difficulties in learning a second language. (E)39. Building vocabulary in a second language can be time - consuming.(F)40. Immersion is an effective way to learn a second language. (H)41. Language exchange can be a fun way to improve language skills. (I)42. The benefits of bilingualism are greater than the difficulties. (G)43. Bilingualism has many benefits for various reasons. (J)Section C.Directions: There are 2 passages in this section. Each passage is followed by some questions or unfinished statements. For each of them there are four choices marked A), B), C) and D). You should decide on the best choice and mark the corresponding letter on Answer Sheet 2.Passage 1.The sharing economy has emerged as a significant economic trend in recent years. Platforms like Airbnb and Uber have revolutionized the way people travel and get around. These platforms allow individuals to share their resources, such as spare rooms or cars, with others in exchange for money.One of the main advantages of the sharing economy is that it can make better use of underutilized resources. For example, many people have spare rooms in their homes that are empty most of the time. By listing these rooms on Airbnb, they can earn extra income while also providing accommodation for travelers. Similarly, cars are often parked and unusedfor long periods. Through Uber or other ride - sharing services, car owners can use their vehicles to earn money when they are not using them.Another advantage is that the sharing economy can provide more affordable options for consumers. Airbnb often offers cheaper accommodationthan hotels, especially in popular tourist destinations. Ride - sharing services can also be less expensive than traditional taxis in some cases.However, the sharing economy also faces some challenges. One issue is regulation. Since these platforms are relatively new, there are often no clear regulations governing their operations. This can lead to problems such as safety concerns and unfair.。
recent findings on the phytoremediation of soils contaminated with environmentally toxic heavy metal

ReviewRecent findings on the phytoremediation of soils contaminated with environmentally toxic heavy metals and metalloids such as zinc,cadmium,lead,and arsenicI.Alkorta 1,J.Hernandez-Allica 2,J.M.Becerril 3,I.Amezaga 3,I.Albizu 2&C.Garbisu 2,*1Unidad de Biofı´s ica,Centro Mixto UPV/EHU,Apdo.644,E-48080Bilbao,Spain;2NEIKER,BasqueInstitute of Agricultural Research and Development,Berreaga 1,E-48160Derio,Spain;3Department of Plant Biology and Ecology,University of the Basque Country,Apdo.644,E-48080Bilbao,Spain (*author for correspondence:phone:+34-94-403-43-00;fax:+34-94-403-43-10;e-mail:cgarbisu@)Key words:metalloids,metals,phytochelatins,phytoextraction,phytoremediation,transgenic plants AbstractDue to their immutable nature,metals are a group of pollutants of much concern.As a result of human activities such as mining and smelting of metalliferous ores,electroplating,gas exhaust,energy and fuel production,fertilizer and pesticide application,etc.,metal pollution has become one of the most serious environmental problems today.Phytoremediation,an emerging cost-effective,non-intrusive,and aesthet-ically pleasing technology,that uses the remarkable ability of plants to concentrate elements and com-pounds from the environment and to metabolize various molecules in their tissues,appears very promising for the removal of pollutants from the environment.Within this field of phytoremediation,the utilization of plants to transport and concentrate metals from the soil into the harvestable parts of roots and above-ground shoots,i.e.,phytoextraction,may be,at present,approaching commercialization.Improvement of the capacity of plants to tolerate and accumulate metals by genetic engineering should open up new possibilities for phytoremediation.The lack of understanding pertaining to metal uptake and translocation mechanisms,enhancement amendments,and external effects of phytoremediation is hindering its full scale application.Due to its great potential as a viable alternative to traditional contaminated land remediation methods,phytoremediation is currently an exciting area of active research.1.Environmental metal pollution and phytoremediationSoil pollution has recently been attracting consid-erable public attention since the magnitude of the problem in our soils calls for immediate action (Garbisu &Alkorta 2003).As a result of human activities such as mining and smelting of metallif-erous,electroplating,gas exhaust,energy and fuel production,fertilizer and pesticide application,etc.,metal pollution has become one of the most serious environmental problems today.Due to their immutable nature,metals are a group of pollutants of much concern.In fact,although several metals are essential for biological systems and must be present within a certain concentration range (Garbisu &Alkorta 2003),at high concen-trations,metals can act in a deleterious manner by blocking essential functional groups,displacing other metal ions,or modifying the active confor-mation of biological molecules (Collins &Stotzky 1989).Metal toxicity for living organisms involves oxidative and/or genotoxic mechanisms (Briat &Lebrun 1999).Based on their chemical and physical proper-ties,three different molecular mechanisms of heavy metal toxicity can be distinguished:(i)pro-duction of reactive species by autooxidation and Fenton reaction (Fe,Cu),(ii)blocking of essential functional groups in biomolecules (Cd,Hg),and (iii)displacement of essential metal ions from biomolecules (Schutzendubel &Polle 2002).Reviews in Environmental Science and Bio/Technology 3:71–90,2004.Ó2004Kluwer Academic Publishers.Printed in the Netherlands.71Metal-contaminated soils are notoriously hard to remediate.Current technologies resort to soil excavation and either landfilling or soil washing followed by physical or chemical separation of the contaminants.Although highly variable and dependent on the contaminants of concern,soil properties,site conditions,and so on,the usually enormous costs associated with the removal of metals from soils by means of traditional physi-cochemical methods explain why most companies tend to ignore the problem.Due to the fact that very often large areas are affected by heavy metal contamination,a removal is certainly difficult. Therefore,some methods are developed to keep the metals in the soil but reduce the risks related to this presence(e.g.,by decreasing bioavailability by in situ immobilisation processes)(Diels et al. 2002).One way to facilitate such immobilisation is by altering the physicochemical properties of the metal-soil complex by introducing a multipurpose anion,such as phosphate,that enhances metal adsorption via anion-induced negative charge and metal precipitation(Bolan et al.2003a).Heavy metals cannot be destroyed biologically (no‘‘degradation’’,change in the nuclear struc-ture of the element,occurs)but are only trans-formed from one oxidation state or organic complex to another(Garbisu&Alkorta2001). Although microorganisms that use metals as ter-minal electron acceptors or reduce them as part of a detoxification mechanism can be used for metal remediation(Garbisu&Alkorta1997), when considering the remediation of metal-pol-luted soil,metal-accumulating plants offer numerous advantages over microbial processes since plants can actually extract metals from the polluted soils,theoretically rendering them clean (metal-free)(Garbisu&Alkorta2001;Garbisu et al.2002).Phytoremediation,the use of plants to extract, sequester,and/or detoxify pollutants,has been reported to be an effective,non-intrusive,inex-pensive,aesthetically pleasing,socially accepted technology to remediate polluted soils(Alkorta& Garbisu2001;Weber et al.2001;Garbisu et al. 2002).Phytoremediation is widely viewed as the ecologically responsible alternative to the envi-ronmentally destructive physical remediation methods currently practiced(Meagher2000).The US phytoremediation market is expected to ex-pand more than ten-fold between1998and2005, to over$214million(Evans2002).In the last few years,some excellent reviews have been published focusing on different aspects of phytoremediation(Salt et al.1995a,1998;Chaney et al.1997;Raskin et al.1997;Chaudhry et al. 1998;Wenzel et al.1999;Meagher2000;Navari-Izzo&Quartacci2001;Lasat2002;McGrath et al. 2002;McGrath&Zhao2003;McIntyre2003;Singh et al.2003).In any case,and in contrast to its many positive aspects,phytoremediation does have cer-tain disadvantages and limitations(Table1).Table1.Advantages and limitations of the phytoremediation technology Advantages LimitationsApplicable to a wide variety of inorganic and organic contaminants.Limited by depth(roots)and solubility and availability of the contaminant.Reduces the amount of waste going to landfills.Although faster than natural attenuation,it requires long timeperiods(several years).Does not require expensive equipment or highlyspecialized personnel.Restricted to sites with low contaminant concentration.It can be applied in situ.Reduces soil disturbance and the spread of contaminants.Plant biomass from phytoextraction requires proper disposal as hazardous waste.Early estimates of the costs indicate that phytoremediation is cheaper than conventional remediation methods.Climate and season dependent.It can also lose its effectiveness when damage occurs to the vegetation from disease or pests.Easy to implement and maintain.Plants are a cheap and renewable resource,easily available.Introduction of inappropriate or invasive plant species should be avoided(non-native species may affect biodiversity).Environmentally friendly,aesthetically pleasing,socially accepted,low-tech alternative.Contaminants may be transferred to another medium,the environment,and/or the food chain.Less noisy than other remediation methods.Actually, trees may reduce noise from industrial activities.Amendments and cultivation practices may have negative consequences on contaminant mobility.72Within thefield of phytoremediation,different categories have been defined such as,among others, phytoextraction,phytofiltration(rhizofiltration, blastofiltration),phytostabilization,phytovolatil-ization,phytodegradation(phytotransformation), plant-assisted bioremediation(plant-assisted deg-radation,plant-aided in situ biodegradation,phyt-ostimulation,enhanced rhizosphere degradation, rhizodegradation),etc.(Table2).Plants for phytoextraction,i.e.,metal removal from soil,should have the following characteris-tics:(i)tolerant to high levels of the metal,(ii) accumulate reasonably high levels of the metal, (iii)rapid growth rate,(iv)produce reasonably high biomass in thefield,and(v)profuse root system(Garbisu et al.2002).The idea of using plants to remediate metal polluted soils came from the discovery of‘‘hyper-accumulators’’(Table3),defined as plants,often endemic to naturally mineralized soils,that accu-mulate high concentrations of metals in their fo-liage(Baker&Brooks1989;Raskin et al.1997; Brooks1998).In fact,plants growing on metal-liferous soils can be grouped into three categories according to Baker(1981):(i)excluders,where metal concentrations in the shoot are maintained, up to a critical value,at a low level across a wide range of soil concentration;(ii)accumulators, where metals are concentrated in above-ground plant parts from low to high soil concentrations; and(iii)indicators,where internal concentration reflects external levels(McGrath et al.2002).The criterion for defining Ni hyperaccumulation is 1000l g Ni g)1on a dry leaf basis(Brooks et al. 1977),whereas for Zn and Mn the threshold is 10,000l g g)1and for Cd100l g Cd g)1.Finally, the criterion for Co,Cu,Pb and Se hyperaccu-mulation is also1,000l g g)1in shoot dry matter (Brooks1998;Baker et al.2000;McGrath et al. 2002).In general terms,metal concentrations in hyperaccumulators are about100–1000-fold high-er than those found in normal plants growing on soils with background metal concentrations,and about10–100-fold higher than most other plants growing on metal-comtaminated soils(McGrath et al.2002).Hyperaccumulators are also charac-terized by a shoot-to-root metal concentration ratio of>1(i.e.,hyperaccumulator plants show a highly efficient transport of metals from roots to shoots),whereas non-hyperaccumulators usually have higher metal concentrations in roots than in shoots(Baker1981;Gabbrielli et al.1990;HomerTable2.Categories of phytoremediationTerm DefinitionPhytoextraction The use of plants to remove pollutants(mostly,metals)from soils.Phytofiltration The use of plants roots(rhizofiltration)or seedlings(blastofiltration)to absorbor adsorb pollutants(mostly,metals)from water.Phytostabilization The use of plants to reduce the bioavailability of pollutants in the environment. Phytovolatilization The use of plants to volatilize pollutants.Phytodegradation The use of plants to degrade organic pollutantsPhytotransformationPhytostimulation The use of plant roots in conjunction with their rhizospheric microorganisms toremediate soils contaminated with organics.Enhanced rhizosphere degradationRhizodegradationPlant-assisted bioremediationPlant-asssisted degradationPlant-aided in situ biodegradationTable3.Examples of hyperaccumulatorsMetal SpeciesZinc(Zn)T.caerulescensCadmium(Cd)T.caerulescensNickel(Ni)Berkheya coddiiSelenium(Se)Astragalus racemosaThallium(Tl)Iberis intermediaCopper(Cu)Ipomoea alpinaCobalt(Co)Haumaniastrum robertiiArsenic(As)P.vittata73et al.1991;Baker et al.1994a,b;Brown et al. 1995;Kra mer et al.1996;Shen et al.1997;Zhao et al.2000;McGrath et al.2002).Hyperaccumulation of heavy metal ions is in-deed a striking phenomenon exhibited by<0.2% of angiosperms(Baker&Whiting2002).Most recently,a plethora of papers are being published in an attempt to dissect the mechanisms of metal uptake,transport and accumulation,both at the physiological and molecular level(Baker& Whiting2002).The‘‘model’’hyperaccumulator Thlaspi caerulescens has been much screened in the search for new and more extreme ecotypes (McGrath et al.2001;Lombi et al.2002).Our understanding of the internal processes that confer the hyperaccumulation phenotype is advancing in leaps and bounds,and the mechanisms of trans-port,tolerance and sequestration in some species, at least in the genera Thlaspi and Alyssum,are partially elucidated(Lasat2002).Trees have also been considered for phyto-remediation of heavy metal-contaminated land, with willow and poplar being promising candi-dates,among others,in this respect(Pulford& Watson2003).According to some authors,trees potentially are the lowest-cost plant type to use for phytoremediation(Stomp et al.1994).Many trees can grow on land of marginal quality,have mas-sive root systems,and their above-ground biomass can be harvested with subsequent resprouting without disturbance of the site(Stomp et al.1994).Following the harvest of metal-enriched plants, the weight and volume of the contaminated material can be further reduced by ashing or composting(Garbisu&Alkorta2001;Garbisu et al.2002).Metal-enriched plants can be disposed of as hazardous material or,if economically fea-sible,used for metal recovery(Salt et al.1998). Recently,some studies have reported on the utili-zation of pyrolysis to separate heavy metals from hyperaccumulators(Koppolu&Clements2003).Although plants acquire essential minerals such as Fe,Cu,Ni,Zn and Se from the soil,for reasons that are not yet clear,they also have the ability to acquire and detoxify non-essential elements such as As,Cd,Cr and Pb(Salt et al.2002).Certain themes in the physiology and biochemistry of trace element accumulation by plants appear common (Salt et al.2002).Most phytoremediation studies have consid-ered metal extraction efficiency in relation to metal concentration of bulk soil samples or metal con-centration of the soil solution,but little is known about the effect of various metal-bearing solids on metal extraction by hyperaccumulators.In fact,it has been shown that it is essential to consider the nature of the metal-bearing solids to better predict the efficiency of plant extraction(Dahmani-Muller et al.2001).Besides,it is also important to con-sider that metal bioavailability changes between the bulk soil and the rhizosphere,the latter being a microbiosphere which has quite different chemical, physical and biological properties from bulk soils (Wang et al.2002b).In this respect,recently,it has been reported that root growth is a more sensitive endpoint of metal availability than chlorophyll assays(Morgan et al.2002).In order to improve phytoremediation of heavy metal polluted sites, the speciation and bioavailability of the metals in the soil,the role of plant-associated soil microor-ganisms and fungi in phytoremediation,and that of plants have to be elucidated(Kamnev&van der Lelie2000).Phytoremediation has been used in mined soil restoration,since these soils are sources of air and water pollution,by means of phytostabilization and phytoextraction techniques to stabilize toxic mine spoils and remove toxic metals from the spoils,respectively(Wong2003).Some higher plant species have developed heavy metal tolerance strategies which enable them to survive and reproduce in highly-metal contam-inated soils.Dahmani-Muller et al.(2000)inves-tigated metal uptake and accumulation strategies of two absolute metallophyte species and one pseudometallophyte.In the former two species, real hyperaccumulation in the leaves as well as metal immobilisation in roots and/or a detoxifi-cation mechanism by leaf fall were found as possible strategies to deal with the high metal concentrations.By contrast,the strategy of the pseudometallophyte,i.e.,Agrostis tenuis,pre-sented a significant metal immobilisation by the roots.Most plants have mycorrhizal fungi associated with them,providing their hosts with an increased capacity to absorb water and nutrients from the soil.The formation and function of mycorrhizal relationships are affected by anthropogenic stressors including metals(Entry et al.2002). Arbuscular mycorrhizal fungi are of interest for their reported roles in alleviation of diverse74soil-associated plant stressors,including those in-duced by metals,so it has been claimed that theevaluation of the efficacy of plant-mycorrhizal associations to remediate metal-polluted soils de-serves increased attention (Entry et al.2002).In addition,phytoextraction practices, e.g.,the choice of plant species and soil amendments,may have a great influence on the quantity and species composition of glomalean propagules as well as on arbuscular mycorrhizal fungi functioning during long-term metal-remediation treatments (Paw-lowska et al.2000).A unique testing system,the target-neighbour method,has been described to allow evaluation of how planting density influences metal uptake,so that the information needed to manipulate plant density for optimization of metal removal could be obtained (Shann 1995).Finally,most recently,phytoremediation has been combined with electrokinetic remediation,applying a constant voltage of 30V across the soil,concluding that the combination of both tech-niques represents a very promising approach to the decontamination of metal polluted soils (O 0Connor et al.2003).2.New findings on the phytoextraction of some of the most relevant environmentally toxic heavy metals (zinc,cadmium,lead)and metalloids (arsenic)This section is divided into four different sub-headings.The first three correspond to three of the most environmentally relevant heavy metals,i.e.,Zn,Cd,and Pb.The fourth sub-heading deals with As,a well-known toxic metalloid.It is important to emphasize here that very often information regarding one metal appears under a different,apparently wrong,sub-heading.Since many of the reviewed publications deal with more than one metal at the same time,it has been preferred to present them as part of the same research,despite the fact that section structure could not be main-tained as desired.2.1.ZincZinc and Cd are ubiquitous pollutants that tend to occur together at many contaminated sites.While Zn is often phytotoxic,Cd rarely inhibits plant growth.In T.caerulescens ,an integrated molecular and physiological investigation of the fundamental mechanisms of heavy metal accumulation was conducted (Pence et al.2000).A metal transporter cDNA,znt1(expressed at very high levels in roots and shoots of this plant),was cloned from T.caerulescens through functional complementa-tion in yeast and was shown to mediate high-affinity Zn uptake as well as low affinity Cd uptake.Alteration in the regulation of znt1gene expression by plant Zn status results in the over-expression of this transporter and in increased Zn influx in roots,even when intracellular Zn levels are high.Thus,specific alterations in Zn-respon-sive elements (e.g.,transcriptional activators)possibly play an important role in Zn hyperaccu-mulation in T.caerulescens (Pence et al.2000).In this respect,Lasat et al.(1998)found that the en-hanced root-to-shoot Zn transport in T.caerules-cens was,at least partly,achieved through an altered Zn compartmentation in the root sym-plasm,which reduces Zn sequestration in root vacuoles.A further step at elucidating the mech-anisms underlying Zn hyperaccumulation was gi-ven thanks to the cloning of metal transporter genes encoding putative vacuolar ion transportproteins in T.caerulescens (Assunc ao et al.2001).Cd NiZnPbAsCuCoTlRoot uptakeTranslocationAccumulationHarvestingDisposal/Recovery Figure 1.Phytoextraction of metals.75In fact,ZTP1(a transporter belonging to the cat-ion-efflux family)has been found to be highly ex-pressed in T.caerulescens,predominantly in leaves (Assunc a o et al.2001).Long et al.(2002)have recently described a large biomass Zn hyperaccumulating plant,i.e., Sedum alfredii.Similarly,Wang et al.(2003)have reported on the discovery of two new plants with potential for phytoremediation of Zn-polluted soils,i.e.,Polygonum hydropiper and Rumex ace-tosa.In the same study,the authors indicate that the consumption of rice grown in paddy soils contaminated with Cd,Cr or Zn may pose a serious risk to human health,because from22to 24%of the total metal content in the rice biomass was concentrated in the rice grain.Interestingly, Platanus acerifolia growing on heavily contami-nated soil accumulated only very low levels of heavy metals,and this mechanism for excluding metal uptake may have value in crop improvement (Wang et al.2003).Holcus lanatus L.genotypes tolerant to Zn toxicity seem to grow better than Zn-sensitive genotypes,even in Zn-deficient soil,because of their greater capacity for taking up Zn from Zn-deficient soil,revealing the coexistence of traits for tolerance to Zn toxicity and Zn deficiency in a single plant genotype(Rengel2000).Metal responses in the metallophyte Arabidop-sis halleri,a close relative to the model plant A.thaliana,that is Cd hypertolerant and Zn hy-peraccumulating,have been studied,and metal-regulated genes isolated and molecularly analyzed as interesting candidate genes for phytoremedia-tion(Bert et al.2000;Dahmani-Muller et al.2000; Clemens2001;Macnair2002).Unlike Thlaspi, A.halleri seems to be largely allogamous,seeds profusely over a longer growing season and can also spread vegetatively by stolons(Baker& Whiting2002).Macnair(2002)demonstrated that the heritability of Zn accumulation was between 25and50%,the highest yet recorded for any hy-peraccumulator,probably because of the out-breeding nature of A.halleri.Intriguingly,the genetic variability of Zn accumulation in A.halleri was manifested more when grown in low-Zn media than in high(Baker&Whiting2002).Zinc-tolerant callus lines of Brassica spp.have been developed,and this might help in the selection and characterization of heavy metal tolerance in plants for breeding programmes(Rout et al.1999).In a paper focusing on soil solution Zn and pH dynamics during phytoextraction using T.cae-rulescens J.&C.Presl,data indicate that the po-tential of this hyperaccumulator to remove Zn from contaminated soil may not be related either to acidification of the rhizosphere(McGrath et al. 1997;Luo et al.2000)or to exudation of specific metal-mobilizing compounds(Zhao et al.2001). Intriguingly enough,some studies have provided evidence suggesting that roots of T.caerulescens are able to sense and actively forage in the Zn rich patches in soil(Schwartz et al.1999;Whiting et al. 2000).A modified glass bead compartment cultiva-tion system for studies on nutrient and trace metal uptake by arbuscular mycorrhiza using two host plant species,maize(Zea mays L.)and red clover(Trifolium pratense L.),and two arbuscu-lar mycorrhizal fungi,Glomus mossae and G. versiforme,found a striking,very high affinity of the fungal mycelium for Zn,suggesting the po-tential use of arbuscular mycorrhiza in the phy-toremediation of Zn-polluted soils(Chen et al. 2001).2.2.CadmiumCadmium is one of the more mobile heavy metals in the soil-plant system,easily taken up by plants and with no essential function known to date (Lehoczky et al.2000).This element can accumu-late in plants without causing toxicity symptoms (Lehoczky et al.1998).In soybean plants,results reveal that the con-tent of Cd in different parts of the plants was roots>>stems>seeds,indicating that the accu-mulation of Cd by roots is much larger than that of any other part of the soybean plant,and might cause deleterious effects to root systems(i.e.,de-creased nodulation,changes in the ultrastructure of root nodule,etc.)(Chen et al.2003).It has been suggested that vetiver grass could be used to remediate Cd-polluted soil,since it accu-mulated218g Cd ha)1at a soil Cd concentration of0.33mg Cd kg)1(Chen et al.2000).Although sequestration of Cd by rhizosphere microorgan-isms may have an important influence on plant Cd uptake,further research is still required to estab-lish whether the accumulation of Cd by rhizobacteria inhibits,or accelerates,Cd uptake by the host plant(Robinson et al.2001).76Recent works have aimed to identify the role of antioxidative metabolism in heavy metal tolerance in T.caerulescens(Boominathan&Doran2003a, b).Hairy roots were used to test the effects of high Cd environments,demonstrating that metal-in-duced oxidative stress occurs in hyperaccumulator tissues even though growth is unaffected by the presence of heavy metals.Superior antioxidant defenses,particularly catalase activity,may play an important role in the hyperaccumulator phe-notype of T.caerulescens.Phragmites australis plants were exposed to a high concentration of Cd,finding out that most of this element accumulated in roots,followed by leaves(Iannelli et al.2002).In roots from Cd-treated plants,both the high amount of glutathione and the parallel increase of glutathione-S-transferase activity seemed to be associated with an induction of the detoxification processes in response to the high Cd concentration.Superoxide dismutase,ascorbate peroxidase,glutathione reductase and catalase activities as well as reduced and oxidized glutathione contents in all samples of leaves,roots and stolons were increased in the presence of Cd.Despite the fact that Cd has a redox characteristic not compatible with the Fenton-type chemistry that produces active oxygen species,Cd tolerance in Phragmites plants might be associated to the efficiency of these mechanisms.The effect of Fe status on the uptake of Cd and Zn by two ecotypes of T.caerulescens,i.e.,Gan-ges and Prayon(the former being far superior in Cd uptake)was studied(Lombi et al.2002). Moreover,the T.caerulescens zip(Zn-regulated transporter/Fe-regulated transporter-like protein) genes,TcZNT1-G and TcIRT1-G,were cloned from Ganges and their expression under Fe-suf-ficient and-deficient conditions analyzed.Cad-mium uptake was significantly enhanced by Fe deficiency in Ganges,while Zn uptake was not influenced by the Fe status of the plants in either of the ecotypes.These results are in agreement with the gene expression study,since the abun-dance of ZNT1-G mRNA was always similar, independently of the Fe status or ecotype,while that of TcIRT1-G mRNA was greatly increased only in Ganges root tissue under Fe-deficient conditions,suggesting a possible relationship with an up-regulation in the expression of genes encoding Fe uptake,possibly TcIRT1-G(Lombi et al.2002).Recently,Song et al.(2003)reported on the utility of the yeast protein YCF1,a protein which detoxifies Cd by transporting it into vacuoles,for the remediation of Cd and Pb contamination,finding out that transgenic Arabidopsis thaliana plants overexpressing YCF1showed enhanced tolerance and accumulated greater amounts of Cd and Pb.Interestingly enough,Lahner et al.(2003) analyzed several essential and nonessential ele-ments in shoots of6000mutagenized A.thaliana plants,demonstrating the utility of genomic scale profiling of nutrients and trace elements as a functional genomics tools.A better understanding of how plants handle mineral elements has the potential of yielding new phytoremediation capa-bilities(Rea,2003).While Cd detoxification is certainly a complex phenomenon,probably under polygenic control, Cd real tolerance found in mine plants seems to be a simpler phenomenon,possibly involving only monogenic/oligogenic control(Sanita di Toppi& Gabbrielli1999).These authors concluded that adaptive tolerance is supported by constitutive detoxification mechanisms,which in turn rely on constitutive homeostatic processes.Symmetric and asymmetric somatic hybridiza-tions have been used to introduce toxic metal-resistant traits from T.caerulescens into Brassica juncea(Dushenkov et al.2002).B.juncea hypo-cotyl protoplasts were fused with T.caerulescens mesophyll protoplasts,and all putative hybrids had morphological characteristics of B.juncea. Hybrid plants,produced by asymmetric somatic hybridization between the two species,demon-strated high metal accumulation potential,toler-ance to toxic metals,and good biomass production.In any case,B.juncea is by itself a good candidate for efficient phytoextraction of heavy metals-such as Cd-from polluted soils (Schneider et al.1999).Two years after the toxic spill caused by the failure of a tailing pond dam at the Aznalcollar pyrite mine(SW Spain)in1998,none of the trace elements measured-As,Cd,Cu,Pb,Tl-reached levels either phytotoxic or toxic for humans or animals in seeds and the above-ground part of spill-affected sunflower plants(Madejon et al. 2003).However,the potential for phytoextraction of these plants is very low,though they might be useful for soil conservation.The production of oil (usable for industrial purposes,whose production77。
Dynamic Transcriptome Landscape of Maize Embryo and

Dynamic Transcriptome Landscape of Maize Embryo and Endosperm Development1[W][OPEN]Jian Chen2,Biao Zeng2,Mei Zhang,Shaojun Xie,Gaokui Wang,Andrew Hauck,and Jinsheng Lai*State Key Laboratory of Agro-biotechnology and National Maize Improvement Center,Department of Plant Genetics and Breeding,China Agricultural University,Beijing100193,People’s Republic of ChinaMaize(Zea mays)is an excellent cereal model for research on seed development because of its relatively large size for both embryo and endosperm.Despite the importance of seed in agriculture,the genome-wide transcriptome pattern throughout seed development has not been well ing high-throughput RNA sequencing,we developed a spatio-temporal transcriptome atlas of B73maize seed development based on53samples from fertilization to maturity for embryo, endosperm,and whole seed tissues.A total of26,105genes were found to be involved in programming seed development,in-cluding1,614transcription factors.Global comparisons of gene expression highlighted the fundamental transcriptomic repro-gramming and the phases of development.Coexpression analysis provided further insight into the dynamic reprogramming of the transcriptome by revealing functional transitions during bined with the published nonseed high-throughput RNA sequencing data,we identified91transcription factors and1,167other seed-specific genes,which should help elucidate key mechanisms and regulatory networks that underlie seed development.In addition,correlation of gene expression with the pattern of DNA methylation revealed that hypomethylation of the gene body region should be an important factor for the expressional activation of seed-specific genes,especially for extremely highly expressed genes such as zeins.This study provides a valuable resource for understanding the genetic control of seed development of monocotyledon plants.Maize(Zea mays)is one of the most important crops and provides resources for food,feed,and biofuel (Godfray et al.,2010).It has also been used as a model system to study diverse biological phenomena,such as transposons,heterosis,imprinting,and genetic diversity (Bennetzen and Hake,2009).The seed is a key organ of maize that consists of the embryo,endosperm,and seed coat.Maize seed development initiates from a double fertilization event in which two pollen sperm fuse with the egg and central cells of the female gametophyte to produce the progenitors of the embryo and endosperm, respectively(Dumas and Mogensen,1993;Chaudhury et al.,2001).The mature embryo inherits the genetic information for the next plant generation(Scanlon and Takacs,2009),whereas the endosperm,which is storage tissue for the embryo,persists throughout seed devel-opment and functions as the site of starch and protein synthesis(Sabelli and Larkins,2009).Elucidation of the genetic regulatory mechanisms involved in maize seed development will facilitate the design of strategies to improve yield and quality,and provide insight that is applicable to other monocotyledon plants.A key means to explore the mechanisms of seed de-velopment is to identify gene activities and functions. Genetic studies have uncovered a number of genes that play major roles in governing embryogenesis and ac-cumulation of endosperm storage compounds,such as Viviparous1,KNOTTED1,Indeterminate gametophyte1, Shrunken1(Sh1),Opaque2(O2),and Defective kernel1 (Chourey and Nelson,1976;McCarty et al.,1991;Smith et al.,1995;Vicente-Carbajosa et al.,1997;Lid et al., 2002;Evans,2007).Furthermore,the activity of some genes has also been extensively studied.Typical exam-ples are zein genes that encode primary storage proteins in endosperm.Woo et al.(2001)examined zein gene expression and showed that they were the most highly expressed genes in endosperm based on EST data,where-as their dynamic expression patterns were revealed in a later study(Feng et al.,2009).Nevertheless,informa-tion on the global gene expression network throughout seed development is still very limited.The transcriptome is the overall set of transcripts, which varies based on cell or tissue type,develop-mental stage,and physiological condition.Analysis of transcriptome dynamics aids in implying the function of unannotated genes,identifying genes that act as critical network hubs,and interpreting the cellular pro-cesses associated with development.In Arabidopsis (Arabidopsis thaliana),the genes expressed in devel-oping seed and its subregions at several develop-ment stages have been analyzed with Affymetrix GeneChips(Le et al.,2010;Belmonte et al.,2013).In1This work was supported by the National High Technology Re-search and Development Program of China(863Project,grant no. 2012AA10A305to J.L.)and the National Natural Science Foundation of China(grant no.31225020to J.L.).2These authors contributed equally to the article.*Address correspondence to jlai@.The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors()is: Jinsheng Lai(jlai@).[W]The online version of this article contains Web-only data.[OPEN]Articles can be viewed online without a subscription./cgi/doi/10.1104/pp.114.240689maize,microarray-based atlases of global transcription have provided insight into the programs controlling development of different organ systems (Sekhon et al.,2011).Compared with microarray,high-throughput RNA sequencing (RNA-seq)is a powerful tool to com-prehensively investigate the transcriptome at a much lower cost,but with higher sensitivity and accuracy (Wang et al.,2009b).Several studies have taken ad-vantage of the RNA-seq strategy to interpret the dy-namic reprogramming of the transcriptome during leaf,shoot apical meristem,and embryonic leaf develop-ment in maize (Li et al.,2010;Takacs et al.,2012;Liu et al.,2013).To date,only two studies have focused on identi-fying important regulators and processes required for embryo,endosperm,and/or whole seed development in maize based on a genome-wide transcriptional pro-file produced by RNA-seq (Liu et al.,2008;Teoh et al.,2013).However,these studies were limited by the low number of samples used and they did not provide an extensive,global view of transcriptome dynamics over the majority of seed development stages.Here,we pres-ent a comprehensive transcriptome study of maize em-bryo,endosperm,and whole seed tissue from fertilization to maturity using RNA-seq,which serves as a valu-able resource for analyzing gene function on a global scale and elucidating the developmental processes of maize seed.RESULTSGeneration and Analysis of the RNA-seq Data SetTo systematically investigate the dynamics of the maize seed transcriptome over development,we generated RNA-seq libraries of B73seed tissues from different developmental stages,including 15embryo,17endo-sperm,and 21whole seed samples (Fig.1).Utilizing paired-end Illumina sequencing technology,we gen-erated around 1.9billion high-quality reads,80.2%of which could be uniquely mapped to the B73referencegenome (Schnable et al.,2009;Supplemental Table S1).The genic distribution of reads was 66.6%exonic,25.6%splice junction,and 2.6%intronic,leaving about 5%from unannotated genomic regions,demonstrating that most of the detected genes have been annotated.Uniquely mapped reads were used to estimate nor-malized transcription level as reads per kilobase per million (RPKM).To reduce the in fluence of transcrip-tion noise,genes from the B73filtered gene set (FGS)were included for analysis only if their RPKM values were $1.Considering that our purpose was not to identify minor differential expression of genes between two time points of development,but to provide an atlas of gene expression pro file across tissues using time se-ries biological samples,we only randomly selected 12samples to have a biological replicate (Supplemental Fig.S1)to assess our data parisons of bio-logical replicates showed that their expression values were highly correlated (average R 2=0.96).For the samples with biological replicates,we took the average RPKM as the expression quantity.To further evaluate the quality of our expression data,we compared the transcript abundance patterns of a number of selected genes with previously measured expression pro files (Supplemental Fig.S2).For example,LEAFY COTYLE-DON1(LEC1),which functions in embryogenesis,was mainly expressed in the early stage of the embryo (Lotan et al.,1998).Globulin2(Glb2)had high expres-sion late in embryogenesis,in accordance with its func-tion as an important storage protein in the embryo (Kriz,1989).Similarly,O2,a transcription factor (TF)that regulates zein synthesis (Vicente-Carbajosa et al.,1997),and Fertilization independent endosperm1(Fie1),a repressor of endosperm development in the absence of fertilization (Danilevskaya et al.,2003),were almost ex-clusively expressed in the endosperm.Expression pat-terns of these selected genes were identi fied exclusively in their known tissue of activity,indicating that the embryo and endosperm samples were processed well.In total,we detected 26,105genes expressed in at least 1of the 53samples (Supplemental Data Set S1).The distribution of these genes is revealed by aVennFigure 1.Overview of the time series maize seed samples used for RNA-seq analysis.The photographs show the changes in maize embryo,endosperm,and whole seed during development.The 53samples shown here were used to generate RNA-seq libraries.Bar =5mm.Transcriptome Dynamics of Maize Seeddiagram (Fig.2A),which shows that 20,360genes were common among all three tissue types.The number of genes detected in endosperm tissue during the devel-opmental stages was lower and much more variable compared with embryo or whole seed tissue,and a greater number of genes were expressed in the tissue during the early and late phases.Several thousand fewer expressed genes were detected 14d after polli-nation (DAP)in the endosperm compared with 6or 8DAP (Fig.2B;Supplemental Data Set S2).In addition,the median expression level in the embryo was roughly 2-fold greater than that of the endosperm from 10to 30DAP (Fig.2C).Of the 1,506genes unique to the whole seed samples,451were present in RNA-seq data of 14and/or 25DAP pericarp tissue (Morohashi et al.,2012).Considering that the maternal tissue is the vast majority of the content of the early seed (Márton et al.,2005;Pennington et al.,2008),we inferred that most of these genes might be expressed exclusively in maternal tissue such as the pericarp or nucellus.Moreover,1,062genes in the embryo and endosperm with low expression were not detected in whole seed samples (Fig.2C).Division of Development Phases by Global Gene Expression PatternsTo gain insight into the relationships among the dif-ferent transcriptomes,we performed principal compo-nent analysis (PCA)on the complete data set,which can graphically display the transcriptional signatures and developmental similarity.The first component(40.7%variance explained)separated samples based on tissue identity and clearly distinguished embryo from endosperm samples,with whole seed samples located in between (Fig.3A).The second component (24.4%variance explained)discriminated early,mid-dle,and late stages of development for all three tissues (Fig.3A).The wider area occupied by endosperm sam-ples than embryo demonstrates stronger transcriptome reprogramming in developing endosperm,which is mainly attributable to drastic changes in the early and late stages.Moreover,whole seed samples of 0to 8DAP and 30to 38DAP clustered closely to the embryo,but 10to 28DAP samples were close to the endosperm.Cluster analysis of the time series data for the tissues grouped samples well along the axis of developmental time (Fig.3,B –D).Embryo samples from 10to 20DAP and 22to 38DAP were the primary clusters,which correspond to morphogenesis and maturation phases of development (Fig.3B).This is consistent with the embryo undergoing active DNA synthesis,cell division,and differentiation,and then switching to synthesis of storage reserve and desiccation (Vernoud et al.,2005).Expression differences in endosperm samples resulted in three primary clusters,which correspond to early,middle,and late phases of development (Fig.3C).The earliest time point (6and 8DAP)is an active period of cell division and cell elongation that terminates at about 20to 25DAP (Duvick,1961).The samples of 10to 24DAP formed one subgroup and 26to 34DAP formed another subgroup,suggesting that they mark the period forming the main cell types and maturation of endo-sperm,respectively (Fig.3C).The two subgroupsformFigure 2.Analysis of global gene expres-sion among different samples.A,Venn diagram of the 26,105genes detected among embryo,endosperm,and whole seed.B,Number of genes expressed in each of the samples.C,Comparison of expression levels of genes detected in embryo and endosperm tissues.Chen et al.a larger cluster for active accumulation of storage com-pounds during 10to 34DAP.The distinct cluster of 36and 38DAP is in accordance with the end of storage compound accumulation in the endosperm and the ac-tivation of biological processes involved in dormancy and dehydration.In whole seed tissue,a primary clus-ter was formed from the earliest time points with 0to 4DAP and 6to 8DAP samples as subgroups,separating the nucellus degradation as well as endosperm syncy-tial and cellularization phases from the rapid expansion of the endosperm and development of embryonic tissues (Fig.3D).After 10DAP,the embryo and endosperm dominate the formation of seed,as shown in Figure 1and morphological observation (Pennington et al.,2008).As effected by both embryo and endosperm,10to 28DAP whole seed samples clustered together and 30to 38DAP formed another group (Fig.3D).These results con firmthat the expression data successfully captured the char-acteristic seed development phases and should there-fore contain valuable insights about corresponding changes in the transcriptome.Integration of Gene Activity and Cellular Function across Development PhasesThe PCA and hierarchical clustering analysis graph-ically display the relationship among different sam-ples,but do not indicate the detailed cellular ing the k-means clustering algorithm,we classi fied the detected genes into 16,14,and 10coexpression modules for embryo,endosperm,and whole seed,re-spectively,each of which contains genes that harbor similar expression patterns (Fig.4).We then used Map-Man annotation to assign genes to functionalcategoriesFigure 3.Global transcriptome relationships among different stages and tissues.A,PCA of the RNA-seq data for the 53seed samples shows five distinct groups:I for embryo (light red),II for endosperm (light blue),and III to V for early (III),middle (IV),and late (V)whole seed (light purple).B to D,Cluster dendrogram showing global transcriptome relationships among time series samples of embryo (B),endosperm (C),and whole seed (D).The y axis measures the degree of variance (see the “Materials and Methods”).The bottom row indicates the developmental phases according to the cluster dendrogram of the time series data.au,Approximately unbiased.Transcriptome Dynamics of Maize Seed(Supplemental Fig.S3).Thus,we can aggregate genes over continuous time points and obtain a view of func-tional transitions along seed development.According to the cluster analysis results,most mod-ules of the embryo can be divided into middle (10–20DAP)and late (22–38DAP)stages (Fig.4A).The middle stage,best represented by modules C1to C7,is typi fied by the overrepresentation of glycolysis,tri-carboxylic acid cycle,mitochondrial electron transport,redox,RNA regulation,DNA and protein synthesis,cell organization,and division-related genes.This is consis-tent with the high requirement of energy during em-bryo formation.The late stage represented by C8to C12exhibited up-regulation of the cell wall,hormone me-tabolism (ethylene and jasmonate),stress,storage pro-teins,and transport-related genes,which coincides with the maturation of the embryo.The modules C13to C16included genes that were broadly expressed across the time points sampled and were related to hormone me-tabolism (brassinosteroid),cold stress,RNA process-ing and regulation,amino acid activation,and protein targeting.All of the 14coexpression modules of endosperm can be roughly divided into early (6–8DAP),middle (10–34DAP),and late (36–38DAP)stages (Fig.4B).The early stage (represented by modules C1to C4)isexempli fied by high expression of hormone metabo-lism (gibberellin),cell wall,cell organization and cycle,amino acid metabolism,DNA,and protein synthesis –related genes,which is consistent with differentiation,mitosis,and endoreduplication.Genes in the tricar-boxylic acid cycle and mitochondrial electron transport are also overrepresented and related to energy de-mands at that time.The middle stage (best represented by C5to C8,in which different modules have distinct pro files)is the active storage accumulation phase and exhibits high expression of carbohydrate metabolism genes,as expected.Increased expression of protein degradation-related genes around 26to 34DAP in C7and C8coincides with the process of endosperm matu-ration.Genes involved in protein degradation,second-ary metabolism,oxidative pentose phosphate,receptor kinase signaling,and transport were up-regulated in the late stage in modules C9to C14during the con-cluding phase of endosperm maturation.Ten DAP and later time points of whole seed sam-ples re flect the additive combination of embryo and endosperm expression.Genes that are active early in development (0–8DAP)in clusters C1to C4are ex-pected to be related to maternal tissue,which is the bulk of the seed at that time.A group of genes are highly expressed in C1at 0DAP,but theirexpressionFigure 4.Coexpression modules.A to C,Expression patterns of coexpression modules of embryo (A),endosperm (B),and whole seed (C),ordered according to the sample time points of their peak expression.For each gene,the RPKM value normalized by the maximum value of all RPKM values of the gene over all time points is shown.Chen et al.drops rapidly by2DAP,suggesting that they have functional roles that precede pollination.This group includes photosynthesis light reaction members and some TFs involved in RNA regulation.Genes related to cell wall and protein degradation,signaling,nucle-otide metabolism,DNA synthesis,cell organization, and mitochondrial electron transport are overrepre-sented in C2to C4,which has increased expression after pollination,in accordance with the degradation of nucellus tissue and development of embryonic tissues. The expression patterns and functional categories of the1,506genes detected only in whole seed samples are shown in Supplemental Figure S4.Because these genes tend to be expressed at high levels mainly before 8DAP,they are presumed to have functions in early seed development.Together,these data show that the transition of major biochemical processes along the developmental time axis of the seed is produced partly by highly coordi-nated transcript dynamics.TF Expression during Seed DevelopmentOf the2,297identified maize TFs(Zhang et al.,2011), 1,614(70%)are included in our analysis(Supplemental Data Set S3),which accounts for6.18%of the total number of genes detected in seed tissue.The num-ber of TFs detected in the different samples is shown in Supplemental Figure S5A.Their proportion to the total genes expressed in each tissue time point was always greater in embryo than endosperm samples (Supplemental Fig.S5B).Shannon entropy has been used to determine the specificity of gene expression, with lower values indicating a more time-specific pro-file(Makarevitch et al.,2013).The Shannon entropy of TFs was significantly lower than all other genes in both embryo(P=2.3310210)and endosperm(P,1.53 1023),indicating that TFs tended to be expressed more time specifically than other genes(Supplemental Fig.S5, C and D).The number of TFs from each family used in the seed development program,along with the proportion of members present in the coexpression modules rel-ative to the total members of the family expressed in the tissue,is shown in Supplemental Figure S6. Enrichment of these TF families in the coexpression modules based on observed numbers was evaluated with Fisher’s exact test.Significant TF family enrich-ment was identified for specific coexpression modules. For example,12auxin-response factor TFs(38.7%) were expressed in embryo module C2during mor-phogenesis and one-half of the detected members of the WRKY family were active in endosperm module C12late in endosperm development.The WRKY family has been reported to be mainly involved in the physiological programs of pathogen defense and se-nescence(Eulgem et al.,2000;Pandey and Somssich, 2009).Twenty-one MIKC family TFs(56.8%)were pres-ent in whole seed module C2,implying an important role in regulating genes involved in response to fertil-ization.The developmental specificity of the detected TFs makes them excellent candidates for reverse ge-netics approaches to investigate their role in grain production.Tissue-Specific Genes of SeedIdentification of uncharacterized tissue-specific genes can help to explain their function and understand the underlying control of tissue or organ identity.To gen-erate a comprehensive catalog of seed-specific genes, results from this study were compared with25pub-lished nonseed RNA-seq data sets(Jia et al.,2009;Wang et al.,2009a;Li et al.,2010;Davidson et al.,2011;Bolduc et al.,2012),including root,shoot,shoot apical meristem, leaf,cob,tassel,and immature ear(Supplemental Table S2).In total,we identified1,258seed-specific genes,in-cluding91TFs from a variety of families(Supplemental Data Set S4).To gain further insight into the spatial expression trend in the developing seed,we divided these genes into four groups:embryo specific,endo-sperm specific,expressed in both embryo and endo-sperm,and other as only expressed in whole seed (Table I).The dynamic expression patterns of these genes reflect their roles in corresponding development stages(Supplemental Fig.S7).The largest numbers of seed-specific genes were observed in the endosperm, consistent with a study in maize using microarrays (Sekhon et al.,2011),perhaps reflecting the specific function of endosperm.We compared the distribution of tissue-specific genes and TFs in embryo and endosperm coexpression mod-ules to identify important phases in the underlying transcription network.Coexpression modules with an enrichment of tissue-specific genes or TFs may provide insight about uncharacterized genes and preparation for subsequent developmental processes.A feature of gene activity is shown in Figure5.Fisher’s exact test (P,0.05)was used to determine modules with sig-nificant enrichment of tissue-specific genes and TFs in the embryo and endosperm.In the embryo,TFs and tissue-specific genes were significantly enriched in the late phase,suggesting a specific process during matu-ration.In the endosperm,we observed that TFs and tissue-specific genes were overrepresented in the mid-dle phase,which conforms to the role of endosperm inTable I.Total number of detected seed-specific genes and TFsData are presented as n.Tissue Type Specific Genes Specific TFs Embryo24923Endosperm74259Embryo and endosperm2196Other a483Total1,25891a These genes only detected expression in whole seeds.Transcriptome Dynamics of Maize Seedstorage compound accumulation and to speci fic pro-gress at this phase.To gain further insight into the functional signi fi-cance of tissue-speci fic genes,overrepresented gene ontology (GO)terms were examined using the WEGO online tool (Ye et al.,2006;Supplemental Fig.S8).All overrepresented GO terms were observed for themiddle embryo development phase,including the bio-synthetic process,cellular metabolic process,macro-molecule,and nitrogen compound metabolic process.Similarly,overrepresented GO terms for the endosperm were mostly observed in the early phase,including the macromolecule,nitrogen compound metabolic pro-cess,DNA binding,and transcription regulator.GenesFigure 5.Distribution and enrichment of genes,tissue-specific genes,TFs,and tissue-specific TFs in coexpression modules of embryo and endosperm.A and B,Bars indicate the percentage of all detected genes (green),tissue-specific genes (blue),TFs (red),and tissue-specific TFs (purple)observed in a coexpression module (C)or in the development phase (Total)relative to the total number of each group detected across samples for embryo (A)and endosperm (B).The number of genes represented by the percentage is shown on the right y axis.Enrichment for tissue-specific genes and TFs was evaluated with Fisher’s exact test based on the number of genes observed in each coexpression module,whereas enrichment for tissue-specific TFs was evaluated based on the number of TFs observed in each coexpression module.Asterisks represent significant enrichment at a false dis-covery rate #0.05.Chen et al.involved in the nutrient reservoir class were enriched in the middle phase.The oxidoreductase class was overrepresented in late phase,and is known to be in-volved in maturation (Zhu and Scandalios,1994).The Expression of Zein Genes in EndospermZeins are the most important storage proteins in maize endosperm and are an important factor in seed quality.According to Xu and Messing (2008),there are 41a ,1b ,3g ,and 2d zein genes.In order to explore their expression pattern,we first con firmed the gene models by mapping publicly available full-length com-plementary DNAs of zein subfamily genes to B73bac-terial arti ficial chromosomes,and then mapped these back to the reference genome.Because some of these zein genes were not assembled in the current B73ref-erence genome or were only annotated in the working gene set,we con firmed a final set of 35zein genes in the FGS of the B73annotation,including 30a ,1b ,3g ,and 1d zein genes (Supplemental Table S3).About three-quarters (26)of these were in the list of the 100most highly expressed genes in the endosperm,based on mean expression across all endosperm samples (Supplemental Table S4).The distribution of these most highly expressed genes clearly showed that the 26zeins and 4starch synthesis genes were actively expressed in the middle phase of endosperm devel-opment,characteristic of storage compound accumu-lation (Fig.6A).Previous research has shown that the zein genes con-stitute approximately 40%to 50%of the total tran-scripts in the endosperm (Marks et al.,1985;Woo et al.,2001),but these results are based on EST data from a single tissue or pooled tissues and only a few zein genes were assessed.Thus,we reevaluated the transcriptomic contribution of zein genes across endosperm develop-ment using our RNA-seq data,which is able to overcome the high structural similarity among them,especially in the a family (Xu and Messing,2008).Zein genes stably accounted for about 65%of transcripts from 10to 34DAP,with 19-kD a zeins (approximately 42%),22-kD a zeins (approximately 8%),and g zeins (approximately 10%)representing the most abundant transcripts (Fig.6B).The expression of different members within agivenFigure 6.Analysis of highly expressed genes in the endosperm.A,The distribution of the 100most highly expressed genes in the endosperm ordered by mean expression in different modules.B,The dynamic transcript levels of different zein gene family members in the endosperm as reflected by their percentage among all detected gene transcript levels.C,Heat map showing RPKM values of 35zein genes in the different development stages of the endosperm.+,Having intact coding regions;2,with premature_stop;N,no;Y ,yes.Transcriptome Dynamics of Maize Seed。
安捷伦产品目录

15
Real-Time PCR
16
Mx3000P QPCR System
17
Brilliant III Ultra-Fast SYBR Green QPCR and QRT-PCR Reagents
18
Brilliant III Ultra-Fast QPCR and QRT-PCR Reagents
Agilent / STRATAGENE
Agilent website: /genomics
Welgene | Agilent Stratagene
威健股份有限公司 | Stratagene 總代理
Table of Content
Table of Contents
/ XL1-Red Competent Cells SoloPack Gold Supercompetent Cells
/ TK Competent Cells Specialty Cells
/ Classic Cells / Fine Chemicals For Competent Cells
適用於 UNG 去汙染或 bisulphite
sequencing
適用於 TA Cloning
最高敏感性
取代傳統 Taq 的好選擇
-
2
威健股份有限公司 | Stratagene 總代理
PCR Enzyme & Instrument
Agilent SureCycler 8800
市場上領先的 cycling 速度和 sample 體積 10 ~ 100 μL 簡易快速可以選擇 96 well 和 384 well 操作盤 優秀的溫控設備讓各個 well 都能保持溫度的穩定 七吋的高解析度觸控螢幕讓操作上更為簡便 可以透過網路遠端操控儀器及監控儀器 Agilent 專業的技術支援可以幫助您應對各種 PCR 的問題
- 1、下载文档前请自行甄别文档内容的完整性,平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益,请联系客服反馈,我们会尽快为您处理(人工客服工作时间:9:00-18:30)。
A Route-directed Hybrid Genetic Approach for the Vehicle Routing Problem withTime WindowsJEAN BERGER, MOHAMED BARKAOUIDefence Research Development Canada - Valcartier, Decision Support Technology Section2459 Pie-XI Blvd. North, Val-Bélair, PQ, Canada, G3J 1X5email: jean.berger@drdc-rddc.gc.ca, barkaoui@videotron.caOLLI BRÄYSYSINTEF Applied MathematicsDepartment of Optimisation, P.O. Box 124 Blindern, N-0314 Oslo,Norway,email: olli.braysy@sintef.noA route-directed hybrid genetic approach to address the Vehicle Routing Problem with Time Windows is presented. The proposed scheme relies on the concept of simultaneous evolution of two populations pursuing different objectives subject to partial constraint relaxation. The first population evolves individuals to minimize total traveled distance while the second focuses on minimizing temporal constraint violation to generate a feasible solution, both subject to a fixed number of tours. Genetic operators have been designed to incorporate key concepts emerging from recent promising techniques such as insertion heuristics and large neighborhood search to further explore the solution space. Results from a computational experiment over common benchmark problems show that the proposed technique matches or outperforms some of the best heuristic routing procedures, providing six new best-known solutions. In comparison, the method proved to be fast, cost-effective and highly competitive.1. INTRODUCTIONVehicle routing problems are important and well-known combinatorial optimization problems occurring in many distribution systems with considerable economic significance. It i s estimated that distribution costs account for almost half of the total logistics costs, and in some industries, such as in the food and drink business, distribution costs can account for up to 70% of the value added costs of goods (De Backer et al., 1997; Golden and Wasil 1987). To get an impression of the magnitude of thefreight transportation, let us mention that the annual cost of excess travel in the United States has been estimated at some USD 45 billion (King and Mast, 1997). In EU the annual turnover of freight transportation is about 150 billion euros, and there are about 500 000 companies and 13 million trucks. Halse (1992) reports that 76,5% of all the transportation of goods is done by vehicles that also underlines the importance of vehicle routing problems. On the other hand, it has been estimated that the capacity utilization with load is only 60%, and the vehicles drive 36% of the time empty (Hasle, 2002) indicating a significant potential for improvement. The Vehicle Routing Problem with Time Windows (VRPTW) has received a lot of attention in the literature recently. This is mostly due to the wide applicability of time window constraints in real-world cases. In VRPTW, customers with known demands are serviced by a homogeneous fleet of vehicles with limited capacity. Routes are assumed to start and end at the central depot. Each customer provides a time interval during which a particular task must be completed such as loading/unloading the vehicle. It is worth noticing that the time window requirement does not prevent any vehicle from arriving before the allowed start of service at a customer location. The objective is to minimize the number of tours or routes, and then for the same number of tours, to minimize the total traveled distance, such that each customer is serviced within its time window and the total load on any vehicle associated with a given route does not exceed vehicle capacity.A variety of algorithms including exact methods and efficient heuristics have already been proposed for VRPTW. For surveys on exact, heuristic and metaheuristic methods, see Desrosiers and al. (1995), Cordeau and al. (2001) and Bräysy and Gendreau (2001a and 2001b) respectively. In particular, evolutionary and genetic algorithms have been among the most s uitable approaches to tackle the VRPTW, and are of particular interest to us.Genetic algorithms (Holland, 1975; De Jong, 1975 and Goldberg, 1989) are adaptive heuristic search methods that mimic evolution through natural selection. They work by combining selection, recombination and mutation operations. The selection pressure drives the population toward bettersolutions while recombination uses genes of selected parents to produce offspring that will form the next generation. Mutation is used to escape from local minima.Blanton and Wainwright (1993) were the first to apply a genetic algorithm to VRPTW. They hybridized a genetic algorithm with a greedy heuristic. Under this scheme, the genetic algorithm searches for a good ordering of customers, while the construction of the feasible solution is handled by the greedy heuristic.Thangiah (1995a and 1995b) uses a genetic algorithm to find good clusters of customers within a “cluster first, route second” problem-solving strategy. Thangiah and al. (1995) test the same approach to solve vehicle routing problems with time deadlines.In the algorithm proposed by Potvin and Bengio (1996), new offspring are created by connecting two route segments from two parent solutions or by replacing the route of the second parent-solution by the route of the first parent-solution. Mutation is then used to reduce the number of routes and to locally optimize the solution. Berger and al. (1998) present a hybrid genetic algorithm based on removing certain customers from their routes and then rescheduling them with well-known route-construction heuristics. The mutation operators are aimed at reducing the number of routes by rescheduling some customers and at locally reordering customers. Bräysy (1999a, 1999b) continues the study of Berger and al. (1998) by performing a comprehensive sensitivity analysis, and by creating new crossover and mutation operators. Also Berger and al. (1999) and Berger and Barkaoui (2000) have further continued the research direction started in Berger and al. (1998).Homberger and Gehring (1999) propose two evolutionary metaheuristics based on the class of evolutionary algorithms called Evolution Strategies and three well-known route improvement procedures Or-opt (Or, 1976), λ-interchanges (Osman, 1993) and 2-opt* (Potvin and Rousseau, 1995). Gehring and Homberger (1999 and 2001) use a similar approach with parallel tabu search implementation. Bräysy and al. (2000) hybridize a genetic algorithm with an evolutionary algorithm consisting of several route construction and improvement heuristics. The recent genetic algorithm by Tan and al. (2001) is based on Solomon’s (1987) insertion heuristic, λ-interchanges and the well-known PMX-crossover operator.In addition to genetic and evolutionary algorithms, tabu searches (Glover, 1986; Glover and Laguna, 1997) have been among the most successful techniques to address VRPTW. The basic idea in tabu search is to allow selection of worse solutions once a local optimum has been reached. Different memory structures are then used to prevent repeating the same solutions (cycling), and to diversify and intensify the search. Rochat and Taillard (1995) present a probabilistic tabu search that uses an adaptive memory to record the best routes produced during the search to create new solutions. Likewise, Taillard and al. (1997) exploit adaptive memory, but employ a different neighborhood structure based on exchanges of consecutive customers between routes. Chiang and Russell (1997) describe a reactive tabu search that dynamically adjusts its parameter values based on the current search status to avoid both cycles as well as an overly constrained search path, and Cordeau and al. (2001) introduce simple tabu searches that allow unfeasible solutions during the search process. Other types of metaheuristics have also been proposed. Gambardella and al. (1999) present an optimization technique based on multiple colonies of artificial ants. Liu and Shen (1999) develop a new route-neighborhood-based metaheuristic that constructs routes in a nested parallel manner. Rousseau and al. (2002) use a variable neighborhood descent scheme (Mladenovic and Hansen, 1997) and new large-neighborhood operators within a constraint-programming framework.Most previously proposed metaheuristics show variability in the average and the best performances, as can be observed from the recent surveys by Bräysy and Gendreau (2001a; 2001b). According to Bräysy and Gendreau (2001b), the reported average performance results do not show any conclusive evidence to support a dominating heuristic over the others. No single method has consistently matched the best-known minimum number of tours over all problem instances examined. Consequently, a more robust, efficient and stable (minimal variance in computed n umber of tours) algorithm is still elusive. The main contribution of this paper is an attempt to develop such a new technique. The proposed route-directed hybrid genetic approach (RHGA) is shown to be fast, cost-effective and highly competitive.The novelty of the proposed approach is based on a new concept that combines constrained simultaneous evolution of two populations and partial temporal constraint relaxation to improvesolution quality. The first population evolves individuals to minimize the total traveled distance while the second focuses on minimizing temporal constraint violation in trying to generate a feasible solution. Imposing a fixed number of tours to solution members of a given population, temporal constraint relaxation allows escaping local minima while progressively moving toward a better solution. Populations interact with one another whenever a new feasible solution emerges, reducing gradually the number of tours imposed on future solutions. Genetic operators have been designed to first maximize the number of customers served within their time intervals, and then temporal constraint relaxation is used to insert remaining unvisited customers. Key features and variants emerging from recent promising techniques are also captured to further diversify and intensify the search.The paper is outlined as follows. Section 2 introduces the main concepts of the proposed genetic algorithm. The basic principles and features of the algorithm are first described. Then, the selection scheme, recombination and mutation operators are presented. The combination of concepts borrowed or derived from well-known heuristics such as large neighborhood search (Shaw, 1998) and route neighborhood-based two-stage metaheuristic (Liu and Shen, 1999) are briefly outlined. Section 3 presents the results of a computational experiment to assess the value of the proposed approach and reports a comparative performance analysis to alternate methods. Finally, some conclusions and future research directions are presented in Section 4.2. ROUTE-DIRECTED HYBRID GENETIC APPROACH2.1 GENERAL DESCRIPTIONThe proposed algorithm mainly relies on the basic principles of genetic algorithms, disregarding explicit solution encoding issues for problem representation. Genetic operators are simply applied to a population of solutions rather than a population of encoded solutions (chromosomes). We refer to these solutions as solution individuals.Our approach relies upon constrained simultaneous evolution and partial constraint relaxation. Two populations Pop1 and Pop2, primarily formed of non-feasible solution individuals, are evolvingconcurrently, each with their own objective functions. Pop 1 contains at least one feasible solution and is used to minimize total traveled distance while Pop 2 focuses on minimizing constraint violation. The number of tours imposed on solution individuals in Pop 1 and Pop 2 are R min and R min – 1, respectively. R min refers to the number of routes found in the best feasible solution obtained so far. As a new f easible solution emerges from Pop 2, population Pop 1 is replaced by Pop 2 (migration), R min is updated and, Pop 2 is mutated with the revised number of tours (R min -1), using the RSR_M mutation operator. In addition, a post-processing procedure (RC_M) aimed at reordering customers is applied to further improve the new best solution. RSR_M and RC_M mutation operators are both described in Section 2.3.2. The evolutionary process is repeated until a predefined stopping condition is met.The proposed approach u ses a steady-state genetic algorithm that involves overlapping populations. At first, new individuals are generated and added to the current population Pop p . The process continues until the overlapping population outnumbers the initial population by n p . Then, the n p worst individuals are eliminated to maintain population size using the following individual evaluation:,i i i CV E Eval +=(1) wherei m i i d r r E γ+−=,(2) i n c c i c c i Viol l b CV βα+−=∑=1},0max{ (3)E i = basic evaluation of individual (solution) i ,r i = number of routes in individual i ,r m = lower bound for number of routes (ratio of total demand over vehicle capacity),d i = total traveled distance related to individual i,γ = relative user-defined w eight parameter controlling the distance contribution. The value of γ isselected to privilege solutions having a smaller number of routes (e.g. γ = 1/d m , where d m refers to the maximum traveled distance over the individuals forming the initial population),CV i= temporal constraint violation associated to individual i,n= number of customers,α= penalty associated with temporal constraint violation c,cib= scheduled time to visit customer c in individual i,cl= latest time to visit customer c,cβ= penalty associated with number of violated temporal constraints,Viol= number of temporal constraints violated in individual i.iThe evaluation function indicates that better individuals generally (but not necessarily) include fewer routes, and smaller total traveled distance, while satisfying temporal constraints. The general algorithm is specified as follows:InitializationRepeatp=1Repeat {evolve population Pop p - new generation}For j =1..n p doSelect two parents from Pop pGenerate a new solution S j using recombination and mutation operators associated with Pop pAdd S j to Pop pend forRemove from Pop p the n p worst individuals using the evaluation function (1)p=p+1Until(all populations Pop p have been visited)if(Pop2 includes a new best feasible solution) thenSet Pop1 = Pop2 {extinct Pop1, migrate Pop2 to Pop1 (duplication)}Modify Pop2 solutions by applying RSR_M {mutate Pop2 individuals, reducing number of routes by one}endifApply RC_M on the best solution {customer reordering}Until(convergence criteria or max number of generations)Feasible solutions for initial populations are first generated using a sequential insertion heuristic in which customers are inserted in random order at randomly chosen insertion positions within routes. This strategy is fast and simple while ensuring unbiased solution generation. The initialization procedure then proceeds as follows:For p = 1..2 do{revisit Pop1 and Pop2}For j = 1..n p doGenerate a new solution S j using the EE_M mutator (defined in Section 2.3.2)Add S j in Pop pend forRemove from Pop p the n p worst individuals using Eval i (equation 1)end forDetermine R min, the minimum number of tours associated with a feasible solution in Pop1 or Pop2.Replicate (if needed) best feasible solution (R min routes) in Pop1.Replace Pop1 individuals with R min-route solutions using the procedure RI(R min).Replace Pop2 members with R min-1 -route solutions using the procedure RI(R min-1).RI(R) is a re-initialization procedure creating an R-route solution. It first generates R one-customer routes formed from randomly selected customers. Then, it uses the insertion procedure proposed by Liu and Shen (1999) to insert at first, as many customers as possible without violating time window constraints. Accordingly, route neighborhoods associated to unvisited customers are repeatedly examined for customer insertion. Introduced by Liu and Shen (1999), this new route-neighborhood structure relates one or multiple routes to individual customers. In our approach the route neighborhood which differs from the one reported by Liu and Shen (1999), is strictly bounded to two tours, comprising routes whose distance separating their centroid from the customer location is minimal. A route centroid corresponds to a virtual site whose coordinates refer to the average position of its specific routed customers. Each feasible customer insertion opportunity is explored over its entir e route neighborhood. The next customer visit is selected by maximizing a so-called regret cost function that accounts for multiple route insertion opportunities:∑∈−=)(*)}()({Cost RegretcRNrccrCrC, (4)where)(cRN=route neighborhood of customer c,)(rC c= minimum insertion cost of customer c within route r,*)(rC c= minimum insertion cost of customer c over its route neighborhood.Remaining unvisited customers (if any) are then inserted in the R -tour solution maximizing an extended insertion regret cost function, in which )(r C c (Liu and Shen, 1999) includes an additional term reflecting temporal constraint violation. This new contribution is given by )(c S r CV (equation 3) where )(c S r refers to the partial solution described by route r in which customer c is inserted.2.2 SELECTIONThe selection process consists in choosing two individuals (parent solutions) within the population for mating purposes. The selection procedure is stochastic and biased toward the best solutions using a roulette-wheel scheme (Goldberg, 1989). In this scheme, the probability t o select an individual is proportional to its fitness value. Individual fitness is computed as follows:Population Pop 1:i n c c i c c i i Viol l b d fitness βα+−+=∑=1},0max{(5)Population Pop 2:i n c c i c c i Viol l b fitness βα+−=∑=1},0max{(6)The notations are the same as in equations (1−3). Better individuals generally (but not necessarily) tend to include short total traveled distance in Pop 1 and satisfy as many temporal constraints as possible in Pop 2.2.3 GENETIC OPERATORSThe proposed genetic operators mostly rely on two basic principles. First, for a given number of tours, an attempt is made to construct feasible solutions with as many customer visits as possible. Second, the remaining customers are inserted within existing routes through temporal constraint relaxation. Constraint violation is used to restrict the total number of routes to a fixed value. The proposed genetic operators incorporate some key features of the best heuristic routing techniques such as Solomon’s10(1987) insertions heuristic I1, large neighborhood search (Shaw, 1998) and the route neighborhood-based two-stage metaheuristic (RNETS) (Liu and Shen, 1999). Details on the recombination and mutation operators used are given in the next sections.2.3.1 RECOMBINATIONTwo recombination operators are considered, namely IB_X(k) and IRN_X(k). The insertion-based IB_X crossover operator creates an offspring by combining, one at a time, k routes of parent solutionP 1 with a subset of customers, formed by nearest-neighbor routes {r2} in parent solution P2. Theneighborhood {r2} includes the routes of P2whose centroid is located within a certain range of r1(centroid). This range corresponds to the average distance separating r1 from the routes defining P2. The k routes ({r1}) are selected either randomly, with a probability proportional to the number of customers characterizing a tour or based on average distance separating consecutive customers over a route. A stochastic remov al procedure is first carried out to remove from r1 customers subject to be migrated to alternate routes. Targeted customers are either selected according to waiting times, distance separating them from their immediate neighbors, or randomly. Then, using a modified insertion heuristic inspired from Solomon (1987) a feasible child tour is constructed, expanding the altered route r1 by inserting customer visit candidates derived from the nearest-neighbor routes {r2} defined earlier. The proposed insertion technique consists in adding a stochastic feature to the standard customer insertion heuristic I1 (Solomon, 1987), by selecting randomly the next customer visit over the three best candidates with a bias toward the best. Once the construction of the child route is completed, and reinsertion is no longer possible, a new route construction cycle is initiated. The overall process isrepeated for the k routes initially selected from P1. Finally, the child inherits the remaining“diminished” routes (if any) of P1. If unrouted customers still remain, additional routes are built using a nearest-neighbor procedure. The whole process is then iterated once more to generate a second child byinterchanging the roles of P1 and P2. Further details of the operator may be found in Berger andBarkaoui (2000). In order to keep the number of routes of a child solution identical to its parents, apost-processing procedure is applied. If the solution has a larger number of tours than expected, it is repeatedly mutated using the RSR_M (Section 2.3.2) procedure to reduce the number of routes. Conversely, for solutions having a smaller number of routes, new feasible routes are constructed repeatedly breaking down the most populated route in two until the targeted number of routes is obtained.IRN_X (k) (insert within route neighborhood) is a variant of the IB_X operator exploiting basic procedures and common features from parent solutions, while constructing offspring differently. IRN_X examines simultaneously rather than separately all customer subsets to be reinserted over a partial solution and, uses an alternate (less myopic) insertion heuristic to generate the complete child, k partial routes r1 are first altered using the same remova l solution. Selected all at once from parent P1procedure presented in IB_X. Then, in contrast to IB_X, the offspring currently described by the kfrom which illegal customer modified partial routes r1 inherits the remaining unaltered routes from P1visits are removed. A customer visit is illegal if i t violates its time window constraint. The removed customers are added to the combination of all customer subsets formed by nearest-neighbor routes {r2} from parent Ptied to the k altered routes r1. Customer subsets are generated as specified in IB_X.2Then, subject to a constraint violation bias, all unrouted customers are reinserted within the child partial routes using the insertion heuristic introduced for the RI re-initialization procedure presented in Section 2.1.2.3.2 MUTATIONA suite of five mutation operators is proposed, namely LNSB_M(d), EE_M, RS_M, RSR_M(I) and RC_M(I). Each mutator is briefly described next.The LNSB_M (d) (large neighborhood search -based) mutation operator relies on the concept of the Large Neighborhood Search (LNS) meth od proposed by Shaw (1998). The LNS consists in exploring the search space by repeatedly removing related customers and reinserting them using constraint-based tree search (constraint programming). Customer relatedness defines a relationship linking twocustomers based upon specific properties (e.g. proximity and/or identical route membership), such that when both customers are considered simultaneously for a visit, they can compete with each other for reinsertion creating new opportunities for solution improvement. Therefore, customers close to one another naturally offer interchange opportunities to improve solution quality. Similarly, solution number of tours is more likely to decrease when customers sharing route membership are removed all together. As stated in Shaw (1998), a set of related customers is first removed. Then, customers violating temporal constraints are taken out as well before proceeding to the reinsertion phase. The proposed customer reinsertion method differs from the procedure proposed by Shaw (1998) in two respects, namely, the insertion cost function used, and the order in which customers are visited for insertion (variable ordering scheme) through the branch -and-bound search process. Insertion cost is defined by the sum of key contributions referring respectively to increased traveled distance, and delayed service time, as prescribed in Solomon’s (1987) procedure I1, as well as to constraint violation (equation (3)). As for customer ordering, customers ({c }) are sorted (CustOrd ) according to a composite ranking, departing from the myopic scheme originally proposed by Shaw. The ranking is defined as an additive combination of two separate rankings, previously achieved over best insertion costs (Rank Cost (c )) on the one hand, and number of feasible insertion positions (Rank |Pos |(c)) on the other hand:))()((c Rank c Rank Sort CustOrd Pos Cost +← (7) The smaller the insertion cost (short total distance, traveled time) and the number of positions (opportunities), the better (smaller) the ranking. The next customer to be visited within the search process is selected according to the following expression:)]([D rand L INTEGER CustOrd customer ← (8) in which L= current number of customers to be inserted,rand = real number over the interval [0,1] (uniform random number generator),D= parameter controlling determinism. If D=1 then selection is purely random (default: D=15).Once a customer is selected, branch-and-bound search is carried out over its different insertion positions, exploiting limited discrepancy search (Harvey and Ginsberg, 1995) as specified in Shaw (1998). However, search tree expansion is achieved using a non-constant discrepancy factor d, selected randomly (uniform probability distribution) over the set {1,2,3}. Despite the implicit contribution of constraint violation in the insertion method, all legal customer visits are first successfully explored. Unvisited customers (if any) are then routed relaxing temporal constraints.The EE_M (edge exchange) and RS_M (repair solution) mutators focus on inter-route improvement. EE_M is inspired from the λ-interchange mechanism of Osman (1993), performing reinsertions of customer sets over two neighboring routes. In the proposed mutation procedure, each customer is explored for reinsertion in alternate neighbor routes. The route neighborhood surrounding a given customer is made up of two routes. Tours are being selected such that the distance separating their centroid from customer location is minimal. Customer exchanges take place as soon as the solution improves, i.e., we use a "first-accept" strategy. A ssuming the notation (x, y) to describe the different sizes of customer sets (λ) issued from the neighboring routes, the current operator explores values running over the range (x=1, y=0,1,2). As for the RS_M mutation operator, it focuses on exchanges involving one illegal customer visit. In that scheme, each illegal visit in a route is exchanged with an alternate legal one or two-customer visit sequence in order to generate a new set of customers with either violated or non-violated temporal constraints. The objective is to further explore the solution space (diversity) while possibly improving quality.The RSR_M (I) (reinsert shortest route) mutation operator eliminates the shortest route of the solution, reducing by one the total number of routes. Customers from the shortest route are first removed. Then, following an iterative process, various solutions are explored, in which unvisited customers are reinserted within existing routes using the insertion heuristic presented for the RI re-。