Influence of cytokinins on in vitro morphogenesis in root cultures of Centaurium erythraea

In?uence of cytokinins on in vitro morphogenesis in root cultures of Centaurium erythraea—Valuable medicinal plant

Angelina Subotic′*,Sla p ana Jevremovic′,Dragoljub Grubisˇic′

Institute for Biological Research‘‘Sinisˇa Stankovic˙’’,Bulevar despota Stefana142,11060Belgrade,Serbia

1.Introduction

Regeneration processes in differentiated or undifferentiated

tissues have attracted the attention for their potential of

application as tools for plant propagation of important plant

species.It is known that the adventitious buds and/or somatic

embryos differ according to a number of endogenous and

exogenous factors.Morphogenetic processes or organ develop-

ment in plants include division,growth and differentiation of cells.

Those processes are genetically determined and regulated by a

number of endogenous and exogenous factors Ezhova(2003).Root

cultures are generally suitable systems from the study and

production of secondary metabolites(Kim et al.,2002;Sudha

et al.,2003).However,they can be used also as model systems in

the research focused on the effect of various substances on

morphogenesis in root culture, e.g.plant growth regulators

(Ba′lva′nyos et al.,2001).

Cytokinins play a fundamental role almost in all plant

developmental process.Some studies showed that synthetic

phenylurea derivates such as TDZ and CPPU have higher cytokinin

activity to induce adventitious buds than adenine-type cytokinins

such kinetin KIN and BA(Huettaman and Preece,1993).It has been

shown that TDZ provided an ef?cient stimulus for the induction of

in vitro shoot regeneration in several plant species(Uchida et al.,

2003;Ipecki and Gozukirmi,2004;Gu and Zhang,2005).

The development and adoption of plant cell culture methods for

a number of medicinal plant species have lead to the production of

useful secondary plant compounds on large scale(Raju et al.,

2004).

Centaurium erythraea is a winter-annual plant of the Gentia-

naceae family,growing throughout Atlantic countries of South

Europe and North Africa.Aerial parts of this plant are a rich source

of bitter secoiridoid glucosides and xanthones and have been put to

numerous uses.In pharmacopoeias of many countries it is known

by the common name Centaury and used as a appetite-stimulating,

digestive,hepatic,febrifugal and tonic(van der Sluis,1985).The

populations of this plant are greatly endangered by collecting for

preparing many preparations like tinctures,decoction,infusion

tonic,lotion,powder,tea etc.C.erythraea inscriptions in the most

red lists of endangered plants.(Holub et al.,1979).Due to

destructive harvesting and lack of proper cultivation,the wild

population of this medicinally important plant has declined very

fast.Therefore,there is an urgent need to develop an appropriate

protocol for mass propagation and conservation of this endangered

Scientia Horticulturae120(2009)386–390

A R T I C L E I N F O

Article history:

Received21February2008

Received in revised form7November2008

Accepted20November2008

Keywords:

Adventitious buds

Cytokinins

Medicinal plant

Organogenesis

Root explant

A B S T R A C T

The purpose of this work was to acquire more information on the capacity of in vitro grown Centaurium

erythraea Gillib.normal and hairy root cultures to simultaneously regenerate adventitious buds and to

evaluate the variations induced on regeneration response by treatments with six cytokinins.Explants

from normal and hairy root cultures were cultured on half-strength MS medium(1/2MS)with kinetin

(KIN),N6-benzylaminopurine(BA),6-g,g-dimethylallylaminopurine(2IP),N-(2-chloro-4-pyridyl)-N0-

phenylurea(CPPU),1-Phenyl-3-(1,2,3-thiadiazol-5-yl)urea(TDZ)and6-[4-Hydroxy-3-methyl-but-2-

enylamino]purine(ZEA),used alone in six different concentrations.The average number of adventitious

buds per explant was promoted by all of cytokinin treatments.Urea-type cytokinins,TDZ and CPPU were

more effective for the induction the morphogenesis of adventitious buds than adenine-type cytokinins.

We found that the1.0m M CPPU induced the largest number(25.6,18.2,respectively)of adventitious

buds in normal and hairy root culture.TDZ-supplemented media induced highest number of

adventitious buds(24.2)from normal root explant,but from hairy root explant average number of buds

is lower(20.5).Regenerated shoots were excised and placed on1/2MS medium without hormone.The

rooted plantlets were successfully acclimatized in greenhouse conditions.

?2008Elsevier B.V.All rights reserved.

*Corresponding author.Tel.:+381112078425;fax:+381112761433.

E-mail address:heroina@ibiss.bg.ac.yu(A.Subotic′).

Contents lists available at ScienceDirect

Scientia Horticulturae

j o u r n a l h o m e p a g e:w w w.e l s e v i e r.c o m/l o c a t e/s c i h o r t i

0304-4238/$–see front matter?2008Elsevier B.V.All rights reserved.

doi:10.1016/j.scienta.2008.11.034

medicinal plant.In general,most reports on induction of organogenesis and/or somatic embryogenesis in C.erythreae involve of leaf,seedlings and callus tissues(Baresˇova,1988).A quick,ef?cient and novel shoot regeneration methodology from root tissue has been developed from C.erythreae.These methods avoid an intermediate callus phase,and may reduce the risk of somaclonal variation.There are only few reports on in vitro morphogenesis from root explants(Vinocur et al.,2000;Chaundhri et al.,2004;Shahzad et al.,2007).Hairy roots,obtained after genetic transformation of host cells by Agrobacterim rhizogenes,are genetically stable and posses high potential to produce the secondary metabolites than the normal roots(Doran,2002).Hairy roots have induced in many plants by transformation with A. rhizogenes Ri T-DNA.This DNA segment carries a set of genes that encode enzymes which control auxin and cytokinin biosynthesis. In this fact,hairy roots are characterized by fast growth,lateral branching and genetic stability on hormone-free medium(Giri and Narasu,2000).On the other hand,normal root cultures need an exogenous plant growth regulators supply and growth very slowly. Also,this balans of plant growth regulators induces different morphogenic responses in hairy root cultures comparing with normal root cultures.Hairy root induction and shoot regeneration from hairy root are important prerequisites for successful production of transgenic plants as well as secondary metabolites using A.rhizogenes(Hu and Du,2006).The advantage of using root cultures in than they growth fast and are relatively easy to prepare and maintain,show a low level of variability and can be easily cloned to produce a large supply of experimental tissue.Previously we have report protocol of A.rhizogenes transformation and regeneration of transgenic plants of C.erythreae(Subotic′et al., 2004).Also,employing the same nutrient medium with addition of different auxins or carbohydrates,Subotic˙et al.(2006)

de?ned

Fig.1.Morphogenetic responses of normal and hairy root culture of C.erythraea on1/2MS medium supplemented with different cytokinins.(A and B)Adventitious buds developed on normal and hairy root explants on medium with3.0m M CPPU,after10and20days in culture,respectively.(C)Hairy root culture with well developed adventitious buds on medium with1.0m M BA.(D)Adventitious buds achieved in normal root culture on medium with3.0m M CPPU.(E)Differentiation of fasciated and distorted buds on medium with3.0m M TDZ.(F)Normal root culture after30days on medium supplemented with3.0m M BA.

A.Subotic′et al./Scientia Horticulturae120(2009)386–390387

some in vitro conditions for development somatic embryos and adventitious buds from normal and hairy root cultures of C. erythreae.

The following investigation was undertaken to study morpho-genic responses of in vitro cultured normal and hairy root explants of C.erythreae as in?uenced by the addition to culture medium six different cytokinins,each at six concentrations.

2.Material and methods

Normal root cultures were established from excised root tips of sterile seedlings in vitro on basal1/2MS medium containing half-strength MS macronutrients,full-strength MS micronutrients and vitamins(Murashige and Skoog,1962),3%(w/v)sucrose,0.7%(w/ v)agar and100mg là1myo-inositol.The pH was adjusted to5.8 prior autoclaving(1148C20min).Hairy root cultures of C. erythreaea were initiated by inoculating of2days explants with A.rhizogenes(strain A4M70GUS)according to procedures described previously(Subotic′et al.,2004).Both,normal and hairy roots were maintained in9cm Petri dishes kept under in photosynthetic photon?ux(50m mol mà2sà1)in16-h light photoperiod at258C?18C on the same hormone-free1/2MS basal medium.They were subcultured every one month by excising15mm apical tips and transferring them to fresh medium.Six cytokinins (KIN,BA,2iP,TDZ,CPPU,ZEA),were compared at6concentrations (0.01;0.03;0.1;0.3;1.0;3.0m M).Growth regulator solutions were introduced into1/2MS medium before autoclaving.ZEA solution was ?lter-sterilized(0.22m m,Milipore),then added to autoclaved culture medium after cooling to558C.Ten root explants were plated per Petri dish containing20ml of a gelled medium.All cultures were grown in growth chamber under16-h photoperiod,with photon?ux density 50m mol mà2sà1(white?uorescent Tesla tubes,Pancevo,Serbia, 60W).At the end of each subculturing period,the average numbers of adventitious shoots were recorded.Each treatment consisted of15 replications(Petri dishes containing15ml medium and10explants).

Statistical analyses were performed using StatGra?cs software version4.2(STSC Inc.,Rockville,MD,USA).Data were subjected to analysis of variance(ANOVA)and comparisons between the mean values of treatments were made by the least signi?cant difference (LSD)test calculated at the con?dence level of p0.05.

3.Results

Small outgrowths were induced at the surface of root explants after7–10days of culture on the medium containing any of the cytokinins tested.They had the capacity to simultaneously regenerate adventitious buds throughout the whole culture period on all media tested(Fig.1A and B).Treatment difference for the number of adventitious buds per explant was noticed within30 days of culture initiation(Tables1and2).

Effect additions of all tested cytokinins supplemented singly on induction of adventitious buds were studied in normal root explants(Table1).An average number of25.61buds per explant was produced on3m M CPPU supplemented medium(Fig.1D), while24.22buds were induced with1m M TDZ.These numbers of buds were much more compared with number of buds induced on medium supplemented with adenine-type cytokinins—KIN (15.43),2iP(14.01)and BA(12.63),(Fig.1F).Addition of ZEA induced fewer adventitious buds(6.21)on normal root explant compared with other cytokinins.

On other hand,in hairy root culture an average number of19.62 buds per explant was produced on1m M BA supplemented medium(Fig.1C),while18.22buds were induced with3m M CPPU.An average number16.75,15.83and13.08of adventitious buds per hairy root explant were produced on1m M TDZ,KIN and 2iP,respectively.Addition of1m M ZEA alone induced more lees adventitious buds7.42in hairy root culture(Table2).Results clearly showed that CPPU and TDZ induced high frequency of shoot regeneration.However,the cultures continuously grown on3m M TDZ containing media resulted in the formation of fasciated and distorted buds(Fig.1E).

Adventitious buds from the different treatments,were excised and transferred to a1/2MS medium,continued to growth and spontaneously developed roots(Fig.2A).All the plants transferred to the greenhouse showed a high homogeneity without obvious morphological evidence of somaclonal variation(Fig.2B).

4.Discussion

In this study,we investigated the effects of six cytokinins(KIN, BA,2iP,CPPU,TDZ,ZEA)on the induction and development of adventitious buds in normal and hairy roots of C.erythraea.We have found that the urea-type cytokinin such CPPU and TDZ were more effective than adenine-type cytokinin for the induction of adventitious buds in normal and hairy roots of C.erythraea. Especially,CPPU was more effective than TDZ for adventitious buds formation and development of shoots with normal morphology.A maximal effect of CPPU on the induction of adventitious buds in Centaury normal root culture can be achieved with 3.0m M concentration.The effect of CPPU on expressing embryogenic and organogenic potentials in culture in Table1

The in?uence of cytokinins on adventitious buds formation from normal root cultures of Centaurium erythreae after3weeks of culture.Values represent mean?SE.Means followed by the same letters within columns are not signi?cantly different according to LSD test at p0.05probability level.(–)No value. Cytokinins(m M)

BA CPPU2iP KIN TDZ ZEA Average number

of shoots

–––––– 3.08?0.45

0.01–––––0.13?0.03abc 0.03–––––0.32?0.05abc 0.1–––––0.59?0.69abc

0.3––––– 2.03?0.23abc

1.0–––––1

2.45?0.59abc

3.0–––––12.63?0.63abc –0.01––––0.61?0.07de

–0.03–––– 3.23?0.33de –0.1–––– 4.23?0.33de –0.3––––16.72?0.42de – 1.0––––17.55?0.36de – 3.0––––25.61?0.53de ––0.01––– 3.01?0.31cde ––0.03–––7.13?0.41cde ––0.1–––11.21?0.61cde ––0.3–––14.01?0.58cde –– 1.0–––13.03?0.61cde –– 3.0–––11.82?0.68cde –––0.01––0.46?0.08ab –––0.03–– 2.03?0.22ab –––0.1–– 1.45?0.17ab –––0.3–– 2.4?0.23ab ––– 1.0–– 4.57?0.32ab ––– 3.0––15.43?0.59ab ––––0.01– 2.09?0.15e

––––0.03–7.98?0.34e

––––0.1–11.69?0.31e

––––0.3–18.18?0.36e

–––– 1.0–24.22?0.45e

–––– 3.0–14.70?0.34e

–––––0.010.54?0.05ab –––––0.030.82?0.09ab –––––0.1 1.92?0.19ab –––––0.3 2.69?0.16ab ––––– 1.0 4.82?0.22ab ––––– 3.0 6.21?0.28ab

A.Subotic′et al./Scientia Horticulturae120(2009)386–390 388

vitro has been studied for a small number of species:Vitis vinifera (Nakano et al.,1997),blackberry(Millan-Menndoza and Graham, 1999),Lavandula vera(Tsuro et al.,2000)and lemon(Fiore et al., 2002).Exogenous CPPU was found in those studies to have a broad morphogenetic effect as it induces callus formation as well as organogenesis and somatic embryogenesis.In lemon,CPPU induces somatic embryogenesis within a concentration range of 0.4–12.0m M.A highest number of adventitious buds is obtained in the presence of4.0m M CPPU in culture.In our work,the application of TDZ,a compound with high cytokinin-like activity, determined a increase in production of adventitious buds.These results are similar with data obtained by D‘Onofrio and Morini (2005)who observed that the application of TDZ determined increase in the production of adventitious shoots in leaf culture of Cydonia oblongata.The high stability of urea-type cytokinin like TDZ due to its resistance to cytokinin oxidase might to be a reason for its ef?cacy(Mok et al.,1987).Another possible reason for the greater effect of TDZ could be substitutive activity for both auxin and cytokinin required for shoot formation(Visser et al.,1992).In our study the cultures grown on high concentrations of TDZ containing media formed fasciated and distorted shoots.Similar deleterious effect of TDZ on development and growth of Cicer arieinum(Murthy et al.,1996),Rauvol?a tetraphyla(Faisal et al., 2005)and Capsicum annum(Ahmad et al.,2006)has been reported.The other tested cytokinins in normal root culture can be ranked on scale of increasing biological activity,from BA to KIN to2iP to ZEA.Of the six cytokinins tested in hairy root culture, BA was most effective in induction morphogenesis in vitro.The stimulating effect of BA on shoots formation was previously reported for some medicinal species Vitex trifolia(Hiregoudar et al.,2006)and Sesbania drummondii(Cheepala et al.,2004). Differences regarding the effect of different cytokinins on the induction and number of adventitious buds may result from their different activities,i.e.different tissue sensitivities to the adenine and urea type of cytokinins(Mok and Mok,2001).Also,differences in the activity of cytokinins can be explained by their various translocation rates to meristematic regions and metabolic processes,in which the cytokinins may be degraded and conjugated with physiologically inert compounds,like sugars or amino acid(Kaminek,1992).Our results demonstrated the different effect of cytokinins on induction of adventitious shoots from normal and hairy root culture of C.erythreae.In our preliminary study,we have established ef?cient protocol for spontaneous plant regeneration in normal and hairy root culture of C.erythraea.Several clones of hairy root were selected for this study based on differences in their morphological potential for induction of adventitious buds.Hairy root clone used in our study cannot regenerate adventitious shoots on hormone-free medium. Adventitious shoots were regenerated from normal roots in the same conditions(Subotic′et al.,2004).Usually hairy root cultures are more ef?cient than normal root cultures,because of their genetic and biochemical stability.Cultures of normal roots would

Table2

The in?uence of cytokinins on adventitious buds formation from hairy of Centaurium erythreae.Values represent mean?SE.Means followed by the same letters within columns are not signi?cantly different according to LSD test at p0.05 probability level.

Cytokinins(m M)

BA CPPU2iP KIN TDZ ZEA Average number

of shoots

0.01–––––0.48?0.06ab 0.03–––––0.83?0.11ab 0.1––––– 3.93?0.21ab

0.3––––– 6.61?0.39ab

1.0–––––19.62?0.62ab 3.0–––––1

2.46?1.23ab –0.01––––0.53?0.08b

–0.03––––0.71?0.09b

–0.1–––– 1.66?0.13b

–0.3–––– 6.93?0.36b

– 1.0––––17.89?0.51b

– 3.0––––18.22?0.65b

––0.01––– 1.93?0.12bc ––0.03––– 3.43?0.25bc ––0.1–––7.03?0.29bc ––0.3–––11.59?0.34bc –– 1.0–––13.08?0.51bc –– 3.0–––12.48?0.42bc –––0.01––0.27?0.04ab –––0.03––0.50?0.06ab –––0.1–– 2.10?0.12ab –––0.3–– 4.19?0.33ab ––– 1.0––15.83?0.73ab ––– 3.0–– 5.91?0.38ab ––––0.01– 1.69?0.13bc ––––0.03– 4.87?0.23bc ––––0.1–11.80?0.39bc ––––0.3–11.43?0.34bc –––– 1.0–16.75?0.59bc –––– 3.0–16.16?0.45bc –––––0.01 1.91?0.09a

–––––0.03 2.02?0.11a

–––––0.1 3.48?0.18a

–––––0.3 6.12?0.26a

––––– 1.07.42?0.36a

––––– 3.07.31?0.31

a Fig.2.Development whole plants from adventitious shoots of C.erythraea(A) rooted plant on1/2MS medium without hormone;(B)potted in vitro plantlets in greenhouse condition.

A.Subotic′et al./Scientia Horticulturae120(2009)386–390389

be bene?cial for the production of active secondary metabolite. However normal root cultures are an alternative method for many plants which are recalcitrant to A.rhizogenes infection(Murthy et al.,2008).

5.Conclusions

An ef?cient and reproducible protocol for high frequency shoots regeneration in normal and hairy root culture of C.erythraea.Our results demonstrated that type and cytokinin concentrations, determine the morphogenesis potential of different root explants. This has considerable practical signi?cance and has been success-fully used to develop a rapid plant production system for this important medical plant.The majority of regenerated plants did not exhibit any visible variations in morphology or growth characteristics compared with plants from wild populations.This is new in vitro propagation methods,requires only root material for plant regeneration,leaving the entire commercially mass produc-tion secondary metabolites from these regenerants.

Acknowledgement

This research was supported by the Serbian Ministry of Science (Grant No.143026B).

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A.Subotic′et al./Scientia Horticulturae120(2009)386–390 390

英语介词口诀

英语介词用法口诀 早、午、晚要用in，at黎明、午夜、点与分。 年、月、年月、季节、周，阳光、灯、影、衣、冒in。 将来时态in...以后，小处at大处in。 有形with无形by，语言、单位、材料in。 特征、方面与方式，心情成语惯用in。 介词at和to表方向，攻击、位置、恶、善分。 日子、日期、年月日，星期加上早、午、晚， 收音、农场、值日on，关于、基础、靠、著论。 着、罢、出售、偷、公、假，故意、支付、相反，准。 特定时日和"一……就"，on后常接动名词。 年、月、日加早、午、晚，of之前on代in。 步行、驴、马、玩笑on，cab，carriage则用in。 at山脚、门口、在当前，速、温、日落、价、核心。 工具、和、同随with，具有、独立、就、原因。 就……来说宾译主，对、有、方状、表细分。 海、陆、空、车、偶、被by，单数、人类know to man。 this、that、tomorrow，yesterday，next、last、one。 接年、月、季、星期、周，介词省略已习惯。 over、under正上下，above、below则不然， 若与数量词连用，混合使用亦无关。' beyond超出、无、不能，against靠着，对与反。 besides，except分内外，among之内along沿。 同类比较except，加for异类记心间。 原状because of,、owing to、due to表语形容词 under后接修、建中，of、from物、化分。 before、after表一点, ago、later表一段。 before能接完成时，ago过去极有限。 since以来during间，since时态多变换。 与之相比beside，除了last but one。 复不定for、找、价、原，对、给、段、去、为、作、赞。 快到、对、向towards，工、学、军、城、北、上、南。 but for否定用虚拟，复合介词待后言。 ing型由于鉴，除了除外与包合。 之后、关于、在......方面，有关介词须记全。 in内to外表位置，山、水、国界to在前。

(完整word版)介词at,in与on的用法与区别

at, in与on的用法 一、表示时间，注意以下用法： ①表示时间的某一点、某一时刻或年龄等用at。如： I get up at six in the morning. 我早上六点钟起床。 He got married at the age of 25. 他25 岁结婚。 ②泛指一般意义的上午、下午或晚上以及月或年等较长的时间，一般用in。如： We watch TV in the evening. 我们晚上看电视。 He went to Japan in 1946. 他于1946 去了日本。 ③若表示星期几或某一特定的日期，则用on。如： He left here on the fifth of May. 他于5 月5 日离开这儿。 二、表示地点、场所、位置等，注意以下用法： ①表示某一点位置，用at。如： We live at No 87 Beijing Road. 我们住在北京路87 号。 The hospital is at the end of the street. 医院在这条街的尽头。 与名词所有格连用表示地点，也用at。如： at my sister's 在我姐姐家at the doctor's 在医务室 ②表示空间或范围，用in。如： What's in the box? 这盒子里有什么? He lives in Paris with his wife. 他同他妻子住在巴黎。 但有时两者可换用。如： The meeting was held at [in] the hotel. 会议在宾馆举行。 ③at 与in 的另一个区别是：at 用于指较小的地方，而in 用于指较大的地方。如： in Shanghai 在上海at the station 在车站 但是，大与小是相对的，有时随着说话者的着眼点不同，大地方也可能用at （比如把一个大地方看作一个点时）。如： Our plane refuelled at London. 我们的飞机在伦敦加油。 We stopped for an hour at Moscow on our way to Paris. 我们在去巴黎的途中在莫斯科停了 1 个小时。 ④介词on 用于地点，主要指在某物的表面。如： What's on the table? 桌上有什么?

浅析《诗经》中的方位词“南”及其文化内涵

龙源期刊网 https://www.wendangku.net/doc/7411537292.html, 浅析《诗经》中的方位词“南”及其文化内涵作者：王春艳 来源：《汉字文化(教育科研卷)》2018年第07期 【提要】本文就《诗经》中出现频率较多的方位词“南”进行辨析，并对具有代表性的“南方”“南亩”“南山”进行具体分析，由此我们可知“南”这一方位词，有着温暖和煦、丰年祈福之愿，有着少妇思君、忧心忡忡之情。随着对“南”与长养、情思、尊愿等方面的分析，可见方位词“南”的文化内涵不断深化并影响深远。通过对方位词“南”的探析，我们可以加深对《诗经》的认识，从而揭开遥远先秦的神秘面纱。 【关键词】诗经方位词“南” 文化内涵 《诗经》作为先秦人民真实的生活写照，其所包含的先民思想、风俗习惯、地理方位等都是我们了解先秦人民生活风貌的重要载体。关于《诗经》中方位词“南”，此前已有学者对此进行研究，譬如张德鑫的《方位词的文化考察》，方经民的《汉语空间方位参照的认知结构经过统计》，金安辉的《诗经》中的“东、西、南、北”及其对后世诗歌的影响等，笔者在前人研究的基础上，旨在更加具体、深刻地分析方位词“南”及其文化内涵。据统计，《诗经》中“南”出现了81次，那么《诗经》中的“南”是仅指方向的方位词呢，还是有着特殊的文化内涵？文化内涵也可以说是文化义，周一农的《词汇的文化蕴涵》中指出“文化义，指的是词在特定文化背景下所获得的反映一个名族风俗习惯、文化背景、宗教信仰和思维方式等诸多文化因素的隐含义”。①因此方位词的文化内涵就是对这些文化因素的分析。下面我们将对《诗经》中的方位词“南”进行逐步探讨，从而还其完整面貌。 一、“南”与长养 万事万物皆有源头，那么方位词源于何处？先秦人民不像现代人一样可以用多种方式指认方向，他们最先感受到的是自身和空间的环境关系，“日出而作，日落而归”，四方就是根据太阳这一参照物来确定的。即太阳升起处定为东方，落下处定为西方，直照处定为南方，背阳处定为北方。这就是人们对方位最初的认识。而我们现在所说的方位词，更加术语化，其实还是指东、西、南、北等。 经统计，我们发现《诗经》中“南”多有表示南方的意思，《说文解字》：“南，木至南方而有枝任也”，即南方是夏天草木繁盛的象征。《诗经》中《周南·樛木》“南有樛木，葛藟累之”；《周南·漢广》“南有乔木，不可休思”；《召南·草虫》“陟彼南山，言采其蕨”这些例子不仅表明这些草木生长在南方，也进一步表明了南方的“樛木”“乔木”“蕨”是何其丰茂，这正如《白虎通·五行》中“南方主长养”的说法，又《黄帝内经·素问·异法方宜论》：“南方，天地所长养，阳之所盛处也”，所以南方是温暖和煦的，也是植物生长的好地方，是生命的象征。

介词in,on,to表示方位的用法

介词in，on，to 表示方位的用法 介词 in，on，to 都可以用来表示某个位置的方向，它们的意义不同，故表示的方向及范围也不同： 1. in 表示方位，含义是“在……之内”，即一个小地方处在一个大地方的范围(疆域)之内。例如： China is in the east of Asia. 中国在亚洲东部。(中国是亚洲的一个国家，处于亚洲的范围之内) Guilin is in the north of Guangxi. 桂林在广西北部。(桂林是广西的一座城市) Taiwan lies in the east of China. 台湾在中国的东部。(台湾是中国东部的一个省份，是中国的领土，在中国的疆域之内) Shanghai lies in the east of China. 上海位于中国的东部。(上海是中国的一个行政区域，在中国的疆域之内) The plant can be seen only in the north of Canada. 那种植物只有在加拿大北部才看得到。(暗指这种植物只生长在加拿大北部地区) The sun rises in the east and sets in the west. 太阳东升西落。 说明：表示某个地方的地理位置时，be，lie 以及 be located 的意义是一样的，可以互换使用。 2. on 表示方位，含义是“在……端/边”，即一个地方在另一个地方的某一端或某一边，两个地方只是相邻或接壤，却互不管辖。例如： Guangdong Province is on the southeast of Guangxi. 广东省在广西的东南边。(广东省与广西在地理位置上是连在一起的，即两者相邻，却互不管辖) China faces the Pacific on the east. 中国东临太平洋。(中国与太平洋相邻) The country is bounded on the west by the sea.那个国家西边与海接界。(暗指该国为沿海国家) Sichuan Province is on the north of Guizhou Province. 四川省在贵州省的北边。(四川省与贵州省在地理上也是连在一起的，但互不管辖) 3. to 表示方位，含义是“在……面”，即一个地方在另一个地方的范围之外，互不管辖。尤其当两个地方相隔较远，且有湖泊、大海等区域相隔时，通常用 to。例如： Japan is to the east of China. 日本在中国的东面。(日本在中国范围之外，且有日本海分隔) Taiwan is to the southeast of Fujian Province. 台湾在福建省的东南面。(台湾在福建省的范围之外，且两者之间有台湾海峡分隔) Jinzhou is to the west of Shenyang. 锦州在沈阳的西面。(锦州和沈阳分别为两座城市，地理位置上互不

in on at的时间用法和地点用法 完全版

in，on，at的时间用法和地点用法 一、in, on, at的时间用法 ①固定短语： in the morning/afternoon/evening在早晨/下午/傍晚, at noon/night在中午/夜晚, （不强调范围，强调的话用during the night） early in the morning=in the early morning在大清早， late at night在深夜 on the weekend在周末（英式用at the weekend在周末，at weekends每逢周末） on weekdays/weekends在工作日/周末， on school days/nights在上学日/上学的当天晚上， ②不加介词 this, that, last, next, every, one, yesterday, today, tomorrow, tonight，all，most等之前一般不加介词。如， this morning 今天早晨 （on）that day在那天（that day更常用些） last week上周 next year明年 the next month第二个月（以过去为起点的第二个月，next month以现在为起点的下个月） every day每天 one morning一天早晨 yesterday afternoon昨天下午 tomorrow morning明天早晨 all day/morning/night整天/整个早晨/整晚（等于the whole day/morning/night） most of the time （在）大多数时间 ③一般规则 除了前两点特殊用法之外，其他≤一天，用on，＞一天用in，在具体时刻或在某时用at（不强调时间范围） 关于on 生日、on my ninth birthday在我九岁生日那天 节日、on Teachers’Day在教师节 （注意：节日里有表人的词汇先复数再加s’所有格，如on Children’s Day, on Women’s Day, on Teachers Day有四个节日强调单数之意思，on Mother’s Day, on Father’s Day, on April Fool’s Day, on Valenti Day） 星期、on Sunday在周日，on Sunday morning在周日早晨 on the last Friday of each month 在每个月的最后一个星期五 日期、on June 2nd在六月二日 on the second (of June 2nd) 在六月的第二天即在六月二日 on the morning of June 2nd在六月二日的早晨，on a rainy morning在一个多雨的早晨 on a certain day 在某天 on the second day在第二天（以过去某天为参照） 注意：on Sunday在周日，on Sundays每逢周日（用复数表每逢之意），every Sunday每个周日，基本一个意思。 on a school day 在某个上学日，on school days每逢上学日。on the weekend在周末，on weekends每逢 周末。 关于in in June在六月 in June, 2010在2010年六月

介词用法口诀

介词用法口诀： 早、牛、晚要用in at黎明、午夜、点与分 年、月、年月、季节、周，阳光、灯、影、衣、帽in 将来时态in表以后，小处at大处in 有形with无形by 语言、单位、材展in 早、午、晚要用in in the morning 在早上 in the afternoon 在下午 in the evening 在晚上 in the day在白天 at黎明、午、夜、点与分 at dawn, at daybreak^ 黎明时候 at noon在中午at night在夜间 at midnight 在午夜 at six o'clock 在6 点钟 at 7:30 （seven thirty）在7 点半 at the weeken血周末 年、月、年月、季节、周都用in in 1986 在1986 年 in March在三月 in July, 1983 1983 年7 月i n spring在春季 in the third week 在第三周 阳光、灯、影（树荫）、衣、冒（雨）in Don't read in dim light.切勿在暗淡的灯光下看书。 They are sitting in the shade of a tree.他们坐在树阴下乘凉。 He went in the rain to meet me at the station. 他冒雨到车站去接我。 the woman in white （black, red, yellow）穿着白（黑、红、黄）色衣服的妇女in uniform 穿着制服 将来时态in表以后 They will come back in 10 days 他们将10 天以后回来。

介词atin与on的用法与区别

at,in与on的用法 一、表示时间，注意以下用法： ①表示时间的某一点、某一时刻或年龄等用at。如： Igetupatsixinthemorning.我早上六点钟起床。 Hegotmarriedattheageof25.他25岁结婚。 ②泛指一般意义的上午、下午或晚上以及月或年等较长的时间，一般用in。如： WewatchTVintheevening.我们晚上看电视。 HewenttoJapanin1946.他于1946去了日本。 ③若表示星期几或某一特定的日期，则用on。如： HelefthereonthefifthofMay.他于5 月 5日离开这儿。 二、表示地点、场所、位置等，注意以下用法： ①表示某一点位置，用at。如： WeliveatNo87BeijingRoad.我们住在北京路87号。 Thehospitalisattheendofthestreet.医院在这条街的尽头。 与名词所有格连用表示地点，也用at。如： atmysister's在我姐姐家atthedoctor's在医务室 ②表示空间或范围，用in。如： What'sinthebox?这盒子里有什么? HelivesinPariswithhiswife.他同他妻子住在巴黎。 但有时两者可换用。如： Themeetingwasheldat[in]thehotel.会议在宾馆举行。 ③at与in的另一个区别是：at用于指较小的地方，而in用于指较大的地方。如： inShanghai在上海atthestation在车站 但是，大与小是相对的，有时随着说话者的着眼点不同，大地方也可能用at（比如把一个大地方看作一个点时）。如： OurplanerefuelledatLondon.我们的飞机在伦敦加油。 WestoppedforanhouratMoscowonourwaytoParis.我们在去巴黎的途中在莫斯科停了1个小时。 ④介词on用于地点，主要指在某物的表面。如： What'sonthetable?桌上有什么? There'sawalletlyingontheground.地上有个钱包。 【注】在少数搭配中，也用介词on。如： Heworksonafarm.他在农场工作。 三、在某些搭配中，三者的区别与英国英语和美国英语有关：

In on at 时间用法及练习

In\ on\ at (time) at 用在具体某一时刻eg at 11:00 at 4:30 在节假日的全部日子里at Christmas 习惯用法at noon at weekends\ at the weekend at night at breakfast\lunch\supper on 具体到某一天；某一天的早晨，中午或晚上on May the first on Sunday morning 对具体某一天的早晨，中午，晚上进行详细的描述on a sunny morning on a windy night 节日的当天；星期on Women?s Day on Monday In 用在年；月；季节in spring in 2012 in August 后面+一段时间表示将来时in two days 习惯用法in the morning\in the afternoon\in the evening “\”以this, that, last, next, some, every, one, any,all开始的时间副词之前的at\on\in 省略在today, tomorrow, yesterday, the day after tomorrow, tomorrow morning，yesterday afternoon，the day before yesterday 之前的介词必须省略 Practice ___ summer ____ 2012 ____ supper ___ 4:00 ___ June the first ___yesterday morning ____ New Year?s Day ___ Women?s Day ___ the morning ____ the morning of July the first ____ 2014 ___ tomorrow morning ____ midnight 1.—What are you doing ____ Sunday? And what is your wife doing ___ the weekend? 2. He?ll see you ____ Monday. And he…ll see your brother ____next Monday. 3. They often go out ___ the evenings. But they don?t go out ____ Sunday evenings. 4. Do you work ____ Fridays? Does she work _____ every Friday? 5. They usually have a long holiday ___ summer. But their son can only have a short holiday ___ Christmas. 6. Paul got married ___ 2010, He got married ___ 9 o?clock ___ 19 May 2010. His brother got married ___ May, 2011. His sister is getting married ___ this year. 1.—When will Mr Black come to Beijing? ---_______ September 5 A. on B. to C. at D. in 2. The twins were born ____ a Friday evening. A. on B. of C. at D. in 3. It?s the best time to plant ____ spring. A. on B. in C. at D.\ 4. ____ the age of twelve, Edison began selling newspaper on train. A. On B. At C. In D.By 5. She has been an English teacher ____ 2000. A. for B. since C. in D.on 6.I have studied English _____ 2003. A. since B. for C. from D.in

at,in与on的用法区别

at, in与on的用法区别 1. 表示时间，注意以下用法： ①表示时间的某一点、某一时刻或年龄等用at： I get up at six in the morning. 我早上六点钟起床。 He got married at the age of 25. 他25 岁结婚。 ②泛指一般意义的上午、下午或晚上以及月或年等较长的时间，一般用in：We watch TV in the evening. 我们晚上看电视。 He went to Japan in 1946. 他于1946 去了日本。 ③若表示星期几或某一特定的日期，则用on： He left here on the fifth of May. 他于5 月5 日离开这儿。 2. 表示地点、场所、位置等，注意以下用法： ①表示某一点位置，用at： We live at No 87 Beijing Road. 我们住在北京路87 号。 The hospital is at the end of the street. 医院在这条街的尽头。 与名词所有格连用表示地点，也用at。如： at my sister’s 在我姐姐家 at the doctor’s 在医务室 ②表示空间或范围，用in： What’s in the box? 这盒子里有什么? He lives in Paris with his wife. 他同他妻子住在巴黎。 但有时两者可换用：

The meeting was held at [in] the hotel. 会议在宾馆举行。 ③at与in的另一个区别是：at多用于指较小的地方，而in多用于指较大的地方：in Shanghai 在上海at the station 在车站 ④介词on 用于地点，主要指在某物的表面： What’s on the table? 桌上有什么? There’s a wallet lying on the ground. 地上有个钱包。 【注】在少数搭配中，也用介词on： He works on a farm. 他在农场工作。 3. 在某些搭配中，三者的区别与英国英语和美国英语有关： in the street (英) / on the street (美) 在街上 in the road (英) / on the road (美) 在路上 in the team (英) / on the team (美) 在这个队 at the weekend (英) / on the weekend (美) 在周末 at weekends (英) / on weekends (美) 在周末 4. 有时三者的差别与搭配习惯和用法有关： in bed / on the bed 在床上 in the tree (多指树外之物) / on the tree (多指树本身之物) 在树上

表示方位的介词用法

Montrer et situer les objets 1.Les mots pour situer les objets. a.contre对着，朝着 b. sur 在…上空 c. a droite (de)在…右边 d. a gauche (de)在…左边 e. sous 在…下面 f. dans在…里面 g. a cote (de)在…旁边h. derriere在…后面i. devant 在…前面 j. au-dessus 在…上方k. au-dessous 在…下方l. entre 在…中间 m. en face de 在…对面n. au bout de 在…尽头o. pres de 在…附近 2.Les mots Cles. a.Vous arrivez 您到达 b. vous prenez 您乘 c. vous allez 您去 d. passez您经过 e. continuez 继续走 f. traversez 穿过 g. tournez 转弯h. entrez走进…i. tout droit 直走 https://www.wendangku.net/doc/7411537292.html, rue 那条路k. par la从那边 3.Transport. a. A pied 走路 b. avelo骑自行车 c. a moto 骑摩托车 d. en rollers滑旱冰 e. en bus 乘公交车 f. en metro乘地铁 g. en voiture 乘汽车h. en taxi乘出租车 -Comment y aller ? -C’est loin. -?a se trouve près d’ici. -C’est situé à 20km de Paris. -Je suis perdue. Je cherche la rueBotonnet pour aller.....vous pouvez m’indiquer....par où il faut passer pour aller à -C’est de quel c?té? Dans quelle direction ? -Continuez jusqu’à un café qui est au bout de la rue. -Vous suivez la Seine. -Vous traversez la rue et passez devant la poste. -C’est à 5 min à pied . -Vous prenez la première à gauche, tout droit.

2时间介词in,on,at的用法

介词in on at 表示时间的用法及区别 Step1 Teaching Aims 教学生掌握时间介词in,on和at的区别及用法。 Step2 Teaching Key and Difficult Points 教学生掌握时间介词in,on和at的区别及用法。 Step3 Teaching Procedures 1.用in的场合后所接的都是较长时间 (1)表示“在某世纪/某年代/特定世纪某年代/年/季节/月”这个含义时，须用介词in Eg: This machine was invented in the eighteenth century. 这台机器是在18世纪发明的。 、 She came to this city in 1980. 他于1980年来到这个城市。 It often rains here in summer. 夏天这里常常下雨。 (2)表示“从现在起一段时间以后”时，须用介词in。(in+段时间表将来) Eg: They will go to see you in a week. 他们将在一周后去看望你。

I will be back in a month. 我将在一个月后回来。 (3)泛指一般意义的上、下午、晚上用in, in the morning / evening / afternoon Eg: They sometimes play games in the afternoon. 他们有时在下午做游戏。 Don't watch TV too much in the evening. 晚上看电视不要太多。（4）A. 当morning, evening, afternoon被of短语修饰，习惯上应用on, 而不用in. Eg: on the afternoon of August 1st & B. 但若前面的修饰词是early, late时，虽有of短语修饰，习惯上应用in, 而不用on. Eg: in the early morning of September 10th 在9月10的清晨； Early in the morning of National Day, I got up to catch the first bus to the zoo. 国庆节一清早，我便起床去赶到动物园的第一班公共汽车。 2.用on的场合后所接的时间多与日期有关 （1）表示“在具体的某一天”或（在具体的某一天的）早上、中午、晚上”，或“在某一天或某一天的上午，下午，晚上”等，须用介

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昆明大观楼长联的语言文化内涵探析

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英语方位词

英语方位词的用法 (一)in the east 与 on the east的区别 1．in the east表示我们生活中和地理位置上的绝对方向。如： The sun rises in the east and sets in the west. 太阳从东边升起，从西边落下。 The Great Wall begins in the east from the Shanghaiguan Pass and ends at the Jiayu guan Pass in the west. 长城东起山海关，西至嘉峪关。 2．on the east表示某事物位于另一事物所朝的方向。这里的方向是相对而言的。如： China faces the Pacific on the east. 中国东临太平洋。 The United States faces the Atlantic on the east and the Pacific on the west. 美国东临大西洋，西濒太平洋。 (二) in (to，on，at) the east of 1．要表示A在B的东部，即：A在B的范围之内时就用"A is in the east of B"，如： Japan is in the east of Asia.日本在亚洲东部。 Italy is in the south of Europe.意大利在欧洲南部。 2．如果A在B的东方，即：A在B的范围之外，且相隔有一定的距离，就用"A lies to the east of B".口语中有时可将to the省去。如： Japan lies (to the) east of China.日本位于中国东方。 France lies (to the) east of England.法国位于英国东方。 3．如果A在B的东边(侧)，即：A与B相邻接。就用"A is on the east of B". 如： Guangdong is on the south of Hunan.广东在湖南南边。

介词in-on-at在表示时间时的用法

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at 表示某个具体时刻。 at eight o’clock 在8点钟 at this time of the year 在一年中的这个时候 at the moment 在那一时刻 at that time 在那时 注意：在英语中，如果时间名词前用this, last, next 等修饰时，像这样的表示，“在某时”的时间短语前，并不需要任何介词。 例如：last month, last week, this year, this week, next year, the next day, the next year 等。 1．What’s the weather like in spring/summer/autumn/winter in your country? 你们国家春天/夏天/秋天/冬天的天气怎么样？ in 在年、月、周较长时间内 in a week 在里面 in the room 用某种语言 in English 穿着 in red on 某日、某日的上下午on Sunday afternoon 在……上面 on the desk 靠吃……为生live on rice 关于 a book on Physics 〔误〕We got to the top of the mountain in daybreak. 〔正〕We got to the top of the mountain at day break. 〔析〕at用于具体时刻之前，如：sunrise, midday, noon, sunset, midnight, night。〔误〕Don't sleep at daytime 〔正〕Don't sleep in daytime. 〔析〕in 要用于较长的一段时间之内，如：in the morning / afternoon, 或in the week / month / year. 或in spring / supper /autumn / winter等等。 〔误〕We visited the old man in Sunday afternoon. 〔正〕We visited the old man on Sunday afternoon. 〔析〕in the morning, in the afternoon 如果在这两个短语中加入任何修饰词其前面的介