Carbanions and Base-catalyzed Formation of Caron

Synthesis,measurements,and theoretical analysis of carbazole derivativeswith high-triplet-energyJianli Li a,Xiaoyun Mi a,Yuchun Wan a,Zhenjun Si a,b,n,Haiying Sun a,Qian Duan a,Xingquan He c,Dong Yan a,Sha Wan aa School of Materials Science and Engineering,Changchun University of Science and Technology,Changchun130022,PR Chinab Institute of Organic Chemistry&Biochemistry,Czech Academy of Science,Praha616610,Czech Republicc School of Chemistry&Environmental Engineering,Changchun University of Science and Technology,Changchun130022,PR Chinaa r t i c l e i n f oArticle history:Received24September2011Received in revised form11December2011Accepted21December2011Available online31December2011Keywords:SynthesisCrystal structureCarbazolePhosphorescenceTheoretical analysesa b s t r a c tIn order to obtain the blue light-emitting organic materials with high triplet state energy,two3,5-diphenyl-4H-1,2,4-triazole(Tz)containing carbazole(Cz)derivatives of9-(4-(3,5-diphenyl-4H-1,2,4-triazol-4-yl)phenyl)-9H-carbazole(TzCz1)and3,6-di-tert-butyl-9-(4-(3,5-diphenyl-4H-1,2,4-triazol-4-yl)phenyl)-9H-carbazole(TzCz2)are synthesized using Cz acting as the starting material,as well as characterized by the1H NMR spectra,ultraviolet–visible(UV–vis)absorption spectra,and theIR absorption spectra.The luminescence quantum yields(LQYs)of TzCz1and TzCz2are measured inCH2Cl2solution to be32.1%and47.5%,respectively.The electrochemical analysis and the photophysicalmeasurements suggest that the triplet energy levels and the energy gaps of the highest-occupied orbitaland the lowest-unoccupied orbital are2.83eV and3.59eV for TzCz1,and2.80eV and3.43eV forTzCz2.At last,the theoretical analyses of their ground state geometries and the simulated UV–visabsorption spectra are carried out at B3LYP1/6-31G*level.The studies mentioned above indicate thatboth TzCz1and TzCz2are suitable for the host materials of blue light-emitting diodes.&2012Elsevier B.V.All rights reserved.1.IntroductionCurrently,the synthesis of the carbazole(Cz)based com-pounds[1]and the studies on their properties[2,3]greatly appealto the researchers because they can be applied in many areas[4–7]including medicament and anticorrosion.For example,Wang et al.reported that the Cz alkaloids,glybomine B andglycoborinine,demonstrated anti-HIV activity with an IC50of9.73mg/mL and4.47mg/mL,respectively[4].Gopia et al.alsoreported that the N-vinyl Cz had a good corrosion inhibitingcharacter[5].At the same time,the application of the Cz and itsderivatives[8–10]in optoelectronic devices becomes another hottopic,because these materials have large triplet energy gap,which is especially important for the improvement of the perfor-mance of the blue phosphorescent organic light-emitting diodes(PhOLEDs)[11,12]by efficiently preventing the back excitationtransfer from the guest materials to the host materials.As a result,more and more host materials based on Cz were synthesized forthe blue PhOLEDs[13–23],for example,Cho and Lee[7]andLee et al.[20]reported the blue PhOLEDs based on TSPC andmCPPO1possess the maximum external quantum efficiencies of22.0%and25.1%,respectively.In2007,Kim et al.[15]synthesized a series of Tz derivatizedgroups containing Cz compounds and found that3,5-diphenyl-4H-1,2,4-triazole(Tz)possesses the electron transporting character-istic,which is helpful to balance the injection and transport of thecharge carrier in OLEDs.Therefore,in this article,we reportthe synthesis and the properties of Tz group containing Czcompounds of9-(4-(3,5-diphenyl-4H-1,2,4-triazol-4-yl)phenyl)-9H-carbazole(TzCz1)and3,6-di-tert-butyl-9-(4-(3,5-diphenyl-4H-1,2,4-triazol-4-yl)phenyl)-9H-carbazole(TzCz2),where TzCz1is a known compound[24],but its crystal structure,electroche-mical properties,and the corresponding theoretical analyses werenot systematically studied.2.Experimental section2.1.MeasurementThe IR spectra were acquired using a FTIR-8400S SHIMADZUspectrophotometer in the4000–400cmÀ1region with KBr pel-lets.Element analyses were performed using a Vario ElementAnalyzer.1H NMR spectra were obtained using a Bruker AVANVEContents lists available at SciVerse ScienceDirectjournal homepage:/locate/jluminJournal of Luminescence0022-2313/$-see front matter&2012Elsevier B.V.All rights reserved.doi:10.1016/j.jlumin.2011.12.062n Corresponding author at:School of Materials Science and Engineering,Changchun University of Science and Technology,Changchun130022,PR China.Tel.:þ8643185583019;fax:þ8643185583015.E-mail address:szj@(Z.Si).Journal of Luminescence132(2012)1200–1206400MHz spectrometer with tetramethylsilane as the internal standard.The Ultraviolet–visible(UV–vis)absorption and photo-luminescent spectra of CH2Cl2solutions with ca.10À4mol/L samples were recorded on a Perkin Elmer Lambda900UV/Vis/ NIR spectrophotometer and a SPEX Fluorolog3spectrometer, respectively.The phosphorescent spectra were measured on Hitachi Spectrophotometer model F-4500at77K.The lumines-cence quantum yields(LQYs)were measured by comparing fluorescence intensities(integrated areas)of a standard sample (quinine sulfate)and the unknown sample according to the following equation:F unk¼F stdðI unk=A unkÞðA std=I stdÞðZunk=Z stdÞ2ð1Þwhere F unk is the LQY of the unknown sample;F std is the LQY of quinine sulfate and taken as0.546[25];I unk and I std are the integratedfluorescence intensities of the unknown sample and quinine sulfate at corresponding excitation wavelength,respec-tively;A unk and A std are the absorbances of the unknown sample and quinine sulfate at the corresponding excitation wavelengths, respectively.The Z unk and Z std are the refractive indices of the corresponding solvents(pure solvents were assumed).The crystals of[TzCz1H]ClÁ2CHCl3were cultured in CHCl3 solution before the product was washed in NaHCO3aqueous solution and measured on a Bruker Smart Apex CCD single-crystal diffractometer using l(Mo K a)radiation,0.7107˚A at293K.The structure was solved using the SHELXL-97program[16–28].The crystallographic refinement parameters of[TzCz1H]ClÁ2CHCl3are summarized in Table1,and the selected bond distances and angles of TzCz1are given in Table2.Cyclic voltammetry measurements were conducted on a YHMEC-3000voltammetric analyzer with a polished Pt plate as the working electrode,Pt mesh as the counter electrode,and a commercially available saturated calomel electrode(SCE)as the reference electrode,at a scan rate of0.1V/s.The voltammograms were recorded using CH3CN solutions with$10À3M sample and 0.1M tetrabutylammonium hexafluorophosphate(TBAPF6)as the supporting electrolyte.Prior to each electrochemical measure-ment,the solution was purged with nitrogen for$10–15min to remove the dissolved O2gas.The energy level of the highest-occupied molecular orbital(E HOMO)and the lowest-unoccupied molecular orbital(E LUMO)was calculated according to Ref.[29] and listed in Table3.2.2.Preparation of the materialsN,N-Dimethylacetamide(DMAc),N,N-dimethylaniline,and toluene were dried with standard procedure[30]and stored under N2,other chemicals are commercially available and used without further purification.9-(4-nitrophenyl)-9H-carbazole (A1),3,6-di-tert-butyl-9-(4-nitrophenyl)-9H-carbazole(A2)[31], 4-(9H-carbazol-9-yl)aniline(B1),4-(3,6-di-tert-butyl-9H-carba-zol-9-yl)aniline(B2)[32],and N0-(chloro(phenyl)methylene)ben-zohydrazonoyl chloride(C)[33]were synthesized according to the reported methods.All reactions and manipulations were carried out under N2with the use of Schlenk techniques.Solvents used in luminescent and electrochemical studies were of spectro-scopic and anhydrous grades,respectively.2.2.1.Synthesis of TzCz1The mixture of compound B1(0.516g,0.2mmol),compound C (0.544g,0.2mmol),and10mL of N,N-dimethylaniline was stir-red at1351C for12h under N2atmosphere.After adding aqueous solution of HCl(30mL,2N),the mixture was stirred for an additional30min.The precipitated solid was collected byfiltra-tion,washed in NaHCO3aqueous solution,dried in vacuo,and recrystallized from EtOH to afford TzCz1(60%);1H NMR(CDCl3): 7.321–7.368(m,2H),7.393(d,2H,J¼3),7.424(d,4H,J¼8), 7.452–7.492(m,4H),7.561(d,2H,J¼8),7.591–7.621(m,4H), 7.698(d,2H,J¼4),8.156(d,2H,J¼8).IR(KBr)/cmÀ1:3066,2356, 1514,1454,1234.Anal.Calcd.for C32H22N4:C,83.09;H,4.79;N, 12.11.Found:C,83.20;H, 4.52;N,12.41.UV–vis:292nm, 323nm,338nm.2.2.2.Synthesis of TzCz2TzCz2was prepared by an analogous procedures used in the preparation of TzCz1with a yield of54%1H NMR(CDCl3):1.471(s 18H),7.342(d,2H,J¼8),7.441–7.461(m,4H),7.495–7.522(m, 4H),7.559–7.606(m,2H),7.660(d,4H,J¼8),7.734(d,2H,J¼8), 8.145(d,2H,J¼1.6).IR(KBr)/cmÀ1:3055,2956,2943,2378, 2320,1517,1471.Anal.Calcd.for C40H38N4:C,83.59;H,6.66;N, 9.75.Found:C,83.72;H,6.49;N,9.86.UV–vis:296nm,332nm, 344nm.Table1Crystal data and structure refinement for[TzCz1H]þClÀ(CHCl3)2[TzCz1H]þClÀ(CHCl3)2Formula C34H25Cl7N4FW737.73T(K)293(2)KWavelength(˚A)0.71073Cryst.syst.OrthorhombicSpace group Pbcaa(˚A)10.2061(5)b(˚A)15.9437(7)c(˚A)42.5960(18)a(deg.)90b(deg.)90g(deg.)90V(˚A3)6931.3(5)Z28r calc.(Mg/m3) 4.949m(mmÀ1) 2.113F(000)(e)10528Range for collection(deg.)0.96–25.04Reflections collected33662Completeness99.9%(y¼25.04)Data/restraints/parameters6123/0/406Goodness-of-fit on F20.985R1,wR2[I42s(I)]0.0716/0.1697R1,wR2(all data)0.1273/0.2053Table2Bond lengths[˚A]and angles[deg.]for[TzCz1H]þClÀ(CHCl3)2C(19)–C(20) 1.467(6)C(19)–N(2) 1.382(6) C(26)–C(27) 1.471(6)C(26)–N(2) 1.358(5) C(1)–N(1) 1.396(6)C(19)–N(3) 1.306(6) C(12)–N(1) 1.402(6)N(3)–N(4) 1.367(5) C(13)–N(1) 1.423(6)N(4)–H(4)0.86C(16)–N(2) 1.444(5)C(26)–N(4) 1.307(6)C(2)–C(1)–N(1)129.8(4)N(4)–C(26)–C(27)125.4(4) N(1)–C(1)–C(6)108.7(4)N(2)–C(26)–C(27)128.8(4) C(11)–C(12)–N(1)129.6(5)C(1)–N(1)–C(12)108.8(4) N(1)–C(12)–C(7)108.4(4)C(1)–N(1)–C(13)125.1(4) C(18)–C(13)–N(1)120.0(4)C(12)–N(1)–C(13)125.5(4) C(14)–C(13)–N(1)119.3(4)C(26)–N(2)–C(19)106.5(4) C(15)–C(16)–N(2)117.8(4)C(26)–N(2)–C(16)127.2(4) C(17)–C(16)–N(2)119.7(4)C(19)–N(2)–C(16)126.3(4) N(3)–C(19)–N(2)110.7(4)C(19)–N(3)–N(4)104.0(4) N(3)–C(19)–C(20)124.5(4)C(26)–N(4)–N(3)113.0(4) N(2)–C(19)–C(20)124.8(4)C(26)–N(4)–H(4)123.5N(4)–C(26)–N(2)105.8(4)N(3)–N(4)–H(4)123.5J.Li et al./Journal of Luminescence132(2012)1200–12061201putational detailsThe geometrical structures of the ground states were opti-mized in gas phase by the density functional theory (DFT)[34]with an B3LYP1exchange-correlation functional calculus (a hybrid method combining five functionals,Becke þSlater þHF exchange,and LYP þVWN1correlation)[35,36].On the basis of the optimized ground state geometry structures,the UV–vis absorption spectral properties in CH 2Cl 2media were calculated by time-dependent DFT (TDDFT)[37],associating with the polar-ized continuum model (PCM).The 6-31G n [38,39]basis set on C,H,N atoms was employed for both TzCz1and TzCz2to ensure that the calculations are performed on the same level.The calculated electronic density plots for frontier molecular orbitals were prepared using the wxMacMolPlt-7.4.2software.All the calculations were performed with the Firefly QC package [40],which is partially based on the GAMESS (US)source code [41].3.Results and discussion 3.1.Structural characterizationAs presented in Scheme 1,the precursor of B1(B2)is prepared from the copper-catalyzed Ullmann reactions between 1-iodo-4-nitrobenzene and Cz (3,6-di-tert-butyl-9H-carbazole,Cz2),which is reduced by the reducing agent of NH 2NH 2ÁH 2O(85%)-Pd (5%)/carbon in EtOH under N 2protection.Meanwhile,the compound Cis synthesized according to the method in literature.At last,the reaction between B1(B2)and C gives the target compounds of TzCz1(TzCz2).The purity and composition of TzCz1and TzCz2are confirmed by 1H NMR,IR,and elemental analyses.At the same time,the colorless needle crystals of [TzCz1H]Cl Á2CHCl 3are cultured from its chloroform solution,and the solid evidence to support the structure of TzCz1is obtained using the single-crystal X-ray diffraction method.An ORTEP diagram of TzCz1is shown in Fig.1.The crystal data and the selected structural data for [TzCz1H]Cl Á2CHCl 3are presented in Tables 1and 2,respectively.The distances of C(13)–N(1)and C(16)–N(2)are 1.423(6)˚Aand 1.444(5)˚A,respectively,and the dihedral angles of C(12)–N(1)–C(13)–C(14)and C(17)–C(16)–N(2)–N(26)are 56.841and 67.011,respectively.These differences should be attributed to the fact that the phenyl groups on 3,5-position of Tz moiety lead to the bigger steric hindrance.At the same time,the existence of the hydrogen ion on the N(4)makes the distance of C(26)–C(27)(1.471(6)˚A)to be longer than that of C(19)–C(20)(1.467(6)˚A).But according to the contribution of Cl À,the dihedral angles of C(28)–C(27)–C(26)–N(4)(26.031)are much smaller than those of C(21)–C(20)–C(19)–N(3)(43.501).3.2.ElectrochemistryThe electrochemical properties of TzCz1and TzCz2are studied in CH 3CN solution through cyclic voltammetry using TBAPF 6as the supporting electrolyte (Fig.2).During the anodic scan,the irreversible anodic waves peaked at þ1.52V with an onsetTable 3Electrochemical and photophysical parameters of TzCz1and plexAbsorption l (nm)aExcitation l (nm)aEmissionE HOMO (eV)E LUMO (eV)l (nm)af (%)l (nm)bE T (eV)TzCz1255,292,324,339290,316,327,339345,35955.66438 2.83À5.84À2.25TzCz2261,296,311,344293,307,336,345353,36960.764432.80À5.88À2.45a The spectra are measured at room temperature.bThe spectra are measured at 77K.Scheme 1.Synthesis route to TzCz1and TzCz2.(i)1-iodo-4-nitrobenzene,DMAc,1701C,24h;(ii)NH 2NH 2ÁH 2O (85%),Pd (5%)/C,EtOH,reflux,10h;(iii)N,N-dimethylaniline,B1or B2,1351C,12h.J.Li et al./Journal of Luminescence 132(2012)1200–12061202oxidation potential(V onset(OX))ofþ1.10V for TzCz1andþ1.25V with a V onset(OX)ofþ1.14V for TzCz2.As can be seen from the insets of Fig.2,the current present somewhat increases when the oxidation potential is gradually shifting to lower potentials during repeated cyclic voltammetry scans.As revealed in the literature,it is important to block the active sites of Cz derivatives when the compounds transport positive charge carriers in PhOLEDs[42]. The values of the E HOMO are obtained according to the following equation:E HOMO¼[V SCEÀV onset(OX)]eV[43],where V SCE is the electrode potential of the SCE,which should beÀ4.74eV in a vacuum,and the values of E HOMO for TzCz1and TzCz2are calculated to beÀ5.84eV andÀ5.88eV,respectively.According to the previous reports,the E HOMO of mCP isÀ5.91eV[6],being lower than those of TzCz1and TzCz2,which reveals that the introduction of the Tz moiety in p-positions of the phenyl unit should lead to the reduction of the hole injection barrier,thereby facilitating the injection of positive charge carriers.At the same time,the values of E LUMO for TzCz1and TzCz2are obtained according to the following equation:E HOMO¼[E LUMOÀ1240/ l(Abs)]eV,where l(Abs)presents the longest absorption wave-length(345nm for TzCz1and362nm for TzCz2),which can be obtained from the corresponding UV–vis absorption spectra (Fig.3).Therefore,the values of E LUMO are calculated to be À2.25eV for TzCz1andÀ2.45eV for TzCz2,and the energy gaps of TzCz1and TzCz2should be3.59eV and3.43eV,respectively.3.3.Photophysical propertiesThe UV–vis absorption spectra of TzCz1and TzCz2in CH2Cl2 are presented in Fig.3,and the selected data of these UV–vis absorption spectra are listed in Table3.As can be seen from Fig.3, the absorption bands of TzCz1and TzCz2in the range of 300–360nm should be assigned to the ICT transitions based on p(Cz)-p n(TzþPh),and the bands with the wavelength being shorter than300nm should be contributed by the mixture of the p(Tz)-p n(Tz)/p(Cz)-p n(Cz)transitions[44].TheseassignmentsFig.1.ORTEP drawing of a crystal of TzCz1with displacement ellipsoids at the50%probability level.Two CHCl3solvent molecules and a ClÀanion is present perunit cell and have been omitted,along with all hydrogen atoms,forclarity.Fig.2.Cyclic voltammograms of complexes TzCz1(a)and TzCz2(b)measured in CH3CN(vs.SCE)at a scan rate of0.1V/s.A polished Pt plate and a Pt mesh were used as the working electrode and the counter electrode,respectively.TBAPF6was taken as supportingelectrolyte.Fig.3.UV–vis absorption spectra and excitation spectra of TzCz1and TzCz2indilute CH2Cl2,along with the UV–vis absorption spectra of Cz,Cz2,and TzMe.J.Li et al./Journal of Luminescence132(2012)1200–12061203are based on the absorption spectra of Cz,Cz2,and 3,5-diphenyl-4-(p -tolyl)-4H-1,2,4-triazole (TzMe)presented in Fig.3and the theoretical studies (vide infra).The PL spectra of TzCz1and TzCz2in CH 2Cl 2solution are presented in Fig.4.Upon UV excitation,the PL spectra of TzCz1and TzCz2present their vibronic structure with the maximum emission band centered at 359nm and 369nm,respectively.Due to the steric effect and the electron donor property of the tert -butyl groups in TzCz2,the emission band of TzCz2is present at longer wavelength than that of TzCz1,and the LQY of TzCz2(47.5%)is almost 1.5times higher than that of TzCz1(32.1%)in dilute CH 2Cl 2solution.The phosphorescent spectra of TzCz1and TzCz2measured in EtOH at 77K are presented in the inset of Fig.4.It is found that the highest energy phosphorescent band peaked at 438nm for TzCz1and 443nm for TzCz2and the triplet energy levels are calculated to be 2.83eV for TzCz1and 2.80eV for TzCz2,which are higher than those of the commonly used triplet blue-emitters FIrpic (2.62eV)and FIr6(2.72eV)[45,46].Therefore,TzCz1and TzCz2can be potentially used as the host materials of the blue PhOLEDs.3.4.Theoretical analysis3.4.1.Frontier molecular orbital propertiesThe selective parameters of the optimized molecular struc-tures of TzCz1and TzCz2are collected in Table 4.As can be seen from Table 4,The bond distance of C(21)–N(24)of TzCz2issimulated to be 1.41˚A,which is 0.01˚A shorter than that of TzCz1(1.41˚A).The bond angles simulated for TzCz2slightly depart from those of TzCz1,which should be attributed to the appearance of the tert -butyl groups in TzCz2.According to the simulated molecular structure of TzCz1,the distances of Cz moiety and Tzmoiety to the phenylene group are calculated to be 1.42˚Aand 1.43˚A,respectively,and the dihedral angles of C(22)–C(21)–N(24)–C(28)and C(7)–N(11)–C(18)–C(19)are 52.971and 67.391,respectively.Meanwhile the dihedral angle of C(17)–C(12)–C(10)–N(11)and the bond distance of C(12)–C(10)are 33.051and 1.47˚A,respectively,which are almost equal to the dihedral angle of C(67)–C(6)–C(7)–N(11)of 33.101and the bond distanceof C(7)–C(5)of 1.47˚A.These results are obviously different to those from the experimental measurements,which should be attributed to the fact that the effect of the H þion and the Cl Àcation on the Tz moiety are ignored during the theoretical studies of the ground state geometry of TzCz1,along with the fact thatthe theoretical studies are optimized in the gas phase and the experimental measurements are in a tight crystal lattice [47].Table 5presents the compositions of the calculated frontier molecular orbitals of TzCz1and TzCz2and the electron density plots of the HOMO and the LUMO of TzCz1and TzCz2are shown in Fig.5.The HOMOs of TzCz1and TzCz2are composed of the p orbitals localized on Cz moiety with Z 85.5%contributions,the HOMO À1of TzCz1is composed of the p orbitals localized on Tz moiety with 99.5%contributions.The HOMO À2of TzCz1and HOMO À1and HOMO À4of TzCz2are composed of the p orbitals localized on Cz moiety with Z 96.0%contributions.Meanwhile,the HOMO À2of TzCz2,the HOMO À3s of TzCz1and TzCz2,and the HOMO À4of TzCz1are contributed by the p orbitals localized on Tz moiety with Z 88.6%distributions.The LUMO of TzCz1is mainly composed of the p n orbitals localized on Tz with 46.6%contributions and Ph moieties with 46.1%contributions.Similar to the LUMO of TzCz1,the LUMO of TzCz2and the LUMO þ2s of TzCz1and TzCz2are also composed of the p n orbitals localized on Tz with 448.5%contributions and Ph moieties with 438.1%contributions.The LUMO þ1s of TzCz1and TzCz2are composed of the p n (Ph)orbitals with 479.2%contributions.The LUMO þ3s of TzCz1and TzCz2are mainly composed of the p n orbitals localized on Cz moiety with 492.0%contributions.Meanwhile,the LUMO þ4are mainly composed of the p n orbitals localized on the Tz moiety with 94.0%contribu-tions for TzCz1and 93.5%contributions for TzCz2.As presented in Table 5,the E HOMO s and the E LUMO s are calculated to be À5.64eV and À1.23eV for TzCz1and À5.44eV and À1.18eV for TzCz2,respectively.Therefore,the energy gaps of the HOMO and the LUMO are simulated to be 4.41eV for TzCz1and 4.26eV for TzCz2,which are bigger than those from the experimental measurements.The difference of the orbital energy betweentheFig.4.PL spectra of TzCz1and TzCz2in CH 2Cl 2at room temperature.Inset:the PL spectra of TzCz1and TzCz2measured in MeOH air at 77K.Table 4Bond lengths [˚A]and angles [deg.]for simulated TzCz1and TzCz2.TzCz1TzCz2C(5)–C(7)1.47 1.47C(10)–C(12) 1.47 1.47C(7)–N(8) 1.32 1.32C(10)–N(9) 1.32 1.32N(8)–N(9) 1.37 1.37C(7)–N(11) 1.39 1.39C(10)–N(11) 1.39 1.39C(18)–N(11) 1.43 1.43C(21)–N(24) 1.42 1.41C(28)–N(24) 1.40 1.40C(25)–N(24) 1.40 1.40C(5)–C(7)–N(8)123.27123.27C(5)–C(7)–N(11)127.36127.35C(7)–N(8)–N(9)108.36108.35N(11)–C(7)–N(8)109.37109.38N(8)–N(9)–C(10)108.35108.35N(9)–C(10)–N(11)109.37109.38C(10)–N(11)–C(7)104.54104.54N(9)–C(10)–N(12)123.27123.29N(11)–C(10)–C(12)127.36127.33C(7)–N(11)–C(18)127.73127.70C(10)–N(11)–C(18)127.73127.75C(19)–C(18)–N(11)119.96119.95C(23)–C(18)–N(11)119.96119.98C(20)–C(21)–N(24)120.28120.26C(22)–C(21)–N(24)120.28120.33C(21)–N(24)–C(28)125.81126.03C(21)–N(24)–C(25)125.81125.84N(24)–C(28)–C(36)129.52130.09N(24)–C(25)–C(29)129.52130.22N(24)–C(28)–C(27)108.86109.12C(28)–N(24)–C(25)108.37108.12N(24)–C(25)–C(26)108.86108.97J.Li et al./Journal of Luminescence 132(2012)1200–12061204experimental measurements and the theoretical calculations should be mainly attributed to the ignorance of the solution effect during the molecular structure optimization.3.4.2.Simulation of the UV–vis absorption spectraThe curves of the theoretically simulated UV–vis absorption spectra of TzCz1and TzCz2presented in Fig.6are nicely fitted to those from the experimental measurements.Similar to the experimental measurements,the simulated UV–vis absorption of TzCz2presents obvious red-shift in accordance with that of TzCz1.This should be assigned to the fact that the energy gap between the HOMO and LUMO of TzCz2is much narrower than that of TzCz1(Tables 3and 5).As presented in Table 6,the lowest lying singlet -singlet absorptions of TzCz1and TzCz2contrib-uted by the configurations of HOMO -LUMO are calculated at 323and 349nm,respectively.According to the simulated com-position of the HOMO and LUMO (Table 5),these lowest lying transition for both TzCz1and TzCz2should be described as the ICT based on the p (Cz)-p n (Tz þPh)transitions.The absorption with the largest oscillator strength at 283nm and 242nm are in agreement with the experimental values of 282nm for TzCz1and 248nm for TzCz2.According to the fact that LUMO þ7of TzCz2is composed of the p n orbital on Cz moiety with ca.97.0%contribution and the other orbital information presented in Table 5,the dominant character of these higher energy absorptions are tentatively assigned to the ICT based on [p (Cz)-p n (Tz þPh)]transitions for TzCz1and the [p (Cz)-p n (Cz)]transitions for TzCz2.At the same time,the absorptions with the moderate oscillator of both TzCz1and TzCz2are calculated to be the mixture transitions of the ICT and the p -p n transfer.4.ConclusionsIn summary,we synthesize two Cz derivatives of TzCz1and TzCz2,which are fully characterized by the 1H NMR,UV–vis absorption spectra,and the IR absorption spectra.It is found that the presence of tert -butyl groups leads to the relatively higher LQY and the lower energy gap of TzCz2.The triplet energy levels,which are deducted from the phosphorescent spectra,are only 2.83eV for TzCz1and 2.80eV for TzCz2.Both of the experimental studies and the theoretical analyses suggest that TzCz1and TzCz2should possess the potential application as the host materials in bluePhOLEDs.Fig.5.Electron density plots of the frontier molecular orbital of TzCz1and TzCz2in gas phase at B3LYP1/6-31G nlevel.Fig.6.Simulated UV–vis absorption spectra of TzCz1and TzCz2in CH 2Cl 2media at B3LYP1/6-31G n level.Table 5Frontier molecular orbital compositions (%)of TzCz1and TzCz2calculated in the gas phase at the DFT/B3LYP1/6-31G n level.OrbitalTzCz1TzCz2E (eV)Bond typeDistribution (%)E (eV)Bond typeDistribution (%)TzPhCzTzPhCzLUMO þ4À0.48p n (Tz)94.0À0.45p n (Tz)93.5LUMO þ3À0.92p n (Cz)92.0À0.85p n (Cz)97.1LUMO þ2À0.94p n (Tz þPh)50.938.1-0.88p n (Tz þPh)48.544.2LUMO þ1À0.99p n (Ph)80.4À0.93p n (Ph)79.2LUMOÀ1.23p n (Tz þPh)46.646.1À1.18p n (Tz þPh)53.040.2Energy gap 4.41 4.26HOMO À5.64p (Cz)85.5À5.44p (Cz)87.3HOMO À1À5.95p (Tz)99.5À5.85p (Cz)99.1HOMO À2À5.99p (Cz)99.4À5.93p (Tz)99.5HOMO À3À6.67p (Tz)93.6À6.63p (Tz)92.2HOMO À4À6.93p (Tz)88.6À6.77p (Cz)96.0J.Li et al./Journal of Luminescence 132(2012)1200–12061205AcknowledgmentsThe authors are grateful to the financial aid from the National Natural Science Foundation of China (Grant No.20901011)and the Undergraduate Teaching Quality Project of Changchun Uni-versity of Science and Technology (Grant No.2010A0635).References[1]M.Ishikura,K.Yamada,T.Abe,Nat.Prod.Rep.27(2010)1630.[2]A.Balan,D.Baran,L.Toppare,Polym.Chem.2(2011)1029.[3]I.P.Singh,H.S.Bodiwala,Nat.Prod.Rep.27(2010)1781.[4]J.Wang,Y.Zheng,T.Efferth,R.Wang,Y.Shen,X.Hao,Phytochemistry 66(2005)697.[5]D.Gopia,indarajua,L.Kavithab,K.Anver Bashac,.Coat.71(2011)11.[6]W.Jiang,L.Duan,J.Qiao,G.Dong,D.Zhang,L.Wang,Y.Qiu,J.Mater.Chem.21(2011)4918.[7]Y.J.Cho,J.Y.Lee,J.Phys.Chem.C 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4.51/2750.1292p (Cz)-p n (Tz)20H -L þ7(46) 5.12/2420.4503248p (Cz)-p n (Cz)28H À3-L þ1(68) 5.33/2330.2722p (Tz)-p n (Ph)44H -L þ9(38)5.79/2140.2409p (Cz)-p n (Cz)J.Li et al./Journal of Luminescence 132(2012)1200–12061206。

2024年3月 热带农业科学第44卷第3期Mar. 2024 CHINESE JOURNAL OF TROPICAL AGRICULTURE Vol.44, No.3收稿日期 2023-03-14；修回日期 2023-05-15基金项目 广西农业科学院科技发展基金（No.桂农科2021JM112，No.桂农科2022JM80）；科技先锋队“强农富民”“六个一”专项行动（No.桂农科盟202412）。

第一作者 孙明艳（1992—），女，硕士，助理研究员，主要研究方向为花卉真菌病害及防治技术，E-mail ：****************。

通讯作者 卜朝阳（1966—），女，硕士，研究员，主要研究方向为主要从事花卉栽培及育种，E-mail ：**************。

一种茉莉炭疽病病原菌的鉴定、生物学特性及药剂筛选孙明艳 刘可丹 李春牛 李先民 喇燕菲 卜朝阳（广西壮族自治区农业科学院花卉研究所 广西南宁 530007）摘 要 在广西横州市发现一种茉莉叶部炭疽病病害，明确其病原菌种类，并对其开展生物学特性和有效杀菌剂筛选研究。

通过病原菌分离纯化、致病性鉴定与形态学观察，运用MEGA v.10.1.5软件对代表菌株HL6-1构建基于ITS ､ACT ､CHS-1､TUB2、GAPDH 和ApMat 多基因测序结果的系统发育树，明确该菌株为暹罗炭疽菌Colletotrichum Siamense ，菌株气生菌丝绒毛状，菌落初为白色，后变为深灰色，有橘色孢子团的产生；分生孢子无色，整体长椭圆形，单胞，平均大小为14.58 μm×5.97 μm ；生物学特性研究结果表明，该菌株最适培养温度为28℃，最适pH 为7，最适碳源为葡萄糖，最适氮源为硝酸钠。

采用菌丝生长速率法进行室内毒力测定，筛选有效杀菌剂，结果表明，吡唑醚菌酯、苯醚甲环唑、咪鲜胺的抑菌效果较好，EC 50值均小于1 mg/L 。

Table Of ContentsTABLE OF CONTENTS (1)MORPHOLOGY OF THE RICE PLANT (2)Germinating seed (2)Seedling (4)Tiller (6)Culm (8)Leaf (12)Panicle and Spikelets (18)Floret (24)Flower (27)Rice grain (29)PRINT VERSION (31)INDEX (32)Morphology of the Rice PlantGerminating seedWhen the seed germinates in well-drained and well-aerated soil, the coleorhiza, a covering enclosing the radicle or primary root, protrudes first.Fig. 1 - The coleorhiza protrudes first.Shortly after the coleorhiza appears, the radicle or primary root breaks through the covering. Fig. 2 - Radicle or primary root breaks through the covering.Two or more sparsely branched seminal roots follow. These roots eventually die and are replaced by many secondary adventitious roots.Fig. 3 - Seminal rootsIf the seed germinates in water, the coleoptile, a covering enclosing the young shoot, emerges ahead of the coleorhiza. The coleoptile emerges as a tapered cylinder.Fig. 4 - Coleoptile emerging as a tapered cylinder.SeedlingThe mesocotyl or basal portion of the coleoptile elongates when the seed germinates in soil, and in darkness. It pushes the coleoptile above the soil surface.Fig. 5 - Mesocotyl pushing the coleoptile above the soil surface.The first seedling leaf, or primary leaf, emerges from the growing seed. It is green and shaped like a cylinder. It has no blade. The second leaf is a complete leaf. It is differentiated into a leaf blade and a leaf sheath.Fig. 6 - First and second seedling leaf.TillerThe seedling will grow and develop branched tillers. Parts of the rice tiller include the roots, culm and leaves. Mature roots of the rice plant are fibrous and produce smaller roots called rootlets. All roots have root hairs to absorb moisture and nutrients.Fig. 7 - Parts of the rice tiller.There are two kinds of mature roots:1. secondary adventitious roots2. adventitious prop roots prop roots.Fig. 8 - Types of roots.Secondary adventitious roots are produced from the underground nodes of young tillers.Fig. 9 - Secondary adventitious roots.As the plant grows, coarse adventitious prop roots often form above the soil surface in whorls from the nodes of the culm.Fig. 10 - Adventitious prop roots.CulmThe culm, or jointed stem of the rice, is made up of a series of nodes and internodes. Fig. 11 - Culm, nodes, and internodes.Young internodes are smooth and solid. Mature internodes are hollow and finely grooved with a smooth outer surface. Generally, internodes increase in length from the lower to the upper portions of the plant. The lower internodes at the plant base are short and thick.Fig. 12 - Young and mature internodes.The node is the solid portion of the culm. The node or nodal region bears a leaf and a bud. The bud is attached to the upper portion of the node and is enclosed by the leaf sheath. The bud may give rise to a leaf or a tiller.Fig. 13 - Leaf, node, and bud.Early tillers arise from the main culm in an alternate pattern. Primary tillers originate from the lowermost nodes and give rise to secondary tillers. Secondary tillers produce tertiary tillers. Fig. 14 - Primary tillers.Fig. 15 - Secondary tillers. Fig. 16 - Tertiary tillers.LeafThe node or nodal region of the culm will bear a leaf. Fig. 17 - Leaf.Leaves are borne alternately on the culm in opposite directions. One leaf is produced at each node. Varieties differ in the number of leaves produced.Fig. 18 - Leaves alternate on the culm in opposite directions.The topmost leaf below the panicle is the flag leaf. The flag leaf contributes largely to the filling of grains because it supplies photosynthetic products, mainly to the panicle.Fig. 19 - Flag leaf.The leaf sheath and leaf blade are continuous.Fig. 20 - Leaf sheath and blade.A circular collar joins the leaf blade and the leaf sheath.Fig. 21 - Leaf collar.The leaf sheath is wrapped around the culm above the node. Fig. 22 - Leaf sheath and culm.The swelling at the base of the leaf sheath, just above the node, is the sheath pulvinus. It is sometimes incorrectly referred to as the node.Fig. 23 - Sheath pulvinus.Leaf blades are generally flat. Varieties differ in blade length, width, thickness, area, shape, color, angle and pubescence.Fig. 24 - Different varieties with varying blade characteristics.With many parallel veins on the upper surface of the leaf, the underside of the leaf blade is smooth with a prominent ridge in the middle; the midrib.Fig. 25 - Parallel veins on upper surface. Fig. 26 - Leaf midrib.Most leaves possess small, paired ear-like appendages on either side of the base of the blade. These appendages are called auricles. Auricles may not be present on older leaves. Another leaf appendage is the ligule, a papery membrane at the inside juncture between the leaf sheath and the blade. It can have either a smooth or hair-like surface. The length, color, and shape of the ligule differ according to variety.Fig. 27 - Ligule and auricle.Although similar, rice seedlings are different from common grasses. While rice plants have both auricles and ligules, common grassy weeds found in rice fields normally do not have these features. These characteristics are often helpful in identifying weeds in rice fields when the plants are young.Fig. 28 - Rice and grassy weed comparison.Panicle and SpikeletsThe terminal component of the rice tiller is an inflorescence call the panicle. The inflorescence or panicle is borne on the uppermost internode of the culm. The panicle bears rice spikelets, which develop into grains.Fig. 29 - Rice panicle.The panicle base often appears as a hairlike ring and is used as a dividing point in measuring culm and panicle length. The panicle base is often called the neck.Fig. 30 - Panicle base (neck).The panicle axis is continuous and hollow except at the nodes where branches are borne. Fig. 31 - Panicle axis.The swellings at the panicle axis where the branches are borne are referred to as the panicle pulvinus.Fig. 32 - Panicle pulvinus.Each node on the main panicle axis gives rise to primary branches which in turn bears secondary branches. Primary branches may be arranged singly or in pairs.Fig. 33 - Secondary and primary branch.The panicles bear spikelets, most of which develop into grains. These spikelets are borne on the primary and secondary branches. The spikelet is the basic unit of the inflorescence and panicle. It consists of the pedicel and the floret.Fig. 34 - Spikelets.The floret is borne on the pedicel.Fig. 35 - Floret and pedicel.The rudimentary glumes are the laterally enlarged, cuplike apex of the pedicel. The rudimentary glumes are the lowermost parts of the spikelet. During threshing, the rudimentary glumes are separated from the rest of the spikelet.The sterile lemmas are small, bractlike projections attached to the floret. The rachilla is a small axis that bears the single floret. It is between the sterile lemmas and the floret.Fig. 36 - Rudimentary glumes, sterile lemmas, and rachilla.FloretThe rachilla, sterile lemmas and the rudimentary glumes all support the floret. The floret includes the lemma, palea, and the flower.Fig. 37 - FloretThe larger protective glume covering the floret is called the lemma and the smaller one is referred to as the palea.Fig. 38 - Palea and lemma.Both the lemma and palea have ridges referred to as nerves. The lemma has five while the palea has three. The middle nerve of the lemma can be either smooth or hairy.Fig. 39 - Nerves.The lemma has a constricted structure at its end called the keel. In some varieties, the keel is elongated into a thin extension, the awn.Fig. 40 - Awn and keel.FlowerThe floret contains a flower. The flower consists of a pistil (female organ) and six stamens (male organs).Fig. 41 - Pistil.Fig. 42 - Stamens.The stamens have two-celled anthers borne on slender filaments.Fig. 43 - Anthers and filaments.The pistil contains one ovule and bears a double-plumed stigma on a short style. Fig. 44 - Stigma, style, and ovule.At the flower’s base near the palea are two transparent structures known as lodicules. The lodicules thrust the lemma and palea apart at flowering to enable the elongating stamens to emerge out of the open floret. The lemma and palea close after the anthers have shed their pollen.Fig. 45 - Lodicule.Rice grainThe rice grain is the ripened ovary, with the lemma, palea, rachilla, sterile lemmas and the awn firmly attached to it.Fig. 46 - Rice grain.The rice hull includes the lemma and palea and their associated structures – the sterile lemmas, rachilla, and awn.Fig. 47 - Rice hulls.The dehulled rice grain is called caryopsis, commonly referred to as brown rice because of three brownish pericarp layers that envelope it. Next to the pericarp layers are the two tegmen layers and the aleurone layers.Fig. 48 - Tagmen, pericap, and aleurone layers.The remaining part of the grain consists of the endosperm and the embryo. The endosperm provides nourishment to the germinating embryo. The embryo lies on the belly side of the grain and is enclosed by the lemma. It is the embryonic organ of the seed.Fig. 49 - Endosperm and embryo.The embryo contains the plumule (embryonic leaves) and the radicle (embryonic primary root).Fig. 50 - Plumule and radicle.Print VersionCultural Control of Rice Insect Pests may also be completely printed, provided you have a printer attached to your computer and Microsoft Word. Click here to launch the entire contents of this course in Microsoft Word.IndexAAdventitious (4)Aleurone (24)Anthers (22)Asian (27)Awn (20)B Bractlike (15)C Caryopsis (24)Characteristics (10)Coleoptile..............................................................................................................................2, 3 portion .. (3)pushes (3)Coleorhiza (2)Contains (24)plumule (24)Culm ................................................................................................................... 4, 6, 10, 15, 27 measuring (15)portion (6)Cuplike (15)D Dehulled (24)Different (10)EEndosperm (24)FFirst (3)Flag (10)Floret ................................................................................................................................ 15, 20 Flower.. (22)Flower’s (22)G Germinating (2)seed (2)Glume (20)Glumes............................................................................................................................. 15, 20 I Index. (27)use (27)L Leaf..................................................................................................................................... 6, 10 Leaves. (10)Ligule (10)shape (10)Ligules (10)Lodicule (22)Lodicules (22)M Measuring (15)culm (15)Mesocotyl (3)Morphology (27)Rice Plant (27)Welcome (27)NNear (22)palea (22)Nerves (20)OOryza sativa (27)P Palea........................................................................................................................... 20, 22, 24 near .. (22)Panicle (15)Panicle pulvinus (15)Parallel (10)Parts ................................................................................................................................... 4, 15 spikelet .. (15)Pedicel (15)Pericap (24)Pericarp (24)Pistil (22)Plumule (24)contains (24)Portion...................................................................................................................................3, 6 coleoptile. (3)culm (6)Primary (6)Pulvinus........................................................................................................................... 10, 15 Pushes .. (3)coleoptile (3)RRachilla ...................................................................................................................... 15, 20, 24 Radicle .. (2)Rest (15)spikelet (15)Rice............................................................................................................................. 10, 15, 24Rice grain (24)Rice Morphology (27)Rice Plant (27)Morphology (27)Rice Production (27)Rudimentary glumes (15)SSearch (27)Secondary...................................................................................................................... 4, 6, 15 Seed. (2)Germinating (2)Seminal (2)Shape (10)ligule (10)Sheath pulvinus (10)Spikelet (15)parts (15)rest (15)Spikelets (15)Stamens (22)Stigma (22)TTagmen (24)Tertiary (6)The coleorhiza (2)These characteristics (10)These spikelets (15)Tiller (4)Types (4)U Use (27)Index (27)WWelcome (27)Morphology (27)Y Young (6)。

1.Volatile flavour components of mango fruitAbstract：An essence of fresh Venezuelan mango fruit obtained by well-established procedures possessed the characteristic aroma of the fruit. It was analysed by GC/MS using both EI and Cl. The fruit produced a relatively small quantity of aroma volatiles (ca60 μg/kg fresh fruit), less than that obtained from many similar tropical fruits. Terpene hydrocarbons comprised ca 68% of the sample, eight monoterpenes contributing ca 54% and four sesquiterpenes contributing ca 14%. Impo rtant constituents included α-pinene, car-3-ene, limonene, γ-terpinene, α-humulene, β-selinene, acetophenone, benzaldehyde and a dimethylstyrene. Car-3-ene (26%) was the major constituent, and on odour evaluation of separated components at an odour port during GC, the peak due to this compound was described as having an aroma of mango leaves. This compound has not previously been detected among mango volatiles. The only other component providing mango aroma was a dimethylstyrene, and this too is a new mango volatile.2.Aroma volati les production during fruit ripening of ‘Kensington Pride’ mangoAbstract：‘Kensington Pride’ mango aroma volatile compounds emitted during ripening were studied using headspace solid-phase microextraction as a sampling method and gas chromatography with a flame ionisation detector as well as gas chromatography mass spectrophotometry for analysis. Fruit were ripe on the seventh day of the ripening period, which corresponded to the fruit being eating soft and a skin colour that was 75% yellow. Ethylene production and respiration reached a peak on the fourth day of ripening. Most of the fatty acids increased during fruit ripening. Sixty-one aroma volatile compounds were identified, of which 35 compounds have not been reported previously in ‘Kensington Pride’ mango. (+)-Spathulenol and β-maaliene were found for the first time in mango fruit. The most abundant group of volatile compounds was hydrocarbons, accounting for about 59% of the total identified compounds, followed by esters (20%). α-Terpinolene was the major compound during the first 7 days of ripening and later ethyl octanoate became the major compound. Except for car-3-ene, the concentration of major monoterpenes increased for the first 3 or 4 days and decreased afterwards. Most of the major sesquiterpenes were intensively synthesised in the early part of the ripening process. The production of three major esters increased quite sharply during fruit ripening. It appeared that production of terpenes was parallel with production of ethylene, whilst production of esters appeared to be associated with production of fatty acids.3.Volatile and quality changes in fresh-cut mangos prepared from firm-ripe and soft-ripe fruit, stored in clamshell containers and passive MAPAbstract：A study was performed to assess volatile and quality changes in stored fresh-cut mangos prepared from “firm-ripe” (FR) and “soft-ripe” (SR) fruit, and to assess whateffect passive modified atmosphere packaging (MAP) may have on cut fruit physiology, overall quality and volatile retention or loss. Florida-grown …Keitt‟ and …Palmer‟ mangos were used, without heat-treatment. Subjective appraisals of fresh-cut mangos based on aroma and cut edge or tissue damage indicated that most SR cubes were unmarketable by day 7 at 4 °C. Both varieties stored in MAP at 4 °C had almost identical O2 consumption, which was independent of ripeness. Percent CO2 and O2 data for cubes stored in passive MAP indicates that the system was inadequate to prevent potential anaerobic respiration after 7 days storage. A significant three-way interaction (container×ripeness×day) was observed for L* (lightness) between stored cubes prepared fro m FR versus SR fruit of both varieties. There was a linear L* decrease for SR …Keitt‟ cubes stored in clamshell containers. δ-3-Carene was the dominant terpene in both varieties in all treatments throughout most of the study, and FR cubes had statistically higher levels of seven terpenes compared with the respective SR treatments. Most terpenes in FR and SR cubes stored in both package types displayed a transient increase, occurring on day 4 or 7, followed by a decline.Translation:1.芒果的挥发性气味成分摘要：通过精心规划的程序得到了新鲜的委内瑞拉芒果果实，此果实本身具有芳香的特征，这是通过GC\MS运用EI和CI两种方式分析得出的。

生物专业外语笔试题目及答案一、选择题（每题2分，共20分）1. The term "gene" was first introduced by which scientist?A. Charles DarwinB. Gregor MendelC. James WatsonD. Francis Crick答案：B2. Which of the following is not a function of DNA?A. Store genetic informationB. Control cell divisionC. Direct protein synthesisD. Provide energy答案：D3. What is the basic unit of a protein?A. CarbohydrateB. LipidC. Amino acidD. Nucleotide答案：C4. The process of DNA replication occurs during which phase of the cell cycle?A. G1 phaseB. S phaseC. G2 phaseD. M phase答案：B5. Which of the following is a type of genetic mutation?A. TranscriptionB. TranslationC. TransversionD. Translocation答案：C二、填空题（每空2分，共20分）6. The central dogma of molecular biology states that genetic information flows from DNA to RNA and then to _______.答案：protein7. In eukaryotic cells, the process of protein synthesis takes place in the _______.答案：cytoplasm8. The term "genome" refers to all the genetic material of an _______.答案：organism9. The process by which a fertilized egg develops into afully formed individual is known as _______.答案：development10. The study of the relationships among various species is known as _______.答案：taxonomy三、简答题（每题10分，共40分）11. Briefly describe the structure of a typical eukaryotic cell.答案：A typical eukaryotic cell has a nucleus that contains the genetic material, a cell membrane that encloses the cell, cytoplasm where organelles such as mitochondria, endoplasmic reticulum, and Golgi apparatus perform various functions, and other structures like lysosomes and a cytoskeleton.12. Explain the concept of natural selection and its importance in evolution.答案：Natural selection is the process by which individuals with traits that are better suited to their environment are more likely to survive and reproduce, passing on those advantageous traits to their offspring. It is a key mechanism of evolution, leading to adaptation and the diversity of life forms.13. What are the main differences between prokaryotic and eukaryotic cells?答案：Prokaryotic cells lack a nucleus and membrane-bound organelles, whereas eukaryotic cells have a defined nucleus and various membrane-bound organelles such as mitochondria and the endoplasmic reticulum. Prokaryotes are generally smaller and simpler in structure compared to eukaryotes.14. Describe the process of photosynthesis and its significance for life on Earth.答案：Photosynthesis is the process by which green plants and some other organisms convert light energy into chemical energy stored in glucose or other organic molecules. It is significant for life on Earth as it provides oxygen and is the primary source of energy for most food chains.四、论述题（每题20分，共20分）15. Discuss the impact of genetic engineering on modern agriculture and medicine.答案：Genetic engineering has revolutionized agriculture by enabling the development of crops with improved resistance to pests and diseases, better tolerance to environmental stresses, and enhanced nutritional content. In medicine, it has facilitated the production of recombinant proteins and vaccines, the development of gene therapies, and the advancement of personalized medicine based on genetic profiles.结束语：本试题旨在考察学生对生物专业外语知识的掌握程度以及应用能力，希望同学们能够通过本试题加深对生物学基本概念和原理的理解，并在实际应用中不断进步。

Pictures of Appetizing Foods Activate Gustatory Cortices for Taste and RewardW.Kyle Simmons 1,Alex Martin 2and Lawrence W.Barsalou 11Department of Psychology,Emory University,Atlanta,GA 30322,USA and 2Cognitive Neuropsychology Section,Laboratory of Brain and Cognition,National Institute of Mental Health,Bethesda,MD,USAIncreasing research indicates that concepts are represented as distributed circuits of property information across the brain’s modality-specific areas.The current study examines the distributed representation of an important but under-explored category,foods.Participants viewed pictures of appetizing foods (along with pictures of locations for comparison)during event-related pared to location pictures,food pictures activated the right insula/operculum and the left orbitofrontal cortex,both gustatory processing areas.Food pictures also activated regions of visual cortex that represent object shape.Together these areas contribute to a distributed neural circuit that represents food knowledge.Not only does this circuit become active during the tasting of actual foods,it also becomes active while viewing food pictures.Via the process of pattern completion,food pictures activate gustatory regions of the circuit to produce conceptual inferences about taste.Consistent with theories that ground knowledge in the modalities,these inferences arise as reenactments of modality-specific processing.Keywords:concepts,fMRI,insula/operculum,knowledge,orbitofrontal cortex IntroductionHow are concepts for everyday objects represented in the brain?Based on accumulating lesion and neuroimaging evi-dence,an object concept is represented as a distributed circuit of property representations across the brain’s modality-specific areas (Martin,2001;Martin and Chao,2001;Thompson-Schill,2003).On encountering a physical object,relevant modalities represent it during perception and action.As the object is processed,association areas partially capture property informa-tion on these modalities,so that this information can later be reactivated during conceptual processing,when the object is absent (Damasio and Damasio,1994;Simmons and Barsalou,2003).Although these conceptual reenactments share import-ant commonalties with mental imagery,there are also important differences.Mental imagery typically results from deliberate attempts to construct conscious vivid images in working memory.In contrast,the perceptual reenactments that underlie conceptual processing often appear to lie outside awareness,resulting instead from relatively automatic and implicit pro-cesses.Of primary interest,these reenactments occur in responses to words and other symbols,and play central roles in the representation of conceptual knowledge (Barsalou,1999,2003a,b;Barsalou et al.,2003a,b).The category of tools illustrates the distribution of property representations across modality-specific brain areas.When people use a hammer,a distributed set of brain areas becomes active to represent the hammer’s properties,including its visual form (ventral occipitotemporal cortex),the physical actionsused to manipulate it (ventral premotor cortex and intraparietal sulcus),and the visual motion that results (middle temporal gyrus)(Beauchamp et al.,2002;Chao et al.,1999;Chao and Martin,2000;Damasio et al.,2001;Grafton et al.,1997;Handy et al.,2003;Johnson-Frey,2004;Martin et al.,1995;Perani et al.,1995).As just described,the brain’s association areas capture this distributed set of modality-specific states for later concep-tual use.On subsequent occasions,when no hammers are present,reenactments of these states represent hammers con-ceptually (e.g.during language comprehension and thought).In the experiment reported here,we explored the distributed property account for the category of foods.Foods constitute a central category for humans,not only in perception and action,but in higher cognition (Ross and Murphy,1999).Previous research on food concepts has addressed the visual properties of fruits and vegetables,relative to the visual properties of other object categories (McRae and Cree,2002).Here,we focus instead on the tastes of high-caloric,high-fat processed foods,such as cheeseburgers and cookies (see Fig.1).We focus on taste properties because the tastes of foods are at least as important as their visual appearances.We focus on processed foods because they are central to the modern diet and because they are associated with strong gustatory and appetitive responses that underlie how people select and consume them.If a distributed circuit of property information represents food knowledge,then viewing a food picture should not only activate brain areas that represent visual properties of the pictured food,but should also activate brain areas that represent how the food is likely to taste and how rewarding it would be to eat.Once one part of the distributed circuit becomes active by viewing the picture,the remainder should become active via the conceptual inference process of pattern completion across the circuit.Given the central role that such inferences play in normal food selection and consumption,it is essential to understand their bases in the brain.Furthermore,given the extensiveness of eating disorders,obesity and other food-related problems,it is important to understand how people generate taste and reward inferences to the broad array of food representations available in modern culture.We presented pictures of food and non-food entities (loca-tion pictures)to subjects undergoing event-related fMRI and predicted that a distributed circuit of brain areas would become active to represent the visual and gustatory properties of the pictured foods.Regarding the visual properties of foods,a large literature demonstrates that ventral temporal regions underlie the representation of objects’visual form properties (Ishai et al.,1999,2000).Thus,we expected regions of the inferior temporal and fusiform gyri to respond to the distinctive visual properties of the pictured foods.Analogously,location pictures shouldÓThe Author 2005.Published by Oxford University Press.All rights reserved.For permissions,please e-mail:journals.permissions@Cerebral Cortex October 2005;15:1602--1608doi:10.1093/cercor/bhi038Advance Access publication February 9,2005at Indian Institute of Science Education and Research, Kolkata (IISER-K) on February 28, 2011 Downloaded fromactivate parahippocampal gyrus,given that this region responds to the visual-spatial properties characteristic of buildings and landmarks(Aguirre et al.,1998;Epstein and Kanwisher,1998; Epstein et al.,1999).Most importantly,the current study attempted to demonstrate that pictures of visual objects,in this case foods,can produce taste inferences.If the distributed account of concept represen-tation is correct,then multiple modality-specific regions should become active when people represent foods conceptually.Not only should visual areas become active to represent a food’s unique visual properties,gustatory areas should become active to represent how the food tastes.Once people access knowledge for a pictured food,an inference is produced about how it tastes. Even though people are not actually tasting the food,their gustatory system becomes active to represent this inference. Specifically,we predicted that simply viewing pictures of appetizing foods(relative to locations)should activate two brain regions that commonly respond to actual taste stimuli in psychophysical neuroimaging studies(Francis et al.,1999;de Araujo et al.,2003a,b;O’Doherty et al.,2001b).Thefirst area, a region in the insula/operculum,is known to represent how foods actually taste(Rolls et al.,1988;Rolls and Scott,2003; Scott et al.,1986).The second area,a region in orbitofrontal cortex(OFC),is known to represent the reward values of tastes (Gottfried et al.,2003;Rolls et al.,1989).Here we demonstrate that simply viewing pictures of processed foods activates both brain regions in much the same way that taste stimulants do in psychophysical studies.Materials and MethodsSubjectsNine right-handed,native-English-speaking volunteers from the Emory University community participated in the scanning study(six female and three male;age range,18--45years).All participants completed a health questionnaire prior to scanning and none of the participants indicated a history of neurological problems.In accordance with protocols prescribed by Emory University’s Institutional Review Board,all partici-pants read and signed an informed consent document describing the procedures and possible risks.Sixteen native-English-speaking volunteers from the Emory commu-nity participated in the stimulus selection study(ten female and six male;age19--46years).None of these volunteers participated in the later brain imaging experiment.As with the imaging participants,all participants read and signed an informed consent document describing the procedures and possible risks in accordance with protocols prescribed by Emory University’s Institutional Review Board. Experimental DesignBefore beginning the brain imaging phase of the study,32types of foods and35types of locations were selected as candidate materials.The foods(e.g.cheeseburger,spaghetti,cookie,etc.)in the list were chosen because they are all encountered frequently in American society.In addition,only processed foods that are relatively high in fat and calories were used.No fruits or vegetables were included.The locations(e.g. house,mall,school,etc.)in the list were chosen because they are all types of places that participants in the study might visit frequently. The foods and locations were equated for familiarity by having volunteers(none of whom participated in the brain imaging experi-ment)provide familiarity ratings for the35types of locations,and32 types of foods.Ratings were made on a1--7scale,with1indicating that a type of food or location was completely unfamiliar and7indicating that it was extremely familiar.Based on these ratings,15food and15 location types were selected such that no reliable familiarity differences existed between the two groups of stimuli.Between six and ten pictures for each type of food and location were then collected.A group of16participants viewed all259pictures and rated each for how typical it was of its respective food or location type.Ratings were made on a1--7scale,with1indicating that a picture was not at all typical of its food or location type and7indicating that it was very typical.For each type of food or location,the three most typical pictures were selected for use in the imaging study,thus yielding a total of90picture stimuli(45foods,45locations)equated for typicality.All of the food and location pictures depicted non-unique entities that would not be individually recognizable to the participants.Finally,23location pictures and22food pictures were randomly selected to create phase-scrambled images that were presented during scanning asfiller items(see Fig.1).During scanning,participants viewed food,location and scrambled pictures.For each picture,participants used a response pad to provide yes/no judgments as to whether it was the same or different as the preceding picture.The pictures were presented in the center of the screen for2s each.Interspersed among picture presentations were variable(‘jittered’)interstimulus intervals(mean=5.7s,range=2--20s) that were included to optimize estimation of the event-related fMRI response.During these interstimulus intervals,participants saw afix-ation cross presented in the center of the screen.Participants were instructed that when they saw thefixation cross they should continue attending to the screen and prepare for the next picture presentation. Prior to beginning data collection,participants performed an abbre-viated practice run to insure that they understood the task instructions. Functional data were collected in three scanning runs.The trial lists for the three runs were counterbalanced across participants.During each run,participants saw16food and16location pictures.Fifteen picture presentations from each category were novel pictures,while one picture was repeated to maintain the participants’attention to the picture repetition detection task.In other words,one location picture and one food picture was repeated in each scanning run.Across the three scanning runs,each subject saw three food picture repetitions and three location picture repetitions.Subjects were told in advance that repeated stimuli would occur in each run.Knowing this and given that the repeated stimuli occurred infrequently,this task requires subjects to pay close attention to each picture presentation to insure that they did not miss a repetition trial.The data from the repetition trials in each run were not analyzed given that they were only included to ensure that participants remained attentive to the task.Subjects were highly accurate at repetition detection(Mean correct=98.8%,SD= 0.94).Each5min8s run consisted of4min48s of the repetition detection task,followed by an additional20s restperiod.Figure1.Examples of location,food,and scrambled image stimuli.Cerebral Cortex October2005,V15N101603at Indian Institute of Science Education and Research, Kolkata (IISER-K) on February 28, Downloaded fromImage Acquisition and AnalysisPictures were back-projected onto a screen located at the head of the scanner and were viewed through a mirror mounted on the head coil. Stimulus presentation and response collection was controlled using Presentation software(v.0.70,).In each of the three imaging runs,154gradient echo recalled MR volumes depicting BOLD contrast were collected with a3T Siemens Trio scanner.Each volume consisted of34contiguous,2mm thick slices in the axial plane(T E=30ms,T R=2000ms,flip angle=90°,FOV= 192mm2,64364matrix).Voxel size at acquisition was33332mm, but was33333mm after spatial normalization.Prior to statistical analyses,image preprocessing was conducted in SPM99(Wellcome Department of Neurology,UK,http://www.fil.ion. ).To reduce motion-related signal changes between volumes, each participant’s scans were realigned and resliced using sinc in-terpolation.Volumes were then normalized to a template EPI scan and finally smoothed in the axial plane using a6mm isotropic Gaussian kernel.Subsequent statistical analyses were also conducted using SPM99. First,individual subjects’data were analyzed using multiple regression. For each subject,event-related changes in neural activity were modeled using afinite impulse response model corresponding to picture stimuli presentation and convolved to the standard SPM hemodynamic re-sponse function.Interstimulusfixation periods having variable durations served as the signal baseline.Global effects were removed by pro-portional scaling and the data were low-passfiltered.Condition effects at the subject level were then assessed with orthogonal contrasts comparing neural activity for food and location pictures.These contrast images,one for each participant,were then analyzed in a second-level random effects analysis of the foods--locations and locations--food contrasts using one sample t-tests.A statistical significance threshold of P<0.005(uncorrected for multiple comparisons)and a spatial extent threshold of at least seven contiguous voxels(corresponding to P<0.05 uncorrected)was used in the random effects analyses.There are at least two reasons why the use of uncorrected P-values in the present study is warranted.First,the activations reported here were identified using random effects analyses which take into account both within-and between-subjects variance.Not only does this allow the results to be generalized to the population from which subjects were drawn,but it also makes the analyses inherently robust statistically. Secondly,based on much previous research reported in the literature (see Introduction and Discussion),we started with a priori hypotheses that the insula/operculum and OFC would be active in the food--location contrasts.Additionally,given that both food and location pictures depicted common objects,both conditions should activate regions in the ventral temporal cortex known to represent objects’visual form properties.More specifically,however,we predicted that the fusiform/ parahippocampal gyrus would be active in the locations--foods contrasts. To be reported here as significant,any other areas of activity would need to be active at the P<0.05level with correction for multiple comparisons.No other areas reached this level of statistical significance. ResultsViewing food pictures for two s in a simple picture-matching task activated gustatory cortex.Specifically,food pictures, relative to location pictures,activated a region of the right insula/operculum,an area that psychophysical research has shown represents the tastes of foods(extent threshold,P= 0.004;see Table1and Fig.2).Importantly,this region was not only significantly more active for food pictures than for location pictures,but it was also reliably activated relative to thefixation baseline(one-tailed,P=0.033).In addition,food pictures,relative to location pictures, activated two regions in the left OFC that psychophysical research has shown represents the reward values of tastes. One of these regions was located in the lateral portion of the OFC(extent threshold,P=0.05;Fig.3);the other,located more superiorly,stretched into the anterior aspect of the cingulate cortex(extent threshold,P=0.01).While the lateral OFC region was reliably activated relative to thefixation baseline(P< 0.001),the more superior OFC/anterior cingulate region was not(one-tailed,P=0.155).Viewing food pictures,relative to location pictures,also produced robust activity in ventral occipitotemporal cortex, bilaterally.Two of these areas were located in the right hemisphere;one extending from the inferior occipital gyrus forward into the inferior temporal gyrus(extent threshold,P= 0.02)and the other located more anteriorly in the inferior temporal gyrus(extent threshold,P=0.035).Additional activity was observed in the left hemisphere,stretching from inferior occipital gyrus into the fusiform and inferior temporal gyri (extent threshold,P=0.001).In addition to producing significantly more activity than location pictures,food pictures reliably activated each of the ventral temporal areas above the signal baseline(P<0.0001).In constrast,and consistent with previous reports(Aguirre et al.,1998;Epstein and Kanwisher,1998;Epstein et al.,1999), location pictures,relative to food pictures,produced bilateral activity extending from the medial portion of the fusiform gyrus into parahippocampal gyrus(see Table1).Activity in these regions was not only greater for locations than foods,but was also reliably activated relative to the signal baseline(P<0.001 for both hemispheres).DiscussionThesefindings support the hypothesis that a distributed circuit of brain regions represents conceptual knowledge about foods.As Figure4a,b illustrates,viewing food pictures activated two brain regions that lie in close proximity to gustatory regions active during psychophysical studies of taste perception (Francis et al.,1999;de Araujo et al.,2003a,b;O’Doherty et al.,2001b).As Figure4a illustrates,food pictures activated the insula/operculum very near regions that become active when people actually taste glucose,sucrose,salt,or umami.As Figure4b similarly illustrates,food pictures also activate OFC very near regions that become active when people experience taste stimuli directly.The close proximity of the regions active for food pictures to well-established gustatory areas suggests that food pictures automatically activate gustatory areas to produce conceptual inferences about taste properties.The two taste areas observed here are associated with dif-ferent functions in the gustatory system.The insula/operculum receives projections from the ventroposterior medial nucleus of Table1Regions showing differential responses to food and location picturesContrast Side/location MNI coordinates Peak T Px y zFoods[locations R insula36ÿ69 5.92\0.001 L OFCÿ2133ÿ18 6.60\0.001L OFC/anterior cingulateÿ1845ÿ6 5.08\0.001aR inferior temporal gyrus48ÿ45ÿ12 5.05\0.001R inferior temporal gyrus48ÿ66ÿ9 5.99\0.001L fusiformÿ48ÿ60ÿ18 4.690.001 Locations[foods L fusiformÿ21ÿ39ÿ1214.50\0.001 R fusiform27ÿ42ÿ159.61\0.001 L,left;R,right.a While this region was significantly active for food pictures relative to location pictures,it was not reliably active relative to thefixation baseline.1604Food Pictures Activate Gustatory Cortex Simmons et al.at Indian Institute of Science Education and Research, Kolkata (IISER-K) on February 28, Downloaded fromthe thalamus (Rolls and Scott,2003),the main subcortical processing area for gustatory input,and has been associated with taste per se .The OFC,in contrast,receives projections from the insula/operculum (Rolls and Scott,2003)and has been associated with the reward values of specific tastes.Specifically,electrophysiological studies in monkeys show that the firing rates of neurons in insula/operculum are not modulated by hunger and satiety,suggesting that they represent taste in-dependent of reward (Rolls et al.,1988).Conversely,the firing rates of neurons in OFC are modulated by hunger and satiety,suggesting that they represent the current reward value of tastes (Rolls et al.,1988).Thus,when a monkey is hungry,the firing rate of OFC neurons is high,given that the reward value of food is high.Similarly in humans,greater activation occurs in gustatory OFC before participants are satiated than after (Gottfried et al.,2003).Taste reward areas are located in a different OFC region than the reward areas for other stimuli (Elliot et al.,2000;O’Doherty et al.,2001a;Rolls,2000).For example,the caudal OFC responds to olfactory rewards (de Araujo et al.,2003b;Zaldand Pardo,2000;O ngu r et al.,2003),whereas the inferiormedial OFC responds to abstract rewards (e.g.money)(O’Doh-erty et al.,2001a).Interestingly,the inferior medial OFC hasa markedly different cytoarchitectonic structure than the morelateral aspect of the OFC where taste activations occur (O ngu ret al.,2003).Thus,the OFC areas active in the present study appear to represent the reward value of tastes,rather than reward in general.As Rolls (2000,p.285)notes,‘it is important to realize that it is not just some general ‘‘reward’’that is represented in the oritofrontal cortex,but instead a very detailed and information-rich representation of which particu-lar reward or punisher is present’.Laterality of the Taste ActivationsFood pictures activated the right insula/operculum,and the left OFC.Our a priori prediction was that food pictures would activate both regions bilaterally.Examination of the psycho-physical taste literature,however,clarifies the laterality of our results.First,consider the insula/operculum.Although many psychophysical taste studies observe bilateral activity in this area,the response is typically stronger and more spatially extensive on the right (Small et al.,1999).This may explain why we only found right insula/operculum activation for food pictures.Indeed,lowering the cluster size threshold in our random effects analysis (but not the P -value threshold)revealed significant activity in a region of the left frontaloperculumFigure 2.Viewing food pictures elicits activity in insula/operculum.A high-resolution anatomical scan showing activity in right insula/operculum associated with viewing pictures of food items.The bar graph displays the average percent signal change in the right insula/operculum cluster for all nine subjects during a period between 4and 14s post-stimulus.The y -axis indicates percent signal change relative to signal baseline,with error bars representing ±1SEM of the subjects.The data shown in the bar graph were obtained in the random effects contrast of foods [locations with P \0.005.Cerebral Cortex October 2005,V 15N 101605at Indian Institute of Science Education and Research, Kolkata (IISER-K) on February 28, 2011 Downloaded from(–48,21,12)that is commonly activate in psychophysical taste studies (Small et al.,1999).Although this cluster of activity was smaller in magnitude and size relative to the activation seen on the right,it suggests that our findings are consistent with the general trend in the psychophysical taste literature for greater insula/operculum activation in the right hemisphere than in the left.With respect to the OFC,we found significant activations only on the left.It is noteworthy that studies in the psycho-physical taste literature are inconsistent with regard to later-ality,with bilateral activity reported only in approximately half of the studies.Again,lowering the cluster size threshold (but not the P -value threshold)on the random effects analysis revealed significant activity in the right OFC (15,45,–3)in nearly the identical location as seen on the left (–18,45,–6).Perhaps the best explanation,however,for why we observe activity in the left OFC comes from a recent finding by Kringelbach et al.(2003).These researchers identified an area in the left OFC where activity was correlated with subjects’ratings of taste pleasantness.Interestingly,the area theyidentified is approximately one centimeter from the activity we observed in the lateral OFC.Given that we only showed pictures of highly appetizing foods,it makes sense that we would observe activity very near the left OFC region that tracks taste pleasantness.ConclusionThe findings reported here indicate that the gustatory system produces taste responses to pictures of foods,not just to actual foods.Other studies have reported similar results.A previous neuroimaging study on pictures of foods found activation in areas near those observed here (insula and OFC),but using a blocked design with fixed-effects analyses (Killgore et al.,2003).Indeed,still other research has found that even words for tastes activate taste areas (Simmons,W.K.,Pecher,D.,Hamann,S.B.,Zeelenberg,R.and Barsalou,L.W.,under review;see Fig.4b ).In general,pictures and words appear to activate property inferences for food tastes and rewards,thus grounding conceptual knowledge in modality-specific brainareas.Figure 3.Viewing pictures of foods elicits activity in left OFC.A high-resolution anatomical scan showing activity in left OFC associated with viewing pictures of food items.The bar graph on the left displays the average percent signal change in the left OFC for all nine subjects during a period between 4and 14s post-stimulus.The bar graph on the right displays the average percentage signal change in the left OFC/anterior cingulate cluster for all nine subjects during a period between 4and 14s post-stimulus.The y -axis indicates percent signal change relative to signal baseline,with error bars representing ±1SEM of the subjects.The data shown in the bar graphs were obtained in the random effects contrast of foods [locations with P \0.005.1606Food Pictures Activate Gustatory CortexSimmons et al.at Indian Institute of Science Education and Research, Kolkata (IISER-K) on February 28, 2011 Downloaded fromIn the experiment reported here,taste inferences arose even when subjects performed fast superficial processing of food stimuli.Subjects were required to only assess whether the current picture exactly matched the previous picture,each presented for only 2s.No categorization or other form of conceptual processing was required.Furthermore,the large majority of trials required the subject to note that the current picture differed from the previous picture,a judgment that could have potentially interfered with making conceptual inferences.In general,the fact that taste inferences were produced under this particular set of task conditions attests to their strength and ubiquity.Consistent with previous findings,the experiment here indicated that conceptual representations are distributed across the brain areas that underlie their processing in perception and action.Because different categories are associated with differ-ent distributions of multimodal properties (McRae and Cree,2002),different categories rely on different configurations of brain areas for conceptual representation.As reviewed earlier,much work has shown that thinking about tools activates brain areas that process visual form,visual motion,and object manipulation.Analogously,we have shown here that thinking about food activates brain areas that process taste,taste reward and food shape.Thus our findings support the view that thebrain areas representing knowledge for a particular category are those typically used to process its physical instances.Besides having implications for theories of distributed con-ceptual representation,these findings have implications for various societal issues related to food,such as eating disorders,obesity and advertising.Taste inferences in the gustatory system,as observed here,arise in response to a wide variety of food stimuli in the environment and in the media.In eating disorders and obesity,the perception of foods and food pictures,as well as thoughts of food,may be associated with dysfunctional inferences about taste and reward.Conversely,behavioral,cognitive and pharmacological interventions may,in part,restore the gustatory activity underlying inferences about taste and reward to more normal forms.NotesThis work was supported by NIMH grant 1F31MH070152-01to K.S.and National Science Foundation grants SBR-9905024and BCS-0212134and Emory University research funds to L.W.B..We are grateful to Melissa Armstrong and Christine Wilson for their assistance in stimulus preparation.Correspondence should be addressed to Lawrence W.Barsalou,Department of Psychology,Emory University,532North Kilgo Circle,Atlanta,GA 30322,USA.Email:barsalou@emory.ed.Figure 4.(a )Locations of peak right hemisphere insula/operculum activations reported in taste perception studies.(b )Locations of peak left OFC activations across various tasks.The squares in the insula/operculum at Z =20and Z =ÿ9represent peak activations observed when participants taste sucrose,whereas the square in the lateral OFC at Z =ÿ10is the peak activation in the area observed to respond to the combination of gustatory and olfactory stimuli,and thus is a likely candidate for being the center of flavor representation (de Araujo et al.,2003).The square in the insula/operculum at Z =13indicates an area of common activation when participants tasted either glucose or salt (O’Doherty et al.,2001).The squares in the insula/operculum at Z =10and in the OFC at Z =ÿ6indicate the peak activations observed when participants taste umami (de Araujo et al.,2003).The squares in the insula/operculum at Z =5and in the OFC at Z =ÿ18represent peak activations when participants tasted glucose (Francis et al.,1999).Diamonds in the inferior medial OFC represent peak activations observed when participants receive abstract rewards (O’Doherty et al.,2001).The circle in the OFC at Z =ÿ10represents peak activation observed when participants verify the taste properties of concepts using strictly linguistic stimuli (Simmons,Pecher,Hamann,Zeelenberg,and Barsalou,under review).Finally,the circles in the insula/operculum at Z =9and in the OFC at Z =ÿ18and Z =ÿ6indicate the activation peaks observed in the present study when participants viewed food pictures.When necessary,coordinates reported in other studies were converted from Talairach to MNI space.Cerebral Cortex October 2005,V 15N 101607at Indian Institute of Science Education and Research, Kolkata (IISER-K) on February 28, 2011 Downloaded from。

Plant Cell, Tissue and Organ Culture 64: 145–157, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.145Oxidative stress and physiological, epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineersAlan C. Cassells & Rosario F. CurryDepartment of Plant Science, National University of Ireland, Cork, Ireland (∗ requests for offprints; Fax: +353-21274420; E-mail: a.cassells@ucc.ie)Received 18 April 2000; accepted in revised form 1 December 2000Key words: DNA repair, free radicals, genetic engineering, hyperhydricity, in vitro culture, juvenility, micropropagation, mutation, reactive oxygen species, somaclonal variation, tissue cultureAbstract A number of well defined problems in physiological, epigenetic and genetic quality are associated with the culture of plant cell, tissue and organs in vitro, namely, absence or loss of organogenic potential (recalcitrance), hyperhydricity (‘vitrification’) and somaclonal variation. These broad terms are used to describe complex phenomena that are known to be genotype and environment dependent. These phenomena affect the practical application of plant tissue culture in plant propagation and in plant genetic manipulation. Here it is hypothesised much of the variability expressed in microplants may be the consequence of, or related to, oxidative stress damage caused to the plant tissues during explant preparation, and in culture, due to media and environmental factors. The characteristics of these phenomena are described and causes discussed in terms of the known effects of oxidative stress on eukaryote genomes. Parameters to characterise the phenomena are described and methods to remediate the causes proposed. Abbreviations: AFLP – amplified fragment length polymorphism; FISH – fluorescent in situ hybridization; HSP – heat shock proteins; PR-proteins – pathogenesis-related proteins; RFLP – restriction fragment length polymorphism; ROS – reactive oxygen species Introduction Those using tissue culture for multiplication or transformation are concerned to produce microplants that are ‘fit for the purpose’, that is, free of specified diseases, vigorous, developmentally normal and genetically true-to-type (Cassells, 2000a, b; Cassells et al., 2000). The exceptions are that the market may exploit altered developmental characteristics, e.g. juvenility in herbaceous or woody plants where this results in greater productivity of the microplants when used for cutting production (George, 1993, 1996) or where tissue protocols give earlier flowering (Cassells, 2000a). In general, genotype-dependent, multiplication via buds tends to be the preferred strategy to maintain genetic stability (Figure 1; George, 1993). Proliferation of side shoots from axillary buds, termed ‘nodal culture’ is preferred over proliferation of precocious axillary buds in shoot tip explants. The basis for this is that apical explants may give rise to basal explant callus from which adventitious buds may arise. The latter has been associated in strawberry with genetic variability in the progeny (Jemmali et al., 1997). It is important to mention here that epigenetic and genetic instability in the tissues used for Agrobacterium transformation (somaclonal variation: see Jain et al., 1998), that is expressed in adventitious shoots, may result in chimeral transformants (Cassells et al., 1987), and in somaclonal variation in the background of the transgenic lines (Sala et al., 2000) which may contribute to the silencing of trangenes (Matzke and Matzke, 1998). In human health the importance of oxidative stress has been long recognised in cancer and ageing studies146Figure 1. The methods of micropropagation least likely to produce plants with genetic variation (reproduced with permission; from George, 1993).(Harman, 1956). It is also recognised how complex the underlying mechanisms and processes are (Halliwell and Aruoma, 1993). The cellular mechanisms to manage stress, namely, constitutive and induced production of radical scavengers, free radical and oxidised-protein enzymatic degradation pathways and DNA repair mechanisms are highly conserved in all eukaryotes (Halliwell and Aruoma, 1993; McKersie and Leshem, 1994). Environmental and pathogeninduced stress have been investigated in detail in plants in vivo (Bolwell et al., 1995; Baker and Orlandi, 1995; Doke et al., 1996; McKersie and Leshem, 1994). Stress-like phenomena expressed in vitro and in microplants have been extensively described but less is known about the underlying causal mechanisms (Ziv, 1991; Jain et al., 1998). Both in initiating cultures and in sub-culturing, explant preparation involves wounding of the tissues which is known to cause oxidative stress (Yahraus et al., 1995). Elicitors of oxidative stress e.g. hypochlorite (Wiseman and Halliwell, 1996) and mercuric salts (Patra et al., 1997), are used to surface sterilize theprimary explants. Many factors associated with aberrations in plant tissue culture such as habituation, hyperhydricity (Gaspar, 1998) are caused by oxidative stresses (Keevers et al., 1995), such as high salt (McKersie and Leshem, 1994), water stress (NavariIzzo et al., 1996), mineral deficiency (Elstner, 1991), excess metal ions (Caro and Puntarulo, 1996) and possible over exposure to auxin (Droog, 1997). Oxidative stress (Gille and Sigler, 1995; Bartosz, 1997) is defined as an imbalance in the pro- versus anti-oxidant ratio in cells and results in elevated levels of pro-oxidants (ROS: reactive oxygen species; including superoxide, hydrogen peroxide hydroxyl, peroxyl and alkoxyl radicals) (Wiseman and Halliwell, 1996) which can cause cell damage (Sies, 1991). ROS (Figure 2) can react with a spectrum of metabolites, proteins including enzymes, and nucleic acid molecules (Gille and Siegler, 1995). Oxidised enzymes which may be inactivated, are degraded by cytosolic proteinases (Laval, 1996). The influence of ROS, through altered cell redox potential, on the cell cycle and oxidative damage to both nuclear and organellar147Figure 2. Reactive oxygen species (ROS) produced constitutively in the cell. The upper section shows the natural antioxidants and enzymes used to minimize the toxic effects of ROS. The lower section gives selected examples of the harmful effects of ROS when the pro- and anti-oxidant balance is perturbed in oxidative stress. (MDE, malondialdehyde; HNE, 4-hydroxynonenal).DNA, may result in mutations (Figure 3; Bohr and Dianov, 1999). Oxidative damage in eukaryote cells is expressed in altered hyper- and hypomethylation of DNA (Kaeppler and Phillips, 1993; Tilghman, 1993; Wiseman and Halliwell, 1996; Cerda and Weitzman, 1997; Wacksman, 1997); changes in chromosome number from polyploidy to aneuploidy, chromosome strand breakage, chromosome rearrangements, and DNA base deletions and substitutions (Gille et al., 1994; Czene and Harms-Ringdahl, 1995; Hagege,1995). Such changes could explain, at least in part, the range of variability found in plant cells, tissues and organs in culture and in microplants, namely, recalcitrance including loss of cell competence (Hagege, 1995; Lambe et al., 1997), hyperhydricity (Olmos et al., 1997) and somaclonal variation including epigenetic and genetic variation (Jain et al., 1998; Joyce et al., 1999; Kowalski and Cassells, 1999). The objective of this review is to discuss tissue culture variability, its causes, detection and remediation148 with emphasis on the possible role of oxidative stress in this phenomenon.Aberrations and variation expressed in vitro At the outset it should be recognised that explants, other than buds, from dicot plants have different characteristics to those from monocots, specifically those of dicots may have a cambium; that there are differences in organogenetic potential between families, genera, species and genotypes; and that different genotypes of a species may show widely different responses (George, 1993, 1996). Further there are differences in the responses of explants from different parts of a plant, which change ontogenetically (George, loc. cit.). Some trends are evident, e.g. increased recalcitrance with advancing age of the cultures (Hagege, 1995) and increased somaclonal variability in microplants with increasing sub-culture number (Brar and Jain, 1998). With a given genotype, wounding of the tissues on cutting (excision), and tissue damage and exposure to sterilants during sterilisation, and suboptimal in vitro factors (Ziv, 1991) are important in relation to genomic damage. So called ‘pre-existing’ genomic diversity at the cell level (D’Amato, 1964; Figure 4) and wound or oxidative damage due to wounding may explain some of the variability subsequently seen in vitro and in the resulting microplants. Possible stress due to unbalanced media, bad culture vessel design and environmental stress may also, or further, contribute to the genetic, epigenetic (developmental) and physiological variability recorded (Ziv, 1991). Wounding or excision per se may be considered both as a trigger for cell division (Sangwan et al., 1992) and as a damaging oxidative burst (Schaaf et al., 1995; Yahraus et al., 1995). As a consequence of the above factors, explants may senesce, fail to respond, undergo cell division and/or produce adventitious organs or somatic embryos. In responding genotypes, the response is generally regulated in a predictable way by manipulation of the auxin to cytokinin ratio and absolute growth regulator concentrations (Skoog and Miller, 1957). In some cases, recalcitrance may be overcome by pulsing in sequence with auxin followed by cytokinin (Christianson and Warnick, 1985). Whether genome variability is ‘pre-existing’, caused by oxidative stress on wounding an/or caused by stress in culture (Figure 4), selection may begin in vitro with the appearance of sectoring in the callus (D’Amato et al., 1980). Cell lineFigure 3. Changes in DNA caused by oxidative stress which can lead to recalcitrance, loss of competence, hyperhydricity and somaclonal variation.selection for in vitro conditions may result in loss of competence; e.g. selection based on fitness of grossly altered genotype(s) may result in the irreversible loss of competence (Hagege, 1995). The main morphological aberration seen in shoots in vitro cultures, both in nodal/bud derived shoots and in adventitious shoots, is hyperhydricity (‘vitrification’) (Debergh et al., 1992). This term is used to describe aberrant morphology, typically hyperhydrated, translucent tissues and physiological dysfunction in plant tissues in vitro (Ziv, 1991). It is also associated with leaf-tip and bud necrosis. The latter often leads to loss of apical dominance in the shoots and is associated with callusing of the stem base. An important characteristic of this condition is impaired stomatal function which causes problems in establishing microplants (Preece and Sutter, 1991). Morphological variability in plants from in vitro culture may be seen in intrapopulation variability (within a population of adventitiously regenerated plants) (Kowalski and Cassells, 1998) and interpopulation variability (between populations of in vitro plants) (Joyce et al., 1999). The latter may arise when plants are propagated on different media or in culture vessels with different characteristics (Joyce et al., 1999). Intrapopulation variability can be a result of the loss of specific viruses, including cryptic viruses, from some of the regenerated plants (Matthews, 1991); chimeral breakdown, rearrangement and/or synthesis of unstable chimeral plants (Tilney-Bassett, 1986). In generally, heritable somaclonal variation (Larkin and149Figure 4. Sources of genetic variation in plants obtained through organogenesis in callus cultures (reproduced with permission; from George, 1993).Scowcroft, 1981) has the characteristics of mutation (Anonymous, 1995; Jain et al., 1998), albeit occurring at higher frequency than occurs spontaneously in seed or vegetative propagules (Preil, 1986). It is genotype-dependent and dependent on the pathway of regeneration (Karp, 1991). Epigenetic changes can occur in vitro culture resulting in ‘apparent rejuvenation’ (Pierik, 1990) affecting woody and herbaceous plants (Huxley and Cartwright, 1994; James and Mantell, 1994; Jemmali et al., 1994; Cassells et al., 1999 a, b). Interpopulation variation is usually cryptic, as control populations are not available for comparison; it is recognised in quality differences in plants produced by different protocols or by different micropropagators (GrunewaldtStoker, 1997). Examples of interpopulation variability are populations differing in degree of hyperhydricity or juvenility (Swartz, 1991). While woody plant propagators are familiar with phase change (Howell, 1998), micropropagators of herbaceous plants appear less conscious of this phenomenon but it has implications for disease susceptibility in that polygenic resistance develops as the plant soma matures (Agrios, 1997) and for time to flowering (Howell, 1998). Plants showing prolonged juvenility (epigen-etic/ontogenetic variability) may be more susceptible to damping-off diseases (Agrios, 1997). This is not always the case, as juvenile tissues are reported to have enhanced resistance to fusaric acid (Barna et al., 1995) and Cassells et al. (1991) have shown that potato crops derived from microplants, showing juvenility compared to a tuber-derived crop, were more resistant to potato blight. In vitro plants may have a longer time to flowering compared to those from vegetative propagules (Cassells et al., 1999a). While morphological intrapopulation variability and ontogenetic and physiological variation, expressed in interpopulation variability, are well recognised phenomena in micropropagation, cryptic intrapopulation variation in juvenility has also been detected in adventitiously regenerated plant populations showing genetic variation (Kowalski and Cassells, 1998) suggesting that genetic and epigenetic variability are not necessarily discrete but can occur in the same population. Somaclonal variation is strongly expressed after the microplant population establishment stage as interplant variation in morphological characters. Some of the plants may show characteristics of chimeral breakdown (Tilney-Bassett, 1986). Somaclonal variation has been extensively reviewed in Jain et al. (1998).150Figure 5. Showing the consequences of oxidative stress from induction of host antioxidant defences (repair, heat shock protein induction, pathogenesis related protein induction) to mutation, programmed cell death and uncontrolled cell death. Figure 6. The relationship between stress inducers e.g. medium salt stress, gene activation and the generation of biomarkers for stress remediation and stress damage repair. Examples of damage exposure are ethylene and ethane; of damage are oxidised bases e.g. 8-oxoguanine; of remediation are glutathione and glutathione reductase and of repair, Poly(ADP-ribose) polymerase (see text for further markers).As discussed above, shifts in characters in populations, e.g. physiological or developmental changes, are not readily recognised unless control populations are available (Cassells et al., 1997). These can, however, be visually expressed in loss of apical dominance, leaf number and leaf size and, more importantly in the time to flowering, and yield quality e.g. tuber number and size distribution in potato seed production (Cassells et al., 1999a).Characterisation of epigenetic and genetic changes in microplants pre- and post- establishment Cytometric analysis of callus has shown variability in chromosome number and ploidy in tissue culturederived plants (Geier, 1991; Gupta, 1998). Investigations indicate more chromosome variability in the callus phase than in adventitious shoots (D’Amato et al., 1980), indicating a loss of competence in the more seriously disturbed genomes (Valente et al., 1998). Cell line selection for secondary product formation also shows differences at the metabolite level (Berglund and Ohlsson, 1995). While occasional albino shoots are observed, the expression of morphological variation is difficult to assess in vitro due to variability between shoots due to temporal differences in shoot initiation and because of the limited leaf expansion in in vitro cultures. Variability in both qualitative and quantitative traits has also been reported (Karp, 1991). The latter expressed in increased standard deviations of the character mean (DeKlerk, 1990) and can be quantified using computerised image analysis (Cassells et al., 1999a). Analysis of DNA-base methylation and various genetic fingerprinting techniques have also been used to confirm and characterise variability in tissue culturederived plants, confirming both morphological and cryptic genetic and epigenetic variability between and within populations (Karp et al., 1998; Cassells et al., 1999b).Current views on the molecular basis of somaclonal variation In recent years plant cell, tissue and organ culture has been developed for applications in plant genetic manipulation (Cassells and Jones, 1995). In this field, somaclonal variation has attracted considerable interest as a means of improving crop plants (Jain et al., 1998). Reviews discussed a number of mechanisms to explain somaclonal variation, these included changes in chromosome number, chromosome breakage and rearrangement, DNA amplification, point mutations, changes in DNA methylation, changes in organellar DNA, activation of transposons (Frahm et al., 1998; Gupta, 1998; Henry, 1998; Jain et al.,151 1998; Kaeppler et al., 1998). The mechanisms appear to be equally applicable to explaining the basis of variation at the cell and callus level and are similar to the variability resulting from oxidative genome damage and induced mutation. Nagl (1990) has discussed the relationship between stress-induced and ontogenetic changes in plant genomes arguing that plant genomes are inherently fluid. In some genomes, e.g. flax (Schneeberger and Cullis, 1991) and banana (Cullis and Kunert, 2000) there are well characterised genomic instabilities associated with somaclonal variation. (Figure 5). In addition to the above ROS, chemical and physical agents can stimulate lipid peroxidation that can become autocalatytic resulting in the production of organic hydroperoxides (Figure 2). ROS in the presence of iron and copper ions may generate highly mutagenic compounds, e.g. peroxyl radicals and alkoxyl radicals (Koh et al., 1997). The primary response to elevated ROS production (Figure 2) is stimulation of production of antioxidant molecules (radical scavengers) such as ascorbic acid and glutathione in the aqueous phase and alpatocopherol and carotenoids in the lipid phase (Gille and Sigler, 1995) and the activation of antioxidant enzyme systems including superoxide dismutase, catalase and glutathione and ascorbic acid peroxidases (Tsang et al., 1991; Larson, 1995; Smirnoff, 1996). Additional responses involve the activation of heat shock proteins to protect enzymes systems against ROS damage (Burdon, 1993) and of proteases to degrade damaged proteins (Stadtman, 1992). More significant is the potential of ROS to cause DNA damage (‘genotoxicity’; Wiseman and Halliwell, 1996). The cell cycle slows or shuts down to minimise the transmission of mutations to daughter cells through mitosis and to facilitate DNA repair (Logemann et al., 1995; Amor et al., 1998; Reichheld et al., 1999) and DNA repair mechanisms, the SOS response, are activated (Laval, 1996; Yamamoto et al., 1997; Vonarx et al., 1998). The outcomes of oxidative stress depend on the balance between pro and anti-oxidants responses. Imbalance may lead to controlled responses (Figure 5) such as induced resistance to pathogens (Ernst et al., 1992), excessive imbalance to cell damage and mutation (Wiseman and Halliwell, 1996), possible programmed cell death (apoptosis) (Polyak et al., 1997) and, in the extreme, to (unprogrammed) cell death (Hippeli and Elstner, 1996). Oxidative stress has been linked to recalcitrance in protoplast culture (Benson and Roubelakis-Angelakis, 1994). Increase in ROS is associated with a range of biotic and abiotic stresses (Gile and Siegler, 1995; Bartosz, 1997). These include salt, drought, heat, and UV-induced stresses. They are also induced by chemical and physical mutagens (Anon, 1977). Their protective and constitutive roles include direct protection against pathogen attack, and involves the role of H2 O2 as a messenger in the induction of host resistance (pathogenesis-related (PR) protein induction) (Lamb and Dixon, 1997). H2 O2 may have a role in xylem formation and other cell-death processes in plants (Howell, 1998). ROS have a role in creatingThe relationship between somaclonal variation and spontaneous mutation The paper of Shepard et al. (1980) on somaclonal variation in potato stimulated interest in the application of this variability in crop improvement but was soon followed by concern about the quality of somaclonal variation and whether it differed qualitatively from spontaneous mutation (Sanford et al., 1984). This issue has been discussed by Karp (1991, 1995) but while it is still controversial, somaclonal variation is used in plant improvement, with induced mutagenesis whose efficiency has been improved by exploiting in vitro plant systems (Cassells, 1998). More importantly here, induced mutation and somaclonal variation result in a qualitatively similar, if not quantitatively identical, spectrum of DNA changes (see Figure 3). The issue is whether somaclonal variation and other tissue culture variability are mechanistically like physically-induced mutation and are caused by reactive oxygen species (Anonymous, 1977; 1995; Micke and Donini, 1993).Oxidative stress and mutation Oxidative stress is caused by the unremediated hyperactivity of reactive oxygen species (Figure 2). ROS such as superoxide, hydrogen peroxide and the hydroxyl radical are metabolic intermediates in respiration and photosynthesis and other metabolic activities in plants (see review by Gille and Sigler, 1995; Bartosz, 1997). Their natural cytoplasmic toxicity and genotoxicity is controlled by antioxidants and enzymic pathways in the cell (Hippeli and Elstner, 1996). Various environmental signals (Bartosz, 1997), which lead to an increase in ROS are associated with induced mechanisms to minimise their harmful effects152 variability in the plant genome by activating transposons (Mhiri et al., 1997), inducing polyploidy, chromosome breakage/rearrangements and base mutations (Figure 2). DNA based changes induced by ROS may inhibit methylating enzymes leading to hypomethylation (Cerda and Weitzman, 1997; Wacksman, 1997), e.g. formation of 8-oxoguanine occurs at high frequency which may lead to mismatch at DNA repair (base mutation, e.g. AT–GC changes), similarly for other oxidative base changes. While much of the ROS-induced effects due to wounding may be localised, some ROS can migrate across membranes, e.g. H2 O2 and cause effects directly, or via membrane lipid peroxidation, at the level of DNA in the stressed cells or in neighbouring cells (Figure 2). A gradient of stress damage from the wound area back into the explant may be hypothesised. ROS (and physical mutagens) have been confirmed in animal cells (Halliwell, 1999) as causing the range of epigenetic and genetic changes in DNA that are problematic in plant tissue culture (Figure 3). number and DNA content (Quicke, 1993; Curry and Cassells, 1998). Techniques including fluorescent in situ hybridisation (FISH) (Maluszynska and HeslopHarrison, 1991) and Giemsa banding (Quicke, 1993) are used to look for somatic recombination, including chromosome breakage and rearrangement and may also be used to detect DNA amplification and reduction. Changes in DNA base methylation can be investigated using methylation-sensitive restrictions in RFLP and AFLP analysis (Karp et al., 1998) or by PCR of bisulphite modified DNA (Joyce et al., 1999).Plant hormones and oxidative stress Plant hormones implicated in hyperhydricity (vitrification) include cytokinins, auxins, and the auxin/cytokinin ratio; gibberellic acid and ethylene (Ziv, 1991; George, 1996). Oxidative stress has been associated with auxin and cytokinin metabolism in Agrobacterium induced tumours (Jia et al., 1996). Ethylene is also strongly linked to oxidative stress (McKersie and Leshem, 1994; Pell et al., 1997). Injury has been shown to activate the oxidation of IAA, while kinetin is reported to be a secondary product of oxidative stress (Barciszewski et al., 1997). In vitro, various media factors have been shown to induce stress, including hormones, and mineral nutrients (Ziv, 1991; George, 1993, 1996) there is evidence that metal toxicities and deficiencies may generate ethylene through oxidative stress (Lynch and Brown, 1997). Oxidative stress also affects cytosolic calcium (Price et al., 1994). Oxidative stress has been suggested as a cause of guard cell malfunction (McAnish et al., 1996). Calcium has been implicated in increased stress tolerance (Gong et al., 1997).Parameters used to characterise oxidative stress A number of methods have been used to monitor/characterise oxidative stress (Figure 6). These include measurement of the redox potential (Reichheld et al., 1999), measurement of stress related metabolites e.g. ascorbic acid (Smirnoff, 1996), glutathione (de Vos et al., 1994), hydrogen peroxide (Schreck et al., 1996). Ethylene has been monitored in hyperhydricity (‘vitrification’) studies (Keevers and Gaspar, 1985) and along with ethane, a marker for lipid peroxidation, has been used to monitor stress in vitro (Cassells et al., 1980; Cassells and Tamma, 1985). Thiobarbituric acid reactive substances are also used to assess lipid peroxidation (Laszczyca et al., 1995). 8oxo-2 -deoxyguanosine (Kasai, 1997; Bialkowski and Olinski, 1999) is considered to be a reliable indicator of genotoxicity as are other bases modified by ROS (Yamamoto et al., 1997). Enzymes of oxidative metabolism (Mehlhorn, 1990), enzymes associated with the cell cycle (Chiatante et al., 1997), enzymes of the SOS response (Laval, 1996; Wiseman and Halliwell, 1996) e.g. poly(ADP-ribose)-polymerase (Amor et al., 1998), screening for heat-shock proteins (HSP) (Burden, 1993) and PR proteins (Glandorf et al., 1997) have also been used as oxidative stress monitors. Karyotyping, flow cytometry and microdensitometry can be used to measure changes in chromosomeRemediation of oxidative and other tissue culture associated stresses Remediation of oxidative stress can be based on several strategies. Genotypes can be screened for their sensitivity to stress and sensitive genotypes avoided; or they can be bred, mutated or engineered for increased stress tolerance (Gupta et al., 1993). The breeding/genetic manipulation options are both relatively long-term and costly and can only be applied to individual genotypes (Jones and Cassells, 1995). An important consideration where plant tissue culture is used for cloning or transformation is the choice153 of the explant. While mature plant tissue may be polysomatic (D’Amato, 1964), this may not be so in the case of the tissues of young plants in vitro (Curry and Cassells, 1998; Curry and Cassells, unpublished). Selection of explants from the latter may avoid the problem of ‘pre-existing’ variation (Figure 4). The main option, however, is the use of stable pathways of multiplication (Figure 1: George, 1993, 1996); albeit, even with these genetic drift may occur (Jemmali et al., 1997). There is evidence e.g. that protoplasts give rise to greater variability than tissue explants as expressed in the greater variability in plants regenerated from the former (Sree Ramulu et al., 1984). This may reflect expression of somatic cell variability (Figure 4) but it also appears to reflect the stress experienced in their isolation and regeneration where they are exposed to osmotic stress and the toxic effects of cell wall degrading enzyme preparations (Cassells and Tamma, 1985) and this has implication for their use in transformation via electroporation or biolistics at the protoplast level (Christou, 1995) as opposed to bombardment of apices (Sautter et al., 1995). The immediacy of the oxidative stress caused by excision and manipulation (Yahraus et al., 1995) would suggest that treatment of the explant after excision would be relatively ineffectual. As an alternative, it is suggested that stress be applied to the donor plant (or in vitro microplant) before excision. Under in vivo conditions, it was been shown that stress exposure induces cross-stress tolerance, e.g. UV treatment not only increases tolerance to further UV exposure but also to pathogen induced stress (Ernst et al., 1992), similarly paclobutrazol which is used in vitro and in weaning treatments (George, 1993, 1996) reduces oxidative stress to high light and high temperature (Mahoney et al., 1998). It has been suggested that induction of heat shock proteins be used to protect against oxidative stress (Banzet et al., 1998; Sebehat et al., 1998). Prevention or reduction of oxidative stress damage in vitro may be possible by manipulating hormone and mineral nutrients using the above oxidative stress parameters to monitor the protocols as has been done in the case of protoplasts (Cassells et al., 1980; Cassells and Tamma, 1985). In vitro calli and tissues are more amenable than intact tissues to permeation techniques. Cells, calli and explants can be infiltrated and bathed with radical scavengers such as ascorbic acid, mannitol and dimethyl sulphoxide. The stress messenger hydrogen peroxide can be broken down by extracellular catalase. Manipulation of media composition, particularly using simple media formulations in autotrophic or microhydroponic culture (Cassells, 2000a) and modification of culture vessel design to facilitate controlled gaseous exchange (Cassells and Walsh, 1998), can greatly influence microplant resistance to hyperhydricity and improve ontogenic development (Cassells, 2000b).Conclusions It is speculated here that problems underlying the application of plant tissue culture systems in plant cloning (micropropagation) and genetic transformation, namely, recalcitrance, hyperhydricity, poor physiological quality, genetic and epigenetic variation, may have a common basis, at least in part, in oxidative stress-induced damage. This damage is caused by an overwhelming of the antioxidative defenses by primary ROS and secondarily, by the production of ROS by lipid autocatalytic peroxidation (Figure 2). While damaged proteins may be broken down by proteinases, damage is fixed in the DNA. Specific levels of DNA damage may result in cell death or programmed cell death, other levels in loss of cell competence, altered methylation patterns may result in epigenetic changes, or in mutations (Figure 3). Somaclonal variation shows a similar spectrum of genetic variation to induced mutation; and oxidative stress and irradiation are known to involve ROS. Oxidative stress damage which it is emphasised is genotype dependent, may also operate at the physiological level since ROS have been shown to influence plant hormones namely, cytokinin, auxin, ethylene metabolism. Oxidative stress also influences calcium metabolism which in turn is involved in auxin transport, guard cell function and as a secondary messenger. Remediation strategies have been proposed and some makers of oxidative stress listed. It is suggested that induction of heat shock proteins may confer cross tolerance to the stress phenomena encountered as problems in establishing plant tissue cultures. Media and environmental manipulations should be carried out aimed at reducing in vitro stress based on adjusting the media composition and the physical culture environment, paying special attention to hormone stress, mineral composition and water and light stress. Reactive nitrogen species (NOS) not discussed here, can。

第一章绪论一简答题1. 21世纪是生命科学的世纪。

20世纪后叶分子生物学的突破性成就，使生命科学在自然科学中的位置起了革命性的变化。

试阐述分子生物学研究领域的三大基本原则，三大支撑学科和研究的三大主要领域？答案：（1）研究领域的三大基本原则：构成生物大分子的单体是相同的；生物遗传信息表达的中心法则相同；生物大分子单体的排列（核苷酸，氨基酸）导致了生物的特异性。

（2）三大支撑学科：细胞学，遗传学和生物化学。

（3）研究的三大主要领域：主要研究生物大分子结构与功能的相互关系，其中包括DNA和蛋白质之间的相互作用；激素和受体之间的相互作用；酶和底物之间的相互作用。

2. 分子生物学的概念是什么？答案：有人把它定义得很广：从分子的形式来研究生物现象的学科。

但是这个定义使分子生物学难以和生物化学区分开来。

另一个定义要严格一些，因此更加有用：从分子水平来研究基因结构和功能。

从分子角度来解释基因的结构和活性是本书的主要内容。

3 二十一世纪生物学的新热点及领域是什么？答案：结构生物学是当前分子生物学中的一个重要前沿学科，它是在分子层次上从结构角度特别是从三维结构的角度来研究和阐明当前生物学中各个前沿领域的重要学科问题，是一个包括生物学、物理学、化学和计算数学等多学科交叉的，以结构（特别是三维结构）测定为手段，以结构与功能关系研究为内容，以阐明生物学功能机制为目的的前沿学科。

这门学科的核心内容是蛋白质及其复合物、组装体和由此形成的细胞各类组分的三维结构、运动和相互作用，以及它们与正常生物学功能和异常病理现象的关系。

分子发育生物学也是当前分子生物学中的一个重要前沿学科。

人类基因组计划，被称为“21世纪生命科学的敲门砖”。

“人类基因组计划”以及“后基因组计划”的全面展开将进入从分子水平阐明生命活动本质的辉煌时代。

目前正迅速发展的生物信息学，被称为“21世纪生命科学迅速发展的推动力”。

尤应指出，建立在生物信息基础上的生物工程制药产业，在21世纪将逐步成为最为重要的新兴产业；从单基因病和多基因病研究现状可以看出，这两种疾病的诊断和治疗在21世纪将取得不同程度的重大进展；遗传信息的进化将成为分子生物学的中心内容”的观点认为，随着人类基因组和许多模式生物基因组序列的测定，通过比较研究，人类将在基因组上读到生物进化的历史，使人类对生物进化的认识从表面深入到本质；研究发育生物学的时机已经成熟。

血管性痴呆（vascular dementia,VD）是一种由脑血管病变导致的疾病，其临床症状包括引起记忆和执行功能障碍等。

它被认为是继阿尔茨海默病之后的第二大常见痴呆类型[1]。

目前，在亚洲和发展中国家的痴呆病例中，VD 约占30%，高于北美和欧洲（15%～20%）[2-3]。

据研究资料显示，我国60岁及以上人群的血管性痴呆发病率为每年每千人中有2.42例[4-5]。

研究表明，我国约有1507万人60岁以上的痴呆患者，其中约有392万人为VD 患者[6]。

VD 会造成日常生活质量不断下降，而且不能扭转，给家庭和社会带来极大的冲击和负担。

复方苁蓉益智胶囊是由王永炎院士多年临床实践研制的具有益智养肝,化浊活血和增智健脑等功效的中成药[7]，主料何首乌、肉苁蓉、荷叶、地龙、漏芦等。

Progress of compound ciYizhi capsule in the treatment of vascular dementia Di Shuai, Zhang Jiapeng, Liu Yixuan, LiYanan, Zhang Jiang, Zhou Fuling. The Affiliated Hospital of North China University of Science and Technology, Tangshan 063000, China【Abstract 】Compound ciYizhi capsule has the effect of nourishing liver,promoting turbidity and activating blood, and increasing wisdom and brain. It is suitable for mild to moderate vascular dementia with liver and kidney deficiency and phlegm stasis blocking collateral syndrome. Recently, it has been widely used in the long-term and synergistic treatment of vascular dementia with remarkable efficacy.To summarizes the clinical and experimental studies of compound ciYizhi capsule. It is found that compound ciYizhi capsule can treat vascular dementia by reducing the expression of MARKS mRNA in hippocampus, inhibiting oxidative stress in brain tissue, protecting mitochondria, reducing the range of cerebral infarction, protecting cerebral ischemic injury and pound ciYizhi capsule combined with other anti-dementia drugs can significantly improve the clinical symptoms of patients with vascular dementia and improve the self-care ability and quality of life.In order to provide some reference for the subsequent study of compound cistanche qianyi capsule.【Key words 】Vascular dementia; Compound ciYizhi capsule; Dementia; Clinical application 复方苁蓉益智胶囊治疗血管性痴呆的研究进展邸帅 张佳朋 刘乙璇 李亚楠 张江* 周福玲作者单位：063000 河北省唐山市，华北理工大学附属医院神内二、四病区*通讯作者【摘要】 复方苁蓉益智胶囊具有益智养肝,化浊活血和增智健脑的功效，适用于肝肾亏虚兼痰瘀阻络证的轻中度血管性痴呆。