Evaluation of transgenic tomato plants ectopically expressing
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Evaluation of transgenic tomato plants ectopically expressing
the rice Osmyb4gene
Candida Vannini a ,*,Manuela Campa a ,Marcello Iriti b ,c ,Annamaria Genga d ,
Franco Faoro b ,c ,Sara Carravieri a ,Giuseppe Rotino e ,Mara Rossoni f ,
Anna Spinardi f ,Marcella Bracale a
a
Dipartimento Ambiente-Salute-Sicurezza,University of Insubria,21100Varese,Italy
b
Istituto di Patologia Vegetale,University of Milan,Milan,Italy
c
Istituto di Virologia Vegetale –CNR,Milan,Italy
d
Istituto di Biologia e Biotecnologia Agraria –CNR,Milan,Italy
e
Istituto Sperimentale per l’Orticoltura –CRA,Montanaso Lombardo (Lodi),Italy
f
Dipartimento di Produzione Vegetale,University of Milan,Milan,Italy
Received 21February 2007;received in revised form 11May 2007;accepted 21May 2007
Abstract
The rice Osmyb4gene,coding for a MYB transcription factor,is expressed at low levels in rice coleoptiles under normal conditions and strongly induced at 48C.Its overexpression in Arabidopsis thaliana plants increases biotic and abiotic stress tolerance and results in the accumulation of several metabolites,essential in defence response.The heterologous expression of the Myb4transcription factor represents a promising potential approach to improve stress tolerance in crops,avoiding endogenous mechanisms that often co-suppress the transgene of interest.
In order to explore the potential of the Osmyb4gene to enhance tolerance toward multiple disease stresses in different host plant genomes,we have generated transgenic tomato (Lycopersicon esculentum Mill cv.Tondino)plants.Like Arabidopsis,tomato plants overexpressing OsMyb4acquired a higher tolerance to drought stress and to virus disease.However,the transgenic plants did not appear to be more cold tolerant than the WT,in any tested condition.The data obtained indicate that the speci?city and the degree of Osmyb4activity depend on the host genomic background.
#2007Elsevier Ireland Ltd.All rights reserved.
Keywords:Osmyb4gene;Abiotic and biotic stresses;Transgenic plants;Lycopersicon esculentum
1.Introduction
Abiotic stresses,such as low temperature and drought conditions,are the main limiting factors involved in de?ning the geographical distribution of crops,the biomass production,the quality and nutritional value of agricultural products.Many plants increase in freezing tolerance in response to low non freezing temperature,a phenomenon known as cold acclima-
tion [1,2].Tolerance to environmental stresses has a complex genetic base.In recent years,research has allowed the identi?cation and characterization of a great number of genes involved in the mechanisms of plant response to abiotic stresses [3,4].Transcription factors represent one of the best targets for engineering plants to achieve enhanced environmental stress tolerance.Several plant transcription factors,regulating the biotic and abiotic stress response,have been identi?ed and the function of some of them (i.e.CBFs/DREBs,SCOF-1,ICE1and Myb4)in freezing and chilling tolerance has been demonstrated [5–9].
The rice Osmyb4gene (GenBank accession no.Y11414),coding for a MYB transcription factor,is expressed at a low level in rice coleoptiles,under normal conditions,and strongly induced at 48C.Its overexpression in Arabidopsis thaliana
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Abbreviations:MES,2-(Nmorpholinoethane)sulfonate;6BAP,6benzil amino purine;IAA,indole-3-acetic acid;MS,Murashige and Skoog
*Corresponding author at:Dipartimento Ambiente-Salute-Sicurezza,Uni-versity of Insubria,Via J.H.Dunant 3,21100Varese,Italy.Tel.:+390332421418;fax:+390332421390.
E-mail address:candida.vannini@uninsubria.it (C.Vannini).
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0168-9452/$–see front matter #2007Elsevier Ireland Ltd.All rights reserved.doi:10.1016/j.plantsci.2007.05.007
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plants induces a high level of tolerance to cold and freezing and results in multiple biochemical changes,commonly observed in plants during cold acclimation [9,10].The microarray analysis of transgenic plants versus the WT showed that the Myb4activates genes involved in tolerance to different abiotic stresses (such as drought and salt),as well as in pathogen resistance,indicating that this transcription factor is able to integrate the activation of multiple components of the stress response [11].In vivo assays demonstrated that Myb4-driven molecular and biochemical changes actually result in an improved tolerance to several abiotic and biotic stresses [11].The ability of the rice Osmyb4gene to activate stress response pathways in the dicotyledonous Arabidopsis suggests the evolutionary conservation of its action.
The heterologous expression of a transcription factor gene represents a potential approach to improve stress tolerance in crops,avoiding endogenous mechanisms that often co-suppress a homologous transgene.The aim of this work was to investigate the possibility to utilize the Osmyb4gene to enhance tolerance towards multiple environmental stresses in tomato (Lycopersi-con esculentum ),one of the major agricultural commodities,characterized by a high sensitivity to https://www.sodocs.net/doc/183428060.html,ly,we analyzed transformed tomato plants for tolerance to chilling,drought and resistance to pathogens.We found that Myb4is able to improve the tolerance to some of the assayed stresses (drought and virus infection),but not to chilling.In addition,to test if the constitutive expression of the Osmyb4modi?es the tomato fruit quality,we analyzed the soluble solids content (SSC),skin colour,lycopene synthesis and ethylene production.2.Materials and methods
2.1.Plant material
Tomato plants (Lycopersicon esculentum Mill.cv.Tondino)were grown in a controlled environment chamber at 258C,with a 16h/8h photoperiod,a light intensity of 120m E m à2s à1and 70%relative humidity.
For transformation,the tomato seeds were surface-sterilized by immersion for 1min in 70%ethanol,followed by 5min in 2.5%NaOCl supplied with 0.1%Tween and washed several times with sterilized distilled water.The seeds were cultured for two days in distilled water and then transferred to Magenta boxes containing 50mL of 1?Murashige and Skoog (MS)salts [12],Gamborg B5vitamins [13],2%sucrose and 0.8%agar,pH 5.8.The cultures were maintained in a growth chamber and,11days after germination,cotyledon sections,with the distal and proximal ends cut off,were used for Agrobacterium-mediated transformation.
2.2.Agrobacterium strain and binary vector
The Agrobacterium tumefaciens strain GV3101was used for tomato transformation.Two different plasmids were used,both deriving from the pGA470binary vector:one (pCaMVmyb4)with the Osmyb4cDNA under the control of the CaMV35S promoter [9]and the other (pCOR15amyb4)with the Osmyb4
cDNA driven by the Arabidopsis COR15a stress-inducible gene promoter.The pCOR15amyb4expression cassette was obtained by inserting the coding region of Myb4in the pUC8containing the COR15a promoter,derived from the pSSBI plasmid,and NOS terminator.2.3.Transformation procedure
Prior to co-cultivation,cotyledon segments were precultured for 48h in Petri dishes containing 25mL of 1?MS salts,Gamborg B5vitamins,3%glucose,500mg/L MES,20m M acetosyringone,0.4mg/L IAA,1mg/L 6-BAP and 0.54%agar,pH 5.5.Bacteria,grown overnight in LB medium with antibiotics (gentamicin 50mg/L and rifampicin 50mg/L)were collected and resuspended in 1?MS medium containing 20m M acetosyringone.The cotyledons were co-cultivated with Agrobacterium harbouring either the plasmid pCaMVmyb4or pCOR15amyb4at 258C in the dark for 48h and then transferred to a modi?ed preculture medium without MES and acetosyringone,but supplemented with kanamycin 50mg/L and timentin 350mg/L.After 4weeks,green calluses were formed and the ?rst shoots appeared.The proliferating shoots were isolated and rooted on 1?MS salts,Gamborg B5vitamins and 2%sucrose supplemented with kanamycin 50mg/L and timentin 350mg/L.The surviving rooted plantlets were transferred to the greenhouse to produce T 1seeds.
2.4.Total RNA extraction and RT-PCR analysis
Total RNA was extracted by using TRIzol 1reagent
(Invitrogen,Carlsbad,CA,USA)from tomato plants grown for 4weeks in the greenhouse.The COR15a promoter was activated by treating plants at 48C for 8h or withholding water for 10days.Material from the control and treated plants was always collected at 9a.m.,in order to avoid variability in the gene expression due to the circadian clock regulation.The plant samples were immediately frozen in liquid nitrogen.Five microgram of DNase treated RNA were retro-transcribed to ?rst-strand cDNA with the SuperScript TM First-Strand Synth-esis System for RT-PCR kit (Invitrogen).PCR reactions were then carried out with the following gene speci?c primers:OsMYB4:
(F)50-CGAGAAGATGGGGCTCAAG-30(R)50-TCGGCTTCTTGTGCTTCTTGC-30;18SrDNA :
(F)50-TTGTGTTGGCTTCGGGATCGGAGTAAT-30(R)50-TGCACCACCACCCATAGAATCAAGAA-30.The samples were subjected to 30ampli?cation cycles under the following annealing conditions:588C for OsMYB4and 658C for 18SrDNA .The ampli?cation products were separated by electrophoresis on 1%agarose gels.
2.5.Chilling treatment and F v /F m measurements
The leaves from 4-week-old plants were layered on the surface of the MS agar medium.The plates were placed in a
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cabinet for photoinhibition treatments consisting of an irradiance of 750m E m à2s à1,provided by a 440-W lamp (HQI-E Powerstar,OSRAM,Munich,Germany),at a temperature of 48C for 5h 30min.The chlorophyll ?uorescence emission,from the upper surface of the leaves,was measured with a plant ef?ciency analyser (Hansatech Instruments,Norfolk,UK),according to Tarantino et al.[14].Before measurements,the leaves were dark-adapted for 30min at room temperature.Each experiment was replicated three times and in each experiment the leaves of one plant were assayed three times at different time intervals.2.6.Drought treatment
To evaluate the drought tolerance of the transgenic tomato lines,the relative water content (RWC)of the transgenic and WT plants leaves was measured.Ten 4-week-old plants,for each genotype,grown individually in 15cm pots,were placed at random in a climatic chamber at 248C,16h/8h light/dark regime,60/70%humidity.The earth of the vases was abundantly watered and left to percolate for 24h.Subsequently,the plants were left dry.From this moment (t 0),every 4days,eight leaves of each genotype were randomly collected and weighed (fresh weight,FW).Afterward,the leaves were left to become impregnated on Petri dishes for 24h on ?lter paper saturated with demineralised water.They were then weighed (turgid weight,TW)and put at 708C for 48h,to determine the dry weight (DW).The RWC was calculated as a percentage of (FW àDW)/(TW àDW).
For in vivo assays,4-week-old wild type (WT)and transgenic T 1plants were grown in 15cm pots and placed at random in a greenhouse (248C,16h/8h light/dark regime,60/70%humidity).The earth of the pots was abundantly watered and,subsequently,plants were left dry without watering for 28days.Then,the plants were watered for 7days and plant survival was evaluated.We have performed two experiments:in the ?rst one we tested WT,12pCaMV and 2pCOR plants;in the second one we used WT,13pCaMV and 10pCOR lines.In each experiment,40plants/genotype were assayed.
2.7.Soluble sugars,proline and amino acid content
Soluble sugars and proline contents were determined on frozen leaves from the same plants used for in vivo drought assays:leaves were detached from WT and transgenic 4-week-old plants grown under normal conditions and from the same plants after 28days of water de?cit.Three individual plants for each genotype were tested.Plant material was extracted using 0.6N perchloric acid,and the supernatant collected after centrifugation at 16000?g for 15min.Sucrose,glucose and fructose were quanti?ed using the Boehringer Mannheim (RBiopharm,Darmstadt,Germany)kit and the reduction of NADP was monitored at 340nm.Standard curves of sucrose,glucose and fructose were prepared.
On the same extracts,proline concentration was determined as described previously [10].Proline content was calculated on
the basis of a standard curve of L-Pro.The amino acid content was determined using the method of Moore [15].2.8.Viral inoculation
The tomato mosaic virus (ToMV)was mechanically inoculated on 300mesh carborundum dusted leaves of 4week-old tomato plants,using a 0.5m g/mL virus suspension.At this growth stage,the plants had developed 5–6pinnately compound leaves and were 15–20cm tall.Virus inoculation was carried out on the ?rst pair of true leaves.The resistance level of the tomato plants to the ToMV was evaluated 7and 20days after inoculation,by analysis of the symptom severity in directly inoculated leaves (local symptoms),and in the apical leaves (systemic symptoms).The systemic symptoms were classi?ed into four classes:no visible symptoms,mild mosaic/chlorotic lesions,intense mosaic,leaf narrowing and distortion.2.9.Soluble solids content in fruits
Juice was squeezed from ripe tomatoes and collected onto a digital Attago N1refractometer.The soluble solids content (SSC)was measured at 208C and the results were expressed as degrees Brix (8Brix).2.10.Lycopene measurement
The lycopene standard was purchased from Sigma.A stock solution,dissolving 5mg (?0.1mg)lycopene in 10mL chloroform,was prepared and sonicated for 20min.Owing to the lycopene instability,the standard purity was checked by a spectrophotometer,with E 1%472nm =3450in hexane.From the stock solution,a 1:250dilution was prepared in hexane,to a ?nal concentration of 2m g/mL,and absorbance was measured at 472nm,using a 1cm path length quartz cuvette after blanking with hexane.The purity was calculated as follows:%Purity =(Abs ?10000/3450?C)?100%,where Abs =ab-sorbance units,and C =standard concentration in m g/mL.The spectrophotometric purity was then used to correct the concentration of the chromatographic calibration solutions.From the 500m g/mL stock solution in chloroform,standard 1:10,1:25,1:50,1:100and 1:250dilutions were prepared with chloroform and methanol (1:1),to create a ?ve point linearity curve for HPLC analysis.
To avoid oxidation during the sample preparation,the whole laboratory procedures were conducted under dim light.Each sample was analyzed in duplicates,with triplicate injections for HPLC.Twenty millilitres of 2N HCl/L -ascorbic acid solution were added to 50mg of sample,into a 50mL glass centrifuge tube wrapped with aluminium foil.The samples were then mixed at room temperature for 30min on a mechanical orbital shaker and,after an addition of 10mL of 0.1%(w/v)Butylated Hydroxytoluene (BHT)-stabilized toluene,an additional 10min-shaking was carried out.After centrifugation,the samples were placed in a refrigerator maintained at 2–68C for 60min.One millilitre aliquot of the toluene upper layer was
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1:25diluted with methanol and tetrahydrofuran (THF)(60:40),and immediately tested with HPLC analysis.
A Shimadzu LC-10ADvp,SIL-10ADvp high-performance liquid chromatograph equipment,with SPD-10Avp and RF-10Axl detectors,was used for sample analyses;the HPLC pumps,auto sampler and detectors were controlled via Class vp 3.4software.The analytical column was a Luna RP C18(4.6mm ?250mm,particle size =5m m),provided with a guard column obtained from Chemtek Analytica.The column temperature was maintained at 308C.The lycopene isomer separation was carried out at a 1.2mL/min ?ow rate,using an isocratic elution with a mobile phase mixture of 90%methanol and 10%THF [16].The injection volume was 10m L and the 472nm wavelength was used for absorbance detection of the lycopene.The data were analysed by using the Statistical Package for the Social Sciences (SPSS)10.0-window program (1999).One-way variance analysis (ANOV A)was employed to identify signi?cant differences between samples at p 0.01signi?cance level.
2.11.Ethylene determination
The ethylene production of ripe fruits was measured using a closed system for 6days,by placing three whole tomatoes,after having been weighed,in air-tight 590mL glass jars,allowing the ethylene to accumulate.One millilitre headspace gas sample was withdrawn with a gas-tight syringe,injected into a gas chromatograph and analysed for ethylene.The ethylene data were collected the ?rst,second and sixth day after the tomato harvest.The data are means of three experiments from three different jars for each tomato set.The jars were sealed for
1h before each measurement,and always ventilated after and during data collection.2.12.Fruit colour measurement
The fruit colour was recorded using the Commission Internationale de l 0Eclairage L*,a*and b*[17]chromatic coordinates,obtained by means of a Minolta CR-200colorimeter.Each fruit was measured twice,at two different sites on opposite sides of the equatorial region.A total of 10tomatoes were assayed,both from WT and each transformed line,and three colour readings were taken from each sample.L*indicates the lightness,ranging from black (0)to white (100);a*speci?es the amount of red and green tonality,on a green (à)to red (+)axis;and b*indicates the amount of blue and yellow tonality,on a blue (à)to yellow (+)axis [18].The results were expressed as the ratio of red to green colour (a*/b*),hue angle value [h =arctan (b*/a*),where 08=red purple;908=yellow;1808=bluish-green and 2708=blue],saturation (or chroma)[C =(a*2+b*2)1/2]and tomato colour index [TCI =a*/L*(a*2+b*2)1/2][19–21].3.Results
3.1.Production of transgenic plants
In order to evaluate the expression effect of the rice Osmyb4gene in tomato,we transformed tomato plants with the CaMVmyb4plasmid carrying the Osmyb4cDNA under the control of the constitutive CaMV35S promoter.Fourteen putative transgenic T 0plants showing resistance to Kanamycin
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Fig.1.Phenotype of transgenic plants and fruits.(a)From left to right:WT,pCaMVmyb4(lines 12and 13)and pCORmyb4(lines 2and 10)plants grown for 1month in the greenhouse;(b)from left to right:tomato fruits of the WT,pCaMVmyb4(line 11)and pCORmyb4(line 2).
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(k r
trait)were further analysed by PCR to screen for the integrity of the inserted transgene.Ten T 0positive lines were transferred on soil to produce T 1seeds.The transgenic plants showed a phenotype similar to the WT:no plant showed a dwarf phenotype,contrarily to what happens in Arabidopsis [11].However,3lines (6,7and 11)produced fruits without seeds (Fig.1).Due to the lack of seeds,we did not include these transgenic lines in further experiments.The T 1seeds were collected and the T 1plantlets were tested for segregation of Kanamycin resistance (k r )and sensitivity (k s )traits.In four different T 1lines,the segregation ratio k r :k s was 3:1,thus con?rming the presence of only one insertion site.For these lines,the Osmyb4expression levels were assayed by RT-PCR (Fig.2a):Osmyb4transcripts were detected only in the transgenic lines,whereas the amount of 18S RNA was similar in the transgenic and WT tomato plants.The two independent 12pCaMV and 13pCaMV lines were selected for further analysis.
To overcome the low transformation ef?ciency obtained (0.8%)and the sterility of some plants,we produced transgenic tomato plants expressing the Osmyb4gene under the control of the cold,drought and ABA inducible promoter of the Arabidopsis COR15a gene [22].We obtained 30transgenic PCR positive lines.The transformation ef?ciency was higher by about ?ve-fold in comparison to the pCaMVMyb4plants.The resulting plants were phenotypically similar to the WT and they were all fertile (Fig.1).
The T 1seeds were collected and the T 1plantlets were tested for segregation of the k r trait.In 12different T 1lines,the segregation ratio k r :k s =3:1con?rmed one insertion site.Two independent transgenic lines,2pCOR and 10pCOR,each with a single insertion,were assayed by RT-PCR.The results showed a low background expression of OsMyb4even under control conditions,indicating that the promoter is leaky.Upon exposure to stress treatments,the Osmyb4expression was elevated with a stronger induction after 8h of cold stress (Fig.2b).
3.2.Effects of OsMyb4expression on chilling tolerance In order to assess cold stress damage on OsMyb4-expressing plants,the Photosystem II (PSII)stability was measured to re?ect the level of cellular damage after chilling treatments.
The ratio between the variable ?uorescence (F v )and the maximum ?uorescence (F m )was used to estimate the quantum yield of the PSII photochemistry.The leaves from control and transformed plants were exposed to an excess of light energy (750m E m à2s à1)at low temperature (48C).F v /F m values were measured at different times during treatment.As shown in Fig.3,at time zero,the OsMyb4-transformed plants exhibited values of F v /F m comparable to that of the WT,indicating that the OsMyb4expression did not affect the PSII ef?ciency.The cold treatment at high light intensity caused a marked inhibition of the PSII for both the WT and transgenic plants,as indicated by a decrease in the F v /F m values from 0.8up to 0.5.A cold acclimation of 8h at 48C did not improve the chilling response of the pCOR lines (data not shown).
3.3.Effects of OsMyb4expression on drought tolerance
To evaluate the resistance of transgenic lines to drought stress,4-week-old plants were not watered for 16days and RWC was measured every 4days.The RWC of leaves in transgenic plants remained high during water de?cit treatments.In contrast,a marked reduction in the water content was observed in the WT plants (Fig.4a).
For the survival rate test,the water de?cit was extended to 28days and then the plants were re-watered for 7days.We have performed two experiments.In the ?rst one,the survival rate was 18/40,29/40and 27/40for the WT and transgenic lines 12pCaMV and 2pCOR,respectively.In the second experiment,the survival rate was 14/40,25/40and 23/40for the WT and transgenic lines 13pCaMVand 10pCOR,respectively (Fig.4b).All results observed indicate that transgenic lines are more resistant to drought stress than WT plants and that there is no signi?cant different behaviour between transgenic plants expressing the Osmyb4gene driven by a constitutive or an inducible promoter.
Since in Arabidopsis the overexpression of Osmyb4increases the level of osmoprotectant compounds [9,10],we also measured the content of soluble sugars and proline in leaves of WT and transgenic 4-week-old tomato plants under normal and water de?cit conditions.In normal growth conditions,the concentrations of sucrose,fructose and glucose were constitutively higher in pCaMV transgenic lines with
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Fig.2.RT-PCR analysis of Osmyb4expression in transgenic tomato plants.(a)OsMyb4gene expression in WT (lane 1)and four independent transgenic pCaMVmyb4T1plants (lane 2–5).(b)OsMyb4gene expression in WT and two independent transgenic pCOR lines (numbers at the top)under normal (N.I.not induced),cold and drought stress conditions.For all RT-PCR analyses,the 18S rRNA was used as the normal
control.
Fig.3.Photosystem II inhibition by cold and high light treatment.Photoinhi-biton (evaluated as F v /F m )induced in detached leaves from WT and tomato transgenic 4-week-old plants treated at 48C and 750m E.The bars represent the S.D.of three independent experiments.
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respect to WT and pCOR lines (Table 1).In response to water de?cit conditions,the soluble sugar content increased in all genotypes.In particular,the WT values were in all cases lower than those of transgenic plants and pCOR lines achieved the same levels of the pCaMV lines.
The proline content in transformed tomato is not much higher than in WT plants under normal conditions;however,there is an increase in response to water stress treatment in both pCOR and pCaMV with respect to WT plants (Table 1).
3.4.Tomato plants’resistance to ToMV
Previously,we demonstrated that,in Arabidopsis,the Osmyb4gene also regulates responses to biotic stresses [11].In order to verify this possibility also in tomatoes,we tested the resistance to ToMV of WT and transgenic plants.We also examined the resistance to ToMVof pCOR plants considering the level of leaky Osmyb4expression driven by the COR15a promoter.The inoculated WT plants developed both local and systemic symptoms of infection after a week (Fig.5).Local symptoms consisted of a mild mosaic or,more rarely,of chlorotic lesions in the inoculated leaves,while systemic symptoms appeared at ?rst as a mild mosaic on the apical leaves.In the same apical leaves the mosaic became more intense in the following days and,after 2weeks,leaf distortion and narrowing were visible.No symptoms were appreciable 7days after inoculation in any of the transgenic tomato plants,except for rare chlorotic spots in the pCOR transformed plants.Furthermore,20days after inoculation,even if both local and systemic symptoms were detectable in the apical leaves in transgenic plants,they were less intense than those recorded in the WT plants.The pCaMV transformed tomatoes appeared less damaged than the pCOR ones (Fig.5).
3.5.Tomato fruit analysis
To test if the use of the constitutive overexpression of the Myb4transcription factor modi?ed the tomato fruit quality,we performed the analysis reported in Table 2.8Brix is a measure of the total soluble solid percentage (chie?y fructose and glucose)increasing in turn with fruit colour and ripeness:their value was higher in the pCaMVMyb4fruits than in the WT ones.
Also the lycopene content,in the pCaMVMyb4freshly harvested ripe tomatoes,was slightly higher than in the WT samples.
Ethylene was detected from the headspace of 1h sealed jars containing freshly harvested ripe WT and pCaMVMyb4tomatoes,according to the procedures explained in Section 2.As shown in Table 2,the ethylene evolution rate peaked the ?rst day after harvesting (1d),on the second day the ethylene evolution decreased slightly and ?nally dropped steadily until day 6in WT and transgenic lines.
The data did not show any signi?cant difference among transformed and untrasformed plants.
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Table 1
Soluble sugar and proline content in WT and transgenic tomato plants before (C)and after 28days of dehydration stress (S)
Glucose (m g/mg FW)Fructose (m g/mg FW)Sucrose (m g/mg FW)Proline/total amino acid%C
S
C
S
C
S
C S
WT 0.92?0.02 1.85?0.01 1.03?0.03 2.27?0.020.34?0.010.42?0.01 1.9?0.05 5.64?0.042pCOR 1.16?0.03 3.94?0.02 1.17?0.04 3.77?0.060.48?0.02 1.86?0.01 2.0?0.05 6.98?0.0210pCOR 1.30?0.03 4.06?0.08 1.27?0.03 3.70?0.020.49?0.01 1.84?0.01 2.3?0.03 6.74?0.0512pCaMV 2.47?0.01 3.99?0.05 2.70?0.01 3.88?0.07 1.70?0.03 1.9?0.02 2.5?0.057.01?0.0313pCaMV
2.55?0.01
4.2?0.04
3.04?0.01
4.42?0.03
1.73?0.01
2.5?0.01
2.6?0.05
6.97?0.05
Each value is the mean ?S.D.(n =3individual
plants).
Fig. 4.The effect of the Osmyb4expression on L.esculentum drought tolerance.(a)The relative water content was calculated after 0,4,8,12,16days of drought stress as described in Section 2.The bars represent the S.D.of ?ve independent experiments.(b)WT and transgenic (12and 13pCaMV;2and 10pCOR)tomato plants grown without watering for 21days.For the survival rate test,the water de?cit was extended to 28days and then plants were re-watered for 7days.The numbers of rescued plants per total number of tested plants are indicated at the bottom of the ?gure.
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Also regarding colour measurements obtained from freshly harvested fruits,the results were not signi?cantly different in the pCaMVMyb4fruits compared to the WT ones (Table 2).
4.Discussion
In a previous work,we demonstrated that the overexpression of the rice Myb4transcription factor in Arabidopsis plants strongly increases cold and freezing tolerance [9].In comparison to the WT,the Arabidopsis transgenic lines also showed a signi?cant tolerance to different environmental (biotic and abiotic)stresses,indicating that Osmyb4is a crucial knot regulating the cross talk of different stress signalling cascades [11].
In this report,we have explored the potential of using the Osmyb4gene to enhance resistance to multiple stresses in tomato.While Arabidopsis can acclimate to cold and survive freezing,by activating multiple biochemical changes,tomato is a chilling sensitive plant.Moreover it is a natural host to a broad spectrum of pathogens.
We produced transgenic tomato plants expressing the Osmyb4gene under the control of the constitutive CaMV35S promoter.The low transformation ef?ciency obtained (0.8%)and the sterility of some transgenic lines are probably due to the detrimental effect of the Osmyb4constitutive overexpression,as we observed in the case of rice and maize [Dr.I.Coraggio,personal communication].In literature,it has been noted that the problems connected to gene expression driven by a constitutive promoter can be often overcome by using a stress-inducible promoter [23].In order to minimize these negative effects,we produced transgenic OsMyb4tomato plants under the control of the Arabidopsis COR15a promoter.We used this promoter,though no COR homologous genes have been reported in tomato,because the COR15a promoter contains CRT/DRE elements for the binding of CBF proteins,induced by low temperature also in tomatoes [24].Moreover,the overexpression of the tomato CBF
coding sequence activates the COR15a expression in transgenic Arabidopsis [24].The molecular analysis of the pCORMyb4plants showed a very low expression of the transgene under normal conditions and its inducibility by cold and drought treatments.The utilisation of the stress-inducible COR15a promoter,for the Myb4overexpression,actually minimizes the negative effects on transformation ef?ciency and seed produc-tion of transgenic plants.
Like Arabidopsis [11],tomato transgenic plants over-expressing OsMyb4acquired a higher tolerance to drought stress.It is known that,under drought stress conditions,plants accumulate several compatible solutes,such as soluble sugars and proline,as a mechanism to improve tolerance to water de?cit [25].Following drought treatment,an increase in the level of these osmolytes was observed in both WT and transgenic tomato plants,but levels accumulated by transgenics were always higher than those observed in WT.As free sugars are concerned,their content in Myb4constitutively expressing plants was higher than in WT plants also under normal growth conditions.These results indicate that the improvement of drought tolerance in Osmyb4transgenics depends,at least partially,on the changes in the accumulated levels of compatible solutes.
Contrary to the results obtained in Arabidopsis,the tomato Myb4transgenic plants did not appear to be more cold tolerant than the WT,in any tested condition.
The different behaviour between Arabidopsis and tomato could lie in the different selective pressure in response to different environments colonised by the two species.The tomato is a tropical plant and selection pressures could have caused the loss of the target genes in the cold pathway controlled by Myb4.The results of Zhang et al.[26]point to the same direction,demonstrating that the tomato CBF regulon involved in freezing tolerance differs considerably from that of Arabidopsis:it is much smaller in size and potentially less diverse in function.
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Fig.5.The effect of the Osmyb4expression on tomato resistance to the ToMV .Number of plants (out of a total 30inoculated ones in 3independent experiments)showing symptoms of virus infection.At 7days after inoculation all symptomatic plants showed only mild mosaic (local or systemic,or both).At 20days after inoculation all plants showed local symptoms of mild mosaic in the inoculated leaves (not reported),while different classes of symptom severity,illustrated in the lower panel,were appreciable in the apical leaves.
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Previously,we demonstrated that in Arabidopsis the Myb4protein was involved in different signal transduction pathways and regulated responses not only to abiotic but also to biotic stresses [11].In order to verify this hypothesis also in tomato,we tested the transgenic line resistance to pathogens.A signi?cant reduction in the symptom severity was observed in tomato plants inoculated with the ToMV .This reduction could be correlated to a limited virus replication and/or to an impaired translocation,being visible systemic symptoms also on transgenic tomatoes.From an epidemiological point of view,a limited virus replication means a signi?cant reduction of the disease in open ?eld,particularly with a virus like the ToMV,a major tomato pathogen that can be transmitted in a non-persistent manner by different vectors.
Owing to the dietary relevance of tomato fruits,we tested the effect of the Myb4transcription factor constitutive over-expression on the quality of transgenic tomato fruits.The results indicate a slight increase both in the lycopene and soluble solids content in transgenic plants.The other tested fruit characteristics were not signi?cantly different between transgenic and control plants.
In conclusion,the obtained data show that the Osmyb4stress-response machinery is only partially conserved between tomato and Arabidopsis.The speci?city and the degree of Osmyb4activity depend on the host genomic background.This fact may represent a constraint in the use of transcriptional factors in the improvement of crop plants.On the other hand,the use of heterologous transcription factors represents a good strategy to minimize possible mechanisms of transgene co-suppression during crop transformation.
Acknowledgments
We thank Dr.Sarah Gilmour for kindly providing the pSSBI plasmid.This work was partly supported by a project grant from the Italian Ministry of University and Scienti?c Research (PRIN 2002,code 2002077233)and by CNR,Plant Virology Institute,Commessa AG.P01.010.References
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520
521522
523
524525
526
527
528
529
530
531
532533
534
535
536
537
538
539
540541
542
543
544
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554
555
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614615616617618619620621622623624625626627628629630631632633
T a b l e 2M a i n a g r o n o m i c t r a i t s o f w i l d t y p e a n d t r a n s f o r m e d t o m a t o e s
W e i g h t (g )
8B r i x
L y c o p e n e (m g /g )
E t h y l e n e (p p m /K g /h )
L *
a *
b *
a */
b *
C
T C I
h 8
1d
2d
6d
W T 10.87?1.015.2?0.119.82?1.86.7?0.25.8?0.52.0?0.240.47?2.4722.31?2.5818.46?2.351.2028.9538.0869.8612p C a M V 10.35?1.045.6?0.524.50?2.8§
9.1?0.4§
8.5?0.6§
2.0?0.439.43?1.8020.41?2.1819.87?
3.641.0428.4836.3559.0613p C a M V 10.02?3.56
6.2?0.3§
23.79?2.05§
7.6?0.5§
7.5?0.4§
1.3?0.5§§
39.28?.86
17.88?4.09
15.39?2.15
1.34
23.59
38.59
66.61
S o l u b l e s o l i d c o n t e n t i s e x p r e s s e d i n B r i x d e g r e e s ;e t h y l e n e p r o d u c t i o n h a s b e e n d e t e c t e d u p t o 6d a y s ;c o l o r i m e t r i c p a r a m e t e r s a r e e x p r e s s e d a s f o l l o w s :L *i n d i c a t e s t h e l i g h t n e s s ,r a n g i n g f r o m b l a c k (0)t o w h i t e (100);a *s p e c i ?e s t h e a m o u n t o f r e d a n d g r e e n t o n a l i t y ,o n a g r e e n (à)t o r e d (+)a x i s ;b *i n d i c a t e s t h e a m o u n t o f b l u e a n d y e l l o w t o n a l i t y ,o n a b l u e (à)t o y e l l o w (+)a x i s ;h =a r c t a n (b */a *),w h e r e 08=r e d p u r p l e ;908=y e l l o w ;1808=b l u i s h -g r e e n a n d 2708=b l u e ;C =(a *2+b *2)1/2;T C I =a */L *(a *2+b *2)1/2.E a c h v a l u e i s t h e ?S .D .m e a n s o f t h r e e i n d e p e n d e n t e x p e r i m e n t s .T h e §s i g n e d v a l u e s w e r e s i g n i ?c a n t l y d i f f e r e n t f r o m t h e W T (t -S t u d e n t ,§P 0.05,§§P 0.01).
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633634635636637638639640641642643644645646647648649650651652653654655656657658659660661662663664
664665666667668669670671672673674675676677678679680681682683684685686687688689690691692693694695
Q2
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番茄高产栽培技术方案
番茄高产栽培技术方案 番茄是种植面积最大的蔬菜品种之一，由于其易管理、产量高、效益好，因此深受广大菜农所喜爱。但从整体管理水平来看，还存在着很大的误区，尤其是在栽培、施肥、植保方面误区最大，要想提高番茄的产量和效益，必须从这几个方面入手。 一、整地、定植 番茄定植前一是注意做好消毒工作，做到无菌苗定植，这步特别关键，也特别被菜农所忽视。二是番茄的底肥问题，菜农不知道应该施多少肥，也不知道施什么样的肥，都是根据经验施肥，造成菜田施肥量过高，许多地块已经出现盐渍化现象（轻的起青苔，重的土壤干后有白霜，再严重的浇水后地面有一块块红色的胶状物），严重影响产量和品质。 1、定植前消毒 由于在育苗过程中，番茄苗极容易带菌，所以为了避免番茄定植后死苗现象（茎基腐病），在定植前2－3天必须药剂预防，即无菌苗处理工作。选用特锐菌1000倍液+培基30ml兑水30斤直接喷淋，要保证药液流到番茄苗的茎基部；或者定植前用特锐菌15克+培基30ml兑水30斤，蘸根1500-2000棵苗。 2、整地施肥 番茄底肥误区最大，菜农不知道应该施多少肥，也不知道施什么样的肥，都是根据经验施肥，造成菜农施肥量过高，许多地块出现板结盐渍化（轻的起青苔，重的土壤干后有白霜，再严重的浇水后地面有一块块红色的胶状物），造成根系不下扎，严重影响产量和品质。 以下是根据河北省土壤推出的施肥配方，每亩施肥量： 复合肥25-40公斤（低氮高磷高钾）；安格力1公斤(和化肥撒施)；培元8-12公斤（拌细土沟施或者穴施）；极护250克（缓苗水）；立沃松1公斤（拌细沙定植后撒施）。 二、番茄病害的防治技术 如果番茄没有病，最好选择保护剂，每桶喜思安30克，10-12天一次，既防病又保叶。 1、茎基腐病、晚疫病 茎基腐病：骏佳750倍液，注意喷药时采用喷淋，使药液流到根茎部。晚疫病：先用骏佳750倍液，压低病原基数，控制住病害发展；5-7天后再喷施一次，抑制病原菌滋生，持效期长达15天以上，效果更佳。 2、早疫病、灰霉病、叶霉病
番茄种植技术完全版(专家解答)
番茄种植技术完全版（专家解答） 番茄种植技术完全版（专家解答）西红柿打叶注意问题问题随着温室中西红柿的生长，下部的老化叶片应逐渐打掉。打掉这些叶片，一方面可以改善通风透光条件，减少病害的发生;另一方面打掉一此感病的老叶，减少病害的传播。同时，打掉老叶可以避免这些叶片无谓地消耗根系吸收的营养，促进植株更好地生长发育。西红柿打叶应遵循如下原则： 1.在西红柿最底部的一穗果达到绿熟期，即果实由绿变白，果实完全长大，种子已经成熟，开始变红时之前，其上部的叶片不能打掉。因为果实上部的2-3 片叶制造营养供给果实生发发育，打掉过早，影响果实生长。 2.凡是颜色浓绿，没 有病虫害、没有黄斑的叶片，不能打掉，因为这些叶片仍然能为西红柿的产量作贡献。 3.那些最底部一穗果实下部的叶色变黄，有严重黄斑或病虫危害的老叶可以打掉。 4.植株中 下部由于病虫危害严重或其他生理性病害造成的严重变黄衰老的叶片，可以打掉。 5.最底部一穗果实下部的叶片可以打掉。 6.打叶时，每次以 2 片为宜，不能过多。7.两次打叶的间隔时间，以10 天以上为宜。8.每次打叶应在晴天的中午12 时左右，不能在早晨露水很大时打叶，以免伤口感染发病。也不能在傍晚打叶，以免伤口未能愈合，而在夜间湿度大时染病。9.打叶后，应喷施天然芸薹素一硕丰481 一次，促进 叶片光合作用的进行，避免影响植株生长发育。如果在打叶时，没有
遵循上述原则，不是造成病害严重，就是因叶片损伤太多，果实的营养积累不足，而严重发生空洞果、减产等损失，或大量落花落果。番茄的精品挂果率如何提高摘除每穗花的第一朵花每穗花的第一朵花一般要比后面的花早开 2 天左右，若点花时，点住此花，容易使营养集中供应此花和果实，会使后面的花因得不到充足的营养而导致果实大小不一致，降低番茄的精品果率。后面的花开放时间基本一致，摘除第一朵花则可减少营养消耗，以积累充足的营养供应后面的花和果实，这样就可使后面的果实大小一致，利于精品果的生产。多点花、少留果、留好果一般情况下，每穗花都能开7~8 朵花，在点花时，根据植株的长势应尽可能地多点花，以备疏果、留果。当幼果坐住后根据需要选留4~5 个大小一致、果形相近且无病虫害的果实，然后将其余的果实摘掉。追施钾肥，促进番茄着色番茄进入结果期后钾肥需求量增大，为氮肥的 2 倍左右。若钾肥不足容易造成番茄着色不良发生筋腐病，影响果实的商品性。因此，当果实坐住如鸡蛋大小时应每亩每次冲施高钾复合肥25~30 公 斤，以促进果实着色，增加果实重量。 科学用药，确保果实正常发育番茄进入结果期后，应预防早疫病、晚疫病、细菌性溃疡病等多种危害果实的病害。在用药时，特别是在使用铜制剂、唑类药剂或抑制剂时一定 要注意使用浓度，以免浓度过大影响果实的膨大或造成药害，降低果实的商品性。番茄树定植管理定植前的准备工作（1）配制营养液 1 营养液配方：采用北京蔬菜研究中心番茄配方。此配方适于北方硬水
中粮概况及笔试面试中粮集团校招招聘
中粮概况及笔试面试中粮集团校招招聘
中粮集团 一、企业发展概况 中粮集团有限公司（简称“中粮集团”，英文简称“COFCO”），是中国最大的粮油食品进出口公司和实力雄厚的食品生产商，享誉国际粮油食品市场，在与大众生活息息相关的农产品贸易、生物质能源开发、食品生产加工、地产、物业、酒店经营以及金融服务等领域成绩卓著。1994年以来，中粮集团一直位列《财富》世界500强企业。 中国最大的进出口公司之一，从事农产品和食品进出口贸易历史最悠久、实力最雄厚的中国企业，连接中国粮油食品市场与国际市场的重要桥梁。 在葡萄酒、精炼食用油、面粉、啤酒麦芽、番茄制品、印铁制罐、金属瓶盖等行业居中国领先地位，旗下的"长城"葡萄酒、"福临门"食用油、"金帝"巧克力、"黄中皇"绍兴酒，“中茶”茶叶等品牌和产品深受消费者喜爱。 中粮集团致力于为13亿中国人民提供营养、健康的优质食品等品牌和产品深受消费者喜爱。中粮集团还是美国可口可乐公司在中国最重要的合作伙伴，生产和销售可口可乐系列饮料。 中粮集团投资开发的海南三亚亚龙湾国家旅游度假开发区被外国游客誉为中国的夏威夷。中粮投资建设的商用写字楼、民用住宅，也在市场上受到欢迎。中粮集团旗下的凯莱国际酒店集团是世界酒店300强之一。 “中国食品0506”“中粮控股0606”是中粮集团在香港的上市公司，中粮集团在国内还拥有“中粮地产000031”、“中粮屯河600737”、“吉生化600893”、“丰原生化000930”等6家上市公司。 二、业务介绍： 粮油食品贸易及物流：经过大米、小麦、玉米等产品的进出口贸易和国内销售，连接粮食、油脂和其它农产品的生产者和用户。 农副食品加工业：向国内国际的食品生产商、食品贸易公司和零售商提供植物油、糖、肉类、蔬菜水果，淀粉制品等农副食品。 食品制造业：以专业技术和先进工艺，为消费者奉献优质健康的品牌食品，包括米、面、糖果、巧克力、罐头、调味品、焙烤食品等。 生物化工业：利用农业副产品，发展新型环保生物质能源。业务包括能源制造
科学减肥知识
科学减肥知识 科学减肥知识 1、新鲜水果水分多，单位热量较低，但如果做成果脯类、蜜饯，经过加工后糖分会大量增加，而且不是天然糖分，减肥时建议最好 不要吃。 2、你有星期一综合症么?运动除了可以减肥之外还能改善肌肤状况。因为锻炼能够加速皮肤下的血流，出汗能清洁毛孔，还能释放 压力。 3、木耳中的脂肪含量仅为0.6%，是为身体排毒的主要成分，建 议您每周吃一次木耳，夏天用醋拌着吃也很凉爽，还有预防便秘的 功效。 4、减肥时切记远离啤酒和烈性酒，它们都有可能提高体内的可 的松，一种将脂肪转移至腹部的荷尔蒙。喝酒时还倾向于食用更多 的食物。 5、纤维不仅能增加饱腹感帮助减重，也可预防便秘，使腹部不 至显得过大。赶快在您的食谱中加入大豆、菠菜、梨、粗粮等高纤 食品吧。 6、规律锻炼不仅能防止肌肉流失，增加肌肉数量，还使我们的 新陈代谢保持在一个较高水平，增强我们的力量和耐力，保持充沛 的精力! 7、时刻保持正确的坐姿：直腰、略微挺胸、沉肩和微收下巴， 久坐的时候每隔1小时就起来活动活动，放松绷紧的肌肉，有助您 上半身塑形。
8、有研究显示大量饮用含糖饮料，比吃任何其他食物可能更易 让腰围变粗喔。就从这个夏天起，赶紧放开可乐瓶，加入泡茶的行 列吧! 9、如果想吃肉就选鸡胸肉吧，是鸡肉中蛋白质含量较多的，脂 肪含量和虾、螃蟹等昂贵海鲜一样。100克的鸡胸肉只有133大卡喔。 10、过多的腹部脂肪组织会产生“饥饿荷尔蒙”，让本来已经肥胖的人吃得更多，导致腹部更加肥胖，由此出现恶性循环，“小腹婆”要警惕喔! 11、运动时皮下血管扩张并大量出汗，不宜马上洗冷水澡，否则体内产生的大量热不能很好地散发，形成内热外冷，破坏人体平衡 易生病。 12、如果最近生理期延迟没来，除了注意保证谷薯和肉蛋类的适量摄入，你也需要重视睡眠。每天7小时以上午夜前睡眠，也就是 23点前睡吧。据测算，青年男女一天的进食量大致如下：粮食500克，蛋1个，瘦肉100克，鱼150克，豆类200克，蔬菜500克， 牛奶200克，植物油25克。 13、单纯节食减肥时肌肉的流失约占所减总重的25%左右，而做 有氧运动同时进行力量训练，既能增加消耗的能量，又能防止肌肉 的减少。 14、热量的品质和数量一样重要，选择自然食品，而非加工食品，新鲜水果比果脯好，蒸土豆泥比薯片好，麦片、全麦面包比蛋糕甜 点好。 15、快走是最易操作的运动之一，但走路的姿势非常重要，挺胸、收小腹，臀部夹紧，千万不要弓腰驼背，这样才能刺激你的腹部肌 肉哦。 16、吃饭时，要避免以汤汁浇饭，因为炒菜尤其是勾芡的的汤汁中含有很多油、盐和调味料，不仅热量高，而且会促进食欲，增加 进食量。
番茄种植技术手册
番茄种植技术手册 第一节概述 一、番茄来源 番茄原产于南美洲安第斯山脉的秘鲁、俄厄瓜多尔、玻利维亚等地形复杂的河谷和山川地带。16 世纪传入欧洲，起初作为庭院观赏用，到17 世纪逐渐为人们食用。大约在17-18 世纪，由西方的传教士、商人或者华侨引进我国，从20 世纪50 年代初迅速发展，栽培面积逐年扩大，成为我国普遍栽培的主要果菜之一。 二、番茄植物学名 茄科番茄属植物番茄Solanum lycopersicum，别名：西红柿，番柿，洋柿子。 第二节番茄生物学特性 番茄为茄科番茄属作物，在我国为一年生草本植物。了解番茄各个器官，如根、茎、叶、花、果实、种子的形态特征和植株生长习性，认识植株及器官生长与环境的关系，把握地上部分和地下部分、营养生长和生殖生长的关系，是制订各项栽培技术措施的依据，是创造适宜的环境条件，满足番茄正常生长发育的要求，获得丰产的前提。 一、根 番茄为深根性作物，根系发达，支根数多，分布深而广。在2 片真叶期时，植株主根即可长达50cm 左右。直播的成年植株主根深达成1m 以上，根幅直径可达2m 以上，
但侧根少；育苗移栽长成的植株，因主根多被切断，促进了侧根生长，相对根数增多，增加了根系吸收面积。在生产中，番茄大多采用育苗移植，根系大部分分布在离土表约20-30cm 深的适用范围内，所以比较不耐干旱，肥水供应不足也容易早衰。 番茄根系的发育和分布，除与种性有关外，因土壤条件和栽培技术而异，如土壤结构、土层深度、排水情况、肥力及土温等，还受地上部生长和栽培密度等的影响。土质粘重，地下水位高，土层薄，都会阻碍根群生长，使得多数根群分布在近地表的土层中。夏季，高温多雨，表土层温、湿度变化大，不利根系生长，甚至会引起根系腐烂死亡，导致病害发生。土层深厚，土壤肥沃疏松，地势高燥，排水良好，有利根群向土壤深层生长，根群发达，分枝多，能增强对养分和水分的吸收能力，提高对不良环境的抵抗力。番茄而旱能力因生长时期而异，果实生长旺盛期，番茄需要肥水最多，只有肥水供给充足，才能获得丰收。土壤温度对番茄根生长也有很大影响。土壤温度低，根系分布趋向表层，温度高则趋向深层。土温在8℃以下，根系活动能力弱；土温高于36℃，生长受到抑制；土温18-24℃最适根系生长分布。栽培密度可以调节地上部生长，从而间接影响根系生长分布。株行距适当，茎叶生长旺盛，根系吸收的养分多，有利于向深广度生长；密植过度，地上部生长不良，根系也不发达，根系生长幅度比疏植小。 番茄根形态 二、茎 番茄按茎的生长习性，可以分为直立型、半直型和蔓生型3 种。直立型植株矮小，高度0.5-1m，茎木质化程度高，分枝少，产量低，栽培较少。大部分栽培品种均属于半直立型和蔓生型。半直立型，如早熟品种早雀钻，株高为1-1.5m；蔓生型，如品种真善美，株高可达2-5m。 幼苗期，番茄顶芽生长处于优势，植株直立，基本上不分枝。主茎第一花序出现后，顶芽生长优势开始减弱，侧芽萌发能力增强，开始发生侧枝，同时茎节间距由短变长。随着茎叶重量不断增加，茎杆支持不住，变成匍匐状。番茄侧芽抽生能力强，生长快，如果不及时去除侧芽，它会很快赶上甚至超过主枝生长。如果侧枝发生过多、长势过旺，就要消耗大量养分，影响果实的正常生长与发育；另一方面，由于枝叶过多通风透光不良，为病菌繁殖提供条件，容易发生病害。所以，栽培番茄时应该经常整打杈，保护枝叶与花果分布合理，以便钻研身合理调节养分的调配，形成破产的株型。 番茄的茎非常容易产生不定根，利用这一特点，可以进行番茄扦插繁殖。在大田栽
西红柿扦插育苗技术
西红柿扦插育苗技术 番茄扦插育苗是利用番茄侧枝进行无性繁殖的育苗方式。这种育苗方式可以较好地保持品种特性；育苗时间短，一般15～20天；结果早，还能节约种子成本。由于番茄扦插育苗结果早，对营养生长抑制较大，加上根系为不定根，植株生长势较弱，容易早衰，栽培上要加强管理，注意在前期补充养分。 一、扦插时间：扦插育苗的时间根据栽培季节而定。露地栽培可于4月底至5月上旬扦插；大棚秋延后栽培可于6月底7月初扦插，最迟在8月上旬扦插；有日光温室可越冬栽培的于8月开始扦插。 二、扦插方式：可选择用营养土扦插或水插的方法来育苗。春季和晚秋采用营养土扦插，以4—5月扦插的成活率最高；7—8月高温高湿季节，苗棚如果没有很好的降温设施，即使覆盖遮阳网，采用营养土扦插也很容易引起插条腐烂，导致育苗失败，因此宜采用室内水插法。 三、插条选择：在生长势好、抗病力强的植株上选择无病、粗壮、叶色深绿、节间短、长15～20厘米、具有4～5节、生长点完好、带花蕾但未开花的侧枝作扦插枝。 四、修剪枝条：将侧枝从基部掰下，去除下部大叶片，保留中上部3～4张叶片即可。番茄的节位处最易生根，可切除侧枝第一节位下部的茎秆。 五、扦插： 1、营养土扦插：将2份无病虫害、没有种过茄科作物的肥沃园土加1份腐熟有机肥混合过筛，喷200毫克/升高锰酸钾溶液消毒，每立方米营养土中加过磷酸钙1公斤、草木灰5～10公斤拌匀，装入营养钵中，摆放于苗床上。为促进发根，可将枝条下端3～4厘米的部分浸入50毫克/升萘乙酸溶液或100毫克/升吲哚乙酸溶液中10分钟，或者用0.3％磷酸二氢钾和0.2％尿素混合液浸泡2～3小时，之后用清水冲洗。营养钵浇透水后扦插，深度为3～5厘米，扦插后立即搭小拱棚，覆盖遮阳网，以保温、保湿和遮光。 2、水插：将1克吲哚乙酸粉剂加入少量酒精溶解，然后加入5公斤清水制成生根原液。量取10毫升原液倒入10公斤清水中即为生根液。取硝酸钾10.2克、硝酸钙4.9克、磷酸二氢钾2.3克、硫酸镁4.9克，分别加少量水溶解，然后依次倒入盛有10公斤清水的容器内搅匀，即成水插育苗营养液。将容积为500毫升的广口瓶消毒，倒入生根液后插入插条，每瓶插10～12条。待插条发根后再用营养液培养。 扦插后管理：（1）营养土扦插：扦插后5～7天是伤口愈合期，这个阶段要避免阳光直射，遮光率以70％～80％为宜，禁止通风，棚温白天保持在25～30℃、夜间保持在17～18℃，地温保持在18～23℃，空气相对湿度保持在90％以上。扦插苗开始萌发不定根后，可早晚揭开覆盖物，适当增加光照时间和强度，适量通风，棚温白天保持在25～28℃、夜间保持在15～17℃，地温保持在18～23℃，每隔5～7天喷一次0.1％～0.2％磷酸二氢钾溶液。扦插后15天，枝条下端萌发出5～7条5厘米以上的新根和许多不定根时进入成苗期，此时可按照正常苗的管理方法进行管理。（2）水插：插入插条后，室温白天保持在22～28℃、夜间保持在15～20℃。隔日换一次水，气温过高或枝条过多时每天换水。插条长成根系发达的幼苗时移入育苗棚炼苗。
番茄栽培技术
番茄种植技术 一、提高幼苗质量 1、选用良种。选用品质好、丰产性高、抗病虫害能力强，耐贮运的新品种，如金都、红宝石1、2号等。 2、苗床选择。选择背风向阳，地势高燥，排灌方便，土质疏松肥沃，交通便利，前茬作物不是种植茄科作物的地块。清除杂草，精耕细作，每个平方米内加入腐熟的农家肥2000克，复合肥1500克，或是磷酸二氢钾1000克，尿素800克，并进行消毒处理。 3、播种。播种时间以定植时间向前推60—80天，每亩用种量40克左右，每平方米播种10-15克。为使苗齐、苗全、苗壮及提高抗病能力，播种前应进行种子处理及催芽。灌足苗床水，把种子均匀撒于床面上，立即覆1厘米厚的过筛细土，厚度均匀、一致。然后盖膜保温保湿，出苗前不能揭膜，出苗后适当揭膜通风炼苗、间苗，提高幼苗的健壮程度，并于移栽前一个星期浇一次清粪水，或是0.2%的尿素及0.2%的磷酸二氢钾叶面喷雾。 4、移栽。当地温稳定在12℃可定植。定植前亩施农家肥2000公斤及草木灰200公斤作基肥。采用规格化栽培，株行距0.4米X0.5米，沟宽0.4米，高墒低埂，便于排水，每亩定植2600株。移栽应选在无风的晴天。先定植后浇水，定植不宜过深或过浅，以保证移栽成活。 二、加强大田管理 1、施肥。每亩施用氮肥10公斤，磷肥5公斤，钾肥25公斤；或亩施清粪水1000公斤，每月一次。 2、植株修整。（1）绑蔓：植株达30厘米时绑蔓；（2）打杈：确定所留主枝后，摘除其余侧枝，并配合适当的摘心。 3.及时防治病虫害。 （1）疫病 75%百菌清可湿性粉剂600倍液喷雾；58%甲霜灵·锰锌可湿性粉剂500倍液喷雾；64%杀毒矾可湿性粉剂500倍液喷雾；代森锌可湿性粉剂500倍液喷雾。 （2）病毒病注意早期防治蚜虫；或1.5%植病灵乳唑1000倍液喷雾；20%病毒A可湿性粉剂500倍液喷雾。 （3）棉铃虫抓住乳化盛期至2龄盛期，功夫（氯氟氰菊酯）2.5%乳油4000倍液喷雾，或天王星2.5%乳油3000倍液喷雾。 （4）红蜘蛛 2.5%功夫乳油4000倍液喷雾；螨触击灭2500-3500倍液喷雾；达螨灵3000—4000倍液喷雾。 （5）蚜虫亩用5%高效大功臣可湿性粉剂10克兑水40公斤喷雾；或10%虱蚜唑可湿性粉剂2500倍液喷雾。 4、适时采收。根据不同用途在不同时期采收。远途运输或贮藏在绿熟期（果实已充分长大，绿色变淡，果肉坚实，经后熟或催熟后可着色）采收；较耐运输或就地供应在转色期（果脐开始变色，采收后1-2天全部着色）采收；宜作就地供应或不宜较远运输在成熟期（果实大部分着色，表现出该品种固有的鲜艳色泽，营养价值高，风味最好）采收；用于留种或加工在完熟期（果实全部着色，果肉变软，含糖量高，种子成熟饱满）采收。
中粮集团企业战略分析
中粮集团企业战略分析 一、中粮集团简介 中粮集团有限公司(简称“中粮集团”,英文简称“COFCO”)，是中国最大的粮油食品进出口公司和实力雄厚的食品生产商，享誉国际粮油食品市场。中粮的历史可以追溯到1952 年，是中国从事农产品和食品进出口贸易历史最悠久、实力最雄厚的企业，几十年一直是国家小麦、玉米、大米、食糖等大宗农产品贸易的主导者。中粮集团目前下设中粮粮油、中国粮油、中国食品、地产酒店、中国土畜、中粮屯河、中粮包装、中粮发展、金融等9大业务板块，拥有中国食品(HK0506)、中粮控股(HK0606)两家香港上市公司，中粮屯河(600737)、中粮地产(000031)和丰原生化(000930)和吉生化(600893)等6家内地上市公司。作为投资控股企业，中粮有效利用自身遍及世界的业务网络来组织、调配各项经营资源，取得稳健快速的业绩增长，名列美国《财富》杂志全球企业500强，居中国食品工业百强之首。 中粮集团积极投身于生物质能源的开发，作为国内生产燃料乙醇的先行者和主要提供者，努力为国家能源安全和环境保护做出贡献。 中粮集团致力于为13亿中国人民提供营养、健康的优质食品，在葡萄酒、精炼食用油、面粉、大米、啤酒麦芽、番茄制品等行业居中国领先地位，旗下的“长城”葡萄酒、“福临门”食用油、“金帝”巧克力、“黄中皇”绍兴酒、“中茶”茶叶等品牌和产品深受消费者喜爱。中粮集团还是美国可口可乐公司在中国最重要的合作伙伴，生产和销售可口可乐系列饮料。但因其大量使用进口转基因粮食用于生产而遭受民众的质疑。 中粮集团投资开发的海南三亚亚龙湾国家旅游度假开发区被外国游客誉为中国的夏威夷。中粮投资建设的商用写字楼、民用住宅，也在市场上受到欢迎。中粮集团旗下的凯莱国际酒店集团是世界酒店300强之一。 中粮集团在香港拥有“中粮控股”“中国食品” “中粮包装” “蒙牛乳业”四家上市公司；在国内还拥有“中粮地产”、“中粮屯河”、“丰原生化”三家上市公司。 由中国红十字会总会、国务院侨务办公室、国家安监总局、中国新闻社
月瘦15斤减肥计划表
月瘦15斤减肥计划表 首先要说的是没有一个健身计划是完美的，每个人的情况不同，不可能有一个适于所有人的健身计划。下面是OK收集的月瘦15斤减肥计划，欢迎阅读。 早餐，午餐必吃，先在跑步机上跑40分钟，(教练说过，跑三十分钟只是消耗的水分，30分钟以上消耗的才是脂肪，所以要跑30分钟以上)然后拍打腿部肌肉，因为跑完容易使大腿变粗，然后锻炼扩胸器，再练仰卧起坐，50个一组，3组，幅度不要太大，不要让手抱着头，(会对颈椎不好)，不要身体着地，然后再哑铃练习，还有晚饭少吃甚至不吃，做不到不吃就少吃，不然消耗的都补回来了，贵在坚持 现在就开始跳操的减肥方法 首先，要先称现在的体重，如果健身房有条件的话，可以进行专业的体脂测定，看体内脂肪的分布情况，然后根据你自己的需要来进行专项减肥。比如说你虽然看起来很瘦，但是其实腰部、背部、小腹的脂肪很厚实，那你就可以通过一般的有氧运动来消耗他，比如说肚皮舞、拉丁操、搏击操、踏板操;也有可能是你的小腿很粗，但是其实脂肪含量并不高，使肌肉成分居多，那么就要选择以拉伸、放松为主的，例如:普拉提、高温瑜伽。
然后，要阶段性的定制减肥计划。最好是自己定制一个目标减重值，我一般定的是:一个月3kg左右。 一般来说运动减肥的周期比较长，一定要有恒心，就我自己来说是6个月，但是也有很多人是3个月这样。 建议从月经干净后的那周开始。经期我会停前两天。 每周6次课，每次75分钟的锻炼时间。如果达不到这个运动量，很抱歉那只能是一个维持现状的运动量。 然后每次运动的时间安拍:20分钟的有氧热身，40分钟的主课程，15分钟的拉伸和放松全身。但是这个会根据教练的不同有所不同。 如果选择在下班后开始锻炼，那么可以在锻炼前1小时补充一瓶250ml的高钙低脂奶，两片全麦面包。运动前20分钟不要进食。 运动时补充水量大约为500ml(夏天可增加200ml，冬天可减 100ml)。在运动过程中补充水量每次不要超过100ml，并且不要一
番茄长季节高产高效栽培技术(南方版)
番茄长季节高产高效栽培技术（南方 版） 番茄又名西红柿，原产于南美洲秘鲁、厄瓜多尔、玻利维亚，约16世纪末至17世纪初传入我国，近几十年发展迅速，在我国山东、辽宁、江苏、浙江、广西等有大面积种植。 番茄是一年生茄科草本植物，器官分为花、茎、叶、根、果实等，番茄果实营养丰富可口，是深受人们喜食的一种蔬菜。 番茄种植已成为一些地区农业主导产业，尤其是利用塑料大棚、日光温室进行反季节栽培，使之能在冬春淡季供应市场，从而提高了经济效益。 番茄不同生育期对温度的要求是： 发芽期适宜温度 25-30℃ 幼苗期适宜温度 白天25-30℃ 夜间10-15℃ 开花坐果期的适宜温度 白天20-28℃ 夜间15-20℃ 结果期的适宜温度 白天24-28℃ 夜间15-20℃ 番茄属中温性蔬菜作物，在适宜的温度下，可以常年生长，根据番茄的生长特性，结合我国南方冬季温暖的有利气候条件，进行番茄 的“六改”超高产长季节栽培。“六改”即：一、改有限生长型品种为无限生长型品种，二、改短季节栽培为长季节栽培，三、改直蔓上架为斜蔓上架；四、改密植为合理密植栽培，五、改短货架品种为长货架品种；六、改6米大棚为连栋竹架简易塑料大棚。使种植期延长至11个月，亩产量在万公斤以上，形成“种一茬，长一年”的省工省本、高产高效的周年种植方式。下面我们就来介绍番茄在浙南地区的具体栽培方法：
（一）品种的选择 番茄育苗期正处在高温多雨季节，而且秋季温度由高到低，后期天气寒冷，不利于番茄生长和果实成熟。因此，应选择较抗病毒病，耐热又耐寒，生长势强，边续座果能力佳，不易裂果，高产果商品性好，结果集中的无限生长类型品种，如好韦斯特、吉朗达、倍盈等。(加品种图片) （二）培育壮苗 1、种子处理。用55-60℃温水浸种10-15分钟，不断搅拌，当水温降至30℃时再浸泡4-6小时，可预防真菌病毒，或用50%多菌灵500倍液浸种2小时，或用100倍福尔马林浸种30分钟。 2、浸种催芽。在28℃水温中浸种8-12小时，种子拌细砂或育苗基质28-33℃催芽72小时，待种子有2/3出芽即可播种。 3、营养土配制： 营养土配制原则： ①床土富含有机质，保肥、保水、透水性好。 ②苗期营养元素要完全，氮、磷、钾、钙、镁等主要元素，还需微量元素。 ③土壤保持中性。 ④床土无病原菌、虫卵、草籽。 4、播种 一般选择在9月上旬播种。一般采用传统育苗或穴苗盘育苗。 ⑴传统育苗。选择地势高的苗床整平，浇透水后每10平方米用过磷酸钙2.5kg打底，种子拌育苗基质或营养土稀播，一般每亩用量2200-2500粒。播种后用育苗基质或营养土覆盖，播后要做好遮荫防曝晒，加强通风降温、防止高温，及时疏苗，育苗期间不浇水，但注意保墒，特别高温干旱时，可适量浇水，同时要预防病毒病。 ⑵穴苗盘育苗。番茄长季节栽培用的是国外进口昂贵的种子，为了便于集中育苗，减少育苗风险（传统育苗芽率不高，苗易受病虫的侵袭，易受秋季田间大水及暴雨影响），采用穴盘育苗是当今番茄育苗发展一大趋势。 穴盘规格有32孔、50孔、128孔等，根据需要选择不同规格，如用128孔育苗以后需要分苗于营养钵中，50孔或32孔则一次育大苗，但要控制苗龄，一般不超过一个月。 具体的育苗操作是：①穴盘育苗法 基质先平铺满穴于穴盘中，用空穴盘孔对孔对准平铺基质的穴盘压实基质至穴孔2/3处。种子放在小碗或盘中，然后用镊子夹取或竹筷粘取一粒种子放入穴中，再覆盖基质至满穴，并用手或空穴盘整平穴面。播种完毕后穴盘整齐摆放于通风阴
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在穴盘上、下各盖一层地膜，温度保持28℃左右，当绝大部分种子破土后撤去上面的地膜，白天温度降至23―25℃，晚上13～15℃。定植前一周育苗室温度降至12―13℃，低温炼苗，增加幼苗抗性。当第一花序现蕾即可定植。 4 适期定植 大棚春提早栽培应尽量提早扣棚，一般定植前20―30 天扣膜，以提高地温。亩施优质腐熟农家肥8～lO方，氮磷钾复合肥50千克。深翻25厘米左右，平整土地，起垄，宽窄行定植。垄高20厘米，宽行80厘米，窄行60厘米。尧都区适宜定植时间为3月中旬（棚内气温稳定在lO℃以上，10厘米低温稳定在15℃以上）。定植前一天窄行浇透水。按株距30―33厘米定植，亩留苗3200―3500株。定植后覆盖黑色地膜，可起到促进根系发育、防治杂草的作用。 5 定植后管理 5.1 温度管理 定植后密闭大棚，使棚温保持280C―30℃，促进缓苗：缓苗后昼温保持22℃―25℃，最高不超过30℃，夜温13℃―15℃左右。开花结果期保持棚内适温低湿，白天25℃―30℃，晚上15℃～18℃，结果期白天25℃―28℃，晚上15℃―17℃。进入5月中旬以后就要昼夜放风，尽量控制白天不超过30℃，夜?g不超过17℃。 5.2 肥水管理
番茄果实结果期技术管理要点
番茄结果期技术管理要点 一、喷花授粉 1、常温授粉药选用果霉宁，药剂使用安全性高。根据药液高温时浓度变小，低温时浓度变大的特点，目前授粉药浓度为每袋兑水2.5斤，在西红柿1－2穗果之间，尤其是第一穗果开花时常温达到37度，浓度低时授粉药性质不稳定。有些棚中出现僵果的原因就是授粉药受高温的影响浓度达不到，以至于果实长到鸡蛋大小时就不再膨果，形成僵果。另外授粉药配置受水质影响大，水中矿物质、重金属和微量元素对授粉药的寿命和稳定性影响特别大，原则要求尽量用纯净水配授粉药，纯净水能起脱盐分和重金属的作用。 2、授粉尽量选择晴天上午，一般日出半小时后至中午11或接近12点，这一段时间叶子进行光合作用药液被吸传导膨果快。下午授粉，叶子不进行光合作用，药液在花朵上存留，第二天才能被吸收传导，容易因温度过高、蒸发量大造成授粉不良出现僵果、畸形果等。 二、调节温度 要想出高产量高品质的果实，必须降低下午的温度，使温度控制在30度以内，28度左右。因为番茄上午叶子进行光合作用的光合产物比较多，下午要进入同化阶段把养分转化贮存，就像人吃饭一样，要把食物消化掉才有力气。同化作用的同时需要低温，温度低养分积累就多，所以下午要把棚口、放风口放大通风降温，不至于在同化过程中消耗太多的养分。如果下午温度过高，同化的同时大部分养分被消耗掉了，到晚上真正需要养分的时候就没了，因为西红柿叶片白天制造养分，晚上把养分运输到果实上去。 要想植株长势好，开花多，昼夜温差必须在10摄氏度以上。为了降低白天温度，可以加遮阳网，或往棚膜上甩泥浆。 三、去下部老叶 叶子和果实的关系是，叶子不向上面的果实上输送养分，而向其下面的果实上输送养分。当最底部的叶子变老变黄时就失去了供养能力，它制造的养分还不如消耗的多，此时可打掉果实下面的老叶。
中粮实习报告
一、实习单位简介 中粮屯河股份有限公司（简称中粮屯河，英文简称COFCO TUNHE）是我国领先的果蔬食品生产供应商，是世界500强企业中粮集团有限公司控股的A股上市公司, 股票代码600737。公司主营农业种植、番茄、食糖、林果、罐头、饮料加工及贸易业务，是全球最大的番茄生产企业之一、全国最大的甜菜糖生产企业、全球最大的杏酱生产企业之一，是国家农产品加工重点龙头企业，是中粮集团九大业务板块之一。公司致力于成为果蔬食品行业的领导者和全球一流的食品企业，奉献绿色营养食品，使客户、股东、员工价值最大化。 中粮屯河股份有限公司（简称“中粮屯河”）主营番茄加工及贸易、制糖及贸易、林果加工及贸易三大业务。中粮屯河是世界第二、亚洲最大的番茄加工企业，在新疆、内蒙古地区拥有50万亩的番茄种植基地，共有21家番茄制品加工企业，现已具备日处理鲜番茄4.62万吨、年产33万吨番茄酱、3000吨番茄粉、10吨番茄红素的生产能力。番茄酱出口量占据了全球贸易量15%的份额，占中国番茄制品出口总量的50%。企业采用先进的番茄育种技术并拥有集食品安全检测、土壤环境监测、食品营养分析及微生物检测功能为一体的专业检验机构，中粮屯河的番茄制品通过了亨氏、联合利华、卡夫、雀巢等知名企业的认证。 图1.1新疆中粮屯河番茄制品股份有限公司 中粮屯河在新疆拥有40万亩丰产高品质甜菜基地，旗下9家糖厂年制糖能力达到45万吨，是中国最大的甜菜糖生产商，生产的白砂糖、绵白糖等产品拥有良好的声誉和优质的客户群，已与可口可乐、卡夫、伊利及蒙牛等知名企业建立了长期合作关系。 旗下拥有4家果品加工企业，10万吨鲜杏的加工能力，具有1.8万吨杏浆的年生产能力。主要产品杏浆远销欧洲、俄罗斯等地区和国家。 中粮屯河成立于1983年， 1996年在上交所上市， 2005年6月，在中粮集团成功重组新疆屯河后，公司进入快速、健康、持续的发展轨道，企业盈利能力、
番茄实用高效栽培及病虫害防治技术
番茄实用高效栽培技术 番茄具有独特的风味和丰富的营养，深受广大消费者的喜爱。其适应性强，全国各地普遍栽培，产品已实现周年供应，创造了巨大的社会和经济效益。番茄实用高效栽培技术要点如下： 1.环境选择 一般土壤都能生长，尤以土层深厚肥沃、保水保肥能力强、土质疏松、地下水位低、排灌良好的土壤为宜。无公害番茄种植区域的土壤、水源、空气需符合《农产品安全质量无公害蔬菜产地环境要求》（GB/T18407·1-2001） 2.品种选择 选用丰产性、抗病性、抗逆性、商品性、适温性等综合性状优良的品种，如斯诺克、超级金刚、格美092、欧蜜等。 3.栽培季节与茬口 在我国，番茄栽培主要以春夏露地生产为主，反季节栽培发展迅猛，主要茬口有以下几种： 3.1早春栽培：10月中下旬育苗，12月中下旬定植。 3.2秋冬栽培：7月中下旬到8月上中旬育苗，8月中下旬到9月上旬定植。 3.3冬春栽培：10月下旬到11月上旬育苗，1月中下旬到2月上旬定植。 4.种子处理与催芽 4.1温汤烫种消毒法：先将番茄种子放在清水中浸泡、揉搓，漂去瘪子及除去杂物，然后放入温水中浸泡3-4小时，再放入55℃恒温热水中烫种10分钟后立即拿出放入清凉温水中冷却，继续浸泡5-6小时，然后用湿毛巾包好，滤去多余水分，然后放到25℃-30℃环境里保湿催芽。 4.2药剂消毒法：将番茄种子放在清水中浸泡、揉搓，漂去瘪子及除去杂物，放入温水中浸泡3-4小时，再放入10%的磷酸三钠溶液中浸泡20分钟，捞出后用清水冲洗干净，温水继续浸泡5-6小时，然后用湿毛巾包好，滤去多余水分，放到25℃-30℃环境里保湿催芽。催芽期间间隔12-24小时冲洗一次。约70%种子露白即可播种。 注：已包衣种子可不用消毒，直接浸泡后催芽。 5.播种育苗 5.1种子约70%露白即可播种。 5.2播种方法：主要有撒播法（苗床或苗盘）、点播法（穴盘/营养钵/营养块），育苗工厂可用播种机播种（不催芽，干籽）。通常采用的流程：穴盘消毒→装填基质→压孔→播种→盖种→浇水→覆膜。 5.3苗期管理：种子70%出芽后及时揭去覆盖物，并及时通风排湿，增加光照，保持育苗基质或营养土湿度适宜，避免干燥或湿度过大。 5.4分苗：撒播法育苗最好采用二次分苗法，在幼苗有2-3片真叶的时候移植到50孔或72孔的穴盘或者10×10厘米的营养钵里。 5.5病虫害防治：苗期主要病虫害有猝倒病、立枯病、蚜虫、斑潜蝇等，需及时防治。 6.定植 6.1建立轮作制度。不能和茄科类作物（辣椒、茄子、马铃薯）连作。 6.2施肥整地做厢：多施腐熟农家肥和有机肥，每亩可施5000kg的优质农家肥和100kg生物菌肥最为基肥。最好进行测土配方施肥，使大量、中量、微量元素肥合理均衡。将基肥均匀撒施土壤表面以后，再