Screening and Expression of a Silicon Transporter Gene in

Research ArticleScreening and Expression of a Silicon TransporterGene (Lsi1) in Wild-Type Indica Rice Cultivars

Mahbod Sahebi1 Mohamed M Hanafi123 M Y Rafii1 Parisa Azizi4

Rambod Abiri5 Nahid Kalhori6 and Narges Atabaki7

1Laboratory of Climate-Smart Food Crop Production Institute of Tropical Agriculture and Food Security Universiti Putra Malaysia43400 Serdang Selangor Malaysia2Department of Land Management Faculty of Agriculture Universiti Putra Malaysia 43400 Serdang Selangor Malaysia3Laboratory of Plantation Science and Technology Institute of Plantation Studies Universiti Putra Malaysia 43400 SerdangSelangor Malaysia4Department of Crop Science Faculty of Agriculture Universiti Putra Malaysia 43400 Serdang Selangor Malaysia5Department of Biochemistry Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia43400 Serdang Selangor Malaysia6Department of Biology Faculty of Science Universiti Putra Malaysia 43400 Serdang Selangor Malaysia7IAU of Tehran Science and Research Branch Tehran Iran

Correspondence should be addressed to Mahbod Sahebi mahbod sahebiyahoocomand Mohamed M Hanafi mmhanafiupmedumy

Received 9 September 2016 Accepted 14 December 2016 Published 16 January 2017

Academic Editor Ravindra N Chibbar

Copyright copy 2017 Mahbod Sahebi et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Silicon (Si) is one of the most prevalent elements in the soil It is beneficial for plant growth and development and it contributes toplant defense against different stresses The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice varietyThis gene was not identified in fourteen Malaysian rice varieties during screening Then a mutant version of Lsi1 was substitutedfor the native version in the three most common Malaysian rice varieties MR219 MR220 and MR276 to evaluate the function ofthe transgene Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties Siliconconcentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants Transgenic varietiesshowed significant increases in the activities of the enzymes SOD POD APX and CAT photosynthesis and chlorophyll contenthowever the highest chlorophyll A and B levels were observed in transgenic MR276 Transgenic varieties have shown a strongerroot and leaf structure as well as hairier roots compared to the wild-type plants This suggests that Lsi1 plays a key role in riceincreasing the absorption and accumulation of Si then alters antioxidant activities and improves morphological properties

1 Introduction

Rice (Oryza sativa L) is one of the most important foodcrops worldwide and it is cultivated in a particular type ofartificial wetland Due to population increases worldwiderice production is expected to increase by over 40 by2030 Hence the production of different rice varieties withgreater yield stability is essential for overcoming reductionsin grain yield as well as the limitations of arable lands [1ndash3] Silicon (Si) is one of the most plentiful macroelements inthe soil and performs an important function in healing plants

under various environmental stresses Si is applied to plantsto induce resistance against various stresses including bothdisease and pests Moreover Si can improve the conditionof the soil which may contain toxic levels of heavy metalsalong with other chemical elements [4] Silicon among allplant nutrient elements inside the soil carries a distinguishedrole in plant formation especially under harsh environmentand poor nutrient condition The role of Si is not limited toplant growth only but also it helps plant to cope with varietyof abiotic and biotic stresses [4 5]

HindawiBioMed Research InternationalVolume 2017 Article ID 9064129 13 pageshttpsdoiorg10115520179064129

2 BioMed Research International

Many insect pests and epidemic diseases affect differentparts of the rice plant resulting in severe rice productionlosses [6] Rice yield stability is affected by various biotic andabiotic stresses Blast and sheath blight fungi bacterial blightroot-knot (caused by nematodes) and rice yellow mottlevirus cause themajority of losses in the annual rice grain yield[7] The brown plant hopper (BPH Nilaparvata lugens) animportant insect pest of cultivated rice has interfered withthe production of rice since the 1960s [8] The plant cell wallis a barrier between host plants and pathogens but the fungusMagnaporthe oryzae uses 30 and 44 different enzymes todegrade cellulose and hemicellulose respectively in plant cellwalls [9 10]

On the other hand the release of chlorofluorocarbongases due to the increased solar ultraviolet-B radiation on thesurface of the earth has reduced the stratospheric ozone (O

3)

layer [11] Depletion of theO3layer decreases plant height and

aboveground biomass and it also increases DNA damage Inthis context it has been shown that increased UV-B radiationhas the same effect on plants grown in the Arctic Antarcticand lower latitudes [12] Therefore it is crucial to introduceeffective ways of improving plant growth and reducingsuch damage The application of various chemical fertilizersshould affect the resistance as well as the tolerance of riceplants to biotic stresses such as the brown plant hopper (BPH)[13] It has also been shown that the risk of BPH outbreaksin hybrid rice could be increased through the application ofhigh levels of nitrogen fertilizer [8 14]

Silicon (Si) is one of the most prevalent elements inthe soil Si is beneficial for plant growth and contributes toplant defense mechanisms against multiple biotic and abioticstresses including tolerance to salt drought and toxic metals[15] Silicon enhancement stimulates plants to produce phy-toalexins and phenolics consequently increasing the plantsrsquocapacity to defend against stress factors [16] Si may reduceoxidative damage in plants encountering environmentalstresses [17] and it plays an important role in enhancingplantsrsquo tolerance to biotic stresses such as fungal diseasesThis increased tolerance is partly attributed to increases inthe defense capacity of plants due to Si-induced increases inantioxidants A Si-cuticle double layer is produced due to Sideposition beneath the cuticle and acts as a barrier to physi-cally prevent the penetration of fungi [15] Si can improve soilconditions in the presence of toxic heavy metals minimizethe toxicity of Mn Al and Fe and enhance P availability[15 18] The Si concentration depends on the plant genotypeas it does in other organisms Therefore plantsrsquo metabolicactivities and physiological mechanisms might be affected bydifferent amounts of Si used in fertilizers [15 19 20]

Rice which can accumulate Si up to 100 of the shootdry weight shows the most effective Si accumulation amongplants Stable production and healthy growth of rice arehighly affected by the Si content of the shoots The high Siaccumulation in rice shoots is attributed to the ability of riceroots to take up large amounts of Si [21] The addition of Si toa susceptible rice variety infected byMagnaporthe grisea wasassociated with the accumulation of momilactone phytoalex-ins [22] and the upregulation of peroxidase PR-1 transcriptsand glucanase [23]

Silicon can increase the tolerance of plants to UV-B stressand increase the amounts of chlorophyll a and b [24 25]Theamount of Si on the adaxial surface of rice-leaves increasestolerance toUV-B radiation suggesting that silicic accumula-tion may help reduce the physical stress caused by UV radia-tion [26] It has been reported that Si supplementation duringthe hydroponic culture of rice enhances UV-B resistancewhich might be attributed to increases in phenolic com-pounds in the plant epidermis [27]

Silicic acid is the form of Si that is absorbable by plantroots it translocates to the shoots through the transpirationstream and is later polymerized and accumulated as silica inthe shoot tissues [28 29] Si uptake in rice is controlled by atleast two transporters Lsi1 (influx) and Lsi2 (efflux) but theaccumulation of Si is mediated only by Lsi1 [30] Therefore itis hypothesized that the transformation of Lsi1 and its expres-sion or overexpression in Indica rice varieties may improvethe resistance of the plant to stress factors In this study wescreened the Lsi1 gene in fourteen Malaysian rice varietiesThree common Malaysian rice varieties were selected forgenetic manipulation and showed morphological improve-ments due to greater Si accumulation

2 Experimental Procedures

21 Plant Material Fourteen important Malaysian rice vari-eties (MR219 MR276 MR220 MR211 MR263 MR269MR185 MR159 MRQ74 MRQ50 Mashuri Jaya Masriaand Milyang) were provided by the Malaysian AgriculturalResearch and Development Institute (MARDI)

22 Screening of the Lsi1 Gene Rice seeds were soaked inwater for three days at 25∘C and germinated in Petri dishesusingWhatmanfilter paper (Sigma-AldrichUSA)moistenedwith a 05mM CaCl

2solution Subsequently seeds with

a bud length of 1-2mm were selected transferred into ahydroponic culture solution [31] and kept in a glasshouse at25ndash30∘C under fluorescent white light with a photoperiod of16 hours light8 hours dark for three weeks Throughout theexperiment the pH of the solution was maintained at 57Thenutrient solution was renewed every 3 days The root tissuesof all varieties were sampled immediately frozen in liquidnitrogen and then kept at minus80∘C for total RNA isolation

23 RNA Extraction and Semiquantitative RT-PCR TotalRNA from the roots of the fourteen varieties was isolated sep-arately using TRIzol Reagent (Invitrogen USA) For all sam-ples DNase I (Qiagen USA) was used to digest contaminat-ing genomic DNA First-strand cDNA was synthesized fromeach sample using SuperScript III (Invitrogen USA)TheTaqDNA polymerase kit (Fermentas Waltham MassachusettsUSA) was used with the following program initial denatura-tion at 95∘C for 5min 35 cycles of 95∘C for 30 sec annealing(55∘C 56∘C 57∘C 58∘C 59∘C 60∘C 61∘C 62∘C 63∘C 64∘C65∘C and 66∘C) for 45 sec extension at 72∘C for 1min anda final extension for 10min at 72∘C The PCR products wereisolated on 15 agarose gels and stained with Midori Green(Nippon Genetics Germany) Two sets of specific primers

BioMed Research International 3

Table 1 Primers for the Lsi1 gene used for screening via semiquantitative RT-PCR

Gene Forward primer (51015840-31015840) Reverse primer (51015840-31015840) Expected PCR product sizeLsi1 (I) CCAGGGCGAACTACTCCAACGA GCTTTGGTTGCTTGGTGGTTCG 1066 bpLsi1 (II) ATGGCCAGCAACAACTCG TCACACTTGGATGTTCTC 897 bp

ATGGCCAGCAACAACTCGAGAACAAACTCCAGGGCGAACTACTCCAACGAGATCCACGATCTCTCCACGGTGCAGAACGGCACCATGCCTACCATGTACTACGGCGAGAAGGCCATCGCCGACTTCTTCCCTCCTCACCTCCTCAAGAAGGTCGTGTCGGAGGTGGTGGCCACGTTCCTGCTGGTGTTCATGACGTGTGGGGCG

CGTGACGGTGATGATCTACGCCGTCGGCCACATCTCCGGCGCCCACATGAACCCCGCCGTGACGCTCGCGTTCGCCGTGTTCAGGCATTTCCCCTGGATTCAGGTTCCGTTCTACTGGGCGGCGCAGTTCACCGGAGCGATATGCGCGTCGTTCGTGCTCAAGGCGGTGATCCACCCGGTGGATGTGATCGGAACCACCACGCCC

GCCGTCGCCACGGACACGAGAGCGGTGGGTGAGTTGGCCGGGTTGGCGGTTGGTTCCGCGGTTTGCATTACGTCCATCTTCGCAGGGGCAATTTCAGGTGGATCGATGAACCCGGCAAGGACGCTGGGGCCGGCGCTGGCGAGCAACAAGTTCGACGGCCTGTGGATCTACTTCCTGGGCCCAGTCATGGGCACGCTCTCGGGA

CTTCAAGCTGCGCCGCTTGCGGAGCCAGCAGTCCATCGCCGCCGACGACGTCGACGAGATGGAGAACATCCAAGTGTGA

GCAGGGATCAGCGGCAGCGACCTGTCTCGCATATCGCAGCTGGGACAGTCGATCGCCGGTGGCCTCAT

GTGGGGCCGCACTGGCACTCGCTCGTCGTCGAGGTCATCGTGACGTTCAACATGATGTTCGTCACGCTC

GCATGGACCTACACCTTCATCCGCTTCGAGGACACCCCCAAGGAAGGCTCCTCCCAGAAGCTCTCCTC

GGGGACAAGTTTGTACAAAAAAGCAGGCTTC

GGGGACCACTTTGTACAAGAAAGCTGGGTC

3998400

3998400

5998400

5998400

Figure 1The CDS of Lsi1with one pair of anchors (required to make the attB PCR product) at the 51015840 and 31015840 ends (nucleotides shown in red)

were designed for the Lsi1 gene based on the sequenceavailable in GenBank using Primer Premier 6 (Table 1)

24 Construction of the Expression Clone

241 Preparation of the attB PCR Product The CDS ofthe Lsi1 gene includes 897 bp (GenBank AB2222721) Onepair of adaptors (anchors) was designed according to theinstructions provided by the manufacturer of the GatewayTechnology with Clonase II (Invitrogen USA) kit based onthe structure of the pDONORZeo vector The kit and vectorwere provided by First BASE Laboratories Sdn Bhd Malaysia(Figure 1)

The template sequence was amplified using the followingspecific primers

Forward primer 51015840 GGGGACAAGTTTGTACAA-AAAAGCAGG 31015840

Reverse primer 51015840 GACCCAGCTTTCTTGTAC-AAAGTGG 31015840

The following PCR (KAPA HiFi Hot Start DNA poly-merase kit USA) program was performed 95∘C for 3minand 30 cycles of 95∘C for 30 sec 62∘C for 30 sec and 72∘Cfor 30 sec followed by a final extension of 10min at 72∘CThe PCR product was isolated on a 15 agarose gel andstained with Midori Green (Nippon Genetics Germany)The expected 958-bp band was purified using a QIAprepSpin miniprep kit (Qiagen Germany) and submitted forsequencing

The Gateway Technology kit (Invitrogen USA) was usedto run the BP and LR recombination reactions according to

the manufacturerrsquos procedures using the expression vectorpFast G02 [32]

242 Transformation of Competent Cells Transformationwas performed using the E coli strain TOP10 which lacksthe ccdA gene and is not sensitive to the effects of the ccdBgene The ccdB gene which is found in the structure of thepDONORZeo vector functions as a negative selectablemarker Zeocin was used for bacterial selection in low-salt LBagar The transformation process was performed accordingto the instructions provided by the manufacturer of the PCRCloningplus Kit (Qiagen Germany)

243 Agrobacterium-Mediated Transformation The Agro-bacterium tumefaciens strain LBA4404 was used to transferthe expression clone containing the CDS of the Lsi1 geneto the calli of three selected varieties MR219 MR220 andMR276 via the freeze-thaw method

25 In Vitro Studies Part One The mature dry seeds of eliteIndica rice varieties (MR219 MR220 and MR276) selectedfrom the screeningwere usedThehulled seedswere sterilizedusing 70 alcohol and shaken at 120 rpm for 20min in a 40solution of sodium hypochlorite containing three drops ofpolyoxyethylene sorbitanmonooleate using an orbital shakerThe seeds were rinsed five times with autoclaved distilledwater Finally the seeds were dried on autoclaved filter paperand transferred to the culture room Ten sterile seeds of eachvariety were placed separately in two Petri dishes (100 times15mm) containing MS-24-D medium pH 58 The culturedseeds were then incubated at 25∘C in the dark for three weeks


Table 2 Primers used to verify the transgenic MR219 MR220 and MR276 plants

Gene Forward primer (51015840-31015840) Reverse primer (51015840-31015840) PCR product sizeLsi1 ATGGCCAGCAACAACTCG TCACACTTGGATGTTCTC 897 bpCaMV35S CCGACAGTGGTCCCAAAGAT TACTCATTTTACTTCTTCG 1198 bp

until a yellowish white embryogenic callus appeared on thescutellar surface

251 Transformation of MR219 MR220 andMR276 Embryo-genic Calli with Agrobacterium Containing the pFast G02Vector The OD of an Agrobacterium suspension culture wasadjusted to 08 using N6-AS medium Then embryogeniccalli from three varieties were placed in the Agrobacteriumsuspension for 30min in three different Petri dishes Sub-sequently bacterial suspension from each Petri dish waspipetted out and the excessAgrobacterium infectionmediumwas absorbed with autoclaved filter paper The transformedcalli of all three varieties were then transferred to N6-ASmedium (solid cocultivation medium) Finally the threeplates were incubated at 28∘C for 72 hours in the dark

Transgenic plants were generated using the followingsteps [3] production of embryogenic calli in MS-24-D at25∘C for 3 weeks under dark conditions proliferation oftransformed calli in MS-24-D + cefotaxime at 25∘C for 10days under dark conditions selection of transformed calliusing MS-24-D + cefotaxime + BASTA (first selection) at25∘C for 2 weeks selection of transformed calli using MS-24-D + cefotaxime + BASTA (second selection) at 25∘C for2 weeks selection of transformed calli using MS-24-D +cefotaxime + BASTA (third selection) at 25∘C for 2 weeksshoot production from surviving and healthy embryogeniccalli in MSKN regeneration medium at 25∘C for 20 daysunder dark conditions plantlet regeneration in regenerationmedium (MSKN) at 27∘C under light conditions (with a 16-hour photoperiod) for 20 days root production in rootingmedium (MSO) at 27∘C under light conditions (with a 16-hour photoperiod) for 20 days

252 Transfer of Transgenic MR219 MR220 and MR276Plants to the Soil The culture medium from the roots ofthe plantlets was gently removed after two weeks whenthe root system was well developed Then the plantletswere placed in Yoshida culture solution and transferred toa transgenic greenhouse with 95 relative humidity and a14-hour photoperiod (160 120583molm2s) and grown at 29∘C forthree weeks Then plants with dynamic root systems wereput into pots containing paddy soil water (initially 1 L perpot) and fertilizers All pots received a basal applicationof 50 kgNhaminus1 70 kg P haminus1 and 70 kgKhaminus1 The sourcesfor N P and K were urea (60N) KH

2PO4(286K and

227P) andmuriate of potash (MoP) (50K) respectivelyN fertilizer top dressing (50 kgNhaminus1N) was performed atthe tillering and flowering stages When the color of thegrains (almost 85) turned straw gold panicles from eachvariety were harvested

253 Analysis of Transgenic MR219 MR220 and MR276Plants Theseeds of transformedplants kept in the transgenicgreenhouse were harvested after full maturationThe putativetransgenic plants were analyzed using the 3G plant PCR kitKAPA (South Africa) with the following program initialdenaturation at 95∘C for 3min 35 cycles of 95∘C for 30 secannealing (54∘C for Lsi1 and 60∘C for CaMV35S) for 45 secextension at 72∘C for 1min and a final extension at 72∘C for10min The PCR amplification was performed using two setsof specific primers (Table 2)

254 GFP Monitoring of MR219 MR220 and MR276 Trans-genic Seeds GFP expression was detected in the transgenicrice seeds obtained from the T

0and T

1generations using

a fluorescence microscope (Leica MZFL111) adjusted with aGFP2 filter Images of transgenic seeds were captured usingthe fluorescence microscope with the Leica DC 200 systemand its related software (Leica DC Viewer)

26 InVitro Studies Part Two Mature dry seeds of transgenicrice varieties (MR219 MR220 and MR276) were used Theprocedure described in part one of the in vitro study wasfollowed Three sterile seeds of each variety were placedseparately in three glass vials containing MS media supple-mented with 2mg Lminus1 24-D and 35mM K

2SiO3 pH 58

Three replicates of each glass were measured thus a totalof 9 glass vials were examined The cultured seeds werethen incubated at 25∘C in the dark for three weeks until ayellowish white embryogenic callus appeared on the scutellarsurface When the regeneration steps of the shoots and rootsof the transgenic plants were completed following the stepsdescribed in part one of the in vitro study the transgenicplants were transferred to the transgenic greenhouse

261 Expression Analysis of the Lsi1 Gene in T1 TransgenicPlants Using Real-Time PCR Total RNA was extracted fromboth wild-type and transformed plants using TRIzol Real-time qRT-PCR (KAPA SYBR FAST One-Step qRT-PCRUSA) was performed to assess the expression level of thecandidate gene (Lsi1) in wild-type and transgenic plants(MR219 MR220 and MR276) The following program wasused for real-time qRT-PCR 42∘C for 5min inactivation ofRT at 95∘C for 5min followed by 40 cycles at 95∘C for 3 sec60∘C for 30 sec and 72∘C for 3 sec The data were obtainedfrom three independent biological replicates Ampliconspecificity was controlled using a melting curve analysisthrough increasing the temperature from 60∘C to 95∘C after40 cycles Lsi1 and two internal reference genes (18S rRNA and120572-Tubulin) were amplified using specific primers (Table 3)The REST software (Qiagen Hilden Germany) was hired to


Table 3 Primers used for real-time qRT-PCR of Lsi1 and two reference genes (18S rRNA and 120572-Tubulin)

Gene Forward 51015840 rarr 31015840 Reverse 51015840 rarr 31015840

Lsi1 ATGGCCAGCAACAACTCG TCACACTTGGATGTTCTC18S rRNA ATGATAACTCGACGGATCGC CTTGGATGTGGTAGCCGTTT120572-Tubulin GGAAATACATGGCTTGCTGCTT TCTCTTCGTCTTGATGGTTGCA

analyze the results of the qRT-PCR and all data were nor-malized using ΔΔCT method The 18S rRNA and 120572-Tubulinreference genes ofO sativawere used as expression referencegenes for normalization The manufacturerrsquos instruction wasfollowed to quantify the relative gene expression

262 Estimation of Antioxidant Enzyme Activity Regener-ated root and leaf tissues (100mg) from each transgenic varietywere homogenized separately in phosphate buffer (50mMpH 70) containing polyvinylpyrrolidone (10mM) Triton X-100 (005) EDTA (10mM) and ascorbate (10mM) Thenthe homogenate was centrifuged at 13500 rpm at 4∘C for15min Next the activities of antioxidant enzymes were mea-sured using the supernatant The activity levels of superoxidedismutase (SOD) peroxidase (POD) ascorbate peroxidase(APX) and catalase (CAT) were determined using previouslypublished methods [33]

263 Chlorophyll Content The leaves of wild-type and trans-genic plants were collected separately for each cultivar Thechlorophyll contents (a b and total) were estimated usingthe method of Arnon [34] Approximately 05 g of plantletleaves was homogenized in liquid nitrogen and then solubi-lized in 80 acetone Then the mixture was centrifuged at10000 rpm for 15min Finally the supernatant was collectedand the absorbance at 663 and 645 nm was measured using ascanning spectrophotometer (AL800 Aqualytic Germany)The chlorophyll contents were measured as mgg of sample

264 Analysis of Photosynthesis The LI-6400XT portablephotosynthesis system (LI-COR Lincoln Nebraska USA)was used tomeasure and compare the photosynthesis of wild-type and transgenic varieties after theywere transferred to thesoil

265 Silicon Concentration Determination of the amountof Si in each of the three transgenic varieties compared tothe wild-type plants was conducted following the methoddescribed by Korndorfer et al [35] The leaves and roots ofthree transgenic varieties (T

1) were dried at 60∘C for 72 hours

using a ventilated ovenWhen the samples reached a constantweight they were ground separately For the digestion step01mg of dried and ground sample was placed in a polyethy-lene tube followed by the addition of 20mL H

2O2and

30mL of NaOH (25mol Lminus1) and incubation at 123∘C and015MPa for 1 hour using an autoclave After the tubes werecooled deionized water was added to fill each tube Finallyan aliquot (10mL) of this extract was taken and 20mL ofdeionized water was added to monitor the Si concentrationusing a spectrophotometric method Silicon reacts with theammonium molybdate in the HCl medium resulting in the

formation of molybdosilicic acid and the emission of a yellowcolor that can be detected by spectrophotometry at 410 nm

266 Si Observations under Scanning Electron MicroscopyThis part of the experiment was performed using the calliand roots of bothwild-type and transgenic rice varieties Calliof wild-type and transgenic varieties cultured in 1 120583M-silicatemedium (MSmedium+K

2SiO3) for 45 days were dehydrated

by filtration through an ethanol series and then freeze-dried(JFD-300 JEOL Tokyo Japan) Similarly the roots of wild-type and transgenic rice varieties obtained from hydroponiccultures supplemented with 15mM SiO

2were dehydrated

and dried using a critical point dryer (CPD2 Pelco CAUSA) Then the dried samples were coated with gold onaluminum stubs using a sputter coater (SC7640 PolaronSussex UK) Samples were observed under scanning elec-tron microscopy (SEM) (LEO 1455 VPSEM New England)Images were obtained at 2000 kV and at magnifications of350 400 and 500x

267 Morphological Characteristics of Transgenic VarietiesIn this part of the experiment we used transgenic varietiesand wild-type rice varieties in dry seeding and wet seedingSeven basic characteristics root length root dry weight atthe maximum tillering stage root dry weight at the panicleinitiation stage root dry weight at the flowering stage shootdry weight at the maximum tillering stage shoot dry weightat the panicle initiation stage and shoot dry weight at theflowering stage weremeasured for comparisons of transgenicand control plants

27 Statistical Analysis The experiments were repeated threetimes for each transgenic variety and wild-type parent Thenormalized data were statistically analyzed (ANOVA andLSD multiple test) using SAS software (version 91 USA)

3 Results of Screening and Expression ofthe Lsi1 Transgene

31 Lsi1 Screening The screening of the Lsi1 betweenMalaysian rice cultivars showed that this gene is not existedin the 14 wild-type varieties using two sets of specific primerswith a series of annealing temperatures

32 Analysis of T1 Transgenic MR219 MR220 and MR276Rice Varieties Three and ten plants of MR219 five andnine plants of MR220 and four and eight plants of MR276were identified as the T

0and T

1transgenic generations

respectively Three transgenic varieties MR219 MR220 andMR276 were submitted to NCBI (KR673322 KT284742and KT284741) The regenerated plant seeds (T

1) continued


(a) (b) (c)

(d)

MR219 MR220 MR276

Figure 2 Regeneration of T1seeds of transgenic MR219 MR220

and MR276 varieties (a) Callus induction (b) and (c) regeneratedtransgenic shoots (d) regenerated transgenic roots Bars = 1 cm

to grow through in vitro culture and after planting in thesoil in the transgenic greenhouse (refer to Figure 2 in themethodology part two of the in vitro study)

The results obtained from the analysis of each T1trans-

genic MR219 MR220 and MR276 plant gave two specificbands of 897 and 1198 bp (Figure 3)

33 GFP Monitoring of the T0 Transgenic Calli and T1 Trans-genic Seeds of MR219 MR220 and MR276 The seeds har-vested from plants of the T

0and T

1generations (Figure 4(a))

and calli obtained from the T1transgenic plants (Figure 4(b))

were monitored under a fluorescence microscope to recordgfp expressionThe seeds of the T

0generation expressing gfp

were selected and used to obtain the T1generation followed

by the selection of T1seeds expressing gfp for further analysis

Meanwhile some of the calli obtained from T1transgenic

plants expressing gfpwere transferred to culture in amediumsupplemented with K

2SiO3

34 Differential Expression of the Lsi1 Gene in TransgenicPlants The real-time qRT-PCR analysis indicated that theexpression level of the Lsi1 gene was higher in all transgenicvarieties compared to their related wild-type or untrans-formed plants To avoid positional effects of transgenesis theexpression level of Lsi1 was considered using a constitutivepromoter (CaMV35S) and the same tissue (roots) in alltransgenic varieties Differences among transgenic varietieswere statistically significant (119875 lt 001) The standard curvesshowed high correlation coefficient of 1198772 = 0994 0995and 0997 presenting that the Ct values were proportionalto the copy numbers of transgene for all three varieties Theamplification efficiencies of transgene in three varieties were

973 984 and 987 The standard curve of transgene foreach variety was within the acceptable range (amplificationefficiency of 90ndash110and1198772 value of 0985)Theupregulationof the Lsi1 gene in transgenic MR276 was higher than that inthe transgenic MR219 and MR220 plants (Figure 5)

35 Si Concentration and Observation under Scanning Elec-tron Microscopy Si concentrations in the roots and leaveswere significantly higher in transgenic plants than in wild-type plants (Table 4) The Si concentrations in the roots andleaves of transgenic MR276 were higher than those in thetransgenic MR219 and MR220 varieties The MR220 varietyamong all the transgenic plants showed the smallest changesin Si concentration extracted from the leaves and rootsTheseresults strongly confirmed the results obtained from real-time qRT-PCR (Figure 6) suggesting that the transgenicMR276 variety with a higher expression level of the Lsi1 geneabsorbedmore Si from the culturemediumaswell as from thehydroponic culture The bar graph shows that all of the wild-type varieties absorbed some amount of Si from the culturemedium or hydroponic culture confirming the ability of riceplants to absorb Si Moreover these results demonstrate thatthe wild-type forms of these three varieties also differed fromeach other with respect to Si absorption It can be concludedthat regardless of the presence of the transgene the MR276plants absorbed more Si than did the corresponding MR219and MR220 plants

Electron microscopy images of the roots and calli ofthe transgenic varieties showed that the Si concentrationwas higher in all transgenic varieties compared to wild-type varieties although the level differed between transgenicvarieties (Figures 7 and 8) Roots and calli obtained from T

1

transgenic MR276 absorbed more Si than did those of thetransgenic MR220 and MR219 varieties The images in Fig-ure 8 strongly confirm the results obtained from examiningthe Si concentration in the roots of plants shown in Figure 6Electron microscopy images show that the MR276 variety(both wild-type and transgenic forms) has a higher capacityfor Si absorption from the media or hydroponic culture thanthe other two varieties withMR220 showing the next-highestlevel of Si absorption

36 Antioxidant Enzyme Activities Chlorophyll Content andPhotosynthesis Analysis The transgenic varieties showed sig-nificant increases in the activities of the enzymes SOD PODAPX and CAT the chlorophyll content and photosynthesiscompared with the wild-type plants (Figure 9 Table 4)Superoxide dismutase catalase and peroxidase activitiesin all three transgenic varieties showed the same changesHowever the transgenic MR276 had the highest ascorbateperoxidase activity of the transgenic varieties

The total chlorophyll content in the wild-type MR276was the same as that in the wild-type MR220 and the totalchlorophyll content in the wild-type MR219 was higher thanthat in the wild-type MR220 However all three transgenicvarieties showed the same increase in total chlorophyllcontent Chlorophyll A and B levels were higher in transgenicMR276 than in transgenic MR219 and MR220 ChlorophyllA in transgenic MR219 was higher than that in transgenic


L1 L2 L3M

1000 bp

(a)

L1 L2 L3M

1000 bp

(b)

Figure 3 Detection of T1putative transgenic plants of MR219 MR220 andMR276 ((a) using specific primers (b) using CaMV35S primers)

M 1 kb DNA ladder lanes 2-3 the Lsi1 gene in transgenic MR219 MR220 and MR276 plants

Table 4 Comparisons of various physiological and morphological traits in transgenic and wild-type rice varieties

SOV df Total ChlCo (mggr) Chl A Chl B Photo Anal SOD POD APX CAT Si (leaf)

Varieties 5 128lowastlowast 76lowastlowast 113lowastlowast 089lowastlowast 05lowastlowast 12lowastlowast 642lowastlowast 081lowastlowast 0007lowastlowast

Replicate 2 010ns 011ns 005ns 061ns 00002ns 001ns 019ns 0003ns 000001ns

Error 10 037 011 033 018 00003 0006 01 0004 000003Total 17 mdash mdash mdash mdash mdash mdash mdash mdash mdashCV mdash 41 71 83 1242 39 455 21 36 156

SOV df Si (root) Root length(cm)

RDW(gplant) MT

RDW(gplant) PI

RDW(gplant)floweringstage

SDW(gplant)

MT

SDW(gplant)

PI

SDW (gplant) floweringstage

Varieties 5 005lowastlowast 334lowastlowast 0071lowastlowast 440lowastlowast 16lowastlowast 27lowastlowast 172lowastlowast 170lowastlowast

Replicate 2 0001ns 015ns 0015ns 008ns 0001ns 0003ns 375ns 349ns

Error 10 0003 013 012 081 0007 002 15 13Total 17 mdash mdash mdash mdash mdash mdash mdash mdashCV mdash 93 58 89 613 147 28 111 103ns and lowastlowast indicate not significant and significance level of 1 respectively Total Chl Co = total chlorophyll content Chl A = chlorophyll A Chl B =chlorophyll B Photo Anal = photosynthesis analysis SOD = superoxide dismutase POD = peroxidase APX = ascorbate peroxidase CAT = catalase Si(leaf) = silicon percentage in leaves Si (root) = silicon percentage in roots RDW (gplant) MT = root dry weight at maximum tillering stage RDW (gplant)PI = root dry weight at panicle initiation stage RDW (gplant) = root dry weight at flowering stage SDW (gplant)MT= shoot dry weight at maximum tilleringstage SDW (gplant) PI = shoot dry weight at panicle initiation stage and SDW (gplant) = shoot dry weight at flowering stage


(a)

MR219 MR220 MR276

(b)

Figure 4 Expression of gfp among harvested seeds (a) and calli (b) obtained from T1transgenic MR219 MR220 and MR276 varieties The

green color observed under fluorescence microscopy is related to expression of green fluorescent protein (GFP) confirming the presence ofthe transgene Bars = 5mm

MR276TransgenicMR220TransgenicTransgenic MR21905

1

2

4

8

16

32

Expr

essio

n ra

tio

Gene

Figure 5 Relative expression levels of the Lsi1 gene were calibratedusing quantitative real-time PCR of two reference genes 18S rRNAand 120572-Tubulin in wild-type and transgenic varieties Expression ofLsi1 in the transgenic cultivar MR276 was significantly higher thanin the transgenic cultivars MR219 and MR220

MR220 however in both transgenic varieties chlorophyllB showed the same changes compared to wild-type plantsThe photosynthetic changes in both transgenic MR276 andtransgenic MR219 were the same and represented greaterchanges in photosynthesis than in transgenic MR220

37 Morphological Characteristics The data presented inFigure 10 and Table 4 demonstrate that the root length rootdry weight at the maximum tillering stage (RDW (gplant)MT) root dry weight at the panicle initiation stage (RDW

e b e c d a

e

b

fc d

a

0010203040506070809

1

219 C 219 T 220 C 220 T 276 C 276 T

Si roots ()Si leaves ()

Figure 6 Comparison of Si concentrations in transgenic and wild-type rice varieties T transgenic C control

(gplant) PI) shoot dry weight at the maximum tilleringstage (SDW (gplant) MT) shoot dry weight at the panicleinitiation stage (SDW (gplant) PI) and shoot dry weightat the flowering stage (SDW (gplant)) were significantlydifferent between transgenic andwild-type varietiesThe rootdry weight at the panicle initiation stage (RDW (gplant)PI) recorded for all transgenic varieties was similar whereasthe transgenic MR276 variety had a markedly higher valuefor the other six morphological traits The values of rootlength root dry weight at the panicle initiation stage (RDW(gplant) PI) root dry weight at the flowering stage (RDW(gplant)) shoot dry weight at the maximum tillering stage(SDW (gplant) MT) and shoot dry weight at the floweringstage (SDW(gplant)) for transgenicMR219were higher than


(a) (b) (c)

(d) (e) (f)

Figure 7 Scanning electron microscopy images of wild-type and transgenic calli (a) Callus of wild-type MR219 (b) Callus of transgenicMR219 (c) Callus of wild-type MR220 (d) Callus of transgenic MR220 (e) Callus of wild-type MR276 (f) Callus of transgenic MR276White trace marked by red arrows silicon Bars = 100 120583m

(a) (b) (c)

(d) (e) (f)

Figure 8 Scanning electron microscopy images of wild-type and transgenic rice roots (a) Roots of wild-typeMR219 (b) Roots of transgenicMR219 (c) Roots of wild-type MR220 (d) Roots of transgenic MR220 (e) Roots of wild-type MR276 (f) Roots of transgenic MR276 Whitespot marked by red circle silicon Bars = 100 120583m


b

d

c

cb a

b

fa

bbc

a

aa b a

c

abc

a

dea

cd

dbc

bc b

b

c

b

bcba

a

a

a

aa

d

ba

c

a

05

1015202530

Ave

rage

()

219 C219 T220 C

220 T276 C276 T

CAT

APX

POD

SOD

Phot

anal

chl B

chl A

Tota

l Chl

Figure 9 Variations in antioxidant activities and physiologicaltraits among transgenic and wild-type rice varietiesThe same smallletter(s) in different columns denote no significant difference at 119875 le005 Total Chl total chlorophyll content Chl A chlorophyll A ChlB chlorophyll B Phot anal photosynthesis analysis SOD superox-ide dismutase POD peroxidase APX ascorbate peroxidase CATcatalase T transgenic C wild-type (control)

c

ab ac

df

b

ac

b a f

b

e

c

c d

e

a a

d

a

ae c

ce

d

dd

d b

b ca

bd

b de

d

02468

1012141618

Root

len (c

m)

RDW

(gp

lant

)M

T

RDW

(gp

lant

) PI

RDW

(gp

lant

)flo

wer

ing

stage

SDW

(gp

lant

)M

T

SDW

(gp

lant

) PI

SDW

(gp

lant

)flo

wer

ing

stage

219 C219 T220 C

220 T276 C276 T

Ave

rage

()

Figure 10 Functional effects of the transgene on various character-istics of rice

those for transgenic MR220 However the shoot dry weightat the panicle initiation stage (SDW (gplant) PI) recordedfor transgenic MR220 was higher than that for transgenicMR219 The root dry weight at the flowering stage did notdiffer between the transgenic MR219 and MR220 varietiesOverall the lowest values were recorded for the transgenicMR220 variety and the highest values were recorded forthe transgenic MR276 variety Transgenic varieties had astronger root and leaf structure as well as hairier rootscompared to the wild-type plants (Figure 11) The transgenicMR220 variety had hairier but shorter roots compared to thetransgenic MR219 and MR276 varieties

4 Discussion

This is the first study to illustrate the effect of Lsi1 expressionon morphological and physiological properties in Indica ricevarieties Our screening assessment revealed that the Lsi1gene is not present in fourteen wild-type Malaysian rice

C CCT TT

MR276 MR219 MR220

MR220-T MR219-T MR276-T

Figure 11 Morphological comparison between wild-type (control)and transgenic plants T transgenic C control

varieties However previous studies have identified the Lsi1gene in the mutant Japonica rice variety and one Indica ricecultivar (Dular) [21 36]The Lsi1 gene has been identified as aSi transporter in rice although we could not detect this geneamong these fourteen varieties in wild-type form Howeverour evaluation confirmed the accumulation of Si among thewild-type Malaysian rice varieties which may be due to theexpression of an unknown gene family Alternatively it can behypothesized that the Lsi1 gene is only expressed in rice underspecific treatments such as sodium azide [21] which explainswhywedid not observe this gene inwild-type rice varieties Inthat casewehave evaluated the overexpression of theLsi1 genein the Indica rice variety It should be noted that Lsi1 plays animportant role in Si accumulation in transgenic varieties ofrice although the effective expression level of this gene wasdifferent between all transgenic rice varieties Moreover theeffects of this gene were not entirely limited to increasing Siin the roots and leaves of transgenic varieties In vitro studiesof the roles of Si in plant tissue culture concluded that Si cansignificantly increase root length chlorophyll content freshand dryweights of roots and shoots and plant height [37ndash39]The ability of Si to enhance plant growth and developmentis based on alterations in antioxidant enzyme activities[37] In this regard it has been reported that Si treatmentconsiderably affects antioxidant enzyme activities in variousplants [40 41] Although most plant scientists believe that Siis not essential for plant growth and development and do notconsider Si to be a plant nutrient the results obtained fromthis study strongly emphasize the crucial role of Si in plantgrowth and development

In our study the expression of the Lsi1 gene in transgenicrice varieties helped these plants to absorb more Si comparedto the wild-type plants as confirmed by morphologicalanalysisThe transgenic plants withmore accumulated Si hadhigher antioxidant enzyme activities and improved morpho-logical traits The morphological analysis of transgenic ricevarieties provided results that strongly confirm the resultsof previous studies We hypothesized that the low Si uptakein wild-type Malaysian rice varieties may be due to eitherthe absence or the low expression of the Lsi1 gene Our


screening observations and the studies of transgenic varietiesconfirmed this hypothesis

Genetic modification is a new technique that has beenused by researchers to increase plant yields via trait improve-ment [42] Instead of using Si as a fertilizer or as a culturemedium supplement in in vitro studies we tried to geneticallymanipulate plants to improve their Si uptake Such transgenicplants should be able to absorb more Si through an improvedroot system We observed a positive correlation between Siaccumulation and photosynthesis components as well asconsequent improvements in morphological characteristicsin transgenic rice plants

Indeed increases in chlorophyll content and photosyn-thesis are typical responses of plants supplemented with Si[24 43] There are many explanations as to how Si accumu-lation leads to alterations in antioxidant enzyme activitiesincreases in chlorophyll content and photosynthesis andimprovements in morphological properties In one studyhigher levels of Si in rice suppressed drought stress byimproving the plantrsquos water status mineral nutrient uptakeand photosynthesis [44] Silicon enhanced resistance to freez-ing stress in wheat which is likely attributable to the protec-tive effects of higher antioxidant levels and lower lipid peroxi-dation via water preservation in leaf tissues [45] An assess-ment of the effect of Si on antioxidant enzymes in cottonshowed that Si altered the activities of guaiacol peroxidase(GPOX) catalase (CAT) and ascorbate peroxidase (APX) inroots and leaves whereas lipid peroxidase activity was notaffected These authors reported that enzymatic activities inthe roots and leaves of plants were changed in parallel withsilicon supplementation [46]

Quantitative real-time PCR is a versatile technique forsensitive reliable precise and high-throughput detectionand quantification of a target sequence in different samples[47] Although the expression of Lsi1 in transgenic plantsleads to improved growth and development of transgenicvarieties based on the results of real-time qRT-PCR it can beconcluded that this gene is differentially expressed in differenttransgenic varieties Therefore the positive results obtainedfrom the expression of the Lsi1 gene among three Indicarice varieties cannot be predicted or expected in other plantspecies and this phenomenon needs to be examined for indi-vidual plant species In this study we performed qRT-PCR ofthe Si transporter response gene (Lsi1) to evaluate its expres-sion levels in the transgenic forms of three commonly grownlocal rice varieties Initially it seemed that all transgenic vari-eties showed the same effects whereas the results of qRT-PCRshowed that the transgenic MR276 rice variety had highestLsi1 expression level We could not find any reasons for thesedifferences between the transgenic varieties We believethat the results obtained from further experiments whichdemonstrated that transgenic MR276 showed high levels ofantioxidant enzyme activity and improved morphologicalproperties are not directly due to the expression of Lsi1 Thetransgenic MR276 variety absorbed more Si than the othertransgenic varieties so it is likely that other properties of thistransgenic variety are related to increased accumulation of Siin the roots and leaves of the plant However the expressionof only one target gene cannot be effective in creating diverse

traits Itmust bementioned that the activation of the Lsi1 geneat the same time promoted Si accumulation and may haveaffected antioxidant enzyme activities by producing planttonoplast intrinsic proteins (TIPs) Various members of theTIP family including alpha (seed) Rt (root) gamma andWsi(water stress induced) may be effective in altering antioxi-dant enzyme activities It has also been predicted that theseprotein family members may allow the diffusion of differentamino acids peptides and water from the tonoplast center tothe cytoplasm On the other hand stimulating the diffusionof some amino acids such as serine and proline should bemore effective for biosilica formation by plants [48]

Our results suggest that the transformation of the Lsi1gene into Indica rice varieties (overexpression or as a foreigngene) is more effective than supplementing the culturemediumor soil with Si Changes in antioxidant enzyme activ-ities chlorophyll content photosynthesis andmorphologicalpropertieswere significantly beneficial in transgenic varietieswhile the wild-type varieties were also treated with the samesource and amount of Si in the culture medium and thehydroponic culture solution Although the transgenic varie-ties did not all show the same variation the transgenicMR220variety which showed the smallest changes still showedbetter characteristics than any of the wild-type varieties

Finally based on the function of the Lsi1 protein in themechanism of diffusion of various amino acids from thetonoplast interior to the cytoplasm and on the results ofour previous study which confirmed the role of serine-richprotein in Si accumulation byArabidopsis thaliana as a modelplant [48] a future study comparing the roles of Lsi1 andserineproline-rich protein in Si uptake by plants is needed

5 Conclusion

Silicon plays an important role in plant growth and develop-ment Although Si is plentiful in the soil and does not needto be applied as fertilizer different plant species differ in Siuptake from the soil The Lsi1 gene has been identified as a Sitransporter gene in the Japonica rice variety Our screeningdid not detect the Lsi1 gene among Indica rice varieties Basedon the important role of this gene in Si uptake by plantsand the crucial role of Si in plant growth and developmentwe tried to increase Si absorption and accumulation insidethe Indica rice varieties via genetic manipulation rather thanusing additional different sources of Si in the culture mediumor soil The rice variety MR276 showed a more positiveresponse to genetic manipulation than the other varietieswhich will aid in future analyses For instance becauseit has been reported that Si can alleviate various stressessuch as drought salinity and pathogen attack in plants thetransgenic MR276 developed here should be more stableunder such harsh environmental conditions In conclusionif we can provide genetically manipulated plants that arecapable of taking up more Si from the soil their improvedmorphological traits and improved resistance against variousbiotic and abiotic stresses will improve their yields


Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The senior author (Mahbod Sahebi) would like to expresssincere gratitude to Universiti Putra Malaysia (UPM) forthe financial support provided through research Grant no9415300 and for the research facilities provided

References

[1] G S Khush ldquoWhat it will take to feed 50 billion rice consumersin 2030rdquo Plant Molecular Biology vol 59 no 1 pp 1ndash6 2005

[2] P Azizi M Y Rafii M Mahmood et al ldquoDifferential geneexpression reflects morphological characteristics and physi-ological processes in rice immunity against blast pathogenMagnaporthe oryzaerdquo PLoS ONE vol 10 no 5 Article IDe0126188 2015

[3] P Azizi M Y Rafii M Mahmood et al ldquoHighly efficientprotocol for callogenesis somagenesis and regeneration ofIndica rice plantsrdquo Comptes RendusmdashBiologies vol 338 no 7pp 463ndash470 2015

[4] M Sahebi M M Hanafi and P Azizi ldquoApplication of siliconin plant tissue culturerdquo In Vitro Cellular amp DevelopmentalBiologymdashPlant vol 52 no 3 pp 226ndash232 2016

[5] M Sahebi MM Hanafi S N A Abdullah et al ldquoIsolation andexpression analysis of novel silicon absorption gene from rootsofmangrove (Rhizophora apiculata) via suppression subtractivehybridizationrdquo BioMed Research International vol 2014 ArticleID 971985 11 pages 2014

[6] J A Litsinger J P Bandong andB L Canapi ldquoEffect ofmultipleinfestations from insect pests and other stresses to irrigatedrice in the Philippines II damage and yield lossrdquo InternationalJournal of Pest Management vol 57 no 2 pp 117ndash131 2011

[7] R N Strange and P R Scott ldquoPlant disease a threat to globalfood securityrdquoAnnual Review of Phytopathology vol 43 pp 83ndash116 2005

[8] D G Bottrell and K G Schoenly ldquoResurrecting the ghost ofgreen revolutions past the brown planthopper as a recurringthreat to high-yielding rice production in tropical Asiardquo Journalof Asia-Pacific Entomology vol 15 no 1 pp 122ndash140 2012

[9] P Azizi M Y Rafii S N Abdullah et al ldquoToward under-standing of rice innate immunity againstMagnaporthe oryzaerdquoCritical Reviews in Biotechnology vol 36 no 1 pp 165ndash174 2014

[10] B C King K D Waxman N V Nenni L P Walker G CBergstrom andDMGibson ldquoArsenal of plant cell wall degrad-ing enzymes reflects host preference among plant pathogenicfungirdquo Biotechnology for Biofuels vol 4 article 4 2011

[11] A R Ganguly K Steinhaeuser D J Erickson III et al ldquoHighertrends but larger uncertainty and geographic variability in21st century temperature and heat wavesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 37 pp 15555ndash15559 2009

[12] K K Newsham and S A Robinson ldquoResponses of plants inpolar regions toUVB exposure ameta-analysisrdquoGlobal ChangeBiology vol 15 no 11 pp 2574ndash2589 2009

[13] W He M Yang Z Li et al ldquoHigh levels of silicon provided asa nutrient in hydroponic culture enhances rice plant resistanceto brown planthopperrdquo Crop Protection vol 67 pp 20ndash25 2015

[14] Z Lu K Heong X Yu and C Hu ldquoEffects of nitrogen on thetolerance of brown planthopperNilaparvata Lugens to adverseenvironmental factorsrdquo Insect Science vol 12 no 2 pp 121ndash1282005

[15] M Sahebi M M Hanafi A Siti Nor Akmar et al ldquoImportanceof silicon and mechanisms of biosilica formation in plantsrdquoBioMed Research International vol 2015 Article ID 396010 16pages 2015

[16] A Fawe M Abou-Zaid J G Menzies and R R BelangerldquoSilicon-mediated accumulation of flavonoid phytoalexins incucumberrdquo Phytopathology vol 88 no 5 pp 396ndash401 1998

[17] M Saqib C Zorb and S Schubert ldquoSilicon-mediated improve-ment in the salt resistance of wheat (Triticum aestivum) resultsfrom increased sodium exclusion and resistance to oxidativestressrdquo Functional Plant Biology vol 35 no 7 pp 633ndash639 2008

[18] K P V Da Cunha and C W A Do Nascimento ldquoSilicon effectson metal tolerance and structural changes in Maize (Zea maysL) grown on a cadmium and zinc enriched soilrdquoWater Air andSoil Pollution vol 197 no 1ndash4 pp 323ndash330 2009

[19] A Hashemi A Abdolzadeh andH R Sadeghipour ldquoBeneficialeffects of silicon nutrition in alleviating salinity stress inhydroponically grown canola Brassica napus L plantsrdquo SoilScience and Plant Nutrition vol 56 no 2 pp 244ndash253 2010

[20] M Ashraf Rahmatullah M Afzal et al ldquoAlleviation of detri-mental effects of NaCl by silicon nutrition in salt-sensitive andsalt-tolerant genotypes of sugarcane (Saccharum officinarumL)rdquo Plant and Soil vol 326 no 1 pp 381ndash391 2010

[21] J FMa K Tamai N Yamaji et al ldquoA silicon transporter in ricerdquoNature vol 440 no 7084 pp 688ndash691 2006

[22] F A Rodrigues N Benhamou L E Datnoff J B Jones andR R Belanger ldquoUltrastructural and cytochemical aspects ofsilicon-mediated rice blast resistancerdquo Phytopathology vol 93no 5 pp 535ndash546 2003

[23] F A Rodrigues W M Jurick II L E Datnoff J B Jonesand J A Rollins ldquoSilicon influences cytological and molecu-lar events in compatible and incompatible rice-MagnaporthegriseainteractionsrdquoPhysiological andMolecular Plant Pathologyvol 66 no 4 pp 144ndash159 2005

[24] X Yao J Chu K Cai L Liu J Shi and W Geng ldquoSiliconimproves the tolerance of wheat seedlings to ultraviolet-BstressrdquoBiological Trace Element Research vol 143 no 1 pp 507ndash517 2011

[25] K Al-aghabary Z Zhu and Q Shi ldquoInfluence of siliconsupply on chlorophyll content chlorophyll fluorescence andantioxidative enzyme activities in tomato plants under saltstressrdquo Journal of Plant Nutrition vol 27 no 12 pp 2101ndash21152004

[26] X-C Wu W-X Lin and Z-L Huang ldquoInfluence of enhancedultraviolet-B radiation on photosynthetic physiologies andultrastructure of leaves in two different resistivity rice cultivarsrdquoActa Ecologica Sinica vol 27 no 2 pp 554ndash564 2007

[27] W-B Li X-H Shi HWang and F-S Zhang ldquoEffects of siliconon rice leaves resistance to ultraviolet-Brdquo Acta Botanica Sinicavol 46 no 6 pp 691ndash697 2004

[28] E Takahashi and K Hino ldquoSilica uptake by rice plant withspecial reference to the forms of dissolved silicardquo Journal of theScience of Soil and Manure 1978 (Japanese)

[29] J F Ma and E Takahashi Soil Fertilizer and Plant SiliconResearch in Japan Elsevier Amsterdam Netherlands 2002

[30] J F Ma N Yamaji N Mitani et al ldquoAn efflux transporter ofsilicon in ricerdquo Nature vol 448 no 7150 pp 209ndash212 2007


[31] K Kamachi T Yamaya T Mae and K Ojima ldquoA role forglutamine synthetase in the remobilization of leaf nitrogenduring natural senescence in rice leavesrdquo Plant Physiology vol96 no 2 pp 411ndash417 1991

[32] M Karimi D Inze and A Depicker ldquoGATEWAY vectors forAgrobacterium-mediated plant transformationrdquo Trends in PlantScience vol 7 no 5 pp 193ndash195 2002

[33] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[34] D I Arnon ldquoCopper enzymes in isolated chloroplasts Polyphe-noloxidase in Beta vulgarisrdquo Plant Physiology vol 24 no 1 pp1ndash15 1949

[35] G Korndorfer H Pereira and A Nola ldquoAnalise de silıcio soloplanta e fertilizanterdquo in Uberlandia UFU vol 2 50 BoletimTecnico 2004

[36] L Wenxiong F Changxun W Qingshui and W XingchunSuppression and Overexpression of Lsi1 Affects Ultraviolet-BTolerance Iin Rice (Oryza sativa L) Institute of AgroecologyFujian Agriculture and Forestry University 2014

[37] I Sivanesan and B R Jeong ldquoSilicon promotes adventitiousshoot regeneration and enhances salinity tolerance of AjugamultifloraBunge by altering activity of antioxidant enzymerdquoTheScientific World Journal vol 2014 Article ID 521703 10 pages2014

[38] K Yamada S Yoshikawa M Ichinomiya A Kuwata MKamiya and K Ohki ldquoEffects of silicon-limitation on growthand morphology of Triparma laevis NIES-2565 (ParmalesHeterokontophyta)rdquo PLoS ONE vol 9 no 7 Article ID e1032892014

[39] J D R Soares M Pasqual A G de Araujo E M deCastro F J Pereira and F T Braga ldquoLeaf anatomy of orchidsmicropropagated with different silicon concentrationsrdquo ActaScientiarummdashAgronomy vol 34 no 4 pp 413ndash421 2012

[40] J F Ma ldquoRole of silicon in enhancing the resistance of plants tobiotic and abiotic stressesrdquo Soil Science and Plant Nutrition vol50 no 1 pp 11ndash18 2004

[41] Y Liang W Sun Y-G Zhu and P Christie ldquoMechanisms ofsilicon-mediated alleviation of abiotic stresses in higher plantsa reviewrdquo Environmental Pollution vol 147 no 2 pp 422ndash4282007

[42] R Abiri A Valdiani M Maziah et al ldquoA critical review ofthe concept of transgenic plants Insights into pharmaceuticalbiotechnology andmolecular farmingrdquoCurrent Issues inMolec-ular Biology vol 18 no 1 pp 21ndash42 2016

[43] S A Asmar E M Castro M Pasqual F J Pereira and JD R Soares ldquoChanges in leaf anatomy and photosynthesisof micropropagated banana plantlets under different siliconsourcesrdquo Scientia Horticulturae vol 161 pp 328ndash332 2013

[44] W Chen X Yao K Cai and J Chen ldquoSilicon alleviatesdrought stress of rice plants by improving plant water statusphotosynthesis and mineral nutrient absorptionrdquo BiologicalTrace Element Research vol 142 no 1 pp 67ndash76 2011

[45] Y Liang J Zhu Z Li et al ldquoRole of silicon in enhancingresistance to freezing stress in two contrasting winter wheatcultivarsrdquo Environmental and Experimental Botany vol 64 no3 pp 286ndash294 2008

[46] C Alberto Moldes O Fontao De Lima Filho J ManuelCamina S Gabriela Kiriachek M Lia Molas and S Mui TsaildquoAssessment of the effect of silicon on antioxidant enzymes incotton plants by multivariate analysisrdquo Journal of Agriculturaland Food Chemistry vol 61 no 47 pp 11243ndash11249 2013

[47] M Kubista J M Andrade M Bengtsson et al ldquoThe real-timepolymerase chain reactionrdquoMolecular Aspects of Medicine vol27 no 2-3 pp 95ndash125 2006

[48] M SahebiMMHanafi A Siti NorAkmarM Y Rafii P Aziziand A S Idris ldquoSerine-rich protein is a novel positive regulatorfor silicon accumulation in mangroverdquoGene vol 556 no 2 pp170ndash181 2015

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Many insect pests and epidemic diseases affect differentparts of the rice plant resulting in severe rice productionlosses [6] Rice yield stability is affected by various biotic andabiotic stresses Blast and sheath blight fungi bacterial blightroot-knot (caused by nematodes) and rice yellow mottlevirus cause themajority of losses in the annual rice grain yield[7] The brown plant hopper (BPH Nilaparvata lugens) animportant insect pest of cultivated rice has interfered withthe production of rice since the 1960s [8] The plant cell wallis a barrier between host plants and pathogens but the fungusMagnaporthe oryzae uses 30 and 44 different enzymes todegrade cellulose and hemicellulose respectively in plant cellwalls [9 10]

On the other hand the release of chlorofluorocarbongases due to the increased solar ultraviolet-B radiation on thesurface of the earth has reduced the stratospheric ozone (O

3)

layer [11] Depletion of theO3layer decreases plant height and

aboveground biomass and it also increases DNA damage Inthis context it has been shown that increased UV-B radiationhas the same effect on plants grown in the Arctic Antarcticand lower latitudes [12] Therefore it is crucial to introduceeffective ways of improving plant growth and reducingsuch damage The application of various chemical fertilizersshould affect the resistance as well as the tolerance of riceplants to biotic stresses such as the brown plant hopper (BPH)[13] It has also been shown that the risk of BPH outbreaksin hybrid rice could be increased through the application ofhigh levels of nitrogen fertilizer [8 14]

Silicon (Si) is one of the most prevalent elements inthe soil Si is beneficial for plant growth and contributes toplant defense mechanisms against multiple biotic and abioticstresses including tolerance to salt drought and toxic metals[15] Silicon enhancement stimulates plants to produce phy-toalexins and phenolics consequently increasing the plantsrsquocapacity to defend against stress factors [16] Si may reduceoxidative damage in plants encountering environmentalstresses [17] and it plays an important role in enhancingplantsrsquo tolerance to biotic stresses such as fungal diseasesThis increased tolerance is partly attributed to increases inthe defense capacity of plants due to Si-induced increases inantioxidants A Si-cuticle double layer is produced due to Sideposition beneath the cuticle and acts as a barrier to physi-cally prevent the penetration of fungi [15] Si can improve soilconditions in the presence of toxic heavy metals minimizethe toxicity of Mn Al and Fe and enhance P availability[15 18] The Si concentration depends on the plant genotypeas it does in other organisms Therefore plantsrsquo metabolicactivities and physiological mechanisms might be affected bydifferent amounts of Si used in fertilizers [15 19 20]

Rice which can accumulate Si up to 100 of the shootdry weight shows the most effective Si accumulation amongplants Stable production and healthy growth of rice arehighly affected by the Si content of the shoots The high Siaccumulation in rice shoots is attributed to the ability of riceroots to take up large amounts of Si [21] The addition of Si toa susceptible rice variety infected byMagnaporthe grisea wasassociated with the accumulation of momilactone phytoalex-ins [22] and the upregulation of peroxidase PR-1 transcriptsand glucanase [23]

Silicon can increase the tolerance of plants to UV-B stressand increase the amounts of chlorophyll a and b [24 25]Theamount of Si on the adaxial surface of rice-leaves increasestolerance toUV-B radiation suggesting that silicic accumula-tion may help reduce the physical stress caused by UV radia-tion [26] It has been reported that Si supplementation duringthe hydroponic culture of rice enhances UV-B resistancewhich might be attributed to increases in phenolic com-pounds in the plant epidermis [27]

Silicic acid is the form of Si that is absorbable by plantroots it translocates to the shoots through the transpirationstream and is later polymerized and accumulated as silica inthe shoot tissues [28 29] Si uptake in rice is controlled by atleast two transporters Lsi1 (influx) and Lsi2 (efflux) but theaccumulation of Si is mediated only by Lsi1 [30] Therefore itis hypothesized that the transformation of Lsi1 and its expres-sion or overexpression in Indica rice varieties may improvethe resistance of the plant to stress factors In this study wescreened the Lsi1 gene in fourteen Malaysian rice varietiesThree common Malaysian rice varieties were selected forgenetic manipulation and showed morphological improve-ments due to greater Si accumulation

2 Experimental Procedures

21 Plant Material Fourteen important Malaysian rice vari-eties (MR219 MR276 MR220 MR211 MR263 MR269MR185 MR159 MRQ74 MRQ50 Mashuri Jaya Masriaand Milyang) were provided by the Malaysian AgriculturalResearch and Development Institute (MARDI)

22 Screening of the Lsi1 Gene Rice seeds were soaked inwater for three days at 25∘C and germinated in Petri dishesusingWhatmanfilter paper (Sigma-AldrichUSA)moistenedwith a 05mM CaCl

2solution Subsequently seeds with

a bud length of 1-2mm were selected transferred into ahydroponic culture solution [31] and kept in a glasshouse at25ndash30∘C under fluorescent white light with a photoperiod of16 hours light8 hours dark for three weeks Throughout theexperiment the pH of the solution was maintained at 57Thenutrient solution was renewed every 3 days The root tissuesof all varieties were sampled immediately frozen in liquidnitrogen and then kept at minus80∘C for total RNA isolation

23 RNA Extraction and Semiquantitative RT-PCR TotalRNA from the roots of the fourteen varieties was isolated sep-arately using TRIzol Reagent (Invitrogen USA) For all sam-ples DNase I (Qiagen USA) was used to digest contaminat-ing genomic DNA First-strand cDNA was synthesized fromeach sample using SuperScript III (Invitrogen USA)TheTaqDNA polymerase kit (Fermentas Waltham MassachusettsUSA) was used with the following program initial denatura-tion at 95∘C for 5min 35 cycles of 95∘C for 30 sec annealing(55∘C 56∘C 57∘C 58∘C 59∘C 60∘C 61∘C 62∘C 63∘C 64∘C65∘C and 66∘C) for 45 sec extension at 72∘C for 1min anda final extension for 10min at 72∘C The PCR products wereisolated on 15 agarose gels and stained with Midori Green(Nippon Genetics Germany) Two sets of specific primers













3998400

3998400

5998400

5998400





















2PO4(286K and




0and T

1generations using



2SiO3 pH 58














2O2and






2were dehydrated







0and T



1) continued


(a) (b) (c)

(d)

MR219 MR220 MR276







0and T









2SiO3





1





L1 L2 L3M

1000 bp

(a)

L1 L2 L3M

1000 bp

(b)









RDW(gplant) MT

RDW(gplant) PI


SDW(gplant)

MT

SDW(gplant)

PI






(a)

MR219 MR220 MR276

(b)




1

2

4

8

16

32

Expr

essio

n ra

tio

Gene




e b e c d a

e

b

fc d

a

0010203040506070809

1

219 C 219 T 220 C 220 T 276 C 276 T





(a) (b) (c)

(d) (e) (f)


(a) (b) (c)

(d) (e) (f)



b

d

c

cb a

b

fa

bbc

a

aa b a

c

abc

a

dea

cd

dbc

bc b

b

c

b

bcba

a

a

a

aa

d

ba

c

a

05

1015202530

Ave

rage

()

219 C219 T220 C

220 T276 C276 T

CAT

APX

POD

SOD

Phot

anal

chl B

chl A

Tota

l Chl


c

ab ac

df

b

ac

b a f

b

e

c

c d

e

a a

d

a

ae c

ce

d

dd

d b

b ca

bd

b de

d

02468

1012141618

Root

len (c

m)

RDW

(gp

lant

)M

T

RDW

(gp

lant

) PI

RDW

(gp

lant

)flo

wer

ing

stage

SDW

(gp

lant

)M

T

SDW

(gp

lant

) PI

SDW

(gp

lant

)flo

wer

ing

stage

219 C219 T220 C

220 T276 C276 T

Ave

rage

()



4 Discussion


C CCT TT

MR276 MR219 MR220

MR220-T MR219-T MR276-T












5 Conclusion



Competing Interests


Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













3998400

3998400

5998400

5998400





















2PO4(286K and




0and T

1generations using



2SiO3 pH 58














2O2and






2were dehydrated







0and T



1) continued


(a) (b) (c)

(d)

MR219 MR220 MR276







0and T









2SiO3





1





L1 L2 L3M

1000 bp

(a)

L1 L2 L3M

1000 bp

(b)









RDW(gplant) MT

RDW(gplant) PI


SDW(gplant)

MT

SDW(gplant)

PI






(a)

MR219 MR220 MR276

(b)




1

2

4

8

16

32

Expr

essio

n ra

tio

Gene




e b e c d a

e

b

fc d

a

0010203040506070809

1

219 C 219 T 220 C 220 T 276 C 276 T





(a) (b) (c)

(d) (e) (f)


(a) (b) (c)

(d) (e) (f)



b

d

c

cb a

b

fa

bbc

a

aa b a

c

abc

a

dea

cd

dbc

bc b

b

c

b

bcba

a

a

a

aa

d

ba

c

a

05

1015202530

Ave

rage

()

219 C219 T220 C

220 T276 C276 T

CAT

APX

POD

SOD

Phot

anal

chl B

chl A

Tota

l Chl


c

ab ac

df

b

ac

b a f

b

e

c

c d

e

a a

d

a

ae c

ce

d

dd

d b

b ca

bd

b de

d

02468

1012141618

Root

len (c

m)

RDW

(gp

lant

)M

T

RDW

(gp

lant

) PI

RDW

(gp

lant

)flo

wer

ing

stage

SDW

(gp

lant

)M

T

SDW

(gp

lant

) PI

SDW

(gp

lant

)flo

wer

ing

stage

219 C219 T220 C

220 T276 C276 T

Ave

rage

()



4 Discussion


C CCT TT

MR276 MR219 MR220

MR220-T MR219-T MR276-T












5 Conclusion



Competing Interests


Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








2PO4(286K and




0and T

1generations using



2SiO3 pH 58














2O2and






2were dehydrated







0and T



1) continued


(a) (b) (c)

(d)

MR219 MR220 MR276







0and T









2SiO3





1





L1 L2 L3M

1000 bp

(a)

L1 L2 L3M

1000 bp

(b)









RDW(gplant) MT

RDW(gplant) PI


SDW(gplant)

MT

SDW(gplant)

PI






(a)

MR219 MR220 MR276

(b)




1

2

4

8

16

32

Expr

essio

n ra

tio

Gene




e b e c d a

e

b

fc d

a

0010203040506070809

1

219 C 219 T 220 C 220 T 276 C 276 T





(a) (b) (c)

(d) (e) (f)


(a) (b) (c)

(d) (e) (f)



b

d

c

cb a

b

fa

bbc

a

aa b a

c

abc

a

dea

cd

dbc

bc b

b

c

b

bcba

a

a

a

aa

d

ba

c

a

05

1015202530

Ave

rage

()

219 C219 T220 C

220 T276 C276 T

CAT

APX

POD

SOD

Phot

anal

chl B

chl A

Tota

l Chl


c

ab ac

df

b

ac

b a f

b

e

c

c d

e

a a

d

a

ae c

ce

d

dd

d b

b ca

bd

b de

d

02468

1012141618

Root

len (c

m)

RDW

(gp

lant

)M

T

RDW

(gp

lant

) PI

RDW

(gp

lant

)flo

wer

ing

stage

SDW

(gp

lant

)M

T

SDW

(gp

lant

) PI

SDW

(gp

lant

)flo

wer

ing

stage

219 C219 T220 C

220 T276 C276 T

Ave

rage

()



4 Discussion


C CCT TT

MR276 MR219 MR220

MR220-T MR219-T MR276-T












5 Conclusion



Competing Interests


Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












2O2and






2were dehydrated







0and T



1) continued


(a) (b) (c)

(d)

MR219 MR220 MR276







0and T









2SiO3





1





L1 L2 L3M

1000 bp

(a)

L1 L2 L3M

1000 bp

(b)









RDW(gplant) MT

RDW(gplant) PI


SDW(gplant)

MT

SDW(gplant)

PI






(a)

MR219 MR220 MR276

(b)




1

2

4

8

16

32

Expr

essio

n ra

tio

Gene




e b e c d a

e

b

fc d

a

0010203040506070809

1

219 C 219 T 220 C 220 T 276 C 276 T





(a) (b) (c)

(d) (e) (f)


(a) (b) (c)

(d) (e) (f)



b

d

c

cb a

b

fa

bbc

a

aa b a

c

abc

a

dea

cd

dbc

bc b

b

c

b

bcba

a

a

a

aa

d

ba

c

a

05

1015202530

Ave

rage

()

219 C219 T220 C

220 T276 C276 T

CAT

APX

POD

SOD

Phot

anal

chl B

chl A

Tota

l Chl


c

ab ac

df

b

ac

b a f

b

e

c

c d

e

a a

d

a

ae c

ce

d

dd

d b

b ca

bd

b de

d

02468

1012141618

Root

len (c

m)

RDW

(gp

lant

)M

T

RDW

(gp

lant

) PI

RDW

(gp

lant

)flo

wer

ing

stage

SDW

(gp

lant

)M

T

SDW

(gp

lant

) PI

SDW

(gp

lant

)flo

wer

ing

stage

219 C219 T220 C

220 T276 C276 T

Ave

rage

()



4 Discussion


C CCT TT

MR276 MR219 MR220

MR220-T MR219-T MR276-T












5 Conclusion



Competing Interests


Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)

(d)

MR219 MR220 MR276







0and T









2SiO3





1





L1 L2 L3M

1000 bp

(a)

L1 L2 L3M

1000 bp

(b)









RDW(gplant) MT

RDW(gplant) PI


SDW(gplant)

MT

SDW(gplant)

PI






(a)

MR219 MR220 MR276

(b)




1

2

4

8

16

32

Expr

essio

n ra

tio

Gene




e b e c d a

e

b

fc d

a

0010203040506070809

1

219 C 219 T 220 C 220 T 276 C 276 T





(a) (b) (c)

(d) (e) (f)


(a) (b) (c)

(d) (e) (f)



b

d

c

cb a

b

fa

bbc

a

aa b a

c

abc

a

dea

cd

dbc

bc b

b

c

b

bcba

a

a

a

aa

d

ba

c

a

05

1015202530

Ave

rage

()

219 C219 T220 C

220 T276 C276 T

CAT

APX

POD

SOD

Phot

anal

chl B

chl A

Tota

l Chl


c

ab ac

df

b

ac

b a f

b

e

c

c d

e

a a

d

a

ae c

ce

d

dd

d b

b ca

bd

b de

d

02468

1012141618

Root

len (c

m)

RDW

(gp

lant

)M

T

RDW

(gp

lant

) PI

RDW

(gp

lant

)flo

wer

ing

stage

SDW

(gp

lant

)M

T

SDW

(gp

lant

) PI

SDW

(gp

lant

)flo

wer

ing

stage

219 C219 T220 C

220 T276 C276 T

Ave

rage

()



4 Discussion


C CCT TT

MR276 MR219 MR220

MR220-T MR219-T MR276-T












5 Conclusion



Competing Interests


Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


L1 L2 L3M

1000 bp

(a)

L1 L2 L3M

1000 bp

(b)









RDW(gplant) MT

RDW(gplant) PI


SDW(gplant)

MT

SDW(gplant)

PI






(a)

MR219 MR220 MR276

(b)




1

2

4

8

16

32

Expr

essio

n ra

tio

Gene




e b e c d a

e

b

fc d

a

0010203040506070809

1

219 C 219 T 220 C 220 T 276 C 276 T





(a) (b) (c)

(d) (e) (f)


(a) (b) (c)

(d) (e) (f)



b

d

c

cb a

b

fa

bbc

a

aa b a

c

abc

a

dea

cd

dbc

bc b

b

c

b

bcba

a

a

a

aa

d

ba

c

a

05

1015202530

Ave

rage

()

219 C219 T220 C

220 T276 C276 T

CAT

APX

POD

SOD

Phot

anal

chl B

chl A

Tota

l Chl


c

ab ac

df

b

ac

b a f

b

e

c

c d

e

a a

d

a

ae c

ce

d

dd

d b

b ca

bd

b de

d

02468

1012141618

Root

len (c

m)

RDW

(gp

lant

)M

T

RDW

(gp

lant

) PI

RDW

(gp

lant

)flo

wer

ing

stage

SDW

(gp

lant

)M

T

SDW

(gp

lant

) PI

SDW

(gp

lant

)flo

wer

ing

stage

219 C219 T220 C

220 T276 C276 T

Ave

rage

()



4 Discussion


C CCT TT

MR276 MR219 MR220

MR220-T MR219-T MR276-T












5 Conclusion



Competing Interests


Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

MR219 MR220 MR276

(b)




1

2

4

8

16

32

Expr

essio

n ra

tio

Gene




e b e c d a

e

b

fc d

a

0010203040506070809

1

219 C 219 T 220 C 220 T 276 C 276 T





(a) (b) (c)

(d) (e) (f)


(a) (b) (c)

(d) (e) (f)



b

d

c

cb a

b

fa

bbc

a

aa b a

c

abc

a

dea

cd

dbc

bc b

b

c

b

bcba

a

a

a

aa

d

ba

c

a

05

1015202530

Ave

rage

()

219 C219 T220 C

220 T276 C276 T

CAT

APX

POD

SOD

Phot

anal

chl B

chl A

Tota

l Chl


c

ab ac

df

b

ac

b a f

b

e

c

c d

e

a a

d

a

ae c

ce

d

dd

d b

b ca

bd

b de

d

02468

1012141618

Root

len (c

m)

RDW

(gp

lant

)M

T

RDW

(gp

lant

) PI

RDW

(gp

lant

)flo

wer

ing

stage

SDW

(gp

lant

)M

T

SDW

(gp

lant

) PI

SDW

(gp

lant

)flo

wer

ing

stage

219 C219 T220 C

220 T276 C276 T

Ave

rage

()



4 Discussion


C CCT TT

MR276 MR219 MR220

MR220-T MR219-T MR276-T












5 Conclusion



Competing Interests


Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)

(d) (e) (f)


(a) (b) (c)

(d) (e) (f)



b

d

c

cb a

b

fa

bbc

a

aa b a

c

abc

a

dea

cd

dbc

bc b

b

c

b

bcba

a

a

a

aa

d

ba

c

a

05

1015202530

Ave

rage

()

219 C219 T220 C

220 T276 C276 T

CAT

APX

POD

SOD

Phot

anal

chl B

chl A

Tota

l Chl


c

ab ac

df

b

ac

b a f

b

e

c

c d

e

a a

d

a

ae c

ce

d

dd

d b

b ca

bd

b de

d

02468

1012141618

Root

len (c

m)

RDW

(gp

lant

)M

T

RDW

(gp

lant

) PI

RDW

(gp

lant

)flo

wer

ing

stage

SDW

(gp

lant

)M

T

SDW

(gp

lant

) PI

SDW

(gp

lant

)flo

wer

ing

stage

219 C219 T220 C

220 T276 C276 T

Ave

rage

()



4 Discussion


C CCT TT

MR276 MR219 MR220

MR220-T MR219-T MR276-T












5 Conclusion



Competing Interests


Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


b

d

c

cb a

b

fa

bbc

a

aa b a

c

abc

a

dea

cd

dbc

bc b

b

c

b

bcba

a

a

a

aa

d

ba

c

a

05

1015202530

Ave

rage

()

219 C219 T220 C

220 T276 C276 T

CAT

APX

POD

SOD

Phot

anal

chl B

chl A

Tota

l Chl


c

ab ac

df

b

ac

b a f

b

e

c

c d

e

a a

d

a

ae c

ce

d

dd

d b

b ca

bd

b de

d

02468

1012141618

Root

len (c

m)

RDW

(gp

lant

)M

T

RDW

(gp

lant

) PI

RDW

(gp

lant

)flo

wer

ing

stage

SDW

(gp

lant

)M

T

SDW

(gp

lant

) PI

SDW

(gp

lant

)flo

wer

ing

stage

219 C219 T220 C

220 T276 C276 T

Ave

rage

()



4 Discussion


C CCT TT

MR276 MR219 MR220

MR220-T MR219-T MR276-T












5 Conclusion



Competing Interests


Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









5 Conclusion



Competing Interests


Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Competing Interests


Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Screening and Expression of a Silicon Transporter Gene in