roots and composite plants in peanut

ORIGINAL PAPEREfficient production of Agrobacterium rhizogenes -transformed roots and composite plants in peanut (Arachis hypogaea L.)Lili Geng •Lihong Niu •Peter M.Gresshoff •Changlong Shu •Fuping Song •Dafang Huang •Jie ZhangReceived:7July 2011/Accepted:30December 2011/Published online:28January 2012ÓSpringer Science+Business Media B.V.2012Abstract Recalcitrance of most large-seeded legumes,such as peanut,to regeneration and genetic transformation has hampered studies on gene function and efforts for genetic improvement.Agrobacterium rhizogenes -mediated transfor-mation provides a system for rapid and efficient transformation of plant tissues.In this study,embryonic axes along with cotyledons of peanut were injected with a suspension culture of A.rhizogenes using microliter syringes.The influence of sev-eral factors such as plant genotype,A.rhizogenes culture stage,co-culture period of A.rhizogenes ,and acetosyringone con-centration in the co-cultivation medium have been evaluated.It is found that A.rhizogenes -mediated transformation of peanut is genotype-independent.Up to 61%transformation was recorded when embryonic axes were co-cultivated with 59107A.rhizogenes cells from logarithmic phase for 2days on co-culture medium containing 50l mol l posite plants with transgenic roots were harvested after 45days of treatment.Furthermore,this method was applied to assess the insecticidal activity of a synthetic cry8Ea1gene against Holotrichia parallela in transgenic roots of peanut.Keywords Agrobacterium rhizogenes ÁPeanut ÁTransformation ÁMicroinjection Ácry8Ea1gene AbbreviationsDIRC Days of induced roots emerging afterco-cultivationNC Non-transgenic control IntroductionWorld peanut production reached 34.42million metric tons in 2010(USDA 2010),being China,India and United states the leading producing countries.Peanuts,which are good sources of proteins,calories,vitamins,and minerals,have shown high potential to reduce hunger and malnutrition in Third World countries (Enserink 2008).However,yields of peanuts are seriously affected by subterranean insects,fungal and bacte-rial diseases (Vargas Gil et al.2008).Recalcitrance of most legumes to regeneration (Molvig et al.1997)and genetic transformation hinders the research the use of transgenic approaches to control pests and diseases in peanuts.Several types of explants,such as cotyledonary nodes (Srinivasan et al.2010),hypocotyls (Matand and Prakash 2007),axillary meristems (Singh and Hazra 2009),and cotyledons (Bhatna-gar et al.2010),are used to develop peanuts regeneration system.Moreover individual genes that confer agronomic traits have been transferred into peanuts via electroporation,particle bombardment (Chu et al.2008)and Agrobacterium -mediated transformation (Bhatnagar et al.2010;Sharma and Bhatnagar-Mathur 2006).Different transformation systems have been reported to require 4–6months or longer.Although great efforts have been made to develop efficient transfor-mation protocols for peanuts,it is still a time consuming and labor intensive process.L.Geng ÁL.Niu ÁC.Shu ÁF.Song ÁJ.Zhang (&)State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,China e-mail:jzhang@P.M.GresshoffARC Centre of Excellence for Integrative Legume Research,The University of Queensland,St.Lucia,Brisbane,QLD 4072,AustraliaD.HuangBiotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,ChinaPlant Cell Tiss Organ Cult (2012)109:491–500DOI 10.1007/s11240-012-0113-1These problems can be solved by using A.rhizogenes, the causative agent of hairy root disease.Transgenic root in vitro induced by A.rhizogenes is proposed as an effective tool for studying gene function in plant.The simplicity and convenience of A.rhizogenes-mediated transformation enhances its application in studies of signal transduction (Kosuta et al.2008),formation of root nodules(Tirichine et al.2007),plant-bacteria interactions(Lefebvre et al. 2009)and nematode resistance genes(Cai et al.1997).A. rhizogenes is used for transformation of more than100 plant species(Georgiev et al.2007),especially those that are difficult to transform,like Linum album(Farkya and Bisaria2008),Plumbago indica(Gangopadhyay et al. 2010),and Pogostemon Cablin(Shi et al.2011).Some utilization of A.rhizogenes in peanuts has also been reported(Akasaka et al.1998;Kim et al.2008;Sinharoy et al.2009),including the investigations of root nodule formation and production of resveratrol.However,little information is available concerning the factors affecting transformation efficiency.In this study,a routine,efficient and genotype-independent method of inducing hairy roots by A.rhizogenes in peanuts was established.A.rhizogenes vitality and concentration,coculture time of A.rhizogenes with explants,acetosyringone concentration of cocultiva-tion medium were assessed and described here.Moreover, a synthetic cry8Ea1gene designed according to codon usage of plant was transferred into peanuts roots using this system to check insecticidal activity against Holotrichia parallela.Our results confirmed that composite plants induced by A.rhizogenes can also be used in the studies of control of subterranean insects.Materials and methodsPlant materialSeven peanut genotypes including Luhua11,Huayu16, Huayu28,Baisha1016,Yuanhua8,Xinhua1,and Xinhua5 were used to assess the effect of plant genotype on A. rhizogenes-mediated transformation.Peanut seeds were sterilized for4min in mercuric chloride,and then washed excessively.Cotyledons of the sterilized seeds were sepa-rated using a scalpel and those with an intact embryo were germinated on basal MS(Murashige and Skoog1962) medium supplemented with30g l-1sucrose and8g l-1 agar.Plants were kept in the growth chamber with a14h/ 35l mol m-2s-1photoperiod at26–28°C.Agrobacterium rhizogenes strain and plasmidA synthetic gene encoding Cry8Ea1protein (AAQ73470.1)(Shu et al.2009)was digested with Bam HI and Sac I,ligated into pCAMBIA2300,and driven by CaMV35S promoter(Fig.4a).The vector pGFP-GUSPlus(Vickers et al.2007)which contained two highly-expressed reporter genes,greenfluorescent pro-tein(GFP)gene and b-glucuronidase gene(GUS)or plasmid pSN8E was introduced into the cucumopine-type A.rhizogenes strain K599by electrotransformation using a Gene Pulser(Bio-Rad).The transformed strains were grown in LB medium containing kanamycin at50mg l-1 and incubated overnight at30°C with shaking at 180rpm.To obtain fresh bacteria,the overnight culture was inoculated(1/100volume)in LB medium and allowed to incubate for about4h until the OD600reached 0.6–0.7.The cultures were resuspended in liquid MS medium for microinjection.Induction of roots by microinjectionFive days after germination(Fig.1a),peanut hypocotyls elongated to about1.5cm.Roots were cut off and dis-carded.The embryonic axes were injected with107cells (OD600=0.6)of A.rhizogenes at four different sites on the cut transverse face using microliter syringe inserted 0.8cm into the hypocotyls(Fig.1b).Yong leaves and cotyledons wounded with a scalpel were soaked in A. rhizogenes for30min.The infected explants were incu-bated on MS medium plus50l mol l-1acetosyringone at 30°C.After3days of co-culture,plantlets were transferred onto basal MS medium supplemented with500mg l-1 carbenicillin and50mg l-1hygromycin(for A.rhizogenes harboring vector pGFPGUSPlus)or70mg l-1kanamycin (for A.rhizogenes harboring vector pSN8E). Assessment of parameters affecting transformationefficiencyTo evaluate the effects of A.rhizogenes‘fitness’on hairy root-induction,a growth curve of A.rhizogenes strain harboring plasmid pGFPGUSPlus was drawn by measur-ing the OD600of samples at different time points for26h. This procedure was repeated three times.Four different growth stages of A.rhizogenes(represented by OD600= 0.2,0.8,3.5or5.0)were selected to inject peanut explants (see above).Each explant was inoculated with107cells resuspended in20l l of liquid MS medium.To investigate the influence of A.rhizogenes cell titre on transformation effectiveness,cells(107,59107,19108 or59108)from the optimal growth phase,resuspended in 20l l of liquid MS medium,were injected into hypocotyls.To assess the effect of co-culture period of A.rhizogenes with the explants on transformation efficiency,injected embryonic axes were kept on MS medium at30°C for1,2, 3or4days.To determine the optimal acetosyringone concentration,embryonic axes injected with A.rhizogenes were kept on MS medium supplemented with 0,5,20,50,or 100l mol l -1acetosyringone at 30°C.For each treatment,30explants were inoculated with A.rhizogenes ,and induced roots were harvested for RT-PCR and GUS detection 25days after injection.Detection of GFP or synthetic cry8Ea1gene in transgenic roots by RT-PCRTotal RNA from induced roots was isolated by the TRIzol (Invitrogen)method and treated with RNase-free DNase (Promega)according to the manufacturer’s instructions.Each RNA sample was amplified with the following two sets of primers to detect genomic DNA contamination from either peanut or A.rhizogenes :primer actF (base pairs 785–804),50-ATGAAGGAGAAGCTAGCTTA-30;and primer actR (base pairs 1,149–1,168),50-AACACTGTACTTCCTCTCTG-30;primer VirHF (base pairs 599–621),50-CCCGCCAAGCGA-CATTCCCTATT-30;primer VirHR (base pairs 1,269–1,291),50-TGTACCGCCTGCCATTCACCTTC-30.Approximately 300ng of total RNA was reverse-transcribed using M-MLV transcriptase (Takara)with oligo (dT)18primer.One micro-liter of first strand cDNA was used as template for PCR amplification.The two sets of primer pairs used in the assay were primers VirHF/VirHR,primer GFPF (base pairs 76–93),50-CTTCTCGTTGGGGTCTTT-30,and primer GFPR (basepairs 627–644),50-ACAAGTTCAGCGTGTCCG-30.Primer jc8EF2,50-GGCGGCAGCATTCAAACTCAA-30,and pri-mer jc8ER2,50-ATCTCCACCAAGATAGTGTCC-30were used to detect the transcription of the synthetic cry8Ea1gene.Histochemical assay for GUS activityRoot segments (2cm long)and hypocotyls were histochemi-cally stained to analyze GUS gene expression according to Jefferson et al.(1987).Both wild-type and putatively trans-formed roots were vacuum-infiltrated with X-Gluc solution and incubated at 37°C in the dark for 12h followed by washing with 70%ethanol.GUSPlus gene contained a catalase intron pre-venting expression in bacteria (Vickers et al.2007).Southern blot analysispGFPGUSplus plasmid DNA digested with Eco RI or/and Nco I served as positive control.About 15l g of roots genomic DNA was digested with Hin dIII.Digested DNA was separated by electrophoresis on a 0.7%agarose gel,transferred to Hybond N ?Nylon membrane,and cross-linked by ultraviolet radiation for 3min.The GFP frag-ment (569bp)amplified from plasmid pGFPGUSPlus with primers GFPF and GFPR was used as bel process and hybridization were carried out using the DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche)according to the manufacturer’sinstructions.Fig.1Peanut hairy-root induction.a 5-day old seedling for micro-injection;b microinjection of A.rhizogenes on hypocotyls at different sides.c Tumor emerging 1week after microinjection.d Induced rootsappearing 2weeks after inoculation.e Transformed root system.Bars :a and e ,1cm;b 5cm;and c and d ,0.5cmInsect bioassayComposite plants which were positive by RT-PCR for the synthetic cry8Ea1gene were transferred to pots(11cm high and13cm in diameter)containing nutritional soil and vermiculite mixture(volume ratio2:1).Two7-day-old Holotrichia parallela larvae were placed in each pot. Number and weight of live larvae,damage degree of composite plants were investigated after30days.Damage degrees were defined as follows:D0,composite plant had strong roots and grew well;D1,aerial parts of the seedling grew normally but the roots were sparse;D2,the seedling bearing no roots wilted or even died.Statistical analysisDIRC was recorded every day after co-cultivation,and the number of induced roots was counted after25days of co-culture.Data were analyzed with SPSS(Version13.0) using LSD Multiple Comparison Test.The frequency of RT-PCR positivity for GFP and percentage of GUS-posi-tive was calculated by dividing the number of positive plants by the number of plants tested.The transformation efficiency referred to either the frequency of RT-PCR positivity for GFP or the percentage of GUS-positive. ResultsEffect of plant genotype on hairy roots inductionLeaves,cotyledons or embryonic axes were inoculated with a suspension culture of A.rhizogenes.Only embryonic axes developed crown gall tumors(Fig.1c)and early forms of roots(Fig.1d)2weeks after injection of A. rhizogenes,and hairy roots were harvested3weeks later (Fig.1e).Seven diverse peanut genotypes including Lu-hua11,Huayu16,Huayu28,Baisha1016,Yuhua8,Xinhua1, and Xinhua5were microinjected with A.rhizogenes strain K599harboring plasmid pGFPGUSPlus to assess the effects of plant genotypes on root induction.It was about 11days of induced roots emerging after co-cultivation (DIRC)for most genotypes,although the DIRC of Hua-yu28was significantly longer than that of the other6 genotypes.The average number of A.rhizogenes-induced roots was less than5in Huayu28,6in Xinhua1and between6and9in the remaining genotypes(Table1). Despite little difference in susceptibility to A.rhizogenes, all the genotypes tested here developed hairy roots after infection.These results showed that A.rhizogenes-medi-ated transformation was not genotypes-dependent.There-fore Baisha1016was randomly chosen for the following experiments.Effects of A.rhizogenes growth phase and cell titreon transformation efficiencyTo evaluate the effect of A.rhizogenes growth stage on hairy-root induction,four samples(OD600of0.2,0.8,3.5 and5.0)were collected.According to the growth curve of A. rhizogenes K599(data not shown),thefirst two samples were from exponential growth phase,and the latter two were from transition phase and stationary phase,respectively.As displayed in Table2,it took more time for embryonic axes to develop hairy roots when injected with A.rhizogenes at stationary phase(OD600=5.5)while it only took9–10days for the other three treatments.In total,12.5and9.5roots (Table2)per plantlet were harvested after injection of A. rhizogenes from the exponential growth phase(OD600=0.2 and0.8,respectively),and they showed significantly more rooting responses than that from the other two phases. Consequently,A.rhizogenes cells in exponential growth phase had good root induction ability,while those of tran-sition phase and stationary phase nearly lost the ability.Transcription of GFP gene and expression of GUS gene were detected via RT-PCR and the GUS histochemical assay,respectively(Fig.2).Because the overgrowth of A. Table1DIRC and number of induced roots of different peanuts genotypesGenotype Number ofplantletsDIRC(d)Number of rootsper plantlets Baisha10165711.1±4.3bc7.2±5.7abc Huayu165310.6±2.7cd8.4±4.5a Huayu285511.6±5.6bc 6.6±4.6bc Xinhua15611.1±4.0bc 6.0±4.6c Yuhua86010.5±2.0cd8.2±3.5ab Luhua115912.0±2.0b8.7±5.0a Xinhua55811.8±2.1b8.4±4.3aNC*2021.7±1.0a0.1±0.3e Numbers followed by any same letters are not significantly different according to a LSD test(P\0.05)*Only2of20explants inoculated with ddH2O developed rootTable2Effect of A.rhizogenes growth stage on DIRC and number of induced roots of peanutsOD600Number ofplantletsDIRC(d)Number of rootsper plantlets 0.2219.5±2.9c12.5±8.4a0.82210.2±2.1c9.5±6.0ba 3.0279.3±1.5c7.3±3.9b 5.52914.9±5.4b 1.9±3.0cNC1018.3±1.5a0.2±0.4c Numbers followed by any same letters are not significantly different according to a LSD test(P\0.05)rhizogenes (OD 600of 3.0and 5.5)inhibited root emer-gence,only samples from the OD 600=0.2and 0.8treat-ments were used for RT-PCR and GUS staining detection;the transformation efficiency were all above 20%(Table 3).These were consistent with results of phenotypic observation.No obvious differences were observed in the transformation efficiency among different stages from the exponential growth phase.Based on the above results,A.rhizogenes in exponential growth phase performed well in induction of hairy roots,but the transformation efficiency was not high.To deter-mine whether a high density of the cells could improve transformation frequency,an A.rhizogenes cell titre gra-dient was established and used to conduct further experi-ments.As the number of inoculated cells increased,DIRC reduced and number of A.rhizogenes -induced roots showed no significant differences (Table 4).The frequency of RT-PCR positivity for GFP reached a plateau when each plantlet was inoculated with 59107cells,and did not continue to rise as the infecting cell number increased.A.rhizogenes was difficult to eliminate due to rapid propa-gation with a large inoculation quantity.Therefore,59107cells from the exponential growth phase in LB medium was chosen an optimal quantity for microinjection.Effects of co-culture period of A.rhizogenes with seedlings on transformation efficiencyFour co-culture periods were examined in this study.A 2-day period of co-cultivation was optimal since hairy roots emerged approximately 5days after injection,and 11roots were obtained per embryonic axe (Table 5).This agreed well with results of the RT-PCR and GUS staining assay (Table 6)that the positive frequency was 52%.Compared with the remaining treatments,the explants with 2-day co-cultivation developed more roots in a shorter time period and exhibited a higher transformation efficiency.Effects of acetosyringone concentration in co-culture medium on transformation efficiencyBecause acetosyringone has been reported to induce the expression of vir genes and thus affecting Agrobacterium -mediated transformation,different concentrations in the co-culture medium were tested.The average number of induced roots and positive frequency of molecular detec-tion peaked at 16and 47%,respectively (Table 7)when explants were placed onto MS medium with 50l mol l -1acetosyringone,and dropped on MS medium with 100l mol l -1acetosyringone.Here,the average number of induced roots was almost twice as many as Sinharoy reported (Sinharoy et al.2009).An efficient and fast system for A.rhizogenes -mediated transformation of peanutsBased on the current results,the optimal procedure for A.rhizogenes -mediated induction of peanuts transformed roots lines was as follow:5-day old seedlings without roots were microinjected with 59107cells from exponential growth phase and placed on MS medium supplemented with 50l mol l -1acetosyringone for 2days.Then explants were transferred to MS medium supplementedwithFig.2GUS staining assay.GUS expression in vertical section (a )and transverse section (c )of hypocotyls injected with K599(containing pGFPGUSplus )and induced roots (e ).Vertical section (b ),transverse section (d )of hypocotyls injected with K599(harboring no vectors)and induced roots (f )as negative control.Bars :a through d ,0.5cm;e and f ,and 0.2cmTable 3Effect of A.rhizogenes vitality on transformation efficiency OD 600Number of samples RT-PCR-positive rate of GFP (%)Percentage of GUS expression (%)0.21822220.81127273.080–5.5–––500mg l-1carbenicillin and50mg l-1hygromycin. Twentyfive days later enough transgenic roots were obtained for further study.Under optimal conditions,61% of89explants were positive for GFP transcription based on RT-PCR assay(Fig.3a)and they were all negative for virH gene which excluded the contamination from A. rhizogenes.Genomic DNA of two root lines was isolated and digested with Hin dIII for Southern blot analysis.A1.0-kb segment containing the GFP gene(Fig.3b)served as positive control.Covalent integration of the GFP gene into the peanut genome was confirmed(Fig.3c),with no sig-nals detected in A.rhizogenes-induced roots without reporter gene plasmid.Assessment of insecticidal activity of peanut roots transformed with the synthetic cry8Ea1geneRT-PCR assay showed that59%of27composite plants were positive(Fig.4)and the positive composite plants carrying the synthetic cry8Ea1gene were transferred to pots.The damage degree of63%of the composite plants was D0.These plants grew well and were not damaged by Holotrichia parallela larvae(Fig.5).Moreover,no larvae survived.The rest of samples suffered varying degrees of damage and the weight of surviving larva increased by23 times(Fig.6).The negative control(peanuts without cry8Ea1gene)wilted or even died(Fig.5).The roots of these peanuts were eaten by larvae and average weight of larva increased from13.5to395.3mg(Fig.6).The results elucidated that the synthetic cry8Ea1gene had certain insecticidal activity to Holotrichia parallela larvae,but further study should be conducted.DiscussionMolecular breeding seems to be the most powerful tool to ensure the improvement of peanut production as well as quality traits.A rapid and efficient transformation system is a prerequisite for investigation of genes confer agronomic traits.Although regeneration and transformation systems of peanuts have been previously investigated in some studiesTable4Effect of A.rhizogenes cell titre on hairy-root inductionInoculated cell number(107)Number ofplantletsDIRC(d)Number of rootsper plantletRT-PCR-positiverate of GFP a(%)13011.6±7.5b7.8±6.9a27 5328.9±4.6c8.0±6.3a50 10307.3±4.3cd7.6±5.8a46 5025 5.5±1.8d8.7±4.3a46 NC1024.2±2.5a0.1±0.3b0Numbers followed by any same letters are not significantly different according to a LSD test(P\0.05)a22samples were analyzed for RT-PCR assay every treatmentTable5Effect of coculture time on DIRC and number of inducedroots of peanutsCoculture time(d)Number ofplantletsDIRC(d)Number of rootsper plantlet124 6.5±1.8cb 6.7±3.7b 223 5.7±1.8c11.4±6.0a 328 6.4±2.5cb8.3±5.4b 4297.1±1.9b 6.0±4.1b NC1024.3±2.2a0.1±0.3cNumbers followed by any same letters are not significantly different according to a LSD test(P\0.05)Table6Effect of coculture time on transformation efficiencyCoculture time(d)Number ofplantletsRT-PCR-positiverate of GFP(%)Percentage ofGUS staining(%)1235239 2235252 3232117 4500Table7Effect of acetosyringone concentration on root induction Acetosyringoneconcentration(l mol l-1)Number ofplantletsNumber of rootsper plantletRT-PCR-positiverate of GFP(%)Percentage of GUSstaining(%) 0209.0±3.3b––5199.9±3.9b322120229.3±7.4b3232501916.4±4.2a47471002211.1±4.4b3232(Ozias-Akins et al.1993;Sharma and Bhatnagar-Mathur 2006;Bhatnagar et al.2010),problems still exist such as the time-consuming and labor-intensive protocol,genotype specificity,and low transformation frequency.Since A.rhizogenes uses a different protein ,GALLS-FL,which acts as an ATP-dependent strand transferase,to pull T-DNA into the host plants nucleus (Ream 2009),it is more effi-cient at transferring exogenous genes.Thus,in this study,A.rhizogenes was chosen to deliver target genes into peanuts.A better protocol that would ensure high efficiency transformation in genotype-independent manner is partic-ularly desirable.Such protocols have been established in some species of crops,like soybean (Ko et al.2003),rice (Yookongkaew et al.2007),potato (Block 1988),and tomato (Anwar et al.2011).But no successful case was reported in peanuts.According to our results,seven com-mercial cultivars grown in China were all susceptible to A.rhizogenes and developed composite plants afterwards.Thus,A.rhizogenes mediated transformation of peanuts which may serve as an alternative strategy to study gene function could be widely used.This method obviated laborious regeneration process and genotype constraints in peanut transformation.The influence of Agrobacterium -inoculation titre has been investigated in previous studies (Gao et al.2009).But the results cannot be unified in that the density of Agrobacterium changes due to different culture conditions and strains.In other words,Agrobacterium samples with the same density are not always in the same growth stage.So the inoculated cell number and growth phase must be considered together.Conclusions drawn from only one aspect are not sufficiently precise.Thus,we drew a growth curve of A.rhizogenes K599and determined the optimal phases for high transformation effectiveness.Our results showed that A.rhizogenes strain K599from exponential growth phase had optimal root induction ability.This was consistent with most studies (Alves et al.2009;Dutt and Grosser 2009;Joyce et al.2010;Ozawa 2009)in preference to low density of Agrobacterium ,but differed from the report of a higher density (OD 600=1.2)being optimal (Jha et al.2011).After confirming expo-nential growth phase as the optimal phase,we had eval-uated the influence of cell number on transformation efficiency.In this study,the transformation frequency peaked at 50%when the number of inoculated cell increased to 59107.An interesting finding is that as the number of inoculated cells increased,DIRC reduced and number of A.rhizogenes -induced roots remained the same.This implied that the expression of rol genes of A.rhizogenes in peanuts might affect the balance of endog-enous phytohormone which accelerated the emergency of hairy roots.6223 bp 925 bp1489 bp 1882 bp 2690 bp 3472 bp 4254 bp 7743 bp 19329 bp M GFP N AP13701 bpABCFig.3RT-PCR analysis from total RNA of induced roots (a ),vector pGFPGUSplus (b ),and Southern blot (c ).a RT-PCR detection of transformed roots.CK were hairy roots induced by A.rhizogenes with no vector.Both the VirH (700bp product)and GFP gene (569bp product)were amplified.DM2000:2,000,1,000,750,500,250bp.b Only one Hin dIII enzyme site in T-DNA region of vector pGFPGUSplus .c Total DNA from induced roots (AP)and hairy roots injected with A.rhizogenes harboring no vector (N)were digested with Hin dIII.GFP:positive control (13,701and 1,022bp).M sizes were indicated to the leftBam H SacCaMV 35SABVector pSN8E (a )and RT-PCR detection of peanut rootsCo-culture plays an important role in gene transfer because vir genes activation and attachment of Agrobac-terium to plant cells occurs during this period (McCullen and Binns 2006).So our study optimized co-culture time and acetosyringone concentration for peanut hairy root transformation.Two day co-culture period was optimal for A.rhizogenes -mediated peanut transformation which agreed well with most studies in Agrobacterium (Baskaran and Jayabalan 2009;Chen et al.2008).Whereas longercoculture period substantially promoted gene delivery of non-Agrobacterium strains (Broothaerts et al.2005).Optimal acetosyringone concentrations reported ranged from 30to 200l mol l -1(Chen et al.2007;Costa et al.2006)for diverse plant species.We believed that 50l mol l -1acetosyringone greatly increased transforma-tion efficiency,while excessively high concentration exerted ‘toxic’effects on host plant cells and inhibited organ formation.White grubs,a kind of subterranean insects,caused severe reduction in peanut yield (Rogers et al.2005).In previous study,we cloned cry8Ea1gene which was toxic to Holotrichia parallela .Due to the high AT content of cry gene,it must be modified according to the codon usage of plants to increase the accumulation of Bt protein in biotech crops.Since A.tumefaciens mediated transformation is time-consuming and labor-intensive,a fast and feasible method is needed to deliver the modified cry genes into interested crops,verify activity of cry genes against sub-terranean insects.In addition,Holotrichia parallela larvae were hard to rear.The procedure for bioassay of Holotri-chia parallela has not been reported until now.Here,the protocol was initially set up and further improvement was needed.Although A.rhizogenes was used in some plant species,no reports focused on factors that affected peanut hairy roots induction.In this study,four parameterswereFig.5Bioassay of peanuts root lines against Holotrichia parallela Motschulsky larvae.a Composite plants with induced roots transformed with synthetic cry8Ea1gene.b Non-transgenic plantlets as negative control.c Live larvae.Bars :a and b ,5cm;and c ,1cmFig.6Weight of Holotrichia parallela Motschulsky larvae in bioassay.D0,D1,D2The damage degree was 0,1,2respectively.CK Non-transgenic plantlets.Numbers followed by any same letters are not significantly different according to a LSD test (P \0.05)。