a chimeric cry1x gene imparts resistance to spodoptera...

Keshamma Entoori1, Rohini Sreevathsa2, Manoj Kumar Arthikala2, PolumetlaAnanda Kumar3, Amrita Raja Vinoda Kumar4, Basavaraj Madhusudhan1,Udayakumar Makarla2*

1Department of Biochemistry, Centre for Nano Science and Technology Reasearch,Kuvempu University, P.G. Centre, Shivagangotri, Davangere 577 002, Karnataka,India2Department of Crop Physiology, University of Agricultural Sciences, GKVK,Bangalore 560 065, Karnataka, India3Centre for Plant Biotechnology, Indian Agricultural Research Institute, New Delhi110012, New Delhi, India4Department of Entomology, University of Agricultural Sciences, GKVK, Bangalore560065, Karnataka, India

* Corresponding author: [email protected]

A chimeric cry1X gene imparts resistance toSpodoptera litura and Helicoverpa armigera in thetransgenic groundnut

AbstractThe transgenic plants of the groundnut (Arachis hypogeae) cv. TMV-2 expressing a chimericBt gene, cry1X, were generated using an Agrobacterium tumefaciens - mediatedtransformation system. A tissue culture-independent transformation method, in planta whichtargets the A. tumefaciens to the apical meristem was used in this study. The protocolinvolves in planta inoculation of the embryo axes of the germinating seeds and allowing themto grow into seedlings ex vitro. PCR analysis indicated the putative transgenic nature of theT1 generation plants. Bioassays against two major pests of the groundnut, Helicoverpaarmigera and Spodoptera litura revealed several T1 plants that perform well against both thelarvae. This revealed that 22% of T1 plants harbor the transgene. The seeds of 27 T1 plantswhen allowed to continued into the next generation amplified the gene of interest in most ofthe plants tested. Enzyme Linked-Immuno Sorbent Assay (ELISA) was used to identify the highexpressing plants. The appearance of the protein band in the quickstix confirmed theexpression of the chimeric Bt toxin. Southern analysis of 10 high expressing plants confirmedthe integration of the transgene. These results suggest that the chimeric Bt gene wasfunctional in the transgenic groundnut and was being expressed. The study also showed thatthe groundnut plants harboring the cry1X gene were resistant to two major insect defoliatorsof the groundnut.

Keywords: Arachis hypogeae, Helicoverpa armigera, transformation, transgenics, in-planta,tissue cultureindependent plant regeneration, Spodoptera litura, synthetic cry gene.

Entoori K, Sreevathsa R, Arthikala MK, Kumar PA, Kumar ARV, Madhusudhan B, Makarla U(2008) A chimeric cry1X gene imparts resistance to Spodoptera litura and Helicoverpaarmigera in the transgenic groundnut. EurAsia J BioSci 2, 7, 53-65.www.ejobios.com/content/2/7/53-65

©EurAsian Journal of BioSciences, 2008 53

EurAsian Journal of BioSciences EurAsia J BioSci 2, 53-65 (2008)

The groundnut, Arachis hypogaea L.(Papilionaceae), is the 13th most importantfood crop of the world. It is the world's 4th

most important source of edible oil and 3rd

most important source of vegetable protein.Groundnut seeds contain a high quality edibleoil (~50%), easily digestible protein (~25%)and carbohydrates (~20%). Major threats for

the production of groundnut are bioticstresses, fungal diseases such as tikka,rhizoctonia and fusarium wilts followed byinsects. A large number of insect pests causeconsiderable damage to the crop (Sridhar andMahato 2000). Some of these such as white

Received: March, 2008Received in revised form: July, 2008

Accepted: July, 2008Printed: September, 2008

INTRODUCTION

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©EurAsian Journal of BioSciences, 200854

EurAsian Journal of BioSciences

grubs can be now managed with suitabletechnologies. But the caterpillar pests remainthe eternal threat. Among these, the redheaded hairy caterpillar (RHHC), Amsactaalbistriga (Walker), is as of now the majorthreat (Ganiger 2006). The leaf miner,Aproaerema modicella (Deventer) isperennially a threat for the production of thiscrop in Asia and Africa, (Reddy 1978,Sumithramma 1998) causing up to a 90%loss. The tobacco caterpillar, Spodopteralitura (Fabricius) is the next most importantproblem in isolated pockets and more sounder irrigated conditions. Occasionally,Helicoverpa armigera (Hubner) occurs on thecrop causing defoliation to a limited extent.All of these caterpillars cause damage to theleaf and result in crop loss. With much of thegroundnut area being under rainfed conditionsin semi arid tracts, the yield potentials aredirectly related to the rainfall during thecropping period. Therefore, the investment onpest management is not an economicallyviable strategy. As a consequence, much ofthe pest and disease problems remain aperennial threat for the production of this cropresulting in poor productivity. Therefore, thereis a need to find reasonably long termsolutions for these pest problems, withoutcalling for increased in put for pestmanagement by the farmers. Insect resistanttransgenics, therefore, would be an ideal seedborne solution in the interest of the groundnutfarmers and the productivity (Sharma et al.2003).

Among the many potential genes are thecry genes from Bacillus thuringiensis(Berliner). The cry1 series of genes aregenerally effective against Lepidoptera, butare likely to be species specific (Chakrabarti etal. 1998, Kumar and Bambawale 2002). Forexample, cry 1Ac is best against H. armigeraand cry1B and cry1C are likely to be good forthe diamondback moth. Similarly, cry1F iseffective against S. litura. As a result,successful incorporation of any of thesenatural gene sequences into a crop is unlikelyto provide good protection against a range ofpests. But, earlier work with hybrid Bt geneshave shown to provide increased protection ofplants against a single insect pest or against a

few closely related insect species (Maagd etal. 1996, Frutos et al. 1999, Ho et al. 2006).Interestingly, Naimov et al. (2003) used ahybrid gene from cry1Ba and cry1la andobtained a transgenic potato resistant to boththe coleopteran and a lepidopteran pest.

Therefore, the synthetic constructs thatincorporate the suitable elements of a numberof Bt genes may provide protection against anumber of crop pests besides being moreeffective against known susceptible pests(Honee et al. 1990). The groundnuttransgenics with such novel hybrid constructstherefore, would be ideal to tackle a range ofcaterpillar pests that attack them in the semiarid tracts of Southern India. Cry1X is asynthetic construct designed and developedby IARI, New Delhi and incorporates theelements of cry1Ac, cry1Ab, cry1Aa3 andcry1F. The novel construct, having beendesigned to act on a wide variety of Btreceptors, is likely to be effective against arange of lepidopteran caterpillars includingsome of the most difficult to manage such as,H. armigera and S. litura.

Many reports of the transformation anddevelopment of groundnut transgenics using avariety of genes such as the bar gene forherbicide tolerance (Brar et al. 1994), cry1A(Singsit et al. 1997), a gene encoding thenucleocapsid protein of the tomato spottedwilt virus (Yang et al. 1998), a gene thatconfers resistance to Indian Peanut Clumpvirus, gus (Venkatachalam 2000, Rohini andRao 2000), chitinase (Rohini and Rao 2001),and DREB1A (Bhatnagar et al. 2007), havebeen reported.

However, the reported successes appear torevolve around the low in vitro cultureresponses seen in groundnut cultivars. As aresult, transformation successes tend to bepoor and call for intensive experimentation.However, to tackle the problems pertaining toregeneration in groundnut and certain otherrecalcitrant crops, alternate methods tominimize or eliminate the steps ofregeneration are being standardized. Researchwith Arabidopsis has benefited from thedevelopment of high throughputtransformation methods that are referred to asin planta transformation protocols and avoid


Entoori et al.EurAsian Journal of BioSciences

the plant tissue culture (Azipiroz-Leehan et al.1997). In particular, the development of theAgrobacterium tumefaciens-mediated vacuuminfiltration method (Bechtold et al. 1993) hashad a major impact on Arabidopsis research.In planta transformation methods have alsobeen standardized for buckwheat (Kojima etal. 2000), mulberry (Ping et al. 2003), kenaf(Kojima et al. 2004), rice (Supartana et al.2005), wheat (Supartana et al. 2006), andmaize (Chumakov et al. 2006). In all thesecrops, Agrobacterium is directed towardseither the apical meristem or the meristems ofaxillary buds. One such viable in plantatransformation protocol has also beenstandardized for other crops (Rao and Rohini1999, Rohini and Rao 2000a, 2000b, Rohiniand Rao 2001). The strategy essentiallyinvolves in planta inoculation of embryo axesof germinating seeds and allowing them togrow into seedlings ex vitro. These in plantatransformation protocols are advantageousover other methods because they do notinvolve regeneration procedures and thereforethe tissue culture-induced somaclonalvariations are avoided. The present workdemonstrates a successful incorporation of achimeric cry gene, cry1X into the groundnutcv. TMV-2 and response of the transgenicsagainst two major groundnut leaf eatingcaterpillars, S. litura and H. armigena.

Plant materialSeeds of the groundnut variety TMV-2

were soaked overnight in distilled water andwere surface sterilized first with 1% Bavistinfor 10 mins and later with 0.1% HgCl2 forfew seconds. After treatment with eachsterilant, the seeds were washed thoroughlywith distilled water. The seeds were laterplaced for germination in petriplates at 30°C.Two-day old seedlings were taken as explants for Agrobacterium infection.

Bacterial strain and vector The Agrobacterium tumefaciens strain,

EHA105, harbouring the binary vector,pBinBt8, was used for transformation. Thevector harbours the cry1X and npt II as thegenes of interest and the selectable marker,

respectively (Fig. 1). The Agrobacteriumstrain EHA105/pBinBt8 was grown overnightat 28°C in LB medium (pH 7.0) containing 50μgml-1 kanamycin. The bacterial culture waslater resuspended in 100 mL of Winans' ABmedium (pH 5.2: Winans et al. 1998) andincubated for 18 h. For the vir gene inductiontreatments, wounded tobacco leaf extract (2g in 2 mL sterile water) was added separatelyto the Agrobacterium suspension in Winans'AB medium, 5 h before infection (Cheng et al.1996).

Transformation and recovery of transfor-mants

The transformation of groundnut cv. TMV-2 and generation of the primary transformantswas accomplished using the tissue-cultureindependent in planta transformationprocedure as standardized earlier (Rohini andRao 2000). The seedlings with just emergingplumule were infected by pricking at themeristem with a sterile needle and subsequentimmersion in the culture of Agrobacterium for16 h. Then, the seedlings were washed brieflywith sterile water and the seedlings were latertransferred onto autoclaved soilrite(vermiculite equivalent) moistened with waterfor germination under aseptic conditions in thegrowth room in wide mouth capped glass jarsof 300 mL capacity with 5 seedlings per jar.After 5 to 6 days, the seedlings weretransferred to soilrite in pots and wereallowed to grow under growth roomconditions for at least 10 days before theywere transferred to the greenhouse. Thegrowth chamber was maintained at 26-28°Cunder a 14 h photoperiod with a florescentlight intensity of 35 μmol m-2 s-1.

Molecular analyses of the putative trans-genic plants

Tissues from the progeny plants wereanalyzed for the presence of the introducedgenes. Genomic DNA was isolated followingthe procedure of Dellaporta et al. (1983) fromthe fresh leaf tissue of the greenhouse-grownT1 generation plants that was used for gridPCR, PCR with individual plant samples andSouthern blot.

MATERIAL AND METHODS

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Grid PCR analyses of putative transgenicplants in T1 generation

Seeds from each individual plant weremaintained as separate lines. T1 groundnutplants were grown in green house followingrecommended package of instructions(Anonymous 2000) and the plants werelabeled with aluminium tags. They weredivided into different grids containing 100plants each so that there were 10 plants eachalong the rows or columns. Samples from the10 plants either along the row or along thecolumn formed a composite sample. As aresult, from each grid of 100 plants numberedfrom 1 to 100, 20 composite samplesoriginated.

Genomic DNA from composite sampleswas isolated and the PCR analysis ofcomposite samples was done using specificprimers under standardized PCR conditions.

PCR was also performed to confirm thepresence of the gene in the plants that wereselected to be advanced further. PCR wasperformed to amplify the 750 bp nptII genefragment in the putative transformants. Inorder to amplify the npt II gene fragment, PCRwas initiated by a hot start at 94°C for 4 minfollowed by 32 cycles of 1 min at 94°C, 1min 30 s at 58°C and 1 min at 72°C. PCRwas also performed with the gene specificprimers (cry1X gene) to amplify a 950 bpfragment. The conditions for the reactionwere the same as above. The product was runon a 1% agarose gel.

Southern analysisIn order to analyse the total genomic DNA

for transgene integration of the cry1X gene,15 μg of total genomic DNA was digestedwith the appropriate restriction enzyme. Boththe digested and uncut DNA samples wereelectrophoresed on a 0.8% agarose gel. Theseparated fragments along with the uncut

DNA were transferred onto a nylon membraneand hybridized with a labeled 950 bp PCRamplified product of the cry1X gene.Hybridization was performed at 65°C inChurch buffer for 18 h. Membranes werewashed for 30 min each in 2X SSC, 0.1%SDS; 0.1X SSC, 0.1% SDS at 65°C(Sambrook et al. 1989). The blots were thenexposed in a phosphorimager.

Expression analyses of the putative trans-genic plants

Enzyme Linked Immuno Sorbent Assay(ELISA)

Qualitative ELISA was used to check thecry protein produced in the transgenicgroundnut plants. A cry1AB/cry1Ac plate kit(Envirologix Inc, Portland, USA) was used forthis purpose. The Sandwich ELISA wasperformed according to the manufacturer'sinstructions.

Quick dip stick detectionQuick detection of the hybrid/fused Bt

protein was accomplished using the"cry1Ab/cry1Ac lateral flow Quickstix Strip"as per the manufacturer's instructions (BtQuant, Nagpur, India). The presence of a testline (second line) on the membrane stripbetween the control line (common to all,including the non-transformed control) and theprotective tape would indicate the expressionof a foreign Bt protein in the transgenics.

In vitro insect bioassayAll bioassays were performed on detached,

fully expanded, groundnut leaves. Twotrifoliate leaves were collected from eachselected plant and washed with distilledwater. The leaves were wiped clean of all dirtand other debris. The stalks of the leaveswere wrapped with wet cotton pieces andplaced in a plastic container. Ten neonatelarvae of H. armigera/S. litura were releasedon to each leaf. Observations were recordeddaily for a period of four days on the numberof dead and live larvae, per cent of leafdamage and the leaf condition. The containerswere wiped clean daily.

Statistical analysesData was analysed using MS excel and

SPSS software. Means and standarddeviations were worked out for all valuesdepending on the need. The mean values of all

Fig. 1. T-DNA map of the binary vector pBinBt8 (13 kb) carrying cry1X and nptII genes.



the plant parameters were subjected toANOVA (Sokal and Rohlf 1969). Correlationand regression analyses were done followingthe method of Snedecor and Cochran (1967).Scatter plots and frequency distributiongraphs were generated where necessary forrepresenting the data.

In planta transformation of groundnutvariety TMV-2 with cry1X gene

Approximately 40 seedlings weresubjected to in planta transformation. Twentyof these plants survived after shifting to thepots in the green house. Under the greenhouse conditions, the plants grew normally,flowered and set pods. These plants weredesignated as the T0 generation plants. Seedswere harvested and used for raising the T1

generation plants.Analysis of the T1 generation plants As many as 600 seeds were harvested

from 20 primary transformants out of which425 T1 plants were established in the greenhouse. Among these, 400 plants wereselected for analysis and were divided intogroups of 100 as grids to form 80 compositesamples for PCR analysis.

Molecular analysis by grid PCRPCR analysis with npt II specific primers of

the 80 grid samples revealed the possibility ofthe presence of the npt II gene in 125 plantsout of the 400 analyzed (Fig. 2a). These 125PCR putative positive plants and 16 morehealthy looking plants from a total of 19primary transformants were further analyzedfor the efficacy of the cryIX gene.

Insect resistance of the transgenic plantsThe efficacy of the cry gene product was

tested against two major pests of groundnutviz., H. armigera and S. litura. Leaves from 4-6 wk old groundnut plants, that were positivefrom grid PCR analysis, were taken forbioassay. Ten neonate larvae were loaded perleaf and were monitored for 96 hrs. Mortalityand percent leaf damage were recorded toasses the effect of the protein on the larvae.Concurrent bioassays were also run on thewild type plants.

The transgenic plants showed significantlyhigher tolerance to the target pests andperformed better when compared to the wildtype (Figs. 2b and 2c). The larvae thatsurvived after feeding on the transgenic plantswere severely stunted when compared to thelarvae that fed on the leaves from the wildtype plants in both the bioassays (Figs. 2dand 2e). Interestingly the efficacy of thechimeric gene appeared similar against boththe larvae as confirmed by the strongcorrelation between the percent mortalities ofthe two larvae by the plants (r= -0.754;p<0.01; n= 141; Fig. 3a) and per centdamages to leaves caused by the two larvae(r= 0.851; p<0.01; n= 141; Fig. 3b).

However, the results suggested a variableresponse of the transgenics towards the twolarvae and a large number of transgenics werefound superior to the wild type plants. Resultsof the experiments revealed that the 141transgenic plants showed a range of 10-80per cent mortality and 5-60 per cent damagein both the bioassays in contrast to 1-10 percent mortality and 60-70 per cent damage inthe wild type leaves. This clearly indicated thevariable expression of the transferred Bt-geneand a consequent variable effectivenessagainst the target insects.

Based on the bioassays against H. armigeraand S. litura, 27 plants that showed > 50 percent mortality and not more than 10 per centdamage were selected to be taken further.Comparison of means by the two tailed 't'test with unequal sample sizes, indicated theselected transgenic plants to be significantlysuperior to the wild type plants in respect tothe percent mortalities of both the H. armigera(t= 18.01 ; p<0.01) and S. litura (t= 14.67; p<0.01). Similarly, the two plants differedsignificantly in respect to leaf damage by boththe H. armigera (t= 17.94; p<0.01) and S.litura (t= 20.14; p<0.01) (Table.1).

Confirmation of the integration andinheritance of the transgene in all 27 selectedplants was obtained by PCR analysis for thenpt II and cry1X gene. Results showamplification at 750 bp for the npt II gene and950 bp for the cry1X gene.

Therefore, the 27 plants confirmed astransgenics based on PCR and bioassays

RESULTS

Entoori et al.



against H. armigera and S. litura, wereselected for further experimentation.

Analysis of the T2 generation groundnutplants harboring the cry1X gene

The seeds of the 27 best transgenics fromthe T1 generation were sown in thegreenhouse and 340 plants were obtained.

The transgenics (340 plants) that weregerminated in the T2 generation weresubjected to PCR by using the primers for thenpt II gene. Results show amplification at 750bp in 322 plants. Further, when all the 340plants were subjected to ELISA, highexpression was observed in 90 plants,showing a 3-16 fold increase over thenegative control (Fig. 4).

High expressing plants based on ELISA (10plants that showed 7-16 fold increase overthe negative control) (Fig. 5a) were selectedfor molecular characterization by PCR andgenomic Southern analysis. A 950 bp cry1Xfragment was amplified in all the 10 plantssuggesting stability and integration (Fig. 5b)and in the strip test (Fig. 5c) the arrow showsexpression. These plants were subjected togenomic Southern analysis. High molecularweight uncut DNA hybridized with the 950bprandom prime labeled cry1X gene fragmentrevealed integration of the transgene (Fig. 5d).In the vector used for raising the transgenicplants, Bam HI releases a 1.9 kb fragmentwhich includes the cry1X gene. Thehybridization signal at the right positionindicates (Fig. 5e) the integration of thetransgene.

The successful cultivation of bollwormresistant Bt cotton varieties has provided agreat impetus for the development of a largenumber of transgenics that are potentiallycapable of alleviating many pest and diseaseproblems in different crops. Engineering forinsect pest resistance in legumes(Ignacimuthu et al. 2006, Sharma et al. 2006)

Fig. 2. Analysis of the T1 generation groundnut plants by grid PCR and insect bioassay: (a) Representative gel showing grid PCR with npt II gene specific primers. Lane M: DNA ladder (1kb); Lane NC: negative control (DNA from untransformed plants); Lane PC: positive control (plasmid DNA); Lanes 1-18: DNA from grid composite samples of putative transformants; Performance of the T1 generation transgenic lines against neonate larvae of (b) Helicoverpa armigera and (c) Spodoptera litura larvae; Larvae fed on leaves of wild type and transgenics (d) Helicoverpa armigera and (e) Spodoptera lutura: C- wild type plants and T- transformed plants.

Fig. 3. Relation between the performance of neonate larvae of Helicoverpa armigera andSpodoptera litura in bioassays of detached leaves of selected putative transgenic plants of T1 generation with respect to a) per cent mortalities of the two larvae and b) per cent leaf damages by the two larvae. The relationship indicated is only for the transgenic plants and the hollow triangles represent the data for control plants.

Table 1. Comparison of mortality and damage to leaf by Helicoverpa armigera and Spodoptera litura larvae in leaf bioassay between T1generation groundnut putative transgenics and wild type control plants. (Two tailed 't' test with unequal sample sizes).

p<0.01, Significance at 1%.

DISCUSSION



has been considered important in the recentyears. However, very little progress is seen inthe improvement of legumes through thetransgenic approach and more so withgroundnut because of the recalcitrancy inregeneration of the crop. There are reports ofregenerability in the groundnut, but with lessfrequency, using leaf discs and leaf sectionexplants (Eapen and George 1994, Cheng etal. 1996), cotyledons (Venkatachalam et al.2000, Sharma and Anjaiah 2000, Bhatnagaret al. 2007) and the cotyledonary node(Anuradha et al. 2006). Recalcitrance andgenotype-dependent regeneration called forstandardization of alternate methods oftransformation that totally avoid tissue cultureand consequent regeneration needs that isoften genotype dependent (Rohini and Rao2000, 2001). The methodology is therefore,readily available for adoption in thetransformation of groundnut with relativelyhigher expectations of success. Nevertheless,low progress has been made with theimprovement of Indian cultivars of groundnutwith respect to insect pest resistance usingtransgenic technology. This work reports thesuccessful transformation of groundnut cv.TMV-2 for insect pest resistance using achimeric cry gene, cry1X.

The method is based on the fact that someof the differentiated embryonic cells that takein DNA can develop into germ cells and

therefore will be transmitted to the nextgeneration. The meristem transformationprotocols are being used as in plantatransformation protocols for the improvementof difficult-to-regenerate species (Rao andRohini 1999, Rohini and Rao 2000, 2001,Keshamma et al. 2007). Earlier, the chitinasegene for fungal disease resistance wasintroduced into the groundnut using the inplanta transformation protocol demonstratingthe feasibility of this protocol to developstable transformants (Rohini and Rao 2000).

In our method, A. tumefaciens is targetedto the wounded apical meristem of the

Fig. 4. Frequency distribution of putative transgenics transformed with cry1X gene for fold increase in ELISA values of transgenics over the negative control among 340 plants of groundnut in T2 generation.

Fig. 5. Analysis of 10 high expressing plants from T2generation by: a) ELISA for cry1X expression -fold increase in ELISA values of selected 10 transgenics over then negative control. b) PCR of the DNA of transgenic plants transformed with cry1X gene in the T2generation groundnut using primers for cry1X gene. Lane M: DNA ladder (1kb); Lane NC: negative control (DNA from untransformed plants); Lane PC: positive control (plasmid pBinAR DNA); Lanes 1-10: DNA from putative transformants. c) Quickstix showing the protein band at the expected position (arrow marked) identifying the chimeric Bt protein in

10 plants and absence in the control. d) Southern hybridization of uncut DNA of T2transformants probed with radiolabelled 950 bp of cry1X gene. Lanes 1-10: DNA from putative transformants; Lane NC: Negative control (DNA from untransformed plants). e) Southern hybridization of DNA of T2transformants digested with Bam HI and probed with radiolabelled 950 bp product of cry1X gene. Lane NC: Negative control (DNA from untransformed plants); Lane PC: Positive control (plasmid DNA); Lanes 1-10: DNA from putative transformants.

Entoori et al.



differentiated seed embryo. Therefore, A.tumefaciens transfers the gene into thegenome of diverse cells which are alreadydestined to develop into specific organs andthe meristematic cells still to be differentiated.This results in the primary transformants (T0)being chimeric in nature. Consequently, thisnecessitates the analysis of the putativetransgenic plants only in the T1 generation.The chimeric plants producing thetransformants in the T1 generation dependson the type of cells that were transformed inthe T0 plants. If the transgene is integratedinto undifferentiated meristematic cells thatare destined to develop into branches, seedsobtained from the reproductive structures ofthese branches are expected to give stabletransformants in T1. The in plantatransformation protocol gives rise to a largenumber of T1 generation plants and apreliminary screening procedure is requiredthat considerably reduces the number ofputative transformants to be taken forward.Grid PCR technique helps eliminate manyplants by using composite DNA samples in aPCR reaction. While all plants eliminated aretruly non-transformants, the methodology,does not help identify the putativetransformants, as the selected plants are amixture of both non-transformants and thetransformants. Therefore, the plantscontributing for a positive composite samplein the grid PCR have to be further checked toidentify the individual putative transgenics.Hence, grid PCR positive composite sampleswere taken ahead for further analysis. Initiallyinsect reactions against two target pests viz.,H. armigera and S. litura based on bioassayswere assessed and subsequently the resistantlines were further analyzed for integration andexpression.

Development of transgenic crops being along lasting and high investment option amajor threat for the long lasting effect of suchcrops is the potentiality of the insects todevelop resistance against such crops.Therefore, it is envisaged that genepyramiding in transgenic plants could be apotentially more viable strategy for delayingthe insect pest evolution leading to resistance

against single cry genes (Greenplate et al.2000, Cao et al. 2002). It is also expectedthat the same option might also help improvethe efficacy range of the introduced genescovering more than one or a set of species(Datta et al. 2002a). An alternative to thegene pyramiding would be to use syntheticgenes of multiple efficacies. Earlier studies inthe transgenic potato with cry1Ba/cry11ahybrid gene encoding a protein consisting ofdomains I and III of cry1Ba and domain II ofcry1Ia demonstrated resistance against bothColeoptera represented by the Coloradopotato beetle larvae and adults, andLepidoptera represented by the potato tubermoth larvae and european corn borer larvae(Naimov et al. 2003).

Similar results were anticipated from ourselection of the cry1X gene in the presentstudy. The chimeric cry gene, cry1X, hasdomains from four cry genes, cry1Ac,cry1Ab, cry1Aa3 and cry1F. Cry1Ac, Ab, andAa3 are effective against H. armigerawhereas, cry1F is best against S. litura. Thusit was anticipated that the novel constructwould provide protection against at least twoentirely differing groups of lepidopteran pestswith different Bt protoxin receptors. Further,due to the fact that the gene encodes theactive domains of four different Bt toxins, it isanticipated that it might work against a fardiverse species of Lepidoptera. Theseexpectations were substantiated by the factthat the efficacy of the cry1X gene wasconfirmed, albeit variable, in bioassaysagainst H. armigera and S. litura.

There were significant differences betweenthe per cent mortality in the transgenics whencompared to those of non-transgenic wildtypes indicating the effectiveness of the Bttoxin in the transgenics. The pattern repeatedwith both the species.

The observed mortality patterns of boththe target pests in the T1 generation clearlysuggested the variable expression levels ofthe transgene in this generation. Further, therange of the variability being sufficiently large,the mortality data was regressed against leafdamage. It was observed that the mortalitywas strongly negatively correlated with the



Aguda RM, Datta K, Tu J, Datta SK, Cohen MB (2001) Expression of Bt genes under control ofdifferent promoters in rice at vegetative and flowering stages. International Rice ResearchNotes 26, 26-27.

extent of leaf damage when groundnut leaveswere challenged with neonates of H. armigeraor S. litura. Clearly, these results indicate thatthe mortality of the larvae is a product of theextent of ingestion of toxin that was inverselyrelated to the total food consumed. Thus theplants with low levels of expression of thetransgene experienced greater extent of leafloss and resulted in better survival of thelarvae. More interestingly, the toxin appearedto be equally effective at levels of expressionagainst the two larvae of disparate Bt toxinprotoxin receptors, as the mortality levels ofthe two larvae against the plants werestrongly and positively correlated. Further, the27 plants that had a high per cent mortalityand per cent damage were selected andcontinued into the next generation andanalyzed for the stability of the transgene.These plants showed mortality in a range of40-80 per cent and damage as low as 5-10per cent which corroborated in the bioassaysagainst both H. armigera and S. litura.

In the progeny of these 27 plants in the T2

generation, 322 plants out of 340 plantsdeveloped in the green house were PCRpositives suggesting segregation in some ofthe lines. However, 90 plants showed veryhigh ELISA values and there was variationbetween the transgenic plants whencompared to the wild type. Such variations inprotein expression levels in differenttransgenic bioassays are quite frequent andcommon due to genotypic, developmental andenvironmental control. Such variations havealso been observed among the progenies of asingle parental line with an identical pattern oftransgene integration; grown under the sameenvironment (Datta et al. 1998, Aguda et al.2001, Alinia et al. 2001, Datta et al. 2002a,Hussain et al. 2002, Marfa et al. 2002). Toconfirm the integration of the transgene in thegroundnut plants (T2 generation) showing

high ELISA values, genomic southern analysiswas performed with the DNA of 10 plantsthat showed high expression. Strips testcarried out in the plants selected for southernanalysis supported the ELISA data.Hybridization of uncut DNA and the release ofa 1.9 kb fragment after digestion with Bam HIand a strong hybridization signal after probingwith a radiolabelled PCR product for the cry1Xgene clearly demonstrated integration,inheritance and stability of the transgene.Nevertheless, copy number studies need to becarried out to select and take further theevents with single copy insertions of thegene.

Transformation efficiency in the presentstudy was calculated using percent mortalityof H. armigera as a parameter. Among theindividual transformants exhibiting >3 (std.dev.) of the wild type plants (std. dev.=6.32), 63.12% of the total 141 transformantsshowed higher values with a mean value of42.42. This resulted in 88 plants performingwell which is 22% of the 400 plants taken forT1 analysis. These plants can be consideredas putative transformants. However, 27plants which showed mortality of >56%were selected for further analysis into thenext generation and were subsequentlyconfirmed as transgenics by molecularanalysis.

This study presents the following: 1)evidence for the production of insect resistantplants using the in planta transformationprotocol. 2) Efficacy of the chimeric Bt-geneagainst lepidopteran pests, H. armigera and S.litura, two major leaf eating caterpillars ofgroundnut. The successful advancement ofthese transgenics containing the cry1X genein groundnut to obtain the stable lines willprovide a seed borne solution to manage somelepidopteran pest of the groundnut.

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Kimerik Bir cry1X Geninin Transgenik Yer Fistigi'nda Spodoptera litura veHelicoverpa armigera'ya Direnc Olusturmasi

Ozet

Agrobacterium tumefaciens kullanilarak, kimerik bir Bt geni olan cry1X'i eksprese eden transgenik cv. TMV-

2 yer fistigi bitkileri (Arachis hypogeae) elde edildi. Bu calismada, A. tumefaciens'de apikal meristemi

hedefleyen ve doku kulturunden bagimsiz bir transformasyon metodu olan in planta metodu kullanildi. Bu

protokol, cimlenmekte olan tohumlarin embriyo eksenlerinin in planta inokulasyonunu ve ex vitro olarak fide

haline getirilmelerini icermektedir. PCR analizi, T1 nesli bitkilerin ongorulen transgenik dogasini gosterdi.

Biyoassayler, bazi T1 bitkilerinin yer fistiginin iki buyuk zararlisi Helicoverpa armigera ve Spodoptera litura

larvalarina karsi iyi performans sergiledigini ortaya cikardi. Calisma T1 bitkilerinin %22'sinin bu transgeni

icerdigini gosterdi. Bir sonraki nesle devam etmelerine izin verildiginde 27 T1 bitkisinin tohumlari, test edilen

bitkilerin cogunda ilgili genin etkisini artirdi. ELISA kullanilarak yuksek oranda eksprese eden bitkiler belirlendi.

Quickstix'de protein bandinin gorunmesi kimerik Bt toksininin ekspresyonunu onaylamaktadir. 10 yuksek

oranda eksprese eden bitkinin Southern analizi transgenin entegrasyonunu onaylamaktadir. Bu sonuclar, Bt

geninin transgenik yer fistiginda islevsel oldugunu ve eksprese edildigini gostermektedir. Calisma ayrica,

cry1X genini iceren yer fistigi bitkilerinin, onemli iki yaprak zararlisina karsi direncli oldugunu gosterdi.

Anahtar Kelimeler: Arachis hypogeae, doku kulturu-bagimsiz bitki yenilenmesi, in-planta, Helicoverpaarmigera, sentetik cry geni, Spodoptera litura, transformasyon, transgenik.

a chimeric cry1x gene imparts resistance to spodoptera...

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