Pyramided rice lines harbouring Allium sativum Galanthus ... JBT Bharathi.pdf · withstand the ravages of various insect pests. Three major sap-sucking pests of rice, viz., brown

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Journal of Biotechnology 152 (2011) 63–71

Contents lists available at ScienceDirect

Journal of Biotechnology

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yramided rice lines harbouring Allium sativum (asal) and Galanthus nivalis (gna)ectin genes impart enhanced resistance against major sap-sucking pests

. Bharathia, S. Vijaya Kumara, I.C. Pasalub, S.M. Balachandranb, V.D. Reddya, K.V. Raoa,∗

Centre for Plant Molecular Biology, Osmania University, University Campus, Hyderabad, Andhra Pradesh 500007, IndiaDirectorate of Rice Research, Rajendranagar, Hyderabad 500030, India

r t i c l e i n f o

rticle history:eceived 21 October 2010eceived in revised form0 December 2010ccepted 24 January 2011vailable online 3 February 2011

a b s t r a c t

We have developed transgene pyramided rice lines, endowed with enhanced resistance to major sap-sucking insects, through sexual crosses made between two stable transgenic rice lines containing Alliumsativum (asal) and Galanthus nivalis (gna) lectin genes. Presence and expression of asal and gna genes inpyramided lines were confirmed by PCR and western blot analyses. Segregation analysis of F2 progeniesdisclosed digenic (9:3:3:1) inheritance of the transgenes. Homozygous F3 plants carrying asal and gnagenes were identified employing genetic and molecular methods besides insect bioassays. Pyramidedlines, infested with brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper

eywords:llium sativum lectinntomotoxic effectsalanthus nivalis lectin

nsect resistanceryza sativayramided linesap-sucking pests

(WBPH), proved more effective in reducing insect survival, fecundity, feeding ability besides delayeddevelopment of insects as compared to the parental transgenics. Under infested conditions, pyramidedlines were found superior to the parental transgenics in their seed yield potential. This study representsfirst report on pyramiding of two lectin genes into rice exhibiting enhanced resistance against majorsucking pests. The pyramided lines appear promising and might serve as a novel genetic resource in ricebreeding aimed at durable and broad based resistance against hoppers.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Rice (Oryza sativa, L.) is one of the foremost cereal crops, whicherves as the primary source of staple food for more than half ofhe global population. By the year 2025, rice production needs toe enhanced by ∼40% to keep pace with the ever-increasing globalopulation (Khush, 2004). A wide range of insects are known to

nfest and cause extensive damage to the rice crop resulting in >20%nnual yield losses (Brookes and Barfoot, 2003). To minimize theamages caused by diverse biotic factors and for sustainable foodroduction, it is essential to design custom-made varieties that canithstand the ravages of various insect pests. Three major sap-

ucking pests of rice, viz., brown planthopper (Nilaparvata lugens,

PH), green leafhopper (Nephotettix virescens, GLH) and white-acked planthopper (Sogatella furcifera, WBPH) are known to causeevere damage to rice plants (Dahal et al., 1997; Foissac et al.,000). These insects cause direct damage to rice plants by sucking

Abbreviations: BPH, brown planthopper; GLH, green leafhopper; WBPH, white-acked planthopper; PCR, polymerase chain reaction; GNA, Galanthus nivalis lectin;SAL, Allium sativum lectin.∗ Corresponding author. Tel.: +91 40 27098087; fax: +91 40 27096170.

E-mail address: rao [email protected] (K.V. Rao).

168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jbiotec.2011.01.021

the sap and also by plugging xylem and phloem with their styletsheaths during exploratory feeding. Continuous feeding by suck-ing insects results in the drying of crop leading to “hopper burn”.Besides causing severe physiological damage to the rice plant, hop-pers also act as vectors for rice tungro, grassy stunt and ragged stuntviruses (Mochida et al., 1979; Saxena and Khan, 1989). Transgenictechnology is known to offer unique opportunities for effectivemanagement of diverse pest populations prevailing in differentagro-climatic zones. In this context, to cope with the problem ofinsect adaptation, it would be ideal to pyramid various resistancegenes that code for multiple resistance factors against target organ-isms (Roush, 1998).

A number of approaches have been identified for introductionof multiple exotic genes into crop plants, such as sexual crossesbetween parental lines containing single transgenes; sequentialretransformation of crop plants; co-transformation with multipleplasmids carrying different genes; use of single plasmids carry-ing linked transgenes; adoption of gateway vector systems; use offusion proteins encoded by different genes; and expression of mul-

tiple proteins from a polyprotein (Halpin et al., 2001). However,each of these methods has its own advantages as well as limita-tions. Among these methods, conventional crosses made betweenselected parents are widely used for pyramiding of transgenes.The major advantages of this method are the avoidance of prob-

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ems associated with the cloning of large multi-gene cassettes andhe high copy number repeats responsible for transgene silencingHalpin, 2005).

Predictions based on models suggest that crop varietiesyramided with two dissimilar insecticidal genes show greaterotential to delay the development of insect resistance than theingle gene transgenics (Roush, 1998). Broccoli plants pyramidedith Bt cry1AC and cry1C genes exhibited increased resistance toiamondback moths, and significantly delayed the developmentf resistance in moths as compared to the plants expressing singleransgenes (Cao et al., 2002; Zhao et al., 2003). Similarly, pyramidedransgenic tobacco lines, expressing cry1Ac and cowpea trypsinnhibitor (CpT1) genes, delayed the development of resistance inelicoverpa armigera when compared to the plants carrying cry1Aclone (Zhao et al., 1999). Second generation transgenic cottons,iz., Bollgard 11 (cry1Ac + cry 2Ab) and Widestrike (cry1Ac + cry1F),xpressing different Bt endotoxins, have been developed to raisehe level of resistance against cotton bollworm (Gahan et al., 2005;ackson et al., 2003). Rice transformants stacked with gna, cry1Acnd cry2A genes exhibited higher levels of insect resistance com-ared to the transgenic plants expressing single genes (Maqboolt al., 2001).

Snowdrop (Galanthus nivalis) lectin gene (gna) has been suc-essfully introduced and expressed in diverse crop plants, such asice (Rao et al., 1998; Foissac et al., 2000; Nagadhara et al., 2003,004), wheat (Stoger et al., 1999), tobacco (Hilder et al., 1995) andotato (Down et al., 1996; Gatehouse et al., 1997; Couty et al.,001), to confer protection against different sucking pests. Sim-

larly, Allium sativum lectin encoding genes (asa and asal), whenxpressed in rice (Saha et al., 2006; Yarasi et al., 2008) and tobaccolants (Bandyopadhyay et al., 2001; Sadeghi et al., 2007), conveyedarked resistance against homopteran and lepidopteran pests.

n our earlier studies, stable transgenic lines, containing gna andsal genes, have been developed in high yielding varieties of rice.ransgenic plants expressing ASAL conferred maximum protectiongainst BPH and GLH followed by WBPH, while GNA-transgenicsroved most effective for WBPH followed by BPH and GLH insectsNagadhara et al., 2003, 2004; Yarasi et al., 2008).

In this investigation, two mannose-specific lectin encodingenes (asal and gna) were pyramided into the elite rice cultivarhaitanya and demonstrated the stable expression of both theransgenes. The pyramided rice lines, compared to the parentalransgenics, exhibited higher levels of resistance against the majorucking pests. This study represents first report on the pyramid-ng of two lectin genes into elite indica rice exhibiting enhancedesistance to BPH, GLH and WBPH insects.

. Materials and methods

.1. Generation of asal + gna pyramided rice lines

In the present study, stable homozygous transgenic rice lines T49T6 generation) and OU-1 (T9 generation) harbouring asal/gna genesave been used as parents (Nagadhara et al., 2003; Yarasi et al.,008). T49 plants, carrying the asal expression cassette, were useds the maternal parent and OU-1 plants, containing the gna expres-ion cassette, served as the male parent. Crosses were made insidehe containment green house at the Directorate of Rice ResearchDRR), Hyderabad, under controlled conditions. When 50% of theanicle emerged from the flag leaf, T49 plants were emasculated

anually, and pollen from the male (OU-1) parent were collected

nd dusted on the emasculated florets of female (T49) parent. All theollinated panicles were bagged to collect the F1 seed. The F1 seedsere germinated in pots to identify the plants containing both the

ransgenes.

hnology 152 (2011) 63–71

2.2. Polymerase chain reaction (PCR) analysis

Genomic DNA was isolated from the parents, F1, F2, F3 and F4plants using the method of McCouch et al. (1988). PCR analysiswas carried out employing the primers corresponding to the cod-ing region of genes asal (5′-ATG GGT CCT ACT ACT TCA TCT CCT-3′;5′-TCA AGC AGC ACC GGT GCC AAC CTT-3′) and gna (5′-GGA TCCGAC AAT ATT TTG TAC TCC G-3′; 5′-CCC GGG TCA TCC GGT GTGAGT TCC AGT AGC-3′). DNA extracted from the parents was usedas comparable controls. The PCR mixture (25 �l), containing tem-plate DNA (100 ng), primers (10 �M), buffer (1×), dNTPs (0.5 mM)and Taq DNA polymerase, was subjected to initial denaturation at94 ◦C for 5 min; followed by repeated denaturation (94 ◦C) for 45 s,annealing (62 ◦C) for 45 s, and elongation (72 ◦C) for 1 min for a totalof 35 cycles on PTC-200 Peltier Thermal Cycler; with final elonga-tion at 72 ◦C for 10 min. The amplified PCR products were subjectedto gel electrophoresis using 1% agarose gel.

2.3. Western blot analysis of pyramided lines

Samples of pyramided homozygous line P17-17, parents anduntransformed control leaf tissue were homogenized in 50 mMTris–HCl buffer (pH 9.0). The extract was centrifuged at 5000 × g for20 min at 4 ◦C, and the supernatant was collected. Protein samples(5 �g) were subjected to 15% SDS–PAGE. Following electrophore-sis, the separated proteins were transferred onto nitrocellulose N−

membrane by electro blotting. After protein transfer, the mem-brane was blocked by incubating in PBS solution containing 10%non fat dried milk and 0.1% Tween 20 for 2 h at room temperature.The membrane was probed with polyclonal rabbit anti-ASAL serum/polyclonal rabbit anti-GNA serum (1:10,000 dilution) and goatanti-rabbit IgG horse-radish peroxidase conjugate as secondaryantibody (1:10,000 dilution). The membrane was washed and ana-lyzed with saturated benzidine solution containing 20% ammoniumchloride and 0.1% H2O2.

2.4. ELISA analysis

Wells of the microtitre plate were coated with 1 �g of crudeprotein extracts of pyramided line P17-17 and kept for overnight at37 ◦C and at 4 ◦C for 1 h. The wells were washed thrice with 20 mMPBS containing 0.05% Tween 20 and were blocked with 10% non-fat dried milk for 2 h at 37 ◦C, subsequently washed six times withPBS-T. The primary antibody (1:10,000) was added to the wells andincubated for 2 h at 4 ◦C. After incubation, the wells were washedthrice with PBS and incubated with secondary antibody (1:10,000)for 1 h at room temperature. The plates were washed thrice withPBS and 0.001% TMB substrate in 0.05 M phosphate citrate bufferwas added along with 0.1% H2O2 and kept in dark for 10 min.The reaction was stopped by 1 N H2SO4 and the absorbance wasrecorded on ELISA reader at 450 nm.

2.5. Insect bioassays

In planta bioassays using BPH, GLH and WBPH insects were car-ried out on F2 and F3 pyramided rice lines, parental transgenic linesand untransformed control plants. The susceptible check TaichungNative 1 (TN-1), and resistant checks PTB33, Vikramarya and MO-1,for BPH, GLH and WBPH, respectively, were also used in bioas-says. All insect bioassays were carried out at the Directorate of RiceResearch (DRR) as described earlier (Nagadhara et al., 2003; Yarasi

et al., 2008). The degree/levels of resistance in mass screeningexperiments exhibited by pyramided rice lines, parental transgeniclines and controls were scored based on a scale of 0–9 as usedin the International rice testing programme (1980). To evaluatethe combined effects of ASAL and GNA on sucking pests, insect

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urvival, developmental and fecundity assays were performed onyramided rice line (P17-17), parents and control plants as describedNagadhara et al., 2003; Yarasi et al., 2008). For insect assays, 20PH/ 20 GLH/ 20 WBPH first instar nymphs were used for infest-

ng separate plants confined in insect proof cages, and mortalityf insects was monitored at 3 day intervals for 24 days. For fecun-ity assays, fourth instar male and female insects were confinedogether on each plant in a 1:1 ratio. Total number of nymphs pro-uced on plants was counted until no nymphs were found emergingrom eggs. All these experiments were repeated thrice using tenlants (replications). The mean, standard deviation and standardrror were calculated and subjected to one way ANOVA.

.6. Honeydew (liquid excreta) assay for estimation of feedingbility of insects

The extent of insect feeding was measured by semi-quantitativessay of the honeydew produced (Pathak et al., 1980). Whatmano.1 filter paper dipped in a solution of bromocresol green (2 mg/ml

n ethanol) was used for honeydew estimation. The filter paper waslaced at the base of each plant and covered with a plastic cup.n each plant five female adult insects of BPH/GLH/WBPH, pre-

tarved for 2 h, were released separately, and allowed to feed for4 h. GLH feeding was confined to the leaf blades by placing thelter paper at the base of leaves. Insect excreta (honeydew) reactith the bromocresol green on the filter paper leading to the devel-

pment of blue colored spots. The area of blue spots developedas measured using the millimeter graph paper and expressed inmm2 units (Nagadhara et al., 2003; Yarasi et al., 2008).

.7. Assessment of yield potential of pyramided lines, transgenicarents and control plants

Traits such as no. of panicles/plant, no. of seeds/panicle andeed yield/plant were evaluated in control, parental transgenicsnd pyramided lines subjected to insect infestation as well as underormal conditions. Three-week old plants were infested for 14 days

ndependently with 20 BPH/20 GLH/20 WBPH insects per plant asescribed above in insect bioassays using 10 plants for each insect.ater, survived plants of parental and pyramided lines were trans-erred to pots and allowed to grow up to maturity, and data wereecorded.

. Results

.1. Identification of F1 plants carrying asal and gna genes

Conventional crosses effected between homozygous T49 andU-1 transgenic Chaitanya lines produced 64 seeds. Out of3 viable plants subjected to PCR analysis using gene-specificrimers, five plants disclosed two expected bands correspondingo that of ∼540 bp of asal and ∼320 bp of gna (Supplementaryig. 1A).

.2. Inheritance of asal and gna genes in F1 and F2 plant progenies

To investigate the inheritance pattern of transgenes, selfed seedarvested from the PCR-positive F1 plants were grown to maturity

n the glass house. PCR analysis of F1 plant progenies (F2 plants)evealed dihybrid segregation of 9:3:3:1 for asal and gna in a nor-

al Mendelian fashion (Supplementary Fig. 1B and Table 1). Out

f 35 F2 plants derived from the P17 line, 18 plants were foundo contain gna and asal genes either in homo-/hemizygous con-ition, and 7 plants were found homo-/hemizygous for gna alonend 8 plants were homo-/hemizygous for asal alone. Whereas, the

hnology 152 (2011) 63–71 65

remaining two F2 plants proved to be azygous for both the trans-genes. The segregation data obtained from insect bioassays on F2plant progenies (F3 plants) corroborated the dihybrid segregationobserved in F2 plants (Supplementary Table 1). The mean insectsurvival rates on 18 F2 plant progenies carrying both the genesdisclosed 2.0 ± 0.6 to 3.0 ± 1.1 (BPH), 2.1 ± 1.0 to 3.8 ± 1.2 (GLH)and 1.2 ± 0.5 to 2.9 ± 0.9 (WBPH) insects/plant. Insect survival on7 progenies containing gna alone ranged from 5.6 ± 1.6 to 6.3 ± 1.0(BPH), 5.9 ± 2.0 to 6.2 ± 1.2 (GLH) and 1.9 ± 1.1 to 2.5 ± 1.2 (WBPH)insects/plant. Similarly, 8 progenies containing asal alone showed2.8 ± 1.2 to 3.8 ± 1.3 (BPH), 2.1 ± 0.8 to 3.5 ± 0.9 (GLH) and 5.5 ± 1.6to 6.0 ± 1.0 (WBPH) insects/plant. Whereas, survival of insects ontwo azygous plant progenies varied from 13.1 ± 1.7 to 13.9 ± 1.3(BPH), 13.9 ± 1.8 to 14.0 ± 1.3 (GLH) and 12.5 ± 1.9 to 12.9 ± 1.1(WBPH) insects/plant.

Insect fecundity on 18 F2 plant progenies containing asal andgna genes revealed a mean number of 89.0 ± 1.9 to 98.0 ± 3.0(BPH), 94.0 ± 1.4 to 104.0 ± 3.9 (GLH) and 34.0 ± 2.9 to 58.0 ± 1.4(WBPH) nymphs/plant. Whereas, a mean number of 156.0 ± 3.3 to169.0 ± 2.3 (BPH), 166.0 ± 4.6 to 173.0 ± 5.1 (GLH) and 53.0 ± 1.7to 63.0 ± 3.2 (WBPH) nymphs/plant were recorded on 7 proge-nies containing gna alone. Similarly, on 8 progenies containing asalalone, a mean number of 92.0 ± 2.5 to 110.0 ± 3.4 (BPH), 103.0 ± 2.1to 119.0 ± 3.0 (GLH) and 83.0 ± 1.6 to 103.0 ± 2.1 (WBPH)nymphs/plant were noticed. On the other hand, a mean num-ber of 351.0 ± 6.3 to 360.0 ± 3.6 (BPH), 362.0 ± 6.6 to 374.0 ± 2.9(GLH) and 350.0 ± 6.5 to 353.0 ± 8.6 (WBPH) nymphs/plant wereobserved on two azygous progenies (Supplementary Table 1).

3.3. Identification and isolation of homozygous pyramided ricelines in F2 plant progenies of P17 line

Selfed seed were collected independently from 18 F2 plantsof P17 containing both asal and gna genes for obtaining F2 plantprogenies. Nine F2 plant progenies were employed to identifyhomozygous lines for gna and asal based on PCR and insect bioas-says (Table 1). From each progeny, 32 plants were used for PCRanalysis to ascertain the presence of both the transgenes. PCRanalyses revealed that all the plants belonging to P17-5 and P17-17progenies contained two expected bands corresponding to that ofasal and gna genes. However, about 75% of the plants from P17-4,P17-12, P17-26 and P17-28 progenies disclosed asal and gna bands,while remaining 25% plants showed single band representing eitherasal or gna (3:1ratio). On the other hand, three progenies, P17-15,P17-19 and P17-21, showed a dihybrid ratio of 9:3:3:1 for asal and gnagenes (Table 1). Progenies derived from the nine F2 plants were alsosubjected to insect bioassays. Based on the resistance/susceptibilityscore (on a 0–9 scale) and number of insects survived/plant, all theplants belonging to P17-5 and P17-17 homozygous lines exhibitedhigher-level resistance to the sucking pests when compared to theremaining progenies. Plants from P17-4, P17-12, P17-26 and P17-28 pro-genies showed monogenic (3:1) segregation for gna or asal, whileP17-15, P17-19 and P17-21 progenies disclosed digenic (9:3:3:1) segre-gation for asal and gna based on the resistance/susceptibility score(Table 1).

3.4. Expression profiles of ASAL and GNA proteins in thepyramided P17-17 line

Western blot analysis of leaf extracts from P17-17 homozy-

gous line, after treatment with anti-ASAL and anti-GNA antibodies,revealed the presence of two polypeptides of ∼12 kDa correspond-ing to the ASAL and GNA proteins of parental transgenics (Fig. 1).From the total soluble leaf proteins of pyramided plants, 1.42% ofASAL and 0.28% of GNA proteins have been detected.

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Table 1Inheritance pattern of transgenes in F2 plant progenies of P17 pyramided line.

F2 progenies (F3 plants) No. of plants analyzed PCR analysis and insect bioassays Segregation ratio (asal/gna) �2

+ve asal + gna +ve asal/−ve gna +ve gna/−ve asal −ve asal + gna

P17-4 32 25R 7R 0 0 3:1 0.166P17-5* 32 32R 0 0 0 – –P17-12 32 24R 0 8R 0 3:1 0P17-15 32 17R 5R 7R 3S 9:3:3:1 0.882P17-17* 32 32R 0 0 0 – –P17-19 32 16R 7R 7R 2S 9:3:3:1 0.541P17-21 32 15R 8R 7R 2S 9:3:3:1 1.320P17-26 32 24R 8R 0 0 3:1 0P17-28 32 25R 0 7R 0 3:1 0.166

+ve, presence of gene; −ve, absence of gene; P17-5* and P17-17* progenies homozygous fodetermined based on the score on a 0–9 scale, besides insect survival and fecundity rates.values were significant at p value 0.05.

Fig. 1. Western blot analysis of leaf extracts of P17-17 pyramided line, parentaltransgenic lines and control plants. Lane 1: protein extract (5 �g) of untransformedcontrol plant treated with ASAL and GNA antibodies; lane 2: protein extract (5 �g)of T49 (asal) transgenic plant treated with ASAL antibodies; lane 3: protein extract(5 �g) of OU-1 (gna) transgenic plant treated with GNA antibodies; lane 4: proteinextract (5 �g) of P17-17 pyramided plant treated with ASAL antibodies; and lane 5:protein extract (5 �g) of P17-17 pyramided plant treated with GNA antibodies.

Fig. 2. Brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthoppecontrol plants. (A) 20-Day old pyramided lines along with parents and respective controls icontrols infested with GLH; (C) 20-day old pyramided lines along with parents and respeccomplete damage; rows 5 and 6: pyramided rice lines (P17-5, P17-17) showing resistance aB: resistant check (var. Vikramarya) for GLH; row 4 C: resistant check (var. MO-1) for WBand WBPH; row 3: OU-1 (gna) transgenic parental line showing resistance against BPH,Photographs were taken after 14 days of infestation.

r both the genes; R (resistance) and S (susceptibility) to BPH, GLH and WBPH wereThe inheritance pattern of transgenes showed dihybrid ratio and the calculated �2

3.5. Conjoint effects of ASAL and GNA on the survival of BPH, GLHand WBPH insects

In planta insect bioassays were performed to test the insectici-dal activity of F3 and F4 plants of P17-5 and P17-17 homozygous linesalong with the parental transgenics and untransformed controlplants. Twenty-day-old plants expressing ASAL and GNA revealedgreater resistance towards BPH, GLH and WBPH insects with min-imal plant damage as compared to the parents (Fig. 2). Pyramidedplants showed a score of ∼1.0 on a scale of 0–9 for resistanceto BPH, GLH and WBPH, and were better than the BPH-resistantPTB33, GLH-resistant Vikramarya and WBPH-resistant MO-1 vari-

eties (1–2 score). Whereas, the susceptible var. TN-1 and theuntransformed control plants showed complete damage (9 score on0–9 scale) caused by the hoppers (Fig. 2). Furthermore, the F3 plantprogenies of P17-17 were subjected to insect bioassays for assessinginsect mortality, developmental delay, fecundity and their feed-

r (WBPH) bioassays on F3 pyramided transgenic rice lines along with parents andnfested with BPH; (B) 20-day old pyramided lines along with parents and respectivetive controls infested with WBPH; rows 1 and 8: control (var. TN-1) plants showinggainst BPH, GLH and WBPH; row 4 A: resistant check (var. PTB33) for BPH; row 4PH; row 2: T49 (asal) transgenic parental line showing resistance against BPH, GLHGLH and WBPH; row 7: untransformed Chaitanya control plants showing damage.

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Y. Bharathi et al. / Journal of Biotechnology 152 (2011) 63–71 67

Fig. 3. Survival of BPH, GLH and WBPH insects on pyramided rice lines expressing ASAL and GNA along with parents and control plants. Twenty 1st – instar nymphs ofBPH (A), GLH (B) and WBPH (C) were released on each plant on day 0. Parental transgenic lines T49 and OU-1 are depicted by triangle and square, respectively; pyramidedrice line P17-17 is depicted by diamond; and control Chaitanya (CC) is depicted by circle. Bioassays were carried out on 10 plants each sampled from pyramided, parentalt n valuv

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ransgenic lines and controls. Bars indicate mean ± SE. Differences between the meas. pyramided lines were significant at p value <0.0001 (one way ANOVA).

ng behaviour. The survival rates of BPH, GLH and WBPH nymphsed on pyramided plants were reduced by ∼86%, ∼85% and ∼90%,espectively, compared to that of T49 (asal) (∼77%, ∼80%, ∼60%),U-1 (gna) (∼57%, ∼56%, ∼83%) parents and susceptible controllants (Fig. 3). During the entire 24-day bioassay period, the sur-ival of BPH fed on pyramided plants was reduced to 2.0 ± 0.6nsects/plant compared to 3.2 ± 1.0 insects on T49, 6.0 ± 1.0 insectsn OU-1 and 13.9 ± 1.3 insects observed on the untransformedontrol plants (Fig. 3A). Likewise, the survival of GLH on pyra-ided plants decreased to 2.1 ± 1.0 insects/plant, while it was

.8 ± 1.4 insects on T49 (asal), 6.1 ± 0.9 insects on OU-1 (gna) and

4.5 ± 1.3 insects on control plants (Fig. 3B). The survival rate ofBPH insects fed on pyramided plants was reduced to 1.2 ± 0.5

nsects/plant compared to 5.0 ± 1.0 insects on T49 (asal), 2.1 ± 1.1nsects on OU-1 (gna) and 12.4 ± 1.1 insects on the control plantsFig. 3C).

ig. 4. Effect of pyramided rice lines expressing ASAL and GNA proteins on the developmnd WBPH (C) were released on pyramided rice lines, parental transgenic plants and untratage and number of nymphs which remained immature because of delayed developmenraph. Bioassays were carried out on 10 plants each sampled from pyramided line, pareifferences between the mean values of control vs. transgenic parents and pyramided lNOVA), respectively. CC: Chaitanya control plants; T49: transgenic (asal) rice line; OU-1:

es of control vs. transgenic parents and pyramided lines and mean values of parents

3.6. Entomotoxic effects of P17-17 pyramided rice line on threesap-sucking pests

First instar nymphs of BPH, GLH and WBPH insects were releasedonto P17-17 F4 plants, parental transgenic lines and control plants,and were monitored for the effect of ASAL and GNA on their growthand development. In comparison with the insects on control plants,insects fed on pyramided plants revealed ∼12–14 days delay forreaching adulthood, while insects fed on parental transgenic plantsshowed a delay of ∼10–12 days for reaching adult stage. AmongBPH survivors, ∼20% insects could reach adult stage on pyramided

plants compared to ∼30% on T49 (asal), ∼38% on OU-1 (gna) and∼80% adults observed on control plants (Fig. 4A). Among GLH sur-vivors, only ∼15% insects could reach the adult stage on pyramidedplants compared to ∼18% on T49 (asal), ∼36% on OU-1 (gna), while∼90% adults were found on control plants (Fig. 4B). In case of WBPH

ent of BPH, GLH and WBPH insects. Twenty 1st – instar nymphs of BPH (A), GLH (B)nsformed controls on day 0. After 24 days, number of nymphs which reached adultt on pyramided lines, single gene transgenic lines and controls were plotted on thental transgenics and control. Bars indicate mean ± SE. * and ** indicate significantines and mean values of parents vs. pyramided lines at p value <0.0001 (one waytransgenic (gna) rice line; P17-17: pyramided rice line.

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Fig. 5. Impact of pyramided rice lines expressing ASAL and GNA on the fecundity of BPH, GLH and WBPH insects. Total number of nymphs produced by a pair of adult BPH( nd wep ndicata 001 (r

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A), GLH (B) and WBPH (C) insects on controls and transgenic plants were counted ayramided line, parental transgenics and control. Bars indicate mean ± SE. * and ** ind pyramided lines and mean values of parents vs. pyramided lines at p value <0.0ice line; OU-1: transgenic (gna) rice line; P17-17: pyramided rice line.

urvivors, ∼10% insects could develop into adults on pyramidedlants compared to ∼23% on T49 (asal), ∼20% on OU-1 (gna) and73% adults on control plants (Fig. 4C).

Concomitant effects of ASAL and GNA on the fecundity ofPH, GLH and WBPH were assessed by counting the total num-er of nymphs produced by insects fed on pyramided plants inomparison with the parental transgenics and control plants. Aean number of 89.0 ± 2.0 BPH nymphs/plant were noticed on

yramided plants compared to 102.0 ± 2.6 nymphs on T49 (asal),65.0 ± 3.2 nymphs on OU-1 (gna) and 360.0 ± 5.9 nymphs onntransformed control plants (Fig. 5A). For GLH, a mean number of4.0 ± 1.4 nymphs/plant were observed on pyramided plants com-ared with 113.0 ± 2.1 nymphs on T49 (asal), 170.0 ± 5.1 nymphs onU-1 (gna) and 375.0 ± 5.7 nymphs on control plants (Fig. 5B). Inase of WBPH, 34.0 ± 2.9 nymphs/plant were scored on pyramidedlants compared to 98.0 ± 5.7 nymphs on T49 (asal), 50.0 ± 4.6ymphs on OU-1 (gna) and 350.0 ± 10.3 nymphs on the susceptibleontrol plants (Fig. 5C).

.7. Impact of P17-17 pyramided line on the feeding behaviour ofPH, GLH and WBPH insects

The feeding ability of insects was evaluated based on the amountf honeydew excreted by the adults. After a lapse of 24 h of feed-

ig. 6. Effect of pyramided transgenic rice lines expressing ASAL and GNA on the feedinoneydew excretion by BPH insects; (B) semi-quantitative estimation of honeydew excrBPH insects. Bars indicate mean ± SE. * and ** indicate significant differences between

alues of parents vs. pyramided lines at p value <0.0001 and =0.0005, respectively (oneransgenic (gna) rice line; P17-17: pyramided rice line.

re plotted on the graph. Bioassays were carried out on 10 plants each sampled frome significant differences between the mean values of control vs. transgenic parentsone way ANOVA), respectively. CC: Chaitanya control plants; T49: transgenic (asal)

ing on pyramided plants/parents/untransformed control plants,the number of honeydew units (blue spots) developed on thebromocresol green paper were counted to estimate the feed-ing capacity of insects. A mean number of 10.1 ± 0.8 honeydewunits/plant were observed for BPH fed on pyramided plants com-pared to 11.2 ± 1.9 honeydew units on T49 (asal), 16.0 ± 2.3 unitson OU-1 (gna) and 157.0 ± 14.0 units on control plants (Fig. 6A). ForGLH, 11.0 ± 0.8 honeydew units/plant were observed on pyramidedplants compared with 14.2 ± 0.9 units on T49 (asal), 21.0 ± 1.2 unitson OU-1 (gna) and 165.0 ± 2.7 units on control plants (Fig. 6B). Sim-ilarly, 9.2 ± 1.6 honeydew units/plant were observed for WBPH fedon pyramided plants compared to 21.0 ± 1.8 units on T49 (asal),15.0 ± 1.4 units on OU-1 (gna) and 167.0 ± 2.9 units on controlplants (Fig. 6C).

3.8. Evaluation of pyramided lines, parental transgenics andcontrol plants under infested and normal conditions

Under uninfested conditions, pyramided lines harbouring asal

and gna lectin genes were found healthy and were on a parwith the untransformed control and parental transgenic lines formean no. of panicles/plant, no. of seeds/panicle and seed yield(g)/plant. However, under infested conditions, the yield potentialof pyramided lines was found superior to that of parental trans-

g behaviour of BPH, GLH and WBPH insects. (A) Semi-quantitative estimation ofetion by GLH insects; (C) semi-quantitative estimation of honeydew excretion bythe mean values of control vs. transgenic parents and pyramided lines and meanway ANOVA). CC: Chaitanya control plants; T49: transgenic (asal) rice line; OU-1:

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Y. Bharathi et al. / Journal of Biotechnology 152 (2011) 63–71 69

Table 2Seed yield potential of pyramided lines, transgenic parents and control plants subjected to BPH, GLH and WBPH infestation.

Lines Uninfested plants Infested plantsa

Mean no. ofpanicles/plant

Mean no. ofseeds/panicle

Seed yield(g)/plant

Mean no. ofpanicles/plant

Mean no. ofseeds/panicle

Seed yield(g)/plant

Chaitanya (control) 7.8 ± 0.51 189 ± 5.55 19.81 ± 0.73 – – –OU-1 (gna) 6.7 ± 0.62 179 ± 4.36 18.72 ± 0.31 6.4 ± 0.32 168 ± 3.14 16.21 ± 5.21T49 (asal) 6.9 ± 0.16 180 ± 5.21 18.82 ± 0.71 6.5 ± 0.24 171 ± 4.32 16.67 ± 6.34Pyramided line (P17-5) (asal + gna) 7.1 ± 0.22 186 ± 5.23 18.98 ± 0.35 6.8 ± 0.31 178 ± 3.25 18.84 ± 1.25Pyramided line (P17-17) (asal + gna) 7.2 ± 0.31 186 ± 6.34 18.87 ± 0.45 6.9 ± 0.14 179 ± 4.02 18.82 ± 2.24

(–) Infested control plants were severely damaged and could not survive.a ted to

a Bars in

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Three-week-old control, pyramided and parental transgenic plants were subjecnd pyramided lines were transferred to pots and allowed to grow up to maturity.

enic lines and was almost similar to uninfested control plantsTable 2).

. Discussion

Prevention of insect populations from developing resistance toultivars poses a major challenge for achieving sustainable croproductivity. For more than a decade, Bt transgenics showingesistance towards various insects belonging to lepidopteran andoleopteran orders, were successfully deployed. However, theseransgenics could not confer any resistance to sap-sucking pestselonging to homopteran group of insects. To analyze and evaluatehe combined effects of two different lectin genes (asal and gna) onap-sucking insects, sexual crosses were made between homozy-ous transgenic rice plants expressing ASAL or GNA proteins. Theresence of ∼540 bp amplified band with asal gene-specific primersnd ∼320 bp band with gna-specific primers in the F1 pyramidedines established the co-existence of both the lectin genes in F1 linesSupplementary Fig. 1A). The segregation pattern of transgenes in2 plants conformed to the dihybrid 9:3:3:1 ratio for asal and gnaenes owing to random assortment, indicating their location atndependent sites in the rice genome (Supplementary Table 1 andig. 1B). Further, in planta insect bioassays on F2 plant progeniesevealed reduced fecundity and survival of BPH, GLH and WBPHnsects (Supplementary Table 1), demonstrating their increasedesistance towards sucking pests. Large scale usage of a single insec-icidal transgene in diverse crops has been found to promote insectesistance in otherwise susceptible populations (Roush, 1998; Ferrend Van Rie, 2002; Tabashnik et al., 2008). Different strategies,uch as the nature of gene constructs and their expression profiles,ifferent combinations of multiple transgenes, fusion constructsetween the domains of toxin and non-toxin chains, adoption ofarious deployment tactics to discourage the development of resis-ant insect populations, etc. have been proposed to ensure theong-term usefulness of transgenic crops expressing various insec-icidal genes (Maqbool et al., 2001; Naimov et al., 2003; Mehlo et al.,005; Manyangarirwa et al., 2006).

Plants belonging to P17-5 and P17-17 progenies homozygous foroth the transgenes (Table 1), when subjected to insect mass-creening bioassays, exhibited greater resistance (∼1 score on a–9 scale), suggesting that ASAL in conjunction with GNA affordnhanced protection against sap-sucking insects (Fig. 2). Fur-hermore, insect survival on homozygous plants was drasticallyeduced to ∼1–2 insects/plant, besides delayed moulting and pro-onged (by ∼5 days) life cycle as compared to the insects fed on

arental lines (Figs. 3 and 4). The higher entomotoxic effects dis-losed by pyramided lines on major sucking insects are attributableo the combined insecticidal effects of ASAL and GNA proteins. Con-ersely, rice lines pyramided with gna, cry1Ac and cry2A proved toe less toxic to BPH nymphs as compared to the plants express-

BPH, GLH and WBPH infestation for 14 days as described in Fig. 3. Later, parentaldicate mean ± SE.

ing GNA alone (Maqbool et al., 2001). Earlier, it was reported thatover-expression of ASAL/GNA in transgenic rice lines decreasedthe survival of BPH by ∼36–74% (Yarasi et al., 2008; Saha et al.,2006; Ramesh et al., 2004; Nagadhara et al., 2003; Rao et al., 1998).Similarly, the survival of GLH was declined by ∼49–53% on GNAtransgenics (Foissac et al., 2000; Nagadhara et al., 2003; Rameshet al., 2004), and by ∼32–79% on ASAL expressing rice plants (Sahaet al., 2006; Yarasi et al., 2008).

Insect bioassays conducted on pyramided lines showed anotable reduction in the nymphal production of BPH (∼76%), GLH(∼75%) and WBPH (∼90%), compared to T49 and OU-1 parents(Fig. 5), implying decreased fecundity of insects fed on pyra-mided plants. In earlier studies, nymphal production of BPH andGLH was reduced by ∼59% and ∼70% (Saha et al., 2006) andby ∼68% and ∼73% (Yarasi et al., 2008), respectively, when fedon ASAL-expressing transgenic rice lines. Furthermore, decreaseswere observed in the honeydew production of BPH (∼93%), GLH(∼93%) and WBPH (∼95%) fed on pyramided rice lines comparedto ∼93% (BPH), ∼91% (GLH) and ∼87% (WBPH) on ASAL lines, and∼90% (BPH), ∼87% (GLH) and ∼91% (WBPH) on GNA plants (Fig. 6),implicating the enhanced effectiveness of pyramided lectins onthe feeding capacity of insects. The overall results on insect bioas-says amply indicate that the presence of ASAL in conjunction withGNA prove more toxic to the sucking insects when compared tosingle proteins. Earlier, it was reported that the mannose-specificGNA and ASAL specifically bind to the luminal surface of themidgut epithelial cells of sap-sucking pests (Bandyopadhyay et al.,2001; Du et al., 2000; Powell et al., 1998). The increased ento-motoxic effects and enhanced insect resistance of the pyramidedlines may be attributed to the differential binding affinities ofASAL and GNA to the receptor proteins at multiple sites on thegut-epithelial cells of insects. Therefore, development of trans-genics bestowed with different combinations of lectin proteinsmight delay the rate of pest adaptation, thereby contributing todurable and broad-based resistance. Plants pyramided with twodistinct toxin genes, deployed either sequentially or in mosaics,exhibited delayed development of insect resistance more effec-tively as compared to the plants with single-toxin genes (Roush,1998; Zhao et al., 2003). Bt toxins Cry1Ac and Cry2Ab, withdifferent binding sites in the larval midgut cells, proved moreeffective in delaying the evolution of insect resistance, presum-ably because insects cannot evolve resistance, so easily, againstboth the toxins as it requires the incidence of simultaneous, inde-pendent mutations in receptor encoding genes (Jackson et al.,2003).

The pyramided lines showed stable and consistent resistanceagainst BPH, GLH and WBPH insects up to F6 generation, imply-ing no sign of transgene silencing. The expression levels of ASAL(1.42%) and GNA (0.28%) proteins in the pyramided lines werefound similar to that of parental transgenics [T49 (ASAL) = 1.45%;

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.U-1(GNA) = 0.30%], suggesting the absence of transgene silencing.ost probably, the single copy nature of transgenes in the parental

ines, low (∼34%) sequence homology between asal and gnaenes and their control under different (CaMV35S/RSs1) promot-rs (Nagadhara et al., 2003; Yarasi et al., 2008), have contributedo the prevention of gene silencing in the pyramided rice lines.he pyramided lines resembled their transgenic parents for var-ous morphological characters and exhibited higher seed yieldotential than that of parental lines under infested conditionsTable 2), owing to the enhanced protection conferred by ASAL andNA.

In planta insect bioassays, reported herein, demonstrate thatyramided (asal + gna) rice lines are more effective in reducing

nsect survival, fecundity, feeding ability and development of threeap-sucking pests. The increased resistance imparted by the pyra-ided lines against these insects is attributable to the differential

inding affinities of ASAL and GNA proteins to the gut-epithelialells of insects. This study represents first report of its kind onhe pyramiding of two plant lectin genes into elite indica ricexhibiting increased resistance against major sucking pests. Thexotic pyramided lines, fortified with enhanced anti-feedant andnti-metabolic effects against sucking pests, appear promising forommercial cultivation in the hopper-prone areas, besides servings a novel genetic resource in recombination breeding aimed aturable resistance.

cknowledgements

We extend our thanks to the Department of Biotechnology, Gov-rnment of India (New Delhi), and Swarna Bharat Biotechnics Pvt.td. (Hyderabad), for providing financial assistance. YB and SVKre thankful to the Council of Scientific and Industrial Research,overnment of India (New Delhi) for the award of Research Fellow-hips. We thank G. Ashok Reddy and K. Ramesh of the Directoratef Rice Research (Hyderabad) for their technical help. Authors arerateful to Prof. T. Papi Reddy of the Department of Genetics, Osma-ia University, for his helpful suggestions and for improving theanuscript.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.jbiotec.2011.01.021.

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