Plants transformed with single Bt gene are liable to develop insect resistance and this has already beenreported in a number of studies carried out around the world where Bt cotton was cultivated on commer-cial scale. Later, it was envisaged to transform plants with more than one Bt genes in order to combat withresistant larvae. This approach seems valid as various Bt genes possess different binding domains whichcould delay the likely hazards of insect resistance against a particular Bt toxin. But it is difficult underfield conditions to develop homozygous plants expressing all Bt genes equally after many generationswithout undergoing recombination effects. A number of researches claiming to transform plants fromthree to seven transgenes in a single plant were reported during the last decade but none has yet appliedfor patent of homozygous transgenic lines. A better strategy might be to use hybrid-Bt gene(s) modifiedfor improved lectin-binding domains to boost Bt receptor sites in insect midgut. These recombinant-Bt gene(s) would express different lectin domains in a single polypeptide and it is relatively easy todevelop homozygous transgenic lines under field conditions. Enhanced chloroplast-localized expressionof hybrid-Bt gene would leave no room for insects to develop resistance. We devised and successfullyapplied this strategy in cotton (Gossypium hirsutum) and data up to T3 generation showed that our trans-genic cotton plants were displaying enhanced chloroplast-targeted Cry1Ac-RB expression. Laboratory
and field bioassays gave promising results against American bollworm (Heliothis armigera), pink boll-worm (Pictinophora scutigera) and fall armyworm (Spodoptera frugiperda) that otherwise, were reportedto have evolved resistance against Cry1Ac toxin. Elevated levels of hybrid-Bt toxin were confirmed byELISA of chloroplast-enriched protein samples extracted from leaves of transgenic cotton lines. While,localization of recombinant Cry1Ac-RB protein in chloroplast was established through confocal laserscanning microscopy.
. Background
Crops genetically engineered to produce Bt toxins for insectontrol may reduce the use of conventional insecticides, but insectesistance could limit the success of this technology (Cohen et al.,000). It was believed in the past that insects would not developesistance to Bt toxins. However, several insect larvae have beeneported which have evolved resistance against Bt crops. Examplesf resistant insects under field conditions are: Diamondback mothsPlutella xylostella] (Cao et al., 2002); cotton bollworm [Helicoverpaea] (Luttrell et al., 2004; Moar et al., 2008); fall armyworm
Spodoptera frugiperda] (Matten et al., 2008; Tabashnik, 2008; Want al., 2008; Liu et al., 2010; Storer et al., 2010); and pink bollworm
[Pectinophora gossypiella] (Tabashnik et al., 2002; Akhurst et al.,2003; Xu et al., 2009; Badiger et al., 2011).
Resistance management strategy generally relies profoundly ontheoretical assumptions by simulating insect population growthunder different conditions (Gould, 2003). It was then proposed thatBt genes should be ‘pyramided’ in order to delay the resistance levelof insects. However, it is a fact that stable expression or manipula-tion of multiple genes in plants is still difficult to achieve. Althougha small proportion of commercial genetically modified (GM) cropspresent ‘stacked’ or ‘pyramided’ traits, only a handful of productshave been developed by introducing three (Maqbool et al., 2001)or more novel genes (Monsanto and Dow AgroSciences, 2007). Onthe research front, a number of conventional and more innova-tive methods have been employed to introduce multiple genes
into plants, but all techniques suffer from certain drawbacks. Thesuccess of gene pyramiding depends upon several critical factors,including the number of genes to be transferred, distance betweenthe target genes and flanking markers, number of genotype selected
n each breeding generation, nature of germplasm etc. (Joshi andayak, 2010). Hence, ‘blind-stacking’ of genes in a single plant is notn ultimate perceptible solution to bring down insect resistance.
Chloroplast transformation technology is a useful strategy thatuarantees localized expression of transgenes in chloroplast buthis technique mainly relies on tissue culture and so far, its efficacys confined to Solanaceae family only (Lössl and Waheed, 2011).obacco is an ideal crop for chloroplast transformation because ofhe comfort to grow it through tissue culture and rapid responseo gene-gun transformation. Since callus generation is challengingn cotton (Hussain et al., 2004; Kamal, 2011); hence, chloroplastransformation seems impractical in cotton crop.
Chloroplast genome codes for only 10% proteins; while 90%roteins are imported from nucleus of the cell (Gatehouse, 2008).uclear proteins carry a specific signal peptide called transit pep-
ide (TP) as N-terminal extension to precursor proteins bound forhloroplast (Steiner et al., 2005) and alone can mediate the importf proteins into the organelle (Lee et al., 2006). Based on this fact,uclear transformation of cotton might be achieved tagging TP at-terminal of Bt gene to transport precursor protein inside chloro-last (Kim et al., 2009; Rawat et al., 2011). The non-toxic B-chainf ricin gene (RB) possesses three lectin domains (Venkatesh andambert, 1997) and can facilitate Bt protein as fusion-partner byroviding three extra binding sites to Bt in insect midgut. Cry1Acnd RB have different binding sites in the larval midgut and are con-idered to be a good combination to delay the evolution of insectesistance (Gatehouse, 2008).
New strategies to circumvent the onset of insect resistancenclude: the expression of multiple Bt toxins at high dosesGryspeirt and Grégoire, 2012), fusing Bt toxins together (Naimovt al., 2003), or Bt toxin tagged with any non-Bt gene having lectinapability to boost the binding domains of Bt in insect midgutMehlo et al., 2005). These approaches can minimize the insectesistance requiring the unlikely acquisition of multiple simulta-eous mutations. Moreover the critical factors, as mentioned by
oshi and Nayak (2010) for the accomplishment of successful geneyramiding to attain stable inheritance are also fulfilled.
Thus, it may be concluded that the targeted larvae of commer-ial crops, including cotton, are likely to develop partial resistancegainst single Bt toxin. Therefore, new strategies should be adaptedo eradicate resistant pests to reduce the yield losses and to improvehe quantity and quality of cotton fiber. In the present investigation,n effort has been made to achieve chloroplast-targeted expressionf recombinant Bt-RB through nuclear transformation of cotton inrder to combat with any likely resistance in pests against Bt tox-ns. Use of the TP sequence for chloroplast-targeting amplified theecombinant cry1Ac-RB transcript and protein levels vividly. Thehloroplast-targeted expression of cry1Ac-RB conferred high levelf resistance against the lepidopteron pests with known resistance.g. fall armyworm and pink bollworm under field conditions.
. Materials and methods
.1. Construction of expression vectors and cotton transformation
The CaM35S-Cry1Ac-NOS was excised from its source plasmid,k2Ac (Rashid et al., 2008), by digestion with ClaI and HindIIInd was sub-cloned into an intermediate vector. This interme-iate recombinant plasmid was digested with NcoI, allowing theub-cloning of TP, previously amplified from Petunia EPSPS gene.irectional cloning of TP with Cry1Ac was confirmed by PCR using
rientation primers. Thus, a second intermediate plasmid vectorPCry was generated in which Bt gene was tagged to the TP at-terminal. This second plasmid was digested with XhoI to lig-te RB, previously amplified from Ricinus communis, at C-terminal
nology 166 (2013) 88– 96 89
of Cry1Ac in such a way that stop codon of Cry1Ac was movedtoward NOS, bringing the Bt- and RB-coding regions in-frame uponre-ligation. The directional fusion of RB with Cry1Ac in third recom-binant plasmid was validated through orientation PCR as well asrestriction digestion with DraI enzyme. This third recombinantplasmid was then digested with ClaI and HindIII, excising the fusioncassette CaM35S-TP-Cry1Ac-RB-NOS for directional cloning intothe plant expression vector pBI121 (name given pKian-1). Paral-lel digestion of pk2Ac plasmid with ClaI and HindIII allowed thecloning of unmodified truncated cry1Ac gene into pBI121 whichwas used as control (name given pKian-0).
Agrobacterium-mediated transformation of cotton (G. hirsutum)local cultivar MNH-786 was carried out as described (Singh et al.,2009). The germinating cotton embryos were injured with ster-ile blade in each transformation process and were treated withAgrobacterium carrying pKian-1 or pKian-0 plasmid and nptIIselectable marker for kanamycin resistance. Afterwards, theseembryos were allowed to co-cultivate on MS-medium withoutantibiotic for 72 h and then were permitted to grow on MS sup-plemented with kanamycin (80 �g/mL) for discriminate selectionof transformants.
2.2. Southern blot
Genomic DNA was extracted from the fresh leaves of transgenicand control cotton plants by using method of Zhang et al. (2000).DNA (10 �g) was digested overnight with 30 units of XhoI at 37 ◦C.The digested DNA was resolved on 0.8% agarose gel, denatured,neutralized, and blotted onto nitrocellulose Hybond-N membrane(Amersham). The 565 bp probe for internal region of Cry1Ac waslabeled by using Biotin DNA Labeling Kit (Fermentas). Southern blotwas hybridized by following the standard procedure provided bythe manufacturer.
2.3. Immunoblot analysis
Immunoblot analysis of transgenic plants was carried outusing whole-leaf as well as chloroplast-enriched protein sam-ples. For extraction of whole-leaf crude protein, leaves werepowdered under liquid nitrogen, dispersed in 1× protein extrac-tion buffer (Wang et al., 2006), and centrifuged at 10,000 × gfor 10 min at 4 ◦C. Chloroplast-enriched protein samples wereprepared by using chloroplast isolation kit (Sigma–Aldrich) accord-ing to the manufacturer’s manual. Protein extracts were thenresolved on 6% SDS–PAGE and blotted onto nitrocellulose Hybond-C membrane (Amersham) using TransBlot semi-dry transferapparatus (Bio-Rad). Transblot was probed with anti-Cry1Ac anti-bodies (Abraxas). Bound anti-Cry1Ac Ab was spotted by usinghorseradish peroxidase-conjugated secondary IgG, and the chemi-luminescence of bound antibodies was spotted by using DAB asHRP-substrate.
2.4. ELISA
50 �L of Cry1Ab/Ac enzyme conjugate was added to each wellof ELISA plate (EnviroLogix). Chloroplast-enriched as well as cyto-solic protein extracts (5 �g) were added to respective wells andincubated at 37 ◦C for 1 h. Then, the contents of the wells wereshuddered vigorously into a sink. After three washings with 1×PBS, 100 �L of substrate was added to each well, mixed and incu-
bated for 30 min at 37 ◦C. After incubation, 100 �L of stop solution(1 N HCl) was added to each well and mixed thoroughly. This turnedthe well contents yellow. The plate was read on ELISA-reader (SPEC-TRAmax Plus 384). For ELISA analysis of subsequent generations,
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rude-leaf protein was extracted to detect recombinant transgenexpression in each transgenic cotton line.
.5. Immuno-histochemical staining and confocal microscopy
Small leaf fragments of transgenic plants were dehydrated,mbedded in paraffin wax and tissue sections 5–15 �m thickere cut using microtome (SORVALL MT-5000). Prior to immuno-istochemical staining, wax was removed and sections wereehydrated. Non-specific binding of primary Ab was blocked byncubating slides in blocking buffer (1% horse serum in PBS)or 30 minutes at room temperature. Sections were incubatedvernight at 2–8 ◦C with anti-Cry1Ac Ab (Abraxas) diluted at:100. Next day, the slides were incubated with FITC-conjugated
econdary IgG diluted 1:100 for 1 h at room temperature.lides were mounted with an anti-fade mounting media andere visualized under confocal laser scanning microscope
Zeiss LSM-510).
ig. 1. (a) Experimental plasmid vector construct pKian-1. Partial map of final plant expref Cry1Ac gene using XhoI site, thus stop codon of Cry1Ac was deliberately moved aftranslation of 98 kDa size polypeptide of recombinant Cry1Ac-RB. (b) Control vector consaMV35S-Cry1Ac-NOS cassette was excised from CEMB pk2Ac plasmid with ClaI and Hior Southern blot analysis of independent cotton transformants, genomic DNA (10 �g perobe specific for internal region of Cry1Ac (shown as ‘probe’ in map) was used to hybrid
nology 166 (2013) 88– 96
2.6. Insect bioassays under laboratory and field conditions
To check the insecticidal activity of transgenic plants, standardlaboratory bioassays with American bollworm (H. armigera) andfield bioassays with armyworm (S. frugiperda) and pink bollworm(P. scutigera) were performed separately. For insecticidal assayswith American bollworm, five replicates were set up for each trans-genic cotton line. Leaves were spread in petri plates on filter paperwet with 100 mg/L benzimidazole solution. One second-instar larvapre-fasted from 4 to 6 h, was released in each plate and the plateswere wrapped with parafilm. The experiment was carried out threetimes under the same conditions. The data on insect survival wasrecorded on daily basis from day 2 up to day 5. The efficacy ofBt-RB in successive generations of transgenic cotton lines was
further tested under field conditions with armyworm and pinkbollworm larvae. The seeds were sown in CEMB trial fields. Nopesticides active for lepidopteron were applied during the wholegrowth period in order to increase the chances of natural outburst of
ssion vector pBI-TP-Cry1Ac-RB (pKian-1). RB was ligated at the end of third domainer RB, bringing the coding regions of Cry1Ac and RB together; thus, allowing fortruct pKian-0. Partial map of control plant expression vector pBI-Cry1Ac (pKian-0).ndIII enzymes and cloned into pBI121 plasmid vector. (c) DNA hybridization blot.r lane) digested with XhoI that cuts once in transforming plasmid region. A 565 bpize with the DNA. Arrow heads indicate hybridization band.
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triplicate were loaded in Cry1Ab/Ac-coated wells of ELISA plate(EnviroLogix). Higher expression of transgene was seen in case ofchloroplast-enriched protein of K1, K2 and K3 transgenic cottonplants; while no chloroplast-localized expression of Cry1Ac was
S. Kiani et al. / Journal of B
rmyworm and pink bollworm. Three replicate block designs weresed with 20 plants per block. A total of 120 plants were scannedor any damage caused by armyworm and pink bollworm and theumbers of affected plants were then scored. The field bioassaysere carried out on three consecutive generations of transgenic
otton lines under almost the same conditions.
. Results
.1. Construction of plant expression vector
Owing to the limitation of restriction sites available, sub-vectorsere used for ligation of TP at N- and RB at C-terminal of Cry1Ac,
enerating pKian-1 plasmid vector carrying TP-Cry1Ac-RB cassetteFig. 1a), and control plasmid pKian-0 with unmodified Cry1Ac geneFig. 1b). Thus, a construct was assembled that encrypted the threeomains of Cry1Ac toxin, fused to the sequence of nontoxic B-chainf ricin possessing three galactose-binding domains. Consequently,
fortified effect of two active domains of Cry1Ac and three lectin-omains of RB, a total of five domains, was wilfully generated.herefore, the complete recombinant polypeptide was 880 aa inength with a predicted Mr of 98,052.
.2. Agrobacterium-mediated transformation of pKian-1 andKian-0
Germinating embryos of cotton (G. hirsutum) were trans-ormed with pKian-1 fusion construct in combination with nptIIs selectable marker using Agrobacterium-mediated shoot-apexethod of transformation. A total of 5000 embryos were trans-
ormed with pKian-1, resulting in the recovery of transgenic cotton
ines carrying the fusion construct. Further transformations werearried out with pKian-0 control construct to generate cotton plantsarrying unmodified Cry1Ac gene. All the transgenic plants werehenotypically normal and grew well in the field.
ig. 2. Transgene expression. Western blot of chloroplast-enriched and whole-leafrotein showed 98 kDa single polypeptide of Cry1Ac-RB in transgenic cotton plants.
n lane 1 contains prestained protein ladder, while trypsin-digested Cry1Ac was runs positive control in lane 2 and Cry1Ac extracted from K0 in lane 3. Lanes 4–7epict Cry1Ac-RB hybrid-protein (98 kDa) isolated from chloroplast of K1, K2 and3 transgenic cotton lines and lanes 8 to 11 represent Cry1Ac-RB extracted fromhole-leaves of K1, K2 and K3 lines.
nology 166 (2013) 88– 96 91
3.3. Transgene integration
Demonstrative Southern blot of genomic DNA extracted fromleaves of transformed cotton plants is shown in Fig. 1c. This dataconfirms that the fusion transgene incorporated into the cottongenome is present in the tested lines. Moreover, copy number ofeach transgene is also evident in Fig. 1c, illustrating one transgene inK2 and three transgene copy numbers in K1 transgenic line. While,K3 transgenic line as well as K0 controls each possess two copiesof the transgene.
3.4. Analysis of recombinant fusion protein
Expression of recombinant Cry1Ac-RB in transgenic cotton lineswas validated by Western blot (Fig. 2) using anti-Cry1Ac Absand ELISA (Fig. 3). For immunoassays, crude-leaf and chloroplast-enriched protein samples were used. Western blot illustrated thepresence of a single polypeptide of predicted 98 kDa size in thetransgenic cotton lines K1, K2 and K3 (Fig. 2). There was no bandequivalent to the size of truncated Cry1Ac (67 kDa), detected inthe transgenic plants but a band of this size was seen in case ofK0 controls expressing unmodified Cry1Ac (Fig. 2). For ELISA ofT0 plants, chloroplast-enriched and cytosolic protein samples in
Fig. 3. (a) Quantification of hybrid-crystal protein by ELISA. Quantification of Bt-RBexpression was made by using ELISA of chloroplast-enriched and cytosolic pro-tein samples extracted from leaves of T0 transgenic and control plants. K1, K2, andK3 transgenic plants expressing Bt-RB under TP showed elevated levels of hybrid-crystal toxin in chloroplast-enriched samples as were expected; while, cytosolicexpression of transgene was found negligible in these transgenic plants. On theother hand, K0 control plant expressing unmodified Cry1Ac without TP showedexpression of crystal toxin in cytosolic samples only. (b) Stable transgene expres-sion studies by ELISA. Stable expression of Bt-RB in transgenic lines of T1, T2, andT3 generations was verified by ELISA using crude-leaf protein extracts from leaves.For generating T2 and T3 cotton lines, T1 transgenic plants of K1, K2 and K3 yieldinghigher transgene expression were discriminately chosen.
92 S. Kiani et al. / Journal of Biotechnology 166 (2013) 88– 96
Fig. 4. Chloroplast-localized expression of hybrid-crystal protein by confocal microscopy. L.S. of leaf section was labeled with FITC-conjugated IgG and was observed underconfocal microscope (Zeiss LSM-510) for FITC and chloroplast-autofluorescence. I = Green FITC fluorescence at 488 nm excitation. II = Red chloroplast auto-fluorescence at580 nm. III = Merged yellow fluorescence by overlapping I & II. Fluorescence of FITC and red auto-fluorescence of chloroplast merged to produce yellow color which impliedthat Cry1Ac-RB was expressing inside chloroplasts of K1, K2 and K3. On the other hand after merging, FITC green fluorescence persisted in K0. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of the article.)
S. Kiani et al. / Journal of Biotechnology 166 (2013) 88– 96 93
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ig. 5. Insect bioassays with American bollworm. Effects of Cry1Ac-RB hybrid-prioassays of transgenic cotton plants. This representative bioassay of T2 generation
een in K0 control (Fig. 3a). While isolating 100% intact chloroplasts, portion of chloroplasts is broken and mixing does occur. Hence,ontaminations of small amounts of organellar proteins in cytosolicxtracts cannot be ruled out completely (Baginsky and Gruissem,004). Which is why, the ELISA results (Fig. 3a) for cytosolic proteinre not ‘zero’ in case of transgenic lines. ELISA of T1 to T3 gener-tions showed enhanced fusion-protein in transgenic cotton lines1, K2 and K3; while, low Bt expression was recorded in K0 control
ine (Fig. 3b).
.5. Chloroplast-localized expression
Confocal microscope images showed green fluorescence of FITCt 488 nm excitation and chloroplast red auto-fluorescence at80 nm excitation. When both images were merged, yellow fluo-escence appeared at the places where green and red fluorescenceverlapped in case of transgenic leaf sections. Therefore, yellowuorescence is the indicator of chloroplast localized expression oft-RB transgene and this is obvious in case of leaf samples of theepresentative K1, K2 and K3 transgenic cotton lines (Fig. 4A–C). Onhe other hand even after merging, the green fluorescence of FITCnd red auto-fluorescence of chloroplast persisted in K0 implyinghe Cry1Ac expression outside chloroplasts (Fig. 4E).
.6. Insect resistivity assays
Bioassays were carried out on fresh leaves from transgenicotton plants, and NT controls. The pKian-1 transgenic plantsemonstrated a high mortality rate of insect killing in bioassaysFigs. 5 and 6) than control plants. Hundred percent mortality ratesere observed with second-instar larvae of American bollworm;hile, pKian-0 controls exhibited ≈55% mortality rates in standard
aboratory bioassays with American bollworm (Fig. 5). On con-rary, all Heliothis larvae grew well on non-transgenic leaves, gainedeight and increased in size. In separate field bioassays, which were
arried out from T1 to T3 generations, the pKian-1 transgenic linesere found resistant against armyworm (S. frugiperda) and pink
ollworm (P. scutigera). In case of transgenic plants, average mortal-ty rates of ≈91% against armyworm (Fig. 6a) and ≈85% against pink
on average mortality of American bollworm (H. armigera) in standard laboratorys 100 percent mortality of 2nd instar Heliothis larvae in transgenic cotton leaves.
bollworm (Fig. 6b) were recorded. While, larvae of armyworm andpink bollworm grew equally well on control and NT plants (Fig. 6aand b).
4. Discussion
Tolerance against Bt toxins by the insects has been observedunder laboratory and field conditions (Moar et al., 2008; Tabashnik,2008); therefore, synchronized application of various crystalproteins seems to be a better option for the pest resistance man-agement. However, crystal proteins expressing under differentpromoters and transformed in different transformation events inone plant are likely to undergo field recombination effects and itwould be difficult to achieve homozygous transgenic plants (Joshiand Nayak, 2010). Therefore, in the present investigation, it wasenvisaged to increase the number of galactose-binding domainsof crystal gene Cry1Ac by tagging it with non-Bt B-chain of ricin(RB). The RB is known to have three lectin domains (Venkateshand Lambert, 1997). Thus, a recombinant toxin Bt-RB was gen-erated after fusion of Cry1Ac and RB that possessed five bindingdomains in insect midgut. Moreover, a transit peptide was taggedto the N-terminal of Cry1Ac to express Bt-RB toxin in chloroplasts.Chloroplast-targeted expression could evade any detrimental effectof Bt toxin on growth of cotton plants as indicated by Rawat et al.(2011) and also, an enhanced chloroplast-localized expression ofhybrid toxin was achieved.
Plasmid vector construct was generated after a series of cloningand ligation steps in sub-vectors because of the limitation of uniquerestriction sites in T-DNA region of pBI121; a significant issue incloning procedures of multiple gene stacking in a single plasmidwhich has been discussed by Ma et al. (2011). Transgenic cot-ton lines K1, K2 and K3 were raised after Agrobacterium-mediatednuclear transformation of cotton. Southern blot of transgeniclines revealed successful integration of fusion-transgene from oneto three copy numbers in the cotton genome (Fig. 1c); while
immunoblot analysis with the whole-leaf as well as chloroplast-enriched protein samples displayed a single hybrid-polypeptide ofpredicted size of 98 kDa (Fig. 2). Confocal microscopy of transgenicleaf sections revealed the expression of transgene in chloroplasts
94 S. Kiani et al. / Journal of Biotechnology 166 (2013) 88– 96
Fig. 6. Field bioassay with fall armyworm (S. frugiperda) and pink bollworm (P. scutigera) on transgenic and control cotton plants. Fall armyworm and pink bollworm toleranceagainst Cry1Ac has already been reported which can be seen in non-transgenic NT and control K0 plants expressing unmodified Cry1Ac under field conditions. Effects ofC wormo sion o
(sttstEf
ry1Ac-RB hybrid-protein and unmodified Cry1Ac on average survival of fall armyf the references to color in this figure legend, the reader is referred to the web ver
Fig. 4) validating the hypothesis that chloroplast-targeted expres-ion could be achieved in a recalcitrant crop after nuclearransformation. Higher levels of transgene expression in quantita-ive ELISA were prominent in case of chloroplast-enriched protein
amples of K1, K2 and K3 transgenic plants (Fig. 3a), which impliedhe localized expression of hybrid toxin in green tissues of cotton.LISA using whole-leaf protein samples in successive generationsrom T1 to T3 showed elevated levels of hybrid toxin in transgenic
and pink bollworm in transgenic cotton plants were recorded. (For interpretationf the article.)
cotton lines (Fig. 3b). Overall quantification studies of ELISArevealed 10–18-fold higher expression of hybrid toxin in K1, K2, K3transgenic lines which, was estimated to be 3% of the total solubleprotein.
The hypothesis that increased number of lectin domains inrecombinant crystal toxin would enhance its efficacy and thehybrid-toxin should kill the tolerant larvae, was tested in bioassays.Insect assays with American bollworm (H. armigera), armyworm
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S. frugiperda) and pink bollworm (P. scutigera) showed promis-ng results. The efficacy of hybrid-toxin protein was outstandingnd higher mortality rates were recorded for American bollworm,rmyworm and pink bollworm larvae. While, larvae of these insectsrew in size and caused considerable damage to the leaves and bollsf controls in artificial laboratory and field assays (Figs. 5 and 6).or K0 cotton plants expressing unmodified Cry1Ac (Fig. 6a and b),urvival of armyworm and pink bollworm larvae was decreasedy ca. 12% and 10%, respectively, compared with NT controls.owever, survival of armyworm and pink bollworm larvae was
urther decreased by ca. 77% and 68%, respectively, in cotton plantsxpressing the hybrid protein, compared with control plants.
Expression of the hybrid-Bt protein affectedly reduced the sur-ival of insects on transgenic cotton plants, with analogous declinesn the level of tissue damage (Figs. 5 and 6). Expression of thenmodified Cry1Ac toxin in K0 controls did not result in high larvalortality in laboratory as well as field assays. It has been reported
y Rajagopal et al. (2009), Li et al. (2007), and Gunning et al. (2005)hat armyworm and pink bollworm have shown resistance againstry1Ac toxin in many areas of the world where Bt cotton waseing cultivated. Our results of bioassays with armyworm and pinkollworm larvae show that, under field conditions, our transgenicotton lines K1, K2 and K3 are resistant against armyworm and pinkollworm. On the other hand, K0 (expressing unmodified Cry1Ac)nd NT controls were found equally susceptible to armyworm andink bollworm verifying the findings of Rajagopal, Li, and Gunning.
Killing of armyworm and pink bollworm in case of transgeniclants expressing hybrid protein was apparently due to RB whichrovides three extra lectin-binding sites to Cry1Ac rendering itore effective against the Bt-tolerant larvae. This is an important
ssue addressing the formulation of future Bt crops that shoulde sophisticated enough to combat with broader range of insectsithout incurring susceptibility because it is highly unlikely for an
nsect to develop mutations against all five binding sites of hybridrotein; in contrast to two active domains of Cry1Ac for whichrmyworm and pink bollworm already have developed resistanceJackson et al., 2003; Bates et al., 2005; Gahan et al., 2005). Thust may be suggested that the fusion-gene strategy would signifi-antly delay the evolution of insect resistance (Zhao et al., 2003;atehouse, 2008).
. Conclusion
Chloroplast-targeted expression of recombinant Bt-RB was suc-essfully accomplished in cotton (G. hirsutum) through nuclearransformation under chloroplast transit peptide. Thus, complexrocedures of chloroplast transformation were avoided whoseuccess is difficult to achieve in cotton crop. Higher levels ofusion-protein were documented in the present study. The hybridoxin was equipped with five lectin-binding domains renderinghe transgenic plants more toxic to a broader range of lepi-opteron pests e.g. American bollworm (H. armigera), armywormS. frugiperda) and pink bollworm (P. scutigera), that otherwise arenown to have developed resistance against single Bt toxin.
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