ORIGINAL PAPER

Transgenic maize lines expressing a cry1C* gene are resistantto insect pests

Dengxiang Du & Cunjuan Geng & Xiaobo Zhang & Zuxin Zhang & Yonglian Zheng &

Fangdong Zhang & Yongjun Lin & Fazhan Qiu

Published online: 19 October 2013# Springer Science+Business Media New York 2013

Abstract The lepidopteran Ostrinia furnacalis is one of themost serious pests of maize production. The Cry1C proteins aregroup of Bacillus thuringiensis (Bt) proteins that are toxic tothe intestine of insects. Overexpression of Cry1C protein hasled to increased resistance to lepidopteran pests in severalcrops. In the present study, the synthetic cry1C* gene thatwas previously tested in rice was introduced into maize Hi-IIgenotypes via biolistic gun-mediated transformation. A total ofnine independent putative callus were obtained and 87 trans-genic plants were positive with cry1C* according to polymer-ase chain reaction (PCR) analysis. Three highly insect-resistanttransgenic plants, ZmKc-2-3, ZmKc-3-2, and ZmKc-3-5, werefurther confirmed by PCR analysis, field assessment, and ge-nomic southern blotting in the T3 generations. Insect bioassayswere conducted in both the field and the laboratory, andshowed that progeny of the three transgenic lines were signif-icantly resistant to lepidopteran maize pests during the wholedevelopment and growth period. The stable integration andexpression of the cry1C* in the three transgenic plants’ prog-eny were confirmed by reverse transcription-PCR (RT-PCR)and enzyme-linked immuno sorbent assay (ELISA) methods.Hybrids were produced by crossing transgenic line ZmKc-2-3with the elite inbred line Zheng58. There was small variationamong the hybrids and backcross offspring, indicating thatthese cry1C* transgenic lines can be used to produce insect-resistant hybrids and served as insect-resistant sources for thedevelopment of Bt maize.

Keywords Maize .Bacillus thuringiensis (Bt) . Cry1C*protein . Insect resistance . Southern blot . ELISA

Abbreviations

Bt Bacillus thuringiensisELISA Enzyme-linked immunosorbent assayRT-PCR Reverse transcription-PCRPCR Polymerase chain reaction

Introduction

Maize (Zea mays L.) is one of the most important crops in theworld. Due to the uses of maize and maize-based products,demand for maize is increasing across the world, and predom-inantly in Asia. Maize cultivation is affected by both bioticand abiotic stresses. O. furnacalis is one of a group of majorlepidopteran pests of maize that cause >10 % of yield loss andhuge economic loss every year worldwide (He et al. 2003). Tolimit the harm of this pest, traditional maize productiondepended chiefly on agrochemicals; however, high prices,environmental pollution, poisoning of beneficial insects, andhealth hazards to farmers now make them unsuitable foreffective control of harmful insects.

One alternative to chemical pesticides is Bacillusthuringiensis (Bt). The insecticidal properties of Bt have longbeen recognized, and applied as an insecticide for severaldecades. Various strains of Bt are known to produce severaldifferent classes of insecticidal proteins, and more than 100different insecticidal genes have been identified to date. Con-siderable research efforts have been invested on the actionmode of the B. thuringiensis (Bt) δ-endotoxin which involvessolubilization of the insect midgut, proteolytic processing ofthe protoxin by midgut proteases, binding of endotoxin tomidgut receptors, and insertion of the toxin into the apicalmembrane to creation channels or pores that lead to disruptionof osmotic processes (Adang 1985). Furthermore, the biolog-ical control function provided by natural predators is regarded

D. Du : C. Geng :X. Zhang : Z. Zhang :Y. Zheng : F. Zhang :Y. Lin : F. Qiu (*)National Key Laboratory of Crop Genetic Improvement, HuazhongAgricultural University, Wuhan 430070, Chinae-mail: qiufazhan@gmail.com

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as a protection goal that should not be harmed by the appli-cation of any new pest management tool. Delta-endotoxinshave been expressed in many important crop plants includingcotton, potato, rice, and maize (Fujimoto et al. 1993; Kozielet al. 1993; Perlak et al. 1990; Perlak et al. 1993; Sawahel2002) and so on. Plants producing Cry proteins from thebacterium B. thuringiensis (Bt) have become a major tacticfor controlling pest Lepidoptera.

Genetic engineering offers the opportunity to developplants with a variety of new traits that may be useful in, forexample, pest control, improvement of crop yields, frost pre-vention, drought resistance, increased nutritional value in foodproducts, sources of medical and industrial biomolecules, anddegradation of a variety of persistent organic molecules, there-by facilitating bioremediation. The rapid deployment of plantbiotechnology has resulted in the development of genetictransformation systems for economically important crops.Transformation has become a basic tool for evaluating foreigngene function in maize and producing genetically modifiedhybrid corn for commercial purposes. Since Koziel et al.(1993) developed insect-resistant transgenic maize withcry1Ab for the first time, transgenic maize that expressesinsecticidal proteins derived from the bacterium B.thuringiensis (Bt) have been used for control of many eco-nomically important lepidopteran, coleopteran, and dipteranpests (Schnepf et al. 1998). Since 1996, Cry1Ab proteinexpressing Bt maize has been commercially available forcontrol of the European maize borer, Ostrinia nubilalis , oneof the most economical damaging pests of maize in the U.S.Maize Belt (Carpenter 2010). The Cry1Ab protein expressionin maize hybrid has proven to be an effective managementoption that also lessens the use of pesticides (Mendelsohnet al. 2003), and reduces the potential adverse effects of thesecompounds on the environment and human health. The ap-proach consisted of the transfer and expression of Bt toxin-encoding genes into plants has attracted much attention. Re-cent advances in plant biotechnology(Ishida et al. 2007;Torney et al. 2007; Vasil 1994) and the rich resources ofcloned cry genes (Höfte and Whiteley 1989) that encode Btδ-endotoxins have provided the possibility to express differentnovel insecticidal proteins in crop plants, including maize(Baumgarte and Tebbe 2005; Li et al. 2011; Munkvold et al.1999; Rhim et al. 1995; Sims and Stone 1991; Stewart et al.2001; Tu et al. 2000; Vaeck et al. 1987; Van Rensburg 2007).They are regarded as environmentally friendly and highlyselective and only a few adverse effects of Bt products onnon-target species have been reported. Today, transgenicmaize with insect and herbicide resistance is in commercialproduction.

In the present study, a codon optimized cry1C gene, re-ferred to as cry1C* , was transformed into maize Hi-II geno-type callus via biolistic gun-mediated transformation.With thegoal of obtaining transgenic lines that may be useful for

developing insect-resistant hybrids, the transgenic plants wereselected and regenerated for herbicide resistance followed thetechnical solutions to securing systems, and then were exam-ined for both insect resistance and agronomic traits under fieldconditions. Three highly insect-resistant lines, ZmKc-2-3,ZmKc-3-2, and ZmKc-3-5, were confirmed for the stableintegration and expression of the transgene in maize transgen-ic plants’ progeny. Hybrids were produced by crossing thesethree lines with the elite inbred line Zheng58. There was littlevariation in the hybrids and backcross offspring, indicatingthat these cry1C* transgenic lines can be used to produceinsect-resistant hybrids and serve as insect-resistant sourcesfor the development of Bt maize.

Materials and methods

Synthetic cry1C* and transformation construct

The transformation construct is displayed in Fig. 1 (Theplasmid was kindly provided by the National Key Laboratoryof Crop Genetic Improvement, Huazhong Agricultural Uni-versity, China). The cry1C* gene was synthesized on the basisof a wild-type cry1Ca5 gene of B. thuringiensis , after itscodons were optimized based on preferred codons in plants.The cry1C* gene was driven by the maize ubiquitin promoter(ubi ) and was cloned into the poly-linker of the plasmidpCAMBIA1300 . The hygromycin phosphotransferase (hph )gene was replaced with the phosphinotricin acetyltransferase(bar ) gene (Tang et al. 2006).

Generation of transgenic plants

Immature zygotic embryos with 1.2–1.8 mm in size weredissected from ears of Hi-II maize plants (A188*B73 origin)at 10–13 days after pollination and cultured to N6 basal media(N6E) medium for initiation of Type II callus (friable, stockedsomatic embryos present). The callus was bombarded forgenetic transformation followed the modified procedures de-scribed by Ishida et al. (2007). After 7–10 days on initiationmedium, the bombarded callus was transferred to selectionmedium (N6S) with 2 mg/L bialaphos to produce herbicide-resistant and highly embryogenic callus. Then, the herbicide-resistant callus was transferred to a regeneration medium toregenerating young plants. The putative transgenic plantsregenerated from the embryogenic callus were grown to ma-turity in a greenhouse. Plantlets with the introduced cry1C*gene (named ZmKc) were tested by polymerase chain reaction(PCR) analysis, the regenerated plants were deemed the T0

generation and crossed with the inbred line Zheng58 (Napet al. 1993).

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Selection of insect-resistant transgenic plantsand the production of cry1C*-containing hybrids

T1 transgenic lines derived from fertile T0 transgenic plantswere grown in spring 2011 on the experimental farm atHuazhong Agricultural University, Wuhan, China. The eliteinbred line Zheng58 was planted as a control and no insecti-cide was applied during the entire growth period. All plantswere tested by PCR assay with the primers in cry1C* , and thePCR positive plants were used for the further studies. Targettransgenic lines in the T1 generation were selected based onthree criteria: the result of molecular detection method, highinsect resistance, and no obvious phenotypic changes. Matureseeds were harvested from individual plants with high resis-tance. The selection of homozygous plants containing thecry1C* gene was carried out by a seed germination assay.The cry1C* gene was transferred into the elite inbred lineZheng58 and some other elite inbred lines by sexual repro-duction from 2010 to 2011.

DNA analyses of the transgenic plants

Genomic DNA of the transgenic plants was extracted by theCTAB method (Stewart and Via 1993) from fresh leaf tissue.PCR and southern blot analyses were used to determine thepresence and the copy number of the cry1C* gene in thetransgenic plants. The PCR analysis was performed usingthe primers Cry1C*-F (5′-gtccgttgaaagtgaatgat-3′) andCry1C*-R (5′-acacttacaagtggactc-3′). A 20-μL mixture of30 ng of template DNA, 2.0 μL of 10× buffer, 1.0 μL of2 mM dNTPs, 1.5 μL of 25 mMMgCl2, 0.4 μl of each of the10 μM primers and 1.5 U Taq DNA polymerase was preparedfor PCR assays. The PCR reaction was performed at 94 °C for5 min, and then for 35 cycles at 94 °C for 40 s, 55 °C for 45 s,72 °C for 50 s, followed by 72 °C for 5 min. The PCRproducts were then checked by electrophoresis on 0.8 %agarose gel.

For southern blot analysis, 15 μg of genomic DNA fromeach sample was digested overnight with BamHI and sepa-rated on a 0.8 % agarose gel for about 16 h, then transferred toa nylon membrane with siphon action. The probe was pre-pared from a PCR-amplified fragment of cry1C* . All proce-dures for the hybridization were conducted as described byLin and Zhang (2005).

Expression analysis of the transgenic plants

Total RNA in leaves of the transgenic plants was isolatedusing TRIzol (Invitrogen, USA) following the manufacturer’srecommendations. The mRNA of the cry1C* gene wereexamined by reverse transcription-PCR (RT-PCR). Reversetranscription of total RNA (5 μg) was performed with an M-MLV RTase cDNA Synthesis Kit (Takara, Japan) followingthe manufacturer’s instructions. The PCR products were sep-arated on 1 % agarose gels for the internal control of thecry1C*gene.

The Cry1C* protein levels of the positively expressingtransgenic plants in fresh leaves at the elongation and fillingstages were respectively measured by ELISAwith the ELISAkit from Enviro-Logix (Portland, ME). The protein assayprocedures were conducted according to the manufacturer’sinstructions. For the protein preparation, approximately 20mgof fresh leaves at the elongation and filling stages from thedifferent transgenic lines were homogenized by grinding in500 μL of extraction/dilution buffer. After 30 min at roomtemperature, a 20 μL aliquot of the supernatant was trans-ferred to a tube to which 480 μL of the buffer was added. Theenzyme-linking reaction was conducted following the manu-facturer’s instructions. The optical density values of the dilut-ed samples were measured using a microplate reader(Multiskan MK3, Labsystem, People’s Republic of China) at450-nm wavelength, and the Cry1C* protein content wascalculated based on the reading. In addition, amount ofCry1C* protein in fresh leaves was contrasted with the back-cross breeding line Zheng58 cry1C*-containing hybrids at theelongation and filling stages by the same method.

The amount of Cry1C* protein in different tissues includ-ing bract, leaf, tassel handle, stem, filament, tassel, female eartip, pollen, and grain at the wax ripeness stage of the homo-zygous transgenic lines were tested by the same procedure.

Field test for insect resistance and agronomic performance

For the evaluation of insect resistance and agronomic perfor-mance, the selected homozygous transgenic lines ZmKc-2-3,ZmKc-3-2, ZmKc-3-5, and the hybrid between ZmKc-2-3with Zheng58, were planted at the experimental farm of theHuazhong Agricultural University in Wuhan, China, in 2011.The homozygous cry1C* transgenic lines, identified by PCR

Fig. 1 T-DNA region of transformation construct cry1C* . The cry1C*gene was driven by a maize ubiquitin promoter and terminated by thenopaline synthase (nos) terminator. The Bar gene as a selectable marker

was under the control of CaMV 35S promoter and tailed by the CaMV35S polyA. LB left border of T-DNA region. RB right border of T-DNAregion

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and southern blot analyses, were tested in the field and withZheng58 as the control. The field layout followed a random-ized complete block design with three replications. Evaluationof insect resistance of transgenic plants in the field wasconducted by natural infestation of O. furnacalis . No chemi-cal insecticides targeted against lepidopteran pests were ap-plied throughout the growth period. The severity of damagecaused by the natural infestation on leaves was recordedwithin 15 days after scrape damage appeared. Leaves withvisible scrapes or folds were considered to be damaged. Deadhearts (the maize stem were damaged by the pest and becomeempty and dead) by the natural infestation were counted at thelate maximum filling stages.

The field testing of agronomic performance of the transgenicplants was conducted as described above and chemical pesti-cides were not applied throughout the development and growthstages. The plant height (PH), ear height (EH), and tasselbranch (TB) were measured at maturity in the field, and 12plants were randomly selected from each plot and harvested formeasurements of ear length (EL), ear width (EW), ear rows(ER), grain traits (GT), and hundred-grain weight (HGW).

Results

Transformation and selection of transgene-positive plants

A total of nine herbicide resistance callus gained from 200bombarded calli, 120 putative regeneration plants from thesecallus were grew to maturity. Of which 87 were positiveaccording to PCR analysis. Southern blot analysis showedone to four copy numbers in the T1 transgenic plants(Fig. 2a). Of the 87 transgenic plants, only one contained asingle copy of the transgene. The ZmKc-2-3 transgenic plant

carried two copies, ZmKc-2-4 had one copy and ZmKc-3-2and ZmKc-3-5 both had four copies. T2 transgenic plants withone or two copies were planted in the field for preliminaryscreening. Single-copy positive transgenic plants were select-ed by Southern blot again in T3 plants (Fig. 2b), and theseparation in ZmKc-2-3 descendant was observed, fromNo.1 to No. 5 in Fig. 2b, were occurred.

Expression of the cry1C* gene

RT-PCR analysis showed that 53 of 87 T0 transgenic plantshad expression of the cry1C* gene. The ears, which expressedmonoclonal grain, were individually harvested and the trans-ferred cry1C* gene was stably expressed in offspring whichwere examined by RT-PCR testing. Independentlyregenerated plant lines such as ZmKc-2-3 and ZmKc-3-5 inT1 expressed the cry1C* gene (Fig. 3, lanes 1–8), the stableexpression of this cry1C* gene was demonstrated by the T3

offspring with an electrophoretic band (Fig. 3, lanes 9–16).

Quantification of the Cry1C* protein

The results of ELISA indicated that the protein contents variedconsiderably between different transgenic lines (Fig. 4a). TheCry1C* protein concentrations in homozygous transgeniclines ZmKc-3-5, ZmKc-2-3, and ZmKc-3-2 were 1.39, 4.03,and 2.97 μg/g leaf fresh weight at the elongation stage, re-spectively. The Cry1C* protein concentrations were in therange of 0.89–1.12 μg/g fresh weight at the filling stage(Fig. 4a). The Cry1C* protein concentration was 4.03 μg/gfresh weight in the homozygous transgenic line ZmKc-2-3compared to 3.42 μg/g in Zheng58 cry1C*-containing hy-brids at the elongation stage (Fig. 4b). The Cry1C* protein

Fig. 2 Southern blot analysis of total genomic DNA of transgenic maizeplants and a set of representative progeny. The DNA samples weredigested withBamHI (no cutting site within cry1C* gene) and hybridizedwith the prepared radioactive probe. The probe size was 2 kb andcontained the whole encoding region of the cry1C* gene and nos termi-nator sequence. λDNA/HindIII was used as a molecular ladder and sizes

are labeled as bp; a M DNA marker, P genome equivalent copy numbercontrols using vector. Lane1 indicate ZmKc-3-2, lane2 ZmKc-2-4, lane3ZmKc-3-5, and lane4 ZmKc-2-3, respectively, in T0. b M DNA marker,P genome equivalent copy number controls using vector. Nos. 1–5indicate plants of ZmKc-2-3 in the T3 generation

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concentrations were in the range of 0.82–1.12 μg/g freshweight at the filling stage (Fig. 4b).

The levels of Cry1C* protein were tested at the filling stagein different organs—the different tissues showed different levelsof expression in the same plant at the same development period(Fig. 4c). In the transgenic line ZmKc-2-3, the highest level was3.43 μg/g fresh leaf weight, and a similar level of 3.36 μg/g wasfound in the other vegetative tissues. However, there were lowerconcentrations of 2.71 μg/g in tassel handles and 0.99 μg/g instems; the Cry1C* protein levels in reproductive tissue were0.79, 0.65, 0.66, and 0.19 μg/g in filaments, tassel, female eartip, and pollen, respectively; the lowest was 0.09 μg/g in thegrain. The same trend of variation was found in line ZmKc-3-2(2.88 μg/g in bract and 0.07 μg/g in grain). In general, the levelof protein declined in order of bract, leaf, tassel handle, stem,filaments, tassel, female ear tip, pollen, and grain (Fig. 4c).

Evaluation of insect resistance and agronomic performancein the field

Families of the selected three homozygous transgenic plantlines (ZmKc-2-3, ZmKc-3-2, and ZmKc-3-5) and the cry1C*containing hybrid were field tested for their resistance to O.furnacalis (Table 1), and also evaluated for agronomicperformance.

Of the wild-type Zheng58 plants, 43.71 % of 100 plantshad various degrees of damage to leaves, with an average of5.75 leaves affected per plant. Stems of Zheng58 were alsoseverely damaged at the end of the filling stages, with 39.36%of the 100 plants having dead hearts (Table 1). There was onlyslight damage on the three selected homozygous transgeniclines (0.00–0.02 leaves per plant in ZmKc-3-5) on the leavesand without the infection on the stem (Fig. 5).

ANOVA for the data collected from the field experimentshowed that there was no significant difference for the traits ofplant type, ear diameter, and hundred-grain weight betweenthe transgenic lines and their control ‘Zheng58’. However, thedifference was significant at the level of p <0.05 for the traitsof plant height and ear rows, and at the level of p <0.01 for thetraits of tassel branch, ear height, ear length, and hundred-

grain weight. The same results were also found between thetransgenic line ZmKc-2-3 and the F1 (ZmKc-2-3×Zheng58)(Table 2).

Discussion

In the present study, the modified novel cry1C* gene wassuccessfully transformed into maize Hi-II genotypes bybiolistic gun method. The results clearly demonstrated thatthe cry1C* gene is effective in developing a transgenic maizeline resistant to O. furnacalis , as shown by bioassay experi-ments and natural infestation trials under field conditions.Based on a range of molecular detection methods, selectedfor field testing for resistance to O. furnacalis and for agro-nomic performance, three transgenic lines were identifiedafter three generations of testing. Consequently, this genemay provide a useful resistance resource for developing trans-genic maize against lepidopteran pests as well as an alternativeto the Cry1A toxins. It should be mentioned that the bioassayof the Cry1C* toxin in maize was consistent with that in riceleaf folders and stem borers (Tang et al. 2006) and cottonHeliothis armigera , the results indicated that the protein wasalso highly toxic to the insects in maize and this gene mayhave broad utility in developing transgenic crops resistant tolepidopteran pests.

Another desirable feature revealed by the present study wasthe complete dominance of the expression level of the Bt toxinin the cry1C* transgenic plants. Although different transgenicevents appeared to express different levels of the transgene,the stable expression of the Cry1C* protein can be detected at1.39, 4.03, and 2.97 μg/g leaf fresh weight at the elongationstage, respectively and 0.89–1.12 μg/g fresh weight at thefilling stage. Indeed, such a system allows the entire plant tobe protected, especially against insects such as borers thatinfect plant parts that sprays often cannot reach. Furthermore,the toxin affects the more susceptible early instar stages of theinsect and the system is environmentally safe as the product isretained within the plant tissues. The hybrid between a homo-zygous transgenic and a non-transgenic plant (Zheng58)

Fig. 3 RT-PCR analysis results of mRNA extracted from generations todetect the transgene. In the early generation the positive expression of theforeign gene was tested by RT-PCR for the internal control of the cry1C*gene. Left T1 generation transgenic plants,M molecular weight marker; Pcry1C*gene; W wild-type maize; C non-positive plants; Lanes 1–4

transgenic plants from transformant ZmKc2-3; Lanes 5–8 transgenicplants from transformant ZmKc3-5; Right T3 generation transgenicplants,M molecular weight marker; P cry1C*gene; Nos. 9–12, transgen-ic plants from transformant ZmKc2-3; Nos. 13–16 transgenic plants fromtransformant ZmKc3-5

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expressed approximately the same amount of the toxin proteinas the transgenic parent. Furthermore, the levels of expressionof cry1C* in the hybrids were highly correlated with theexpression levels in the transgenic parents. This is extremelyimportant for the development of hybrid maize, as resistanceis critically dependent on the concentration of the toxinprotein.

In addition, there was a declining trend of Cry1C* proteinconcentration between vegetative and reproductive tissues.The changes of the Cry1C* protein concentration differed indifferent tissues, in general, the Cry1C* protein level washigher in vegetative than in reproductive tissue, and decreasedafter maturity. These results indicated that the selected trans-genic lines ZmKc-2-3, ZmKc-3-2, and ZmKc-3-5 possessedgreat potential for use in maize production. The homozygoustransgenic lines not only had high resistance to lepidopteranpests and normal agronomic performance for natural infesta-tion trials under field conditions, but also the concentration ofCry1C* protein was only 0.09 μg/g in endosperm, i.e., it wasnearly absent from the endosperm.

A large number of studies have reported the developmentof insect-resistant transgenic maize (Carrillo et al. 2011; Collet al. 2009; Faria et al. 2007). Concerns have been expressedfor potential emergence of insect resistance to the Bt toxinsfollowing large-scale commercial cultivation (Bates et al.2005; Frutos et al. 1999; Roush 1998). However, it is worthcommenting on the strategies for pyramiding the Bt genes foractual production-transgenic cotton plants with two differentBt genes had stronger resistance to insects than those with a

single Bt gene (Greenplate et al. 2003; Stewart et al. 2001).The cultivation of transgenic crops with two or more differentforms of resistance would significantly increase their insectresistance (Zhao et al. 2003). Our next step in developing linesof multiple resistances is to combine the genes by sexualcrossing, similar to the approach of Cao et al. (1999; 2002)in broccoli, to achieve the stacking of two Bt genes and someother positive genes. Compared with the strategies of havingthe two genes in the same transformation construct or the co-transformation of two constructs (Daley et al. 1998; Zhao et al.2005), this strategy may have certain advantages. More

Fig. 5 The performance of ZmKc-2-3 against O. furnacalis under lab-oratory and field conditions. a The field performance of the transgenicline ZmKc-2-3 against O. furnacalis at the filling stage under fieldcondition. Left no visible affected leaf was observed on the transgenicpositive plant at the filling stage; Right the control, Zheng58, was heavilydamaged by theO. furnacalis . b , c Insect bioassays performance with thehomozygous transgenic line ZmKc-2-3 in the insect-rearing room. b Leftextensive damage was visible on the control leaf, some faces were left onthe surface of the leaf, and larvae were much larger than those originallypresent at the start of the bioassay. Right no visible damage was observedon the transgenic plant leaf. c Transgenic positive plants: no visibledamage or living larvae were found on the inner surface of the cut stemswith the homozygous transgenic ZmKc-2-3. Zheng58 (control): exten-sive damage was visible, some faces were left on the surface of the innertissue, and larvae were survived in the stem;Arrows point to the site showthe parts injured by pests

Table 1 Resistance of the three transgenic lines and ZmKc-2-3 derived hybrids against natural infestations of O. furnacalis under field conditions(Wuhan, China, 2011)

Maize genotype Damage at leaf Damage at stem

Number of plants affected (%) Number of damaged leaves per plant Number of plants affected (%) Number of dead stems

ZmKc-2-3×Zheng58 0.00±0.0a 0.00±0.0 0.00±0.0 0.00±0.0

ZmKc-2-3 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0

ZmKc-3-2 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0

ZmKc-3-5 0.62±0.0 0.02±0.0 0.00±0.0 0.00±0.0

Zheng58 43.71±0.0 5.75±0.0 39.36±0.0 2.04±0.0

a Data expressed as mean±standard deviation

�Fig. 4 Protein assay for transgenic lines and their hybrids. The Cry1C*protein contents varied considerably from one line to another. The threetransgenic lines and their hybrids had higher Cry1C* protein contentsthan the other lines and the corresponding hybrids. a The Cry1C* proteinconcentrations in different transgenic lines ZmKc-2-3, ZmKc-3-2, andZmKc-3-5 at the elongation stage and the filling stage, respectively. bThe Cry1C* protein concentrations of the homozygous transgenic linescontrasted with the backcross breeding lines such as Zheng58 cry1C*-containing hybrids at the elongation stage and the filling stage, respec-tively. c The contents of Cry1C* protein in different organizations fromdifferent transgenic plants at the filling stage

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importantly, this strategy of a single-gene construct offers theflexibility of utilizing the gene in various ways, either indi-vidually or in any of a number of desired combinations.

Acknowledgments This research was supported by the GeneticallyModified Organisms Breeding Major Projects, China (No.2009ZX08003-016B) and the National High-Technology Research andDevelopment Program of China (Program 863, No. 2012AA101104).The vector plasmid was kindly provided by Dr. Lin Yongjun and Dr.Zhang Qifa from the National Key Laboratory of Crop Genetic Improve-ment, Huazhong Agricultural University, Wuhan, China.

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Table 2 Comparison of agronomic traits of the homozygous transgenic line ZmKc-2-3 and its derived hybrid ZmKc-2-3×Zheng58 under fieldconditions (Wuhan, China, 2011)

Maize genotype PT PH (cm) EH (cm) TB EL (cm) ED (cm) ER HGW (g)

ZmKc-3-2 Horizontal 192b 73.6C 6–10 15.7C 5.9B 12–18 31.7B

Zmkc-3-5 Horizontal 171a 47.9A 4–6 17.7A 5.2A 12–16 33.3A

ZmKc-2-3 Horizontal 187b 61.4B 6–10 16.2C 5.2A 12–18 33.6 B

Zheng58 Half-compact 168a 47.8A 4–6 18.3A 5.2A 14 32.8 A

ZmKc-2-3×Zheng58 Half-compact 174a 48.4A 4–6 17.6B 5.6A 12–16 33.1 A

a, b and A, B, and C: ranks resolved by Duncan’s multiple range test at the 0.05 and 0.01 probability levels, respectively

PT plant type, PH plant height, EH ear height, TB tassel branch, EL ear length, ED ear diameter, ER ear rows, HGW hundred-grain weight

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