novel indica basmati line (b-370) expressing two unrelated genes of bacillus thuringiensis is highly...
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doi:10.1016/j.cr
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School of Agri
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Crop Protection 24 (2005) 870–879
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Novel indica basmati line (B-370) expressing two unrelated genes ofBacillus thuringiensis is highly resistant to two lepidopteran
insects in the field
Khurram Bashir1, Tayyab Husnain�, Tahira Fatima, Naveeda Riaz,Rahat Makhdoom, Sheikh Riazuddin
National Center of Excellence in Molecular Biology, 87-W, Canal Bank Road, University of the Punjab, Lahore, Pakistan
Received 9 August 2004; received in revised form 23 December 2004; accepted 7 January 2005
Abstract
We report the first field trials of indica basmati rice expressing two Bt genes, cry1Ac and cry2A simultaneously. Different
transgenic lines were sown under field conditions for two consecutive years (2001 and 2002). Artificial infestation of yellow stem
borer (YSB, Scirpophaga incertulas) and natural infestation of rice leaf folder (RLF, Cnaphalochrocus medinalis) were studied.
Transgenic lines showed up to 100% and 98% resistance against YSB at vegetative and flowering stages, respectively, with 98%
additional resistant against RLF as compared with the control. Variation in some morphological characteristics, e.g., the average
number of tillers, plant height and maturity, were also observed. Transgenic lines produced up to 59% more grains than control
plants under artificially augmented conditions, while up to an 8% increase was recorded under natural infestations. All lines
expressed high level of Cry proteins when compared with commercially released cultivars of Bt cotton, maize and potato. It was also
observed that although toxin titer substantially decreased with increasing age of the plants, it remained well within the limits
necessary to kill the target insects. It was also observed that the transgenic lines released Bt toxins from roots into Murashige and
Skoog basal medium, hydroponic cultures and soil, which could be detected through sandwich ELISA. On the basis of these results
these lines seem good candidates to be released as the first commercial cultivars of Bt indica basmati rice.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: Oryza sativa L.; Bacillus thuringiensis; Insect resistance; Yellow stem borer; Rice leaf folder; Gene pyramid; Morphological variation
1. Introduction
Rice is one of the most important food crops, feedingover 2 billion people in developing countries (Food andAgriculture Organization, 1995). Rice productivity isseverely affected by several abiotic and biotic factors,including damage caused by different insect pests anddiseases. Larvae of Lepidoptera are, perhaps, the most
e front matter r 2005 Elsevier Ltd. All rights reserved.
opro.2005.01.008
ing author. Tel.: +92425421235; fax: +92 425421316.
esses: [email protected] (K. Bashir),
yahoo.com (T. Husnain).
ress. Laboratory of Plant Biotechnology, Graduate
cultural and Life Sciences, The University of Tokyo,
destructive rice pests in the world (Khan et al., 1991),with the yellow stem borer (YSB, Scirpophaga incertu-
las) and the rice leaf folder (RLF, Cnaphalochrocus
medinalis) particularly important among them. Ricestem borers alone are responsible for annual damage of5–10% to the rice crop, and damage from these insectsoccasionally reaches up to 60% under favorableconditions of insect attack (Pathak and Khan, 1994,pp. 15–20). Rice YSB attacks the crop right from theseedling to the harvest stage and thus causes completeloss of affected tillers (Salim and Masih, 1987). The YSBbore into the rice stem and hollow out the stemcompletely. Physical symptoms like dead hearts andwhite heads are produced by stem borer larvae when the
ARTICLE IN PRESSK. Bashir et al. / Crop Protection 24 (2005) 870–879 871
tillers are at vegetative stage and at the time of headingrespectively. The RLF also causes serious damage to ricecrop, i.e. the young larvae feed on tender leaves and asingle larva may damage a number of leaves as itmigrates from one leaf to another.
Transformation of rice with genes from a soilbacterium Bacillus thuringiensis (Bt) is a commonapproach to confer resistance to insect infestations andmany groups have produced transgenic Bt rice resultingin built-in insect resistance (Datta et al., 1998; Fujimotoet al., 1993; Maqbool et al., 1998). Several groupshave also demonstrated successful use of Bt rice in fieldtrials to combat resistance against devastating Lepidop-teran insects (Bashir et al., 2004; Shu et al., 2000; Tuet al., 2000; Ye et al., 2001a,b). Transgenic lines mostlyused in field trials expressed either cry1Ab, cry1Ac,cry2A or a fused gene from cry1Ab/cry1Ac. Althoughthese lines provided high level of resistance againstLepidopterans, Bt cultivars have the same weakness asmany other insect control technologies e.g., insects canevolve resistance to them, thus eliminating their effec-tiveness. Insects have already evolved resistance to allclasses of widely used insecticides, including Bt productsthat are applied as sprays (Frutos et al., 1999). Insectshave also adapted to numerous resistant crop varietiesproduced through conventional breeding. Notable ex-amples in rice include the brown plant hopper,Nilaparvata lugens (Heinrichs, 1986), and the Asian ricegall midge, Orseolia oryzae (Bennett et al., 2000).Considering these issues, it is recommended that onlythose lines expressing two Bt genes should be releasedinto environment (Cohen et al., 2000a). It is alsostrongly recommended that any two Bt toxins that areused in combination must not be too similar to eachother; otherwise a single mutation may confer cross-resistance to both toxins. More than 100 Bt toxin geneshave been cloned and sequenced. Many of these toxinsare highly divergent in amino acid sequence andbiochemical properties (Frutos et al., 1999). Studiesdone by Fiuza et al. (1996) and Lee et al. (1997) showedthat Cry1Ac with Cry2A is good toxin combination forLepidopterans.
It has been reported (Saxena et al., 1999; Saxena andStotzky, 2000; Saxena et al., 2002) that Bt toxin isreleased in root exudates from hybrids of Bt corn insterile hydroponic cultures, in sterile and non sterile soilunder controlled conditions and in the field. Moreover,the phenomenon is not cultivar specific as release of Bt
toxin from roots was observed in 12 transgenic cornhybrids representing three transformation events in vitroand in situ. The toxin released through root exudates, aswell as from the biomass of Bt corn adsorbs and bindsrapidly on surface-active particles (e.g. clays and humicsubstances) in soil and remain larvicidal for at least 180days after harvest or incorporation in to soil (Saxenaand Stotzky, 2001). Thus Bt toxins may accumulate in
soil (Tapp and Stotzky, 1998; Saxena et al., 1999). Thisaccumulated protein is larvicidal and could accelerateconsumption by larvae, abiotic inactivation, and degra-dation by microbes. This would result in amount oftoxins that could constitute a hazard to non-targetorganisms and enhance the selection of toxin-resistanttarget insects especially if the toxins are bound on soilconstituents (Tapp and Stotzky, 1995). To our knowl-edge the release of Bt toxins from transgenic rice eitherin MS media, hydroponic solution or soil has not beenpreviously reported.
Commercial success of a cultivar is dependent onmorphological and agronomic characteristics. Schuh etal. (1993) observed tremendous heritable variation in theT2 transgenic populations transformed by Polyethyleneglycol or protoplast electroporation. Several otherreports also confirmed that transgenic lines of riceexhibit considerable variation in agronomic and mor-phological characteristics (Jiang et al., 2000; Shu et al.,2002) in agrobacterium and particle mediated transfor-mation. Genomic changes were more common intransgenic rice plants transformed with agrobacteriumas compared with biolistic transformation, easily identi-fied through AFLP and PCR analysis, but the data forphenotypic changes were not presented (Labra et al.,2001). Ideally, there should not be any compromise formorphological traits like average number of tillers perplant, average plant height, days to maturity and yieldduring selection of desirable lines.
To address all these concerns, the present studies wereconducted at the National Centre of Excellence inMolecular Biology, Lahore, Pakistan (CEMB) for twoconsecutive years to determine potential of locallytransformed lines of B-370 against yellow stem borerand rice leaf folder. Additionally studies were under-taken to address the bio-safety concerns of Bt toxinrelease into the soil.
2. Material and methods
2.1. Plant material and experimental design
Basmati 370 (B-370), a commercial cultivar of indicabasmati rice famous for its long grain and aroma, isextensively used in different breeding programs inPakistan. Different transgenic plants of B-370 expres-sing either cry1Ac and cry2A alone or in combinationwere generated and screened for the presence of Bt genesthrough PCR and Southern and Western Blot analysisand insecticidal activity through lab based insectbioassays (Husnain et al., 2003). Three lines expressingboth cry1Ac and cry2A, along with one line expressingcry1Ac and one line expressing cry2A, were selectedfrom the 3rd and 4th generations of transgenic plants onthe basis of molecular analysis for cry genes and insect
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Table 1
Characteristics of transgenic lines used in field trials (2001)
Line no. Gene PCR Southern Western Insect bioassays
L-4-311 hpha+gusb+cry2A + + + +c
L-3-382 hph +gus+cry1Ac + + + +
L-8-22 hph+cry1Ac+cry2A + + + +
L-26-3 hph+cry1Ac+cry2A + + + +
L-26-8 hph+cry1Ac+cry2A + + + +
Control (B-370) — � � � �
ahph: hygromycin phosphotransferase.bgus: b glucuronidase.cThese plants gave more than 50% mortality against YSB.
K. Bashir et al. / Crop Protection 24 (2005) 870–879872
bioassays (Table 1). Untransformed parental B-370 wasused as control in all experiments. Maize ubiquitinpromoter controlled the expression of cry1Ac while theexpression of cry2A was driven by CaMV 35S promoter.During the first year (2001) 66 plants of each line weresown in a randomized complete block design with threereplications. Distance between plants was adjusted to45 cm to limit the movement of insect larvae from oneplant to another and to provide better cultural practices.Details about field sizes and other cultural practices aresummarized in Table 2. For trials in 2002, the progenyof six plants from experimental line L-8-22 and 7 plantsfrom L-26-3 was grown in the green house and thehomozygous status of these plants was confirmed byELISA on the basis of progeny analysis. All plants fromL-8-22 proved homozygous while none of the plantsfrom L-26-3 proved homozygous. Finally four indivi-dual plants, three from L-8-22 (L-8-22-2, L-8-22-32 andL-8-22-35) and one from L-26-3 (L-26-3-1), wereselected on the basis of insect bioassays as well asmolecular and morphological performance. The pro-geny of these plants was sown (2002) along with the splitplot design at two locations.
The main plot factor was infestations (natural or naturalplus artificial); main plots were arranged according torandomized complete block design with four replications.The subplot factor consisted of four transgenic lines alongwith the control. One main plot in each replication wasartificially infested while the other was not. Six hundredplants of each line were sown at each location and plant-to-plant distance was reduced to 22 cm to accommodatethe conditions provided in the farmer cooperator’s fieldand to provide the opportunity for YSB larvae to movefrom one plant to another as they do in field (Cohen et al.,2000b). Fields were surrounded with seven rows of thecontrol all around to serve as refugia and to trap pollen.All recommended practices of Department of AgriculturalExtension, Pakistan, including bio-safety guidelines fromthe National Bio-safety Committee (National BiosafetyCommittee, 1999) were followed. To maximize naturalpest damages, no pesticide/herbicides were applied duringthe entire period of rice development at the test areas.
2.2. Insect infestations and data recording
Yellow stem borers (YSB) were provided by theEntomology Division, CEMB that were initially col-lected from different rice growing areas in Lahore. Thesemoths were reared on rice plants covered by muslincloths at 2872 1C, 60–70% relative humidity with a 14 hphoto phase. The egg masses were collected and hatchedin glass vials under same conditions. During the firstyear of the study neonate insects were starved forapproximately 12 h and then twenty insects per plant perinfestation were placed on each using a camel’s hairbrush (Table 2). In 2001 egg masses were collected andhatched into 10 cm long plastic tubes held at 2872 1C,60–70% relative humidity with a 14-h photo phase tillhatching. These tubes were placed between tillers ofalternate plants within 6–14 h of hatching. In thismanner, 150 plants of each line were infested at eachlocation. One egg mass contains between 60 and 150larvae. On an average about 67,500 larvae were releasedduring single infestation on 1500 plants while no insectwas released on another 1500 plants to compare thedifference in yield under both conditions (i.e. one mainplot in each replication was infested). In total, threeinfestations were done during each year, two atvegetative stage while the third was done at paniclebearing stage (Table 2). Same plants were infestedduring all three infestations. Although naturally, YSBdamage the rice plants from seedling to harvestdepending upon the environmental conditions, artificialinfestations were done at critical stages of plant growthi.e., after tillering stage and at panicle bearing stage. Thedamage caused by YSB to the rice plants, at these stagesis difficult to compensate. To fulfill the requirements ofrefugia, 20% of total insects released were raised on thecontrol surrounding the experimental field. Rice Naturalinfestations of YSB and RLF were observed at bothlocations during the second year only. Leaf folder wasnot infested artificially and the data was recorded onlyfor natural infestations.
Data for insect damage and morphological character-istics were recorded according to instructions described
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Table
2
Summary
ofculturalpractices
fortransgenic
lines
sownbasedonexperim
entaldesign
Exp.design
Year
No.ofplots
Plotsize
(m2)
No.ofplants
Date
of
sowing
Date
of
transplanting
Insectsper
infestation
Date
of1st
infestation
Date
of2nd
infestation
Date
of3rd
infestation
RCBD
2001
324.2
396
June23
July
25
20/Plant
Sep.6
Sep.18
Oct.7
Splitplot
(CEMB)
2002
439.5
3000
June20
July
22
Oneeggmass
per
twoplants
(60–150)
Aug.30
Sep.15
Sep.25
Splitplot
(RRIK
SK)
2002
439.5
3000
June20
July
23
Oneeggmass
per
twoplants
(60–150)
Sep.1
Sep.16
Sep.26
Exp.¼
Experim
ental,Aug.:August,Sep.:September,Oct.:October.
No.ofplots¼
number
ofreplicationforeach
design.
No.ofplants¼
totalplants
usedduringaparticularyear.
Plotsize
¼Refer
tothesize
ofoneplotforRCBD
andsize
ofeach
main
plotforsplitplotdesign.
K. Bashir et al. / Crop Protection 24 (2005) 870–879 873
by Lancashire et al. (1980). Dead hearts and white headswere counted for each plant and shown as a percentageof the total tillers and panicles respectively. To accessthe damage caused by RLF, number of infested leaveswere counted and expressed as percentage of totalleaves.
Data was analyzed according to RCBD and split plotanalysis of variance during first and second yearrespectively using CoStat version 6.204 (CoHort Soft-ware, Monterey, CA, USA. Treatment means werecompared by Student-Newman-Kuels (SNK) test.
2.3. Protein quantification and bio-safety studies
ELISA was used to confirm the presence andexpression of cry genes and to confirm the homozygousstatus of different lines. The concentration of Cry1Acand Cry2A were determined in different plant parts suchas stem, panicle, root, straw, husk, kernel and seeds byELISA using Envirologix kits AP 003 and AP 005(Envirologix, Maine, USA). Protein was extracted asdescribed by Koziel et al. (1993) using the bufferprovided by manufacturer (Envirologix, Maine, USA).Protein was extracted and processed from leaves at 30,60, and 90 days after transplantation as well as 15 and30 days after harvest from three different transgenicplants in duplicate. All these samples were processed forsandwich ELISA (Crowther, 1995) according to themanufacturer’s instruction. Samples were collected fromprimary tillers of plants sown in filed in every case. Allplant parts were collected from the field and stored inliquid nitrogen before use. After harvest, three plantsused for protein quantification earlier were marked andleft in the field under natural conditions to check theexistence of Bt protein in dead parts and usedimmediately after collection (15 and 30 days afterharvest) in liquid nitrogen. So the same plants wereused for protein quantification at different growth stagesand after harvest.
For bio-safety studies seeds of two homozygoustransgenic lines of basmati rice, L-8-22-2 & L-8-22-32,were sterilized and sown on MS media in threereplicates. The resulting plantlets were kept on the samemedia for 25 days and then media adhering to the rootsof transgenic plants was collected from all replicates andprotein was extracted using alkalic buffer (10mM DTT,50mM Na2CO3, pH410.5). Samples were mixed withbuffer and incubated at 37 1C for 30min and super-natant was collected after centrifugation. Incubation at37 1C was replaced with incubation with 4 1C when thebuffer provided by Envirologix was used. All sampleswere concentrated with the help of a Centricon(10 kDa). Thirty transgenic plants of L-8-22-2 and L-8-22-32 were grown in 200ml of hydroponic culture; thepH of the solution was adjusted to 5.8 after 3 daysinterval. After 20 days 50ml of hydroponic solution was
ARTICLE IN PRESSK. Bashir et al. / Crop Protection 24 (2005) 870–879874
collected and concentrated up to 500 ml with the help ofa Centricon that keep the fragment above 10 kDa.Protein was extracted using the buffer provided byEnvirologix. Protein was also extracted from soiladhering to the roots of transgenic plants. All theseprotein samples along with appropriate positive andnegative controls were subjected to ELISA using cry1AcEnvirologix plate kits (AP 005).
3. Results
3.1. Insect resistance
Transgenic lines showed high and stable levels ofresistance against both insects i.e. YSB and RFL
Table 3
Damage caused by artificially augmented infestations of yellow stem
borer in indica basmati rice (2001)
Line no. Dead heartsa White headsa Yieldb
L-4-311 13.4572.1b 14.1472.5b 708718.5b
L-3-382 11.6071.8b 11.2371.7b 711712.4b
L-8-22 00.4570.3a 00.2570.1a 864723.5a
L-26-3 03.7570.9a 02.8270.4a 826721.4a
L-26-8 06.2971.8ab 04.2770.9a 718723.8b
Control 37.8872.6c 45.4573.1c 543725.3c
Prob4 F o 0.00001 o0.00001 o0.00001
Values followed by 7 represent the standard error of the mean.
Means followed by the similar letters within a column do not differ
according to Students-Newman K test (po 0.05).aDead hearts and whiteheads are expressed as percentage of total
tillers and panicles, respectively.bYield of each line is expressed as grams per plot containing 22
plants of each line.
Table 4
Damage caused by artificially augmented infestations of yellow stem borer a
Line no. Gene Planted at CEMBa
Deadc Whitec RLFd
hearts heads
L-8-22-2 hph+cry1Ac+cry2A 0.070.0a 1.370.4a 0.070.0a
L-8-22-32 hph+cry1Ac+cry2A 0.070.0a 1.470.4a 1.070.4a
L-8-22-35 hph+cry1Ac+cry2A 0.070.0a 0.970.3a 0.570.3a
L-26-3-1 hph+cry1Ac+cry2A 6.370.7b 7.371.0b 7.070.6b
Control — 43.171.3c 37.372.5c 47.072.1c
Prob4 F o0.00001 o0.00001 o0.00001
Means followed by the similar letters within a column do not differ accordiaPlanted at National Center of Excellence in molecular Biology, Lahore.bPlanted at Rice Research Institute Kala Shah Kaku.cDead hearts and white heads are expressed as percentage of total tillers adDamage of RLF is expressed as percentage of total leaves damaged.eYield of each line is expressed as grams per main-plot containing 75 plants
design and then according to Randomized Complete Block Design for infes
(po0.05). During the first year (2001), L-8-22 was themost desirable line with only 0.45% dead hearts ascompared with 37.88% dead hearts in the control, whileother lines produced dead hearts within the range of3.75–13.45%. All lines also showed resistance againsttarget insects at the flowering stage (po0.05), with L-8-22 showing only 0.25% whiteheads as compared with45.45% in the control (Table 3). White heads in otherlines ranged between 2.82% in L-26-3 to 14.14% in L-4-311. L-8-22 was the only homozygous line among 16different transgenic lines tested in different field experi-ments. Three individual plants from this line numberedas L-8-22-2, L-8-22-32 and L-8-22-35 along with oneplant from L-26-3 numbered as L-26-3-1 was selectedfor second year field trials.
During the 2002 L-8-22-2, L-8-22-32 and L-8-22-35had no dead heart at either location. L-26-3-1 showed6.26% and 5.18% dead heart as compared with 43.10%and 43.48% in the control at CEMB and RRIKSKrespectively (po0.05). This line proved heterozygous onthe basis of ELISA (data not shown). A slight reductionin resistance was observed at the flowering stage and alow frequency of white heads was observed in all lines.L-8-22-2, L-8-22-32 and L-8-22-35 showed 1.31%,1.39% and 0.88% damage, respectively, whereas L-26-3-1 produced 7.30% white heads at CEMB as comparedwith 37.34% in the control (po0.05). A similar patternof resistance was observed at RRIKSK (Table 4). Threetillers from each replication with white heads from linesL-8-22-2, L-8-22-32 and L-8-22-35 at both locationswere cut to recover the larvae of YSB but no livinginsect was recovered from these lines. On the Livinglarvae of YSB were recovered from L-26-3-1 (7) andfrom the control (17). RLF were not artificially infestedin any case and data was recorded for naturalinfestations only. L-8-22-2 showed 0% and 1% damage
nd rice leaf folder in indica basmati rice (2002)
Planted at RRIKSKb Yielde (g/plot)
Dead White RLF Infested Not infested
hearts heads
0.070.0a 1.270.4a 1.070.2a 1840763.5a 1887742.5a
0.070.0a 4.870.5a 1.870.3a 1852730.9a 1911726.6a
0.070.0a 2.070.2a 0.770.3a 1826762.3a 1874738.2a
5.271.5b 10.571.1b 9.070.9b 1654752.8a 1817741.4a
43.573.6c 52.972.6c 39.072.1c 1165746.0b 1776735.3a
o0.00001 o0.00001 o0.00001 o0.00001 0.1570
ng to Student-Newman-Kuels test (po 0.05).
nd panicles, respectively.
of each line. Data was first subjected to ANOVA according to split plot
ted and not infested conditions.
ARTICLE IN PRESSK. Bashir et al. / Crop Protection 24 (2005) 870–879 875
of RLF at CEMB and RRIKSK, respectively. Otherlines also showed considerable resistance to RLF withdamage within the range of 1–9% (po0.05; Table 4).
The damage caused by natural infestation of YSB incontrol plant was 3% and 5.1% at CEMB andRRIKSK, respectively, with respect to dead heartswhile transgenics had no dead hearts. At flowering stage4.1% and 5.5% white heads were recorded in the controlat CEMB and RRIKSK while transgenic plants showedless than 0.2% dead hearts (po0.05).
3.2. Agronomic characteristics
Significant differences were observed between linescontaining one and two Bt genes, as well as the controlfor the average number of tillers (po0.05). The smallestnumber of tillers (27) was recorded for lines L-4-311 andL-3-383 which were statistically similar to a value of 28for the control (po0.05). The average number of tillersfor L-8-22, L-26-3 and L-26-8 was 36, 34 and 35,respectively which were statistically superior to thecontrol as well as to the lines expressing only one Bt
gene (po0.05; Table 5). During the second year, themaximum number of tillers (28) was recorded for linesL-8-22-2 and L-26-3-3 as compared with 21 in thecontrol (po0.05). Significant variation (po0.05) wasalso observed for plant height (101 cm for L-26-8 to 161for the control). It was also observed that linescontaining two Bt genes were shorter as compared with
Table 5
Morphological properties of transgenic lines sown based on experimental de
Line no. Meana Meanb Meanc Meand
No. of tillers Plant height (cm) Days to flower Panicle length (
2001
L-4-311 2771.2b 13572.9c 9871.2b 2671.4a
L-3-382 2671.2b 12472.3b 11172.2c 2871.2a
L-8-22 3670.6a 10371.9a 8670.9a 2571.3a
L-26-3 3471.2a 10271.4a 8471.2a 2671.2a
L-26-8 3571.0a 10172.6a 8471.8a 2770.7a
Control 2870.9b 16174.1d 10272.3b 3071.9a
Prob4 F 0.0002 o0.00001 o0.00001 0.214
2002
L-8-22-2 2870.7a 10970.9a 8570.9a 2871.8a
L-8-22-32 2670.5a 10471.8a 8771.6a 2771.8a
L-8-22-35 2770.5a 10771.6a 8271.9a 2971.7a
L-26-3-1 2870.7a 10571.9a 8371.8a 2771.7a
Control 2170.7b 16172.4b 10271.9b 3072.7a
Prob4 F o0.00001 o0.00001 o0.00001 0.0606
Means followed by the similar letters within a column do not differ accordiaMean number of tillers, number of tillers was recorded 45 days after tranbMean plant height from soil to the top of highest panicle excluding awncMean days to flower, data for days to flowers was recorded on daily basdMean panicle length, Panicle length was recorded 12 days after ear deveeMean flag leaf area, flag leaf area was recorded 10 days after ear developfMean lodging incidence, lodging incidence was recorded 15 days after eagMean spikelet fertility, spikelet fertility was recorded 12 days after ear de
lines containing either cry1Ac or cry2A alone or thecontrol. The plant heights of L-26-8, L-26-3 and L-8-22were 101, 102 and 103 cm respectively as compared with161 cm for the control (po0.05). During the second yearaverage plant height was 104–161 cm for L-8-22-32 andthe control, respectively. It was also observed that linescontaining two Bt genes were shorter as compared withlines containing either cry1Ac or cry2A alone or thecontrol (po0.05). L-26-3 and L-26-8 were 18 days earlyas compared with the parental control as they took only84 days to flower (po0.05). Similar patterns wereobserved during the second year (Table 5). During thefirst year panicle lengths were 25 and 30 cm for L-8-22and L-26-3, where as 27 and 30 cm for L-8-22-32 and L-26-3-3 and the control. Although transgenic plants wereinferior to the control for panicle length, flag leaf areaand spikelet fertility, results were not significantlydifferent from the control (po0.05, Table 5). Similarpatterns for these characteristics were also recorded forthese lines planted at RRIKSK (data not shown).
Transgenic lines expressing two Bt genes were highlyresistant to lodging with only 2% plants lodged in L-8-22 and L-26-3 at maturity as compared with 29%lodging in the control and 23–28% in lines expressingeither of the genes (po0.05). A similar pattern wasobserved during the second year with 2–4% lodging fortransgenic lines and 23% for the control.
Yield data showed that transgenic lines produced upto 59% higher yield as compared with the control during
sign
Meane Meanf Meang
cm) Flag leaf area (cm) Lodging incidence (%) Spikelet fertility (%)
3171.8a 2874.1b 8971.2a
2870.3a 2373.3b 8971.4a
2571.3a 271.2a 9071.4a
2970.6a 271.2a 8671.8a
3071.2a 271.7a 8971.8a
3271.9a 2973.9b 9270.9a
0.0537 0.0001 0.2840
2970.6a 270.6a 9170.8a
2970.8a 270.6a 9070.9a
3070.8a 270.5a 9370.8a
3070.5a 270.5a 8970.8a
3270.9a 2371.7b 9271.6a
0.0864 o0.00001 0.1628
ng to Student-Newman-Kuels test (po 0.05).
splanting.
s. Plant height was recorded after ear development.
is.
lopment.
ment.
r development.
velopment by counting the number of filled and empty spikelets.
ARTICLE IN PRESSK. Bashir et al. / Crop Protection 24 (2005) 870–879876
first year. L-8-22 had the greater yield advantage of59.0% (864 g/plot) followed by L-26-3 (826 g/plot) andL-26-8 (712 g/plot) with 52% and 31% additional yieldas compared with the grain yield of 543 g/plot forcontrol (po 0.05; Table 3). In 2001, a split plot designwas used to check the yield potential of transgenic linesunder artificially plus naturally and naturally infestedconditions. One main plot in each replication was notinfested artificially but natural infestations of YSB andRLF were observed at both locations. L-8-22-32produced 59% greater yield (1852 g/plot) as comparedwith the control (1165 g/plot) under artificially plusnaturally infested conditions. L-8-22-2, L-8-22-35 andL-26-3-1 were 58%, 57% and 42% greater yieldcompared with the control (po0.05). Under naturalinfestations alone at difference of 8% was recorded forL-8-22-32 while an increase of 6%, 6% and 2% wasrecorded for L-8-22-2, L-8-22-35 and L-26-3-1 respec-tively (Table 4). These differences were not statisticallysignificant (po0.05).
3.3. Protein quantification and risk assessment studies
Sandwich ELISA was used to quantify the amount ofprotein in transgenic plants at different growth stages as
To
xin
Tit
er (
ug
/g)
Cry2A
Cry1Ac
H 15H 30H
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
0 15 30 45 60 75 90 105 120 135 150
Age of Plant
Cry
2A
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Cry
1Ac
Fig. 1. Effect of age of plant on expression of Bt proteins.
Age of the plant is expressed as day after transplanting H ¼ harvest;15H ¼ 15 days after harvest; 30H ¼ 30 days after harvest. Bars show
standard error of the mean, n ¼ 6:
Table 6
Bt protein concentration (ng/g of tissue) in different parts of transgenic plan
Gene Stema Rootsa Panicleb
cry1AC 3075 300719 110714
cry2A 0772 100711 00772
Values followed by the 7 represent the standard error of the mean ðn ¼ 4Þ:aProtein was extracted and processed 60 days after transplanting.bProtein was extracted and processed 5 days after panicle initiation.cProtein was extracted and processed 5 days after harvesting.dProtein was extracted and processed 10 days after harvesting.
well as in different parts of the transgenic plants. It wasobserved that the toxin titer for Cry1Ac declinedsubstantially with increasing age of the plant, from6.10 mg g�1 of leaf fresh weight at 30 days to 4.54 and3.42 mg g�1 at 60 and 90 days after transplanting,respectively (Fig. 1). The titer for Cry2A also declinedwith increasing age and was quantified as 0.72 mg g�1 ofleaf fresh weight at 30 days after sowing to 0.43 and0.42 mg g�1 at 60 and 90 days, respectively. The Cryproteins were also detected in plant debris 15 daysafter harvesting though no Bt proteins were foundin rice straw 30 days after harvest. The level ofexpression in different parts of transgenic plants wasalso compared and expression of Cry1Ac was thought tobe higher than that of Cry2A. Both genes gaveconstitutive expression; Cry proteins were quantified inroots, stem, panicle, straw, husk and seeds. Cry proteinswere also present in the rice kernel at a proportion of300 and 260 ng g�1 for Cry1Ac and Cry2A, respectively(Table 6). Transgenic plants secreted Cry1Ac proteininto the growth media, as quantified by sandwichELISA. The protein was present in MS media(1.5 ngml�1), hydroponic cultures (1.6 ngml�1), and soil(in traces) (Table 7).
4. Discussion
Engineering of genes encoding insecticidal crystallineproteins from Bacillus thuringiensis into crop plantscould dramatically reduce the use of conventionalbroad-spectrum pesticides against insect pests. There isalways a risk that insects could become resistant to Bt
toxins after prolonged and repeated field exposure. AsBt toxins are valuable natural resources, good manage-ment is essential for preserving the natural effectivenessof these toxins. The most practical approach to prolongthe effectiveness of Bt crops is a high dose/refugiastrategy and stacking of two or more genes into the samecultivar (Cohen et al., 2000a). Here we report the resultsof the first effort to check the potential of transgenicbasmati rice expressing two Bt genes under fieldconditions.
ts, planted at CEMB (2001)
Strawc Huskd Seedd Kerneld
570727 2073 490712 300717
9076 1072 400713 26079
ARTICLE IN PRESS
Table 7
Excretion of Bt protein in growing media
Gene Ms media
(ngml�1)
Hydroponic
culture (ngml�1)
Soil (ng g�1)
Cry1AC 1.570.2 1.670.2 Traces
Cry2A N/A N/A N/A
Values followed by the 7 represent the standard error of the mean
ðn ¼ 3Þ:N/A: Not applicable, these tests was done only for Cry1Ac only.
K. Bashir et al. / Crop Protection 24 (2005) 870–879 877
Different transgenic lines provided up to 100% and97.68% additional resistance against YSB at vegetativeand flowering stage, respectively, while 98% additionalresistance against RLF. Although whiteheads wereobserved in all transgenic lines expressing the two Bt
genes, we failed to recover any live larvae from stemcuttings. All the larvae recovered were dead and showedsymptoms of typical Bt toxicity. (Johnson andMcGaughey, 1996; Prieto-Samso0nov et al., 1997). Theefforts to recover the dead larvae were undertakenduring the second year only.
These lines provided built-in resistance against targetinsects at all stages of plant growth, in two seasons andat two different locations. The resistance of these lineswas similar at both locations except L-8-22-32, whichhad 4.81% whiteheads at RRIKSK as compared with avalue of 1.39% at CEMB. Resistance to differentLepidopterans has been reported in Japonica and indicarice expressing Bt toxins (Tu et al., 2000; Ye et al.,2001a,b), and one report described resistance againsteight Lepidopterans through expression of the cry1Abgene (Shu et al., 2000). Present results showed that linesexpressing two Bt genes are not only important forresistance management but also provide better protec-tion against economically important Lepidopterans ascompared with lines expressing one Bt gene. Recently,Zhao et al. (2003), reported results from a greenhousestudy showing that stacking two toxin genes withdifferent modes of action into plants offers a means ofachieving longer delays in the development of resistanceand mostly fit the predictions of simple genetic models.
The selection of lines expressing high dose titers isextremely important to delay resistance development.Toxin titer differs substantially among plant lines thatare transformed with the same Bt gene construct (Chenget al., 1998; Datta et al., 1998) and decline substantiallyat reproductive stages in rice (Alinia et al., 2000). As nocommercial Bt rice exists, (High et al., 2004) compar-isons with commercially released Bt cotton, maize andpotato cultivars can provide guidance about high toxintiter in Bt rice. Toxin titer in commercial high dosecultivars of Bt cotton, maize and potato range between 1and 11 mg g�1 of leaf fresh weight or 0.1–0.2% of totalleaf soluble proteins. Three released cultivars in USA
are considered to have a high toxin titer against alltarget insects, based on extensive experimental evalua-tion and experience in farmer fields (reviewed by Cohenet al., 2000a). For successful resistance management, it isimportant that the toxin titer be maintained throughoutthe growth season. It is also important that toxin titershould not be too high as it may affect agronomicperformance (Maqbool et al., 1998). Rice YSB attacksthe crop right from the seedling to the harvest stage.Rice crops have the capacity to compensate for stemborer injury at the vegetative stage through formation ofnew tillers (Rubia et al., 1990) but after the tilleringstage damage is difficult to compensate. Although thetoxin titer declined substantially in our lines, it remainedwell above the level required to reduce the number of thetarget insects and would be effective to control thedevelopment of resistance against Bt toxins. Toxin titerfor Cry1Ac was 6.06 mg g�1 of leaf fresh weight at 30days after transplanting, 4.54 mg g�1 at 60 days, and3.42 mg g�1 at 90 days. Expression of Cry2A was0.72 mg g�1 of leaf fresh weight at 30 days, 0.43 mg g�1
at 60 days, and 0.42 mg g�1 at 90 days. The level ofCry2A protein was up to 8.5 times less as compared withCry1Ac. The gene expression was constitutive as toxinswere quantified in different parts of the plants,specifically leaves, stem, roots, panicle, seeds and kernel.Although the promoters were not tissue specific,significant variation was recorded for Bt toxin quanti-fied in different parts of the plants. This may be due tothe difference in water and protein contents of differentparts. Some variation in different parts of the plants hasbeen reported (Bashir et al., 2004; Husnain et al., 2002).Low expression of Cry2A may be due to the fact that itwas under the control of CaMV 35S promoter.Ubiquitin promoter is reported to be more active thanCaMV 35S in transgenic rice plants (Koziel et al., 1993).It is also reported that younger tissue has higher levels ofprotein expression with use of the 35S promoter(Willianson et al., 1989).
Lines harboring two Bt genes were superior to linescontaining one Bt gene and the control with respect tothe average number of tillers, average plant height, daysto flowering and lodging incidence. The resistance tolodging may be due to increased insect resistancecoupled with reduction in plant height. The slightreduction in average number of tillers during the secondyear could be due to the fact that the plant-to-plant androw-to-row distance was decreased from 45 to 22 cm.Lines L-8-22, L-26-3 and L-26-8 had a 36–37%reduction in plant height. This growth pattern in linescontaining two Bt genes was similar to growth reductionrecorded in a greenhouse in earlier generations (data notshown). Reduction in plant height also contributedsignificantly to resistance to lodging in these lines. Shortplants are desirable from the breeding point of view andseveral reports indicated that transformation inherently
ARTICLE IN PRESSK. Bashir et al. / Crop Protection 24 (2005) 870–879878
reduced plant height (Jiang et al., 2000; Shu et al., 2002).Lines L-8-22, L-26-3 and L-26-8, all of which containedboth Bt genes, were up to 18 days earlier in flowering.On the other hand lines with only cry1Ac or cry2A werelate as compared with the control. It has already beendocumented that transgenic lines may differ in maturityfrom conventional control (Jiang et al., 2000). As insectattack is a continuous process during rice development,lines early in maturity are also important as these plantsare exposed to insect attack for comparatively shortperiod. Possible reasons for this morphological varia-tion may be somaclonal variation (Larkin and Scow-croft, 1981), breakdown of plant genes caused bytransgene insertion or insertion mutagenesis (Van etal., 1991), pleiotropy or transgene induced endogenoussilencing (Matzke et al., 2000). Somaclonal variationseems the most likely cause of these changes. The longerthe tissue culture time the higher the frequency ofsomaclonal variation as it took more time to producetransgenic plants as compared with normal tissueculture procedure (Kaeppler et al., 2000). Antibodies,such as hygromycin in this case, might also inducemutations in rice (Wu et al., 2000). No significantvariation was observed for other characteristics, such aspanicle length, spikelet fertility and flag leaf area. It wasappeared that the most affected characteristics were theaverage number of tillers, plant height and maturity.Yield of all transgenic lines was significantly superior tothe control in all infested plots, with the control showinga negative correlation between borer infestation andyield. Transgenics were up to 8% superior for yieldunder natural infestations alone, due to low frequencyof YSB and RLF. It is interesting that although thetransgenic lines were superior for total tillers andpanicles per plant, the yield potential seems to becomparable with the control. This may be due to thecombined effect of a reduction in panicle length andspikelet fertility coupled with a reduction in grain weight(data not shown).
The transgenic Bt rice released Bt toxins in MS media,hydroponic cultures and soil, which could be detectedthrough immunoassays. Although this phenomenon iswell characterized in transgenic Bt maize, the persistenceof these toxins should be studied to carefully address thebio-safety questions.
In summary, transgenic basmati lines expressed twounrelated Bt genes, cry1Ac and cry2A throughout theentire growth period. Three homozygous lines (L-8-22-2,L-8-22-32 and L-8-22-35) derived from a single parentalline provided high resistance against Lepidopterans untilharvest and were desirable in morphological character-istics, specifically, the average number of tillers, averageplant height, days to maturity and lodging incidence.Similar results were found at two different locations,which increase the reliability of data. Although thesecretion of Bt proteins from plants into media was
detected, the chances of uptake of these proteins werenegligible. On the basis of these data, these lines aresuitable candidates to be released as the first indicabasmati rice expressing two Bt genes.
References
Alinia, F., Ghareyazie, B., Rubia, L.G., Bennett, J., Cohen, M.B.,
2000. Effect of plant age, larval age, and fertilizer treatment on
resistance of a cry1Ab transformed aromatic rice to lepidopterous
stem borers and foliage feeders. J. Econ. Entomol. 93, 484–493.
Bashir, K., Husnain, T., Fatima, T., Latif, Z., Mehdi, S.A., Riazuddin,
S., 2004. Field evaluation and risk assessment of transgenic indica
basmati rice. Mol. Breed. 13, 301–312.
Bennett, J., Bentur, J.S., Pasalu, I.C., Krishnaiah, K. (Eds.), 2000.
New Approaches to Gall Midge Resistance in Rice. International
Rice Research Institute, Makati City (Philippines).
Cheng, X., Sardana, R., Kaplan, H., Altosaar, I., 1998. Agrobacter-
ium-transformed rice plants expressing synthetic cryIA(b) and
cryIA(c) genes are highly toxic to striped stem borer and yellow
stem borer. Proc. Natl. Acad. Sci. USA 95, 2767–2772.
Cohen, M.B., Gould, F., Bentur, J.S., 2000a. Bt. rice: practical steps to
sustainable use. Int. Rice Res. Notes 25 (2), 4–10.
Cohen, M.B., Romena, A.M., Gould, F., 2000b. Dispersal by larvae of
the stem borers Scirpophaga incertulas (Lepidoptera: Pyralidae)
and Chilo suppressalis (Lepidoptera:Crambidae) in plots of
transplanted rice. Environ. Entomol. 29 (5), 958–971.
Crowther, J.R., 1995. ELISA; Theory and Practice in Method in
Molecular Biology, vol. 42. Humana Press, Totowa, N.J., p. 223.
Datta, K., Vasquez, A., Tu, J., Torrizo, L., Alam, M.F., Oliva, N.,
Abrigo, E., Khush, G.S., Datta, S.K., 1998. Constitutive and
tissue-specific differential expression of the cryIA(b) gene in
transgenic rice plants conferring resistance to rice insect pests.
Theor. Appl. Gene 97, 20–30.
Fiuza, L.M., Nielsen-Leroux, C., Goz, E., Frutos, R., Charles, J.F.,
1996. Binding of Bacillus thuringiensis Cry1 toxins to the midgut
brush border membrane vesicles of Chilo suppressalis (Lepidop-
tera: Pyralidae): evidence of shared binding sites. Appl. Environ.
Microbiol. 62, 1544–1549.
Food and Agriculture Organization of the United Nations, 1995. FAO
Quarterly Bulletin of Statistics 8, 1–2.
Frutos, R., Rang, C., Royer, M., 1999. Managing insect resistance to
plants producing Bacillus thuringiensis toxins. Crit. Rev. Biotech-
nol. 19, 227–276.
Fujimoto, H., Itoh, K., Yamamoto, M., Kayozuka, J., Shimamoto,
K., 1993. Insect resistant rice generated by a modified delta
endotoxin genes of Bacillus thuringiensis. Biotechnology 11,
1151–1155.
Heinrichs, E.A., 1986. Perspectives and directions for the continued
development of insect-resistant rice varieties. Agric. Ecosyst.
Environ. 18, 9–36.
High, S.M., Cohen, M.B., Shu, Q.Y., Altosaar, I., 2004. Achieving
successful deployment of Bt rice. Trend. Plant Sci. 9 (6), 286–292.
Husnain, T., Jan, A., Maqbool, S.B., Datta, S.K., Riazuddin, S., 2002.
Variability in expression of insecticidal Cry1Ab gene in indica
basmati rice. Euphytica 128, 121–128.
Husnain, T., Bokhari, S.M., Riaz, N., Fatima, T., Shahid, A.A.,
Bashir, K., Jan, A., Riazuddin, S., 2003. Pesticidal genes of Bacillus
thuringiensis in transgenic rice technology to breed insect
resistance. Pak. J. Biochem. Mol. Biol. 36 (3), 133–142.
Jiang, J., Linscombe, S.D., Wang, J., Oard, J.H., 2000. Field
evaluation of transgenic rice (Oryza sativa L.) produced by
agrobacterium and particle bombardment methods. Plant and
Animal Genome VIII Conference, Town and Country Hotel, San
Diego, CA, January 9–12.
ARTICLE IN PRESSK. Bashir et al. / Crop Protection 24 (2005) 870–879 879
Johnson, D.E., McGaughey, W.H., 1996. Contribution of Bacillus
thuringiensis spores to toxicity of purified cry proteins towards
Indianmeal moth larvae. Curr. Microbiol. 33, 54–60.
Kaeppler, S.M., Kaeppler, H.F., Rhee, Y., 2000. Epigenetic aspects of
somaclonal variation in plants. Plant. Mol. Biol. 43, 179–188.
Khan, Z.R., Litsinger, J.A., Barrion, A.T., Villanueva, F.F.D.,
Fernandez, N.J., Taylor, L.D., 1991. World Bibliography of Rice
Stem Borers. IRRI, Los Banos, Philippines, pp. 1794–1990.
Koziel, M.G., Beland, G.L., Bowman, C., Carozzi, N.B., Crenshaw,
R., Crossland, L., Dawson, J., Desai, N., Hill, M., Kadwell, S.,
Launis, K., Lewis, K., Maddox, D., McPherson, K., Meghji, M.R.,
Merlin, E., Rhodes, R., Warren, G.W., Wright, M., Evola, S.V.,
1993. Field performance of elite transgenic maize plants expressing
an insecticidal protein derived from Bacillus thuringiensis. Biotech-
nology 11, 194–200.
Labra, M., Savini, C., Bracale, M., Pelucchi, N., Columbo, L.,
Bardini, M., Sala, F., 2001. Genomic changes in transgenic rice
(Oryza sativa L.) plants produced by infecting calli with
Agrobacterium tumefaciens. Plant Cell Rep. 20, 325–330.
Lancashire, P.D., Bleiholder, H., Van Den Boom, T., Langeluddeke,
P., Stauss, R., Weber, E., Witzenberger, A., 1980. Standard
Evaluation System for Rice. IRRI, Los Banos, Philippines.
Larkin, P.J., Scowcroft, W.R., 1981. Somaclonal variation—A novel
source of variability from cell culture for plant improvement.
Theor. Appl. Genet. 60, 197–214.
Lee, M.K., Aguda, R., Cohen, M.B., Gould, F.L., Dean, D.H., 1997.
Determination of receptor binding properties of Bacillus thur-
ingiensis d-endotoxins to rice stem borer midguts. Appl. Environ.
Microbiol. 63, 1453–1459.
Maqbool, S.B., Husnain, T., Raizuddin, S., Christou, P., 1998. Effective
control of yellow rice stem borer and rice leaf folder in transgenic
rice indica varieties Basmati 370 and M 7 using novel q-endotoxincry2A Bacillus thuringiensis gene. Mol. Breed. 4, 501–507.
Matzke, M.A., Mette, M.F., Matzke, A.J.M., 2000. Transgene
silencing by the host genome defense: implications for the evolution
of epigenetic control mechanism in plants and vertebrates. Plant.
Mol. Biol. 43, 401–415.
National Biosafety Committee, Pakistan, 1999. Biosafety Guidelines in
Genetic Engineering and Biotechnology. Ministry of Environ.,
Local Govt. Rural Develop. Govt. Pakistan.
Pathak, M.D., Khan, Z.R., 1994. Insect Pests of Rice. International
Rice Research Institute, Los Banos, Philippines, pp. 15–20.
Prieto-Samso0nov, D.L., Va0zquez-Padro0n, R.I., Ayra-Pardo, C.,
Gonza0lez-Cabrera, J., de la Riva, G.A., 1997. Bacillus thuringien-
sis: from biodiversity to biotechnology. J. Indust. Microbiol.
Biotechnol. 19, 202–219.
Rubia, E.G., Shepard, B.M., Yambao, E.B., Ingram, K.T., Arida,
G.S., Penning de Vries, F.W.T., 1990. Stem borer damage and
grain yield of flooded rice. J. Plant Protec. Tropics 6, 205–211.
Salim, M., Masih, R., 1987. Efficacy of insecticides against rice stem
borer at NARC, Islamabad. Pak. J. Agric. Res. 4, 477–479.
Saxena, D., Stotzky, G., 2000. Insecticidal toxin from Bacillus
thuringiensis is released from the roots of transgenic corn in vitro
and in situ. FEMS Microbiol. Ecol. 33, 35–39.
Saxena, D., Stotzky, G., 2001. Bt toxin uptake from soil by plants.
Nature Biotech. 19, 199.
Saxena, D., Flores, S., Stotzky, G., 1999. Insecticidal toxin in root
exudates from Bt corn. Nature 402, 480.
Saxena, D., Flores, S., Stotzky, G., 2002. Bt toxin is released in root
exudates from 12 transgenic corn hybrids representing three
transformation events. Soil Biol. Biochem. 34, 133–137.
Schuh, W., Nelson, M.R., Bigelow, D.M., Orum, T.D., Orth, C.E.,
Lynch, P.T., Eyles, P.S., Blackhall, N.W., Jones, J., Cocking, E.C.,
Davey, M.R., 1993. The phenotypic characterization of R2
generation transgenic rice plants under field conditions. Plant Sci.
89, 69–79.
Shu, Q., Ye, G., Cui, H., Cheng, X., Xiang, Y., Wu, D., Gao, M., Xia,
Y., Hu Cui, Sardana, R., Altossar, I., 2000. Transgenic rice plants
with a synthetic cry1Ab gene from Bacillus thuringiensis were
highly resistant to eight Lepidopteran rice pest species. Mol. Breed.
6, 433–439.
Shu, Q., Cui, H.R., Ye, G., Wu, D., Xia, Y.W., Gao, M.W., Altosaar,
I., 2002. Agronomic and morphological characterization of
Agrobacterium-transformed Bt rice plants. Europhysics 127 (3),
345–352.
Tapp, H., Stotzky, G., 1995. Insecticidal activity of the toxin from
Bacillus thuringiensis subsp kurstaki and tenebrionis adsorbed and
bound on pure and soil clays. Appl. Environ. Microbiol. 61,
1786–1790.
Tapp, H., Stotzky, G., 1998. Persistence of the insecticidal toxin from
Bacillus thuringiensis subsp kurstaki in soil. Soil Biol. Biochem. 30,
471–476.
Tu, J., Zhang, G., Datta, K., Xu, C., He, Y., Zhang, Q., Khush, G.S.,
Datta, S.K., 2000. Field performance of transgenic elite commer-
cial hybrid rice expressing Bacillus thuringiensis q-endotoxin.Nature Biotech. 18, 1101–1104.
Van, L.M., Vanderhaeghen, R., van, M.M., 1991. Insertional
mutagenesis in Arabidopsis thaliana Isolation of a T-DNA-linked
mutation that alters leaf morphology. Theor. Appl. Genet. 81,
277–284.
Willianson, J.D., Hirsch-Wyncott, M.E., Larkins, B.A., Gelvin, S.B.,
1989. Differential accumulation of a transcript driven by the
CaMV 35S promoter in transgenic tobacco. Plant Physiol. 90,
1570–1576.
Wu, G., Cui, H.R., Shu, Q., Xia, Y.W., Xiang, Y.B., Gao, M.W.,
Cheng, X., Altosaar, I., 2000. Striped stem borer (Chilo suppressa-
lis) resistant transgenic rice with a cry1Ab gene from Bt (Bacillus
thuringiensis) and its rapid screening. J. Zhejiang Univ. 19 (3),
15–18.
Ye, G., Shu, Q., Yao, H., Cui, H.R., Cheng, X., Hu, C., Xia, Y., Gao,
M.W., Altosaar, I., 2001a. Field evaluation of resistance of
transgenic rice containing a synthetic cryIAb gene from Bacillus
thuringiensis Berliner to two stem borers. J. Econ. Entomol. 94 (1),
271–276.
Ye, G.Y., Tu, J., Datta, K., Datta, S.K., 2001b. Transgenic IR72 with
fused Bt Gene cry1Ab/cry1Ac from Bacillus thuringiensis is
resistant against four Lepidopteran species under field conditions.
Plant Biotech. 18 (2), 125–133.
Zhao, J., Cao, J., Li, Y., Collins, H.L., Roush, R.T., Earle, E.D.,
Shelton, A.M., 2003. Transgenic plants expressing two Bacillus
thuringiensis toxins delay insect resistance evolution. Nature
Biotech. 21, 1493–1497.