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Normal Expression of Insect-resistant Transgene in Progeny

Normal Expression of Insect-resistant Transgene in Progeny

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Theor Appl Genet (2009) 119:635–644DOI 10.1007/s00122-009-1075-5
 1 3
ORIGINAL PAPER
Normal expression of insect-resistant transgene in progeny of common wild rice crossed with genetically modi
W
ed rice: its implication in ecological biosafety assessment
Hui Xia · Bao-Rong Lu · Jun Su · Rui Chen · Jun Rong · Zhiping Song · Feng Wang
Received: 7 June 2008 / Accepted: 15 May 2009 / Published online: 6 June 2009
󰂩
 Springer-Verlag 2009
Abstract
Transgene out
X
ow from genetically modi
W
ed(GM) rice to its wild relatives may cause undesirable eco-logical consequences. Understanding the level of transgeneexpression in wild rice following gene
X
ow is important forassessing such consequences, providing that transgeneescape from GM rice cannot be prevented. To determinethe expression of a transgene in common wild rice (
Oryzaru
 W
 pogon
), we analyzed the content of Cry1Ac protein inthree GM rice lines containing a
 Bt 
 transgene, their F
1
hybrids with common wild rice and F
2
 progeny at di
erentgrowth stages, using the sandwich enzyme-linked immuno-sorbent assay. The average content of Cry1Ac protein inleaf samples of the wild rice lines ranged between 0.016and 0.069% during the entire growth period, whereas thatin stems varied between 0.12 and 0.39%. A great variationin Cry1Ac protein content was detected among individualsof F
1
 hybrids and F
2
 progeny, with some wild individualsshowing higher level of
 Bt 
 toxin than the cultivated GMrice. The results suggest that the
 Bt 
 transgene can expressnormally in the interspeci
W
c hybrids between insect-resistantGM rice and common wild rice, and may have similare
ects on the target insects as in GM rice.
Introduction
The extensive environmental release and commercial pro-duction of genetically modi
W
ed (GM) crops with noveltraits have increasingly aroused ecological biosafety con-cerns and debates worldwide (Snow 2002; Lu and Snow2005; Hails and Morley 2005). One of the major ecological biosafety concerns is the escape of genetically engineeredgenes (transgenes) through cross-pollination into wild orweedy relatives of GM crops. Transgenes with evolutionaryselective advantage dispersed into weedy or wild popula-tions may result in
W
tness changes of individuals that havepicked up the transgenes, causing unwanted ecological con-sequences (Snow and Palma 1997; Riches and Valverde2002). These include the promotion of weed populations inagro-ecosystems (Darmency 1994; Halfhill etal. 2005), in
X
uences on genetic integrity of wild gene pool (Gepts andPapa 2003), and non-target e
ects (Poppy 2000; Hellmichetal. 2001). To understand the potential ecological conse-quences caused by transgene escape, questions concerningthree successive steps should be addressed: (1) At what fre-quency can a transgene disperse from GM crops into theirwild relative populations through gene
X
ow? (2) At whatlevel will a transgene express and inherit after being incor-porated by wild relatives? (3) How will a transgene changethe
W
tness of wild individuals that have incorporated thetransgene, and the surviving/competitive ability of wildpopulations? Knowledge about each of these questions willprovide the scienti
W
c bases for biosafety assessment of potential ecological consequences caused by transgeneout
X
ow to wild relatives (Lu and Snow 2005).
Communicated by M. Bohn.H. Xia · B.-R. Lu (
&
) · J. Rong · Z. SongThe Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, Fudan University, 200433 Shanghai, Chinae-mail: brlu@fudan.edu.cnJ. Su · R. Chen · F. Wang (
&
)Fujian Province Key Laboratory of Genetic Engineering for Agriculture, Fujian Academy of Agricultural Sciences, 350003 Fuzhou, Chinae-mail: wf@fjage.org
 
636Theor Appl Genet (2009) 119:635644
 1 3
Rice (
Oryza sativa
 L.) is an important world cereal cropthat provides staple food for nearly one-half of the globalpopulation (Lu and Snow 2005). Biotechnology researchand development have long been applied in rice and greatprogress has been made in the past decades. Many coun-tries (e.g., China, India, Vietnam, and Iran) have investedenormously in research and development of GM rice, andconsequently, a large number of GM rice lines have beendeveloped (Lu and Snow 2005; Wang and Johnston 2007). In 2005, Iran became the
W
rst country in the world thatcommercialized
 Bt 
 transgenic rice (James 2006). Recently,the USA has approved the non-regulated cultivation of twoherbicide-tolerant rice varieties, “Liberty Link 
®
 Rice”(LLRICE06 and LLRICE62), containing a
bar 
 gene (see:US Regulatory Agencies Uni
W
ed Biotechnology Website,http://usbiotechreg.nbii.gov/database_pub.asp). As thelargest rice producing and consuming country, China isalso actively exploring transgenic biotechnology for theimprovement of rice varieties (Wang and Johnston 2007).In fact, a large number of GM rice lines have been devel-oped in China, and some are under preparation (includingbiosafety assessment) for commercialization (Xiong 2004).However, due to the strict biosafety assessment protocols,commercial production of GM rice has been signi
W
cantlydelayed in China. As one of the centers of origin and diver-sity for cultivated rice and the northernmost distributionregion of wild
Oryza
 species, China has taken a precautiousbiosafety approach to assess potential ecological conse-quences caused by transgene out
X
ow. Given that gene
X
owfrom cultivated rice to wild or weedy rice is unavoidableunder normal cultivation practices where wild rice exists(Ellstrand etal. 1999; Song etal. 2003; Chen etal. 2004), it is important to determine subsequent levels of transgeneexpression in wild rice and thereafter the
W
tness change inwild rice populations that contain a transgene, as a part of the biosafety assessment for ecological consequences.The insect-resistance
 Bt 
 (
 Bacillus thuringiesis
) gene hasbeen widely used in crop improvement through geneticengineering. Insect-resistant GM crops include
 Bt 
 cotton(Hilder and Boulter 1999; Pray etal. 2002),
 Bt 
 maize(Alcalde and Camara 2007; Andersen etal. 2007),
 Bt 
 cab-bage (Bhattacharya etal. 2002), and
 Bt 
 rice (Nayak etal.1997; Tu etal. 2000; High etal. 2004). One type of
 Bt 
transgenes that encodes the Cry1Ac toxin is e
ective to killthe larvae of a number of rice lepidopteran insects, such asrice stem borers (
Sesamia nonagrioides
,
Scirpophagaincertulas
, and
Chilo
 
suppressalis
) and rice lea
olders(
Cnaphalocrocis medinalis
). These target rice herbivoresoccur frequently in cultivated rice and they are also foundin some populations of common wild rice (Cohen etal.2008). These pests mainly attack stems (by rice stemborers) and leaves (by lea
olders) of rice plants after the jointing stage (when plants stop major tilling and start toelongate rapidly), causing yield losses up to
»
20% for cul-tivated rice (Muralidharan and Pasalu 2006). Studies havedemonstrated that transgenic
 Bt 
 rice can e
ectively reducedamage from the target insect pests (Khanna and Raina2002; Huang etal. 2005; Han etal. 2007). If the
 Bt 
 trans-gene disperses from GM rice into wild rice individuals andexpressed normally in wild rice plants, it may provide aselective advantage to the wild rice populations exposed tolepidopteran insects, resulting in unwanted ecological con-sequences (Cohen etal. 2008). In addition, from the germ-plasm point of view, wild rice is the reservoir of manybene
W
cial genes for cultivated rice improvement. If thetransgene spreads into wild rice populations, providingresistance against these insects, other useful genes, forexample those giving resistance to the insects naturally,may be lost over time in the wild rice populations.Evidence has shown that
 Bt 
 transgenes can express sta-bly in wild
 Brassica
 hybrids after the transgenes wereincorporated into wild relatives (Halfhill etal. 2002; Zhuetal. 2004; Ammitzbøll etal. 2005). Whether the
 Bt 
 trans-gene that introgresses into individuals of common wild rice(
Oryza ru
 W
 pogon
) will express normally is still unknown,and this knowledge gap will hinder the appropriate predic-tion of potential ecological consequences caused by
 Bt 
transgene escape to wild rice relatives. Common wild riceis the putative ancestor of cultivated rice, and widely dis-tributed in most tropical regions in Asia where cultivatedrice is grown. It is also found in northern Australia, LatinAmerica, and Africa as an introduced species (Naredo etal.1997; Lu and Snow 2005; Vaughan etal. 2005a). In some rice growing countries such as India, Vietnam, Cambodia,and Malaysia, common wild rice occurs abundantly and is anoxious weed infesting rice
W
elds (Vaughan etal. 2005b).In this study, we applied the sandwich enzyme-linkedimmunosorbent assay (ELISA) method to measure thecontent of Cry1Ac protein in F
1
 hybrids of common wildrice crossed with
 Bt 
 GM rice lines, and their F
2
 progenyproduced in the course of our GM rice biosafety research.The sandwich ELISA has been proven to be an e
ectivemethod for quantifying the
 Bt 
 Cry1Ac protein (Sims andBerberich 1996; Takahashi etal. 1998) and estimating
 Bt 
transgene expression in vivo organism, such as cotton(Adamczyk and Sumerford 2001) and rice (Qin etal.2003; Bashirn etal. 2005). The objectives of this study were to determine the level and variation pattern betweentissues and growth stages of
 Bt 
 
Cry1Ac
 transgene expres-sion in hybrid lines of common wild rice crossed with cul-tivated GM rice, compared to the GM rice parents. Thisknowledge will facilitate our decision making on furtherecological risk assessment of transgene out
X
ow from GMrice to its wild relatives.
 
Theor Appl Genet (2009) 119:635644637
 1 3
Materials and methods
Plant materialsThree double-inserted insect-resistant GM rice lines(KeFeng6, Ba23, and Ba28) were used in the experiment totest the same target transgenes in three di
erent geneticbackgrounds (Table1). Two insect-resistance transgenes,i.e.,
 Bacillus thuringiesis
 (
 Bt 
) and cowpea trypsin inhibitor(
CpTI 
), were transferred into the three GM rice lines andtightly linked with the hygromycin resistance gene (
hpt 
),used as the selectable marker.
 Bt 
 was under the control of the maize ubiquitin (
Ubi
) promoter and encodes theCry1Ac protein (toxin) that kills lepidopteran larvae.KeFeng6 was produced from Minghui-86, a traditional ricevariety, using
 Agrobacterium
-mediated transformationtechnology and sel
W
ng to the T7 generation until individu-als of the line with stable insect resistance and geneticinheritance (Chen etal. 2006). Ba23 was bred from thebackcrossed lines of KeFeng6
£
F96110 (a commercialrice variety), and Ba28 was selected from the backcrossedlines of KeFeng6 with a complex rice variety having threeparents (Duoxi1/W311/Minghui-69). These two transgeniclines (Ba23 and Ba28) were homozygous for
 Bt 
, whichshowed stable inheritance and agronomic performance.Four genotypes of common wild rice (
O.
 
ru
 W
 pogon
),coded as R1, R2, R3, and R4, and collected from di
erentlocalities (Table1), were used for producing F
1
 hybrids andF
2
 progeny with the GM rice lines. The four genotypes of common wild rice were unrelated, and therefore repre-sented di
erent genetic backgrounds based on the analysisof simple sequence repeat (SSR) molecular markers(unpublished data). One GM parent and its hybrid descen-dents developed from the same wild rice genotypes werede
W
ned as a family (e.g., KF-6, F
1
-K/R1, and F
2
-K/R1). Atotal of 13 lines (one type of GM parent or F
1
 hybrid or F
2
progeny) belonged to
W
ve families including GM rice par-ents, F
1
 hybrids, and F
2
 progeny were obtained for thetransgene expression experiment (Table1). To guaranteeall the hybrids and F
2
 progeny included in the experimentcontained the
 Bt 
 transgene, the experimental materials weresubjected to PCR analyses using the speci
W
c primer pairsfor amplifying the
 Bt 
 transgene, according to Rong etal.(2005).Experimental designA total of 14 experimental lines (treatments), each withthree replicates, were included in the experiment: three GMrice parents,
W
ve F
1
 hybrids,
W
ve F
2
 progeny, and one
Table1
Genetically modi
W
ed (GM) insect-resistant rice lines (containing
 Bt/CpTI 
 transgenes) and interspeci
W
c hybrids and F
2
 progeny with common wild rice (
Oryza ru
 W
 pogon
) from di
erent localities in the ELISA (enzyme-linked immunosorbent assay) experiment for transgene (
 Bt 
) expressionParent and hybrid progenyCodeNotesParentsKeFeng-6KF-6This material is T7 generation, containing tightly linked double-inserted homozygous
 Bt 
 / 
CpTI 
 genesBa-23Ba-23Bred from KF-6, homozygous for
 Bt 
 / 
CpTI 
Ba-28Ba-28Bred from KF-6, homozygous for
 Bt 
 / 
CpTI 
Minghui-86M-86Non-transgenic cultivated rice, used as blank control
Oryza ru
 W
 pogon
R1, R2, R3, R4Four plant individuals with di
erent genotypes are included. They were coded as R1, R2, R3 and R4F
1
 hybrid (
#
£
)KF-6
£
R1F
1
-K/R1R1 was collected from Suixi, Guangdong Province, China, hemizygous for
 Bt 
 / 
CpTI 
KF-6
£
R2F
1
-K/R2R2 was collected from Huilai, Guangdong Province, China, hemizygous for
 Bt 
 / 
CpTI 
KF-6
£
R3F
1
-K/R3R3 was collected from Dongxiang, Jiangxi Province, China, hemizygous for
 Bt 
 / 
CpTI 
Ba-23
£
R1F
1
-Ba23/R4Hemizygous of
 Bt 
 / 
CpTI 
Ba-28
£
R4F
1
-Ba28/R1R4 was collected from Suixi, Guangdong Province, China, hemizygous for
 Bt 
 / 
CpTI 
F
2
 progenyKF-6
£
R1F
2
-K/R1Mixed with homozygous and hemizygous individualsKF-6
£
R2F
2
-K/R2KF-6
£
R3F
2
-K/R3Ba-23
£
R1F
2
-Ba23/R4Ba-28
£
R4F
2
-Ba28/R1A non-GM rice parental variety (Minghui-86) was used as blank control

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