Molecular Plant Breeding (online), 2011, Vol. 2 http://mpb ...

Molecular Plant Breeding (online), 2011, Vol. 2http://mpb.sophiapublisher.com

Molecular Plant Breeding (online), 2011, Vol. 2ISSN 1923-8266http://mpb.sophiapublisher.comLatest ContentGenetic Diversity of Involved Varieties and Improvement of Elite Restorer of Indica Rice (Oryza sativa L.) UsingBackcross Introgression 1-7Jinteng Cui, Bingxu Chen, Yingyao Shi, Rong Zhang, Hui Wang, Yiliang Qian, Haiyan Liu, Linghua Zhu, Zhikang Li, YongmingGaoRapid Acquirement of Transgenic Rice Plants Derived from Callus of Mature Embryos Transformed byAgrobacterium Mediation 8-13Gang Guo, Jing Yu, Degang ZhaoEctopic expression of an AGAMOUS homolog NTAG1 from Chinese narcissus accelerated earlier flowering andsenescence in Arabidopsis 14-21Xinjie Deng, Lijun Xiong, Yang Wang, Xiaofang LiGenetic Diversity of the Selected 64 Potato Germplasms Revealed by AFLP Markers 22-29Fang Wang, Fangdi Lim Jian Wang, Yun Zhou, Haihong SunIntegrating the hrap Gene from Sweet Pepper into Potato Enhances Resistance to Phytopthora infestans 30-36Xianqun Huang, Minhua Jiang, Jian Chen, Zhenyu Zhu, Li Li, Yingping Dong, Zhongping LinPreliminary Mapping of Soybean Dominant Locus Hrcs7 Conferring Resistance to Cerocospara sojina Race 737-40Zhimin Dong, Shuming Wang, Jia Liu, Zhi Li, Zhigang YiGenetic Diversity of 30 Cai-xins (Brassica rapa var. parachinensis) Evaluated Based on AFLP Molecular Data41-47Weidong Shi, Ruikui Huang, Shengmao Zhou, Faqian XiongIntegration and Expression Stability of Transgenes in Hybriding Transmission of Transgenic Rice Plants Producedby Particle Bombardment 48-59Yan Zhao, Longbiao Guo, Huizhong Wang, Danian HuangEvolution of the Genes Encoding Starch Synthase in Sorghum and Common Wheat 60-67Xiaoxue Pan, Hongbo Yan, Meiru Li, Guojiang Wu, Huawu JiangQTL Detection for Water-soluble Oligosaccharide Content of Grain in Common Wheat 68-74Xiyang Fu, Zhaoliang Qi, Sishen LiNERICA: A Hope for Fighting Hunger and Poverty in Africa 75-82Tondi Yacouba Nassirou, Yuqing HeGenetic Diversity of Coconut Cultivars in China by Microsatellite (SSR) Markers 83-91Xiaolei Liu, Hua Tang, Dongdong Li, Liheng Hou

Molecular Plant Breeding (online), 2011, Vol. 2ISSN 1923-8266http://mpb.sophiapublisher.comQTL Analysis of Plant Height based on Doubled Haploid (DH) Population derived from PTSMS Wheat 92-97Liping Zhang, Xiaoqin Xu, Chanping Zhao, Fuhua Shan, Shaohua Yuan, Qun XiangOMICS Based Strategies for Efficient Accumulation of Silicon in Rice to Enhance Its Tolerance againstEnvironmental Stresses 98-100Sajad Majeed Zargar, Muslima Nazir, Ganesh Kumar Agarwal, Randeep RakwalBioinformatics Analysis on Ribulose-1,5-bisphosphate Carboxylase/ Oxygenase Large Subunits in DifferentPlants 101-108Biaojin Zhang, Linguang Luo, Xiangxi Zhang, Ruili Li, Yan Song, Dawen Zhang, Yuanyuan Nie, Yanbing Zeng, Qiegen Liao, YihuaWeiCloning of SCA Gene Related to Pollen Tube Adhesion and Oriented Growth and Analysis of Gene Diversity inLilium spp. 109-118Yuejuan Wu, Jinteng Cui, Kezhong Zhang, Yuehui JiaRecent Advances on in vitro Fertilization of Gamineae 119-122Zhong-an Wang

Research LetterMolecular Plant Breeding 2011, Vol.2, No.08, 48-59http://mpb.sophiapublisher.comOpen AccessIntegration and Expression Stability of Transgenes in Hybriding Transmissionof Transgenic Rice Plants Produced by Particle BombardmentYan Zhao 1,2 , Longbiao Guo 2 , Huizhong Wang 3 , Danian Huang 21. College of Food Science and Biotechnology Engineering, Zhejiang Gongshang University, Hangzhou, 310035, P.R. China2. State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, 310006, P.R. China;3. College of Life and Environmental Science, Hangzhou Normal University, Hangzhou, 310018, P.R. ChinaCorresponding author email: yanzhao9918@163.com; AuthorMolecular Plant Breeding, 2011, Vol.2 No.08 doi: 10.5376/mpb.2011.02.0008Received: 26, Apr., 2011Accepted: 14, Jun., 2011Published: 20, Jun., 2011This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Preferred citation for this article:Zhao et al., 2011, Integration and Expression Stability of Transgenes in Hybriding Transmission of Transgenic Rice Plants Produced by Particle Bombardment,Molecular Plant Breeding Vol.2 No.08 (doi: 10.5376/mpb.2011.02.0008)Abstract Four transgenic rice lines TR 5, TR 6, Ming B and Jingyin 119 obtained via particle bombardment were used as transgenedonors to create hybrids. The integration and expression stability of exotic bar and cecropin B gene in conventional hybridingtransmission were investigated by Southern and Northern blotting analyses. The selection marker bar gene was transferred to allhybrids under selection of Basta herbicide. Loss or gain of small hybridization bands (no more than 2.0 kb) of bar gene occurred insome hybrids, but the difference in integration sites of bar gene copies did not influence their stable expression. The non-selectiongene cecropin B was stably transferred from the four transgene donors to their resulting hybrids, but expression level was verydifferent. Silencing of cecropin B gene occurred in some hybrids from TR 5, TR 6 and Ming B. In the transgene donor Jingyin 119and all its resulting hybrids, cecropin B and bar gene were stably expressed. We concluded that the stability of transgene duringcrossbreeding transmission is mainly determined by the primary transgenic donors and may be affected by recombination.Keywords Transgene; Heredity and expression; Hybriding transmission; Particle bombardment; RiceBackgroundRice is the world’s most important food crop and aprimary source of food for more than half the world’spopulation (Khush, 2005). Improvements in crop yieldhave been achieved through conventional hybridizationand selection procedures (Peng et al., 2007). Currently,rice hybrids are cultivated in about 55% of therice-growing areas in China and contribute to 66% ofthe total rice production of the whole country (Wu andLuo, 2007). The breakthrough in plant biotechnologyhas provided tools to develop more elite transgenicdonors for crop crossbreeding. In the transformationprocess, ideally, a single gene with its appropriatecontrolling elements is added to the genome instead ofmoving a whole chromosome or chromosomesegment with many known and unknown genes asdone with the traditional hybridizations (Horvath et al.,2001). Since protocols for rice transformation havebeen well established, genetic transformation iscurrently complementing conventional breedingprograms in the development of advanced germplasm(Popelka and Altpeter, 2003). In 1996, the herbicideresistantbar gene was transformed into Japonica riceJingyin 119 via particle bombardment (Huang et al.,1996), and then the transgenic plants weresuccessfully crossed to the male-sterile line Pei-ai 64Sof a two-line crossbreeding system for creatingtransgenic rice hybrids. Based on the herbicideresistance of the marker gene bar, a new method wascreated to examine and improve the purity of hybridrice (Huang et al., 1998). More recently, Chen et al.introduced the synthetic cryl2A* and crylC* gene intoan elite Indica cytoplasm male-sterile restorer lineMinghui 63 and crossed the transgenic lines toZhenshan 97A to produce insect-resistant hybrid rice(Chen et al., 2005; Tang and Lin, 2007). Transgenictechnology is providing new tools for the conventionalcrossbreeding of rice.The main methods used for rice transformation areparticle bombardment and Agrobacterium-mediated48

Molecular Plant Breeding 2011, Vol.2, No.8, 48-59http://mpb.sophiapublisher.comtransformation. Although the generation of transgenicplants is relatively easy for many rice varieties, thetransformation frequency is usually low and rathergenotype-dependent. Also, these gene deliverytechniques need undergo the obligatory processes oftissue culture, which often results in phenotypicabnormalities and reduced fertility of the transgenicplants obtained (Zhang et al., 2005). Like mutantsproviding useful traits, transgenic plants often have tobe used for relocating the gene in more suitablegenotypes (Horvath et al., 2001). Thus, the success ofplant genetic manipulation not only requires the stableinheritance and expression of transgenes in thetransgenic plants across generations, but also dependson whether the transgenic plant can be used as atransgene donor in recombination crossbreeding.Many studies have analysesed the progenies of theprimary rice transformants, revealing that transgenestability was significantly related to differences intransgene structure and expression levels betweentransgenic lines, particularly in transgenic plantsderived from direct DNA transfer such as particlebombardment (Vain et al., 2002; Altpeter et al., 2005).In transgenic cereals, more than 50% of transgenescan be inactivated over successive generations (Iyer etal., 2000). These problems make molecular geneticstudies difficult, and frustrate attempts at cropimprovement through genetic engineering. Additionally,they create difficulties in predicting transgenebehavior when transgene needs to be transferred byconventional crossing (Vain et al., 2002). Altpeter etal. (2005) speculated that particle bombardment mightbe advantageous over Agrobacterium-mediated transformationin respect of transferring the transgenes intoa new genetic background via traditional breeding,because by particle bombardment multiple transgenesare tend to be integrated into the same locus. But thereare few direct evidences for this question up to date.We introduced the plasmid pCB 1 carrying the selectedherbicide-resistant bar gene and the non-selectedcecropin B gene into four Japonica rice varieties viaparticle bombardment between 1996 and 1998. Bargene was introduced into rice plant for resistance tophosphinothricin (the active component of theherbicide Basta) and the cecropin B gene was used toresist a range of plant pathogenic bacteria includingXanthmomonas compestris pv oryzae, which leads torice leaf bacterial blight disease. With obviousphenotype and convenient detection, bar gene hasbeen proved to be a very useful marker to screentransgenic hybrids. In the past ten years, the elitetransgenic rice plants harboring bar and cecropin Bgene were selected as transgene donors to cross todifferent rice varieties. We constructed a population ofrice hybrids derived from multiple conventionalcrosses. Here we report the inheritance and expressionbehaviours of the foreign bar and cecropin B genesduring rice crossbreeding transfer.1 Results and Analysis1.1 Stability of transgene integration patterns inmono-cross transmissionThe stability of integration patterns for the selectedbar gene and non-selected cecropin B gene in thetransmission from transgenic donors to hybrid riceplants was investigated using genomic DNA Southernblotting analysis. Three transgene donors includingTR 5, TR 6, Ming B were used to produce hybrids, inwhich several bar gene copies were inherited as asingle transgenic locus when tested by Bastaresistance (Hua et al., 2003). The transgene integrationpatterns of transgene donor TR 5, TR 6, Ming B andtheir corresponding hybrids were analysed and shownin Figure 1A and 1B. Southern blotting resultsrevealed as follows: (i) The tightly linked bar andcecropin B gene in the original plasmid exhibiteddifferent integration patterns in the three transgenedonors, when hybridized with bar and cecropin Bprobe respectively, after genomic DNA was digestedwith Hind III, which cut once in plasmid pCB 1 . Therewere two hybridization bands of bar gene and threebands of cecropin B gene in transgene donor TR 5.Transgene donor Ming B had four bar genehybridization bands and three cecropin B genehybridization bands. TR 6 donor plant possessed sixhybridization bands of bar gene and five bands ofcecropin B gene. These indicated that bar andcecropin B might have different copies integrated inthe receptor genomes. (ii) In the self-pollinatedprogenies of transgenic rice hybrids, the integrationpatterns of non-selected cecropin B gene remained the49

Molecular Plant Breeding 2011, Vol.2, No.8, 48-59http://mpb.sophiapublisher.comsame as that of their corresponding transgene donors(Figure 1B). However, the integration patterns of bargene were changed in some hybrids (Figure 1A). Forexample, two bands of 1.5 kb and 2.0 kb in lengthwere lost from the progeny plants of cross lines TR6/CJN 2 and TR 6/Bing 95-13, comparing with theirtransgene donor TR 6. Two new hybridization bandsof bar gene, 1.5 kb and 2.0 kb in length, emerged inthe progeny plant of cross line TR5/CJN 3, comparingwith its transgene donor TR5. Although the TR5 lanewas loaded with less DNA, giving possibility of the1.5 kb and 2.0 kb bands of bar gene coming from itsparent TR5, these two new hybridiztion bands did notappear in the remaining other three hybrids from TR5,whoes lanes were loaded with more DNA (Figure 1A).These confirmed the conclusion that the 1.5 kb and2.0 kb hybridization bands of bar gene were created incross line TR5/CJN 3.Figure1 Integration and expression analyses of selected bar andnon-selected cecropin B gene in rice in mono crossing transmissionNote: A: Southern blot of bar gene: genomic DNA wasdigested with Hind III, which cut once in the plasmid pCB 1 andhybridized with DIG-labeled bar probe, comprising bar genecoding region and nos teminator (0.9 kb); B: Southern blot ofcecropin B gene: genomic DNA was digested with HindⅢ andhybridized with DIG- labeled cecropin B probe, comprisingcecropin B coding region and Pin terminator (1.12 kb); C:Southern blot of the intact of cecropin B gene: genomic DNAwas digested with HindⅢ and PstⅠ and hybridized withcecropin B probe, which generated a 1.12 kb fragment ascecropin B probe sequence; D: Northern blot analysis of thenon-selected cecropin B gene expression; Lane M: DNAmolecular weight markerIII (Roche); Lane P: Plasmid pCB 1control; Lane U: Untransformed rice plant control; Lane T:Jin-yin 119 transgenic rice plant positive control; Lane 1: TR 5transgene donor, Lane 2: TR 5/CJN 3; Lane 3: TR 5/CJ 601;Lane 4: TR 5/CJ 683; Lane 5: TR 5/Bing 97-267; Lane 6: MingB transgene donor; Lane 7: Ming B/Jia 59; Lane 8: Ming B/Jia60; Lane 9: Ming B/Xuzao; Lane 10: TR 6 transgene donor;Lane 11: TR 6/CJN 2; Lane 12: TR 6/Bing 95-13; Lane 13: TR5/TR 61.2 Stability of transgene integration patterns inmultiple crosses transmissionThe inheritance of transgene bar and cecropin B in thecourse of multiple crosses was revealed by DNASouthern blotting analysis, using Jingyin 119 line astransgene donor. This Jingyin 119 transgenic line hadfour bar gene loci and two cecropin B gene loci whenhybridized with their probe respectively, aftergenomic DNA was digested with HindⅢ, which cutonce in the plasmid pCB 1 (Figure 2A and 2B).Southern blotting results demonstrated that theintegration pattern of non-selected cecropin B genewas very stable in mono- and multiple crossbreedingtransmission (Figure 2B). All the progenies of hybridsshowed two hybridization bands of cecropin B gene,exactly the same as that of Jingyin 119 donor. But theintegration pattern of selected bar gene did not alwaysremain stable during crossbreeding transmission.Among the seventeen rice crosses, the integrationpattern of bar gene remained stable in eight hybridsand changed in the other nine lines, which lost twosmaller hybridization bands of bar gene, 1.6 kb and1.0 kb in length respectively (Figure 2A). Our formerresearch revealed that the transgenic integrationpatterns of Jingyin 119 transgene donor kept stable inself-pollination across generations, but bar andcecropin B gene showed different integration patterns50

Molecular Plant Breeding 2011, Vol.2, No.8, 48-59http://mpb.sophiapublisher.comwhen Southern-blotting analyses were conducted aftergenomic DNA digested with different enzymes (Huaet al., 2003). During crossing transmission the twosmaller hybridization bands of bar gene were lost insome hybrids while the other two bigger bands weretransmitted stably (Figure 2A and 2B).Figure 2 Integration and expression analyses of selected bar and nonselectedcecropin B gene in rice in multiple crossing transmissionNote: A: Southern blot of bar gene: genomic DNA wasdigested with HindIII and hybridized with DIG-labeled barprobe, comprising bar gene coding region and nos teminator(0.9 kb); B: Southern blot of cecropin B gene: genomic DNAwas digested with HindIII and hybridized with DIG- labeledcecropin B probe, comprising cecropin B coding region and Pinterminator (1.12 kb); C: Southern blot of the intact of cecropinB gene: genomic DNA was digested with HindⅢ and PstⅠand hybridized with cecropin B probe, which generated a 1.12kb fragment as cecropin B probe sequence; D: Northern blotanalysis of the non-selected cecropin B gene expression. Lane M:DNA molecular weight marker Ⅲ (Roche); Lane U:untransformed rice plant control; Lane 1: Jingyin 119 transgenedonor; Lane 2: C20/ Jingyin 119; Lane 3: Jingyin 119/57; Lane4: Jingyin 119/Bing 94-02; Lane 5: Jingyin 119/59; Lane 6:Jingyin 119/104; Lane 7: Jingyin 119/59//L97-55; Lane 8:Jingyin119/57//9522; Lane 9: Jingyin 119/390//S1; Lane 10:Jingyin 119/63//T951; Lane 11: Jingyin 119/Bing 94-02//T951;Lane 12: Jingyin 119/02//T951; Lane 13: Jingyin 119/63//390;Lane 14: Jingyin 119/59//DS4; Lane 15: Jingyin 119/503//T951;Lane 16: Jingyin 119/59//T951; Lane 17: Jingyin 119/57//DS4///L97-55; Lane 18: Jingyin 119/59//DS4/// Jingyin 119/31//9522We also noticed that disappearance of the two smallerfragments (1.6 kb and 1.0 kb) of bar gene did notdepend on the cross turns (Fig. 2A). For example, thecross hybrid of 119/Bing 94-02 kept the sameintegration pattern of bar gene as that of its transgenedonor Jingyin 119, but in its related re-cross hybridJingyin 119/Bing 94-02//T951, the 1.6 kb and 1.0 kbhybridization bands of bar gene were lost. Anotherhybrid rice line of cross Jingyin 119/59 lost the twosmaller fragments of bar gene, but in progenies of itsfour related multiple crossing hybrids, three crossingcombinations including Jingyin 119/59//L97-55,Jingyin 119/59//DS4 and Jingyin 119/59//DS4///Jingyin 119/31//9522 lost the two smaller fragments ofbar gene, the remaining one cross Jingyin119/59//T951 exhibited the same bar gene integrationpattern as that of the original Jingyin 119 donor. Therewas also one hybrid line Jingyin 119/57//DS4///L97-55 that carrying all the original four bar gene lociof its transgene donor after three crossing turns. Thesesuggested that loss of bar gene fragments intransmission of multiple crosses was not related tocrossing turns, but mainly depended on the primaryhybrid plants that were randomly selected as transgenedonors for the next crosses.Moreover, disappearance of the bar gene smallerfragments occurred in both hybrids when transgenedonor Jingyin 119 was male parent (C20/Jingyin 119)and female parent (e.g. Jingyin 119/59). Thisdemonstrated the bar gene smaller fragmentsharbouring in donor plant had equal chance to be lostthrough pollen and egg. Therefore, we speculated thatthe two smaller hybridization bands of bar gene inJingyin 119 transgene donor might integrate in oneseparate genomic locus from the other bigger ones andmore possibly, possess no expression activity.51

Molecular Plant Breeding 2011, Vol.2, No.8, 48-59http://mpb.sophiapublisher.com1.3 Stability of transgene expression in crossingtransmissionDuring the course of crossbreeding for generatingdifferent transgenic hybrid rice lines, all the hybridswere subject to Basta-resistance assay and only theresistant plants were selected. These ensured theexistence and expression of selected bar gene in allhybrids. What about the fate of the non-selectedcecropin B gene? The completeness of cecropin Bgene expression cassette in hybrid rice lines wasexamined by Southern blotting analyses and theexpression status of cecropin B in crossingtransmission was revealed by Northern blottinganalyses.To detect the completeness of cecropin B geneexpression cassette in rice genome, Southern blot wasconducted after genomic DNA was digested with PstⅠand HindⅢ, which released a 1.12 kb fragmentcomprising of the coding region of cecropin B geneand its pin terminator (Figure 3). Results showed thatpresence of the predicted 1.12 kb fragment in hybridlines mainly depended on their original transgenicdonors (Figure 1C and 2C). Transgene donor TR 5,TR 6 and their derived hybrids did not exhibit theexpected 1.12 kb fragment, illustrating there was nointact cecropin B copies, or more probably, the cutsites of restriction enzymes (Pst Ⅰ and Hind Ⅲ) incecropin B gene expression cassette were modified.Transgene donor Ming B, Jingyin 119 and theirhybrids generated the 1.12 kb fragment as expected,revealing that there was at least one intact copy ofcecropin B gene in these transgenic lines.Northern blot results showed the expression behaviourof non-selected cecropin B gene varied significantlyamong transgenic donors and their hybrid lines(Figure 1D and Figure 2D). The expression ofcecropin B gene in the primary transformants (T0generation) of all the four transgene donors includingTR 5, Ming B, TR 6 and Jingyin 119 was proved byNorthern blot analysis (data not shown). However, intheir self-pollinated offspring, gene silence ofcecrropin B occurred in TR 5 and Ming B donor(Figure 1D), both harbouring 3 hybridization bands ofcecropin B (Figure 1B). The TR 6 and Jingyin 119donors, with 5 and 2 hybridization bands of cecropinB gene respectively (Figure 1B and 2B), expressedcecropin B gene stably arcoss 6 and 12 generations,respectively. This indicated that silencing ofnon-selected cecropin B over generations was relevantto different transformation events rather than thenumber of integrated hybridization bands. In view ofthe Southern blot results, we also concluded that therewas no certain relationship between the cecropin Bgene expression status and the emergence of theexpected 1.12 kb fragment, which was used to predictthe completeness of cecropin B gene copy.The expression behaviour of cecropin B gene in crosstransmission was revealed by comparing the Northernblot results of transgenic hybrids with that of theircorresponding donors. Gene silence of cecropin Boccurred in both self-pollinated progeny of TR 5donor (T5) and all its derived hybrid lines (F 3 ) (Figure1D). In TR 6 transgene donor, cecropin B gene wasexpressed at mRNA level over 6 generations, butsilenced in its two hybrid lines (F 3 generation).Interestingly, cecropin B gene did not express in theselfed progenies (T6) of transgene donor Ming B, butexpressed in its two out of three cross lines MingB/Jia 59 and Ming B/Xuzao, till F3 generation (Figure1D). The stable expression of cecropin B gene wasobserved in Jinyin 119 donor and all its hybrids,ignoring the complex cross combinations and multiplecross turns. We analysed the cecropin B expression inthe progeny plants of 14 out of 17 hybrids fromJingyin 119 donor (Figure 2D). Whether transgnenedonor Jinyin 119 was female parent or male parent(C20 / Jingyin 119), whether transgenes in Jingyin 119were transferred through one cross turn (Jingyin119/57, Jingyin 119/Bing 94-02, Jingyin 119/59), twocross turns (Jingyin 119/59//L97-55, Jingyin 119/57//9522, Jingyin 119/390//S1, Jingyin 119/63//T951,Jingyin 119/Bing 94-02//T951, Jingyin 119/02//T951,Jingyin 119/63//390, Jingyin 119/59//DS4 and Jingyin119/503//T951) or three cross turns (Jingyin 119/ 57 //DS4///L97-55), or stacked by crossing betweentransgenic hybrids (Jingyin 119/59//DS4///Jingyin119/31//9522), the non-selected cecropin B geneexpressed stably in all hybrids over 6 to 8 generations.In general, the expression behaviour of non-selectedcecropin B gene was complex in crossbreeding52

Molecular Plant Breeding 2011, Vol.2, No.8, 48-59http://mpb.sophiapublisher.compromoted the plasmid rearrangements during integration,but might exert effects on the DNA recombinationevents of bar gene loci in the subsequent crossingtransmission. In deed, the presence of new transgenicfragments of bar gene in hybrid TR 5/CJN 3 furtherconfirmed the rearrangement of bar gene integrationloci. More importantly, we found that the varianceexisting in integration patterns of bar gene did notinterfere with its stable expression and the successfulselection of the corresponding hybrids. This indicatedthat transgenes introduced by particle bombardmentcould be successfully transferred in conventionalcrossbreeding.2.2 Stability of transgene expression in crossingtransmissionDifferent integration sites, copy numbers andtransgenic locus configurations, as well as epigeneticsilencing mechanisms are revealed to be the mainfactors influencing transgene expression by previousresearchers (Iyer et al., 2000; Meyer, 1995; Matzkeand Matzke, 1998). However, documents on therelationship between transgene expression andcrossing transmission are very limited up to date.Several reports described the expression behaviour oftransgenes in rice crossbreeding in recent years, whichgiving the opinion that at least the same transgeneexpression level in hybrids, if no more than that of thetransgenic parents, could be expected (Chen etal.,2005; Tang et al., 2006; 2007; Wang and Lin, 2007).But the above results were established on the base oftransgenic donors containing single-copy exotic geneand transformed by Agrobacterium tumefaciensmethod. Our results revealed the different fate oftransgenes in crossbreeding transmission that wasintroduced by DNA direct delivery system.The selected bar gene is expressed in all hybrids andtheir corresponding transgenic parents acrossgenerations in spite of its variability in integrationpatterns. However, the co-expression behaviour ofnon-selected cecropin B gene was very complex. Bothinactivation (e.g. in hybrids of TR 6) and maintainingof successive expression activity (e.g. in hybrids ofMing B) of cecropin B gene were observed throughcrossing transmission. The stable expression ofcecropin B gene were also observed after kinds ofcross combinations across several generations (e.g. inhybrids of Jingyin 119 donor). We confirmed thatexpression status of selected bar and non-selectedcecropin B was independent within the same genomiclocations. This means the inactivation of cecropin Bdoes not spread over to the adjacent bar gene, whichis consistent with the conclusion of some previousreports (Vain et al., 2002; Kohli et al., 2003) butargues against others (Lindsay et al., 1996).As expression status of non-selected cecropin B genewas very different among the four transgenic donorsin crossing transfer, we concluded that the structure oftransgenic loci and the chromosomal locations wheretransgenes integrated are main factors influencinggene expression in sexual reproduction as well as inconventional crossbreeding. TR 5 is a typicaltransgenic line that non-selected cecropin B geneexpressed in primary transformant but silenced inprogenies of both self-pollination and crosses. Thisindicated the integration structure and/or transgenicloci of cecropin B in TR 5 are prone to trigger genesilence. The instability of selected bar geneintegration pattern in TR 5 hybrids illustrated thattransgenic loci of TR 5 line were recombinationtriggering. Silencing of transgene is often aroused byhomologous sequences between multiple transgenecopies or between exotic DNA and endogenous DNAsequences of the host plant, either the homologoussequences at allelic or nonallelic chromosomallocations (Kumpatla et al., 1997). It is probable thathomologous sequences of different copy or fragmentof cecropin B gene caused its own inactivation. Thepossibility of interactions between actin promoter andendogenous homologous sequences in rice genomicDNA could not be ruled out, for the coding region ofcecropin B gene is driven by actin promoter from rice.Homology-mediated gene silencing is based onDNA-DNA pairing, which might be involved in thecase where longer time and more generations areneeded. It could be strongly triggered when transgenesare arranged in palindromic manner or in invertedrepeat (IR) (Fojtová et al., 2006). Whether IR exists intransgenic loci of TR 5 needs to be revealed byextensive research on the sequence of transgenic lociin this line.54

Molecular Plant Breeding 2011, Vol.2, No.8, 48-59http://mpb.sophiapublisher.comNorthern blotting analysis showed the inactivation ofcecropin B gene occurred at transcriptional level.Transcriptional gene silencing in plant is oftenassociated with DNA methylation in the promoterregion and the 5’ un-translated region. DNAmethylatin-correlated gene inactivation can bereleased after treatment with methylation inhibitorssuch as 5-azacytidine (Kohli et al., 1999; Kumpatla etal., 1997). Further experiments need to be conductedfor investigating whether DNA methylation isinvolved in the cecropin B gene silence.An interesting result of the present study is that wefound Jingyin 119 is a special transgenic line fortransgenes to be expressed stably. Both cecropin B andbar gene were stably expressed in Jingyin 119 donorand all its resulting hybrids, ignoring the complexcrossbreeding transmission and across generations.Previously, the stable inheritance and expression ofexotic bar and cecropin B gene in this Jingyin 119transgenic line was confirmed over six successivegenerations (Kumpatla et al., 2001). Here wepresented the foreign bar and cecropin B gene in thistransgenic line kept expressing across long-termgenerations of self-pollination (T12) and after multiplecrossing transmission. It is interesting to inquire intothe following questions: is the integration structure ofexotic bar and cecropin B in Jingyin 119 donor verysuitable for their expression? Are there safechromosomal locations in rice genome for transgenesto reside and escape from the rice genome supervisionand modification system, just as the locations inJingyin 119 genome where exotic bar and cecropin Bintegrated? Meyer (1995) proposed that endogenous,transcriptional active sequences contain cis-actingflanking regions are necessary for adequatefunctioning of genetic machinery, for these sequencescan facilitate DNA bending or elicit differenttrans-acting factors. Such ‘isochore’ sequences arespecial sequences. Any deviation from isochorestructure is recognized as a foreign element, whichmay lead to elimination or silencing of transgene.Thus, how and why foreign bar and cecropin B inJingyin 119 could escape from the recognition andattack of the rice genome defending system?Information regarding the chromosomal locationsfavouring transgene expression is very limited so far.The excellent performance of transgenes in Jingyin119 transgenic line provides a promising hint to seekfor these suitable chromosomal locations for transgeneintegration. Further experiments are necessary todiscover the transgene structure and integrationpositions on rice chromosome of bar and cecropin Bgene in Jingyin 119 transgenic.Another phenomenon we found in present study isthat conventional crossing transmission can affect theexpression status of non-selected cecropin B gene. Theinactivation of cecropin B gene in crosses of TR 6 andthe maintaining successive expression activity ofcecropin B gene in some crosses of Ming B impliedthat effect from host genotypic constitution ontransgene expression can not be ignored. Under theseconditions, the effects from integration position, copynumber and rearrangement of transgenic loci oncecropin B expression could be ruled out, because theintegration patterns of cecropin B gene in hybrids areexactly the same to their corresponding transgenicdonors. The mechanism to explain how host genotypicconstitution influenced transgene expression ispossible at epigenetic level, such as change ofmethylation status of cecropin B gene expressioncassette. The alteration in genotype backgrounds byconventional crosses might prevent, or at least delay,the epigenetic modification process of transgenes insome circumstances, which were illustrated by theexpression activity of cecropin B in some hybrids ofMing B.On the other hand, we used the progenies oftransgenic donors and hybrids as materials toinvestigate the transgene expression behaviour incrossing transmission. It remains unclear whethertransgene inactivation in hybrids occurred immediatelyafter cross (in F 1 generation) or throughgenerations. The results of F1 hybrid of TR 5/TR 6may throw some light on this question. Cecropin Bgene silenced in TR 5 donor but expressed normally inTR 6 donor. The subsequent F1 hybrid of TR 5/TR 6lost its express activity of cecropin B (Figure 1D).This implied the inactivation of cecropin B geneoccurred immediately after cross. Fojtová et al (2006)revealed in tobacco that meiosis could not alter the55

Molecular Plant Breeding 2011, Vol.2, No.8, 48-59http://mpb.sophiapublisher.comexpression and methylation patterns established in thehybrid plants. Thus we speculated the inactivation ofcecropin B gene occurred in F 1 hybrids and thesilencing status was passed on through sexualreproduction.In conclusion, transgenes in rice genome introducedby particle bombardment could be easily transferredby conventional crossbreeding. The stability oftransgene integration patterns and expression status incrossbreeding transmission was mainly determined bythe primary transgenic donor plant. In the course ofcross transmission, the variance in integration patternsof selected marker bar gene did not influence its stableexpression under selection, whereas the expressionstatus of non-selected cecropin B gene is affected bycross recombination. And more importantly, transgenicplant can be produced by particle bombardment inwhich transgenes sustains inheritance and expressionactivity over long-term generations in both sexualreproduction and conventional crossbreeding.3 Materials and methods3.1 Generation of transgenic plantsFour transgenic rice lines TR 5, TR 6, Ming B andJingyin 119 transformed via particle bombardmentwere used as transgene donors to create transgenichybrids. All the above transgenic donors weregenerated as described in previous reports, harboringplasmid pCB 1 , which contains the selectedmarker-gene bar and non-selected cecropin B gene.The bar gene is controlled by cauliflower mosaic virus35S (CaMV 35S) promoter, cecropin B gene is controlledby the rice actin-1 promoter (Huang et al., 1996).3.2 CrossesThe transgenic plants of each rice variety wereidentified by both Basta-resistant phenotype analysesand Southern-blotting hybridization. Bagged seeds oftransgenic T0 plants of Jingyin 119, TR 5, TR 6 andMing B variety were planted in the experimental farmof China National Rice Research Institute between1996 and 1998. Seeds of each plant were harvestedindividually and the elite homozygous transgeniclines were selected as transgene donors for crossexperiments.All cross experiments were conducted in the field inthe rice growing seasons between 1996 and 2009 bothin Zhejiang Province and Hainan Province. A crossbetween transgene donor TR 5 (T5) and TR 6 (T6)was made to investigate the relationship betweenintegration and expression of transgenes in differenttransgene donors. The homozygous transgenic lineJingyin 119 (T3 generation) was used as transgenedonor to cross with different Japonica rice varieties.Some of the resulting F 1 hybrids were used as newtransgene donors for next crosses to produce hybridsof multiple crosses. All the selected transgene donorsand hybrids were subjected to Basta resistant assay inevery generation. We sprayed 0.32% Basta to wholeplant at their seedling stage. After one week, thesusceptible plants were dead. The selected transgenicplants were grown in the field for seeding or crossexperiment. The transgenic hybrids were self-crossedto produce homozygotes for the bar gene, andprogenies were selected based on Basta resistance andSouthern hybridization. All homozygous transgenichybrids and their transgenic donors were planted inthe experimental farm of China National RiceResearch Institute. The information of rice materialsfor experiments was detailed in Table 1.3.3 Southern blot hybridizationGenomic DNA was isolated from rice leaves using theSDS DNA extraction method as described by Lu andZheng (1992) Aliquots (5 μg) were digested overnightwith appropriate restriction enzymes, fractionated by0.8% agarose gel electrophoresis and blotted ontoAmersham N + Hybond membranes according to theSouthern blotting method (Sambrook et al., 1989).The linear fragment (0.9 kb) comprising the openreading frame of bar gene and nos terminator frompCB 1 digested with EcoRⅤ was used as probe for bargene. The linear fragment (1.12 kb) from pCB 1 afterdigestion with HindⅢ and PstⅠ, which comprisescecropin B coding region and Pin terminator was usedas probe for cecropin B gene. The probe structure andsites on pCB 1 are illustrated in Figure 3.The probe labeled by random priming was conductedusing DIG DNA Labeling Kit (Roche Company)according to the manufacture’s instructions. Southern56

Molecular Plant Breeding 2011, Vol.2, No.8, 48-59http://mpb.sophiapublisher.comTable 1 Pedigree of transgenic rice cross lines used for transgene integration and expression analysesTransgenic donors Cross combinations * (Female parent (generation) /Male parent (generation))Plant generation for Southern andNorthern analysisTR 5T5TR 5 (T2) / CJN3F3TR 5 (T2) / CJ601F3TR 5 (T2) / CJ683F3TR 5 (T2) / Bing 97-264F3Ming BT6Ming B (T2) / Jia 59F3Ming B (T2) / Jia 60F3Ming B (T2) / XuzaoF3TR 6T6TR 6(T2) / CJN2F3TR 6(T2) / Bing 95-13F3TR 5(T5) / TR 6 (T6)F1Jingyin 119T12C20 / Jingyin 119 (T3)F8Jingyin 119 (T3) /57F8Jingyin 119 (T3) / Bing 94-02F8Jingyin 119 (T3) / 59F8Jingyin 119 (T3) / 104F8Jingyin 119 (T3) /59 // L97-55F7Jingyin 119 (T3) /57 // 9522F7Jingyin 119 (T3) / 390 // S1F7Jingyin 119(T3) / 63 // T951F7Jingyin 119(T3) / Bing 94-02 // T951F7Jingyin 119(T3) / 02 // T951F7Jingyin 119(T3) / 63 // 390F7Jingyin 119 (T3) / 59 // DS4F7Jingyin 119(T3) / 503 // T951F7Jingyin 119(T3) / 59 // T951F7Jingyin 119(T3) / 57 // DS4 /// L97-55F5Jingyin 119 (T3) / 59 // DS4 /// Jingyin 119 (T3) / 31 // 9522 F4*Note: ‘/’ stands for the first turn cross; ‘//’ stands for the second turn cross; ‘///’ stands for the third turn cross. The F 1 hybrid plantswere used as transgenic donors when multiple cross experiments were conductedblot hybridization and detection was carried out usingDIG Luminescent Detection Kit (Roche Company)according to the manufacture’s instructions.Figure 3 The probe structure and site on pCB 1Note: Diagrams of the pCB 1 are not to scale; Probe 1: bar geneprobe used for Southern and Northern blotting; Probe 2:cecropin B gene probe used for Southern and Northern blotting;Act: Rice actin-1 promoter; CB: cecropin B gene encodingsequence; Pin: Potato proteinase inhibitor II terminator; 35S:Cauliflower mosaic virus 35S promoter; bar: bar geneencoding sequence; nos: A. tumefaciens Ti-plasmid nopalinesynthase terminator3.3 Southern blot hybridizationGenomic DNA was isolated from rice leaves using theSDS DNA extraction method as described by Lu andZheng (1992) Aliquots (5 μg) were digested overnightwith appropriate restriction enzymes, fractionated by0.8% agarose gel electrophoresis and blotted ontoAmersham N + Hybond membranes according to theSouthern blotting method (Sambrook et al., 1989).The linear fragment (0.9 kb) comprising the open57

Molecular Plant Breeding 2011, Vol.2, No.8, 48-59http://mpb.sophiapublisher.comreading frame of bar gene and nos terminator frompCB 1 digested with EcoRⅤ was used as probe forbar gene. The linear fragment (1.12 kb) from pCB 1after digestion with Hind Ⅲ and Pst Ⅰ , whichcomprises cecropin B coding region and Pinterminator was used as probe for cecropin B gene. Theprobe structure and sites on pCB 1 are illustrated inFigure 3.The probe labeled by random priming was conductedusing DIG DNA Labeling Kit (Roche Company)according to the manufacture’s instructions. Southernblot hybridization and detection was carried out usingDIG Luminescent Detection Kit (Roche Company)according to the manufacture’s instructions.3.4 Northern blot hybridizationLeaf tissue (0.5~1g) was ground in liquid nitrogen.The dispersed tissue was used for total RNAextraction using TRIzol Kit (Invitrogen Company)according to the manufacture’s instructions. ExtractedRNA was initially checked for quality and quantity onnormal 1% agarose gel. After electrophoresis on 1.2%formaldehyde-agrose gel, the gel was washed for10 min in sterile water to remove the formaldehyde.The RNA was denatured in 0.05 mol/L NaOH andblotted onto Amersham N + Hybond membranes in20×SSC according to the Southern blotting method(Meyer et al., 1995). The DNA probe of cecropin Bgene described as above was used for Northernhybridization.Northern blot hybridization and detection was carriedout using DIG Luminescent Detection Kit (RocheCompany) according to the manufacture’s instructions.Authors’ contributionsYan Zhao conducted the major part of this study including experimentaldesign, Southern and Northern hybridization experiment, and manuscriptpreparation. Longbiao Guo and Huizhong Wang conducted the rice materialhybrid design and the crossing experiment. Danian Huang participated inexperimental design.AcknowledgementsThis work was supported by grants from the National Nature ScienceFoundation of China (No: 30871511&30771317), the key program from theMinistry of Agriculture of China for Creation of New Transgenic OrganismVarieties (Nos: 2008ZX0810-003 &2009ZX08001-022B), the post-doctoralfoundation of China (No.20090450477).ReferencesAltpeter F., Baisakh N., Beachy R., Bock R., Capell T., Christou P., Daniell H.,Datta K., Datta S.J., Dix P., Fauquet C., Huang N., Kohli A., Mooibroek H.,Nicholson L., Nguyen T.T., Nugent G., Raemakers K., Romano A.,Somers D., Stoger E., Taylor N., and Visser R., 2005, Particlebombardment and the genetic enhancement of crops: myths and realities,Mol. Breed., 15(3): 305-327 doi:10.1007/s11032-004-8001-yChen H., Tang W., Xu C.G., Li X.H., and Zhang Q., 2005, Transgenic indicarice plants harbouring a synthetic cry2A* gene of Bacillus thuringiensisexhibit enhanced resistance against rice lepidopteran pests, Thero. Appl.Genet., 111(7): 1330-1337 doi:10.1007/s00122-005-0062-8 PMid:16187120Fojtová M., Bleys A., Bedřichová J., Houdt H.V., Křížová K., Depicker A., andKovařík A., 2006, The transsilencing capacity of invertedly repeatedtransgenes dependends on their epigenetic state in tobacco, Nucleic AcidsRes., 34(8): 2280-2293 doi:10.1093/nar/gkl180 PMid:16670434 PMCid:1456325Horvath H., Jensen L.G., Wong Q.T., Kohl E., Ullrich S.E., Cochran J.,Kannangara C.G., and Von Wettstein D., 2001, Stability of transgeneexpression, field performance and recombination breeding of transformedbarley lines, Thero Appl Genet, 102(1): 1-11 doi:10.1007/s001220051612Hua Z.H., Zhu X.F., Wu M.G., Yu Y.C., Zhao Y., Gao Z.Y., Yan M.X., andQian Q., 2003, Inheritance of exo-genes integrated into the rice genomes,Acta Agronomica Sinica, 29(1): 44-48Huang D.N, Li J.Y., Zhang S.Q., Xue R., Yang W., Hua Z.H., Xie X.B., andWang X.L., 1998, New technology to examine and improve the purity ofhybrid rice with herbicide resistant gene, Chin. Sci. Bull., 43(9): 784-787doi:10.1007/BF02898961Huang D.N., Zhu B., Yang W., Xue R., Xiao H., Tian W.Z., Li L.C., and DaiS.H., 1996, Introduction of cecropin B gene into rice (Oryza sativa L) byparticle gun bombardment and analysis of transgenic plants, Science inChina (Series C), 39(6): 652-661Iyer L.M., Kumpatla S.P., Chandrasekharan M.B., and Hall T.C., 2000,Transgene silence in monocots, Plant Mol. Biol., 43(2-3): 323-346doi:10.1023/A:1006412318311 PMid:10999414Khush G.S., 2005, What it will take to feed 5.0 billion rice consumers in 2030,Plant Mol. Biol., 59(1): 1-6 doi:10.1007/s11103-005-2159-5 PMid: 16217597Kohli A., Griffiths S., and Palacios N., 1999, Molecular characterization oftransforming plasmid rearrangements in transgenic rice reveals arecombination hotspot in the CaMV35S promoter and confirms thepredominance of microhomology mediated recombination, Plant J., 17(6):591-601 doi:10.1046/j.1365-313X.1999.00399.x PMid:10230059Kohli A., Twyman R.M., Abranches A., Wegel E., Stager E., Christou P., andStoger E., 2003, Transgene integration, organization and interaction inplants, Plant Mol. Biol., 52(2): 247-258 doi:10.1023/A:1023941407376PMid:12856933Kumpatla S.P., Teng W., Buchholz W.G., and Hall T.C., 1997, Epigenetictranscriptional silencing and 5-azacytidine-mediated reaction of a complextransgene in rice, Plant Physiol., 115: 361-373 doi:10.1104/pp.115.2.361PMid:9342860 PMCid:158494Lindsay H., Adams R.L., 1996, Spreading of methylation along DNA, Biochem.J., 320(Pt2): 473-478 PMid:8973555 PMCid:1217954Lu Y.J., and Zheng K.L., 1992, A simple method for isolation of rice DNA,Chinese J. Rice Sci., 1: 47-48 (in Chinese with an English abstract)Matzke A.J.M., and Matzke M.A., 1998, Position effect and epigenetic silencingof plant transgenes, Curr. Opin. Plant Biol., 1(2): 142-148 doi:10.1016/S1369-5266(98)80016-2Meyer P., 1995, Understanding and controlling transgene expression, TrendsBiotechnol., 13(9): 332-337 doi:10.1016/S0167-7799(00)88977-5Peng S., Laza R.C., Visperas R.M., Sanico A.L., Gassman K.G., and KhushG.S., 2000, Grain yield of rice cultivars and lines developed in Philippinessince 1996, Crop Sci., 40(2): 307-314 doi:10.2135/cropsci2000.402307xPopelka J.C., and Altpeter P., 2003, Evaluation of rye (Secale cereale L.) inbredlines and their crosses f or tissue culture response and stable genetictransformation of homozygous rye inbred line L22 by biolistic genetransfer, Thero. Appl. Genet., 107(4): 583-590 doi:10.1007/s00122-58

Molecular Plant Breeding 2011, Vol.2, No.8, 48-59http://mpb.sophiapublisher.com003-1314-0 PMid:12830384Register J.C. 3 rd , Peterson J., Bell P.J., Bullock W.P., Evans I.J., Frame B.,Greenland A.J., Higgs N.S., Jepson I., Jiao S., Lewnau C.J., Sillick J.M.,and Wilson H.M., 1994, Structure and function of selectable andnon-selected transgenes in maize after introduction by particlebombardment, Plant Mol. Biol., 25(6): 951-961 doi:10.1007/BF00014669PMid:7919215Sambrook J., Fritsch E.F., and Maniatis T., 1989, Molecular Cloning: ALaboratory Manual, 3 nd ed., Cold Spring Harbor Laboratory Press,Plainview, NY, pp.9.31-9.62Tang W., Chen H., Xu C., Li X.H., Lin Y., and Zhang Q., 2006, Developmentof insect-resistant transgenic indica rice with a synthetic cry1C* gene; Mol.Breed., 18(1): 1-8 doi:10.1007/s11032-006-9002-9Tang W., and Lin Y.J., 2007, Field experiment of transgenic crylAb insectresistant rice, HERIDITA (Bingjing), 29(8): 1008-1012 (in Chinese withan English abstract) doi:10.1360/yc-007-1008 PMid:17681932Vain P., James V.A., Worland B., and Snape J.W., 2002, Transgene behaviouracross two generations in a large random population of transgenic riceplants produced by particle bombardment, Thero. Appl. Genet., 105(6-7):878-889 doi:10.1007/s00122-002-1039-5 PMid:12582913Wang W., and Lin Y.J., 2007, Field experiment of transgenic cry1Ab insectresistant rice. Yi Chuan (Heriditas), 8: 1008-1012 (in Chinese with anEnglish abstract)Wu J.H., and Luo R.L., 2007, Current situation and strategy on internationaldevelopment of Chinese hybrid rice, Chinese Rice, 1: 11-13, 62 (inChinese with an English abstract)Zhang Y., Yin X., Yang A., Li G., Zhang J., 2005, Stability of inheritance oftransgenes in maize (Zea mays L.) lines produced using differenttransformation methods, Euphytica, 144(1-2): 11-22 doi:10.1007/s10681-005-4560-159