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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
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