Molecular Breeding 8: 285–293, 2001.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Breeding bacterial blight-resistant hybrid rice with the cloned bacterialblight resistance gene Xa21

Wenxue Zhai1, Wenming Wang1, Yongli Zhou2, Xiaobing Li1, Xianwu Zheng1, Qi Zhang2,Guoliang Wang1,3 and Lihuang Zhu1,∗1Institute of Genetics, Chinese Academy of Sciences, Beijing 100101, China; 2Institute of Crop Breeding andCultivation, Chinese Academy of Agricultural Sciences, Beijing 100080, China; 3Present address: Departmentof Plant Pathology, 201 Kottman Hall, The Ohio State University, Columbus, OH 43210, USA; ∗Author forcorrespondence (fax: 086-10-64873428; e-mail: lhzhu@igtp.ac.cn)

Received 14 March 2000; accepted in revised form 29 September 2000

Key words: blight disease resistance, Indica hybrid rice, Marker-assisted selection, Transgenic restorer line, Xa21

Abstract

The cloned bacterial blight (BB) resistance gene Xa21 was transferred into Minghui63, a widely used restorerline of indica hybrid rice in China, through an Agrobacterium-mediated system. Molecular and resistance analysesrevealed that the Xa21 gene was integrated in the genomes of transgenic plants and their progeny inherited resis-tance stably. For the purpose of hybrid breeding, Xa21 transgenic homozygous restorer lines were selected through‘within-lane’ dosage comparison of hybridization signal in combination with PCR and resistance analyses. Theselected transgenic restorer lines were then crossed with a commonly used sterile line, Zhenshan97A, to produceXa21 transgenic hybrid rice, Shanyou63-Xa21. The hybrid rice plants with Xa21 displayed high broad-spectrumresistance to Xanthomonas oryzae pv. oryzae (Xoo) races and maintained elite agronomic characters of Shanyou63.The propagation of this BB-resistant hybrid variety with Xa21 will benefit rice production.

Introduction

The utilization of hybrid vigor has become an effectiveapproach to increase rice yield. Hybrid rice varietiescan normally produce nearly 20% higher yields thaninbred semi-dwarf varieties (Virmani 1996). Currentlyrice hybrids are being widely used in the rice produc-tion throughout the world, particularly in China, wherehybrid rice accounts for half of the rice acreage andcontributes 60% of the total rice yield of the country(Lin and Min 1991; Virmani 1996). The productionof hybrid rice involves three-line systems: cytoplas-mic male sterile (CMS), maintainer and restorer lines.The narrow genetic base of the three lines often makestheir hybrids susceptible to many diseases (Virmani1996). For example, Shanyou63, one of the mostpopular hybrid cultivars produced through the crossbetween a CMS line, Zhenshan97A, and a restorerline, Minghui63, is easily damaged by bacterial blightdisease (Lin and Min 1991).

Bacterial blight (BB) caused by Xanthomonasoryzae pv. oryzae (Xoo) is one of the most destruc-tive diseases of both inbred and hybrid rice throughoutthe world (Mew 1987). The loss in rice productioncaused by BB in some areas of Asia can be as highas 50%. The incorporation of resistance genes intocultivars is the most economic and effective method tocontrol this disease (Ogawa 1996). More than twentygenes for resistance to Xoo have been identified in rice(Khush et al. 1990; Kinishita 1995; Zhang et al. 1999).Some of these genes have been incorporated into eliterice varieties by using traditional breeding approaches(Khush et al. 1989) and pyramided into breedinglines through marker-assisted selection (MAS; Huanget al. 1997). However, traditional backcross breedingis generally a tedious and time-consuming task andoften produces linkage drags in spite of utilization ofMAS (Tanksley et al. 1989). Fortunately, two resis-tance genes, Xa21 and Xa1, were cloned by positional

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cloning (Song et al. 1995; Yoshimura et al. 1998). Theavailability of the cloned resistance genes, particularlythe complete dominant gene Xa21 that has a widespectrum of resistance to Xoo (Khush et al. 1990), nowprovides an important opportunity for improving BBresistance of rice by genetic transformation (Ronald1997).

The cloned Xa21 gene has been transferred intoseveral rice varieties through particle bombardment(Wang et al. 1996; Tu et al. 1998; Zhang et al.1998), but in some transgenic progeny variation ofagronomic traits and possible silence of the transgeneXa21 were observed (Tu et al. 1998; Zhang et al. 1998;Zhang et al. personal communication), which mayattribute to multiple-copy insertion of the transgene.In rice, besides particle bombardment, an effectiveAgrobacterium-mediated transformation system hasbeen established (Hiei et al. 1994; Rashid et al. 1996).Recently we used this system to transfer Xa21 geneinto five Chinese rice varieties successfully (Zhai et al.2000). In this study, the Xa21 gene was introduced intoMinghui63, a widely used hybrid restorer line fromwhich up to 60% of the total hybrid seeds in China areproduced each year. From the transgenic Minghui63plants homozygous lines with a single copy of trans-gene Xa21 were selected through ‘within-lane’ dosagecomparison of hybridization signal (Yu et al. 1994).The homozygous Xa21 transgenic Minhui63 restorerplants were crossed with Zhenshan97A to breed a BB-resistant hybrid, Shanyou63, with the transgene Xa21.The transgenic hybrid rice plants not only displayedhigh broad-spectrum resistance to Xoo races but alsomaintained all the elite agronomic characters of thehybrid Shanyou63.

Materials and methods

Plasmids and rice transformation

The plasmid pCXK1301 (Zhai et al. 2000) contain-ing the Xa21 gene was derived from pC822 (Songet al. 1995) and pCAMBIA1301 (Cambia, Australia).It contains the whole Xa21 gene in the 9.6 kb KpnIfragment and two selectable markers HYG and GUSin the T-DNA region (Figure 1). This plasmid canbe directly used in rice transformation through parti-cle bombardment or introduced into Agrabacteriumtumefaciens strains. In this study, pCXK1301 wasintroduced into the A. tumefaciens strain EHA105through electroporation. The strain containing Xa21

Figure 1. Diagram of the plasmid pCXK1301. Abbreviations:T-BORDER (L), T-DNA left border; T-BORDER (R), T-DNAright border; HYG, hygromycin phosphotransferase; GUS,β-glucuronidase; LAC Z, β-galactosidase alpha; Kan, kanamycinresistance gene; Xa21, rice Xa21 gene. The primers (Z2, Z3, U1,and I1) and the probe used in this study were also donated.

was then used in rice transformation. Rice transfor-mation was carried out according to the previouslydescribed method (Hiei et al. 1994; Rashid et al. 1996;Zhai et al. 2000). Tissues were cultured according tothe procedure described by Zhang et al. (1998).

Rice varieties

Minghui63, the restorer line in hybrid rice, wasused as the recipient of the Xa21 gene. The trans-genic Minghui63 plants were then crossed with Zhen-shan97A, a rice CMS line, to produce transgenic hy-brid rice Shanyou63. The seeds of Minghui63, Zhen-shan97A and Shanyou63 were kindly provided by DrQian Qian of the Chinese Rice Institute in Hangzhou,China.

Strategy of breeding BB-resistant hybrid rice with thecloned Xa21 gene

Transgenic restorer plants were selected in T0, T1andT2 generations by PCR, Southern and resistanceanalysis. The selected homozygous transgenic restorerlines (T1and T2) were crossed with a CMS line toproduce BB resistance hybrid F1. The procedure isoutlined in Figure 2.

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Figure 2. Strategy for selecting homozygous Xa21 transgenic re-storer and cultivating BB-resistant hybrid rice.

PCR analysis

One pair of primers, Z2and Z3, as indicated in Fig-ure 1, was designed according to the sequence of thegene for Xa21-specific PCR amplification (Zhai et al.2000), which amplified a 1.4 kb DNA fragment fromthe transgene Xa21 but no product from the genomicDNAs of the controls, Minghui63 and Shanyou63. Theother pair of primers, U1 and I1, as indicated in Fig-ure 1, which had been designed by Dr G.L. Wang(Wang et al. 1996), was used to amplify a 1.4 kbDNA fragment from the transgene Xa21, but it couldalso amplify a 1.3 kb DNA fragment from the controlgenomic DNAs. Sequences of the primers are as fol-lows: Z2 5′-ATTGAATAATTCACTGGGTATTGG-3′, Z3 5′-GTCTTGCCTTGCACTTCTGCACGA-3′;U1 5′-CGATCGGTATAACAGCAAAAC-3′, I1 5′-ATAGCAACTGATTGCTTGG-3′. Each 25 µl PCRreaction mixture contained 50 ng genomic DNA,10 mM Tris-HCl pH 9.0, 50 mM KCl, 1.8 mM MgCl2,0.1% TritonX-100, 200 µM dNTPs, 50 ng each of theprimer pair, 1 unit Taq DNA polymerase. PCR reac-tions were performed in a PE-480 thermocycler usingthe following file: predetermination 94 ◦C, 5 min; then94 ◦C, 1 min; 56 ◦C, 1 min; and 72 ◦C, 2 min for 35cycles; followed by 10 min at 72 ◦C. PCR productswere electrophoresed in a 1.4% agarose gel in 1× TAEbuffer.

DNA extraction and Southern hybridization

Genomic DNA was extracted by the procedure de-scribed by McCouch et al. (1988). DNA (5 µg) was

digested with HindIII and electrophoresed on a 0.8%agarose gel. Southern hybridization was carried outas described by the manufacturer (Amersham). Theprobe for Xa21 was the 1.4 kb PCR fragment amplifiedfrom pCXK1301 with the primer pair Z2 and Z3.

Resistance analysis

Transgenic restorer and hybrid plants were grown inthe greenhouse or net house on the experimental farmof the Institute of Genetics, Chinese Academy of Sci-ences in Beijing. At maximum tillering stage, thefully expanded leaves were inoculated with race 6and/or other strains of Xoo by the leaf-clipping method(Kauffman et al. 1973). For resistance analysis of allthe transgenic plants with Xa21, the Xa21-specific dif-ferential race 6 was used. For analysis of the resistancespectrum of Xa21 transgenic plants, race 6 and 18other strains of Xoo, as listed in Table 3, were used.The inoculum was grown on PSA medium at 28 ◦Cfor 72 h and adjusted to a concentration of about 109

cells per milliliter. Two weeks after inoculation, whenthe susceptible check lesion became obvious and sta-ble, individual plants were scored for their diseaseresistance.

Evaluation of agronomic traits

The Xa21 transgenic lines Minghui63-Xa21 andShanyou63-Xa21 as well as control varieties wereplanted in the test field. The plants were evaluatedfor their seven agronomic traits: growth duration,plant height, stooling ability, amount of pollens, pan-icle length, grains per panicle and 1000-grain weight.Twenty individual plants for each line or variety wereinvestigated. The means are listed in Table 2.

Results

Generation of primary Minhui63 transformants witha single copy of the transgene Xa21

Minghui63 was transformed with plasmid pCXK1301containing the Xa21 gene through an Agrobacterium-mediated system established previously (Zhai et al.2000). Primary transgenic plants were identifiedthrough PCR and resistance analyses. The expected1.4 kb Xa21-specific fragment was amplified by theprimer pair of Z2 and Z3 from transgenic plants,whereas no identical fragment was obtained from non-transgenic control plants (Figure 3A). All the PCR-positive transgenic plants were resistant to the 19 BB

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Figure 3. PCR (A) and Southern (B) analyses of Xa21 transgenic Minghui63 T0 plants. M: molecular size markers; C: non-transgenicMinghui63 control; 1-26: different resistant T0 plants. A. DNAs were amplified with the primer pair of Z2 and Z3. B. Genomic DNAswere digested with HindIII. The probe was designated in Figure 1. M1 to M5 were identified resistant T0 plants with a single copy of Xa21.

Figure 4. PCR (A) and Southern (B) analyses of T1 plants from transgenic Minghui63 (M1). M, molecular size markers; others, different T1plants from transgenic Minghui63 (M1). R, BB-resistant plant; S, BB-susceptible plant. A. PCR was carried out with the primer pair U1 andI1. B. Southern analysis was carried out as that in Figure 3B. DNAs were digested with HindIII. The homozygous T1 plants with the transgeneXa21 are denoted with ∗.

Table 1. Segregation of five transformants with a single copy of the transgene in selfed T1generation.

Primary PCR and resistance analyses of Southern analysis of resistant

transformant T1 plants T1 plants

(T0) number of plants ratio number of plants ratio of R(+−):

Total R(++) S(− −) of R:S R(+−) R(++) R(++)

or +−)

M1 79 59 20 3.0 40 19 2.1

M2 39 29 10 2.9 19 10 1.9

M3 77 58 19 3.1 38 20 1.9

M4 74 55 19 2.9 37 18 2.1

M5 34 25 9 2.8 16 9 1.8

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strains including race 6, the differential race for Xa21(data not shown). However, PCR and resistance analy-ses can not disclose the number of integrated copiesof the transgene in transgenic plants. To breed a hy-brid rice variety with the transgene, the transgenicrestorer plants with a single copy of transgene shouldbe preferably selected. In this study, a Southern hy-bridization method was used to detect the integratedcopies and sites of the transgene Xa21 in primarytransgenic plants for selection of transgenic plants thathad only one copy of the transgene Xa21 integrated intheir genomes. The restriction endonuclease HindIII,which cuts the Xa21 coding sequence at 4.5 kb inpCXK1301 (Figure 1), was used to digest genomicDNAs of transgenic plants. When HindIII-digestedDNA from a transgenic plant hybridized with the Xa21probe, more than one hybridization band could be de-tected. Except for a 4.0 kb non-transgenic hybridiza-tion band, which could be used as a suitable internalcontrol, and some weak background bands, other hy-bridization bands should be the Xa21-integrated frag-ments. If there is no obvious deletion in the T-DNAregion, the Xa21-integrated fragments detected shouldbe larger than 6.8 kb, which is a minimum size ofthe hybridization fragments expected from the mapof pCXK1301 (Figure 1). Compared with that of theinternal control, the number of the Xa21-integratedhybridization bands and their signal strength couldgrossly reflect the copy number of integrated Xa21genes in the transgenic plants. In this study a total of26 primary resistant plants of Minhui63 were analyzedwith the HindIII-digested Southern hybridization (Fig-ure 3B), of which five were considered as transgenicplants with a single copy of the transgene. These plantswere maintained for further genetic analyses.

Identification of homozygous resistant T1 plants withthe transgene Xa21

The segregation of the transgene Xa21 in the T1 gen-eration was analyzed through specific PCR and BBresistance expression. PCR amplification was con-ducted with the primer pair of U1 and I1 (Wang et al.1996). There were two PCR patterns in transgenicT1 plants: homozygous resistant and heterozygous re-sistant plants produced both 1.3 kb and 1.4 kb PCRfragments, whereas homozygous susceptible plantsproduced only one fragment of 1.3 kb. For single-copy-integrated T0 transformants, the segregation ra-tio of 3:1 in the T1 generation was revealed by PCRanalysis (Figure 4A), and the result of resistance

analysis was consistent with that of PCR analysis (Ta-ble 1). No inactivation or silence of the transgene wasobserved in T1 generation including a total of 303plants. However, PCR and resistance analyses couldnot distinguish homozygous resistant plants from het-erozygous ones. To screen Xa21 homozygous resistantplants, Southern analysis with ‘within-lane’ dosagecomparison was carried out in T1 generation (Fig-ure 4B). Since the hybridization signal intensity couldreflect the relative dosage of the detected fragment, re-sistant homozygotes with two copies of the transgenewere distinguishable from resistant heterozygotes withonly one copy of the transgene according to the dosagecomparison between the integrated Xa21 fragment (ca.11 kb in M1) and the within-lane control (ca. 4.0 kb),which is a Xa21 homologous fragment with two copiescorresponding to a pair of alleles of the Xa21 ho-mologous member in Minhui63, whereas in the BBsusceptible homozygotes no integrated Xa21 fragment(ca. 11 kb in M1) could be detected (Figure 4B).According to the results from PCR, Southern and re-sistance analyses, the segregation ratios of T1 plantswere calculated (Table 1). The segregation ratio of 3:1between resistant and susceptible, and the ratio of 2:1between heterozygous resistant and homozygous re-sistant were both observed. A total of 60 homozygousresistant plants were selected, which could be usedas breeding stocks for production of transgenic hybridrice with the Xa21 gene.

Confirmation of homozygous resistant lines with thetransgene Xa21 in the T2 generation

In the selfed T2 generation, three different types of T2lines were derived: homozygous resistance lines withthe transgene Xa21, homozygous susceptible lineswithout Xa21 and transgene Xa21 segregating lines.Both PCR and resistance analyses were conducted inT2 lines, and ten randomly chosen plants for each T2line were tested. Three different PCR patterns weredisplayed among the T2 lines (Figure 5). In the ho-mozygous resistant line each of the ten plants testedproduced a 1.4 kb Xa21-specific PCR fragment in ad-dition to a 1.3 kb background control PCR fragment,while in the homozygous susceptible line no 1.4 kbfragment was obtained. However there was a PCR pat-tern segregation in the heterozygous line (Figure 5).Resistance analysis with Xoo race 6 gave a consistentresult with that by PCR analysis (data not shown).When these analyses were carried out in the T2 gen-eration of all the resistant homozygous T1 plants, no

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Figure 5. PCR analysis of T2 plants from transgenic Minghui63 (M1). R, BB-resistant plant; S, BB-susceptible plant; M, molecular sizemarkers. Lanes 1–10, T2 plants from a Xa21 homozygous T1 plant; lanes 11–20, T2 plants from a susceptible T1 plant without the transgeneXa21; lanes 21–30, T2 plants from a Xa21 heterozygous T1 plant. PCR was carried out with the primer pair U1 and I1.

Table 2. Agronomic traits and BB resistance of Xa21 transgenic restorer lines and hybrid varieties.

Varieties Growth Plant Stooling Amount Panicle Grains/panicle 1000 grain Lesion length to

duration height ability of length weight (g) race 6 in % ∗(day) (cm) pollens (cm)

Minghui63 127.5 ± 2.5 95.9 ± 3.1 high large 25.4 ± 0.7 144.5 ± 21.0 27.4 ± 0.2 57.9 ± 2.4

Minghui 127.6 ± 2.2 96.0 ± 2.7 high large 25.5 ± 0.6 144.7 ± 20.1 27.5 ± 0.2 6.1 ± 2.

63-Xa21

Shanyou63 137.5 ± 2.3 102.5 ± 2.5 high large 29.2 ± 0.5 155.3 ± 25.6 27.9 ± 0.3 49.5 ± 4.9

Shanyou 137.7 ± 2.5 102.6 ± 2.8 high large 29.4 ± 0.6 155.4 ± 26.5 27.8 ± 0.2 7.9 ± 2.2

63-Xa21

∗This is the ratio of the lesion length to the whole length of leaf inoculated. The data was the average of at least five plants from whichthree infected leaves were scored.

PCR-negative and susceptible plants were discoveredduring the tests. This gave a confirmation of the ho-mozygous resistant T1 plants identified by Southernhybridization with HindIII digestion. These recon-firmed Xa21 homozygous resistant restorer lines werenamed as Minghui63-Xa21. In addition to resistanceto Xoo race 6 (Table 2), the Minghui63-Xa21 lineswere proved to maintain the wide resistance spectrumof the Xa21 donor to other Xoo races or pathotypes aswell (data not shown). Furthermore, we evaluated theMinghui63-Xa21 plants for their agronomic traits, andno variations were observed in the seven investigatedtraits such as growth duration, plant height, stoolingability, amount of pollens, panicle length, grains perpanicle and 1000-grain weight (Table 2).

Introduction of the transgene Xa21 into hybrid riceby a cross between a CMS line and Minghui63-Xa21

To produce BB-resistant hybrid rice with the trans-gene Xa21 different CMS lines including Zhen-shan97A, W-qingA, K17A, W17A, G46A, D297A,XinshanA (with rice blast resistance), D62A, D-shanA and D702A were crossed with the transgenicrestorer line Minghui63-Xa21. All the cross combina-tions could produce normal hybrids (data not shown).

The resultant hybrid rice should carry a copy ofthe transgene Xa21. In the present study, the trans-genic hybrid produced by a cross combination ofZhenshan97A/Minghui63-Xa21, named Shanyou63-Xa21, was further investigated in detail. To confirmthe presence of the transgene Xa21 in the genome ofShanyou63-Xa21, the transgenic hybrid plants wereanalyzed by PCR and Southern hybridization (Fig-ure 6). The expected 1.4 kb Xa21-specific fragmentswere amplified from all the tested Shanyou63-Xa21plants in the PCR reactions with the primer pair ofU1 and I1, whereas no identical fragment was ob-tained from control hybrid plants in addition to a1.3 kb control amplification fragment (Figure 6A).In Southern hybridization with a probe of 1.4 kbXa21 fragment amplified from pCXK1301, the 11 kbHindIII-digested Xa21-specific fragments, as expectedfrom the Minghui63-Xa21 lines (see Figure 4B), weredetected from all the Shanyou63-Xa21 plants testedin addition to background bands, whereas no suchfragments were detected from control hybrid plants(Figure 6B). The hybridization pattern also revealedonly one copy of the transgene Xa21 existed in thehybrid rice Shanyou63-Xa21 plants.

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Figure 6. PCR (A) and Southern (B) analyses of transgenic hybrid rice Shanyou63-Xa21. M, molecular size markers; C, Shanyou63hybrid plants. Lanes 1–10, different Shanyou63-Xa21 plants produced from T1 plants of Minghui63-Xa21 (M1); lanes 11–20, differentShanyou63-Xa21 plants produced from T2 plants of Minghui63-Xa21 (M1). PCR and Southern hybridization were carried out as in Figure 4.

Investigation of BB resistance and other agronomictraits of Shanyou63-Xa21

To evaluate resistance of transgenic hybrid rice plantsto BB pathogen, Shanyou63-Xa21 plants were inocu-lated with race 6 at tillering stage. It was noted that allthe Xa21 transgenic hybrid plants ested were highlyresistant and, on average, the lesion length was lessthan 10% of the whole leaf inoculated (Table 2). Bycontrast the lesion length of non-transgenic control hy-brid plants was about half of the whole leaf inoculated(Table 2). The resistance spectrum of Shanyou63-Xa21 plants was also evaluated by inoculation with atotal of 19 BB strains, including nine Philippine races,three Japanese races and seven Chinese pathotypes.The tested transgenic plants were highly resistant to allthe 19 BB strains (Table 3). This result indicates thatthe transgene Xa21 maintains its wide resistance spec-trum in a hybrid genetic background. In addition, nopartial resistance was observed in any of the transgenicplants, and no detectable difference was observed onthe level of resistance to race 6 among the differentShanyou63-Xa21 plants indicating that the transgeneXa21 expresses its full dominant resistance to Xoo in ahybrid genetic background. This will promote the pop-ularization of Shanyou63-Xa21 as a new BB-resistanthybrid variety.

In addition to the BB resistance phenotype, theShanyou63-Xa21 plants were also evaluated for theirimportant agronomic traits. As shown in Table 2,the evaluation data obtained from both T1 and T2plants of the Minghui63-Xa21 lines from M1to M5andtheir hybrids in comparison with their control coun-

terparts demonstrated that no variations in the testedagronomic traits occurred in the generation of theMinghui63-Xa21 and Shanyou63-Xa21. In addition,the similar results were also obtained in other hy-brids made from Minghui63-Xa21 crossed with otherCMS lines including W-qingA, K17A, W17A, G46A,D297A, XinshanA (with rice blast resistance), D62A,D-shanA and D702A (data not shown). The cross testsalso proved that Minghui63-Xa21 maintained the nor-mal restoring ability of Minghui63 and could be usedas a new restorer in place of Minghui63 to produceBB-resistant hybrid rice.

Discussion

Xa21 is the first resistance gene cloned by a map-based cloning strategy in the cereal crops. Because ofits wide spectrum of BB resistance, Xa21 is of greatvalue in breeding rice varieties for BB resistance. Thecloned Xa21 gene has been introduced into several ricevarieties recently (Wang et al. 1996; Tu et al. 1998;Zhang et al. 1998; Zhai et al. 2000). However, inthese studies, no agronomic traits other than resistanceof the transgenic plants with Xa21 have been evalu-ated, and those transgenic lines have not been usedin hybrid breeding either. In this study we focused onselecting homozygous Xa21 transgenic lines withoutvariations on other agronomic traits and breeding BB-resistant hybrid rice. A strategy for breeding bacterialblight resistant hybrid rice with the cloned Xa21 genewas established on the basis of selfing, cross, andmarker-assisted selection as outlined in Figure 1. Thegenerated hybrid, Shanyou63-Xa21, was BB-resistant

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Table 3. Resistance reactions of Xa21 transgenic hybrid rice to 19 races or pathotypes of Xoo.

Strains Races/Pathotypes Origin Lesion length of Lesion length

Shanyou63-Xa21 (cm) of Shanyou63 (cm)

PXO61 Race 1 Philippines 3.4 ± 0.5 22.3 ± 2.3

PXO68 Race 2 Philippines 2.8 ± 1.1 17.2 ± 2.4

PXO79 Race 3 Philippines 2.9 ± 0.8 16.7 ± 2.4

PXO71 Race 4 Philippines 3.3 ± 0.5 19.3 ± 2.8

PXO112 Race 5 Philippines 2.6 ± 0.6 15.6 ± 2.2

PXO99 Race 6 Philippines 4.0 ± 1.1 24.8 ± 2.5

PXO280 Race 7 Philippines 2.4 ± 0.6 17.5 ± 2.9

PXO145 Race 8 Philippines 1.9 ± 0.5 15.4 ± 2.0

PXO87 Race 9 Philippines 2.8 ± 0.7 18.5 ± 1.7

T7174 T1 Japan 3.0 ± 1.0 17.7 ± 2.7

T7147 T2 Japan 2.8 ± 0.4 18.2 ± 2.4

T7133 T3 Japan 3.5 ± 1.2 23.0 ± 3.2

HLJ72 CI China 3.2 ± 0.5 18.1 ± 2.9

HB17 CII China 3.0 ± 0.7 17.6 ± 2.8

NX42 CIII China 3.4 ± 1.2 20.2 ± 3.1

Z173 CIV China 2.3 ± 0.8 17.5 ± 2.0

GD1358 CV China 3.9 ± 1.2 24.5 ± 2.6

LN57 CVI China 2.2 ± 0.6 16.1 ± 2.8

JS49-6 CVII China 2.7 ± 0.9 15.5 ± 1.9

The data were the average of at least five plants from which three infected leaves were scored.

and maintained the normal hybrid vigor. This studynot only improved the resistance of the important hy-brid Shanyou63 to Xoo, but also testified the geneticstability of the transgene Xa21 in a hybrid background.Therefore, the present study demonstrates the effec-tive approach to breed transgenic hybrid crops bycombining genetic engineering with marker-assistedselection.

From the resistance analysis to race 6, it wasobserved that the level of resistance of Minghui63-Xa21 plants was higher than that of Shanyou63-Xa21plants (Table 2). This difference on the level of resis-tance to race 6 may be attributed to the differences incopy number of the transgene Xa21 and genetic back-ground between Minghui63-Xa21 with two copies ofXa21 in a single genetic background and Shanyou63-Xa21 with only one copy of Xa21 in a hybrid geneticbackground. Which factor - copy number or geneticbackground – imposes greater effect on the resis-tance expression of Xa21 is still to be studied. Itseems that genetic background imposed greater ef-fect on the resistance expression of Xa21 than copynumber as there was no obvious resistance differenceamong Minghui63 transformants (T0) with differentXa21 copies (data not shown) while there was indeed

some resistance difference among Xa21 transformantsof different varieties (Zhai et al. 2000).

It was noted that no silence or inactivation of thetransgene was observed in the Minghui63-Xa21 (T2)and Shanyou63-Xa21 (T2 or T3) plants though fur-ther analysis in more generations would be requiredto access the stable expression of the transgene. Thiswas possibly due to only one copy of Xa21 integratedin transgenic plants in addition to an endogenoushomologue. One of the possible reasons for the trans-gene silence is the interaction between the integratedgene and its homologue (Flavell 1994). Therefore, alower degree of the transgene silence in single-copy-integrated plants than multiple-copy-integrated plantsis expected. Moreover, from single-copy-integratedplants the homozygous transgenic progenies are eas-ily identified, which can be directly used in transgenichybrid rice breeding.

Based on genetic engineering and marker-assistedselection of homozygous transgenic restorer lines, wenot only successfully transferred the Xa21 into hybridrice, but also established a effective strategy of molec-ular breeding of hybrid rice. Presently the Minghui63-Xa21 and Shanyou63-Xa21 plants have been releasedinto the fields with the approval of the Chinese Min-

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istry of Agriculture. Both varieties were resistant toXoo and maintained their normal elite traits under fieldconditions. In the near future they will be directlypropagated for rice production.

Acknowledgements

We thank Dr P. Ronald and Dr Wenyuan Song of theUniversity of California, Davis, USA for their assis-tance and suggestions; Dr R. A. Jefferson of CAM-BIA, Canberra, Australia for providing EHA105 andpCAMBIA1301; Dr Qian Qian of the Chinese RiceInstitute for providing rice varieties. This project wassupported by grants from the Chinese 863 High Tech-nology Program and the Rockefeller Foundation’sRice Biotechnology Program.

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