Crop Science 42:2072-2079 (2002)
© 2002 Crop Science Society of America
CELL BIOLOGY & MOLECULAR GENETICS
Molecular Breeding for the Development of Blast and Bacterial Blight Resistance in Rice cv. IR50
N. N. Narayanana,
N. Baisakha,
C. M. Vera Cruza,
S. S. Gnanamanickamb,
K. Dattaa and
S. K. Datta*,a
a Entomology and Plant Pathology Div., IRRI, DAPO Box 7777, Metro Manila, The Philippines
b Center for Advanced Studies in Botany, Univ. of Madras, Guindy Campus, Chennai-600025, India
* Corresponding author (s.datta{at}cgiar.org)
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ABSTRACT
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The objective of this study was to improve IR50, an elite Indica rice line, by molecular breeding approach involving marker-aided selection (MAS) and genetic transformation for resistance against blast (BL) and bacterial blight (BB). On the basis of BL pathogen population dynamics and lineage exclusion assays in southern India, the major BL resistance gene Piz-5 can exclude most Pyricularia grisea Sacc. {= P. oryzae Cavara [teleomorph: Magnaporthe grisea (Hebert) Barr]} lineages. Resistant CO39 near-isogenic line (NIL) C101A51 carrying Piz-5 was used as the donor parent and IR50 as the recurrent parent in backcrossing up to four generations. BC4F1 plants were finally selfed to produce BC4F2 seeds. A polymerase chain reaction (PCR)-based sequence-tagged site (STS) marker RG64 was used to identify Piz-5 in the segregating population and the resultant resistant progenies were obtained through phenotypic assays and MAS. To have further resistance to bacterial blight in this cultivar, these blast-resistant isolines were transformed with Xa21, which is known to confer resistance to all races of Xanthomonas oryzae pv. Oryzae [= X. campestris pv. Oryza (Ishiyama 1922) Dye 1978] (Xoo). Stable integration and inheritance of the Xa21 gene were demonstrated by PCR and Southern blot analysis from three independent transformants. Bioassay data showed that transgenic IR50 is resistant to pathogens, M. grisea and Xoo. Apart from that, pedigree analysis and phenotypic studies with IR50 revealed the endogenous presence of the Xa4 gene, which may show an increased level of resistance to the BB pathogen along with the transformed Xa21 gene. This is the first report documenting the stacking of two major genes (Piz-5 + Xa21) in rice using molecular breeding through MAS and transformation.
Abbreviations: BB, bacterial blight • BL, blast • bp, base pair • DAS, days after sowing • DLA, diseased leaf area • kb, kilobase • MAS, marker-aided selection • NIL, near-isogenic line • PCR, polymerase chain reaction • STS, sequence-tagged site • Xoo, Xanthomonas oryzae pv. Oryzae [= X. campestris pv. Oryza (Ishiyama 1922) Dye 1978]
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INTRODUCTION
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RICE BLAST caused by the heterothallic ascomycete fungus M. grisea and BB caused by Xoo (ex. Ishiyama, 1922) (Swings et al., 1991) are the most widespread and devastating diseases of rice. These diseases have plagued rice farmers since the beginning of recorded rice cultivation (Ou, 1985, p. 111–201) and can cause yield loss as high as 50% in some areas of Asia. Genetic and molecular studies of rice and these two pathogens have allowed a holistic view of the interactions of disease symptom production, host metabolic response, and resistance.
The severity and significance of damage caused by both diseases in rice have necessitated the development of strategies to control and manage them to reduce crop loss and to avert an epidemic. Though Bordeaux mixture, antibiotics, and other copper and mercurial compounds were used in the early 1950s, environmentally safe and stable chemical control agents rendering control at very low concentrations have yet to be developed. Today, the exploitation of host resistance appears to be the only reliable method of disease management. Fortunately, modern tools such as MAS and transformation (transgenics) for their manifold advantages can be effectively used to complement conventional breeding for the development of built-in resistance in rice cultivars.
In this study, an elite indica rice cultivar IR50, popular in southern India, that is quite vulnerable to both diseases, was selected for improvement. On the basis of the BL pathogen population dynamics and lineage exclusion assays (Gnanamanickam et al., 1998, 1999), we found that the major BL resistance gene Piz-5 can exclude most of the M. grisea lineages (Sivaraj et al., 1996; Lavanya and Gnanamanickam, 2000). To achieve multiple resistance for BL and BB in this cultivar, these blast-resistant isolines were transformed with the cloned BB resistance gene Xa21, as it is known to confer resistance to all known races of Xoo (Khush et al., 1990; Ikeda et al., 1990).
The objective of this study is to improve cultivar IR50 against BL and BB by introgressing Piz-5 using MAS in the process of recurrent backcrossing and introgressing Xa21 by genetic transformation via particle bombardment. Another objective is to obtain stable homozygous transgenic lines and evaluate them with biological assays for their resistance to both BL and BB diseases.
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MATERIALS AND METHODS
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Plant Materials, Phenotyping, and Selection Scheme
IR50, a high-yielding cultivar quite vulnerable to BL and BB, was used as the recurrent parent, and the resistant CO39 NIL C101A51 carrying the major BL resistance gene Piz-5 (developed at IRRI by Mackill and Bonman, 1992) was used as the donor parent for backcrossing up to four generations. BC4F1 plants were finally selfed to produce BC4F2 seeds. Starting from the BC1F1, through subsequent generations, resistant progenies were phenotyped upon inoculation with BL Race IK81-3 (International Race 101). These were also genotyped using selected STS marker RG64 for the gene Piz-5 and, from these, plants carrying the resistant alleles of the target resistance genes (based on their marker genotypes) were selected. To confirm the accuracy of MAS for single target genes, 25 lines of BC4F2 were finally used for further genotyping and phenotyping experiments.
PCR Identification of the Piz-5 Gene by the STS Marker RG64
The DNA of the parents and the crosses was subjected to PCR amplification for confirmation of the Piz-5 gene using the codominant RG64 STS marker (Hittalmani et al., 2000). Extraction and other protocols for PCR-based MAS were followed as per Zheng et al. (1995) and Hittalmani et al. (1995). The amount and quality of PCR amplification were first monitored by running 5-µL aliquots of the reaction mixture on a 1.5% agarose gel using 1X TAE buffer.
Restriction Digestion of PCR Products
The monomorphic PCR products were restricted at 37°C for 3 to 4 h with HaeIII to generate sequence amplified polymorphism (SAP) fragments (as described by Williams et al., 1991; Kleinhofs et al., 1993; Hittalmani et al., 1995). The digested products were run on a 1.5% agarose gel to resolve polymorphism.
Disease Evaluation
Twenty-five individual plants of the BC4F2 family were grown in a plastic tray under standard greenhouse conditions for phenotypic disease scoring with parental resistant and susceptible cultivars as controls. Twenty-one-day-old seedlings (at about the four-leaf stage) were inoculated with BL isolates IK81-3, IK81-25, and C9240-5 in two replicates. The inoculum was prepared as described by Bonman et al. (1986). The rice seedlings were sprayed with 50 mL of BL inoculum suspension per tray (5 x 104 conidia mL-1) and incubated for 24 h in a controlled dew chamber maintained at 25°C. The seedlings were then transferred to a temperature- and humidity-controlled chamber. Plants were monitored for BL lesions 1 wk after inoculation and scored for resistance and susceptibility when the typical BL lesions developed in the susceptible controls IR50 and CO39. Scoring and evaluation were done using the scale given by Bonman et al. (1986).
Blast Nursery
Near-isogenic line parents, recurrent parents, and the resistant progenies were tested in the rice BL nursery at IRRI, Metro Manila, The Philippines. The densely sown seedlings were surrounded by spreader rows of cultivars IR50 and IR72. Good disease development was ensured by planting infected seedlings with spreader rows. Observations on the disease reaction of the seedlings were recorded at 15, 20, 25, and 30 d after sowing (DAS). Disease scoring was expressed as percentage diseased leaf area (DLA) and length of lesions (0–9 scale, standard evaluation system of rice, IRRI).
Endogenous Presence of Xa4 in IR50
Seeds of IR24 (susceptible), IRBB4 (NIL for Xa4 gene), and IR50 were sown in plastic trays in rows and each row contained 10 single plants. Isolates PXO61 (Philippine Race 1) and PXO86 (Philippine Race 2) were stored in skim milk at -10°C for further future use. They were revived in modified Wakimoto's medium (Karganilla et al., 1973), subcultured on slants, and incubated at 30°C for 3 d. Inoculum was prepared by suspending the bacterial cells of each slant in sterile water and adjusting the concentration to 
109 cfu (colonies forming units) mL-1. Colored twine was used to divide the individual plant into two groups to be inoculated with PXO61 and PXO86. Clip inoculation was done on fully expanded leaves at the maximum tillering stage (45 DAS) (Kauffman et al., 1973). Lesion length of three to five leaves per plant per isolate were measured 14 d after inoculation.
Plasmid Constructs Used
Two plasmids were used for cotransformation: pC822, containing the Xa21 coding sequence, and pROB5, containing the selectable marker gene hph driven by the CaMV 35S promoter (Tu et al., 1998).
Transformation, Selection, and Regeneration Procedure
Sterile immature embryos of blast-resistant IR50 introgressed with the Piz-5 gene were used as the starting material for biolistic transformation. The transformation procedures involving explant preparation, selection of resistant calli on hygromycin-supplemented medium, subculturing, regeneration, rooting, and transfer to nutrient solution were essentially the same as described by Datta et al. (1997)(1998). The putative primary transformants were grown to maturity in the transgenic greenhouse under a day:night temperature regime of 29:23°C, and subsequent generations were advanced through seed progenies.
DNA Extraction, PCR, and Southern Blot Analysis
Total genomic DNA was extracted according to the procedure of Dellaporta et al. (1983). PCR analysis of the DNA from T0 and T1 progenies was done using STS primers U1 and I1 (Wang et al., 1996). For Southern analysis, 6 to 8 µg of DNA were digested overnight with EcoRV (Gibco-BRL, Gaithersburg, MD) in a final volume of 40 µL. Southern blotting and hybridization were done as per Baisakh et al. (2001). The 1.4-kilobase (kb) PCR amplified fragment of pC822 was labeled with 
-[32P]-dCTP using the Rediprime Labeling Kit (Amersham Heights, IL) and was used as the hybridization probe.
Bioassay
Transgenic T1 progenies of a single T0 plant (T13) were inoculated with three diagnostic races of BB pathogen: PXO86 (Race 2), PXO99 (Race 6), and PXO341 (Race 10). The inoculum preparation and inoculation methods were the same as those described earlier for Xa4. At the maximum tillering stage, each plant was inoculated with the above three strains of Xoo using the leaf clipping method (Kauffman et al., 1973). Plant reaction to each race of Xoo was scored after 7 and 14 d of inoculation.
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RESULTS AND DISCUSSION
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Crossing and Phenotypic Evaluation using Various Diagnostic Strains
After three BC generations, 308 BC3F1 seeds were obtained and selected by phenotypic evaluation using international BL Race IK81-3. Plants segregated in a 1:1 ratio of resistant:susceptible (Table 1)
, further confirming their resistance was governed by a single dominant gene (Piz-5). The resistant progenies were advanced to the next generation and selfed. The disease reactions of the 25 plants (selected from the next generation) were screened with various diagnostic strains of M. grisea pathogens (Fig. 1, 2)
. The donor C101A51 showed a high level of resistance to all three races of M. grisea, whereas IR50 and CO39 showed susceptible symptoms. The reactions of the recurrent parent lines toward IK81-3 and IK81-25 were found to be highly resistant but they were susceptible to Race C9240-5, as this isolate is compatible to the gene Piz-5. The performance of the lines challenged with isolate C9240-5 was intriguing. This isolate, belonging to the genetic Lineage 44, is the only isolate in the Philippines known to be compatible with the Piz-5 gene (Chen et al., 1995; 1996). Figure 1 clearly shows a characteristic susceptible reaction of C101A51 when inoculated with C9240-5. This isolate might have yielded a virulence pattern fully consistent and compatible with that of the individual genes of the parental NIL. It appears that some isolates may carry factors that modify the interactions of their avirulence genes with major resistance genes (Hittalmani et al., 2000).
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Table 1. Reaction of the BC3 population represented by three individual lines A1, B1, and C1 segregating for Piz-5 to blast isolate IK81-3 from the cross C101A51 x IR50.
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Fig. 1. Phenotypic reaction of Piz-5 introgressed IR50 line against rice blast isolates C9240-5, IK81-3, and IK81-25.
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Fig. 2. Phenotypic evaluation of blast resistant IR50 containing the Piz-5 gene using three diagnostic strains following the procedure of Bonman et al., 1986.
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Identification of Piz-5 Gene by STS Marker RG64
To examine the validity of STS as genetic markers, genomic DNA from the 25 BC4F2 individuals of the cross between the susceptible recurrent parent (IR50) and the resistant isogenic line (C101A51) was PCR-amplified using RG64 STS primers. Polymorphism was not revealed (Fig. 3A)
, but cleavage of the PCR product with restriction enzyme HaeIII revealed a clear polymorphism between susceptible and resistant plants when resolved on 1.5% agarose gel (Fig. 3B). The banding pattern of the progenies was classified as homozygous for CO39 or any susceptible marker [650-base pairs (bp)], homozygous for C101A51, the 750-bp marker associated with resistance or as a heterozygote (displaying both fragments). The marker genotypes were then used to predict the presence of the Piz-5 gene in the progenies. Since RG64 is located 2.8 cM from the Piz-5 gene, it was predicted that those markers carrying only the 750-bp fragment were considered as homozygotes for the Piz-5 locus. From the results, we found that all 25 lines were homozygous-resistant (RR), showing the 750-bp fragment. The accuracy of identifying individuals carrying the resistance gene by selecting for the linked marker was examined by genotyping and phenotyping experiments. Our results from the disease scoring and marker analysis data were consistent, emphasizing the presence of the resistance allele Piz-5 in the cultivar. This is further evidenced by studies that showed 97% matching of the identity of the Piz-5 gene based on the genotype with the linked RG64 STS marker (Hittalmani et al., 1995). Furthermore, the accuracy of identification can be improved when two markers flanking the Piz-5 gene are used simultaneously. This emphasizes the usefulness of marker-assisted selection in precisely identifying the genotype of a linked target gene in a segregating population.
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Fig. 3. The generation of sequence-specific amplicon polymorphism between resistant (C101A51) and susceptible (IR50) rice varieties for marker-aided selection. (A) Gel analysis of polymerase chain reaction (PCR) products before restriction digestion. (B) Gel analysis of PCR products after digestion with restriction enzyme HaeIII. Lanes 1 and 2 represent the susceptible parent IR50 and donor resistant line C101A51, respectively. The molecular weight of PCR products as banding patterns is indicated in kb (kilobases). M = Molecular weight marker (1-kb ladder).
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These lines produced by MAS provide excellent tools for plant pathologists to study the reactions of different isolates across lineages to different combinations of genes. Further studies on the resistance genes for their durability and wide applicability are in progress. With the availability of all these PCR-based primers, candidate genes, and RFLP-linked genes, simple and rapid marker-assisted selection could be easy and efficient in rice breeding programs.
Disease Reaction in the Blast Nursery
Fig. 4
presents the reaction of IR50 blast-resistant lines with Piz-5 to BL under natural conditions (Fig. 5)
. On the 30th day after sowing, results showed that the susceptible checks (CO39 and IR50) had maximum infection (> 65% DLA) and most of the isolines with Piz-5 showed less infection, 5 to 10% DLA (Fig. 5). Certain lines showed susceptible reactions of 25 to 30% DLA, perhaps because this gene (Piz-5) is susceptible to some isolates studied in the Philippines. This is in agreement with the report that field infection always reflects compatibility of isolates with the host (Chen et al., 1995). However, such isolates compatible with Piz-5 under laboratory-controlled conditions might not prevail or survive in natural field conditions. Studies carried out by other researchers show the relative and absolute fitness of strains carrying all compatibility combinations (Silue et al., 1992; Tohme et al., 1993).
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Fig. 4. Evaluation of isogenic lines and gene stacked lines after 15 to 30 d with 5-d interval in the rice blast nursery at IRRI, The Philippines, based on the standard evaluation system of rice 15, 20, 25, and 30 d after inoculation.
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Fig. 5. Blast nursery showing higher infection in susceptible check (IR50) and less infection on isolines of IR50 with Piz-5 gene.
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Endogenous Presence of Xa-4 in IR50
Table 2
presents the lesion length of lines IR24, IR50, and IRBB4 with mean values. The mean lesion length of IRBB4 and IR50 of all the plants was always shorter when inoculated with isolate PXO61 than with PXO86. The pattern of inheritance of lesion length following inoculation with PXO61 is explained on the basis of a single dominant gene for a high level of resistance to Xoo, but the pattern of inheritance of lesion length following inoculation with PXO86 is not clear (Koch and Parlevliet, 1991). Xa4 is clearly expressed against PXO61, in both IRBB4 and IR50, since they have almost the same lesion length, but the mean lesion length of IR50 is still shorter than that of IRBB4. When inoculated with PXO86, IR50 has a mean lesion length shorter than that of IRBB4 (Fig. 6)
. Comparative data on IR50 showed the same pattern of inheritance of the gene Xa4 and its resistance to these isolates like that of resistant cultivar IRBB4. Xa4 is considered to be a defeated major resistance gene that plays a key role in rice–Xoo interaction. Riley (1973) introduced the term defeated or ghost genes, which may contribute in a minor way to resistance, and visualized them as contributing to polygenically determined quantitative resistance. In such reports, host lines carrying a major race-specific resistance gene were compared with near-isogenic host lines not carrying this gene. When exposed to a race virulent to the resistance gene, the NIL should show identical disease responses because the resistance gene is no longer effective. In some cases this was observed, but in others the NIL carrying the resistance gene showed some resistance. This indicated the endogenous presence of Xa4 in cultivar IR50, which showed the same reaction and made it resistant to these isolates of Xoo. It was presumed that IR50 might carry one or more minor genes for quantitative resistance, which is in close linkage with Xa4, making the resistance more apparent and dominant to act against these virulent isolates. IRBB4 also exhibited variation when it reacted with different races and isolates. Isolate PXO61 of Race 1 is avirulent to Xa4, whereas PXO86 of Race 2 is virulent to this gene (Vera Cruz and Mew, 1989; Koch and Parlevliet, 1991). From the pedigree background of cultivar IR50, we suspect that the quantitative resistance gene Xa4 may have some endogenous polygenic factor. This gene may play an important role in additive and synergistic resistance to BB along with the transformed Xa21 gene. For example, transformed IR50 (containing both Xa21 + Xa4) showed enhanced resistance to Race 10 (PXO341). Race 10 (PXO341) is virulent to Xa21 but moderately avirulent to Xa4.
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Table 2. Origin and background of rice cultivars/lines and lesion length measure 14 d after clip inoculation with two races of Xanthomonas oryzae pv. oryzae to identify the endogenous presence of the Xa4 gene in genotype IR50.
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Fig. 6. Bacterial blight reactions to (A) Race PXO61 and (B) PXO86 in IR24 (1), IRBB4 (2), and IR50 (3) to examine endogenous Xa-4 gene in rice cv. IR50.
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Transformation and Molecular Analysis
Blast-resistant IR50 lines were transformed with Xa21 by particle bombardment. Out of 16 regenerants, three were found to be transgenic as revealed by the 1.4-kb fragment from PCR analysis (Fig. 7)
. These were further confirmed by Southern blot analysis showing the presence of a 3.8-kb Xa21 fragment corresponding to most of its coding region and its complete intron sequence (Fig. 8) . Apart from this, there were two rearranged fragments of the transgene in two of the lines. The band common to all the transgenics and the nontransformed control correspond to the endogenous Xa gene having homology with Xa21 (Tu et al., 1998). T1 progenies of 20 plants each from three transgenic plants (IR50-13, 14, and 15) were grown in the containment greenhouse and were used for further analysis. The inheritance of the transgene in the next generation shown by PCR analysis (data not shown) and Southern analysis (Fig. 9)
indicated the stable integration of Xa21 in the IR50 genome. T1 progenies showed the Mendelian segregation ratio of 3:1, again confirming a single locus integration of the Xa21 transgene. Earlier observations also confirmed those of this study (Tu et al., 1998).
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Fig. 7. Polymerase chain reaction analysis of transgenic IR50 T0 Plants 13, 14, and 15 showing a 1.4-kilobase (marked with arrow) Xa-21-specific DNA fragment amplified by primers U1 and I1. M = molecular weight marker; Lanes 1, 2, and 3 represent nontransformed IR50, IRBB21, pC822, and transgenic IR50 Plants 13, 14, and 15, respectively.
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Fig. 8. Southern analysis of transgenic T0 plants. The arrow represents the expected 3.8-kilobase (kb) band corresponding to the Xa21 transgene. Lanes 1 to 5 represent nontransformed IR50, transgenic T0 IR50-13, 14, and 15, and plasmid pC822, respectively. Five- to 7-µg plant genomic DNA and 30 pg of plasmid DNA were digested with EcoRV. The 1.4-kb polymerase chain reaction amplified fragment of pC822 was labeled with -[32P]-dCTP using the Rediprime Labeling Kit (Amersham Heights, IL) and was used as the hybridization probe.
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Fig. 9. Southern analysis of transgenic T1 plants. A total of 5 to 7 µg of plant genomic DNA and 30 pg of plasmid DNA were digested with EcoRV and hybridized with the same enzyme-digested plasmid DNA fragment. The arrow marks the expected 3.8-kilobase hybridizing band corresponding to Xa21 transgene in T0 and T1 plants. T0 = primary transgenic plant, T1 = selfed progenies of T13 (T0).
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Xa21 is the first resistance gene to be cloned (Song et al., 1995; Wang et al., 1996) and transferred into rice, showing higher resistance of the transgenics to the BB pathogen (Song et al., 1995; Tu et al., 1998; Zhang et al., 1998; Tang et al., 1999). All these studies demonstrated the tremendous potential of transgenic Xa21 rice plants for inclusion in molecular breeding programs. Several reports of field evaluation in cereals have been documented for different agronomic traits such as Bt gene in maize (Armstrong et al., 1995), herbicide resistant gene in rice (Jiang et al., 2000), and Xa21 in rice (Tu et al., 2000). We chose the transgenic approach to introduce Xa21 into rice, keeping in view the time advantage in developing the homozygous lines in 2 generations (T2) instead of 3 to 4 backcrossing generations followed in MAS. Moreover, Xa21 transgene itself could be used as a marker.
Bioassay and Molecular Marker Analysis of Transgenic T1 Plants for Blast
The reaction of these transgenic plants to leaf BL was evaluated by inoculation with Race IK81-3 of M. grisea (Fig. 10)
. The scores represent the overall mean of the two replicates from two independent challenge experiments performed in the transgenic greenhouse at IRRI. The response of the 10 individual plants from each experiment to BL infection (Table 3)
revealed that all transformants exhibited enhanced resistance to rice BL (1–2 mm), whereas the controls CO39 and IR50 showed higher susceptible reactions with a maximum lesion size of 7 to 12 mm. The increased resistance in these transgenic plants against rice BL was further confirmed by STS RG64 analysis showing the presence of the Piz-5 gene (data not shown). This provides evidence of the stability of the Piz-5 MAS introgressed gene even after transformation with Xa21.
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Fig. 10. Reactions of transgenic T1 IR50 plants carrying Xa21 and Piz-5 introgressed for blast resistance when inoculated with IK81-3. Leaves 1 to 4 represent CO39, IR50, C101A51 (Piz-5), and transgenic IR50 (Piz-5 + Xa-21), respectively.
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Table 3. Screening of transgenic plants (T1) against the rice blast fungus Magnaporthe grisea race IK81-3 in the greenhouse.
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Bioassay of T1 Plants for Bacterial Blight Resistance
The reaction of the transgenic T1 plants to the BB pathogen was studied with three different Races: 2, 6, and 10 (Fig. 11)
. The bioassay data also corresponded to the molecular analysis. Among 20 plants, 15 were completely resistant and five were susceptible. The lesion length of 13 T1 plants and the other donor IRBB21 was <4 cm (Table 4)
, whereas the lesion length of the control IR24 ranged from 12 to 17 cm. It is interesting to find that nontransformed IR50 showed a lesion length equal to that of the resistant reaction when inoculated with Race 10, as IR50 contains the endogenous Xa4 gene. Transgenic IR50 showed complete resistance to all three races, emphasizing the synergistic effect of transformed Xa21 with the endogenous Xa4 gene.
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Fig. 11. Resistant reactions of transgenic T1 IR50 plants introgressed with Xa-21 for bacterial blight resistance inoculated with Races (A) PXO99, (B) PXO86, and (C) PXO341. Leaves 1 to 5 represent IR24, IR50, IRBB4, IRBB21, and transgenic IR50 (T13) (Piz-5 + Xa-21), respectively, scored 14 d after inoculation.
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Earlier studies have also shown that the transgenic lines carrying the Xa21 gene had enhanced resistance to multi-isolates of Xoo (Wang et al., 1996). Our results also clearly demonstrate that the transgenic line of IR50 showed higher resistance than the donor line IRBB21, as observed earlier with IR72 (Tu et al., 1998). This may be due to the additive effect or pyramiding of the endogenous Xa4 gene along with the transformed Xa21 gene. Similar results were also reported from several rice lines with more than one gene for resistance to Xoo such as Xa4, xa5, xa13 and Xa21 (Huang et al., 1997; Chen et al., 2000; Singh et al., 2001).
Although the line in question contained the hph gene used as the antibiotic selectable marker gene (data not shown), this study shows great potential of transgenic approach, which could be used in future, using only the expression cassette (minimal vector approach) with nonantibiotic selection system and by Agrobacterium tumefaciens-mediated transformation to meet the regulatory issues with low copy integration. These stacked lines are currently being advanced in the transgenic greenhouse to achieve homozygosity for the Xa21 transgene. Efforts are under way to extend the stacked homozygous blast- and blight-resistant transgenic IR50 (Piz-5 + Xa21) lines for field-testing in India.
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ACKNOWLEDGMENTS
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Financial support from the Rockefeller Foundation is gratefully acknowledged. We are grateful to Dr. P.C. Ronald of the University of California, Davis, CA, for providing the plasmid pC822, and to Dr. S. Hittalmani, University of Agricultural Sciences, Bangalore, India, for providing the primer sequence of RG64. Thanks go to colleagues Jumin Tu, Sena Balachandran, Lina Torrizo, Norman Oliva, Michelle Viray, and Reynaldo Garcia for their research and technical assistance and to Bill Hardy for editorial suggestions.
Received for publication July 12, 2001.
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INTRODUCTION
