INTRODUCTION
Rice tungro disease (RTD) causes a significant yield loss in rice-growing areas of South and South East Asia. It is believed to be responsible for 0·05–0·10 of annual losses of rice yield in Asia (Dai & Beachy Reference Dai and Beachy2009) with the level in India being c. 0·02, although losses could be substantial at the regional levels (Muralidharan et al. Reference Muralidharan, Krishnaveni, Rajarajeswari and Prasad2003). RTD is caused by simultaneous infection of rice tungro bacilliform virus (RTBV), a pararetrovirus having a double-stranded DNA (dsDNA), belonging to the genus Tungrovirus (Hull et al. Reference Hull, Geering, Harper, Lockhart, Scholez, Fauquet, Mayo, Maniloff, Desselberger and Ball2005), and rice tungro spherical virus (RTSV), a positive single-stranded RNA (ssRNA) virus of the genus Wailkavirus (Shen et al. Reference Shen, Kaniewska, Smith and Beachy1993; Hull Reference Hull1996). The virus complex is transmitted by several species of green leafhoppers (GLH, Nephotettix virescens), the major vector over much of South East Asia (Hibino & Cabauatan Reference Hibino and Cabauatan1987). RTBV is responsible for the development of tungro symptoms, as shown by artificial introduction of the cloned viral DNA into rice (Dasgupta et al. Reference Dasgupta, Hull, Eastop, Poggi-Pollini, Blakebrough, Boulton and Davies1991). For most rice cultivars, infection with the virus complex induces the economically important RTD (Hibino et al. Reference Hibino, Roechan and Sudarisman1978). Typical RTD symptoms include severe stunting and yellow–orange discolouration of infected plants, reduced tillering and incomplete panicle emergence with sterile florets (Azzam & Chancellor Reference Azzam and Chancellor2002).
Because of the economic significance of RTD, incorporation of tungro resistance has been an important breeding objective in rice improvement programmes in Asia. The initial breeding efforts during the 1960s and early 1970s were aimed at developing RTD resistant lines by increasing resistance to insect transmission (Mew et al. Reference Mew, Leung, Savary, Cruz and Leach2004). During the late 1970s, several sources of resistance to RTSV were identified. Far fewer varieties were identified for RTBV, although a few RTBV-tolerant varieties have been developed (Khush et al. Reference Khush, Angeles, Virk and Brar2004). Most attempts to control RTD by classical breeding have not been sustainable, because the improved rice varieties were bred for elevated resistance to the GLH rather than the viruses, and the continual release of new GLH-resistant varieties resulted in a boom–bust cycle (Dahal et al. Reference Dahal, Hibino, Cabunagan, Tiongco, Flores and Aguiero1990; Azzam & Chancellor Reference Azzam and Chancellor2002).
Transgenic resistance against RTD has also been reported, with most reports targeting RTBV, as it is the agent responsible for most symptoms (Dai et al. Reference Dai, Wei, Alfonso, Pei, Duque, Zhang, Babb and Beachy2008; Tyagi et al. Reference Tyagi, Rajasubramaniam, Rajam and Dasgupta2008; Ganesan et al. Reference Ganesan, Suri, Rajasubramaniam, Rajam and Dasgupta2009); although an early report indicated that targeting RTSV also results in tungro resistance (Sivamani et al. Reference Sivamani, Huet, Shen, Ong, Dekochko, Fauquet and Beachy1999). These developments were achieved either in japonica rice cultivars or in Pusa Basmati-1 (PB-1), an indica rice cultivar most conducive to transgenesis. Diversification of transgenic resistance by a backcross (BC) breeding approach can be useful in managing RTD through the incorporation of the transgene construct to several high-yielding rice varieties.
RNA interference (RNAi) has been used recently as a tool for developing virus resistance in plants against a variety of viruses (Abhary et al. Reference Abhary, Anfoka, Nakhla and Maxwell2006; Lennefors et al. Reference Lennefors, Savenkov, Bensefelt, Wremerth-Weich, Van Roggen, Tuvesson, Valkonen and Gielen2006; Bonfim et al. Reference Bonfim, Faria, Nogueira, Mendes and Aragão2007; Ramesh et al. Reference Ramesh, Mishra and Praveen2007; Aragao & Faria Reference Aragao and Faria2009; Ma et al. Reference Ma, Song, Wu, Jiang, Li, Zhu and Wen2011), including RTBV (Tyagi et al. Reference Tyagi, Rajasubramaniam, Rajam and Dasgupta2008). RNAi results in the modulation of gene expression by degrading transcripts, preventing translation of transcripts or by interfering with transcription, in which small RNA molecules play an active role (Chicas & Macino Reference Chicas and Macino2001; Herr Reference Herr2005; Mazo et al. Reference Mazo, Hodgson, Petruk, Sedkov and Brock2007). In the present study, an RNAi construct, consisting ORF IV of RTBV in both sense and antisense orientation, was transferred from a transgenic PB-1 line into two commercial, high-yielding but tungro-susceptible, rice cultivars (IET4094 and IET4786) popular in West Bengal, India. Repeated BCs were performed and the progenies were evaluated for the presence of transgene and their reaction to artificial virus inoculation.
MATERIALS AND METHODS
Plant materials and growth conditions
The transgenic PB-1 line (PB1/RTBV-O-Ds2) described in Tyagi et al. (Reference Tyagi, Rajasubramaniam, Rajam and Dasgupta2008) was used as the donor of resistance to RTBV. Two popular high-yielding rice cultivars, IET4094 (cvar Khitish) and IET4786 (cvar Satabdi) of West Bengal, India were used as recipient of the transgene. The transgene cassette present in the donor parent PB1/RTBV-O-Ds2 consisted of an RTBV DNA fragment between residues 5700 and 7026 encoding the ORF IV of an Indian isolate, RTBV-AP in both sense as well as anti-sense orientation with 35S promoter and a selectable marker hygromycin phosphotransferase (hpt). The seed sowing, planting and crossing were performed in a containment polyhouse facility. Seeds of the parental lines and crossed progenies were first germinated in plastic trays and transplanted at 30 days after seeding into pots filled with 5 kg of clay loam soil and supplemented with chemical fertilizers (1·1 g N in the form of urea, 0·2 g P2O5 in the form of single super phosphate and 0·7 g K2O in the form of muriate of potash.) The temperature of the polyhouse was kept between 28 and 31°C and the photoperiod was maintained at 14/10 h light/dark using supplementary lighting.
Backcrossing
The BC breeding method was followed to transfer the RNAi construct. Initially two sets of crosses, viz. IET4094×PB1/RTBV-O-DS2 (set-I) and IET4786×PB1/RTBV-O-DS2 (set-II), were made using transgenic plants as the pollen parent. The F1 seeds of both crosses were raised and seedlings were screened for the presence of transgenes by polymerase chain reaction (PCR) analysis using gene-specific primers. Screening was conducted for both the ORF IV and hpt-coding regions within the construct. The transgene-positive F1 plants were used as pollen parents in the backcrossing with the female recurrent parents (IET4094 and IET4786). The BC1F1 plants were screened for the presence of transgenes at the seedling stage and positive plants were used as pollen parents in next BC. Subsequent BCs were performed similarly.
DNA and RNA isolation and PCR analysis on transgenic plants
DNA was extracted from rice leaf blades 400–500 mm long, as described by Dellaporta et al. (Reference Dellaporta, Wood and Hicks1983). The extracted DNA was dissolved in 20 μl of water and used in PCR. Total RNA was isolated from 100 mg of leaf tissue using TRIZOL reagent (a ready-to-use, monophasic solution of phenol and guanidine isothiocyanate; Genei, India) according to the manufacturer's protocol. PCR amplifications were performed in an Eppendorf (Master cycler) thermal cycler with Taq DNA polymerase (Fermentas, India) and cycling conditions of initial denaturation (94°C) for 5 min, 35 cycles of 94°C for 30 s, respective annealing temperatures for 30 s and 72°C for 1 min, followed by final extension of 72°C for 7 min. The amplified PCR products were resolved in 10 g/l of TBE-agarose gels. The details of the gene-specific primers used in the present study are summarized in Table 1.
Table 1. List of primers used in the present study
Measurement of morphological and quality parameters
Observations of grain yield, yield component traits and grain quality parameters were recorded at maturity in F1 and subsequent BC generations. Data were collected from individual plants. The days to heading was defined as when 0·50 panicles of a plant reached the heading stage. Amylose content, alkali spreading value and kernel elongation ratio of rice grains were determined following the standard evaluation system for rice from the International Rice Research Institute (IRRI 1996). The protein content of grains was estimated as described by Bradford (Reference Bradford1976).
Co-inoculation of RTBV and RTSV with GLH
RTBV and RTSV were maintained in a containment polyhouse facility, as described earlier, on the RTD-susceptible rice cultivar TN1 by GLH-mediated serial transfer initiated using naturally infected plants. GLH were reared on healthy rice plants, and were then enclosed in a mylar cage for 24 h with symptomatic rice plants for virus acquisition. The test plants were inoculated for 24 h with three viruliferous GLH/plant obtained as above. Altogether, six batches, each containing ten plants from BC2F1 generation of set-I and set-II, recurrent parents, transgenic donor parent and the susceptible line TN1 were subjected to GLH-mediated virus inoculation. Another group of six batches of un-inoculated plants (ten plants from each line) were also taken as control.
Disease scoring
Disease incidence ratings were recorded according to Dai et al. (Reference Dai, Wei, Alfonso, Pei, Duque, Zhang, Babb and Beachy2008) based on a modified Standard Evaluation System for Rice (developed by IRRI, The Philippines). The plants inoculated with virus were scored for their reaction to tungro at 21 days after inoculation (DAI) when symptoms began to appear. Disease scoring was conducted on the basis of percent reduction in plant height as compared with un-inoculated plants and symptoms of leaf yellowing: 1, No symptoms; 2, 1–5% height reduction, no distinct yellowing symptoms, 3, 6–10% height reduction, no distinct yellowing symptoms; 4, 11–20% height reduction, no distinct leaf symptoms; 5, 21–30% height reduction, no distinct leaf symptoms; 6, 31–40% height reduction with distinct yellow leaf discolouration; 7, 41–50% height reduction, with distinct yellow leaf discolouration; 8, >50% height reduction with distinct yellow leaf discolouration; 9, >50% height reduction, with distinct yellow–orange leaf discolouration. Plant reaction to disease incidence was scored as: 0–3, resistant/tolerant; 4–6, moderately tolerant; 7–9, susceptible.
Detection of RTBV and RTSV
RTBV and RTSV were detected in inoculated plants by PCR at 20 DAI using total DNA and RNA isolated from rice leaves. Sequences of primers employed for PCR are given in Table 1. The published sequences of RTBV (accession number: AJ314596; Nath et al. Reference Nath, Mathur and Dasgupta2002) and RTSV (accession number: AM234049; Verma & Dasgupta Reference Verma and Dasgupta2007) were used to design the primers. The primers for RTBV were designed to amplify a fragment of c. 1·1 kb RTBV genome covering the parts of RT/RNase H and ORF IV (nt 5444–6553). For RTSV, the primers were expected to amplify an 848 bp region of the large polyprotein (nt 3675–4522). For RTSV, cDNA was prepared from 5 μg of total RNA isolated from the infected plants using a first-strand cDNA synthesis kit (Fermentas Life Sciences, India) following the manufacturer's instructions. The cDNA was used as the template in reverse-transcription PCR (RT–PCR). The PCR cycles were similar as described earlier.
Statistical analysis
Mean values and standard errors were computed using Microsoft Excel. The patterns of similarity and dissimilarity of the progenies based on morphological and grain quality parameters were assessed through cluster analysis using Ward's method (Ward Reference Ward1963) in SPSS version 16.0 for windows (SPSS Inc., Chicago, IL, U.S.A.;SPSS Inc. 2007). Observations on plant height under tungro-infected and uninfected conditions were analysed in two factorial completely randomized designs (CRD) and were also subjected to mean comparison by paired sample t test. Analyses were done in SPSS.
RESULTS
Backcrossing and progeny analysis
A large number of seeds were produced from the two sets of crosses, i.e. set-I: IET4094 (female)×PB1/RTBV-O-Ds2 (male) and set-II: IET4786 (female)×PB1/RTBV-O-Ds2 (male). Out of 80 F1 plants tested from set-I, 68 plants carried the transgene and 84 out of 100 F1 plants from set-II (Table 2). PCR analysis of genomic DNA from F1 plants using primers specific to ORF IV and hpt gave amplification of 883 and 531 bp fragments that strongly indicated the presence of the transgene (Fig. 1).
Fig. 1. PCR analyses showing the amplicons for RTBV ORF IV (883 bp) and hpt (531 bp) from representative BC2F1 progenies, transgenic donor parent and non-transgenic parent (IET4094). M, molecular weight marker; lanes 1–6, BC2F1 plants from set-I (recurrent parent IET4094); lanes 7–12, BC2F1 plants from set-II (recurrent parent IET4786); lanes 13–14, transgenic donor parent PB1/RTBV-O-Ds2 (TP); lanes 15–16, non-transgenic (NT) parent IET4094.
Table 2. Plants carrying the RTBV ORF IV gene and the marker gene hygromycin phosphotransferase (hpt) in different generations*
* Numbers of plants negative for both RTBV ORF IV and hpt are not shown.
† Equal number of progenies each from five randomly selected transgene positive (ORF IV and hpt) families were assayed in BC2F2 and BC2F3 generations.
Five healthy transgene positive F1 plants from each set of crosses were used as pollen parents for backcrossing (BC1) with the recurrent parent (IET4094 and IET4786). A total of 120 BC1F1 seeds from each of the two sets of crosses were obtained. The BC1F1 plants derived from the seeds were then screened for the presence of transgene. Out of 120 plants, 55 and 70 plants from set-I and set-II, respectively, contained both ORF IV and the hpt-marker gene. The BC1F1 plants were evaluated for their morphological similarity to the respective recurrent parent and 5 BC1F1 plants from set-I and from set-II, containing the transgenes and showing the highest phenotypic similarity to the recurrent parents, were backcrossed. In the BC2F1 generation, 100 plants of each set were assayed for the presence of the transgene. A total of 62 plants of set-I and 54 plants of set-II contained the transgene. The result of PCR analysis on the BC2F2 and BC2F3 generations indicated the stable segregation of the transgene (Table 2).
Phenotyping of the progenies in crossed generation
The mean values for phenotypic characters in the generations are given in Table 3. The F1 plants from the first set of crosses were superior to the recurrent parent (IET4094) for panicles/plant, grain length, grain L/B ratio, mean grain weight and grain yield/plant. The F1 plants from set-II of crosses were superior to the recurrent parent (IET4786) for panicles/plant, panicle weight, grains/panicle, mean grain weight and grain yield/plant. However, the proportion of fertile tillers in F1 plants was reduced considerably compared with that of the corresponding recurrent parents. Comparison of mean performance of backcrossed plants revealed that in the case of set-I, the mean plant height in BC1F1 and BC2F1 generations remained similar to that of the recurrent parent. The mean values for panicle weight, panicle length, grains/panicle, days to heading, grain L/B ratio, proportion of fertile tillers and grain yield/plant in BC2F1 generation were also similar to that of the recurrent parent (Table 3). In set-II, the phenotypes of the BC1F1 plants were similar to the recurrent parent, but in BC2F1 generation, the plants were similar for panicle length and grain characteristics.
Table 3. Mean performance of parental lines and the progenies in F1 and BC generations
* Represents means±s.e.
A total of 15 and 11 phenotypically similar BC2F1 plants from set-I and set-II, respectively, were evaluated for morphological characteristics and some important grain quality traits such as amylose content, alkali spreading values, kernel elongation ratio and protein content (Supplementary Tables S1 and S2 available online at http://journals.cambridge.org/ags). Some of the BC2F1 plants were similar to the recurrent parent for most of the traits studied. Cluster analysis on the basis of the morphological and quality traits indicated that out of 15 BC2F1 in the case of progenies for IET4094, six BC2F1 plants were most similar to IET4094 (Supplementary Fig. 1a available online at http://journals.cambridge.org/ags). However, the BC2F1 of set-II formed two major clusters and five plants exhibited higher similarity to IET4786 than the others (Supplementary Fig. 1b).
Assessment of RTD resistance in BC2F1 progenies
The RTD resistance in BC2F1 generation was assessed by inoculating test plants with the rice tungro viruses through GLH-mediated inoculation by taking TN1, an RTD-susceptible rice cultivar, as a susceptible control. The development of symptoms in the recurrent parents was typical of RTD, whereas those in the PB1/RTBV-O-Ds2 and BC2F1 plants were very mild (Fig. 2 a). However, it was observed that the yellowing of leaves in the recurrent parents started at a later stage of infection (45–50 DAI). The presence of tungro viruses in the source plants as well as in the inoculated plants was confirmed by the presence of 1·11 kb and 848 bp PCR fragments specific to RTBV and RTSV, respectively (Fig. 2 b, c).
Fig. 2. Assessment of RTD resistance in BC2F1 progenies. (a) Representative infected transgenic BC2F1 plants of set-I and set-II, recurrent parents and donor parent (PB1/RTBV-O-Ds2). The seedlings were subjected to GLH-mediated forced inoculation with RTBV and RTSV at 10 days after planting. The image was taken at 35 days post-inoculation. (b, c) PCR analysis of the inoculated plants for the detection of RTBV and RTSV, respectively, at 20 DAI. M, molecular weight marker; lanes 1–6, representative inoculated plants showing the presence of amplicons specific to RTBV (1·11 kb) and RTSV (850 bp). The sizes of some of the standard size marker are shown indicated by arrows. (d) Height of inoculated (grey bars) and un-inoculated plants (white bars) at 70 DAI. Each bar represents mean height of 10 plants. Error bars indicate standard errors of means. TN1, an RTD-susceptible rice variety was included in the disease resistance assay as a susceptible control. (e) Percent reduction in plant height under inoculated condition at different DAI. Each line represents the average percentile reduction in height for ten plants at different DAI. (f) Disease scoring on the basis of height reduction and tungro-like leaf yellowing in infected lines. Each line represents the average disease incidence index (score) of ten infected plants at different days intervals. (▵) BC2F1 plants of set-I, (▲) IET4094, (□) BC2F1 plants of set-II, (▪) IET4786, (⧫) PB1/RTBV-O-Ds2, (●) TN1.
Observations on plant height and leaf yellowing in the inoculated plants indicated that inoculation with rice tungro viruses resulted in the reduction in height of parental plants as well as of the BC2F1 plants (Fig. 2 d). The reduction in plant height and leaf yellowing were most prominent in the case of non-transgenic parental lines and TN1. The inoculated plants showed reduced growth rate starting from 21 DAI. Significant reduction (P⩽0·01) in the growth of inoculated plants was observed at 35 DAI and at later stages the reduction in plant height became more prominent. In BC2F1 plants of set-I, the reduction in plant height increased from an initial level of 0·11% at 21 DAI to 9·8% at 70 DAI (Fig. 2 e). A similar trend was also noted in the case of set-II, and at 70 DAI the height reduction was only 7·3% in inoculated plants (Fig. 2 e). In contrast, the height reduction in recurrent parents and TN1 at 70 DAI was as high as 38% (IET4094), 36% (IET4786) and 50% (TN1). A similar pattern of disease incidence was noted in BC2F1 progenies and transgenic donor parent. In these plants, the virus inoculation resulted in a reduction in plant height without inducing the other deleterious effects of RTD. The plants showed normal panicle emergence and grain formation. Therefore, the disease scores were recorded between 3 and 4 in these plants. In contrast, the non-transgenic control plants showed leaf yellowing, stunting and incomplete panicle emergence and scored between 7 and 8 (Fig. 2 f).
DISCUSSION
BC breeding is a popular method of introgressing a resistance gene from a donor to recipient lines. Conventional crossing involving transgene-based marker-aided selection has been previously allowed the combination of multiple transgenes for disease and insect resistance (Datta et al. Reference Datta, Baisakh, Thet, Tu and Datta2002) and to transfer marker-free transgene into desirable cultivars (Matthews et al. Reference Matthews, Wang, Waterhouse, Thornton, Fieg, Gubler and Jacobsen2001; Baisakh et al. Reference Baisakh, Rehana, Rai, Oliva, Tan, Mackill, Khush, Datta and Datta2006). In the present study, an RTBV-derived RNAi construct, imparting resistance against RTBV, was transferred from PB-1 to two popular indica rice varieties by repeated backcrossing. The selection of progenies was based on morphological similarities to the recurrent parent coupled with molecular markers. In parallel, inheritance of the viral transgene (ORF IV) and the selection marker (hpt) were also monitored. Almost all the crossed progenies tested revealed the presence of ORF IV and hpt-marker genes as in the donor plant. However, the segregation patterns of ORF IV and hpt genes in the BC generations showed that in some progenies the marker gene might have been lost during cross-breeding (Table 2). An in-depth investigation on these probable marker-free transgenic progenies could confirm the true nature of these plants, which would be extremely valuable before any future release of these lines for cultivation, keeping in mind the current regulations on the release of genetically modified crops in various countries.
The phenotypes of the progenies were evaluated in comparison with the parents used in the present experiment and it was observed that the fertility in F1 hybrids in comparison with the recurrent parents was compromised. It is obvious that stable introgression through the development of BC generations depends on F1 hybrid fitness, i.e. the ability to set viable seeds and growth vigour. The first-generation hybrids are often genetically unstable with reduced fertility that must be overcome in subsequent BC generations for stable introgression to occur. In the present experiment, the fertility of the progenies in BC1 and subsequent generations was comparable to the recurrent parent (Table 3). Since the combination and segregation of the genes in different crosses are random in two different parents, the nature of the loci for different characteristics will be different. This has been shown by Shao et al. (Reference Shao, Rush, Wu, Groth, Kang and Linscombe2009), who noted significant variation among the progenies and recurrent parent for some of the characteristics such as plant height, number of spikelet, seed set rate etc. in advanced BC generations. For this reason, the similarities of the progenies in comparison with the recurrent parents for 16 phenotypic characters were evaluated to select the progenies for backcrossing.
The levels of accumulation of RTBV and RTSV in the progeny plants were not determined in the present study, although the presence of both the viral nucleic acids was clearly demonstrated in the inoculated plants by PCR (Fig. 2(b)). Despite the presence of the viruses, the RTD symptoms in infected BC2F1 progenies and transgenic donor parent were very mild, which is consistent with the report of Tyagi et al. (Reference Tyagi, Rajasubramaniam, Rajam and Dasgupta2008). The significant amelioration of tungro symptoms in the BC2F1 progenies strongly indicates that RTBV resistance has been transferred from the transgenic donor plant. Tyagi et al. (Reference Tyagi, Rajasubramaniam, Rajam and Dasgupta2008) reported a direct correlation between the severity of tungro symptoms in transgenic plants and the levels of RTBV in the first few weeks following inoculation. Thus, it would be reasonable to assume that the levels of RTBV were low in the BC2F1 progenies showing mild tungro symptoms on challenge inoculation, although they were not measured in the present study. It would be interesting to investigate the levels of both RTBV and RTSV in the plants to reveal the interactions between the two viruses, if any.
The overall results of the present study indicate that transgenic viral resistance, based on RNAi, can be stably transferred from the transgenic line into popular rice cultivars via repeated backcrossing, resulting in the diversification of tungro resistance to several rice cultivars. This has resulted in further value-addition to the two popular rice varieties used and illustrates an important step towards ensuring food security in regions of the World, where RTD is prevalent.
This work was funded by the Department of Biotechnology (DBT) Government of India (Grant No. BT/PR5906/AGR/02/301/2005). S. R. and A. B. are grateful to DBT for providing the senior research fellowship. The authors acknowledge Shri Buddhadev Pal for his assistance in conducting greenhouse and laboratory experiments. We thank Dr P. Bandopadhyay, Plant Pathologist, Rice Research Station, Government of West Bengal, Chinsura for providing the tungro infected rice plants.
