The polyembryo gene (OsPE) in rice

T-DNA insertional mutagenesis is one of the most important approaches for gene discovery and cloning. A fertile polyembryo mutant generated by T-DNA/Ds insertion in Oryza sativa, cv. Basmati 370 showed twin or triple seedlings at a frequency of 15–20%. T-DNA insertion was confirmed by 950 bp hpt gene amplification in the promoter region of the candidate gene. The annotated protein corresponding to the OsPE candidate gene has been reported as a hypothetical protein in O. sativa. OsPE gene lacked functional homologs in other species. No OsPE paralog was found in rice. No conserved domains were found in the protein coded by OsPE. RT-PCR showed the expression of OsPE gene in Basmati 370 shoots. Full-length OsPE gene was cloned in Basmati 370. The combined use of Southern blot, genome walking, TAIL-PCR, RT-PCR techniques, and bioinformatics led to the identification of a candidate gene controlling the multiple embryos in rice. There is gain of function, i.e., multiple embryos in the seeds in the knockout mutant OsPE whereas its wild-type allele strictly controls single embryo per seed. The seeds with multiple embryos are distributed at random in the rice mutant panicle. The origin of multiple embryos, whether apomictic, zygotic or both is under investigation.     

Transgenic indica Rice Variety Pusa Basmati 1 Constitutively Expressing a Rice Chitinase Gene Exhibits Enhanced Resistance to Rhizoctonia solani

Agrobacterium-mediated transformation of an elite indica rice variety, Pusa Basmati 1, was performed using LBA4404 (pSB1, pMKU-RF2) that harbours a rice chitinase gene (chi11) under the control of the maize ubiquitin (Ubi1) promoter-intron. Right border (gus) and left border (hph) flanking sequences and the transgene (chi11) in the middle of the T-DNA were used as probes in Southern analysis. Out of eleven independent T0 plants regenerated, three had single copy T-DNA insertions and eight had multiple T-DNA insertions. Nine T₀ plants carried the complete T-DNA with the chitinase transgene. Two T₀ plants did not carry chi11, though they had other T-DNA portions. Three plants harbouring single copy insertions and one plant harbouring two inserted copies were analyzed in detail. A segregation ratio of 3:1, reflecting T-DNA insertion at a single locus, was observed in the progeny of all the four T₀ plants. Northern and western blot analyses of T₁ plants revealed constitutive expression of chitinase at high levels. Bioassays of T₁ plants indicated enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, in comparison to control plants. A homozygous transgenic line was established from one T₀ line, which exhibited the maximum resistance to R. solani.     

Assessment of Genetic Diversity Among Rice Genotypes with Differential Adaptations to Salinity Using Physio-morphological and Molecular Markers

Breeding for salt tolerance using traditional screening and selection methods have been limited by the complex and polygenic nature of salt tolerance trait. This study was designed to evaluate some of the premium Basmati rice varieties for salt tolerance and to characterize genetic diversity among the rice varieties with different adaptations to saline soils using microsatellite (SSR) and ISSR markers. Plants of nine rice varieties including salt tolerant, salt sensitive and traditional Basmati, were grown in hydroponics using Yoshida solution containing 0 (control, pH 5.0) and 30 mM NaCl (Electrical conductivity 4.8 d/S, pH 5.0) and assessed for salinity tolerance on 1–9 scale as per IRRI standard evaluation system using seedling growth parameters, visual salt injuries and Na-K ratio. Physio-morphological studies showed that traditional Basmati rice varieties (Basmati 370 and HBC19) were more sensitive than the salt sensitive control variety, MI-48. SSR as well as ISSR marker systems generated higher levels of polymorphism and could distinguish between all the 9 rice cultivars. A total of 299 (225 polymorphic) and 437 (430 polymorphic) bands were detected using 28 UBC ISSR primers and 100 welldistributed mapped SSR markers, respectively. ISSR and SSR marker data-sets showed moderate levels of positive correlation (Mantel test, r = 0.43). The ISSR and SSR marker data analyzed using clustering algorithms showed two distinct clusters separating the Basmati (Basmati 370, HBC19 and CSR-30) from other non-aromatic indica (IR36, Pokkali, CSR10 and MI-48) rice varieties indicating greater divergence between Basmati and non-aromatic indica rice genotypes. Marker analysis showed a close relationship among the two traditional (Basmati 370 and HBC19) and cross-bred (CSR30) Basmati rice varieties and greater diversity between the two salt-tolerant genotypes, Pokkali and BR4-10.     

High frequency androgenesis in indica × Basmati rice hybrids using liquid culture media

An improved procedure has been developed for high frequency androgenesis in indica × Basmati rice hybrids using a liquid culture medium. Anthers from fourteen genotypes comprising of indica × Basmati rice F₁ hybrids, F₂ plants and the parental rice cultivars, were floated in liquid RZM, N6M, and Heh5M media. Anther culture frequencies (percentage of anthers forming calluses) in most of the genotypes were significantly higher in RZM medium (16–75%) compared to those obtained in N6M (7–29%) and Heh5M (7–41%) media. Agarose (1.0% w/v)-solidified MSR1 medium containing 3.0% (w/v) maltose, 1 mg l⁻¹ kinetin, 1 mg l⁻¹ 6-benzyladenine (BA) and 0.5 mg l⁻¹α-naphthalene acetic acid (NAA) induced green shoot regeneration at high frequencies compared to the medium (MSR2) lacking BA. In all the genotypes, microspore calluses initiated in RZM medium regenerated green shoots with over tenfold higher frequencies compared to the calluses initiated in other two media. High plant regeneration frequencies (up to 270 green plants/1000 anthers) were obtained from microspore-derived calluses of some of the F₁ hybrids (Gobind × Basmati 370, Gobind × Taraori Basmati) and F₂ plants (Gobind × Basmati 370, Gobind × Taraori Basmati, HKR86-3 × Taraori Basmati) as compared to their actual parents. Cytological analysis of the root tips of the progeny seedlings of the microspore-derived plants revealed haploids at a frequency of about 50%; 22% of the microspore- derived plants had > 5% spikelet fertility and were diploid. Use of RZM liquid and MSR1 media, respectively for anther culture and plant regeneration resulted in several fold increase in the recovery of green plants from recalcitrant indica × Basmati rice F₁ hybrids/F₂ plants which were comparable to those reported for japonica rice varieties/hybrids leading to the improved feasibility of using doubled haploids in genetic, breeding and mapping research with indica rice. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular dissection of QTL governing grain size traits employing association and linkage mapping in Basmati rice

Grain size is one of the key traits that determines the quality of Basmati rice from the consumers’ as well as the traders’ point of view. Though many genes governing grain size have been identified in indica and japonica, little work has been done in Basmati rice. The present study aims at dissection of a QTL region governing grain size traits in Basmati employing association and linkage mapping approaches. Association mapping revealed that three markers, i.e., RM 6024 (grain breadth), RM1237 and RM18582 (grain length-breadth ratio), which cover 889 kb in the targeted QTL region have been significantly associated with grain size traits. Using linkage mapping, the targeted QTL region has been further delimited to a physical distance of 268 kb that comprises 24 annotated genes. The gene expression analysis of parents, revealed 19 genes differentially expressing within the QTL. Of them, 15 genes showed high expression in Basmati370, while four were expressed in Jaya, and whereas five genes did not show any differential expression between parents. Among differentially expressed genes, a highly expressed gene in Basmati370, Os05g0374200 (Cytokinin dehydrogenase 1 precursor) seems to be involved in accumulation of cytokinins, thus affecting the grain size. Therefore, our findings demonstrated that by complimenting association and linkage mapping, it is likely to dissect a QTL governing grain size traits in Basmati rice and also the QTL could be a potential target for marker-assisted breeding and map-based cloning studies.     

Knockout of the VPS22 component of the ESCRT-II complex in rice (Oryza sativa L.) causes chalky endosperm and early seedling lethality

In both yeast and mammals, the major constituent of the endosomal sorting complex required for transport-II (ESCRT-II) is the VPS22/EAP30 protein, which plays an important role in ubiquitin-mediated degradation of membrane proteins through the multivesicular body pathway. However, the functions of ESCRT-II subunits in plants are largely unknown. In this work, we report the genetic analysis and phenotypic characterization of mutants in OsVPS22 gene, which encodes a functional VPS22 homolog in rice. On the basis of a collection of T-DNA lines, we identified a T-DNA insertion mutant, which showed abnormal segregation ratios; we then found that the T-DNA insertion is located within the sixth intron of the OsVPS22 gene. Compared with the wild type, this vps22 mutant exhibited seedling lethality and severe reduction in shoot and root growth. In addition, the vps22 mutant had a chalky endosperm in the grain. In summary, our data suggest that OsVPS22 may be required for seedling viability and grain filling in rice, thus providing a valuable resource for further exploration of the functions of the ESCRTing machinery in plants.     

Characterization and fine mapping of <Emphasis Type="Italic">osh15</Emphasis>(<Emphasis Type="Italic">t</Emphasis>), a novel dwarf mutant gene in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plant height is one of the most important agronomic traits of plant architecture, and also affects grain yield in rice. In this study, we obtained a novel dwarf rice mutant of japonica variety Shennong9816, designated Shennong9816d. Compared with wild-type, the Shennong9816d plant height was significantly reduced, and the tiller number significantly increased. Additionally, the mutant yield component, and the number of large and small vascular bundles were significantly decreased compared with wild-type. Genetic analysis indicated that the Shennong9816d dwarf phenotype was controlled by a recessive nuclear gene, while the plant was shown to be sensitive to gibberellic acid. Using a large F₂ population derived from a cross between Shennong9816d and the indica rice variety Habataki, the osh15(t) gene was fine mapped between RM20891 and RM20898, within a physical distance of 73.78 kb. Sequencing analysis showed that Shennong9816d carries a 1 bp mutation and a 30 bp insertion in the OSH15 region. These results suggest that osh15(t) is a novel allelic mutant originally derived from japonica variety Shennong9816, which may be useful for introducing the semi-dwarf phenotype to improve plant architecture in rice breeding practice.     

SDR7-6, a short-chain alcohol dehydrogenase/reductase family protein,regulates light-dependent cell death and defence responses in rice

Lesion mimic mutants resembling the hypersensitive response without pathogen attack are an ideal material to understand programmed cell death, the defence response, and the cross-talk between defence response and development in plants. In this study, mic, a lesion mimic mutant from cultivar Yunyin treated with ethyl methanesulphonate (EMS), was screened. By map-based cloning, a short-chain alcohol dehydrogenase/reductase with an atypical active site HxxxK was isolated and designated as SDR7-6. It functions as a homomultimer in rice and is localized at the endoplasmic reticulum. The lesion mimic phenotype of the mutant is light-dependent. The mutant displayed an increased resistance response to bacterial blight, but reduced resistance to rice blast disease. The mutant and knockout lines showed increased reactive oxygen species, jasmonic acid content, antioxidant enzyme activity, and expression of pathogenicity-related genes, while chlorophyll content was significantly reduced. The knockout lines showed significant reduction in grain size, seed setting rate, 1000-grain weight, grain weight per plant, panicle length, and plant height. SDR7-6 is a new lesion mimic gene that encodes a short-chain alcohol dehydrogenase with atypical catalytic site. Disruption of SDR7-6 led to cell death and had adverse effects on multiple agricultural characters. SDR7-6 may act at the interface of the two defence pathways of bacterial blight and rice blast disease in rice.     

Role of Cysteine in Enhancing Androgenesis and Regeneration of Indica Rice (Oryza sativa L.)

The effects of amino acid cysteine to culture systems of microspore-derived callus induction as well as plantlet regeneration were studied. Isolated pollen along with anther walls of basmati cultivars, Pusa basmati 1, Basmati 370 and Basmati 386 were cultured in a medium based on N₆ salts supplemented with or without cysteine following pollen embedment in agarose. The induction and regeneration medium with cysteine gave twice as effective androgenesis and plantlet regeneration in recalcitrant basmati rice cultivars as compared with medium lacking cysteine. Unlike the highly responsive model systems, most of the indica cultivars responded rather poorly in anther culture. So the study may accelerate the introgression of desirable genes into basmati rice using anther culture as a breeding tool. Response of microspores in androgenesis, plant regeneration and albinism was genotype specific. Regeneration of Indica rice varieties remains a limiting factor for researchers undertaking transformation experiments.     

Characterization of an Abc1 kinase family gene OsABC1-2 conferring enhanced tolerance to dark-induced stress in rice

Leaf senescence is a complex and highly organized process resulting in numerous changes of gene expression and metabolic procedures. However, the exact mechanisms underlying these changes are not well understood. In this study, we reported a rice (Oryza sativa) T-DNA insertion mutant impaired in an Abc1 kinase family gene with a dwarf and pale-green phenotype. The mutant showed reduced pigment content and photosynthetic efficiency and increased superoxide dismutase activity in leaves. The mutated gene, designated OsABC1-2, is expressed primarily in green tissues and/or organs and encodes a protein localized in chloroplast envelope. Expression of the gene was drastically suppressed by dark treatment. Overexpression of the gene in rice enhanced tolerance to prolonged dark-induced stress. Phylogenetic analysis revealed that the plant Abc1 proteins could be divided into three subgroups and OsAbc1-2 resides in a subgroup with potential chloroplast origin. Our results suggest that divergence has occurred among plant Abc1 family and chloroplast Abc1 kinases play potential roles in regulating dark-induced senescence of plants.     

Identification of a novel candidate gene for rolled leaf in rice

A semi-narrow and adaxially rolled leaf mutant, rl15(t), was induced from Korean japonica rice cultivar Ilpum by chemical mutagenesis using ethyl methanesulfonate. We characterized the mutant and identified the novel gene causing the mutant phenotype. Cytological analysis of mutant leaves indicated that the adaxial leaf-rolling phenotype is due to the reduced size and number of bulliform cells in the mutant. Genetic analysis showed that the rolled leaf trait is controlled by a single recessive gene, designated rl15(t). Using an F₂ mapping population generated from a cross between Milyang23 and the mutant, we mapped the candidate region to a 174 kb interval on the long arm of chromosome 1 near the centromeric region. Through whole genome sequencing in bulk and MutMap analysis, we identified the causal SNP within the candidate region. The results of RT-PCR analysis indicated that a splicing error occurred due to a base change from G to A at the beginning of the fifth intron of LOC_Os01g37837, which encodes a putative seryl-tRNA synthetase, resulting in the mutant phenotype. Further study of the rl15(t) gene will facilitate analysis of leaf architecture and morphogenesis in rice plants.     

Increased polyamine biosynthesis enhances stress tolerance by preventing the accumulation of reactive oxygen species: T-DNA mutational analysis of Oryza sativa lysine decarboxylase-like protein 1

A highly oxidative stress-tolerant japonica rice line was isolated by T-DNA insertion mutation followed by screening in the presence of 50 mM H₂O₂. The T-DNA insertion was mapped to locus Os09g0547500, the gene product of which was annotated as lysine decarboxylase-like protein (GenBank accession No. AK062595). We termed this gene OsLDC-like 1, for Oryza sativa lysine decarboxylase-like 1. The insertion site was in the second exon and resulted in a 27 amino acid N-terminal deletion. Despite this defect in OsLDC-like 1, the mutant line exhibited enhanced accumulation of the polyamines (PAs) putrescine, spermidine, and spermine under conditions of oxidative stress. The generation of reactive oxygen species (ROS) in the mutant line was assessed by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), and by DCFH-DA staining. Cellular levels of ROS in osldc-like 1 leaves were significantly lower than those in the wild-type (WT) rice after exposure to oxidative, high salt and acid stresses. Exogenouslyapplied PAs such as spermidine and spermine significantly inhibited the stress-induced accumulation of ROS and cell damage in WT leaves. Additionally, the activities of ROS-detoxifying enzymes were increased in the homozygous mutant line in the presence or absence of H₂O₂. Thus, mutation of OsLDC-like 1 conferred an oxidative stress-tolerant phenotype. These results suggest that increased cellular PA levels have a physiological role in preventing stress-induced ROS and ethylene accumulation and the resultant cell damage.     

Fine mapping of grain length QTLs on chromosomes 1 and 7 in Basmati rice (Oryza sativa L.)

Grain dimensions (length, breadth and length/breadth ratio) are important quality attributes of Basmati rice for its high consumer acceptance. Earlier we identified two significant quantitative trait loci (QTL) intervals on chromosomes 1 and 7 for grain dimensions in Basmati rice using a population of recombinant inbred lines (RILs) from cross between Basmati variety Pusa 1121 and a short grain non-aromatic variety Pusa 1342. For fine mapping of these QTLs, 184 F₆ RILs were grown and phenotyped in the normal rice growing season at two different locations. Forty-nine new SSR markers targeting these QTL intervals were tested and nine were found polymorphic between the parents. Using revised genetic maps adding new markers, the grain length QTL qGRL1.1 on chromosome 1 was narrowed down to 108?kbp from the earlier reported 6,133?kbp. There were total 13 predicted gene models in this interval which includes the probable candidate gene for the exceptionally high grain length of Basmati variety Pusa 1121. Similarly, two tandem QTL intervals qGRL7.1 and qGRL7.2 on chromosome 7 were merged into a single one narrowed down to 2,390?kbp from the earlier reported length of 5,269?kbp. This region of chromosome 7 also has co-localized QTLs for grain breadth and length to breadth ratio. SSR markers tightly linked to the QTL at a map distance of ??0.2?cM were developed for the qGRL1.1 and qGRL7.1 loci that could be used for marker-assisted breeding. Validation of earlier published markers for the grain length gene GS3 on chromosome 3 showed no difference between Pusa 1121 and Pusa 1342, highlighting the significance of qGRL1.1 and qGRL7.1 for the extra grain length of Basmati variety Pusa 1121.     

Isolation and Characterization of a Rice Cysteine Protease Gene, OsCP1, Using T-DNA Gene-Trap System

The T-DNA gene-trap system has been efficiently used to elucidate gene functions in plants. We report here a functional analysis of a cysteine protease gene, OsCP1, isolated from a pool of T-DNA insertional rice. GUS assay with the T-DNA tagged line indicated that the OsCP1 promoter was highly active in the rice anther. Sequence analysis revealed that the deduced amino acid sequence of OsCP1 was homologous to those of papain family cysteine proteases containing the highly conserved interspersed amino acid motif, ERFNIN. This result suggested that the gene encodes a cysteine protease in rice. We also identified a suppressed mutant from T2 progeny of the T-DNA tagged line. The mutant showed a significant defect in pollen development. Taken together, the results demonstrated that OsCP1 is a cysteine protease gene that might play an important role in pollen development.     

Improvement of two traditional Basmati rice varieties for bacterial blight resistance and plant stature through morphological and marker-assisted selection

Bacterial blight (BB) is a major production threat to Basmati, the aromatic rice prized for its unique quality. In order to improve the BB resistance of two elite, traditional BB-susceptible Basmati varieties (Taraori Basmati and Basmati 386), we utilized the strategy of limited marker-assisted backcrossing for introgression of two major BB resistance genes, Xa21 and xa13, coupled with phenotype-based selection for improvement of their plant type and yield. Improved Samba Mahsuri, an elite high-yielding, fine-grain-type BB-resistant rice variety served as donor for BB resistance. Backcross-derived improved Basmati lines at BC1F5 possessing a single resistance gene (i.e. either Xa21 or xa13) displayed moderate resistance to BB, while lines possessing both Xa21 and xa13 showed significantly higher levels of resistance. Two-gene pyramid lines (Xa21 + xa13) possessing good grain and cooking quality similar to their respective traditional Basmati parents, short plant stature (<110 cm plant height) and higher grain yield than the recurrent parent(s) were identified and advanced. This work demonstrates the successful application of marker-assisted selection in conjunction with phenotype-based selection for targeted introgression of multiple resistance genes into traditional Basmati varieties along with improvement of their plant stature and yield.     

A key ABA hydrolase gene,OsABA8ox3 is involved in rice resistance to Nilaparvata lugens by affecting callose deposition

Abscisic acid (ABA) is an important plant hormone in regulating abiotic and biotic stresses. OsABA8ox3 is the key gene in ABA hydrolase genes, and plays an important role in controlling ABA level, but little is known in rice resistance to insects. We used rice osaba8ox3 T-DNA insertion mutant (knocking down the OsABA8ox3 gene) to elucidate rice resistance to the insect. There were obvious phenotype differences between the osaba8ox3 T-DNA insertion mutant and wild-type (WT), and the relative expression of synthetase genes in the osaba8ox3 mutant was higher, while the relative expression of hydrolase genes was lower than that of WT, respectively. The electrical penetration graph (EPG) recording indicated that the osaba8ox3 mutant had the less sucking phloem sap duration compared with WT, which indicated a significant increase in rice resistance to brown planthopper (Nilaparvata lugens; BPH). The callose deposition in the osaba8ox3 mutant increased by 60.39%, 52.2%, 26.6% and 31.7% than that of WT after BPH feeding for 0, 24, 48, and 72 h, respectively. These results showed OsABA8ox3 gene played an important role in rice resistance to BPH, and also provided new insights into the mechanism of callose deposition regulation in response to the piercing-sucking pest.