RFLP mapping of QTLs for yield and related characters in rice (Oryza sativa L.)

Quantitative triat loci (QTLs) for yield and related traits in rice were mapped based on RFLP maps from two indica/indica F₂ populations, Tesanai 2/CB and Waiyin 2/CB. In Tesanai 2/CB, 14 intervals carrying QTLs for eight traits were detected, including 3 for grain weight per plant (GWT), 2 for number of panicles per plant (NP), 2 for number of grains per panicle (NG), 1 for total number of spikelets per panicle (TNS), 1 for spikelet fertility (SF), 3 for 1000-grain weight (TGWT), 1 for spikelet density (SD), and 1 for number of first branches per main panicle. The 3 QTLs for GWT were located on chromosomes 1, 2, and 4, with 1 in each chromosome. The additive effect of the single locus ranged from 2.0 g to 9.1 g. A major gene (np4) for NP was detected on chromosome 4 within the interval of RG143–RG214, about 4cM for RG143, and this locus explained 26.1% of the observed phenotypic variance for NP. The paternal allele of this locus was responsible for reduced panicles per plant (3 panicles per plant). In another population, Waiyin 2/CB, 12 intervals containing QTLs for six of the above-mentioned traits were detected, including 3 for GWT, 2 for each of NP, TNS, TGWT and SD, 1 for SF. Three QTLs for GWT were located on chromosome 1, 4, and 5, respectively. The additive effect of the single locus for GWT ranged from 6.7 g to 8.8 g, while the dominance effect was 1.7–11.5 g. QTL mapping in two populations with a common male parent is compared and discussed.     

Generation and characterization of low phytic acid germplasm in rice (Oryza sativa L.)

Phytic acid (PA, myo-inositol 1,2,3,4,5,6-hexakisphosphate), or its salt form, phytate, is commonly regarded as the major anti-nutritional component in cereal and legume grains. Breeding of low phytic acid (lpa) crops has recently been considered as a potential way to increase nutritional quality of crop products. In this study, eight independent lpa rice mutant lines from both indica and japonica subspecies were developed through physical and chemical mutagenesis. Among them, five are non-lethal while the other three are homozygous lethal. None of the lethal lines could produce homozygous lpa plants through seed germination and growth under field conditions, but two of them could be rescued through in vitro culture of mature embryos. The non-lethal lpa mutants had lower PA content ranging from 34 to 64% that of their corresponding parent and four of them had an unchanged total P level. All the lpa mutations were inherited in a single recessive gene model and at least four lpa mutations were identified mutually non-allelic, while the other two remain to be verified. One mutation was mapped on chromosome 2 between microsatellite locus RM3542 and RM482, falling in the same region as the previously mapped lpa1-1 locus did; another lpa mutation was mapped on chromosome 3, tightly linked to RM3199 with a genetic distance of 1.198 cM. The latter mutation was very likely to have happened to the LOC_Os03g52760, a homolog of the maize myo-inositol kinase (EC 2.7.1.64) gene. The present work greatly expands the number of loci that could influence the biosynthesis of PA in rice, making rice an excellent model system for research in this area.     

Sink anatomy in relation to solute movement in rice (Oryza sativa L.): A summary of findings

The results of studies on assimilate and water transport in the developing caryopsis of rice are summarised. Evidence is presented for a symplastic movement of solutes as far as the aleurone layer. However, transport into the apoplast at the nucellus/aleurone interface appears to be a necessary step due to the absence of plasmodesmata at this site. It is suggested that water leaves the caryopsis during grain filling by the isolated cell walls of the pigment strand, the suberised walls of these cells functioning to isolate the apoplast from the symplast and thereby allowing opposing fluxes of water and assimilates to occur in the dorsal region of the grain.     

Genotypic variability for callus formation and plant regeneration in rice (Oryza sativa L.)

Summary Sixty rice varieties (Oryza sativa L.), belonging to three subspecies, japonica, indica and javanica (some japonicaXindica hybrids were included), were compared for their capacity for callus growth and plant regeneration. Tissue cultures initiated from mature seeds on Murashige and Skoog's (1962) medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were transferred to a medium containing 0.02 mg/l 2,4-D and 10 mg/l kinetin, from which plantlets were regenerated. Large variabilities in callus growth and plant regeneration potentials were revealed among the varieties tested. Most japonica varieties formed a callus that weighed more than 100 mg per seed 30 days after inoculation, and showed a relatively high regenerative potential, whereas indica varieties, japonica-indica hybrids and javanica varieties showed poor callus growth and plant regeneration, although considerable varietal variation was observed in each subspecies. The callus growth potential was not correlated with the plant regeneration potential. Histological observations revealed that the epithelium cells of the scutellum mainly proliferated to form a callus, from which shoot and root primordia were differentiated independently from each other. The shoot primordia developed into plantlets when roots were formed adventitiously. In a few cases, shoots and roots were bilaterally initiated from a single primordium.     

Production of doubled haploid rice plants (Oryza sativa L.) by anther culture

The results of anther culture of F₂ pollen issued from 23 single crosses are presented. A relation between the morphology of the panicle and the microspore stage was established. After cold-pretreatment (8 days at 4°C), the anthers were cultured on the callus-induction medium N6 supplemented with 1 mg l^–1 naphthaleneacetic acid. The calli were transferred to MS plant regeneration medium supplemented with 3 mg l^–1 kinetin + 0.5 mg l^–1 naphthaleneacetic acid. The induction frequency varied from 0.22% to 29% and the regeneration frequency from 0% to 144.4%, dependent upon the crosses used. On average, 27% of the plants obtained were albinos and 59% of the green plants underwent spontaneous chromosome doubling. Thirtynine doubled haploid lines were evaluated and multiplied in the field. Lines with an excellent behaviour in upland culture conditions were selected from two crosses.     

Screening of rice (Oryza sativa L.) genotypes for physiological characters contributing to salinity resistance,and their relationship to overall performance

Summary Phenotypic resistance of salinity is expressed as the ability to survive and grow in a salinised medium. Some subjective measure of overall performance has normally been used in plant breeding programmes aimed at increasing salinity resistance, not only to evaluate progeny, but to select parents. Salinity resistance has, at least implicitly, been treated as a single trait. Physiological studies of rice suggest that a range of characteristics (such as low shoot sodium concentration, compartmentation of salt in older rather than younger leaves, tolerance to salt within leaves and plant vigour) would increase the ability of the plant to cope with salinity. We describe the screening of a large number of rice genotypes for overall performance (using an objective measure based on survival) and for the aforementioned physiological traits. There was wide variation in all the characters studied, but only vigour was strongly correlated with survival. Shoot sodium concentration, which a priori is expected to be important, accounted for only a small proportion of the variability in the survival of salinity. Tissue tolerance (the cellular component of resistance reflecting the ability to compartmentalise salt within leaves) revealed a fivefold range between genotypes in the tolerance of their leaves to salt, but this was not correlated positively with survival. On the basis of such (lack of) correlation, these traits would be rejected in normal plant breeding practice, but we discuss the fallacies involved in attempting correlation between individual traits and the overall performance of a salt-sensitive species in saline conditions. We conclude that whilst overall performance (survival) can be used to evaluate the salt resistance of a genotype, it is not the basis on which parents should be selected to construct a complex character through breeding. It was the norm for varieties which had one good characteristic affecting salt resistance to be unexceptional or poor in the others. This constitutes experimental evidence that the potential for salt resistance present in the rice genome has not been realised in genotypes currently extant. The results are discussed in relation to the use of physiological traits in plant breeding, with particular reference to environmental stresses that do not affect a significant part of a species' ecological range.     

QTLs and epistasis underlying activity of acid phosphatase under phosphorus sufficient and deficient condition in rice (Oryza sativa L.)

Eighty-four selected lines from a recombinant inbred (RI) line population of 284 lines derived from a cross between the indica varieties IR20 and IR55178-3B-9-3 were used in a hydroponic culture experiment with sufficient P supply (10 mg P L^–1) and P-deficient stress (0.05 mg P L^–1). After 2 weeks, the activity of acid phosphatase (AAP) in roots of each parent and each line from both normal culture and P-deficient stress was determined. QTLs for AAP, P-deficiency stress induced AAP (Psi-AAP) and relative AAP (RAAP) were detected using 178 molecular markers mapped on all 12 chromosomes based on single marker analysis and interval mapping. One QTL for AAP and three QTLs for Psi-AAP were detected on chromosome 1, 6 and 12, respectively. Two QTLs for RAAP were identical with these for Psi-AAP on chromosome 6 and 12. The results in this case indicated that the genetic system for Psi-AAP was different with that for AAP under normal culture. The AAP was mainly influenced by interaction among muti-factors, while Psi-AAP was controlled by a Psi genetic system.     

Simple dehydration treatment promotes plantlet regeneration of rice (Oryza sativa L.) callus

Summary To increase plantlet regeneration frequency, rice callus was dehydrated in a Petri dish with a single layer of filter paper prior to transfer to the regeneration medium. With a 24 h dehydration treatment, the regeneration frequency was increased to 47 %, while the regeneration frequency of the untreated control was less than 5 %. This relatively simple method provides an alternative method for improving the regeneration frequency of rice callus.     

RFLP analysis of rice (Oryza sativa L.) introgression lines

Summary Fifty-two introgression lines (BC₂F₈) from crosses between two Oryza sativa parents and five accessions of O. officinalis were analyzed for the introgression of O. officinalis chromosome segments. DNA from the parents and introgression lines was analyzed with 177 RFLP markers located at approximately 10-cM intervals over the rice chromosomes. Most probe/enzyme combinations detected RFLPs between the parents. Of the 174 informative markers, 28 identified putative O. officinalis introgressed chromosome segments in 1 or more of the introgression lines. Introgressed segments were found on 11 of the 12 rice chromosomes. In most cases of introgression, O. sativa RFLP alleles were replaced by O. officinalis alleles. Introgressed segments were very small in size and similar in plants derived from early and later generations. Some nonconventional recombination mechanism may be involved in the transfer of such small chromosomal segments from O. officinalis chromosomes to those of O. sativa. Some of the introgressed segments show association with genes for brown planthopper (BPH) resistance in some introgressed lines, but not in others. Thus, none of the RFLP markers could be unambiguously associated with BPH resistance.     

Inheritance of two endosperm protein loci in rice (Oryza sativa L.)

Summary Previous studies indicated two types of phenotypic protein markers as two minor bands of SDS-PAGE for rice storage protein. A variant derived from a Pakistani variety, Dular, was found to show a mobility variant with Band 11, a relatively faster-moving band as compared to Band 10, while most of the other cultivated rices exhibited Band 10 at a molecular weight of around 100–110 K. Band 11 was also observed in several wild rice species. How this variant occurred is not known. Another marker is characterized by the presence of either Band 56 (slower-migrating band) or Band 57 (faster-migrating band) in most cultivars at a molecular weight of about 28–27 K. Most indica varieties developed in Taiwan have Band 57 and japonica varieties have Band 56. Genetic analysis of F₁, F₂ and F₃ seeds from interstrain crosses indicated that Band 10 versus Band 11 and Band 56 versus Band 57 are due to codominant alleles at two loci. Tests of independent inheritance between these two loci (Band 10/11 versus Band 56/57) indicated that there is no linkage between them. Both of these two protein loci encode for endosperm proteins and mostly belong to the minor polypeptide subunits of the glutelin fraction of rice seed proteins. Studies on reciprocal crosses indicate dosage effects as exhibited in band patterns. Variations in band intensity were frequently observed when the maternal genotype was different.     

Characterization of factors affecting plantlet regeneration from rice (Oryza sativa L.) callus

Various components of culture media were tested to characterize factors affecting plantlet regeneration from rice (Oryza sativa L.) callus. It was found that plantlet regeneration from rice callus was affected by concentrations of gelling agents, osmoticum, and the combination of hormones in the regeneration medium. High concentrations (4–6 g/l gellan gum, 10–16 g/l agar) of gelling agents promoted regeneration frequency. However, the total number of plantlets decreased with gellan gum concentrations above 4 g/l. Addition of sorbitol (15–75 g/l) promoted plantlet regeneration. However, the addition of mannitol was inhibitory and no regeneration was observed at concentrations above 30 g/l. This difference in the effects on regeneration suggests that sorbitol had another function besides as a osmoticum. High regeneration frequency was obtained with combinations of NAA (0.05–0.5 g/l) and kinetin (0.5–2 mg/l). However, higher concentrations (2 mg/l) of NAA are preferred to increase the total number of regenerated plantlets.     

Distribution of mitochondrial plasmid-like DNA in cultivated rice (Oryza sativa L.) and its relationship with varietal groups

Summary Mitochondrial (mt) plasmid-like DNA was found in most of more than 100 rice cultivars (Oryza sativa L.) by the use of 0.7% agarose gel electrophoresis (AGE). The DNA varied in molecular weight and number. By electron microscopy, small circular DNAs of different sizes could be detected in addition to the DNAs of high molecular weight, even in cultivars in which mt plasmid-like DNA was not detected by AGE. The detection of the mt plasmid-like DNAs by AGE did not depend on their presence or absence, but on their high stoichiometry. The relationship between cytoplasms with mt plasmid-like DNAs and varietal (for example, Indica rice) groups was close. The geographical distribution of cytoplasms is discussed.     

Diallel analysis of heading date in rice (Oryza sativa L.)

Summary The genetics of heading date was investigated in an 8×8 diallel set of crosses involving diverse rice cultivars. Wr, Vr graph analysis revealed the presence of a complementary type of non-allelic interaction which apparently affected the position and slope of the regression line such as if there were overdominance. Omission of two interacting parents resulted in a 6×6 subset of diallel crosses from which, as observed in the Wr, Vr graph, the non-allelic interaction had disappeared and the regression line exhibited partial dominance. Estimates of the genetic components of variation were in close conformity with the results obtained from the Wr, Vr graph: the average degree of dominance, as measured by (H₁/D)^1/2, was in overdominance range in the interacting 8×8 set of diallel crosses whereas it was reduced to partial dominance in the non-interacting 6×6 set of crosses. Further analysis by a standardized deviations graph indicated that earliness was controlled, on the average, by an excess of dominant alleles.     

Regulation of leaf senescence, grain-filling and yield of rice by kinetin and abscisic acid

Senescence of rice ( Oryza sativa L. cv. Jaya) leaves was regulated with kinetin and abscisic acid (ABA) sprays at the reproductive stage. The effect of such sprays on grain-filling and yield was analyzed. Spraying 100-day-old plants with kinetin solution (100 μg ml^-1) significantly delayed senescence as indicated by higher total chlorophyll and protein levels in the three uppermost leaves compared with the controls. In contrast, spraying with ABA (15 μg ml^-1) significantly promoted foliar senescence. The number of spikelets per panicle, number of panicles, percentage filled grains, panicle weight and grain yield per plant and the mobilization and harvest indices were significantly increased by kinetin treatment, while ABA decreased most of them. The possibility of increased grain-filling and thus, yield due to delayed foliar senescence by kinetin treatment and decreased grain-filling due to hastening of senescence by ABA is discussed.     

Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification

Summary Rice cells were precultured for 10 d in medium containing 60 g/L sucrose and subsequently for 1 d in medium supplemented with 0. 4 M sorbitol. After loading with 25%PVS2 at 22°C for 10 min and dehydration in 100%PVS2 at 0°C for 7. 5 min,they were plunged into liquid nitrogen directly. Survival was 45. 0 ±5.1% (based on the reduction of triphenyl tetrazolium chloride)following warming and unloading. For regrowth, cells were plated on semi-solid medium replenished with 40%(w/v) starch for 2d prior to reculture. Cell suspensions were reestablished and plants were regenerated from recovered cells. Twenty eight plants set seeds in the greenhouse.Abbreviations PVS plant vitrification solution - P preculture - LN liquid nitrogen - TTC triphenyl tetrazolium chloride - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - EG ethylene glycol - BSA bovine serum albumin     

Nuclear and cell migration during pollen development in rice (Oryza sativa L.)

Nuclear and cell migration during pollen development in rice were studied using semi-thin section light microscopy, differential interference contrast microscopy and epifluorescence microscopy. Four migrations of nuclei and cells were observed and described in detail here. The first nuclear migration occurs at the uninucleate microspore stage, when the nucleus of the microspore migrates from the center to the periphery of the cell, and then to the wall opposite the pollen aperture where pollen mitosis I takes place. The second migration occurs at the early bicellular pollen stage, with the vegetative nucleus migrating three-quarters of the circumference of the pollen wall, finally locating at the periphery of the wall where the microspore cell nucleus is positioned. The third migration occurs at the late bicellular pollen stage, with the vegetative nucleus migrating from the periphery of the cell to the central part of the pollen and the generative cell migrating from the opposite side of the aperture to a position between the aperture and the vegetative nucleus where pollen mitosis II takes place. The fourth migration appears at the mature pollen stage when the two sperm cells and the vegetative nucleus migrate to the opposite side of the aperture, finally becoming positioned in the cytoplasm of the vegetative cell distal to the aperture where the male germ unit forms. Cytological observations of pollen abortion resulting from allelic interaction at the S-a, S-b and S-c loci show that abnormalities in the first or second nuclear migration result in the formation of empty abortive pollen, whereas abnormalities in the third or fourth migrations cause production of stainable abortive pollen.     

Family 19 chitinase from rice (Oryza sativa L.): substrate-binding subsites demonstrated by kinetic and molecular modeling studies

A family 19 chitinase (OsChia1c, class I) from rice, Oryza sativa L., and its chitin-binding domain-truncated mutant (OsChia1cCBD, class II) were produced by the Pichia expression system, and the hydrolytic mechanism toward N-acetylglucosamine hexasaccharide [(GlcNAc)₆] was investigated by HPLC analysis of the reaction products. The profile of the time-course of (GlcNAc)₆ degradation obtained by OsChia1c was identical to that obtained by OsChia1cCBD, indicating that the chitin-binding domain does not significantly participate in oligosaccharide hydrolysis. From the theoretical analysis of the reaction time-course of OsChia1cCBD, the free energy changes of sugar residue binding were estimated to be –0.4, –4.7, +3.4, –0.5, –2.3, and –1.0 kcal/mol for the individual subsites of (–3), (–2), (–1), (+1), (+2), and (+3), respectively. The hexasaccharide substrate appears to bind to the enzyme through interactions at the high-affinity sites, (–2) and (+2), and the sugar residues at both ends more loosely bind to the corresponding subsites, (–3) and (+3). The docking study of (GlcNAc)₆ with the modeled structure of OsChia1cCBD supported the subsite structure estimated from the experimental time-course of hexasaccharide degradation. Since the class II chitinase from barley seeds was reported to possess a similar subsite structure from (–3) to (+3) and a similar free energy distribution, substrate-binding mode of plant chitinases of this class would be similar to each other.     

High amylose mutants of rice,Oryza sativa L.

Summary Five mutant lines of rice with increased amylose content in starch granules were identified among floury endosperm mutants. The amylose contents of the mutants ranged from 29.4% to 35.4% and were about twice as high as that of the normal counterpart. Starch properties of the high amylose mutants were analyzed by column chromatography, X-ray diffractometry, photopastegraphy and scanning electron microscopy. The high amylose mutants produced longer unit chains of amylopectin than those of the normal counterpart as well as an increased amount of amylose. A X-ray diffractogram of starch in the mutant was characterized by a type B pattern, while that in the normal counterpart showed a type A pattern which is typical for starches of common cereals. The temperatures at the initiation of gelatinization of the mutants were much higher than that for the normal counterpart. The endosperm cells of the mutant were loosely packed with irregular round-shaped starch granules, whereas those of the normal counterpart were densely packed with polyhedral starch granules. Judging from the results obtained, it was concluded that starch properties of the high amylose mutants of rice were similar to those of the amylose-extender (ae) mutant of maize.     

Structural changes in the plastid DNA of rice (Oryza sativa L.) during tissue culture

To investigate the rearrangement of the plastid genome during tissue culture, DNA from rice callus lines, which had been derived individually from single protoplasts isolated from seed or pollen callus (protoclones), was analyzed by Southern hybridization with rice chloroplast DNA (ctDNA) clones as probes. Among 44 long-term cultured protoclones, maintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large region of the plastid genome. The deletions could be classified into 15 types from their respective sizes and positions. By contrast, no deletions were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments revealed that plastid genomes with deletions in several protoclones were organized as head-to-head or tail-to-tail structures. Furthermore, ptDNAs retained during long-term culture all had a common terminus at one end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed the accumulation of starch granules in plastids and amyloplasts in protoclones in which the plastid genome had undergone deletion. Our observations indicated that novel structural changes in the plastid genome and morphological changes in the plastid had occurred in rice cells during long-term tissue culture. Moreover, the morphological changes in plastids were associated with deletions in the plastid genome.     

Response of excised embryos of rice (Oryza sativa L.) to X-rays

Summary The response of rice (Oryza sativa L.) embryos to X-rays (M₁ to M₃) was studied. By means of irradiating excised embryos, both chlorophyll and macromutation were successfully induced in three genotypes of rice. However, differential responses in terms of mutation frequency, mutation spectrum and optimal levels of X-rays required for induction of mutation (chlorophyll as well as morphological) were found to exist between cultivars. In Satika and Ashkhata, LD₅₀ values and maximum induced seed sterility are concomitant to optimum level of radiation required for triggering chlorophyll mutation. However, optimum dose for induction of macromutation in Satika and Kerangserang is independent of either LD₅₀ and/or induced seed sterility.Chances of obtaining both dominant and locus specific recessive mutations in the immediate X-ray treated generation (M₁) are large. This indicates the very high degree of effectiveness of the excised embryo irradiation technique with rice.