Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary To determine the time and duration of the first and second DNA synthetic phases in fertilized egg cells and central cells of rice, a total of 753 ovules were sampled at 2 h intervals during the first 30 h after pollination and exposed to ³H-thymidine for 2 h at 25 °C. Autoradiographic observation of labeled nuclei was made for fertilized egg cells, as well as for central and antipodal cells. The first and second DNA synthetic phases in fertilized egg cells were found 8–12 h and 21–25 h after pollination, respectively. The durations of each cell-cycle phase in the egg cell were estimated to be 4–6 h for G₁, 4 h vor S and for G₂, and 2 h for M. In the central cell, the first DNA synthesis took place at 3–4 h after pollination, i.e., immediately after fertilization, followed by the formation of the primary endosperm nucleus. Antipodal cells also showed labeled nuclei in the early stages after fertilization. The first divisions of fertilized egg cell and primary endosperm nucleus were observed at 16–18h and at 4–6 h after pollination, respectively. The present observations suggest that sperm and egg nuclei participate in fertilization with haploid amount (1C) of DNA and fertilized egg cell originates thus in 2C state.     

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     

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.     

Isolation of gametes and central cells from Oryza sativa L.

In vitro fertilization system of higher plants has been well established using maize gametes and central cells, which can produce embryos and endosperms. In the present study, procedures for isolating gametes and central cells from rice (Oryza sativa L. cv. Nipponbare), a model plant, are reported with the goal of establishing rice in vitro fertilization system. Egg cells and central cells were isolated by manual manipulation of enzyme-treated unpollinated ovules, and an alternative direct isolation method for egg cells that does not use enzymatic treatment was also established. Fluorescent visualization of the granular structures in the cytoplasm of isolated egg cells and the nucleoli in two polar nuclei of isolated central cells suggest that these cells are reliable gametes and central cells. For sperm cell isolation, the contents of rice pollen grains were released by osmotic pressure-induced bursting of the grains. In addition, electrofusion with isolated gametes was successfully conducted.     

Genetic analysis of endosperm mutants in rice Oryza sativa L.

Summary Sugary, shrunken, floury, white core, amylose extender and dull mutants induced in japonica varieties were used in this study. The results of an allelic analysis conducted in japonica background indicated that the two sugary mutants 82GF and EM5 are allelic. The two amylose extender mutants 2064 and EM16 are also allelic. The opaque mutant ESD7-3(0) and floury mutants 2047, EM17 and EM28 are allelic as well and have the flo-1 gene. The three white core mutants EM3, EM24 and EM66 were found to be non-allelic. Eleven dull mutants were investigated. Dull mutants 2057, 2083, 2091 and EM15 were found to be allelic to each other. Similarly, dull mutants 2077, 2078 and 2120 have allelic genes. Dull mutants 2035, EM12, EM47, and EM98 are non-allelic to the above loci. Dull genes in EM12, EM15, and EM98 were designated earlier as du-1, du-2 and du-4, respectively.The mutant genes were transferred to indica background by two backcrosses to IR36. Some of the mutant genes were located to respective chromosomes through trisomic analysis using primary trisomics of IR36. In this way the amylose extender gene ae was located to chromosome 2, the flo-1 was located to chromosome 5 and the flo-2 to chromosome 4. Dull genes of EM47, 2120, and 2035 were assigned to chromosomes 6, 9, and 6, respectively.     

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.     

Nuclear DNA amplification in cultured cells of Oryza sativa L.

Summary Highly repeated nuclear DNA sequences from suspension cultured cells of Oryza sativa L. cv. Roncarolo have been cloned in pBR322. Ten clones with specific digestion patterns have been randomly selected. Nine sequences appear to be organized in a clustered tandem array while one is interpersed in the rice genome. The clones have been used to gather information on: (a) their modulation in cultured cells as compared to whole plant and (b) their distribution in different rice cultivars belonging to the Japonica or Indica subspecies of Oryza sativa L. Hybridization with nuclear DNA isolated either from suspension or from seedlings of the Roncarolo cultivar revealed extensive quantitative variations, with most cloned sequences showing amplification (up to 75-fold) in cultured cells. Hybridization with nuclear DNA isolated from seedlings or suspension cultured cells from different cultivars belonging to the Japonica or to the Indica sub-species of O. sativa have shown that (a) amplification also occurs in a similar pattern in the case of DNA from the other tested suspension cultured cell types but not in the case of DNA from seedlings; (b) in some cases the tested sequences show minor but significant variations in different rice accessions.On leave from China National Rice Research Institute, Hangzhou, China     

Growth and respiration of rice (Oryza sativa L.) Cells in suspension culture

Studies on the growth and respiration of batch suspension cultures of rice (Oryza sativa L.) in a reference medium containing Murashige-Skoog salts, 2% (w/v) sucrose and yeast extract are reported. It was found that the yeast extract contributed 70% of the phosphate in this medium, and that the cells grew equally well in continued subculture in a defined medium which contained 6 mM phosphate and 3% (w/v) sucrose and the remaining Murashige-Skoog salts. Cell clumps (up to 1.5 mm diameter) were prevalent in the initial cultures in the reference medium. In such cultures the critical O₂ pressure of cell respiration was high (125 M), and ethanol accumulated. When cell clumps were routinely removed during several weekly subcultures on the defined medium cultures were obtained in which no clumps were present, the critical O₂ pressures was decreased to 40 M and no ethanol accumulated.This work was supported by grant PCM-84-03542 from the U.S. National Science Foundation.     

Genetic variation detected by DNA fingerprinting with a rice minisatellite probe in Oryza sativa L.

A rice minisatellite probe detecting DNA fingerprints was used to assess genetic variation in cultivated rice (Oryza sativa L.). Fifty-seven cultivars of rice, including 40 closely related cultivars released in the US, were studied. Rice DNA fingerprinting revealed high levels of polymorphism among distantly related cultivars. The variability of fingerprinting pattern was reduced in the closely related cultivars. A genetic similarity index (S) was computed based on shared fragments between each pair of cultivars, and genetic distance (D) was used to construct the dendrograms depicting genetic relationships among rice cultivars. Cluster analysis of genetic distance tended to group rice cultivars into different units corresponding with their varietal types and breeding pedigrees. However, by comparison with the coefficients of parentage, the criterion of relatedness based on DNA fingerprints appeared to overestimate the genetic relationships between some of the closely related US cultivars. Although this may reduce the power of fingerprints for genetic analysis, we were able to demonstrate that DNA fingerprinting with minisatellite sequences is simpler and more sensitive than most other types of marker systems in detecting genetic variation in rice.This paper reports the results of research only. Mention of a proprietary product does not consititute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the US Department of Agriculture, Agricultural Research Service, Plant Genetics Research Unit, and the University of Missouri Agricultural Experiment Station Journal Series No. 12178.     

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.     

Interspecific hybridization of cultivated rice, Oryza sativa L. with the wild rice, O. minuta Presl.

Crosses were made between four varieties (Mahsuri, Setanjung, MR84 and MR103) of Oryza sativa L. (2n=24, AA) and one accession of O. minuta (2n= 8, BBCC). The seed set obtained ranged between 9.5% and 25.1% depending on the rice variety used. By rescuing 14-day-old embryos and culturing them on 25%-strength MS medium we obtained a total of 414 F₁ hybrids. The F₁s were vigorous, tillered profusely, were perennial and male-sterile. The hybrids were triploid (ABC) with 36 chromosomes and showed irregular meiosis. The average frequency and range of chromosome associations at metaphase I or early anaphase I pollen mother cells of F₁ plants were 29.31(16–36) Is +3.32(0–10) IIs+0.016(0–1) IIIs+0.002(0–1) IVs. Upon backcrossing the original triploid hybrids and colchicine-treated hybrids to their respective recurrent parents, and further embryo rescue, 17 backcross-1 (BC₁) plants were obtained. Of all the crosses using MR84, no BC₁ plant was obtained even after pollinating 13 894 spikelets of the triploid hybrid. The BC₁s were similar in appearence to the F₁s and were male-sterile, their chromosome number ranged from 44 to 48. By backcrossing these BC₁s and nurturing them through embryo rescue, we obtained 32 BC₂ plants. Of these, however, only 18 plants grew vigorously. One of these plants has 24 chromosomes and the other 17 have chromosome numbers ranging between 30 and 37. The 24-chromosome plant was morphologically similar to the O. sativa parent and was partially fertile with a pollen and spikelet fertility of 58.8% and 12.5% respectively. All of the F₁ and BC₁ plants were found to be resistant to five Malaysian isolates (XO66, XO99, XO100, XO257 and XO319) of Xanthomonas campestris pv oryzae. Amongst the BC₂s, the reaction varied from resistant to moderately susceptible. The 24-chromosome BC₂ plant was resistant to the four isolates and moderately resistant to isolate XO100 to which the O. sativa parent was susceptible.Part of PhD thesis submitted by first author to Universiti Kebangsaan Malaysia, Bangi     

Genotypic effects on the callus formation from different explants of rice,Oryza sativa L.

A total of 108 rice varieties were examined for their tissue culture responses. Callus tissues were initiated from the seed, radicle, coleoptile and anther explants. Our results indicated that genotypes differed in the ability to develop vigorously growing callus. The callus growth responses in seed, radicle and coleoptile cultures were intercorrelated, but were not correlated with that in anther culture.     

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.     

Identification and genetic analysis of semidwarfism-related proteins in rice (Oryza sativa L.)

Summary By transferring a semidwarf gene (sd-1) from Taichung Native 1 into a tall Japanese cultivar, Norin 29, through seven backcrosses, a semidwarf near-isogenic line SC-TN1 was obtained. The proteins of the embryo in Norin 29 and SC-TN1 were separated by two-dimensional electrophoresis. Most of the proteins showed the same electrophoretic pattern. However, it was found that there was a difference in the appearance of two basic glycoproteins designated as SRP-1 and SRP-2. These proteins exhibited the same molecular mass, but different isoelectric points. Hybridization results indicated that a single locus controls SRP-1 and SRP-2 with codominant alleles. The gene symbol Srp was given to this locus, with alleles Srp-1 and Srp-2 responsible for SRP-1 and SRP-2, respectively. Srp-2 was found in all of the semidwarf cultivars and lines having sd-1, except a tall cultivar Tsaiyuan-chung. This finding suggests that Srp-2 may be closely linked with sd-1. The amounts of these proteins markedly increased after water absorption of the seed, suggesting that these proteins may be related to the early development of the plant.     

Somatic embryogenesis and plant regeneration from cultured young inflorescences of Oryza sativa L. (rice)

Compact calli initiated from young inflorescences of Oryza sativa L. (rice) on the Linsmaier and Skoog's (LS) medium containing 1 to 2.5mg/l of 2,4-dichlorophen-oxyacetic acid (2,4-D) were used for regeneration studies. After smooth and compact nodules appeared, these calli were transferred to the regeneration medium containing indole-3-acetic acid (IAA) and either kinetin or 6-benzylaminopurine (BAP). Somatic embryos developed in ten days and were examined by histological studies. Some of the embryos showed scutellum-like structures and a coleoptile-coleorhiza bipolar organization. Regenerated plants had the normal chromosome number of 2n=24.     

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.     

The serine carboxypeptidase like gene family of rice (Oryza sativa L. ssp. japonica)

Serine carboxypeptidases (SCPs) comprise a large family of protein hydrolyzing enzymes and have roles ranging from protein turnover and C-terminal processing to wound responses and xenobiotic metabolism. The proteins can be classified into three groups, namely carboxypeptidase I, II and III, based on their coding protein sequences and the fact that each family is characterized by a central catalytic domain of unique topology designated as the “α/β hydrolase fold”. The available SCP protein sequences have been utilized as datasets to build a HMM (hidden Markov model) profile, which is used to search the rice (Oryza sativa L. ssp. japonica) proteome. A total of 71 SCP and serine carboxypeptidase-like (SCPL) protein-coding genes exist in rice. The intron-exon structure, chromosome localization, expression and characteristics of encoded protein sequences of the 71 putative genes are reviewed.     

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.