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.     

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.     

Characterization of a rice (Oryza sativa L.) mutant deficient in the heme domain of nitrate reductase

Summary Biochemical and genetical characterization of a rice nitrate reductase (NR)-deficient mutant, M819, which had been isolated as a chlorate-resistant mutant, was carried out. In M819, leaf NADH-NR activity was found to be about 10% of that of the wild-type cv Norin 8, while NADPH-NR activity was higher than that in the wild-type; FMNH₂-NR and MV-NR activities were also 10% of those of the wild type; BPB-NR activity was higher than that of the wild type; and xanthine dehydrogenase activity was revealed to be present in both. These results suggest that the mutant line M819 lacks the functional heme domain of the NADH-NR polypeptide due to a point mutation or a small deletion within the coding region of the structural gene. Chlorate resistance in M819 was transmitted by a single recessive nuclear gene.Abbreviations NR Nitrate reductase - NiR nitrite reductase - FMNH₂ reduced flavin nucleotide - MV reduced methyl viologen - BPB reduced bromphenol blue - XDH xanthine dehydrogenase     

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.     

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.     

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.     

Ecological significance of root tip rotation for seedling establishment of Oryza sativa L

How plant seeds secure root penetration into soil to obtain good seedling establishment is one of the basic ecological problems. In this study, seminal root growth was investigated to clarify the cause of varietal difference of seedling establishment in direct seeding of rice in flooded paddy fields, with special reference to root tip rotation. In a field experiment, seedling establishment percentage had a weak correlation with seminal root elongation rate but was not correlated with apparent seedling weight in water, which has been reported to be the cause of floating seedlings resulting in poor seedling establishment. Root tip rotation was analyzed for indoor-grown seedlings using spectrum analysis: the maximum entropy method (MEM) was used. Maximum entropy method power spectrum analysis clarified that maximum MEM power density (practically corresponds to spiral angle) detected in the frequency range above 0.1 cycles mm^-1 was highly and positively correlated to seedling establishment percentage in the field experiment. Maximum MEM power density in high correlation with seedling establishment was mostly found around frequencies of 0.2 cycles mm^–1, which corresponded to 2.0–3.4 cycles of root tip rotation per day. From these results, root tip rotation (circumnutation) with a larger spiral angle was suggested to play an important role in the establishment of rice seedlings on flooded and very soft soil. A possible explanation for why a larger spiral angle was advantageous for seedling establishment is that if buoyancy and seedling weight are constant, a larger pushing force of the seminal root is available without causing floating of a seedling, due to the upward force being a reaction of the seminal root pushing force.     

Selection of higher regenerative callus and change in isozyme pattern in rice (Oryza sativa L.)

Summary Calli were initiated from seedling roots in rice (Oryza sativa L. var. Tadukan) and subcultured at 45-day intervals on MS medium supplemented with 2 mg/l 2,4-D. Sectors of callus which differentiated shoot meristems (green spots) under the same 2,4-D concentration were selected from the calli subcultured 90 days after initiation. The selection was continued for about 2 years. Responses to 2,4-D between original and selected lines differed considerably, although differentiation was not generally seen in rice callus in the presence of 2 mg/l 2,4-D. After 180 days, calli of the selected line differentiated into numerous shoot-bud primordia and grew out new callus tissues under 2 mg/l 2,4-D concentration; the frequency of the differentiation exceeded 90%. On the other hand, no calli of non-selected line differentiated into shootbuds under 2 mg/l 2,4-D, and the frequency of the shootbud was only about 50% under lower 2,4-D concentration (0.1 mg/l). The pattern and activity of peroxidase isozyme varied markedly between calli of the selected and non-selected lines. First, two strong peroxidase bands which show fast mobility and one intermediate peroxidase band with slow mobility were detected only in the calli of selected line. Secondly, changes in band pattern of proteins separated by SDS-PAGE were observed. In the calli of selected line, there was a loss of the polypeptide bands with molecular weight of 24 and 42 K in the selected calli, but they were clearly present in the unselected line. The appearance of new peroxidase isozyme bands and loss of polypeptide bands, change in response to auxin and increased ability for shoot bud differentiation are closely correlated to each other.     

Chromosomal location of genes conditioning low amylose content of endosperm starches in rice,Oryza sativa L.

Summary Eight dull mutants that lower the amylose content of rice endosperm as well as waxy mutant and a cultivar with common grains were crossed in a diallele manner. The amylose content of F₁ and F₂ seeds was determined on the basis of single grain analysis. It was concluded that the low amylose content of dull mutants is under monogenic recessive control. Alleles for low amylose content are located at five loci designated as du-1, du-2, du-3, du-4 and du-5. These loci are independent of wx locus located on chromosome 6. The five du loci have an additive effect in lowering the amylose content. Two loci, du-1 and du-4, were found to be located on chromosomes 7 and 4, respectively.     

Effect of age of seedling and phytohormones on micropropagation of indica rice (Oryza sativa L.) from Meristem Culture.

An efficient protocol forin vitro micropropagation of seven indica rice varieties was developed from meristem culture. Meristem (leaf base) was isolated from different age of seedlings and cultured on MS medium without hormones and supplemented with different concentrations of NAA and BAP. Regeneration of plantlets from meristem was observed within five days of culture. The meristem isolated from 4-day old seedlings gave highest regeneration on hormone free MS medium. Histological study of meristem (leaf base) from 4-day old seedlings confirmed the presence of meristematic cells. Regenerated plants were multiplied on MS medium supplemented with 0.05 mg/L NAA and 5 mg/L BAP. An average of five plants were obtained from single regenerated meristem. The plants regenerated from meristem showed morphological uniformity.     

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.     

Effect of culture methods on the regeneration of albino rice (Oryza sativa L.) plantlets

In our study, we investigated the effects of regeneration conditions on both green and albino rice plants (Oryza sativa L.). The regeneration frequency of an albino cell line was compared to a normal cell line obtained from mature seed under two kinds of culture conditions; namely, the static culture on semi-solid regeneration medium and the suspension culture in liquid regeneration medium. The albino cell line, from which only albino plantlets were regenerated, was induced from the albino leaf segments. There were no significant differences in the regeneration frequencies between normal and albino calli on the semisolid regeneration medium. On the other hand, the frequency of regeneration of albino calli was significantly lower than that of the control specifically in the liquid regeneration medium.     

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.     

Mode of inheritance of dwarf stature and allelic relationships among various spontaneous and induced dwarfs of cultivated rice Oryza sativa L.

Summary Genetic study of spontaneous and induced dwarfs included the mode of inheritance of dwarf stature and the allelic relationships among various dwarfs. Qualitative genetic analysis involving crosses of fourteen dwarfs with a common tall variety IARI 11124 showed that the degree of dominance in the F₁ hybrids varied with the cross. With the exception of the crosses of IARI 6579 and IARI 10560 with the tall variety, all crosses exhibited incomplete dominance. The segregation pattern in F₂ populations of height classes showed dwarfness to be a monogenic recessive trait functioning, however, in association with modifier complexes of varied strength. From F₂ behaviour of all possible crosses involving the fourteen different dwarfs, the allelic relationships were deduced. Three major groups of dwarfs could be recognised. Group I, comprised of FF 36, IARI 5842, IARI 5906-2B, IARI 5923, IARI 10061, IARI 10560 and IARI 11445, was allelic to I-geo-tse and Dee-Gee-Woo-Gen with modifiers of predominantly negative effects, while group-2, comprised of dwarfs IARI 5901-2, IARI 5924, IARI 6579 and IARI 7312B, was also allelic to Dee-Gee-Woo-Gen and I-geo-tse but with large and equal number of modifiers of positive and negative effects. The induced mutant, Central Africa Mutant (CAM) which constituted the third group seemed to possess a dwarfing gene that was non-allelic to those of the above mentioned two groups of dwarfs, with equal strength of modifiers of plus and minus effects. Unlike the dwarfs of spontaneous origin, which are invariably allelic to Dee-Gee-Woo-Gen, the induced dwarf was nonallelic. Thus, induced mutagenesis appears to give rise to dwarfing genes different from those found in the naturally occurring dwarfs.     

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.     

Analysis of glucose phosphate isomerase in near-isogenic lines,and cultivars of rice (Oryza sativa L.)

Using the near-isogenic lines, the possible location of glucose phosphate isomeras-2 (phosphoglucose isomerase-2) locus (Pgi-2) in relation to photoperiod sensitivity locus (Se-1) and blast resistance locus (Pi-z) was investigated. The recombination frequency data indicate thatPgi-2 locus locates betweenSe-1 andPi-z loci. Furthermore, 15 Indica cultivars possessed two types of glucose phosphate isomerase-2 (GPI-2) isozyme, whereas only one type of GPI-2 isozyme was found in 30 Japonica cultivars.     

Efficient plant regeneration through somatic embryogenesis from callus induced on mature rice embryos (Oryza sativa L.)

To obtain a reproducible efficient procedure for regeneration of rice plants through somatic embryogenesis from callus four published methods of callus induction and regeneration were compared. Callus was initiated from mature embryos of the Japonica cultivar Taipei 309 of rice (Oryza sativa L.). The number, mass and morphology of the callus formed on the scutellum were dependent on the medium used. A limited humidity and an optimal aeration of the culture vessels enhanced the frequency of embryogenesis and plant regeneration. A method described by Poonsapaya et al. (1989) was found to be the most efficient and was slightly modified. As a result 98% of the T309 embryos formed callus, of which 63% regenerated into plants. Each callus yielded an average of 6 plants. Plant morphology, fertility and seed set of the regenerants were found to be normal.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - IAA 3-indole-acetic acid - BA 6-benzyladeninepurine - S.E.M. standard error of mean     

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     

Expression of functional proteins of cDNA encoding rice nucleoside diphosphate kinase (NDK) in Escherichia coli and organ-related alteration of NDK activities during rice seed germination (Oryza sativa L.)

The GST (glutathione S-transferase)-NDK (nucleoside diphosphate kinase) fusion protein was expressed in Escherichia coli. The GST-NDK protein was capable of transferring -phosphate from ATP to nucleoside diphosphates such as GDP, CDP, TDP and UDP. Western blot analysis using anti-NDK antibody indicated that NDK in endosperm gradually decreased during 36 h of imbibition. On the contrary, NDK in embryo increased during the same period. NDK activities in both tissues were in accord with these observations. Whereas the NDK protein in roots of rice seedlings during 7 days of imbibition remained constant, in shoots it declined after 5 days of imbibition. Thus, NDK may play a significant role in the cellular event modulated by adenylate energy charge level.     

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.