Plant regeneration from indica rice (Oryza sativa L.) protoplasts

Rice (Oryza sativa L.) plants of the indica cultivar IR54 were regenerated from protoplasts. Conditions were developed for isolating and purifying protoplasts from suspension cultures with protoplast yields ranging from 1·10⁶ to 15·10⁶ viable protoplasts/1 g fresh weight. Protoplast viability after purification was generally over 90%. Protoplasts were cultured in a slightly modified Kao medium in a Petri plate by placing them onto a Millipore filter positioned on top of a feeder (nurse) culture containing cells from a suspension culture of the japonica rice, Calrose 76. Plating efficiencies of protoplasts ranged from 0.5 to 3.0%; it was zero in the absence of the nurse culture. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the protoplasts. After three weeks the Millipore filter with callus colonies were transferred off feeder cells and onto a Linsmaier and Skoog-type medium for an additional three weeks. Selected callus colonies that had embryo-like structures were then transferred to regeneration medium containing cytokinins, and regeneration frequencies up to 80% were obtained. Small shoots emerged and were transferred to jars for root development prior to transferring to pots of soil and growing the plants to maturity in growth chambers. Of the cytokinins evaluated, N⁶-benzylaminopurine was the most effective in promoting shoot formation; however, kinetin was also somewhat effective. Regeneration medium could be either an N₆ or Murashige and Skoog basal medium. Of 76 plants grown to maturity, 62 were fertile, and the plant heights averaged about three-fourths the height of seed-grown plants.Two other suspension cultures of IR54, one developed from the protoplast callus of the initial IR54 line, and the other developed from callus produced by mature seeds, have yielded protoplasts capable of regenerating plants when using cells of the Calrose 76 suspension as a nurse culture. In addition, protoplasts obtained from three-week-old primary callus of immature embryos of IR54 were capable of regenerating plants when using the same culture conditions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pcy packed cell volume - BAP N⁶-benzylaminopurine - FDA fluorescein diacetate - FW fresh weight - IAA indole-3-acetic acid Media AA Muller and Grafe (1978) - CPW Frearson et al. (1973) - Kao^* Kao (1977) - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962) - N₆ Chu et al. (1975) - PCM Ludwig et al. (1985)     

Identification of a mutable slender glume gene in rice (Oryza sativa L.)

The segregation pattern and chromosomal location of a slender glume mutation, induced by gamma-ray irradiation, was investigated. The mutation is genetically unstable: in the selfed progenies of slender glumed plants, not only plants with normal glumes but also plants that are chimeric for glume shape almost always appear at low frequency. The results showed that the mutation is controlled by a single recessive, mutable mutant gene slg. The frequency of reversion of slg to its wild-type state was little affected by crossing, backcrossing, genetic background or cytoplasmic factors. Conventional trisomic and linkage analyses revealed that the slg locus was located close to the rfs (rolled fine stripe leaf) locus on chromosome 7. In a subsequent RFLP analysis, slg was found to be located between the two RFLP loci XNpb20 and XNpb33, with recombination values of 3.0 and 3.2%, respectively. Southern analysis indicated that the mutability of slg is caused by none of the known transposable elements in rice. From these results, we infer that slg has a novel transposable DNA insert in its vicinity, which was possibly activated by gamma-ray irradiation.     

Molecular mapping of genes conferring aluminum tolerance in rice (Oryza sativa L.)

Crop productivity on acid soil is restricted by multiple abiotic stress factors. Aluminum (Al) tolerance seems to be a key to productivity on soil with a pH below 5.0, but other factors such as Mn toxicity and the deficiency of P, Ca and Mg also play a role. The development of Al-tolerant genotypes of rice is an urgent necessity for improving crop productivity in developing countries. Inhibition of root growth is a primary and early symptom of Al toxicity. The present study was conducted to identify genetic factors controlling the aluminum tolerance of rice. Several parameters related to Al tolerance, most importantly the relative root growth under Al stress versus non-stress conditions, were scored in 188 F₃ selfed families from a cross between an Al-tolerant Vietnamese local variety, Chiembau, and an Al-susceptible improved variety, Omon269–65. The two varieties are both Oryza sativa ssp. indica, but showed a relatively high level of DNA polymorphism, permitting the assembly of an RFLP map consisting of 164 loci spanning 1,715.8 cM, and covering most of the rice genome. A total of nine different genomic regions on eight chromosomes have been implicated in the genetic control of root and shoot growth under aluminum stress. By far the greatest effects on aluminum tolerance were associated with the region near WG110 on chromosome 1. This region does not seem to correspond to most of the genes that have been mapped for aluminum tolerance in other species, nor do they correspond closely to one another. Most results, both from physiological studies and from molecular mapping studies, tend to suggest that aluminum tolerance is a complex multi-genic trait. The identification of DNA markers (such as WG110) that are diagnostic for aluminum tolerance in particular gene pools provides an important starting point for transferring and pyramiding genes that may contribute to the sustainable improvement of crop productivity in aluminum-rich soils. The isolation of genes responsible for aluminum tolerance is likely to be necessary to gain a comprehensive understanding of this complex trait. Received: 29 March 2000 / Accepted: 16 August 2000     

Mapping of QTLs conferring extremely early heading in rice (Oryza sativa L.)

Two genes related to extremely early heading were identified in populations derived from crosses between Hoshinoyume, a variety adapted to the northernmost limit of rice cultivation (Hokkaido), and Nipponbare, a variety adapted to the temperate region of Japan. The segregations for heading date clearly revealed that a two-gene model determined the extremely early heading in the F₂ and BC₁F₁ populations under natural field conditions in Hokkaido. Using molecular markers corresponding to ten known quantitative trait loci (QTLs) for heading date, we carried out QTL analysis in the BC₁F₁ population and detected two QTLs, qDTH-7-1 and qDTH-7-2, both on chromosome 7, and observed epistatic interaction between them. We conclude that the recessive alleles of these two genes contribute to extremely early heading for the adaptation to Hokkaido environment and to stable rice production in Hokkaido. The relationships between the two QTLs identified in this study and known QTLs are discussed.     

Identification, cDNA cloning, and gene expression of soluble starch synthase in rice (Oryza sativa L.) immature seeds.

Three forms of soluble starch synthase were resolved by anion-exchange chromatography of soluble extracts from immature rice (Oryza sativa L.) seeds, and each of these forms was further purified by affinity chromatograph. The 55-, 57-, and 57-kD proteins in the three preparations were identified as candidates for soluble starch synthase by western blot analysis using an antiserum against rice granule-bound starch synthase. It is interesting that the amino-terminal amino acid sequence was identical among the three proteins, except that the 55-kD protein lacked eight amino acids at the amino terminus. Thus, these three proteins are products of the same gene. The cDNA clones coding for this protein have been isolated from an immature rice seed library in lambda gt11 using synthetic oligonucleotides as probes. The deduced amino acid sequence of this protein contains a lysine-X-glycine-glycine consensus sequence for the ADP-glucose-binding site of starch and glycogen synthases. Therefore, we conclude that this protein corresponds to a form of soluble starch synthase in immature rice seeds. The precursor of the enzyme contains 626 amino acids, including a 113-residue transit peptide at the amino terminus. The mature form of soluble starch synthase shares a significant but low sequence identity with rice granule-bound starch synthase and Escherichia coli glycogen synthase. However, several regions, including the substrate-binding site, are highly conserved among these three enzymes. Blot hybridization analysis demonstrates that the gene encoding soluble starch synthase is a single-copy gene in the rice genome and is expressed in both leaves and immature seeds. These results suggest that soluble and granule-bound starch synthases play distinct roles in starch biosynthesis of plant.     

High-frequency plant regeneration from coleoptile tissue of indica rice (Oryza sativa L.)

Summary An efficient method was established for high-frequency embryogenic callus induction and plant regeneration from 3-,4-, 5- and 7-d-old coleoptile segments of Indica rice (Oryza sativa L. cv. Kasturi), Compact and friable callus developed from the cut ends and also on the entire length of the coleoptile segments cultured on Murashige and Skoog (MS) basal medium (1962) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 4.50–18.0 μM), kinetin (2.32 μM) and sucrose (3%, w/v). High frequency embryogenic callus induction and somatic embryo development was achieved when embryogenic calluses were transferred to MS medium supplemented with 2.25 μM 2,4-D, 2.32 μM kinetin, 490 μM L-tryptophan and 3% (w/v) sucrose. Plant regeneration was achieved by transferring clumps of embryogenic callus onto MS medium containing 2.85 μM indole-3-acetic acid (IAA), 17.77 μM 6-benzylaminopurine (BA) and 3% (w/v) sucrose. Histological observations of embryogenic calluses revealed the presence of somatic embryos and also plant regeneration via multiple shoot bud formation. Three, 4- and 5-d-old coleoptile segments showed a significantly (P<0.05) higher frequency of plant regeneration and mean number of plantlets per explant in comparison to 7-d-old coleoptile segments. The highest frequency (73.5%) of plant regeneration and mean number of plantlets (11.9±1.0) was obtained from 4-d-old coleoptile segments. Regenerated shoots were rooted on MS basal medium containing 4.92 μM indole-3-butyric acid (IBA) and plants were successfully transferred to soil and grown to maturity.     

Genetic control of paste viscosity characteristics in indica rice (Oryza sativa L.)

 Paste viscosity parameters play an important role in estimating the eating, cooking and processing quality of rice. Four cytoplasmic male-sterile (CMS) lines and eight restorer (R) lines were employed in an incomplete diallel cross to analyze seed effects, cytoplasmic effects and maternal gene effects on the viscosity profiles of indica rice. The results indicated that the viscosity profiles of rice were controlled by the direct effects of the seed, by the cytoplasm and by maternal plant. The seed-direct effects (V _A +V _D ) accounted for over 51% of the total genetic variances (V _A +V _D +V _C +V _Am +V _Dm ) for all the traits, suggesting that seed direct effects were more important than maternal effects and cytoplasmic effects. The additive variances (V _A +V _Am ) were much larger than the dominance variances (V _D +V _Dm ), which revealed that additive genetic effects were the major contributors of genetic variation for the paste viscosity profiles, and that selection could be applied for viscosity traits in the early generations. Significant cytoplasmic variance (V _C ) was detected for hot paste viscosity (HPV), cool paste viscosity (CPV) and consistency viscosity (CSV). The cytoplasmic effects for these three traits can, therefore, not be neglected in rice breeding. It was also shown that seed heritabilities (h ² _o ) tended to be larger than maternal heritabilities (h ² _m ) and cytoplasmic heritabilities (h ² _c ). Prediction of the main genetic effects for 12 parents showed that CMS lines had highly positive effects on all the traits except for the breakdown viscosity (BDV), and that R lines had both positive and negative effects on the paste viscosity characteristics. Received: 3 August 1998 / Accepted: 28 November 1998     

In vitro flowering of indica rice (Oryza sativa L. spp. indica)

Nodal explants of rice cultivar Pathumthani 1 (PT1; short-day photoperiod insensitive) were collected, surface-disinfected, and cultured on modified MS medium under in vitro conditions for 90 d. A total of 60% nodal explants generated flowering plantlets (with one inflorescence per cluster). The net photosynthetic rate was greater, and soluble sugars (including glucose, fructose, and sucrose) accumulated to higher levels in the leaves of flowering as compared to non-flowering plants. In contrast, chlorophyll a, chlorophyll b, total chlorophyll, and total carotenoid content were enriched to a greater degree in the leaves of non-flowering as compared to flowering plants. Also, growth performance parameters, including plant height, number of leaves per plant, leaf area, fresh weight, and dry weight of plantlets derived from seedlings were superior to those of plantlets derived from nodal explants. In addition, the protocol proved to successfully induce flowering in KDML 105, a short-day photoperiod-sensitive rice cultivar.     

Cloning,transformation and expression of cell cycle-associated protein kinase OsWee1 in indica rice (Oryza sativa L.)

The development process of seed in plants is a cycle of cells which occur gradually and regularly. One of the genes involved in controling this stage is the Wee1 gene. Wee1 encode protein kinase which plays an important role in phosphorylation, inactivation of cyclin-dependent kinase 1 (CDK1)-cyclin (CYC) and inhibiting cell division at mitotic phase. The Overexpression of Wee1 leads to delaying entry into mitotic phase, resulting in enlargement of cell size due to suppression of cell division. Accordingly, the cloning and overexpressing of Wee1 in rice plant is important aim of this research in achieving better quantity and quality of future rice. The main objective of this present study is to cloning and generate transgenic rice plants overexpressing of Wee1 gene. Wee1 was isolated from cDNA of indica rice (Oryza sativa), called OsWee1. The full length of OsWee1 was 1239?bp in size and successfully inserted into plant expression vector pRI101ON. Seven-day-old rice seedlings were prepared for transformation of OsWee1 gene using Agrobacterium-mediated transformation method. Four positive transgenic lines were identified through the presence of kanamycin resistance gene (nptII) using genomic PCR analysis. Southern blot analysis result provides evidence that four independent rice transformants contained one to three rearranged transgene copies. Further screening in transgenic rice generation is needed in order to obtain stable expression of OsWee1.     

Proteomics characterization of different bran proteins between aromatic and nonaromatic rice (Oryza sativa L. ssp. indica)

Proteomic approach is applied for the analysis of seed brans of 14 rice varieties (Oryza sativa L. ssp. indica) which can classify to five aromatic rice and nine nonaromatic rice. The two-dimensional electrophoresis (2-DE) protein patterns for 14 rice varieties were similar within pH ranges of 3-10 and 4-7. To characterize aromatic group-specific proteins, we compared 2-D gels of aromatic rice to nonaromatic rice using PDQUEST image analysis. Four out of six differential spots were identified as hypothetical proteins, but one (SSP 7003) was identified by matrix assisted laser desoption/ionization-quardrupole-time of fight (MALDI-Q-TOF) as prolamin with three matching peptides based on NCBI database. Prolamin is a class of storage proteins with three different polypeptides of 10, 13, and 16 kDa. Spot SSP7003 was identified as a 13 kDa polypeptide of prolamin by combination of mass spectroscopy and N-terminal sequence analyses. In contrast, one sulfur-rich 16 kDa polypeptide of prolamin was found in extremely high intensity in brans of deep-water rice compared to nondeep-water rice. Our results suggest that proteomics is a powerful step to open the way for the identification of rice varieties.     

Identification, genetic characterization, GA response and molecular mapping of Sdt97: a dominant mutant gene conferring semi-dwarfism in rice (Oryza sativa L.)

Semi-dwarfism is an important agronomic trait in rice breeding programmes. sd-1, termed the 'Green Revolution gene', confers semi-dwarf stature, increases harvest index, improves lodging resistance, and is associated with increased responsiveness to nitrogen fertilizer. It has contributed substantially to the significant increase in rice production. In this paper, a novel semi-dwarf mutant in rice is reported. Genetic analysis revealed that only a single dominant gene locus non-allelic to sd-1, temporarily designated Sdt97, is involved in the control of semi-dwarfism of the mutant. The semi-dwarfism of the mutant could be partly restored to the tall wild-type by application of exogenous GA3, suggesting that the mutant gene Sdt97 may be involved in the gibberellin (GA) synthesis pathway and not the GA response pathway in rice. A residual heterozygous line (RHL) population derived from a recombinant inbred line (RIL) was developed. Simple sequence repeat (SSR) and bulked segregation analysis (BSA) combined with recessive class analysis (RCA) techniques were used to map Sdt97 to the long arm of chromosome 6 at the interval between two STS markers, N6 and TX5, with a genetic distance of 0.2 cM and 0.8 cM, respectively. A contig map was constructed based on the reference sequence aligned by the Sdt97 linked markers. The physical map of the Sdt97 locus was defined to a 118 kb interval, and 19 candidate genes were detected in the target region. This is the first time that a dominant semi-dwarf gene has been reported in rice. Cloning and functional analysis of gene Sdt97 will help us to learn more about molecular mechanism of rice semi-dwarfism.     

A PCR-based marker for a locus conferring the aroma in Myanmar rice (Oryza sativa L.)

Aromatic rice is an important commodity for international trade, which has encouraged the interest of rice breeders to identify the genetic control of rice aroma. The recessive Os2AP gene, which is located on chromosome 8, has been reported to be associated with rice aroma. The 8-bp deletion in exon 7 is an aromatic allele that is present in most aromatic accessions, including the most popular aromatic rice varieties, Jasmine and Basmati. However, other mutations associated with aroma have been detected, but the other mutations are less frequent. In this study, we report an aromatic allele, a 3-bp insertion in exon 13 of Os2AP, as a major allele found in aromatic rice varieties from Myanmar. The insertion is in frame and causes an additional tyrosine (Y) in the amino acid sequence. However, the mutation does not affect the expression of the Os2AP gene. A functional marker for detecting this allele was developed and tested in an aroma-segregating F(2) population. The aroma phenotypes and genotypes showed perfect co-segregation of this population. The marker was also used for screening a collection of aromatic rice varieties collected from different geographical sites of Myanmar. Twice as many aromatic Myanmar rice varieties containing the 3-bp insertion allele were found as the varieties containing the 8-bp deletion allele, which suggested that the 3-bp insertion allele originated in regions of Myanmar.     

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.