Indica-type Rice Research Articles

Optimum Cooking Conditions for Indica Type Rice by Using Random Centroid Optimization

Optimum Cooking Conditions for Indica Type Rice by Using Random Centroid Optimization

Evidence for genomic changes in transgenic rice (Oryza sativa L.) recovered from protoplasts.

The occurrence of genomic modifications in transgenic rice plants recovered from protoplasts and their transmission to the self-pollination progeny has been verified with the random amplified polymorphic DNA (RAPD) approach. The plant was the Indica-type rice (Oryza sativa L.) cultivar Chinsurah Boro II. The analysed material was: (1) microspore-derived embryogenic rice cells grown in suspension culture, (2) transgenic plants recovered from protoplasts produced from the cultured cells and (3) the self-pollination progeny (two successive generations) of the transgenic plants. DNA purified from samples of these materials was PCR-amplified with different random oligonucleotide primers and the amplification products were analysed by agarose gel electrophoresis. Band polymorphism was scored and used in band-sharing analyses to produce a similarity matrix. Relationships among the analysed genomes were expressed in a dendrogram. The extensive DNA changes evidenced in cultured cells demonstrate the occurrence of somaclonal variation in the material used to produce protoplasts for gene transfer. Quantitatively reduced DNA changes were also found in the resulting transgenic plants and in their self-pollination progenies. While confirming the stability of the foreign gene in transgenic plants, this work gives molecular evidence for the occurrence of stable genomic changes in transgenic plants and points to in vitro cell culture as the causative agent. RAPDs are shown to be a convenient tool to detect and estimate the phenomenon at the molecular level. The methodology is also proposed as a fast tool to select those transgenic individuals that retain the most balanced genomic structure and to control the result of back-crosses planned to restore the original genome.

Identification and Isolation of Blast Resistance Genes in Three indica-type Rice Varieties.

Two methods were used to identify blast resistance genes in indica -type varieties and isolate the genes found: one was an inoculation of F3 Iines between indica -type and japonica varieties (Suweon 262 x Reimei) with a common fungus strain (Ken 54-20) and a fungus strain having many virulence genes (AO 14-20) and selection of F3 Iines showing different reactions to the two fungus strains, and the other was selection of F3 Iines showing a 3 : I segregation ratio in the hybrid (Norin 29 x Hokuriku 129 and Norin 22 x sifeng 43) for Ken 54-20. The former method was used to identify and to select the resistance gene brokendown by Ao 14-20 having several number of virulence genes. The latter was to identify and isolate all major genes included in the resistant variety. It was demon-strated that Suweon 262 had Pi-i and an unidentified major gene and at least a minor one, and Hokuriku 129 had two major genes, Pi-i and Pi-b : Sifeng 43 had one major gene Pi-b and at least one minor gene. Minor genes dis-turbed the isolation of major genes in cross combinations of Suweon 262 x Reimei and Norin 29 x Hokuriku 129. It was considered that Hokuriku 129 is heterogeneous at least for Pi-i and Pi-b.

Plant Regeneration through Direct Culture of Isolated Pollen Grains in Rice.

Immature pollen grains of an Indica type rice cultivar IR24 were isolated and cultured to regenerate green plants without preculture of anthers. Either low-temperature pretreatment of panicles at 10'C in the dark or exposure to sugar starvation in the beginning of culture were necessary for induction of cell division of the grains. Low-temperature pretreatment was more effective treatuent than exposure to sugar starvation. It was found that there was a complementary relationship between the effects of low-temperature pretreatuent and of exposure to sugar starvation the pollen grains required shorter period of exposure to sugar starvation to start cell division as low-temperature pretreatnent was prolonged. The optimal condition for induction of cell division of the pollen grains was 21 days of the pretreatuent with I day of sugar starvation or 28 days of the pretreatment without sugar starvation. Though 2, 4-D was not necessary for induction of cell division, the development of the derived colonies followed by shoot regeneration was observed only in the presence of 2, 4-D. Reduced nitrogen source in the induction medium also affected the frequency of both colony formation and plant regeneration. The medjum containing 20mM KN03 and 5mM glutamine as nitrogen sources gave the highest frequency of colony formation at 0.20/0 and its frequency reached 2.0 times higher than the control R2 medium containing 40mM KN03 and 2.5mM (NH4)2S04. On the other hand, the calli which were formed in the medium containing 20mM KN03 and 5mM alanine had the highest regenerating ability. In addition, the calli which were formed in the medium containing 20mM KN03 and 5mM alanine showed marked higher frequency of green plant regeneration than ones in any other media tested, when 21 days of low-temperature pretreatment without exposure to sugar starvation was applied as induction condition.

Reducing the Stale Flavor of Cooked Rice by Treating with Cells of Acetic Acid Bacteria

Japonica-type rice grains stored at 40°C for 2 months were treated with a freeze-dried powder of acetic acid bacteria and then cooked. It was found that the originally existing n-hexanal were decreased by more than 35%. Moreover, the stale flavor had been significantly reduced compared to that of non-treated rice grains. Simultaneous treatment with freeze-dried cells of the acetic acid bacteria and a surfactant was 1.3–1.9 times more effective for reducing the amount of n-hexanal than with the cells of acetic acid bacteria alone.The optimum conditions for reducing the stale flavor were investigated by using a combination of the acetic acid bacterial cells and the surfactant. The optimum conditions were as follows: soaking temperature, 50°C; soaking time, 30–180 min; pH 4.5–7.0, ptimally at pH 6.0.Treating Indica-type rice grains with the freeze-dried cells of acetic acid bacteria was also effective for reducing the specific off-flavor of the rice.

Expression of human ?-interferon cDNA in transgenic rice plants

The plasmid pIG3031 containing human α-interferon cDNA and the neomycin phosphotransferase II coding sequence was successfully transferred into rice protoplasts (Indica type rice) by Lipofectinmediated transformation. Assays for NPT II enzyme activity indicated that the transformation frequency was 10%. Transgenic plants were regenerated from transformed calli. Southern blots showed that the human α-interferon cDNA sequence was present in rice DNA. RNA slot blots indicated apparent transcription of human α-interferon cDNA under the control of the plant-active promoter PI'. Extracts of transgenic cell cultures and plants contained apparent interferon activity as measured by resistance of a human amniotic cell line to viral infection in the presence of plant extracts. These studies demonstrate that human α-interferon cDNA may be correctly expressed in rice cells.

Fertile plants regenerated from suspension culture-derived protoplasts of an indica type rice (Oryza sativa L.)

Calluses were induced from immature embryos of an indica type rice and finely dispersed cell suspension cultures were initiated from the callus using modified AA medium (S1 medium). The suspension cultures were maintained alternatively (1–2 passages in each medium) in S1 medium and S2 medium, the latter containing KNO3, NH4NO3, proline and glutamine as nitrogen source. Protoplasts of high quality were isolated form suspension cells cultured in S2 medium supplemented with ABA. Embedding the protoplasts in agarose blocks containing NH4NO3-free modified KM8P(PM1) medium and immersing the blocks in NH4NO3-containing modified KM8P(PM3) medium were most effective for obtaining protoplast division and callus formation. The protoplast-derived calluses were precultured in potato extract-aand/or ABA-containing N6(D1, D2 or D3) media and many embryo-like structures were formed. These structures developed into plantlets after being transferred to N6 differentiation (D4) medium. The regenerated plantlets grew into mature plants and beard seeds normally.

Efficient fertile plant regeneration from protoplasts of the Indica rice breeding line IR72 (Oryza sativa L.)

Protoplasts were isolated from embryogenic cell suspensions obtained from mature seed derived embryogenic callus of the advanced Indica type rice breeding line IR72 available from IRRI (International Rice Research Institute), Manila. Culture of protoplasts with the agarose bead type method without nurse culture led to sustained proliferation of protoplast derived clones. A simple culture protocol was developed which stimulated embryogenic development. Germination of somatic embryos has so far produced 277 green plants from 6 independent experiments. 117 plants have been transferred to soil and are growing in the greenhouse. A few of them have already flowered and set seeds.

Plant Regeneration from Isolated Pollen Grains in Indica Type Rice(Oryza sativa L.).

Plant Regeneration from Isolated Pollen Grains in Indica Type Rice(Oryza sativa L.).

Fertile Indica rice plants regenerated from protoplasts isolated from microspore derived cell suspensions

Rice plants (Oryza sativa L., Chinsurah Boro II var. Indica) were regenerated from protoplasts isolated from microspore derived cell suspensions. A simple procedure for the establishment of such cell suspension cultures from embryogenic microcallus derived from cultured isolated microspores of Indica-type rice is described. Regenerating protoplasts could readily be isolated from 5-12 months old cell suspensions showing visible colony formation in the range of 180-1050 colonies/10(6) protoplasts after about one month in culture. More than 100 independent green plantlets were regenerated via secondary embryogenesis from ca 20×10(6) protoplasts. Out of 32 plants grown to maturity under greenhouse conditions 24 were fertile.

Genetically Engineered Fertile Indica-Rice Recovered from Protoplasts

We have established an efficient protocol for plant regeneration from haploid Indica-type rice protoplasts. Incubation of these protoplasts with the selectable hygromycin phosphotransferase (hph) gene expressed under control of the 35S promoter of cauliflower mosaic virus (CaMV) and polyethyleneglycol (PEG), and subsequent culture in the presence of hygromycin B, led to the recovery of numerous resistant clones from which 77 plants were regenerated. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed, and that it was inherited in offspring.

Plant regeneration from protoplasts of Indica type rice and CMS rice

Cell suspensions were established from 12 rice genotypes including Indica type, Japonica type and cytoplasmic male sterile (cms) rice lines of both types. 133 green plants were regenerated from protoplasts isolated from an Indica type rice and 15 plants were obtained from a Japonica type cms rice. A cell suspension, from which protoplasts can be isolated (about 10(6)/ml) but from which cell division has not yet been induced, has been established from the most widely used Indica type cms rice which contains Wild Abortive (WA) cytoplasm.

Studies on Evapotranspiration from Crops

The problems are whether evapotranspiration (ET) from paddy field would be affected by growth stage and by rice varieties, and whether the radiation method and Priestley-Taylor method for estimating ET would be applicable to paddy field.ET was measured with microlysimeter (Tomar and O'Toole, 1980A) at the eight test plots (N1-N8) in which cultivar “Nipponbare” (Japonica rice) was grown under eight different cropping seasons, and at the two plots (K5 and R5) in which cultivars “Milyang 23” (Japonica-Indica cross variety) and “IR-36” (Indica rice) were grown under the ordinary cropping season (see Horie and Sakuratani, 1985, Table 1). Three stainless pans (30cm long, 20cm wide and 60cm deep) were installed in the center part of the each plot: the two with the hills of rice plant and the other without crop were used as evapotranspirometer and evaporimeter, respectively. ET and transpiration (T) from each plot were determined, respectively, by averaging the values of ET from the two pans, and by subtracting the evaporation (E) from the ET. Meteorological data were also collected.In each cropping season of “Nipponbare” rice, the E at the early growth stage in which the leaf area index (LAI) was small, was comparable to the ET at the maximum LAI stage (Fig. 2A). Thus, there was no appreciable difference in ET between the two stages. This also caused the noticeable increase of total E for entire growing periods in the plots of the earlier cropping seasons such as N1 and N2 in which the initial growth was delayed by lower air temperature (Table 1). The mean value of daily ET ranged between 3.5 and 4.2mm day-1 for the eight cropping seasons, averaging 4.0mm day-1.The ratio of ET to reference ET (ETR) calculated from modified Penman method (Doorenbos and Pruitt, 1977) was fairly constant throughout the main growth period except mid-September to October and averaged 1.05 for all the cropping season (Fig. 2B). The overestimate in the ET in the fall seemed to be owing to the enrichment of ET by advective energy and to the underestimate in the ETR. On the other hand, the ET/ETR for “Milyang 23” and “IR-36” tended to increase slightly around the heading stage (Fig. 4). These varieties also tended to show higher T than “Nipponbare” under the LAI above 2.0 and high radiation conditions (Fig. 5). These results suggested that crop factor such as canopy resistance is somewhat important in controlling ET of Indica type rice, whereas not so important for Japonica rice.The variation of the daily value of a in the radiation method (Eq. 7) was relatively small throughout the main growth period (Fig. 6). The linear regression technique for all the cropping seasons yielded a=0.88, and five-day ET was estimated with high correlation coefficient (r=0.912).The daily value of α in Priestley-Taylor method (Eq. 8) greatly fluctuated over the growing season (Fig. 6). Apparently, this variation of α was attributed to that of daily net radiation caused by long wave radiation exchange at nighttime. Priestley-Taylor method seemed to be less effective in estimating ET of paddy field in Japan when daily values of net radiation are used.

Dinitrogen Fixation of Rice‐Klebsiella Associations1

We report N2 fixation by Klebsiella oxytoca strain NG13, isolated from the rhizosphere of a rice (Oryza sativa L.) plant, in association with Indica type rice strain, C5444. The C2H2‐reducation assays, biomass measurements, N content determinations, and 15N isotopic dilution assays indicated that inoculation of C5444 with NG13 resulted in a 6% increase of total plant+soil N content, and a 19% 15N isotopic dilution of plant N after 120 days of cultivation. Our results demonstrated significant levels of N2 fixation by K. oxytoca when associated with rice. By optimizing such rice plant‐bacterial N2 fixation capacity, the N uptake by rice could be enhance.

Effect of seed presoaking in acetone containing ABA on plumule elongation of rice seedlings.

When rice plants of japonica type were grown on the agar medium containing ABA in darkness, the elongation of mesocotyl was strikingly simulated but that of coleoptile and seminal root was inhibited. In this experiment, the rice seeds presoaked in acetone containing ABA were used for ascertaining the direct effect of ABA on the elongation of mesocotyl and coleoptile. A japonica type variety "Koshijiwase" and an indica type variety "Peta" were uscd. After presoaking seeds in acetone containing ABA at 25°C, they were dried for two hours in a vacuum desiccator and stored for two days at room temperature. In all the experiments, each lot consisted of 60-80 plants. The results obtained were as follows: 1. In the plants grown from the presoaked seeds in acetone contatining 10-2M or 2×10-3M of ABA, not only the elongation of mesocotyl but also that of coleoptile was sitmulated, reaching the plateau at the soaking of 24 hours. Thus the soaking for about 48 hours may be enough to examine the effect of ABA (Fig. 1). 2. By the 48 hours soaking in ABA, the stimulation of mesocotyl elongation occurred between the concentration of 10-3M to 2×10-2M. The length of mesocotyl in ABA treated plants was from 3 to 16 times longer than that of control treated with pure acetone, while the coleoptile length was from 1.0 to 1.5 times longer than that of the control. Accordingly, the morphology of plumules was almost similar those from the high temperature pre-treated seed and those of indica type rice. On the other hand, elongation of seminal root was somewhat inhibited even at 10-3M of ABA, and the inhibitory effect increased with increasing ABA concentration (Fig. 2). 3. When the seeds presoaked with acetone containing ABA were again soaked in pure acetone, the stimulatory effect of ABA on the elongation of mesocotyl and coleoptile and inhibitory effect on that of seminal root disappeared. When the seeds of high temperature pre-treated japonica rice and those of nontreated indica rice were soaked in pure acetone, whereas, no effect was detected in the elongation of either mesocotyl or coleoptile (Fig. 3). 4. Parenchymatous cell length and number in a longitudinal section of mesocotyl with about 23 mm long in each experimental lot were measured. The plumules grown from the presoaked seeds in acetone containing 2×10-2M of ABA were almost similar to those from the high temperature pre-treated seeds, 40°C for 10 days. On the other hand, the plumules grown on agar medium containing 2×10-6M ABA were almost similar to those from immature seeds, harvested 15 days after anthesis (Table 1). 5. Considering the similarities of plumules between those originated from seeds presoaked with 2×10-2M ABA and those originated from seeds pre-treated with 40°C for 10 days, about 1.6 μg of ABA was estimated to be synthesized in a seedling originated from the latter, if the assumption that the mesocotyl elongation is based only on ABA is adopted (Fig. 4).

Effect of low water potential of the culture medium on mesocotyl elongation of rice seedlings.

At a soil water content of water potential of ca. -3.1 to -6.2 bars, mesocotyl elongation of both indica and japonica type rice was extremely stimulated. It was also stimulated in an aseptic culture medium (2.5-4.0% mannitol, water potential ca. -3.5 to -5.5 bars). The stimulation of the mesocotyl may be caused by cell multiplication.

Environmental Differences in Leaf Stomatal Frequency of Rice

1. Stomatal frequency (stomatal number per unit leaf area, abbreviated Sf) of a flag leaf was high at the middle of the leaf and low at the leaf margin. It was low at the leaf base and the leaf apex (Fig. 1, 2). Sf in a leaf in upper leaf position on a stem was higher than that in lower leaf position. Sf in lower leaf surface was higher than that in upper leaf surface (Fig. 3). 2. Sf of upland rice cultivars was lower than that of lowland rice cultivars (Fig. 3). Sf of Indica type rice was higher than that of Japonica rice (Fig. 4, 6). Sf of leaves grown in different years (Fig. 4), in different transplanting dates (Table 4) and in low-land and upland field (Table 3) correlated highly in positive way. Sf of L2 and Lf (Table 2) and Sf in lower and upper leaf surface (Table 3) correlated highly also in positive way. 3. Fertilizer application at lag stage caused an increase of Sf leaf size and total stomates of a flag leaf (Table 1, Fig. 5). Sf decreased with a shortening of the growing period (Table 5, 7). In comparison among cultivars of lowland rice, Sf of cultivars having a large leaf was high (Table 5). But in comparison among cultivars containing upland rice, Sf of cultivars having a large leaf was low (Table 7). Values of genetic correlation between Sf and leaf size (Table 6) explained this tendency quite well. Values of environmental correlation between them were low (Table 6). Sf of lowland rice grown in lowland field was higher than that in upland field. In case of upland rice, however, difference between Sf grown in lowland and upland field was not so clear (Fig. 3).

Ripening of Japonica and Indica Type Rice as Influenced by Temperature During Ripening Period

Ripening of Japonica and Indica Type Rice as Influenced by Temperature During Ripening Period

Flavonoid Pigments in Rice Leaves and the Isolation of Glucotricin

The distribution of flavonoid pigments in the leaves of indica and japonica type rice plants was studied by paper chromatographic and electrophoretic techniques. A fluorescent flavoniod compound prominent in the leaves of indica was isolated in a crystalline form and identified as glucotricin (tricin-7-β-glucoside), the structure of which was characterized by Kuwatsuka in 1962.

Flavonoid Pigments in Rice Leaves and the Isolation of Glucotricin

The distribution of flavonoid pigments in the leaves of indica and japonica type rice plants was studied by paper chromatographic and electrophoretic techniques. A fluorescent flavoniod compound prominent in the leaves of indica was isolated in a crystalline form and identified as glucotricin (tricin-7-β-glucoside), the structure of which was characterized by Kuwatsuka in 1962.