Investigating Pollen and Gene Flow of WYMV-Resistant Transgenic Wheat N12-1 Using a Dwarf Male-Sterile Line as the Pollen Receptor

Pollen-mediated gene flow (PMGF) is the main mode of transgene flow in flowering plants. The study of pollen and gene flow of transgenic wheat can help to establish the corresponding strategy for preventing transgene escape and contamination between compatible genotypes in wheat. To investigate the pollen dispersal and gene flow frequency in various directions and distances around the pollen source and detect the association between frequency of transgene flow and pollen density from transgenic wheat, a concentric circle design was adopted to conduct a field experiment using transgenic wheat with resistance to wheat yellow mosaic virus (WYMV) as the pollen donor and dwarf male-sterile wheat as the pollen receptor. The results showed that the pollen and gene flow of transgenic wheat varied significantly among the different compass sectors. A higher pollen density and gene flow frequency was observed in the downwind SW and W sectors, with average frequencies of transgene flow of 26.37 and 23.69% respectively. The pollen and gene flow of transgenic wheat declined dramatically with increasing distance from its source. Most of the pollen grains concentrated within 5 m and only a few pollen grains were detected beyond 30 m. The percentage of transgene flow was the highest where adjacent to the pollen source, with an average of 48.24% for all eight compass directions at 0 m distance. Transgene flow was reduced to 50% and 95% between 1.61 to 3.15 m, and 10.71 to 20.93 m, respectively. Our results suggest that climate conditions, especially wind direction, may significantly affect pollen dispersal and gene flow of wheat. The isolation-by-distance model is one of the most effective methods for achieving stringent transgene confinement in wheat. The frequency of transgene flow is directly correlated with the relative density of GM pollen grains in air currents, and pollen competition may be a major factor influencing transgene flow.     

Modelling pollen‐mediated gene flow in rice: risk assessment and management of transgene escape

Fast development and commercialization of genetically modified plants have aroused concerns of transgene escape and its environmental consequences. A model that can effectively predict pollen‐mediated gene flow (PMGF) is essential for assessing and managing risks from transgene escape. A pollen‐trap method was used to measure the wind‐borne pollen dispersal in cultivated rice and common wild rice, and effects of relative humidity, temperature and wind speed on pollen dispersal were estimated. A PMGF model was constructed based on the pollen dispersal pattern in rice, taking outcrossing rates of recipients and cross‐compatibility between rice and its wild relatives into consideration. Published rice gene flow data were used to validate the model. Pollen density decreased in a simple exponential pattern with distances to the rice field. High relative humidity reduced pollen dispersal distances. Model simulation showed an increased PMGF frequency with the increase of pollen source size (the area of a rice field), but this effect levelled off with a large pollen‐source size. Cross‐compatibility is essential when modelling PMGF from rice to its wild relatives. The model fits the data well, including PMGF from rice to its wild relatives. Therefore, it can be used to predict PMGF in rice under diverse conditions (e.g. different outcrossing rates and cross‐compatibilities), facilitating the determination of isolation distances to minimize transgene escape. The PMGF model may be extended to other wind‐pollinated plant species such as wheat and barley.     

Low frequency of transgene flow from Bt/CpTI rice to its non-transgenic counterparts planted at close spacing

Crop-to-crop transgene flow will affect seed purity of non-GM rice varieties, leading to unwanted consequences. To assess the maximum probability of transgene outflow in rice (Oryza sativa), gene flow experiments were conducted with three cultivation patterns with different mixed-planting proportions of adjacent GM and non-GM rice at two sites in Fujian and Hainan Provinces of China. Three GM rice lines containing two insect-resistance genes (Bt/CpTI) and their non-GM counterparts were used in the experiments to allow natural hybridization to occur. A hygromycin resistance gene was used as a selective marker for identifying hybrids. Based on the examination of > 645 700 geminated seeds, the result showed low frequencies (0.05-0.79%) of transgene flow from GM to non-GM rice at close spacing, although with significant variation among mixed-planting proportions. It is concluded that rice transgene flow will occur at a very low frequency (< 1.0%), even if the GM rice is planted at close spacing with non-GM rice, and high densities of GM rice cultivated in the neighborhood of non-GM rice will increase the probability of outcrossing with the non-GM rice.     

A field study of pollen-mediated gene flow from Mediterranean GM rice to conventional rice and the red rice weed

The objective of this study was to assess the frequency of pollen-mediated gene flow from a transgenic rice line, harbouring the gusA and the bar genes encoding respectively, -glucuronidase and phosphinothricin acetyl transferase as markers, to the red rice weed and conventional rice in the Spanish japonica cultivar Senia. A circular field trial design was set up to investigate the influence of the wind on the frequency of pollination of red rice and conventional rice recipient plants with the transgenic pollen. Frequencies of gene flow based on detection of herbicide resistant, GUS positive seedlings among seed progenies of recipient plants averaged over all wind directions were 0.036 ± 0.006% and 0.086 ± 0.007 for red rice and conventional rice, respectively. However, for both red rice and conventional rice, a clear asymmetric distribution was observed with pollination frequency favoured in plants placed under the local prevailing winds. Southern analyses confirmed the hemizygous status and the origin of the transgenes in progenies of surviving, GUS positive plants. Gene flow detected in conventional rice planted at 1, 2, 5 and 10 m distance revealed a clear decrease with increasing distance which was less dramatic under the prevailing wind direction. Consequences of these findings for containment of gene flow from transgenic rice crops to the red rice weed are discussed. The precise determination of the local wind conditions at flowering time and pollination day time appear to be of primary importance for setting up suitable isolation distances.     

Establishment of a rice transgene flow model for predicting maximum distances of gene flow in southern China

We aimed to establish a rice gene flow model based on (i) the Gaussian plume model, (ii) data from a three-location x 3-yr field experiment on transgene flow to common rice cultivars (Oryza sativa), male sterile (ms) lines (O. sativa) and common wild rice (Oryza rufipogon), and (iii) 32-yr historical meteorological data collected from 38 meteorological stations in southern China during the rice flowering period. The concept of the gene flow coefficient (GFC) is proposed; that is, the ratio of the transgene flow frequency (G%) obtained from field experiments to the aggregated pollen dispersal frequency (P%) calculated based on the pollen dispersal model. The maximum distances of gene flow (MDGF) to traditional rice cultivars, ms lines, and common wild rice at a threshold value of either 1.0 or 0.1% were determined. The MDGF and its spatial distribution in southern China show that the gene flow pattern is significantly affected by the monsoon climate, the topography, and the outcrossing ability of recipients. We believe that the information provided in this study will be useful for the risk assessment of transgenic rice in other rice-growing regions.     

Pollen flow of cultivated rice measured under experimental conditions

The pollen flow pattern of a cultivated rice variety, Minghui-63, was studied at horizontal and vertical levels under experimental conditions. Data obtained from pollen traps for six designed populations (as pollen sources) at different intervals showed that the dispersal of rice pollen decreased with the increase of distance from pollen sources and that the rice pollen flow was significantly influenced by weather conditions, particularly by wind direction and speed. For a mean wind speed of 2.52 m/s in a downwind direction, the observed distance of rice pollen dispersal was 38.4 m, indicating that rice pollen grains normally disperse at a relatively small range. However, the maximum distance of rice pollen flow could be up to 110 m, using regression analysis of pollen flow and wind speed, when the wind speed reached 10 m/s in this study. The frequency of pollen flow was positively correlated with pollen source size within a given range, suggesting that pollen flow will occur effectively at a considerable rate in rice fields with sufficiently large pollen sources. In addition, many more pollen grains were detected at the height of 1.0–1.5 m than at 2.0 m, indicating that rice pollen mainly disperses at relatively low heights. Results from this study are useful both for minimizing transgene escape from transgenic rice and in situ conservation of wild relatives of rice, as well as for hybrid seed production, where an effective isolation buffer zone needs to be established.     

Transgene flow to hybrid rice and its male-sterile lines

Gene flow from genetically modified (GM) crops to the same species or wild relatives is a major concern in risk assessment. Transgenic rice with insect and/or disease resistance, herbicide, salt and/or drought tolerance and improved quality has been successfully developed. However, data on rice gene flow from environmental risk assessment studies are currently insufficient for the large-scale commercialization of GM rice. We have provided data on the gene flow frequency at 17 distances between a GM japonica line containing the bar gene as a pollen donor and two indica hybrid rice varieties and four male-sterile (ms) lines. The GM line was planted in a 640m² in an isolated experimental plot (2.4 ha), which simulates actual conditions of rice production with pollen competition. Results showed that: (1) under parallel plantation at the 0-m zone, the transgene flow frequency to the ms lines ranged from 3.145 to 36.116% and was significantly higher than that to hybrid rice cultivars (0.037–0.045%). (2) Gene flow frequency decreased as the distance increased, with a sharp cutoff point at about 1–2 m; (3) The maximum distance of transgene flow was 30–40 m to rice cultivars and 40–150 m to ms lines. We believe that these data will be useful for the risk assessment and management of transgenic rice lines, especially in Asia where 90% of world's rice is produced and hybrid rice varieties are extensively used. Shirong Jia, Feng Wang and Lei Shi contributed equally to this investigation.     

Pollen-mediated transgene flow in the wind-pollinated grass species tall fescue (Festuca arundinacea Schreb.)

Information regarding gene flow in wind-pollinated, outcrossing forage grasses is essential for any future releases of value-added transgenic cultivars. Experiments on pollen dispersal was carried out by growing transgenic tall fescue (Festuca arundinacea) in a central plot, surrounded by exclosures containing recipient plants up to a distance of 200 m from the central source plants in eight directions. The central transgenic tall fescue plants carried a chimeric hygromycin phosphotransferase gene (hph) and a chimeric -glucuronidase gene (gusA). Seeds were collected from the recipient plants and germinated seedlings were used for high throughput DNA isolation and polymerase chain reaction (PCR) analysis. More than 21,000 seedlings were PCR analyzed for the experiments conducted in three years. Transgenes were detected in recipient plants at up to 150 m from the central transgenic plot. The highest transgene frequencies, 5% at 50 m, 4.12% at 100 m and 0.96% at 150 m, were observed north of the central plot, the prevailing wind direction. Lower transgene frequencies were detected in other directions, particularly at 100 m and 150 m distances. No transgene was detected at 200 m distance in any direction. Transgene flow was less effective or ineffective when recipient plants were further away from the central donor plants. Southern blot hybridization analysis confirmed the transgenic nature of the PCR positive plants. A supplementary experiment demonstrated that transgene flow can be controlled by placing transgenic plantings downwind and long distances from non-transgenic seed increases, thus allowing tall fescue breeding and transgene development programs to be conducted concurrently at the same research station.     

Gene flow in genetically modified wheat

Understanding gene flow in genetically modified (GM) crops is critical to answering questions regarding risk-assessment and the coexistence of GM and non-GM crops. In two field experiments, we tested whether rates of cross-pollination differed between GM and non-GM lines of the predominantly self-pollinating wheat Triticum aestivum. In the first experiment, outcrossing was studied within the field by planting "phytometers" of one line into stands of another line. In the second experiment, outcrossing was studied over distances of 0.5-2.5 m from a central patch of pollen donors to adjacent patches of pollen recipients. Cross-pollination and outcrossing was detected when offspring of a pollen recipient without a particular transgene contained this transgene in heterozygous condition. The GM lines had been produced from the varieties Bobwhite or Frisal and contained Pm3b or chitinase/glucanase transgenes, respectively, in homozygous condition. These transgenes increase plant resistance against pathogenic fungi. Although the overall outcrossing rate in the first experiment was only 3.4%, Bobwhite GM lines containing the Pm3b transgene were six times more likely than non-GM control lines to produce outcrossed offspring. There was additional variation in outcrossing rate among the four GM-lines, presumably due to the different transgene insertion events. Among the pollen donors, the Frisal GM line expressing a chitinase transgene caused more outcrossing than the GM line expressing both a chitinase and a glucanase transgene. In the second experiment, outcrossing after cross-pollination declined from 0.7-0.03% over the test distances of 0.5-2.5 m. Our results suggest that pollen-mediated gene flow between GM and non-GM wheat might only be a concern if it occurs within fields, e.g. due to seed contamination. Methodologically our study demonstrates that outcrossing rates between transgenic and other lines within crops can be assessed using a phytometer approach and that gene-flow distances can be efficiently estimated with population-level PCR analyses.     

A large-scale field study of transgene flow from cultivated rice (Oryza sativa) to common wild rice (O. rufipogon) and barnyard grass (Echinochloa crusgalli)

The introgression of transgenes into wild relatives or weeds through pollen-mediated gene flow is a major concern in environmental risk assessment of transgenic crops. A large-scale (1.3–1.8 ha) rice gene flow study was conducted using transgenic rice containing the bar gene as a pollen donor and Oryza rufipogon as a recipient. There was a high frequency of transgene flow (11%−18%) at 0–1 m, with a steep decline with increasing distance to a detection limit of 0.01% by 250 m. To our knowledge, this is the highest frequency and longest distance of gene flow from transgenic rice to O. rufipogon reported so far. On the basis of these data, an adequate isolation distance from both conventional and transgenic rice should be taken for in situ conservation of common wild rice. Meanwhile, there is no evidence of transgene introgression into barnyard grass, even when it has coexisted with transgenic rice containing the bar gene for five successive years. Thus, the environmental risk of gene flow to this weedy species is of little concern.     

The mating system of an hydrophilous angiosperm Posidonia australis (Posidoniaceae)

The hydrophilous seagrass Posidonia australis has a wide range of multilocus outcrossing rates (t), which vary from 0 to 0.89, with "apparent' outcrossing rates varying from 0 to 0.42 among the seven populations sampled. This pattern of outcrossing rate indicates that water pollination (hydrophily) is less uniform than wind pollination and more similar to animal pollination in its variability. Variation in levels of outcrossing between populations may be due to differences in water movement; for example, open bays have greater pollen dispersal and higher outcrossing rates. Considerable pollen movement within meadows was inferred from a high frequency of nonmaternal alleles in the pollen pool. The distribution of genetic diversity among populations (GST = 0.229) suggests moderate gene flow on the local scale. These results demonstrate that successful submarine cross-pollination occurs in the hydrophile P. australis, which has a diverse mating system with populations that range from predominantly inbred to predominantly outcrossed.     

Scale effect on rice pollen‐mediated gene flow: implications in assessing transgene flow from genetically engineered plants

Transgene flow from genetically engineered (GE) crops to non‐GE varieties and wild relatives via pollen‐mediated gene flow (PMGF) may create food and environmental biosafety concerns. Assessing the level of PMGF from GE crops is required before commercialization. Whether the level of PMGF estimated at relatively small scales can sufficiently represent the actual scenario at large production scales remains unresolved. Here, we estimated average PMGF frequencies from three insect‐resistant GE rice lines to their non‐GE counterparts at four scales ranging from 9 to 576 m², having the number of GE to non‐GE plants constantly at the ratio of 8:1. Based on nearly 1.3 million examined seedlings from non‐GE rice plots, very low frequencies (<0.1%) of transgene flow were detected. The highest frequencies were found in plots at the smallest scales. Scale had a significantly negative effect on the frequency of PMGF in rice, with decreased gene flow at increased scale. An extended PMGF model could well represent the experimental data. Field experiments at relatively small scales combined with mathematical modelling could provide useful prediction on the level of rice transgene flow at large production scales. This is probably applicable for other crop species with wind‐ and self‐pollination.     

家蝇介导的水稻基因逃逸风险评估(英文)

【目的】对转基因作物进行生态风险评估是大面积种植前的一个必要步骤,水稻Oryza sativa访花昆虫有上百种,包括家蝇Musca domestica。本研究旨在明确访花昆虫家蝇介导转基因水稻外源基因逃逸的风险。【方法】2010年,我们使用转基因水稻B1, B6和G8-7作为父本(花粉供体),用同源非转基因水稻Jiazao 935和Wuyunjing 7作为母本（花粉受体）,并用家蝇作为授粉昆虫,在浙江大学华家池和长兴试验基地开展了田间种植试验,对收割的后代水稻种子进行室内种植培养,对种苗用潮霉素B和草甘膦处理进行转基因杂交种检测,对存活植株进行潮霉素和草甘膦抗性基因PCR检测,测试家蝇介导的转基因水稻外源基因逃逸频率。【结果】对浙江两个测试基地3个转基因水稻品种共计超过216 500粒后代水稻种子进行的检测及结果表明,在毗邻区域杂交种少,家蝇授粉区和无家蝇授粉区转基因水稻外源基因向非转基因水稻逃逸频率均较低(0~0.64%)。【结论】家蝇介导的转基因水稻外源基因逃逸频率较低,家蝇没有增加转基因水稻外源基因逃逸的风险。     

Defective pollen wall is required for anther and microspore development in rice and encodes a fatty acyl carrier protein reductase

Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.     

Gene flow in maize fields with different local pollen densities

The development of maize (Zea mays L.) varieties as factories of pharmaceutical and industrial compounds has renewed interest in controlling pollen dispersal. The objective of this study was to compare gene flow into maize fields of different local pollen densities under the same environmental conditions. Two fields of approximately 36 ha were planted with a nontransgenic, white hybrid, in Ankeny, Iowa, USA. In the center of both fields, a 1-ha plot of a yellow-seeded stacked RR/Bt transgenic hybrid was planted as a pollen source. Before flowering, the white receiver maize of one field was detasseled in a 4:1 ratio to reduce the local pollen density (RPD). The percentage of outcross in the field with RPD was 42.2%, 6.3%, and 1.3% at 1, 10, and 35 m from the central plot, respectively. The percentage of outcross in the white maize with normal pollen density (NPD) was 30.1%, 2.7%, and 0.4%, respectively, at these distances. At distances greater than 100 m, the outcross frequency decreased below 0.1 and 0.03% in the field with RPD and NPD, respectively. A statistical model was used to compare pollen dispersal based on observed outcross percentages. The likelihood ratio test confirmed that the models of outcrossing in the two fields were significantly different (P is practically 0). Results indicated that when local pollen is low, the incoming pollen has a competitive advantage and the level of outcross is significantly greater than when the local pollen is abundant. This journal paper of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa, Project No. 3638, was supported by Hatch Act, State of Iowa fund and by a USDA-CREES BRA Grant.     

Historical and contemporary gene dispersal in wild carrot (Daucus carota ssp. carota) populations

Background and Aims

Wild carrot is the ancestor of cultivated carrot and is the most important gene pool for carrot breeding. Transgenic carrot may be released into the environment in the future. The aim of the present study was to determine how far a gene can disperse in wild carrot populations, facilitating risk assessment and management of transgene introgression from cultivated to wild carrots and helping to design sampling strategies for germplasm collections.

Methods

Wild carrots were sampled from Meijendel and Alkmaar in The Netherlands and genotyped with 12 microsatellite markers. Spatial autocorrelation analyses were used to detect spatial genetic structures (SGSs). Historical gene dispersal estimates were based on an isolation by distance model. Mating system and contemporary pollen dispersal were estimated using 437 offspring of 20 mothers with different spatial distances and a correlated paternity analysis in the Meijendel population.

Key Results

Significant SGSs are found in both populations and they are not significantly different from each other. Combined SGS analysis indicated significant positive genetic correlations up to 27 m. Historical gene dispersal σ_g and neighbourhood size N_b were estimated to be 4–12 m [95 % confidence interval (CI): 3–25] and 42–73 plants (95 % CI: 28–322) in Meijendel and 10–31 m (95 % CI: 7–∞) and 57–198 plants (95 % CI: 28–∞) in Alkmaar with longer gene dispersal in lower density populations. Contemporary pollen dispersal follows a fat-tailed exponential-power distribution, implying pollen of wild carrots could be dispersed by insects over long distance. The estimated outcrossing rate was 96 %.

Conclusions

SGSs in wild carrots may be the result of high outcrossing, restricted seed dispersal and long-distance pollen dispersal. High outcrossing and long-distance pollen dispersal suggest high frequency of transgene flow might occur from cultivated to wild carrots and that they could easily spread within and between populations.     

Transgenic GFP as a molecular marker for approaches to quantify pollination mechanism and gene flow in Arabidopsis thaliana

Arabidopsis thaliana is commonly regarded as a self-pollinated plant. We observed that the stigma in each flower of A. thaliana cannot be pollinated by its own pollen in the early phases of the flowering process, when the anthers had dehisced but the filaments were still too short for the pollen to be deposited on the stigma. In the later stages, after elongation of the filaments, self-pollination can occur. After artificial pollination of the flower of a wild plant with GFP transgenic pollen grains in earlier stages of flowering, GFP expressed within epidermal cells was detected in some of the offspring (26.1-57.1 %). Wind-mediated pollen dispersal was poor but is likely to exist in natural habitats, while insects were observed visiting flowers of A. thaliana in natural and experimental populations. We constructed an experimental population consisting of 28 GFP transgenic plants and 240 wild plants and examined gene flow in the population. The result was that the distance of gene flow was limited to 0.5 m. 22 offspring with expressed GFP were found in 28,299 filial individuals examined, which suggested a relatively low outcrossing rate (0.74%). We conclude that outcrossing in populations of A. thaliana is mainly due to insect pollination. The data on gene flow could be useful to assess the ecological hazards of experimental transgene combinations.     

Wind pollination and reproductive assurance in Linanthus parviflorus (Polemoniaceae), a self-incompatible annual

Wind pollination was experimentally demonstrated in Linanthus parviflorus (Polemoniaceae), a predominantly beefly-pollinated, self-incompatible annual. Seed set in plants enclosed in mesh tents that excluded pollinators but allowed airborne pollen flow provided evidence for wind pollination, and the extent of seed set due to wind pollination was compared to that in open-pollinated controls and pollen-supplemented treatments. Additional controls were included to test for possible confounding effects of the mesh tent. Mean seed number in open-pollinated plants was 72.8–81.1% of that in pollen-supplemented plants, while wind pollination alone produced 49.5–52.2%, a smaller but substantial proportion of seed set with pollen supplementation. Further evidence for wind pollination was found in a comparison of sites differing in the extent of wind exposure in two populations of L. parviflorus. Airborne pollen counts were higher in exposed sites than in protected sites, and the difference was marginally significant. Seed set was significantly pollen limited in protected sites, but not in exposed sites. Taken together, the data suggest that wind pollination provides some reproductive assurance in this obligately outcrossing species. Wind pollination is hypothesized to represent an alternative to selfing as an evolutionary solution to the problem of temporal or spatial variation in pollination visitation.     

Rice transgene flow: its patterns,model and risk management

Progress has been made in a 12 year's systemic study on the rice transgene flow including (i) with experiments conducted at multiple locations and years using up to 21 pollen recipients, we have elucidated the patterns of transgene flow to different types of rice. The frequency to male sterile lines is 10¹ and 10³ higher than that to O. rufipogon and rice cultivars. Wind speed and direction are the key meteorological factors affecting rice transgene flow. (ii) A regional applicable rice gene flow model is established and used to predict the maximum threshold distances (MTDs) of gene flow during 30 years in 993 major rice producing counties of southern China. The MTD_0.1% for rice cultivars is basically ≤5 m in the whole region, despite climate differs significantly at diverse locations and years. This figure is particularly valuable for the commercialization and regulation of transgenic rice. (iii) The long‐term fate of transgene integrated into common wild rice was investigated. Results demonstrated that the F₁ hybrids of transgenic rice/O. rufipogon gradually disappeared within 3–5 years, and the Bt or bar gene was not detectable in the mixed population, suggesting the O. rufipogon may possess a strong mechanism of exclusiveness for self‐protection. (iv) The flowering time isolation and a 2‐m‐high cloth‐screen protection were proved to be effective in reducing transgene flow. We have proposed to use a principle of classification and threshold management for different types of rice.