Spikelet Positions Research Articles

Genotypic variation in spike fertility traits and ovary size as determinants of floret and grain survival rate in wheat.

Spike fertility traits are critical attributes for grain yield in wheat (Triticum aestivum L.). Here, we examine the genotypic variation in three important traits: maximum number of floret primordia, number of fertile florets, and number of grains. We determine their relationship in determining spike fertility in 30 genotypes grown under two contrasting conditions: field and greenhouse. The maximum number of floret primordia per spikelet (MFS), fertile florets per spikelet (FFS), and number of grains per spikelet (GS) not only exhibited large genotypic variation in both growth conditions and across all spikelet positions studied, but also displayed moderate levels of heritability. FFS was closely associated with floret survival and only weakly related to MFS. We also found that the post-anthesis process of grain set/abortion was important in determining genotypic variation in GS; an increase in GS was mainly associated with improved grain survival. Ovary size at anthesis was associated with both floret survival (pre-anthesis) and grain survival (post-anthesis), and was thus believed to 'connect' the two traits. In this work, proximal florets (i.e. the first three florets from the base of a spikelet: F1, F2, and F3) produced fertile florets and set grains in most cases. The ovary size of more distal florets (F4 and beyond) seemed to act as a decisive factor for grain setting and effectively reflected pre-anthesis floret development. In both growth conditions, GS positively correlated with ovary size of florets in the distal position (F4), suggesting that assimilates allocated to distal florets may play a critical role in regulating grain set.

Wheat Grain Yield Increase in Response to Pre-Anthesis Foliar Application of 6-Benzylaminopurine Is Dependent on Floret Development.

Wheat yield is largely determined during the period prior to flowering, when the final numbers of fertile florets and grains per spike are established. The aim of this study was to assess the dynamics of floret primordia development in winter wheat in response to pre-anthesis application of a synthetic cytokinin, 6-benzylaminopurine (6-BA). We conducted an experiment in which two foliar spray treatments were applied (water or 6-BA) to Chinese winter wheat at 25 days after jointing during two growing seasons (2012–2013 and 2013–2014). Both the final grain number per spike and grain yield at maturity exhibited remarkable increases in response to the 6-BA treatment. Application of 6-BA increased the number of fertile florets in basal spikelets and, to a greater extent, in central spikelets. The mechanism by which 6-BA application affected the final number of fertile florets primarily involved suppression of the floret abortion rates. Application of 6-BA considerably reduced the abortion rates of basal, central and apical spikelet florets (by as much as 77% compared with the control), as well as the degeneration rates of basal and central spikelet florets, albeit to a lesser degree. The effect of 6-BA application on the likelihood of proximal florets being set was limited to the distal florets in the whole spike, whereas obvious increases in the likelihood of grain set under 6-BA treatment were observed in distal florets, primarily in central spikelet positions. The results of this study provide important evidence that 6-BA application to florets (final fertile floret production) results in an increased grain yield.

Near‐Infrared Spectroscopic Evaluation of Single‐Kernel Deoxynivalenol Accumulation and Fusarium Head Blight Resistance Components in Wheat

Fusarium head blight (FHB) symptoms, single‐kernel deoxynivalenol (DON) levels, and distribution of DON levels among kernels in response to artificial FHB inoculation were investigated in three selected wheat cultivars that had different reported levels of FHB resistance. DON levels were estimated with near‐infrared spectroscopy. The percentages of DON‐containing spikelets per spike of 15.2, 49.7, and 89.1% were significantly different among point‐inoculated spikes of Everest, Karl 92, and Overley, respectively. The percentage of visually Fusarium‐damaged kernels in point‐inoculated Karl 92 and Overley spikes was significantly higher than for Everest. However, the DON‐containing spikelets per spike and visually Fusarium‐damaged kernels values for spray‐inoculated spikes were not significantly different among the three cultivars. In spray‐inoculated spikes, DON levels in kernels ranged from 0 to 291.3 ppm, whereas the variation of DON levels in spikelet positions was random. In contrast, DON levels in spikelets below the inoculated spikelet in point‐inoculated spikes showed marked differences among the three cultivars. Overley had the highest DON accumulation in kernels. This near‐infrared spectroscopy method may be used as a novel way to evaluate wheat cultivars for FHB resistance to toxin accumulation. Other resistance components such as resistance to pathogen infection and resistance to pathogen spread may also be evaluated.

Grain growth of different rice cultivars under elevated CO2 concentrations affects yield and quality

Elevated CO2 concentrations (E-[CO2]) will likely increase grain yield but decrease grain protein content and increase grain chalkiness, thus degrading the grain quality of rice. Grain growth is the key process that determines both grain yield and quality, hence its responses to E-[CO2] were studied in a free-air CO2 enrichment (FACE) with the focus on variability among the spikelet positions within a panicle. We investigated the changes in grain growth in superior spikelets (SS) and inferior spikelets (IS) for three cultivars of rice differing in grain size and grain number: Akita 63, Koshihikari and Takanari. E-[CO2] increased the grain yield by 15% across 2 years in the three cultivars. The grain growth responses were dependent on spikelet position: the grain mass was increased only in IS, while grain N content was reduced in SS but slightly increased in IS. The increase in IS grain mass was greatest in Takanari followed by Koshihikari with Akita 63 showing a slight decrease. E-[CO2] significantly changed the occurrence of grain chalkiness, and the extent of the increased occurrence of basal white and white back kernels was greatest in Koshihikari followed by Akita 63 with Takanari being least affected. The varietal difference in the responses to E-[CO2] should be studied further with respect to the variability of grain growth and distribution of carbon and nitrogen within a panicle for taking better advantages of the increasing [CO2] for rice grain yield and quality in the future.

Effects of Nitrogen Application on Spikelet Position in High-Yielding Rice Cultivar and Lines Bred for Iwate Prefecture

Effects of Nitrogen Application on Spikelet Position in High-Yielding Rice Cultivar and Lines Bred for Iwate Prefecture

Dynamics of floret development determining differences in spike fertility in an elite population of wheat

Further increases in wheat yield potential could be achieved through a better understanding of the dynamics of floret primordia generation/degeneration, a process which has received little attention. We quantified genotypic variation among elite genotypes of the CIMCOG panel assembled by CIMMYT for its usefulness for wheat breeding. Ten genotypes, representing the range of variation for yield and its components of the whole panel, were grown under high-yielding conditions in NW Mexico for two growing seasons. The stage of development of floret primordia was determined 2–3 times weekly during stem elongation for apical, central and basal spikelets within the spike. The dynamics of floret initiation/death, and the resulting number of fertile florets, were determined for each spikelet position. We found that the variation in number of fertile florets within this elite germplasm was much more related to the survival of floret primordia than to the maximum number of florets initiated. As the two floret primordia most proximal to the rachis were almost always fertile and most distal florets (florets 6–8) were never fertile, the differences in number of fertile florets were clearly attributed to the differential developmental patterns of intermediate florets (floret primordia 3, 4 and 5, counted from the rachis, depending on the spikelet position). We found significant differences among elite germplasm in dynamics of floret development. Differences in floret survival seemed positively related to those in the length of the period of floret development: the longer the duration of floret development the higher the likelihood of that floret becoming fertile. It is proposed that this type of study may be instrumental for identifying prospective parents for further raising yield potential wheat breeding programmes.

Differences of starch granule distribution in grains from different spikelet positions in winter wheat.

Wheat starch development is a complex process and is markedly difference by changes in spikelet spatial position. The present study deals with endosperm starch granule distribution and spatial position during filling development. The study was conducted with pure starch isolated from wheat (Triticum aestivum L.), Jimai20 and Shannong1391, at 7–35 days after anthesis (DAA). The results showed that grain number, spikelet weight and grain weight per spikelet in different spatial position showed parabolic changes. Upper spikelets had highest starch and amylose content followed by basal spikelets, then middle spikelets. The paper also suggested the volume percents of B-type and A-type granule in grain of middle spikelets were remarkably higher and lower than those of basal and upper spikelets, respectively. However, no significant difference occurred in the number percents of the two type granule. The ratio of amylase to amylopectin was positively correlated with the volume proportion of 22.8–42.8 µm, but was negatively related to the volume proportion of <9.9 µm. The results indicated that the formation and distribution of starch granules were affected significantly by spikelet position, and grains at upper and basal spikelet had the potential of increasing grain weight through increasing the volume of B-type granules.

Floret development and grain setting characteristics in winter wheat in response to pre-anthesis applications of 6-benzylaminopurine and boron

The number of grains per unit land area is the most important component to determine grain yield in wheat. Survival of floret primordia seems to be critical for the determination of grain number, which is related to the number of fertile florets at anthesis and the growth of the florets have been frequently considered the determinant key to establish grain number. The objective of this study was to explore the dynamics pattern of floret generation/degeneration/abortion and the grain setting characteristics in winter wheat under contrasting conditions of foliar spraying. Field experiments were carried out during the 2011–2012 and 2012–2013 growing seasons at the Experiment Station of Henan Agricultural University. The experiment involved the wheat cultivar Yumai 49-198 and three foliar applications; water, 6-benzylaminopurine (6-BA), and boron, which were applied 25 days after the jointing stage. This study analysed in detail floret development in wheat as affected by resource availability (mainly foliar spraying), and found that the degeneration rates of the basal spikelet florets decreased by 24% and 6%, respectively, following application of 6-BA and boron. Similarly, the floret abortion rates were reduced by 73% (6-BA) and 22% (boron) when compared to the water control. The degeneration rates of florets from central spikelets decreased by 15% and 8%, after treatment with 6-BA and boron, respectively, while the abortion rates were reduced by 76% and 44%, respectively, when compared to the control. Spraying 6-BA significantly enhances the process by which florets became grains at different floret and spikelet positions, and especially promoted the grain setting of distal florets, respect to the rachis, into the spikelets (i.e., Florets 3 and 4).

山口で栽培したコムギの九州育成品種ダイチノミノリと 北海道育成品種ハルユタカの粒ならびに これらの雑種第1代に着生したF&lt;sub&gt;2&lt;/sub&gt;粒の着生小穂・小花位置別の一粒重の違い

山口で栽培したコムギの九州育成品種ダイチノミノリと 北海道育成品種ハルユタカの粒ならびに これらの雑種第1代に着生したF&lt;sub&gt;2&lt;/sub&gt;粒の着生小穂・小花位置別の一粒重の違い

Effect of spikelet position on starch proportion, granule distribution and related enzymes activity in wheat grain

The starch proportion, starch granule distribution, and activities of enzymes involved in starch synthesis in different spikelet positions were examined during grain development in two high-yielding winter wheat cultivars. The results showed that grain number and weight per spikelet in different spatial position showed a single-peak curve from the base to the top in a wheat spike. Upper spikelets had the highest starch and amylose proportion followed by basal spikelets, whereas middle spikelets had the lowest. Starch and amylose absolute content was in opposition to their proportion. The volume of B- and A-type granule in grain of middle spikelets was remarkably higher and lower than those of basal and upper spikelets, respectively. However, no significant difference occurred in the number of A- and B-type granule in grains among different spikelet position. Compared with the basal and upper spikelets, the middle spikelets showed higher sucrose and ATP content and activities of starch biosynthetic enzymes, and subsequently higher starch absolute content. The results suggested that superior sucrose providing and degradation capacity and the high activities of enzymes involved in starch synthesis resulted in development of B-type starch granule in grain of middle spikelets.

Differences of Starch Granules in Grains from Different Spikelet Positions and Their Correlation with Grain Weight in Winter Wheat

The purpose of this study was to reveal the differences in development and size-distribution of starch granules in endosperm of wheat grains in different grain positions and spikelet positions. The endosperm cell size, and the number, volume and surface area distribution of starch granules in mature caryopsis, as well as the changes of starch granule number during endosperm development, were studied using wheat cultivar Jimai 20 grown in the field. The results showed that the development of starch granule was closely related with grain position and spikelet position. In the same spikelet position, starch grains formed 4–5 d earlier in superior grains (the first and the second grains) than in inferior grains (the third and the fourth grains).At the same grain position, starch granules developed first in the grains of the middle spikelets, and then in grains of upper and lower spikelets. The amount of starch granules was the highest in the middle spikelet grains all the time. The lower spikelet grains had the least starch granules at the beginning of grain filling, however, the number of starch granules increased gradually and surpassed that of the upper spikelet grains as caryopsis matured. Correlation coefficients between grain weight and starch granule amount became more significant with grain filling, which reached 0.88 ** (large starch granule) and 0.78 ** (small starch granule) at mature stage. Correlation coefficients between grain weight and large starch granule amount were higher than 0.96 ** , and had no difference among different spikelet positions. Correlation coefficients between grain weight and small starch granule amount were higher than 0.93 ** , and had a trend of upperlowermiddle in different spikelet positions. The trend of size distribution was BL-typeBS-typeA-type. There were more BS-type granules in superior grains than in inferior grains. The number of BS-type granules increased with the spikelet position rising, whereas the number of BL-type granules showed a reverse trend. The size distribution of starch granules had similar changes to volume and surface area. The formation and size distribution of starch granules were affectedsignificantly by grain position and spikelet position, and inferior grains had the potential of increasing grain weight through increasing the quantity of starch granules.

Floret development and grain setting differences between modern durum wheats under contrasting nitrogen availability.

Wheat yield depends on the number of grains per square metre, which in turn is related to the number of fertile florets at anthesis. The dynamics of floret generation/degeneration were studied in contrasting conditions of nitrogen (N) and water availability of modern, well-adapted, durum wheats in order to understand further the bases for grain number determination. Experiments were carried out during the 2008–2009 and 2009–2010 growing seasons at Lleida (NE Spain). The first experiment involved four cultivars (Claudio, Donduro, Simeto, and Vitron) and two contrasting N availabilities (50 kgN ha–1 and 250 kgN ha–1; N50 and N250) while experiment 2 included the two cultivars most contrasting in grain setting responsiveness to N in experiment 1, and two levels of N (N50 and N250), under irrigated (IR) and rainfed (RF) conditions. In addition, a detillering treatment was imposed on both cultivars under the IR+N250 condition. The number of fertile florets at anthesis was increased by ~30% in response to N fertilization (averaging across treatments and spikelet positions). The effect of N and water availability was evident on floret developmental rates from the third floret primordium onwards, as these florets in the central spikelets of all genotypes reached the stage of a fertile floret in N250 while in N50 they did not. In this study, clear differences were found between the cultivars in their responsiveness to N by producing more fertile florets at anthesis (through accelerating developmental rates of floret primordia), by increasing the likelihood of particular grains to be set, or by both traits.

Effects of Nitrogen Fertilization on Seed-setting Characteristics of Spike and Grain Yield in Winter Wheat under Waterlogging at Booting Stage

Waterlogging has great influence on yield reduction in winter wheat production in some areas of China. Nitrogen (N) application is believed to be able to improve dry matter translocation in wheat, as a result to increase final yield. In a field experiment across two growing seasons, we used winter wheat cultivar "Wanmai 54" to investigate the effect of waterlogging at booting stage and N fertilizing strategy on number and weight of grain at different spikelet positions and grain positions. Under both waterlogging and control (normal watering) conditions, we implemented four N application treatments with different N application ratios (total N amount of 240 kg ha 1 in all treatments) at land preparation, jointing, and booting stages (10:0:0 for N1, 7:3:0 for N2, 5:5:0 for N3, and 3:5:2 for N4). The results showed that seed-setting characteristics of main stem were superior to tiller spike. In the N1 treatment, the sterile spikelet per spike was significantly increased, and the grain number per spike was significantly decreased. Compared with N4 treatment, sterile spikelet per spike of N1 treatment increased by 25.5% and 29.8% in 2008-2009 and 2009-2010 growing seasons, respectively. The grain number per spike of N1 treatment decreased by 5.7%. Waterlogging had greater effect on the tiller spike than the main stem spike. Waterlogging at booting stage significantly increased sterile spikelet per spike by 10.6% and 4.5%, and the grain number per spike decreased by 2.8% and 1.4% in the two growing seasons, especially for spike with four grains on main stem and three grains per spike of tiller spike, the grain weight in the third and the fourth grain positions, the contribution rate of the third and the fourth grain weights to the grain weight per spike. With the delay of N fertilization, spikelet per spike, single weight per spike increased significantly, the sterile spikelet per spike decreased compared to the prior N fertilization treatments. Postponed N fertilization could compensate the decrease of spike with three and four grains and increase the grain weight in third and the fourth grain positions under waterlogged environment at booting stage, increase the contribution rate of the third and the fourth grains weight to the grain weight per spike. Therefore, with the postponed N fertilization, grain yield and spikelet grain number and grain weight at different spikelet of wheat were enhanced. Results indicate that postponed N fertilization alleviates the effect of waterlogging at booting stage on the seed-setting characterisitics of spike and single grain weight.

Sporisorium reilianumInfection Changes Inflorescence and Branching Architectures of Maize

Sporisorium reilianum is a biotrophic maize (Zea mays) pathogen of increasing economic importance. Symptoms become obvious at flowering time, when the fungus causes spore formation and phyllody in the inflorescences. To understand how S. reilianum changes the inflorescence and floral developmental program of its host plant, we investigated the induced morphological and transcriptional alterations. S. reilianum infection promoted the outgrowth of subapical ears, suggesting that fungal presence suppressed apical dominance. Female inflorescences showed two distinct morphologies, here termed "leafy ear" and "eary ear." In leafy ears, all floral organs were replaced by vegetative organs. In eary ears, modified carpels enclosed a new female inflorescence harboring additional female inflorescences at every spikelet position. Similar changes in meristem fate and organ identity were observed in the tassel of infected plants, which formed male inflorescences at spikelet positions. Thus, S. reilianum triggered a loss of organ and meristem identity and a loss of meristem determinacy in male and female inflorescences and flowers. Microarray analysis showed that these developmental changes were accompanied by transcriptional regulation of genes proposed to regulate floral organ and meristem identity as well as meristem determinacy in maize. S. reilianum colonization also led to a 30% increase in the total auxin content of the inflorescence as well as a dramatic accumulation of reactive oxygen species. We propose a model describing the architectural changes of infected inflorescence as a consequence of transcriptional, hormonal, and redox modulation, which will be the basis for further molecular investigation of the underlying mechanism of S. reilianum-induced alteration of floral development.

Quality Response of Grains in Different Spikelet Positions to Temperature Stress During Grain Filling of Rice

High temperature after anthesis may degrade the grain quality of rice ( Oryza sativa L.). To understand the effect of stress duration (initial and mid grain filling stages) and the influence to grain quality at different spikelet positions, an indica cultivar and a japonica cultivar, Yangdao 6 and Yangjing 9538, were used in a pot experiment under 35 °C during the initial (8–18 d after anthesis) and mid (18–28 d after anthesis) terms of grain filling. The treatment under normal atmospheric temperature (25–27 °C) served as the control. The high-temperature treatment at initial filling stage had larger influence on grain quality than the stress imposed at mid filling stage, and the grain quality varied with cultivars and spikelet positions. Both treatments with high temperature reduced milled rice rate and head milled rice rate, but increased crude protein content; grains on the primary branches received larger effect than those on the secondary branches. In the same branch, later-flowered grains were affected greater than early-flowered grains. When high temperature was imposed at initial grain filling stage, the chalkiness degree and chalky grain percentage were significantly increased in the early-flowered grains, whereas the amylose content was significantly decreased; such variations were larger in grains on the primary branches than those on the secondary branches. Besides, the 2 cultivars showed different responses of grain quality to high-temperature stress, which indicated the fact of phenotypic difference on temperature sensitivity. Generally, the influence of temperature on rice quality was greater when the stress imposed at the earlier stage of grain filling.

Effects of Night Temperature, Spikelet Position and Salicylic Acid on Yield and Yield-Related Parameters of Rice (Oryza sativa L.) Plants

Periodic episodes of heat stress and seasonally high night temperatures (HNT) are predicted to occur more frequently in the current changing weather environment. These events affect aspects of crop growth and development, including oxidative-stress damage, reducing crop yield and quality. Salicylic acid (SA), a naturally occurring phenolic compound, associated with thermo-tolerance, prevents oxidative damage by detoxifying superoxide radicals and altering antioxidant capacity in plants. A study was conducted to determine the effects of HNT and SA on multiple rice yield-determining parameters with special emphasis on effects of HNT and SA at different spikelet positions in the panicle. Plants were grown under ambient night temperature (27 °C) or HNT (32 °C) in the greenhouse. They were subjected to HNT through use of continuously controlled infrared heaters, starting from 2000 h to 0600 h. The HNT did not affect productive tillers, main-stem panicle length or number of primary branches per panicle; however, reduced yield resulted from significant negative effects on spikelet fertility (SF), grain length and width. The grains located at the base of the panicle showed decreased fertility, length and width. Application of SA increased antioxidant capacity, thereby preventing damage to membranes, hence increasing yield by predominantly affecting SF in rice plants.

Effect of seed position in spikelet on life history of Eremopyrum distans (Poaceae) from the cold desert of north-west China

Most studies on seed position-dependent effects have focused on germination characteristics. Our aim was to determine the effects of seed position in the spikelet on differences in timing of germination and on the ecological life history of the grass Eremopyrum distans in its cold desert habitat. For seeds in three spikelet positions, morphology, mass and dormancy/germination characteristics were determined in the laboratory, and seeds planted in field plots with and without watering were followed to reproduction to investigate seedling emergence and survival, plant size and seed production. After maturation, of the seeds within the spikelet, basal ones (group 1) are the largest and have the highest proportion with physiological dormancy, while distal ones (group 3) are the smallest and have the highest proportion of non-dormant seeds. A higher percentage of seeds after-ripened in groups 2 and 3 than in group 1. Seeds sown in the field in early summer and watered at short, regular intervals germinated primarily in autumn, while those under natural soil moisture conditions germinated only in spring. Both cohorts completed their life cycle in early summer. Seeds in group 1 had lower percentages of seedling emergence and higher percentages of seedling survival than those in groups 2 and 3. Also, plants from group 1 seeds were larger and produced more seeds per plant than those from groups 2 and 3. Seed position-dependent mass was associated with quantitative differences in several life history traits of E. distans. The environmentally enforced (low soil moisture) delay of germination from autumn to spring results in a reduction in fitness via reduction in number of seeds produced per plant.

Effects of high night temperature and spikelet position on yield-related parameters of rice ( Oryza sativa L.) plants

In subtropical rice-growing regions, occurrence of seasonally high night temperatures (HNT) during the critical stages of development reduces rice ( Oryza sativa L.) yield and quality. A study was conducted to determine the effects of night temperatures and spikelet position in the panicle on multiple rice yield-determining parameters, including number of productive tillers, spikelet sterility, and grain length, width and weight. Plants were grown under ambient night temperature (ANT) (27 °C) or HNT (32 °C) in the greenhouse. They were subjected to a HNT through use of continuously controlled infrared heaters, starting from 2000 h until 0600 h. High night temperatures decreased yield (90%) by affecting spikelet sterility (61%), and grain length (2%), width (2%), and weight. High night temperature increased grain nitrogen concentration (44%), which was inversely related to grain weight. The increase in spikelet sterility under HNT was associated with decreased pollen germination (36%) and increased respiration rates (28%) and relative membrane injury (86%), but not with photosynthesis (production). In addition to HNT, position of the spikelet in the panicle also played an important role in determining its weight and nitrogen concentration. The spikelets at the tip of the panicle weigh more than the spikelets at the base of the panicle.

Variation in radiosensitivity and the dose effect relationship in the low dose region

In the present communication a study of the variation in radiosensitivity of different stages of development was undertaken by the aid of waxy mutants. The main purpose of the investigation was to study the dose-effect relationship in the low dose region. In barley it was shown that irradiation during meiosis caused a considerably higher induction of waxy mutants than during subsequent developmental stages. Furthermore, the amount of mutants was shown to be dependent on the height of tiller at irradiation and on the spikelet position within the spike. In maize a pronounced difference in mutation rate of the pollen grains from individual plants fixed on five consecutive days was observed. This constitutes a complication for the determination of the dose-effect relationship. This complication was circumvented by constructing one dose-effect relationship for the maximum mutation rate of each plant, as well as one dose-effect relationship for the average mutation rate of five fixing days. Regardless of the way in which the dose-effect relationship was computed it was shown to be linear. The entire investigation comprised the analysis of more than 40 million pollen grains composing more than 30,000 pollen samples.

THE RELATIVE DURATION OF THE MEIOTIC STAGES IN POLLEN MOTHER CELLS OF BARLEY

HE present communication is an extension of a previous one (EKT BERG and ERIKSSON, 1965), in which a method for calculating the average meiotic stage of a certain spikelet position was presented. The method was based on the assumption that all meiotic stages from pachytene to tetrads were of equal duration which evidently is an oversimplification. Therefore, it was necessary to develop more precise methods for determining the duration of individual stages. This is of particular interest since it has been shown that some of the meiotic stages may be highly radiosensitive compared to other stages (for references cf. ERIKSSON and TAVRIN, 1965).