| Experiments were conducted in the field of Shandong Agricultural University Research Farm (36°10'19"N, 117°9'03"E) and the state key laboratory of crop biology from 2004 to 2007, in Taian, Shandong Province, China. Field experiment method and physiological- biochemical analysis were used. The variety comparative test was carried out in 2004 in order to discuss the relationships among test weight (TW), yield and quality, at the same time, the nitrogen fertilizer experiment was combined. Three different maize types, normal maize, flint maize and pop maize were used to investigate the effect of nitrogen fertilizer on TW of maize. On this basis, ND108 (normal maize, NM) and FY4 (flint maize) were selected and used in 2005. In this year, three different planting densities were set to study the response of planting density to TW. Based on the correlation analysis in 2004, four maize hybrids ND108, ZD958 (NM), FY3 and ZD18 (high starch maize, HSM) were used in 2006. This study was focused on the starch analysis, associated with the effect of sowing date on TW. Supplement experiment was carried out in 2007.In addition, we discussed the relationship between TW and kernel development focusing on starch analysis. The accumulation of starch, the shape, size, arrangement of starch granules and the starch granule size distribution were studied. Finally, we summarized the effect of cultivation measures (nitrogen fertilizer, planting density and sowing date) on TW of maize. The maize results as followed:1 Correlation Analysis on Test Weight with Yield and Quality in Maize1.1 Relationships among TW, yield and quality of maize The correlation analysis indicated that the TW was significantly and positively correlated with kernel weight, kernel specific gravity and yield. The floating percent was negatively correlated with TW (P<0.05). TW values of apical, middle and basal kernels were significantly and negatively correlated with percent water content after harvest. With the percent water content decreasing, TW was increasing. Each decreasing of percent water would resulting in 4.86, 5.33 and 5.50 g/L increasing of TW in apical, middle and basal maize kernels. There was a negative correlation between TW and gross fat contents.TW was correlated positively and significantly with protein (r=0.573, P<0.05) and starch contents (r=0.719, P<0.01) respectively. The pass analysis also indicated that starch content was determinated factor for TW, because of its largest decision coefficient (R(4)2=68.52%).Therefore, it is more important to improve TW with an eye to the increasing of starch contents on account of the less constraint effect of lysine to starch contents.1.2 Sources of variations of test weight in maizeClustering analysis was used in 2004. Twenty-nine summer maize hybrids released for commercial production in Shandong province were divided into three types: high-test weight (H-TW), medium-test weight type (M-TW) and low-test weight type (L-TW). Both sources of variations of genotypes and experiment locations were significant. Kernel type was the first important factor to determine the TW, and the difference of TW between kernel types was significant. The value was ordered as Pop> Flint >Dent. Results of 2006 indicated that seeding date and genotype played important and determinant roles on TW. The mean squares for sowing date-by-hybrid interaction, position factor, and hybrid-by-position interaction were significant different both for TW and kernel size traits. Basal kernels had the highest TW, apical kernels had intermediate, and middle kernels had lowest TW. Finally, a significant sowing date-by-hybrid-by-position interaction was observed for TW even though the meaningfulness of the interaction could be ascribed to hybrid factor, because the mean square was much greater than that of the seeding date and position factor.2. Correlation between Test Weight and Kernel DevelopmentDue to the positively and significantly correlation between test weight and kernel specific gravity, it was reasonable to discuss the changes of specific gravity instead of test weight during grain filling. Fresh and dry specific gravities of maize kernels were studied after pollination till to the maturity. The fresh specific gravity increased regularly and trended to stable in the maturity. Quadratic equation of the simulation curve was y=1.368-0.0232x+0.000696x2-0.000006x3, (F=8.172, P<0.01, R=0.6706). However, the dry specific gravity shoed two-peak curve. Cubic equation of the simulation curve was y=1.368-0.0232x+0.000696x2-0.000006x3(F=8.172, P<0.01, R=0.6706)。100-kernel weight, kernel volume and dry matter accumulation per volume changing curves could be simulated by Logistic equations suitably. The percent water content decreased rapidly with the development of kernels. The⊿volume per kernel showed typical single curve, reached its maximum at about 29 days after pollination, and decreased later. Regression analysis between specific gravity and each index of grain filling indicated that the fast-increasing period of filling was the key period of specific gravity forming. During the period, measures which influenced grain filling greatly would affect specific gravity similarly.3. Correlation between Test Weight and Starch Accumulation3.1 Test weight and starch granule size distributionFour maize hybrids ND108, ZD958 (NM), FY3 and ZD18 (HSM) were used in 2006. The purpose of this study was focused on starch granule size distribution and the relationship between starch granule size and grain quality properties. The starch granule in matured grain was 0.37-31.5μm in diameter. Taking 2μm and 15μm as limit, we divided the starch particles into three types: small starch granule group (SSG, <2μm), middle starch granule group (MSG, 2μm-15μm) and large starch granule group (LSG, >15μm). The distribution showed typical single peak curve in number of starch granule, with the peak value was about 0.829μm. The SSG granule accounted for over 95% of the total starch granule. The distribution showed two-peak curve in starch granule volume, and the peak value occurred at 1.45μm and 16.4μm around. The distribution showed typical three-peak curve in starch granule surface area, and the peak value occurred at 1.2μm, 4.88μm and 14.94μm around.The percent of volume of SSG and MSG were higher than that of LSG in the high starch maize, but in the normal maize, the percent of volume, number and surface area of LSG were higher than those in high starch maize. The percent of number and surface area of SSG and MSG were different in hybrids and years. In addition, the percent of volume of LSG was higher than that of SSG and MSG in the apical and basal maize kernels, but on the contrary in middle maize kernels.Correlation analysis indicated that test weight was positively correlated with the volume of 0.8-2μm, 2-10μm and >20μm starch granules, respectively, but negatively correlated to other size ranges, and all the correlation coefficients were not significant. The starch content was positively correlated with the volume of 0.8-2μm(r=0.777, P<0.05), 2-10μm (r=0.735, P<0.05) and >20μm starch granules, but on the contrary to the protein content. The content of starch and protein had no correlation with volume percentage of starch granules with other size ranges. Therefore, based on the positively and significantly correlation between TW and starch content, we would focus on the 0.8-2μm and 2-10μm starch granules.3.2 Starch accumulation and enzyme activities responsible for starch biosynthesis in developing grainsAt early grain filling stage, there was no difference between high starch maize and normal maize in percent starch content. After about 20 DAP, with the increasing of kernel weight, the content of amylose of high starch maize was higher than normal maize, but not significantly. At maturity, the contents of amylopectin and starch were higher than those of normal maize significantly. High starch maize had higher accumulation rates of amylopection and starch and lower accumulation rate of amylase than those of normal maize. The average diameters of starch granules relative to volume and surface area increased quickly during 30 DAP then increased slowly at last stage of growth. Comparatively, the average diameter of starch granules relative to number decreased rapidly in the early and stabilized at about 14 days after pollination. High starch maize had lower average diameter (12.41μm and 6.81μm) and larger average diameter (1.10μm) of starch granules, which were relative to volume, surface area and number respectively than those of normal maize (12.98μm, 6.40μm and 1.08μm). The volume percent of starch granules of each size range changed differently in the grain filling. The volume of <0.8μm starch granules increased in the early then decreased. The volume of 0.8-2μm and 2-10μm starch granules decreased in the early, and the volume of >10μm starch granules increased in the early, all the changes stabilized in the middle and later period of grain filling. The number and surface area of starch granules of each size range had similar trends. The activities of adenosine diphosphate glucose pyrophosphorylase (ADPGPPase), uridine diphosphate glucose pyrophosphorylase (UDPGPPase), and soluble starch synthase (SSS) of high starch maize were lower than those of normal maize, increased in the middle and later grain filling stage, then kept relative and high activities at last stage of growth. Comparatively, the activity of granule-bound starch synthase (GBSS) of high starch maize was lower than that of normal maize during the whole grain filling stage.3.3 Test weight and the shape, size and arrangement of starch granulesThe shape of starch granules was spherical, elliptical or polyhedral. According to the surface traits, we divided the starch granules into two types: the"uncovered endosperm starch"with smooth surface and the"covered endosperm starch"attached with much matrix protein and matrix granules. The average starch granule diameter observed with Scanning Electron Microscope (SEM) was within the range from 7.891μm to 20.472μm. That is to say, the middle and large starch granule groups were in the majority, and it was lack of the small starch granule group. This result was different from the analysis with starch granule size distribution. It was possibly resulted from the different accuracy of SEM relative to particle distribution analysis. Flint type maize had the highest average starch granule diameter either in the central or in the edge endosperm, normal maize had intermediate, and high starch maize had the lowest average starch granule diameter.Starch granule arrangement had three types: loosely, tightly relatively and tightly. As to different maize hybrids, starch granules of flint type maize in endosperm arranged tightly, the next was high starch maize, and starch granules of normal maize arranged loosely. In addition, as to the different kernel positions with the same hybrid, the apical, middle and basal kernel of high starch maize had the similar arrangement of starch granules. Comparatively, as to normal maize, starch granules arrangement of apical, middle and basal kernels in endosperm had a little difference. Thus, the size of starch granules was not the decisive factor to the difference of test weight, but the shape and structure of starch granules, especially the varies of arrangement were the internal basic reasons.4 The Effect of Cultivation Measures on Test Weight in Maize4.1 The effect of nitrogen fertilizer and planting density on test weight The effect of nitrogen fertilizer on test weight in maize was not significant. Test weight was negatively correlated with planting density to some extent. Test weight decreased with planting density increased. But there was no significant difference among treatments. Thus, we can conclude that the effect of planting density on test weight in maize was also not significant.With the increasing of planting density, the yield per plant and its component factors of ND108 and FY4 all decreased. Only the ear diameter and row number kept relatively stable. Grain filling was affected by planting density, with the grain filling duration decreased. At the early grain filling stage, specific gravity was significantly different form each treatment. With the kernel weight increasing, the differences declined gradually. At the maturity, the specific gravity value was D1>D2>D3, but no significant difference.The ratio of amylase and amylopectin was sensitivest to planting density, the soluble sugar content was intermediate, and planting density had the smallest influence at the total starch content. As planting density increasing, the ratio of amylase and amylopectin and crude protein content decreased, but soluble sugar content increased. Increasing planting density was beneficial to the promotion of globulin content, but the total protein content decreased.4.2 The effect of sowing date on test weightSowing date had more impact in high starch maize (FY3 and ZD18) than on normal maize (ND108 and ZD958). Proper and late sowing was beneficial to the promotion of test weight.Sowing date affected spike differentiation and grain filling of maize directly. With delaying of sowing date, seeding and growth periods were shorter, but different in hybrids. The bare tip length was sensitivest to sowing date, the next were ear length and kernel numbers per row. As to kernel features, kernel volume was affected greater by sowing date than kernel weight, and the effect of sowing date on the ratio of kernel length and kernel width was significant.Sowing date influenced grain filling mainly through affecting the grain filling rate rather than grain filling duration. Correlation analysis indicated that grain filling duration was significantly and negatively related to daily mean temperature and precipitation. The correlation coefficients were -0.451 and -0.583, respectively. On the contrary, there were significantly and positively correlation between the average filling rate with daily mean temperature (r=0.689) and precipitation (r=0.625), however, the sunshine hours (r=-0.608, P<0.05) and daily range (r=-0.697, P<0.05) were negatively correlated with the average filling rate.
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