Determination of seed oil content and fatty acid composition in sunflower through the analysis of intact seeds, husked seeds, meal and oil by near-infrared reflectance spectroscopy

A methodological study was conducted to test the potential of near-infrared reflectance spectroscopy (NIRS) to estimate the oil content and fatty acid composition of sunflower seeds. A set of 387 intact-seed samples, each from a single plant, were scanned by NIRS, and 120 of them were selected and further scanned as husked seed, meal, and oil. All samples were analyzed for oil content (nuclear magnetic resonance) and fatty acid composition (gas chromatography), and calibration equations for oil content and individual fatty acids (C_16:0, C_16:1, C_18:0, C_18:1, and C_18:2) were developed for intact seed, husked seed, meal, and oil. For intact seed, the performance of the calibration equations was evaluated through both cross- and external validation, while cross-validation was used in the rest. The results showed that NIRS is a reliable and accurate technique to estimate these traits in sunflower oil (validation r ² ranged from 0.97 to 0.99), meal (r ² from 0.92 to 0.98), and husked seeds (r ² from 0.90 to 0.97). According to these results, there is no need to grind the seeds to scan the meal; similarly accurate results are obtained by analyzing husked seeds. The analysis of intact seeds was less accurate (r ² from 0.76 to 0.85), although it is reliable enough to use for pre-screening purposes to identify variants with significantly different fatty acid compositions from standard phenotypes. Screening of intact sunflower seeds by NIRS represents a rapid, simple, and cost-effective alternative that may be of great utility for users who need to analyze a large number of samples.     

The composition of safflower seed

Safflower has some interesting variations in composition. Current commercial seed types have about 40% hull, 37% oil, and 23% meal. Varities also exist with from 59-18% hull and inversely varying oil and meal percentages. The fatty acid composition of the linoleic acid type oils is quite constant at about 78% linoleic, 11% oleic, 3% stearic, 6% palmitic. Experimental types have been described with about 45% oleic: 45% linoleic, 80% oleic: 10% linoleic, and with 10% stearic. Compositional data are reviewed with particular attention to major and minor constituents (especially linolenic acid) that influence safflower use. W. Utiliz. Res. Dev. Div., ARS, USDA.     

Development of Rigorous Fatty Acid Near-Infrared Spectroscopy Quantitation Methods in Support of Soybean Oil Improvement

The mature seeds of soybean (Glycine max L. Merr) are a valuable source of high‐quality edible lipids and protein. Despite dramatic breeding gains over the past 80 years, soybean oil continues to be oxidatively unstable, due to a high proportion of polyunsaturated triacylglycerols. Until recently, the majority of soybean oil underwent partial chemical hydrogenation. Mounting health concerns over trans fats, however, has increased breeding efforts to introgress mutant and biotechnological genetic alterations of soybean oil composition into high‐yielding lines. As a result, there is an ongoing need to characterize fatty acid composition in a rapid, inexpensive and accurate manner. Gas chromatography is the most commonly used method, but near‐infrared reflectance spectroscopy (NIRS) can be calibrated to non‐destructively phenotype various seed compositions accurately and at a high throughput. Here we detail development of NIRS calibrations using intact seeds for every major soybean fatty acid breeding goal over an unprecedented range of oil composition. The NIRS calibrations were shown to be equivalent to destructive chemical analysis, and incorporation into a soybean phenotyping operation has the potential to dramatically reduce cost and accelerate phenotypic analysis.     

Nondestructive Estimation of Fat Constituents of Sesame (Sesamum indicum L.) Seeds by Near‐Infrared Reflectance Spectroscopy

A rapid and efficient method for oil constituent estimation in intact sesame seeds was developed through near‐infrared reflectance spectroscopy (NIRS) and was used to evaluate a sesame germplasm collection conserved in China. A total of 342 samples were scanned by reflectance NIR in a range of 950–1650 nm, and the reference values for oil content and fatty acid (FA) profiles were measured by Soxhlet and gas chromatograph methods. Useful chemometric models were developed using partial least squares regression with full cross‐validation. The equations had low standard errors of cross‐validation, and high coefficient of determination of cross‐validation (R_c²) values (>0.8) except for stearic acid (0.794). In external validation, r² values of oil and FA composition equations ranged from 0.815 (arachidonic acid) to 0.877 (linoleic acid). The relative predictive determinant (RPD_v) values for all equations were more than 2.0. The whole‐seed NIR spectroscopy equations for oil content and FA profiles can be used for sesame seed quality rapid evaluation. The background information of the 4399 germplasm resources and accessions with high linoleic acid content identified in this study should be useful for developing new sesame cultivars with desirable FA compositions in future breeding programs.     

Analyzing seed weight,fatty acid composition,oil, and protein contents in <Emphasis Type="Italic">Vernonia galamensis</Emphasis> germplasm by near-infrared reflectance spectroscopy

Vernonia galamensis is a potential new industrial oilseed crop from the Asteraceae family. The interest in this species is due to the presence of a high vernolic acid content of its seed oil, which is useful in the oleochemical industry for paints and coatings. The development of a rapid, precise, robust, nondestructive, and economical method to evaluate quality components is of major interest to growers, processors, and breeders. NIR reflectance spectroscopy (NIRS) is routinely used for the prediction of quality traits in many crops. This study was conducted to establish a rapid analytical method for determining the quality of intact seeds of V. galamensis. A total of 114 Vernonia accessions were scanned to determine seed weight, FA composition, oil, and protein contents using NIRS. Conventional chemical analysis for FA composition, total oil, and protein contents were performed by GC, Soxhlet extraction, and the Dumas combustion method, respectively. Calibration equations were developed and tested through cross-validation. The coefficient of determination in cross-validation for FA ranged from 0.47 (linoleic acid) to 0.55 (vernolic acid), and for oil, protein, and seed weight from 0.71 (oil) to 0.86 (seed protein). It was concluded that NIRS calibration equations developed for seed weight and seed quality traits can be satisfactorily used as early screening methods in V. galamensis breeding programs.     

Composition of Catalpa ovata Seed Oil and Flavonoids in Seed Meal as Well as Their Antioxidant Activities

In view of the growing demand for vegetable oil, currently exploration of some non‐conventional oils is of great concern. This study firstly analyzed the contents of fatty acids, phytosterols, and tocopherols in Catalpa ovata seed oil collected from four different Provinces in China. Then the composition of flavonoids as well as their antioxidant activities in defatted seed meal was determined. The results showed that the relative oil content in C. ovata seeds ranged from 24.0 to 36.0 % and seed oil was mainly composed of fatty acids linoleic acid (43.4–50.1 %), α‐linolenic acid (23.8–24.4 %), and oleic acid (13.1–16.2 %). The content of unsaturated fatty acids was up to 85.0 %. Sterol in seed oil mainly contained campesterol, stigmasterol, and β‐sitosterol. β‐sitosterol accounted for 74.0 % of the total sterol. The tocopherol content was 173.0–225.7 mg/100 g. Defatted seed meal from Hubei Province showed the highest content of total flavonoids (11 mg/g) and the strongest activities for DPPH radicals scavenging, ABTS radicals scavenging, and ferric reducing antioxidant power compared with other defatted seed meal in this study. Seven flavonoids were identified from C. ovata seed meal. These results suggest that C. ovata seeds may be developed as a new source of oil and can also be properly used in pharmaceuticals and cosmetics.     

Performance of near-infrared reflectance spectroscopy (NIRS) in routine analysis of C18 unsaturated fatty acids in intact rapeseed

This study was aimed to evaluate the performance of near-infrared reflectance spectroscopy (NIRS) in the analysis of the oil composition for fatty acids like oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) in zero-erucic acid rapeseed (Brassica napus L.). Intact-seed samples of 1094 lines from a breeding programme for high-oleic acid rapeseed were analyzed by both NIRS and gas chromatography (GC). Previously developed calibration equations were initially used for NIRS analyses. The accuracy of NIRS was considerably improved by including some samples of the actual breeding population into the original calibration set and developing new calibration equations. The inclusion of twenty randomly selected samples led to a reduction of the standard error of performance (SEP) from 2.6% to 1.9% for oleic, from 3.8% to 2.0% for linoleic, and from 1.1% to 0.9% for linolenic acid. The application of the new equations to the remaining population of 1074 samples resulted in coefficients of correlation between NIRS and GC values of 0.95 for oleic, 0.92 for linoleic, and 0.90 for linolenic acid. Furthermore, the effectiveness of a selection for high oleic, high linoleic, or low linolenic acid content based on NIRS data was demonstrated. The results of this study will help potential users to choose the optimal selection strategy in routine analysis of C18 unsaturated fatty acids by NIRS within a breeding programme.     

Determination of the fatty acid composition of the oil in intact-seed mustard by near-infrared reflectance spectroscopy

Near-infrared reflectance spectroscopy (NIRS) was used to estimate the fatty acid composition of the oil in intact-seed samples of Ethiopian mustard (Brassica carinata Braun) within a mutation breeding program that produced seeds with variable fatty acid compositions. Five populations, from 1992 to 1996 crops, were included in this study; and NIRS calibration equations for major fatty acids (palmitic, stearic, oleic, linoleic, linolenic, eicosenoic, and erucic) were developed within each single population. Furthermore, global calibration equations, including samples from the five populations, were developed. After external validation, the NIRS technique permitted us to obtain a reliable and accurate nondestructive estimation of the fatty acid composition of the oil, especially for the major acids—oleic, linoleic, linolenic, and erucic. For these, the r ² in external validation was higher than 0.95 by using both single-and multipopulation equations, and higher than 0.85 for the remaining fatty acids. Moreover, the multipopulation equations provided an accurate estimation of samples from a population not represented in the calibration data set, with values of coefficient of determination in validation (r ²) from 0.80 (palmitic and eicosenoic acids) to 0.97 (erucic acid). The ability of NIRS to discriminate among different fatty acid profiles was mainly due to changes within six spectral regions, 1140–1240, 1350–1400, 1650–1800, 1880–1920, 2140–2200, and 2240–2380 nm, all of them associated with fatty acid absorbers. Thus, NIRS can be used to estimate the fatty acid composition of Ethiopian mustard seeds with a high degree of accuracy, provided that calibration equations be developed from calibration sets that include large variability for the fatty acid composition of the oil.     

Calibration of NIRS for the Estimation of Fatty Acids in Brassica Juncea

This study was done to test whether near infrared reflectance spectroscopy (NIRS) could be used as a quick substitute for measuring quality characteristics, i.e., a fatty acid profile over gas liquid chromatography. Near infrared reflectance spectroscopy is a multi‐trait technique. In the present study, the fatty acid profiles of seeds of 200 genotypes of Brassica juncea were analysed by gas liquid chromatography. Near infrared reflectance spectra of intact seeds of the same samples were collected (400–2500 nm) on a NIR systems Model 6500 spectrophotometer. The spectra were subjected to scanning, mathematical processing, and statistical analysis using Win ISI software. Data were scored to remove redundancy. Spectra were treated as outliers with H > 3.0 (global H) and similar samples with H < 0.6 (neighbourhood H). Cross validation of the spectra was done using a modified partial least‐square method to develop the calibration equation. The calibration equation had good R² values for oleic acid (R² = 0.91), linoleic acid (R² = 0.83), and erucic acid (R² = 0.88). The internal validation was done to test the goodness of fit of the developed equation. The equation provided reliable estimations of these traits in internal validation with R² values of 0.77, 0.68, 0.81 for the above quoted fatty acids, respectively. The external validation results also showed higher R² values for oleic acid (0.89), linoleic acid (0.69), and erucic acid (0.90). The equation was less reliable for linolenic acid, which had R² values of 0.53 in cross validation, 0.25 for internal validation, and 0.20 for external validation. The results indicated that NIRS could be used to rapidly determine oleic acid, linoleic acid, and erucic acid in intact B. juncea seeds.     

Determination of the Fatty Acid Composition in Tree Peony Seeds Using Near-Infrared Spectroscopy

Tree peonies (Paeonia Sect Moutan DC) are an emerging oil crop because of their high oil and α‐linolenic acid (ALA) content. This research was to investigate the potential use of near infrared reflectance spectroscopy (NIRS) for estimating the major fatty acids contents, such as palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3) in tree peonies. A total of 115 small seed samples and 447 single seeds were selected to calibrate the predictive models. NIRS absorbance spectra were collected using a Fourier transform near infrared (FT‐NIR) spectrometer for the small seed samples, and acousto‐optic tunable filter‐near infrared (AOTF‐NIR) for the single seed samples. Statistical analysis was performed with partial least squares (PLS). For the husked samples, C18:3, C18:1 and C18:2 showed the highest correlation coefficient of validation (R_v; = 0.9756, 0.9467 and 0.8485, respectively) and the ratio of performance to deviation (RPD; = 3.58, 1.91 and 2.17, respectively); however, C16:0 did not reach expectations (R_v = 0.7783, RPD = 1.32). For intact samples, C18:3 showed the best prediction (R_v = 0.9096, RPD = 3.14), followed by C18:2 (R_v = 0.8479, RPD = 1.96). The results for C18:1 were poor (R_v = 0.7237, RPD = 1.70). For single seeds, only the results for C18:3 (R = 0.9150, RPD = 1.73) were good in the husked seed samples. It was concluded that NIRS can be used to rapidly assess the content of the major fatty acids in small samples.     

Veränderungen verschiedener Inhaltsstoffe in einzelnen Sonnenblumenfrüchten nach mutagener Behandlung in M2 und M3

Changes in Various Components in Single Sunflower Fruits after Mutagenous Treatment in M₂ and M₃ These investigations had the goal to find two different oil types with high linoleic acid content for dietfood at the one side and with high oleic acid content for special purposes for example as frying oils at the other side. By mutagenic treatment with Ethyl-methane-sulfonate and radiation the variability of fatty acid composition should be increased. After EMS-treatment a variation range from 36.6 to 83.4% linoleic acid and from 10.6 to 44.8% oleic acid was found by means of single seed investigations. After radiation with 10 KR x-rays linoleic acid content varied from 43.5 to 84.9% and oleic acid content varied from 9.2 to 42.4%. Oil and protein content as well as hull percentage of single seeds had wide variation, too. Correlation calculations showed highly negative correlations between contents of oleic and linoleic acid in all cases and partially relations between fruit size and oil and protein content.     

Near‐infrared spectroscopy for analysis of oil content and fatty acid profile in almond flour

Almond kernels show large variability for oil content and fatty acid profile. The objective of this research was to evaluate the potential of near infrared (NIR) reflectance spectroscopy (NIRS) for the analysis of these traits in almond flour. Ground kernels of 181 accessions collected in 2009 were used for developing calibration equations for oil content and concentrations of individual fatty acids. Calibration equations were developed using second derivative transformation and modified partial least squares regression. They were validated with samples from 179 accessions collected in 2010. The accuracy of calibration equations was measured through the coefficient of determination (r²) in external validation and the ratio of the SD in the validation set to the standard error of prediction (RPD). Both r² and RPD were high for oil content (r² = 0.99; RPD = 9.24) and concentrations of oleic (r² = 0.97; RPD = 5.37) and linoleic acids (r² = 0.98; RPD = 7.35), revealing that calibration equations for these traits are highly accurate. Conversely, the accuracy of the calibration equations for palmitic (r² = 0.54; RPD = 1.41) and stearic acids (r² = 0.52; RPD = 1.44) was too low for allowing their application in practice. NIRS discrimination of oil content and concentrations of oleic and linoleic acids was mainly based on the spectral region from 2240 to 2380 nm. Practical applications : NIRS is a high‐throughput analytical technique that allows fast measurement of several traits in a single analysis without using chemical reagents. We evaluated the feasibility of analyzing oil content and concentrations of palmitic, stearic, oleic, and linoleic acids in almond flour using fruits collected during 2 years from a world germplasm collection. The fruits collected in 2009 were used for NIRS calibration, whereas the fruits collected in 2010 were used for validation. NIRS equations were highly accurate for measuring oil content and concentrations of oleic and linoleic acids, which are important traits defining the quality of almond flour for specific uses in the food industry. These results have applications both in the research laboratory and the food industry, where NIRS is becoming a widely used technique for quality control.     

Oxidative stability of safflower oil

Oils from a number of varieties of safflower (Carthamus tinctorius L.) seeds (achene) were measured for oxidative stability by the gain in weight method. The induction periods of oils containing 75% to 80% linoleic acid ranged from 288 to 715 hr. Safflower oils containing 79% to 80% oleic acid and only 11% to 15% linoleic acid had induction periods ranging from 1274 to 2374 hr. No correlation between induction period and total tocopherol content was observed. However, there were indications that oils from pigmented seeds were less stable than oils from pigmentless seeds. Blending of an oil containing a high amount of linoleic acid with an oil containing a high amount of oleic acid gave a blend with an induction period intermediate between the two. However, the induction period was considerably less than the theoretical average calculated for the mixture. Arizona Agricultural Experiment Station Technical Paper No. 1389.     

Composition of several types of Safflower seed

The residue remaining after commercial extraction of oil from safflower seed has a greater potential as a source of animal feed or human diet supplement than is presently being realized. Safflower seed hull, kernel, and meal were analyzed to provide more information regarding their nutritive possibilities. Commercial and experimental normal hull varieties and experimental thin hull and striped hull varieties were hand separated into hull and kernel fractions and both fractions analyzed for protein, fat, fiber, ash, and amino acids. Samples of partially decorticated commercial meal and undecorticated meal, hulls, and defatted kernel from striped hull seeds were analyzed for protein, fat, fiber, ash, lignin, pentosans, anhydrouronic acid, total and reducing sugars, and amino acids. Cellulose was calculated by difference. A new factor for converting nitrogen to protein for summative analyses of safflower seed was calculated. These analyses indicate that about 15% of the nonfiber, nonash, nonprotein part of the defatted safflower kernel is of unknown composition. W. Utiliz. Res. Dev. Div., ARS, USDA.     

Studies on marigold seed oil and seed meal

The fixed oil (26%) of the seeds of Marigold (Calendula officinalis) locally known as “Gul-e-Ashrafi” growing around the laboratories, was studied for its physico-chemical properties. Fatty acid composition of the seed oil as determined by gaschromatography and UV-spectroscopy showed the presence of lauric (3.90%), myristic (3.58%), palmitic (14.96%), stearic (10.13%), palmito-oleic (4.55%), oleic (16.26%), linoleic (39.45%) and linolenic (7.15%) acids. In the seed oil, conjugated acid is present to the extent of 4.5% whereas the percentage of non-conjugated acid (linolenic acid) is only 2.65%. The residual meal after the extraction of oil was also studied for its proteins (18%) and amino acids composition.     

Genetic Analysis of Oil Content and Fatty Acid Composition in Safflower (<Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L.)

The F₁ and F₂ progenies of eight-parent diallel crosses were used to investigate the mode of inheritance of fatty acids, oil, and protein in safflower (Carthamus tinctorius L.) seeds. The results indicated significant differences among the parents for general (GCA) and specific combining ability (SCA). Relatively high narrow-sense heritability was estimated for fatty acids including linoleic (0.84), oleic (0.77), palmitic (0.61), and stearic (0.6) acids. The high narrow-sense heritability and the high ratio of GCA/SCA mean squares for all the fatty acids investigated indicate the important role of additive gene action in controlling these traits. In our experiment, however, low narrow-sense heritability was obtained for oil (0.37) and protein (0.28) contents. Furthermore, the estimates of genetic variance components proposed the importance of non-additive genetic effects that contribute to variation in oil and protein content. The overall results indicated that K₂₁ × Mex.22-191 cross could be employed for the production of high oil yielding and high oleic acid safflower lines in breeding programs.     

Phosphatides isolated from seeds of commercial and experimental safflower varieties

Studies on phosphatides from several safflower varieties show the following five major results. The total phosphatide contents of the various safflower seeds are quite similar (0.48% for a commercial and 0.58% for a brown-striped variety). The same three major and one minor phosphatides were present in all varieties: phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS). The amounts of these lipids present in the crude phosphatide mixture were quite similar in all varieties tested (}36% for PC, }15% for PE, }23% for PI, and less than 2% for PS). The fatty acid composition of the phosphatides of UC-1 high oleic safflower is very different from that of the other varieties, but it reflects the composition of the corresponding oil triglycerides as far as the major acid is concerned. All other safflower seed phosphatides investigated have linoleic acid as the major fatty acid constituent. A simple and very sensitive color test has been found which can differentiate phosphatides of the high linoleic from the high oleic type. W. Utiliz. Res. Dev. Div., ARS, USDA.     

Estimation of Protein and Fatty Acid Composition in Shell-Intact Cottonseed by Near Infrared Reflectance Spectroscopy

Rapid and accurate analysis of cottonseed protein content and the composition of fatty acids (especially, saturated fatty acids) is often required in cotton production and breeding programs. This study aimed to establish a set of effective estimation models for these parameters. Near infrared reflectance spectroscopy (NIRS) calibration equations using partial least-squares regression for protein concentration, oil concentration, and five fatty acids of shell-intact cottonseeds were established based on 90 varieties, and the prediction abilities of the calibration models were verified using 45 other varieties. The prediction abilities of the NIRS calibration equations were basically consistent with external validation results. Each equation was assessed based on the ratio of performance to deviation (RPD_p). Protein content and seed total fatty acid (STA) content had high RPD_p values (3.687 and 3.530, respectively), whereas cottonseed kernel total fatty acid (KTA) content, linoleic acid (18:2), stearic acid (18:0), myristic acid (14:0), and palmitic acid (16:0) exhibited relatively high RPD_p (2.866, 2.836, 2.697, 2.676, and 2.506, respectively). The calibration model for oleic acid (18:1) had a low RPD_p (1.945). The results indicated that NIRS can be used to rapidly determine contents of STA, KTA, protein, stearic acid (18:0), myristic acid (14:0), and palmitic acid (16:0) in shell-intact cottonseed.     

Oil Content and Fatty Acid Composition in Seeds of Three Safflower Species

Seeds of six safflower (C. tinctorius L.) genotypes and 19 accessions of two wild species were analyzed for oil and fatty acid composition. Oil content ranged from 29.20 to 34.00, 20.04 to 30.80 and 15.30 to 20.80% in C. tinctorius, C. oxyacantha Bieb. and C. lanatus L., respectively. The main fatty acids of oleic, linoleic, palmitic and stearic acids composed 96–99% of the total fatty acids in all species. The sum of myristic, palmitoleic, arachidic, and behenic fatty acids in oil of the species ranged from 0.43 to 0.57%. The oleic acid in seed oil of C. tinctorius, C. oxyacantha and C. lanatus ranged from 12.24 to 15.43, 14.11 to 19.28 and 16.70 to 19.77%, respectively. The corresponding ranges for linoleic acid were 71.05 to 76.12, 63.90 to 75.43 and 62.47 to 71.08%. Palmitic acid in seed oil varied from 5.48 to 7.59% in C. tinctorius, 6.09 to 8.33% in C. oxyacantha and 7.44 to 8.78% in C. lanatus. The stearic acid of the seed oil showed a variation of 1.72 to 2.86, 2.50 to 4.87 and 3.14 to 4.79% in genotypes of these species, respectively. The fatty acids composition of oil among the cultivated and wild species were not considerably different, indicating that seed oil of the wild safflower is possibly suitable for human consumption and industrial purposes.     

Lipid changes in maturing oil-bearing plants. II. Changes in fatty acid composition of flax and safflower seed oils

Changes in the fatty acids composition of the oil in flax and safflower seed that occur during the seed-ripening period have been measured. Concentrations of lipid or of specific fatty acid, expressed on a weight-per-seed basis, have been plotted as functions of days after fertilization and of percentage of oil development. Relations between these two independent variables have been established, and limitations to the unsefulness of the latter variable have been pointed out. Days after fertilization proved to be the more useful abscissa. Nonpolar solvents were used to remove free lipid from the tissue, and the total extractable matter was separated into true lipid and nonlipid components. With both flax and safflower, weight of true free lipid per seed and total unsaturation increased during the same period of growth. Nonlipid extractable matter was an inverse function of the extent of development. In developing flax seed, oleic, linoleic, and linolenic acids all increased continuously; oil in immature seed however was more saturated than oil in more mature seed. Nevertheless the ratio of linolenic acid to linoleic acid that characterizes linseed oil was established by the 20th day after fertilization during a normal growing season. In developing safflower seed, oleic acid concentration increased slowly during the first 30 days after fertilization and then appeared to level off in some cases as maturity was approached. Initially linoleic acid was present in almost the same amount as oleic acid, but by the 20th day after fertilization its concentration was three times that of oleic acid. This ratio of linoleic to oleic acid tended to increase steadily during the latter part of seed development.