Comparison of Policosanol Profiles of the Sprouts of Wheat Mutant Lines and the Effect of Differential LED Lights on Selected Lines

Policosanols (PCs) are long-chain linear aliphatic alcohols that are present in the primary leaves of cereal crops, such as barley and wheat, sugar cane wax, and beeswax. PCs have been used as a nutraceutical for improving hyperlipidemia and hypercholesterolemia. However, the PC content in mutant wheat lines has not been investigated. To select highly functional wheat sprouts with a high content of PCs in wheat mutant lines developed via gamma-irradiated mutation breeding, we cultivated the sprouts of wheat mutant lines in a growth chamber with white LED light (6000 K) and analyzed the PC content in these samples using GC-MS. We studied the PC content in 91 wheat sprout samples: the original variety (Woori-mil × D-7; WS01), commercially available cv. Geumgang (WS87) and cv. Cheongwoo (WS91), and mutant lines (WS02–WS86 and WS88–WS90) developed from WS01 and WS87. Compared to WS01, 18 mutant lines exhibited a high total PC content (506.08–873.24 mg/100 g dry weight). Among them, the top 10 mutant lines were evaluated for their PC production after cultivating under blue (440 nm), green (520 nm), and red (660 nm) LED light irradiation; however, these colored LED lights reduced the total PC production by 35.8–49.7%, suggesting that the cultivation with white LED lights was more efficient in promoting PCs’ yield, compared to different LED lights. Therefore, our findings show the potential of radiation-bred wheat varieties as functional foods against hyperlipidemia and obesity and the optimal light conditions for high PC production.


Introduction
Wheat (Triticum aestivum L.) is a staple crop that ranks third in global grain production and serves as a source of energy and an important nutrient in the human diet [1].Wheat contains not only nutrients, including vitamins, proteins, minerals, and dietary fiber, but also health-promoting compounds, such as flavonoids, lignans, phenolic acids, alkylresorcinols, benzoxazinoids, steroids, sphingolipids, fatty acids, and glycolipids [2].Wheat flour is a staple food worldwide.However, wheat sprouts have recently attracted considerable attention as a functional food [3], as sprouts can improve their nutritional value and produce more health-promoting compounds than grains [4].Flavonoid C-glycosides and policosanols (PCs) are major bioactive components in wheat sprouts [5][6][7].Wheat sprout extract has been reported to exhibit antioxidant [8], anticancer [9], antimutagenic [10], anti-osteoporotic [11], hepatoprotective [12], and hyperlipidemic activities [6].
PCs are mixtures of long-chain aliphatic primary alcohols derived from plant wax [13,14] or insect wax [15,16].Previous studies have demonstrated that PCs can inhibit 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity by adenosine 5 -monophosphateactivated protein kinase (AMPK) phosphorylation, which inhibits cholesterol synthesis [17], Plants 2023, 12, 3377 2 of 19 and clinical studies on PCs have shown that it has lipid-lowering, LDL-cholesterol-lowering, and HDL-cholesterol-increasing effects [18].However, results from several randomized controlled trials conducted in Europe and the United States provided insufficient evidence of PCs' significant effect on plasma cholesterol levels [19].For this reason, the European Food Safety Authority has rejected claims about the beneficial effects of PCs [20].Research on the health beneficial effects of PCs has been continuously conducted, including a study on the in vivo mechanism of policosanols on hypercholesterolemia induced by a high-fat and high-cholesterol diet in rats [21].In addition, PCs attenuate hepatic lipid accumulation [22] and vascular calcification [23] and stimulate osteoblast differentiation [24].These effects have been attributed to the inhibition of sterol biosynthesis via the regulation of AMPK [21][22][23][24].Recently, several in vivo studies have also shown that PCs have protective effects against Alzheimer's [14,15] and Parkinson's diseases [16].Hence, researchers investigated the composition and content of PCs in cereal sprouts, including wheat, barley, and oats [6,7,25,26].However, the PC content in wheat mutant lines developed via gamma-irradiated mutation breeding has not been studied thus far.
Environmental factors such as artificial light, water, temperature, and carbon dioxide influence the increased production of various secondary metabolites in plants [27][28][29].Several studies of sprout cultivation in controlled environments using diverse wavelengths of light emitting diodes (LEDs), including red, blue, and green, and their combinations, have been reported to enhance the accumulation of health-promoting compounds and biomass production [7,[30][31][32].The environment controlled by different wavelengths, intensities, and cycles of LED light can modify the photosynthetic process in plants [33].Photomorphogenic responses are regulated by plant photoreceptors such as phytochromes and cryptochromes [34].Phytochromes regulate plants' physiological responses and synthesize phytochemicals such as phenolic substances [35].Cryptochromes regulate biomass production and the biosynthesis of anthocyanins, carotenoids, and chlorophyll [36,37].PCs, very long-chain fatty acid alcohols, were present in wheat leaf cuticle wax, and fatty acyl coenzyme A reductase (FAR) is known to be involved in the biosynthesis of long-chain primary alcohols [38].In diploid Aegilops tauschii, the D-genome donor for hexaploid wheat (T.aestivum), five FARs are found to be responsible for primary alcohol (C16-C28) biosynthesis [39].Recently, the policosanol content and the expression pattern of its biosynthesis-related genes in sprouts of barley and wheat cultivated under LED light irradiation were studied [7].
In this study, the composition and content of PCs in the original variety and its mutant lines, cultivated in an environmentally controlled growth chamber, were determined using gas chromatography-mass spectrometry (GC-MS).The individual and total PC contents in these samples were compared to the selected mutant lines with a high PC content.In addition, we evaluated the effects of different LED light sources on the production of PCs and the growth quality of the 10 selected mutant lines under controlled conditions.

Comparison of Policosanol Contents in Different Wheat Sprout Samples
Several studies have analyzed the PC contents of different varieties of wheat and barley sprouts cultivated at different growth times and under different LED light conditions [6,7,25,26].However, the PC contents of wheat mutant lines developed via gamma-irradiated mutation breeding have not yet been evaluated.Initially, we confirmed the production of PCs in the wheat sprout samples cultivated in a growth chamber with white LED light (6000 K).This analytical method was then used to investigate the contents of the PC-TMS derivatives in wheat sprout samples.The individual and total PCs in the wheat sprout samples differed considerably (Table 2).Octacosanol (C28-OH) was the most abundant PC in the wheat sprout samples, followed by triacontanol (C30-OH) and hexacosanol (C26-OH).Moreover, the contents of heptacosanol (C27-OH), hexacosanol (C26-OH), tetracosanol (C24-OH), tricosanol (C23-OH), and docosanol (C22-OH) were lower than those of the three aforementioned compounds.Notably, eicosanol (C21-OH) and heneicosanol (C20-OH) were not detected in the GC-MS analysis of the wheat sprout samples (Figure S2).
Among the 85 sprout samples of the wheat mutant lines (WS02-WS86) derived from the original variety (WS01), 19 mutant lines showed a higher total PC content than that of WS01.WS74 exhibited the highest total PC content, followed by WS37, WS69, WS72, WS76, WS57, WS81, WS78, WS49, WS75, WS31, WS79, WS51, WS46, WS63, WS48, WS70, WS66, and WS40.Octacosanol (C28-OH) was the most abundant PC in all the mutant lines, accounting for approximately 75% of the average total PC content.Triacontanol (C30-OH) and hexacosanol (C26-OH) were the second and third major components, accounting for ~13.5% and ~6.5% of the average total PC content, respectively.The remaining PCs accounted for <2% of the average total PC content, indicating that they are very minor components.
The concentration values of the PCs of the 91 wheat sprout samples were exported for hierarchical clustering analysis.HCA with a heatmap was performed to determine the sample classification and to highlight variations in wheat sprout samples' PC composition (Figure 1).Among the improved 19 mutant lines, WS37 and WS74 were clustered at the top with the highest quantification of octacosanol (C28-OH) and triacontanol (C30-OH).Similar to these, eight mutant lines, WS78, WS81, WS40, WS48, WS70, WS63, WS75, and WS79, showed a relatively high quantification of octacosanol (C28-OH) and triacontanol (C30-OH) compared to the other components and were clustered together.Four mutant lines, WS66, WS76, WS69, and WS72, with a high quantification of octacosanol (C28-OH) and triacontanol (C30-OH) as well as a high quantification of docosanol (C22-OH) and tricosanol (C23-OH) were clustered together.Five mutant lines, WS57, WS49, WS46, WS51, and WS31, with a high quantification of hexacosanol (C26-OH), heptacosanol (C27-OH), octacosanol (C28-OH) were clustered together.These two clusters were merged into one supergroup.To develop new varieties of T. aestivum for sprouts containing high contents of functional ingredients, the top 10 mutant lines with a >20% higher total PC content than that of the original cultivar were selected.The GC-MS chromatograms for each sample are shown in Figure S2.The accumulation patterns of individual components differed owing to different radiation breeding methods (Figure 2).The octacosanol (C28-OH) content increased in the top 10 mutant lines but showed a varied trend in the other 75 mutant lines.Moreover, the triacontanol (C30-OH) content increased in only 7 out of the top 10 mutant lines, except for three lines (WS49, WS57, and WS75) but decreased in the other 75 mutant lines.The tricosanol (C23-OH) content increased in all the mutant lines except for WS37 To develop new varieties of T. aestivum for sprouts containing high contents of functional ingredients, the top 10 mutant lines with a >20% higher total PC content than that of the original cultivar were selected.The GC-MS chromatograms for each sample are shown in Figure S2.The accumulation patterns of individual components differed owing to different radiation breeding methods (Figure 2).The octacosanol (C28-OH) content increased in the top 10 mutant lines but showed a varied trend in the other 75 mutant lines.Moreover, the triacontanol (C30-OH) content increased in only 7 out of the top 10 mutant lines, except for three lines (WS49, WS57, and WS75) but decreased in the other 75 mutant lines.The tricosanol (C23-OH) content increased in all the mutant lines except for WS37 and were similar to those in WS01 or decreased.WS76 had the highest contents of docosanol (C22-OH; 14.10 mg/100 g) and tetracosanol (C24-OH; 7.46 mg/100 g), which were more than three times higher than those in WS01.Furthermore, WS53 contained the highest contents of hexacosanol (C26-OH; 70.85 mg/100 g) and heptacosanol (C27-OH; 16.25 mg/100 g), which were twice as high as those in WS01.The highest tricosanol (C23-OH) content (17.07 mg/100 g) was observed in WS49.These results suggest that γ-irradiation breeding influences genetic mutation for the increased accumulation of beneficial metabolites, as previously reported for wheat and perilla [5,40].Compared to the commercially available cultivars cv.Geumkang (WS87) and cv.Cheongwoo (WS91), the original cultivar (Woori-mil × D-7; WS01), had a higher total PC content.However, compared to those in WS01 and WS87, WS91 showed 60-70% lower octacosanol (C28-OH) and triacontanol (C30-OH) contents, which are major components, and slightly higher tricosanol (C23-OH), tetracosanol (C24-OH), and heptacosanol (C27-OH) contents.The three mutant lines (WS88-WS90) derived from cv.Geumkang (WS87) had a slightly lower total PC content than that in WS87.WS88 and WS90 showed a similar PC content distribution to that in WS87.However, WS89 had 1.85 times the tricosanol (C23-OH) present in WS87, but the contents of the other components decreased compared to those in WS87.Among the 10 selected mutant lines, the highest total PC content was observed in WS74 (873.24mg/100 g).The octacosanol (C28-OH; 686.38 mg/100 g), tricosanol (C23-OH; 13.61 mg/100 g), and docosanol (C22-OH; 9.94 mg/100 g) contents in WS74 were more than twice as high as those in WS01, and the content of the remaining four components slightly increased (<1.3 times) or decreased.WS37 ranked second in the total PC content, and its octacosanol (C28-OH; 636.02 mg/100 g), hexacosanol (C26-OH; 58.12 mg/100 g), and triacontanol (C30-OH; 121.20 mg/100 g) contents increased by ~1.8, 1.7, and 1.3 times, respectively, compared to those in WS01; however, the contents of the other compounds were similar to those in WS01 or decreased.WS76 had the highest contents of docosanol (C22-OH; 14.10 mg/100 g) and tetracosanol (C24-OH; 7.46 mg/100 g), which were more than three times higher than those in WS01.Furthermore, WS53 contained the highest contents of hexacosanol (C26-OH; 70.85 mg/100 g) and heptacosanol (C27-OH; 16.25 mg/100 g), which were twice as high as those in WS01.The highest tricosanol (C23-OH) content (17.07 mg/100 g) was observed in WS49.These results suggest that γ-irradiation breeding influences genetic mutation for the increased accumulation of beneficial metabolites, as previously reported for wheat and perilla [5,40].

Effects of Different LED Conditions on the Policosanol Content in the Sprouts of Wheat
Compared to the commercially available cultivars cv.Geumkang (WS87) and cv.Cheongwoo (WS91), the original cultivar (Woori-mil × D-7; WS01), had a higher total PC content.However, compared to those in WS01 and WS87, WS91 showed 60-70% lower octacosanol (C28-OH) and triacontanol (C30-OH) contents, which are major components, and slightly higher tricosanol (C23-OH), tetracosanol (C24-OH), and heptacosanol (C27-OH) contents.The three mutant lines (WS88-WS90) derived from cv.Geumkang (WS87) had a slightly lower total PC content than that in WS87.WS88 and WS90 showed a similar PC content distribution to that in WS87.However, WS89 had 1.85 times the tricosanol (C23-OH) present in WS87, but the contents of the other components decreased compared to those in WS87.

Effects of Different LED Conditions on the Policosanol Content in the Sprouts of Wheat Mutant Lines
To evaluate the effects of different LED conditions on PC accumulation in wheat sprouts, we quantified the PC content in the top 10 wheat mutant lines and the original variety, which were grown in a well-controlled growth chamber under white (6000 K), blue (440 nm), green (520 nm), and red (660 nm) LED light irradiation for 7 d, using GC-MS (Tables 3-5).The GC-MS chromatograms for the PCs and each sample are shown in Figures S3-S6.The total and individual PC contents significantly decreased in the wheat sprout samples irradiated with blue, green, and red LED lights compared to the PC contents in the samples irradiated with a white LED light; in particular, the total PC contents in the sample irradiated with blue, green, and red light decreased by 35.8, 46.9, and 49.7%, respectively (Figure 3).
The total PC content in the original variety WS01 decreased more under green-light irradiation than under blue-or red-light irradiation.WS78 and WS81 exhibited similar patterns to WS01, whereas the total PC content in WS37, WS49, WS57, WS74, and WS76 decreased slightly as the wavelength of the colored LED lights increased in the order of blue (440 nm) < green (520 nm) < red (660 nm).In contrast, the total PC content in WS72 showed a slight decreasing trend as the wavelength of the colored LED lights decreased from red to green to blue.The total PC content in WS69 and WS75 decreased slightly under green LED light compared to that under blue and red LED lights.Hence, the individual and total PC content in wheat sprout samples varied without a specific trend under different LED light conditions, and white LED light was the best condition for PC accumulation.The inconsistent or negative changes in the PC content under LED cultivation observed in this study were similar to those reported in previous studies on barley and wheat sprouts [7].Irregular trends of hexacosanol (C26-OH) and octacosanol (C28-OH) accumulation were observed in barley and wheat sprout samples, respectively, cultivated under fluorescent and white, blue, and red LED irradiation on the 7th day of growth.This suggests that light irradiation is not the only factor that influences PC biosynthesis.The total PC content in the original variety WS01 decreased more under green-light irradiation than under blue-or red-light irradiation.WS78 and WS81 exhibited similar patterns to WS01, whereas the total PC content in WS37, WS49, WS57, WS74, and WS76 decreased slightly as the wavelength of the colored LED lights increased in the order of blue (440 nm) < green (520 nm) < red (660 nm).In contrast, the total PC content in WS72 showed a slight decreasing trend as the wavelength of the colored LED lights decreased from red to green to blue.The total PC content in WS69 and WS75 decreased slightly un-
The content of octacosanol (C28-OH), which was the major constituent of wheat sprouts in this study, increased in the top 10 mutant lines (W74, WS37, WS69, WS72, WS76, WS57, WS81, WS78, WS49, and WS75) along with an increased total PC content, compared to the original variety.Octacosanol (C28-OH) induces a dose-dependent activation of AMPK phosphorylation in human hepatoma HepG2 cells, suggesting it is a lipid/cholesterol-lowering agent [6,48].In addition, new molecular mechanisms of octacosanol (C28-OH) have recently been discovered, including insulin-resistance management by the regulation of the gut microbiota and inflammatory signaling pathway [49], lipid-decreasing effects through a modulation of the lipid metabolism-related signaling pathway [50], protective effects on the integrity of the gut barrier through a modulation of the intestinal flora and its metabolism [51], and anti-fatigue effects in an exercise-induced fatigue model [52].Therefore, mutant lines exhibiting a high octacosanol (C28-OH) accumulation could have potential applications as functional foods and promote new cultivars' development.
Red/far-red light-sensing phytochromes and blue light-sensing cryptochromes play important roles in regulating light-mediated physiological responses through regulated transcriptional networks [53].Phytochromes are photoreceptors that display a photoreversible conformational change between two spectrally distinct forms: red light absorbing Pr and far-red light-absorbing Pfr.Red light converts Pr to biologically active Pfr, inducing reactions such as seed germination and desulfurization, while far-infrared light converts Pfr back to Pr, physically inactivating it [27,53].Cryptochromes with B-light photoreceptor functions have two members, cry1 and cry2.Both cryptochromes undergo B light-dependent phosphorylation, and homodimerization is required for these cryptochromes' phosphorylation and physiological activity [27,53].In particular, it was revealed that cry2 plays a role in forming homodimers in response to blue light [54].Therefore, applying light resources is promising to improve crop biomass and quality.The light environment is a factor that has a significant impact on the production of secondary metabolites in plants [55].
Additionally, growing plants under different wavelengths of light exposure results in physiological changes [56].For example, it has been reported that the type and intensity of the wavelength can affect the enhancement of carotenoid pigment and glucosinolate concentrations [57,58].Conversely, a GC-MS analysis of the PC content in sprouts of barley (H.vulgare) and wheat (T.aestivum) subjected to differential LED light conditions showed that the hexacosanol (C26-OH) content in barley and the octacosanol (C28-OH) content in wheat were not consistent with light qualities [7].In this study, we attempted to enhance the policosanol content of wheat sprouts using different wavelengths of light.The top 10 mutant lines with the highest increase in total PC content were cultivated under blue, green, and red LED light irradiation to investigate the effect of LED irradiation on the production of PCs.However, the total PC content decreased or showed irregular trends, suggesting that the white LED light irradiation was the most effective condition for increasing PC production.In addition, it indicated that LED responses may be crop-or secondary metabolite-dependent and that a colored LED light is not a factor in amplifying PC biosynthesis in wheat sprouts.
In conclusion, the differences in the individual and total PC contents of the wheat sprout samples, including the original variety (WS01), commercially available varieties cv.Geumgang (WS87) and cv.Cheongwoo (WS91), and 85 mutant lines (WS02-WS86 and WS88-WS90) were evaluated for the first time in the literature.Compared to WS01, 18 mutant lines exhibited a higher total PC content with an increased octacosanol (C28-OH) content, suggesting that these mutant lines can serve as a functional source of PCs.Moreover, the top 10 mutant lines with the highest range of a total PC were subjected to differential LED light conditions (blue, green, and red).However, their individual and total PC contents were reduced compared to those with the white LED light irradiation, indicating that the correlation between colored LED lights and PC biosynthesis in wheat sprouts is negative.Our study reveals the beneficial effect of radiation breeding on increasing the metabolite accumulation in wheat sprouts.However, further studies on the genetic factors controlling radiation-induced PC biosynthesis in wheat sprouts are required.

Plant Materials
Wheat mutant lines were developed by radiation breeding of the original cultivar of colored wheat using seeds treated with 200 Gy of gamma ( 60 Co) irradiation.The original cultivar of colored wheat was developed by a cross-breeding of Woori-mil (Korea RDA accession no.IT172221) and D-7 (a wheat line developed by Korea University) [59].The mutant lines selected according to the phenotypic variants stably inherited their phenotype for over 4 years and were bred by Drs.Jin-Beak Kim and Min-Jeong Hong (Korea Atomic Energy Research Institute).Voucher specimens were deposited at the Radiation Breeding Research Center, the Advanced Radiation Technology Institute, and the Korea Atomic Energy Research Institute.In this study, seeds of the selected mutants were sown in 50-cell seeding plug trays with soil and water and were germinated and grown in a well-controlled growth chamber (DS-50TPLH-3Light; Dasol Science, Hwaseongsi, Gyeonggi-dom, Republic of Korea) at 22 • C with 60% relative humidity.The growth chambers were irradiated with white (6000 K), blue (440 nm), green (520 nm), and red (660 nm) lights with a photoperiod of 16 h of light and 8 h of darkness.Sprouts of the wheat mutant lines were collected 7 d after sowing.Each sample was freeze-dried, chopped, and stored at −20 • C in polyethylene plastic bags until further analysis.

GC-MS Analysis
The GC-MS analysis of wheat sprout samples cultivated in a growth chamber under a white LED light (6000 K) was performed on a GCMS-QP2010 Ultra (Shimadzu, Kyoto, Japan).The GC-MS analysis of wheat sprout samples cultivated in a growth chamber with blue, green, and red LED lights was performed using a GCMS-QP2020 NX (Shimadzu).An HP-5 MS capillary GC column (30 m length × 0.25 mm i.d.× 0.25 µm film thickness, Agilent Technologies Co., Santa Clara, CA, USA) was used with 99.99% high-purity helium at a flow rate of 1.2 mL/min.The sample (1 µL) was injected in split mode (1:5 ratio).The oven temperature was initially set to 230 • C, was increased to 260 • C at a heating rate of 25 • C/min, and was maintained at this temperature for 10 min.The transfer line temperature was set to 280 • C. The MS data were acquired in the electron ionization (EI) mode with an ionization voltage of 70 eV, an ion source temperature of 230 • C, and a scan range of m/z 50-500.The MS data were collected using a mass spectra database (National Institute of Standards and Technology, MassSpectra Libraries, Gaithersburg, MD, USA).The PCs were identified by comparing the retention time and fragmented mass values of the peaks with those of the PC standards.

Statistical Analysis
All experiments were conducted in triplicate, and the mean values were reported.One-way analysis of variance was performed using GraphPad Prism 9 software (GraphPad Software, La Jolla, CA, USA) to assess significant differences (* p < 0.05, ** p < 0.005, *** p < 0.001, and **** p < 0.0001).The data set of the quantification of the PCs obtained for the 91 wheat sprout samples was normalized and subjected to a hierarchical clustering analysis (HCA) with a heatmap using "pheatmap" in R software (version 4.0.2) [60].

Supplementary Materials:
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants12193377/s1, Figure S1: Chromatogram and mass spectrum of policosanols (20 ppm) using a GCMS-QP2010 Ultra (Shimadzu, Kyoto, Japan).Figure S2: Representative chromatogram of the sprout extract of the original variety and the selected 10 mutant lines of wheat, which were cultivated in a growth camber exposed to a white LED light.Figure S3: Chromatogram and mass spectrum of policosanols (20 ppm) using a GCMS-QP2020 NX (Shimadzu).Figure S4: Representative chromatogram of the sprout extract of the original variety and the selected 10 mutant lines of wheat, which were cultivated in a growth camber exposed to a blue LED light.Figure S5: Representative chromatogram of the sprout extract of the original variety and the selected 10 mutant lines of wheat, which were cultivated in a growth camber exposed to a green LED light.Figure S6: Representative chromatogram of the sprout extract of the original variety and the selected 10 mutant lines of wheat, which were cultivated in a growth camber exposed to a red LED light.

Figure 1 .
Figure 1.Hierarchical clustering analysis (HCA) with a heatmap for the wheat sprout samples.

Figure 1 .
Figure 1.Hierarchical clustering analysis (HCA) with a heatmap for the wheat sprout samples.
Plants 2023, 12, 3377 8 of 19 WS56.The contents of the other four compounds showed varied increasing and decreasing trends.

Table 1 .
GC-MS retention times and mass spectral data of the policosanols.

Table 2 .
Individual and total policosanol contents in the sprouts of wheat mutant lines.
a All values are presented as the mean ± standard deviation (SD) of triplicate measurements.b Eicosanol and heneicosanol were not detected.c

Table 3 .
Individual and total policosanol contents in the sprout samples of the top 10 wheat mutant lines cultivated under blue (440 nm) LED light irradiation.

Table 4 .
Individual and total policosanol contents in the sprout samples of the top 10 wheat mutant lines cultivated under green (520 nm) LED light irradiation.

Table 5 .
Individual and total policosanol contents in the sprout samples of the top 10 wheat mutant lines cultivated under red (660 nm) LED light irradiation.