Scientia Agricultura Sinica

The Dilemma and Way Out of Patent Regulation for Gene-Edited Crops

XU YiHeng

Scientia Agricultura Sinica. 2025, 58(5): 831-839. doi:10.3864/j.issn.0578-1752.2025.05.001

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Gene-edited crops, the product of the intersection between biotechnology and agricultural science, represent a crucial direction in the development of modern agriculture. With the rapid advancement of the CRISPR-Cas9 system, the scientific research and commercial development of crop trait improvement have gradually shifted towards a “technology-driven” path, which has not only overturned traditional crop cultivation methods but also fundamentally propelled humanity’s exploration of crop research. Nevertheless, the phenomenon of patenting fundamental research tools has sparked widespread controversy within academia and profoundly impacted the sharing and utilization of crop resources. Private entities patenting CRISPR-Cas9 technology restrict other researchers and farmers’ opportunities to explore and harness genetic resources. This practice not only hinders scientific progress but also violates the fundamental consensus that genetic resources should be shared by all humanity. The sharing and openness of crop resources are crucial for the sustainable development of global agriculture and ecological balance, serving as a necessary condition for safeguarding public interests. A key issue that the governance of biotechnology patents urgently needs to address is how to reasonably allocate benefits and risks among traditional communities, researchers, research investors, and the public. This is also essential for constructing a new scientific ethics framework and regulating emerging technologies. However, China’s policy responses in this area are still insufficient. To mitigate the negative effects stemming from the exclusivity of patents, it is imperative to reassess and reconstruct the framework of relevant systems. Firstly, we should adhere to the principle of moral utility, emphasizing the public nature of scientific research and its social responsibilities, while carefully considering the “harmful” nature of inventions to social morality. Secondly, implementing a mandatory disclosure system for biological genetic resources is a crucial step towards achieving transparency and fairness, with “applicants truthfully disclosing the actual origin of crop genes based on the principle of good faith” elevated to a mandatory norm. Lastly, the open licensing of fundamental patented technologies can draw inspiration from the experience of open-source software, encouraging more researchers to participate in the exploration of crop resources through the open sharing of research tools, thereby facilitating broader scientific collaboration and the transformation of research outcomes.

Cloning of the Promoters and Analysis of Expression Patterns of Maturity Genes E1 and E2 in Soybean

LIU LuPing, HU XueJie, QI Jin, CHEN Qiang, LIU Zhi, ZHAO TianTian, SHI XiaoLei, LIU BingQiang, MENG QingMin, ZHANG MengChen, HAN TianFu, YANG ChunYan

Scientia Agricultura Sinica. 2025, 58(5): 840-850. doi:10.3864/j.issn.0578-1752.2025.05.002

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【Objective】Maturity time is an essential phenotypic measure of ecological adaptability of soybean and an important trait related to its yield formation. The study of promoters and expression patterns of major maturity genes E1 and E2 would provide basis for the study of gene function and molecular regulatory network of maturity time and lay foundation for adaptability improvement and yield increase in soybean.【Method】The promoter sequences of major maturity genes E1 and E2 were analyzed through the promoter cis-element analysis website PlantCARE, and the important regulatory elements were detected. The promoters of E1 and E2 were cloned, the GUS vectors were constructed, and transformation of Arabidopsis was performed to detect GUS activity in different tissues and organs of transgenic plants. Under low light and strong light conditions, the expression levels of E1 and E2 were compared between long day and short day conditions. The expression levels of E1 and E2 were detected in soybean varieties of different maturity groups, which is for the analysis of correlation between expression levels and maturity time of soybean varieties.【Result】Both E1 and E2 promoters contained multiple photoresponsive elements such as AE-box, Box4 and G-box, E1 promoter also contained auxin-response, abolic acid-response elements, and E2 promoter also contained low temperature-response, drought-response elements and meristem expression elements. In GUS activity detection of transgenic Arabidopsis, E1 promoter had strong transcriptional activity in all organs of the plant, and transcriptional activity of E2 promoter in fibrovascular tissues of seedling hypocotyl, leaf and root was relatively strong. Under both low light and strong light conditions, the expression level of E1 was significantly higher in long day than in short day. Under low light conditions, the expression level of E2 was higher in short day than in long day. Under strong light conditions, the expression level of E2 was higher in long day than in short day. With the increase of maturity time of different soybean varieties, expression level of E1 increased gradually, while E2 expression level did not change regularly.【Conclusion】The promoter of E1 gene was a widely expressed promoter, and its expression level was significantly regulated by photoperiod and significantly correlated with the maturity time of soybean varieties. The promoter of E2 was strongly expressed in vascular tissues of various organs, the photoperiodic regulation mode of this gene was different under strong light and low light conditions, and there was no significant correlation between expression level of E2 and maturity time.

Genome-Wide Survey and Development of Novel SSR Markers in Erianthus fulvus

LUO ZhengYing, HU Xin, WU ZhuanDi, QIAN ZhenFeng, TIAN ChunYan, LIU XinLong, LI FuSheng

Scientia Agricultura Sinica. 2025, 58(5): 851-863. doi:10.3864/j.issn.0578-1752.2025.05.003

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【Objective】 Erianthus fulvus, serving as a crucial wild resource for sugarcane, is capable of enhancing the stress tolerance and yield of varieties. In order to utilize E. fulvus for sugarcane breeding, it is important to systematically identify and develop simple sequence repeat (SSR) loci in the E. fulvus genome, screen for polymorphic SSR markers, analyse the genetic diversity characteristics of E. fulvus resources and then develop SSR markers associated with important traits. 【Method】Using the SSRminer module in the software TBtools, a comprehensive exploration of SSR loci was conducted on the diploid E. fulvus whole genome sequence. The obtained data were statistically analyzed to reveal their distribution patterns and regularities within the genome. The Batch Target Region Primer Design function was employed for batch designing SSR primers, and the specificity of the primers was evaluated using the Primer check tool. To comparethe SSR polymorphism betweenE. fulvus and sugarcane, amplification experiments were performed on 50 pairs of randomly synthesized SSR primers and 14 pairs of SSR primers sourced from sugarcane across 6 E. fulvus germplasms. 【Result】A total of 152 707 SSR loci, which were distributed on E. fulvus genome with an average density of 5.64 kb/locus, were identified. The majority were located in intergenic regions. In terms of SSR type distribution, mononucleotide, dinucleotide, and trinucleotide had the highest density. Dinucleotide SSR types exhibited the greatest variation in motif repeat numbers, while pentanucleotide motif repeat variations were the least. Across the entire genome, 883 distinct SSR motif repeat types were identified, with A/T and AT/TA being the most abundant. A total of 144 692 pairs of SSR primers, of which 85 025 pairs exhibited high specificity, were designed. These specific primers displayed a distribution characteristic of dense ends and sparse middles on the genome. Amplification experiments showed that 42 out of the 50 randomly synthesized SSR primer pairs yielded stable and clear bands in E. fulvus, with 32 exhibiting polymorphisms, yielding a polymorphism rate of 64.0%. In contrast to the 14 sugarcane SSR primers, the E. fulvus SSR primers demonstrated superior amplification efficacy and greater polymorphism. After screening, 16 pairs of SSR primers with good polymorphism and clear amplification bands were determined from the 32 effective SSR primer pairs. These 16 pairs of primers amplified a total of 72 bands, with polymorphism information content (PIC) ranging from 0.63 to 0.83, and an average PIC value of 0.74, indicating their effectiveness and practicality in polymorphism analysis and molecular marker research of E. fulvus germplasm resources. 【Conclusion】This study comprehensively identified SSR loci in the E. fulvus genome, revealing the high abundance and diversity of SSR distribution features. Sixteen pairs of highly specific and polymorphic SSR primers were successfully screened.

Characteristics and Technical Approaches of Integrated Unmanned High-Yield Cultivation of Wheat

ZHANG HongCheng, XING ZhiPeng, ZHANG RuiHong, SHAN Xiang, XI XiaoBo, CHENG Shuang, WENG WenAn, HU Qun, CUI PeiYuan, WEI HaiYan

Scientia Agricultura Sinica. 2025, 58(5): 864-876. doi:10.3864/j.issn.0578-1752.2025.05.004

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【Objective】The aim of this study was to provide the theoretical and technical support for the innovation of green, high-yield, high-quality and high-efficient unmanned cultivation technology system of wheat. 【Method】 According to the situation of accelerating land transfer and large-scale operation, decreasing labor force engaged in agricultural production, and more efficient and comfortable farming methods, the integrated unmanned cultivation technology of wheat was put forward through the integration study of “agronomy-machinery-intelligence”, that is, using new technology, new product and new equipment to simplify and integrate the whole process of wheat production, and complete wheat production with the least number of operations, the least number of machines and unmanned operations. On the basis of exploratory experimental research, the integrated unmanned cultivation technology of wheat (IU) and conventional mechanized high-yield cultivation techniques of wheat in experimental area (CK) were set up as treatments in Dazhong Farm of Yancheng, Jiangsu Province in 2019-2020, 2020-2021 and 2021-2022, to study the traits and differences of wheat yield formation among different technology treatments, analyze the high-yield traits of IU, and put forward the technical approaches of IU. 【Result】 The IU increased wheat yield by 3.0%-5.9% compared with CK, and significant differences were observed between treatments of some varieties or some growing seasons. In terms of yield components, the spike number was IU>CK (significant differences were observed between treatments of some varieties or some growing seasons), the grains per spike were IU>CK (P>0.05), the total grains were IU>CK (P<0.05), and the 1000-kernels weight was IU<CK (P>0.05), indicating that the IU increased wheat yield by stabilizing the grains per spike and 1000-kernels weight, and increasing the spike number. In the production of photosynthetic matter, the culm number, leaf area index, dry matter accumulation at the main growth stages, the leaf area duration and crop growth rate in the main growth periods, and the culm fertility and grain leaf ratio were all expressed as IU>CK (significant differences were observed between treatments of some varieties or some growing seasons), which laid a material foundation for the yield increase of the IU. This paper not only summarized the technical approaches and basic technologies of IU but also discussed the development of IU from the aspects of integrated cultivation, unmanned cultivation, “agronomy-machinery-intelligence” fusion degree, key agronomy technology and comprehensive evaluation. 【Conclusion】 The yield under IU was equivalent or significantly increased to that under CK. And the high-yield cultivation of wheat was realized with less agricultural machinery and labor and unmanned operation, which was an effective way for the development of agricultural modernization production. In the future, multi-faceted collaborative innovation and investment should be strengthened to accelerate the application and large-scale promotion of this technology.

Changes in Seed Vigor and Physiological Index of Winter Wheat Under Natural Aging Condition

ZHANG Ling, CAO Lei, CAI Cheng, YAN XinYi, XIANG BoCai, AI Jia, ZHAN XinYang, SONG YouHong, ZHU YuLei

Scientia Agricultura Sinica. 2025, 58(5): 877-889. doi:10.3864/j.issn.0578-1752.2025.05.005

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【Objective】This study aimed to examine changes in seed vitality and physiological mechanisms under natural aging conditions in winter wheat, to analyze how different storage durations affect seed germination vitality, root growth, and antioxidant capacity, and to identify key physiological indicators of seed aging, so as to provide a basis for breeding winter wheat varieties with improved storability. 【Method】 The newly bred winter wheat cultivars, including Annong1589 (AN1589), Annong1687 (AN1687), and Annong179 (AN179), were used as experimental materials. The seeds were stored at room temperature to simulate natural aging condition. Samples were taken from seeds stored for 6 months, 18 months, and 30 months to measure seed vigor indices, such as germination rate, germination potential, germination index, vigor index, field emergence rate, and seedling root characteristics. Additionally, those key physiological indicators during the seed and seedling stages were analyzed, including electrical conductivity (Con), malondialdehyde (MDA) content, proline (Pro), superoxide dismutase (SOD) activity, catalase (CAT) activity, peroxidase (POD) activity, and the content of soluble sugars and soluble proteins in the seeds.【Result】Genotype had a significant effect on seed vigor and physiological indicators within the same storage period, with the interaction of cultivar and aging time having a notable impact on seed vigor. As storage time increased, the germination potential and root vigor of winter wheat seeds significantly declined. Germination index, vigor index, field emergence rate, and seedling stage indicators such as seedling height, fresh weight, and root fresh weight also showed a decreasing trend. Germination potential decreased most rapidly, with all three cultivars maintaining over 90% germination potential after 6 months of storage. After 18 months, the germination potential of AN1589, AN1687, and AN179 declined to 85.88%, 81.70%, and 88.58%, respectively. After 30 months of storage, the germination potential of all cultivars dropped below 80%, to 75.42%, 74.04%, and 79.17% of AN1589, AN1687, and AN179, respectively. This indicated a significant decline in seed vigor during natural aging. The study found that as the aging process progressed, the three cultivars continuously accumulated MDA and Pro, indicating increasing oxidative damage to the seed cell membranes. The initial MDA content in AN1589 was the lowest among the three cultivars, at 0.0427 μmol·g^-1. Moreover, AN1589 had the smallest increase in Pro content after 30 months of storage, at 22.43%. The activities of antioxidant enzymes of SOD, CAT, and POD, as well as the contents of soluble sugars and soluble proteins, decreased with increasing aging, indicating a gradual loss of antioxidant capacity and consumption of internal substances to support seed germination and seedling growth. 【Conclusion】Under natural aging conditions, the vigor of winter wheat seeds gradually decreased, closely related to the consumption of internal substances, increased oxidative damage, and weakened antioxidant capacity. The increase in MDA content and changes in antioxidant enzyme activity were important physiological indicators reflecting seed aging. AN1589 exhibited relatively stable vigor and physiological indicators under natural aging conditions, indicating strong storage tolerance.

Accumulation Characteristics of Anthocyanins in Black Rice Under Different Sowing Dates and Its Relationship with Meteorological Factors

XIAO ChangChun, WEI XinYu, ZENG YueHui, HUANG JianHong, XU XuMing

Scientia Agricultura Sinica. 2025, 58(5): 890-906. doi:10.3864/j.issn.0578-1752.2025.05.006

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【Objective】The dynamic process of anthocyanin accumulation in black rice grains under different sowing dates and its relationship with meteorological factors were studied to provide reference for the breeding of high anthocyanin black rice varieties and adjusting sowing dates to optimize favorable meteorological conditions for black rice growth, thereby enhancing anthocyanin content in grains.【Method】Three black rice varieties (Heinuo, Lirenzi and Yanziuo 35) with significant difference in anthocyanin content in grains were selected as materials, and 5 sowing dates (B1-B5) were set for each from 2022 to 2023. Samples were taken every 5 days after flowering to determine anthocyanin content in grains. The anthocyanin accumulation characteristics of black rice in grains at each sowing date were analyzed based on Logistic equation. The response mechanism of anthocyanin accumulation characteristics to meteorological factors was discussed by path analysis and regression analysis. 【Result】The sowing date changed the meteorological factors during the grain filling period of black rice, and shortened overall growth period of black rice by shortening the initial heading date. The genotype was found to be the primary determinant of anthocyanin content in black rice grains, and its accumulation characteristics (Wa, MRa, MeRa, T_MRa, W_MRa, Da) were most significantly influenced by variations in sowing dates. There is an observed trend of decreasing followed by increasing anthocyanin accumulation with delayed sowing dates. Latesowing is more conducive to the accumulation of anthocyanins in grains than early sowing. The process of anthocyanin accumulation in grains exhibited a “slow-fast-slow” pattern with peak values reached 9.79-15.35 days after flowering; this process was regulated by year type, sowing date and intervariety factors. Path analysis results indicated that Wa is mainly determined by MeRa and Da, with MeRa contributing the most to Wa. Furthermore, characteristic parameters of anthocyanin accumulation in grains showed significant negative correlations with light and temperature climatic factors during the effective filling period. Notably, air temperature had a significant influence on anthocyanin accumulation: as T_amax, D_heat and S_avg increased, Da decreased along with MeRa and Wa; an average temperature range of 25.0-27.4 ℃during the filling period was found to be optimal for anthocyanin accumulation in grains.【Conclusion】The effect of sowing date on anthocyanin accumulation in black rice grains reflects the comprehensive effect of meteorological factors, with temperature being the primary factor influencing anthocyanin accumulation. This, in turn, affects both the rate and duration of anthocyanin accumulation. Based on this, it is advisable to select black rice varieties with high anthocyanin content according to the planting system.Timely seeding from late June to early July can optimize the adaptation of meteorological elements during the grouting period, leading to an increase in grain anthocyanin content.

PGRP6 Negatively Regulates Symbiotic Bacteria to Prevent the Transovarial Transmission of RDV in Nephotettix cincticeps

XU YuanYuan, JIA DongSheng, BIN Yu, WEI TaiYun

Scientia Agricultura Sinica. 2025, 58(5): 907-917. doi:10.3864/j.issn.0578-1752.2025.05.007

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【Objective】Rice dwarf virus (RDV) causes rice dwarf disease, which is a significant threat to rice production in southern China. This study builds on prior research and employs molecular biology techniques to investigate the role of PGRP6 in regulating the vertical transmission of RDV from symbiotic bacteria within the vector insect Nephotettix cincticeps, thereby establishing a foundation for biological control strategies against rice viral diseases.【Method】Yeast two-hybrid technology was used to screen peptidoglycan recognition proteins (PGRPs) that interact with three proteins (prp, Nasuia porin, and P8) related to transovarial transmission, and the interaction was further confirmed by GST pull-down assay. Real-time fluorescence quantitative PCR technology was employed to assess the differential expression of related genes in nonviruliferous and viruliferous insects, as well as the changes in transcription levels of symbiotic bacteria following interference with PGRP6 and prp. Western blot experiments further confirmed the impact of RNAi treatment on symbiotic bacterial membrane proteins in insects. Following the reduction of PGRP6 expression by RNAi, variations were observed in the survival rate of N. cincticeps, the virus infection rate of the offspring, and the viral nucleic acid level of the infected offspring. Fluorescence in situ hybridization (FISH) and fluorescent immunolabeling techniques were employed to visualize the distribution of prp, PGRP6, Nasuia, and RDV. The functionality of the PGRP6 protein was assessed through inhibition zone assays and experiments on peptidoglycan degradation.【Result】PGRP6 interacted with prp, Nasuia porin, and P8. The expression levels of PGRP6 and prp were significantly elevated in RDV-infected N. cincticeps. Suppression of PGRP6 expression could facilitate the proliferation of symbiotic bacteria Nasuia and Sulcia, while inhibition of prp reduced the proliferation of symbiotic bacteria, acting oppositely to PGRP6. When both were interfered simultaneously, there was no significant effect on the proliferation of symbiotic bacteria. Treatment with dsPGRP6 significantly decreased the survival rate of N. cincticeps and enhanced the transmission of RDV to their offspring, resulting in a substantially higher viral load in the progeny compared to the control group. Immunofluorescence analysis revealed that PGRP6 co-localized with prp, Nasuia, and RDV within ovarian tissue, where prp and Nasuia exhibited a wrapping relationship. Ultimately, PGPR6 was identified as possessing antibacterial properties that inhibited the proliferation of symbiotic bacteria.【Conclusion】PGRP6 maintains the homeostasis of ovarian tissue in N. cincticeps by inhibiting the proliferation of symbiotic bacteria, which functionally antagonizes prp. This mechanism not only ensures the vitality of N. cincticeps but also regulates the transovarial transmission of RDV associated with bacterial symbionts.

The Roles of Heat Shock Protein Genes CfHsp70-1 and CfHsp70-2 in Enhancing the High-Temperature Tolerance after Heat Acclimation in Cryptolestes ferrugineus

CHEN ErHu, TANG JingJie, HU ShunJie, TANG PeiAn

Scientia Agricultura Sinica. 2025, 58(5): 918-928. doi:10.3864/j.issn.0578-1752.2025.05.008

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【Objective】Heat shock proteins (Hsps) are essential molecular chaperones in organisms and play the crucial roles in resisting adverse environmental stresses. Cryptolestes ferrugineus is a cosmopolitan pest of stored grains with strong environmental adaptability. This study aims to elucidate the crucial roles of heat shock protein genes CfHsp70-1 and CfHsp70-2 in the development of high-temperature tolerance in this pest.【Method】C. ferrugineus was acclimated to sub-lethal temperatures (37 and 42 ℃) for 2 h to examine the tolerance changes to lethal high temperature (50 ℃). Two key heat shock protein genes (CfHsp70-1 and CfHsp70-2) were identified based on the transcriptome data of C. ferrugineus, and the amino acid sequences and phylogenetic analysis of Hsp70 proteins were further conducted. The quantitative real-time PCR method was employed to analyze the expression patterns of CfHsp70-1 and CfHsp70-2 in response to sub-lethal heat stress. The RNA interference (RNAi) technology was used to silence CfHsp70-1 and CfHsp70-2, and then the changes in high-temperature tolerance of C. ferrugineus under different conditions were analyzed.【Result】The median lethal time (LT₅₀) of different C. ferrugineus populations was significantly increased under lethal heat temperature conditions (50 ℃) after acclimation of insects to sub-lethal temperatures (37 and 42 ℃) for 2 h, indicating a substantial enhancement of the high-temperature tolerance. The further sequence and phylogenetic analysis revealed that the amino acid sequences of CfHsp70-1 and CfHsp70-2 contained three conserved Hsp70 family signature motifs, and they clustered together with Hsp70 proteins of other Coleoptera insects. The results of quantitative real-time PCR analysis suggested that the expression levels of heat shock protein genes CfHsp70-1 and CfHsp70-2 were significantly up-regulated after acclimation to 37 and 42 ℃ for 2 h in C. ferrugineus. The gene functional analysis revealed that the high-temperature tolerance of C. ferrugineus was significantly reduced after the effective silencing of CfHsp70-1 and CfHsp70-2 via RNAi, that is, the mortality of the test insects at 50 ℃ increased significantly.【Conclusion】The heat shock protein genes CfHsp70-1 and CfHsp70-2 are involved in enhancing the high-temperature tolerance after heat acclimation of C. ferrugineus.

Effects of Water-Nitrogen Coupling on the Mineralization of Organic Carbon and Nitrogen for Mulched Farmland Soils in the Arid Regions of Northwest China

ZHAO TongTong, GU XiaoBo, TAN ChuanDong, YAN TingLin, LI XiaoYan, CHANG Tian, DU YaDan

Scientia Agricultura Sinica. 2025, 58(5): 929-942. doi:10.3864/j.issn.0578-1752.2025.05.009

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【Objective】This study aimed to clarify the effects of irrigation and nitrogen application on soil fertility, to explore the characteristics of organic carbon and nitrogen mineralization and their influencing factors in mulched farmland soils under different water and nitrogen conditions, so as to provide the theoretical basis for water and nitrogen control measures for mulched farmland crops in the Northwest of China.【Method】In this study, on the basis of five consecutive years of field trials of winter wheat-summer maize with nitrogen application in mulching, the soil samples were collected under three nitrogen application levels of 0 (N0), 180 kg·hm^-2 (N1) and 360 kg·hm^-2 (N2), and three soil moisture gradients, namely, 40% of the field holding capacity (W0), 60% of the field holding capacity (W1), and 100% of the field holding capacity (W2), were set up for indoor organic carbon and nitrogen mineralization. Then, the effect of water-nitrogen coupling on soil organic carbon and nitrogen mineralisation in mulched farmland were analyzed.【Result】Increasing water content significantly increased the cumulative soil carbon mineralization (C_min), carbon mineralization rate, cumulative net soil nitrogen mineralization (N_min), nitrogen mineralization rate, and potential mineralized nitrogen (N_p). C_min, N_min and N_p all showed a tendency to increase and then decrease with increasing nitrogen application. At the end of incubation, C_min was the highest under N1W1 treatment (1 781.00 mg·kg^-1), which was significantly higher than that under other treatments(N0W0, N0W1, N0W2, N1W0, N1W2, N2W0, N2W1, N2W2) by 8.8% to 51.8%, respectively, and its N_min was also maintained at a relatively high level (29.52 mg·kg^-1), while the potential mineralized carbon (5 883.79 mg·kg^-1) and N_p(30.74 mg·kg^-1) were also maintained at a relatively high level. The random forest algorithm indicated that soil microbial carbon (MBC), soil microbial nitrogen, dissolved organic carbon, organic carbon, and total dissolved nitrogen were the important factors affecting C_min and N_min. MBC showed a tendency of increasing and then decreasing with the increase of soil moisture, and the MBC content under W1 significantly increased by 60.1%-340.0% and 3.1%-6.7%, respectively. The structural equations showed that soil moisture had a direct positive effect (0.70) and an indirect positive effect (0.55) on soil carbon mineralization, while the nitrogen application had a direct positive effect (0.90) and an indirect negative effect (0.24) on soil nitrogen mineralization.【Conclusion】From the perspective of soil carbon and nitrogen mineralization, this study recommended 60% field capacity and 180 kg N·hm^-2 as suitable water and nitrogen regulation strategies for mulched farmland in the dryland of Northwest China.

Effects of Long-Term Chemical Phosphorus Application on Phosphorus Morphology and Phosphatase Activity of Different Aggregates Sizes in Calcareous Brown Soil

HUANG ShaoHui, YANG HuiMin, YANG JunFang, YANG WenFang, NIE HaoLiang, ZHANG Jing, XING SuLi, WANG JingXia, YANG YunMa, JIA LiangLiang

Scientia Agricultura Sinica. 2025, 58(5): 943-955. doi:10.3864/j.issn.0578-1752.2025.05.010

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【Objective】This study aimed to explore the differences of phosphorus (P) morphology and phosphatase activity in different aggregates sizes, and to clarify the change mechanism of soil P component contents and availability under different long-term chemical P application rates, so as to provide the theoretical support for the efficient utilization of soil P and sustainable agricultural development. 【Method】Long-term field experiment with different chemical P fertilizer application rates were established in calcareous brown soil. Soil samples in the topsoil (0-20 cm) were collected, which were treated with three gradients of P fertilizer application rates of 0 (P0, control), 120 (P120) and 210 (P210) kg P₂O₅·hm^-2 annually. The soil aggregate content, P component contents and alkaline phosphatase activity of different soil aggregate sizes were determined. The effects of long-term chemical P application on P morphology and phosphatase activity of different aggregates sizes in calcareous brown soil were analyzed. 【Result】Compared with P0, the stability and P component content of different aggregates in calcareous brown soil were significantly improved after long-term P application. The content of acid-soluble inorganic P (Pi-HCl) was the highest in different P components, while the content of water-soluble P (Pi-H₂O), sodium bicarbonate organic P (Po-NaHCO₃) and sodium hydroxide inorganic P (Pi-NaOH) were relatively low. The changes of inorganic P pools in all aggregates were higher than those in organic P pools under different treatments. Compared with P120 treatment, the inorganic P content in large aggregate (>2 mm), small aggregate (0.25-2 mm) and micro-aggregate (<0.25 mm) were reduced by 21.5%, 27.0% and 18.7%, respectively, and the organic P content decreased by 15.6%, 12.8% and 12.2%, respectively. There were significant differences in organic P contents among different aggregate sizes and P application rates. The labile P (LP) content changed largest in different P availability forms. There were extremely significant differences among different particle size and P application rate. The contribution rate of inorganic P in large aggregates (Pi, >2 mm) was the highest, ranging from 27.6% to 38.3%, while that of organic P in small aggregates (Po, 0.25-2 mm) was the lowest, ranging from 2.9% to 4.9%. The contribution rate of stable P (SP) content to total P content was the highest, accounting for 84.3-91.2%. The contribution rate of SP in large aggregates (SP, >2 mm) was the highest, ranging from 52.6% to 55.2%. Soil phosphatase activity was significantly different in soil aggregates, which increased with the increase of aggregate size. In large aggregates, the phosphatase activity was significantly increased with the increase of P application rate. In small aggregates, the phosphatase activity of P120 treatment was the highest, and it was a significant difference between them. However, there was no significant difference in phosphatase activity of micro-aggregates between different treatments. The results of correlation analysis showed that the aggregate size was negatively correlated with the content of organic P content significantly, and positively correlated with the activity of alkaline phosphatase significantly. Structural equation model (SEM) analysis results showed that the P application rate could directly affect the inorganic P content in soil and then affect the P availability in soil. Soil aggregate structural could direct influence on the organic P content and alkaline phosphatase activities, and indirectly affect the inorganic P content and P availability. 【Conclusion】 Long-term application of chemical P fertilizer significantly increased the content of aggregates, the content of P components in each size aggregates, and the activity of alkaline phosphatase in calcareous brown soil. The large aggregates contributed the most to the soil P availability. P application rate and soil aggregate regulated soil P availability synergistically. Therefore, the scientific P application and increasing the proportion of soil large aggregates were important to improve the availability of soil P utilization.

Sulfur Concentration and Distribution in Wheat Grain Sampled from Farmers’ Fields in Main Wheat Production Regions of China and Its Affecting Factors

SHE WenTing, SUN RuiQing, DANG HaiYan, LI WenHu, ZHANG Feng, TIAN Yi, XU JunFeng, DING YuLan, WANG ZhaoHui

Scientia Agricultura Sinica. 2025, 58(5): 956-974. doi:10.3864/j.issn.0578-1752.2025.05.011

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【Objective】It was of great significance to clarify the grain sulfur (S) concentration, its distribution in flour and bran, and relevant affecting agronomy, nutrition, soil physicochemical and fertilization factors for regulating grain S nutrition, and optimizing S management regionally over major wheat production regions of China.【Method】During the two wheat growing seasons from 2021 to 2023, 445 wheat plants and soil samples were collected from typical farmers’ fields in 18 major wheat production provinces (municipalities or districts) in China, to analyze the relationship of grain, flour and bran S concentrations with yield, biomass, harvest index and yield components, S accumulation and distribution in shoot, as well as soil physicochemical properties and fertilization.【Result】Grain S concentrations of farmers in main wheat production regions of China ranged from 1.00 to 2.31 g·kg^-1, with an average of 1.59 g·kg^-1. The flour and bran S concentration were 0.55-2.05 and 0.54-4.26 g·kg^-1, respectively, with an average of 1.33 and 2.03 g·kg^-1, respectively. About half of the samples were at low grain S concentration level. Sulfur concentration in wheat grain and flour from wheat-maize and dryland wheat production regions were higher than that from spring wheat and rice-wheat production regions. The bran S concentration was higher than that of flour, both increased with the grain S concentration. The increase of wheat yield and thousand kernel weight was beneficial to the improvement of grain S concentration. For each 1.0 t·hm^-2 increase of yield, the grain S concentration increased by 0.01 g·kg^-1; and for each 1.0 g increase of the thousand kernel weight, the grain S concentration increased by 0.003 g·kg^-1. A positive correlation was found between the grain and flour S concentration and S accumulation in all wheat organs. With the increase of grain S concentration, its distribution ability to flour decreased while the distribution to bran developed. However, in the dryland wheat production region, the increase of grain S accumulation was lower than that of yield, thus the grain S concentration decreased with the yield increase. Soil pH, nitrate nitrogen (N) and phosphorus fertilizer application all showed significant effects on grain S concentration. Grain S concentration increased by 0.02, 0.0004 and 0.0004 g·kg^-1, respectively, with each unit increase of these three parameters. The main soil factors affecting flour S concentration were pH, ammonium N, and available iron (Fe). For each 1.0 unit increase of pH, the flour S concentration increased by 0.04 g·kg^-1; while for each 1.0 mg·kg^-1 increase of soil ammonium N and available Fe, the flour S concentration decreased by 0.0003 and 0.0005 g·kg^-1, respectively. 【Conclusion】In brief, the grain S concentrations were of great increase potentials for farmers’ wheat in main wheat production regions of China. It was obviously a vital approach to regulate grain S concentration and its distribution in flour and bran, by optimizing wheat grain yield components, adjusting soil pH, coordinating the soil nitrate N, ammonium N and available Fe supply, and applying nitrogen and phosphorus fertilizers rationally together with increased S fertilizer application, for improving soil S availability and balancing wheat S uptake and yield formation to harvest high yield with optimized grain S nutrition.

Effects of LED Supplementary Lighting on Production and Leaf Physiological Properties of Substrate-Cultivated Strawberry in Chinese Solar Greenhouse

ZHANG Han, ZHANG YuQi, LI JingLai, XU Hong, LI WeiHuan, LI Tao

Scientia Agricultura Sinica. 2025, 58(5): 975-990. doi:10.3864/j.issn.0578-1752.2025.05.012

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【Objective】The objective of this research is to clarify the effects of LED supplementary lighting on production and leaf physiological characteristics of substrate-cultivated strawberry, and develop a light control strategy for strawberry cultivation in Chinese solar greenhouses, which will provide theoretical basis and technical support for improving the quality and efficiency of strawberry cultivation in winter and spring seasons in China when solar radiation is low.【Method】Strawberry cultivar ‘HongYan’ was grown in a Chinese solar greenhouse with substrate cultivation, and LED supplementary lighting was provided during the early stage of flower bud differentiation (lamps were installed approximately 15 cm above the canopy). The experiments were set up with different light intensity experiments (photosynthetic photon flux density (PPFD) of 254, 367, and 492 μmol·m^-2·s^-1, corresponding to the power of 80, 120 and 160 W, respectively), the different light quality experiments (red/blue 9/1, red/blue 1/1, and white light, PPFD of 360-390 μmol·m^-2·s^-1, with the same power of 120 W), and the different supplementary lighting duration and control strategy experiments (i.e. dynamic supplementary lighting for 10 h and continuous supplementary lighting for 5 h, referred to as DL10 and CL5 hereafter, respectively, both using 120 W white LED, PPFD of 367 μmol·m^-2·s^-1, lamp on/off strategy of DL10 treatment was the same as the light intensity and quality experiments, lamp of CL5 treatment was continuously turned on during the time period of 8: 00-13: 00), and the control was no supplementary lighting treatment. During the experiment, strawberry production, physiological and biochemical index of leaves and fruits, as well as the leaf photosynthetic parameters were measured, and the power usage efficiency was also analyzed.【Result】Compared with the control, all supplementary lighting treatments increased strawberry yield and accelerated harvest time by ~10 d. In the light intensity experiment, the yield of 160 W treatment increased by 41.9%, which was slightly but not significantly higher than that of 80 W and 120 W treatments. In the light quality experiment, the yield of red/blue 9/1, red/blue 1/1 and white light treatments increased by 55.9%, 44.1%, and 33.1%, respectively, compared to the control. In addition, the yield of DL10 treatment increased by 16% compared to CL5 treatment. Supplementary lighting increased yield due to the higher number of fruits per plant. Supplementary lighting reduced fruit water content and increased leaf thickness, but had no significant effect on leaf physiological and biochemical parameters. Supplementary lighting in the morning and afternoon significantly improved stomatal conductance, which was beneficial for photosynthesis. However, in the light intensity experiment, the maximum photosynthetic capacity of the leaves treated with 160 W was significantly lower than that of 120 W treatment, and the stomatal conductance was also lower than that of the control. Regarding the power usage efficiency, red/blue 9/1 (120 W) treatment was the highest, while the 160 W white light was the lowest among all treatments. The power usage efficiency of DL10 treatment was 2.6 times that of CL5 treatment.【Conclusion】Supplementary lighting can significantly improve strawberry production and accelerate harvest time in winter and spring seasons when solar light is limited, appropriate supplementary light intensity is crucial for yield formation, and supplementing with a high fraction of red light has the best effect on strawberry production, dynamic supplementary light control strategy can significantly improve the power usage efficiency.

Cloning and Expression of BcDET2 Gene and Functional of Its Regulatory Effect on Bolting and Flowering in Wucai (Brassica campestris L.)

ZHENG YaQin, LIU XueQing, WU SiWen, TANG XiaoYan, YANG DanNi, WANG YongKang, AHMAD Aftab, KHAN Afrsyab, WANG ChengGang, CHEN GuoHu

Scientia Agricultura Sinica. 2025, 58(5): 991-1003. doi:10.3864/j.issn.0578-1752.2025.05.013

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【Objective】 The steroid 5α-reductase gene BcDET2 (DE-ETIOLATED 2), involved in the brassinosteroid (BR) biosynthesis pathway, was cloned and analyzed in Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen). The functions of BcDET2 in regulating bolting and flowering were investigated through genetic transformation, thereby providing a molecular basis for the genetic breeding of Wucai. 【Method】The BcDET2 was obtained via homologous cloning, based on the DET2 (BraA10g023600.3C) sequence from the Chinese cabbage (B. rapa) genome. Bioinformatics analyses were performed using online tools such as Expasy, SOPMA, SWISS-MODEL, and TMHMM. The expression pattern of BcDET2 was analyzed using quantitative real-time PCR (qRT-PCR). Subcellular localization of BcDET2 was determined through Agrobacterium-mediated transient transformation in tobacco (Nicotiana tabacum) cells. The role of the BcDET2 in regulating bolting and flowering was studied through genetic transformation of Wucai. Yeast two-hybrid (Y2H) assay was utilized to screen and identify BcDET2-interacting proteins, followed by qRT-PCR analysis to examine the response of BrbHLH96 to vernalization and its expression levels in BcDET2 transgenic plants. 【Result】The cDNA sequence of BcDET2 (825 bp), encoding 274 amino acids, was successfully obtained through homologous cloning. Bioinformatics analysis indicated that the BcDET2 protein predominantly consists of α-helices, is predicted to be a weakly hydrophilic, non-secretory membrane protein, and contains 39 phosphorylation sites and 5 transmembrane domains. Phylogenetic analysis revealed that BcDET2 shares a close evolutionary relationship with DET2 from Brassica rapa and Brassica napus. Subcellular localization showed fluorescent signals of BcDET2 in both the nucleus and cell membrane. qRT-PCR analysis indicated the highest expression level of BcDET2 at 15 days of vernalization, suggesting its responsiveness to the vernalization process. Functional assay demonstrated that overexpression of BcDET2 significantly promoted early flowering in transgenic Wucai plants. Y2H analysis revealed that BcDET2 interacts with the transcription factor BcBHLH96, which exhibited high transcription levels in the late vernalization stage and significantly increased expression in BcDET2 transgenic plants. 【Conclusion】In this study, the cDNA of BcDET2 from Wucai was cloned, and it is likely involved in the regulation of bolting and flowering in Wucai via the vernalization pathway.

Formation and Structure of Wheat Bran Polysaccharide-Golden Threadfin Bream Surimi Blended Gel

ZHANG Tao, WANG Huan, XIE HongKai, CHEN YinJi

Scientia Agricultura Sinica. 2025, 58(5): 1004-1016. doi:10.3864/j.issn.0578-1752.2025.05.014

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【Objective】This study aimed to investigate the impact of wheat bran polysaccharide on the physical properties and microstructure of surimi gel, and to elucidate the mechanism by which the concentration of wheat bran polysaccharide affects surimi gel characteristics, so as to provide the theoretical support for enhancing surimi gel quality through the utilization of wheat bran by-products.【Method】Different concentrations of wheat bran polysaccharide were added to threadfin bream (Nemipterus virgatus) surimi to prepare composite gels. The effects of wheat bran polysaccharide on the characteristics and structures of surimi gel were investigated using texture analysis, rheological test, molecular interaction measurement, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM).【Result】The impact of wheat bran polysaccharide on the macroscopic physical properties and microstructures of surimi gel exhibited a pronounced concentration dependence. As the amount of wheat bran polysaccharide increased, the rheological parameters (G' and G" values), water holding capacity, texture profile analysis (TPA) values, gel strength, and water distributions of the blended system were all initially elevated and then declined, achieving the maximum improvement at the addition level of 1.0%. FTIR revealed that the secondary structure of proteins during the heat-induced gelation of surimi was closely related to the polysaccharide content: with increasing polysaccharide content, the α-helix content gradually decreased, while β-turn and β-sheet content showed an upward trend (P<0.05). However, when the polysaccharide addition exceeded 1.0%, the α-helix content gradually increased again, while β-turn and β-sheet content exhibited downtrends. This may be due to the concentration-dependent effect of polysaccharides on the disulfide bonds and electrostatic interactions, which were the primary intermolecular forces within the protein gel, leading to the changes in its secondary structure. SDS-PAGE electrophoresis showed that when the polysaccharide addition was over 1.0%, the actin (AC) and myosin heavy chain (MHC) bands became significantly lighter, with dark bands accumulating at the top of the stacking gel. SEM observations of the microstructure of surimi gels revealed that as the polysaccharide added, the pore size within the surimi gel network gradually reduced. When the polysaccharide addition reached 1.0%, the surimi gel network structure was the most compact, with the fractal dimension (D_f) reaching a maximum value of 2.8657. Further addition of the polysaccharide leaded to phase separation in the gel system, and extensive self-aggregation of polysaccharides disrupted the surimi protein gel network, resulting in a decrease of D_f.【Conclusion】Wheat bran polysaccharide with the concentration of 1.0% to the surimi could be used as a filler as well we water retention agent in the three-dimensional network structure of surimi protein, and induce the formation of a more uniform and dense gel matrix, which could effectively improve the gel quality of surimi.

Research Progress on Mechanisms Interpretation and Prediction Methods for Heterosis of Livestock

SUN YanYan, NI AiXin, YANG HanHan, YUAN JingWei, CHEN JiLan

Scientia Agricultura Sinica. 2025, 58(5): 1017-1031. doi:10.3864/j.issn.0578-1752.2025.05.015

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Heterosis is a phenomenon where the offspring of genetically distinct populations exhibit superior vitality, reproductive capacity, and adaptability compared with the average of their parent populations, which is an important genetic resource. Heterosis plays a significant role in modern agriculture, contributing to increase yields and quality of livestock and crops, rapidly improve traits, accelerate the breeding of new varieties, and enhance genetic diversity, thereby efficiently boosting the production of animal husbandry and agriculture while reducing costs. Despite the discovery of heterosis is over a century ago, the elucidation of its genetic basis lags far behind its application in agricultural production. The study of the complex formation mechanism of heterosis is a classic and an active topic in the field of genetics and breeding, but the clear conclusions remain limited. In response to the characteristics of heterosis, scientists have successively proposed various hypotheses for its formation, such as the dominance hypothesis, overdominance hypothesis, and epistasis hypothesis, revealing that the genetic basis of heterosis was non-additive genetic effects. However, these hypotheses are based on the effects of single genes, which are overly idealized and simplistic. Explorations at different levels, such as DNA, RNA, and proteins, have successively discovered the coexistence of multiple genetic effects. Particularly in hybrid crops like rice and corn, the related researches have been continuously identified the loci of heterosis effects, enriched the understanding of the formation mechanism for heterosis in crops, and promoted the transformation of crop breeding technologies, such as precise molecular design breeding. Heterosis is also widely applied in the breeding of livestock and poultry. In developed countries with advanced animal husbandry, over 80% of commercial pork, chicken, and eggs are obtained from hybrid breeds. To efficiently apply heterosis in production for animal husbandry, it is necessary to predict heterosis in advance. New methods, such as the inter- and intra-group phenotypic variance ratio prediction, hybrid heritability prediction, and molecular marker prediction, have been developed to solve the long experimental cycle, environmental sensitivity, and high human and financial costs associated with traditional hybridization experiments for predicting heterosis. However, the accuracy of these prediction methods is limited. Heterosis involves in interaction of multiple levels, and because of the complex genetic background and long breeding cycle, it is still a big challenge for the study of the heterosis formation mechanism and accurate prediction methods. In recent years, the gradual application of sequencing technology has provided a new perspective for understanding the molecular regulatory network of heterosis in livestock and poultry. QTL mapping and genome-wide association study reveal the molecular mechanism of heterosis at the genomic level, and the identified molecular makers are applied in selection and breeding. Combined with multi-omics researches, such as transcriptomics and metabolomics, the key functional genes, variations, and metabolites affecting heterosis can be more precisely located, which facilitate hybrid improvement. This review elaborated the research progress in the formation mechanism and prediction methods for heterosis in the field of livestock and poultry. For looking forward to future, the researches will gradually clarify the complex mechanism of heterosis by integrating multi-omics sequencing data and bioinformatics analysis, in order to identify genes and molecular markers related to heterosis, and innovate new prediction methods, which will provide a more accurate direction for the utilization of heterosis.

Establishment and Application of Sandwich ELISA Method for Detecting Lawsonia intracellularis

HONG RunJing, ZHOU Hong, LIN HuiXing, FAN HongJie

Scientia Agricultura Sinica. 2025, 58(5): 1032-1042. doi:10.3864/j.issn.0578-1752.2025.05.016

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【Objective】This study established a sandwich ELISA method for detecting Lawsonia intracellularis (L. intracellularis), which could be used to measure antigen content in the development of porcine proliferative enteropathy (PPE) inactivated vaccines.【Method】L. intracellularis was used as the immunogen, and polyclonal antibody against L. intracellularis was obtained by immunizing New Zealand rabbit. Additionally, 6-week-old BALB/c mice were immunized to prepare monoclonal antibodies against L. intracellularis by hybridoma cell technique. The antibodies were identified by Western Blot, indirect immunofluorescence (IFA) and immunoperoxidase monolayer assay (IPMA). A sandwich ELISA method was established for detecting L. intracellularis, using monoclonal antibody as capture antibody and polyclonal antibody as detection antibody. the optimized reaction conditions and the specificity, sensitivity and reproducibility of the method were evaluated. The optimized sandwich ELISA was used to quantify inactivated L. intracellularis.【Result】The polyclonal antibody titer reached 1﹕409 600. After three rounds of subcloning, three positive hybridoma cell lines were screened, named 1B7, 3C7, and 4F10, respectively, and the titer of ascites were all 1﹕204 800. All three monoclonal antibodies had specific reactions with L. intracellularis and showed no cross-reactivity with E.coli O157:H7, S. choleraesuis, and S. typhimurium. The optimal conditions for the sandwich ELISA were as follows: capture antibody was 4F10; working concentrations for capture and detection antibodies were 1.25 and 0.625 μg·mL^-1, respectively. The coating solution was phosphate buffer, and the coating condition was 4 ℃ for 14 h. Blocking solution was 1% gelatin and blocking condition was 37 ℃ for 1.5 h. The reaction condition of antigen was 37 ℃ for 1 h, and the reaction condition for detection antibody was 37 ℃ for 0.5 h. HRP-antibody dilution was 1﹕16 000 and reaction condition of HRP-antibody was 37 ℃ for 1.5 h. The developing time was 10 min at room temperature. This method was negative for other common intestinal pathogens, with a minimum detection limit of 1×10⁵ L. intracellularis/mL. The coefficient of variation for intra and inter assay were less than 10%. A linear relationship was observed between L. intracellularis concentration and P/N values in the range of 1×10⁵ - 1×10⁸, with the standard equation : y=2.7349x-11.643 (R²=0.9966). This method was used to quantify the antigen content of PPE inactivated vaccines in our laboratory. The quantitative results indicated that the antigen content remained basically unchanged before and after inactivation, with two groups of vaccine samples demonstrating good intra-assay reproducibility. The results of 120 clinical fecal samples of swine showed that 62 positive samples and 58 negative samples were detected using the sandwich ELISA method; 67 positive samples and 53 negative samples were detected using nested PCR method. The positive coincidence rate of the two methods was 86.6% and the negative coincidence rate was 92.5%, the total coincidence rate of the two methods was 89.2%, and the kappa value was 0.78.【Conclusion】Monoclonal antibody and polyclonal antibody against L.intracellularis were successfully prepared, and a sandwich ELISA method for detecting L.intracellularis was successfully established and optimized. This method had good specificity and reproducibility, with a detection limit of 1×10⁵mL^-1 for L.intracellularis, which could be used for the quantification of antigen content in the production of PPE inactivated vaccine and detection of PPE clinical samples.

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