Dissecting the genetic basis of heterosis in elite super-hybrid rice.

Y900 is one of the top hybrid rice (Oryza sativa) varieties, with its yield exceeding 15 t·hm-2. To dissect the mechanism of heterosis, we sequenced the male parent line R900 and female parent line Y58S using long-read and Hi-C technology. High-quality reference genomes of 396.41 Mb and 398.24 Mb were obtained for R900 and Y58S, respectively. Genome-wide variations between the parents were systematically identified, including 1,367,758 single-nucleotide polymorphisms, 299,149 insertions/deletions, and 4,757 structural variations. The level of variation between Y58S and R900 was the lowest among the comparisons of Y58S with other rice genomes. More than 75% of genes exhibited variation between the two parents. Compared with other two-line hybrids sharing the same female parent, the portion of Geng/japonica (GJ)-type genetic components from different male parents increased with yield increasing in their corresponding hybrids. Transcriptome analysis revealed that the partial dominance effect was the main genetic effect that constituted the heterosis of Y900. In the hybrid, both alleles from the two parents were expressed, and their expression patterns were dynamically regulated in different tissues. The cis-regulation was dominant for young panicle tissues, while trans-regulation was more common in leaf tissues. Overdominance was surprisingly prevalent in stems and more likely regulated by the trans-regulation mechanism. Additionally, R900 contained many excellent GJ haplotypes, such as NARROW LEAF1, Oryza sativa SQUAMOSA PROMOTER BINDING PROTEIN-LIKE13, and Grain number, plant height, and heading date8, making it a good complement to Y58S. The fine-tuned mechanism of heterosis involves genome-wide variation, GJ introgression, key functional genes, and dynamic gene/allele expression and regulation pattern changes in different tissues and growth stages.

OsPDCD5 negatively regulates plant architecture and grain yield in rice.

Plant architecture is an important agronomic trait that affects crop yield. Here, we report that a gene involved in programmed cell death, OsPDCD5, negatively regulates plant architecture and grain yield in rice. We used the CRISPR/Cas9 system to introduce loss-of-function mutations into OsPDCD5 in 11 rice cultivars. Targeted mutagenesis of OsPDCD5 enhanced grain yield and improved plant architecture by increasing plant height and optimizing panicle type and grain shape. Transcriptome analysis showed that OsPDCD5 knockout affected auxin biosynthesis, as well as the gibberellin and cytokinin biosynthesis and signaling pathways. OsPDCD5 interacted directly with OsAGAP, and OsAGAP positively regulated plant architecture and grain yield in rice. Collectively, these findings demonstrate that OsPDCD5 is a promising candidate gene for breeding super rice cultivars with increased yield potential and superior quality.

The OsWRKY72-OsAAT30/OsGSTU26 module mediates reactive oxygen species scavenging to drive heterosis for salt tolerance in hybrid rice.

Hybrid rice (Oryza sativa) generally outperforms its inbred parents in yield and stress tolerance, a phenomenon termed heterosis, but the underlying mechanism is not completely understood. Here, we combined transcriptome, proteome, physiological, and heterosis analyses to examine the salt response of super hybrid rice Chaoyou1000 (CY1000). In addition to surpassing the mean values for its two parents (mid-parent heterosis), CY1000 exhibited a higher reactive oxygen species scavenging ability than both its parents (over-parent heterosis or heterobeltiosis). Nonadditive expression and allele-specific gene expression assays showed that the glutathione S-transferase gene OsGSTU26 and the amino acid transporter gene OsAAT30 may have major roles in heterosis for salt tolerance, acting in an overdominant fashion in CY1000. Furthermore, we identified OsWRKY72 as a common transcription factor that binds and regulates OsGSTU26 and OsAAT30. The salt-sensitive phenotypes were associated with the OsWRKY72paternal genotype or the OsAAT30maternal genotype in core rice germplasm varieties. OsWRKY72paternal specifically repressed the expression of OsGSTU26 under salt stress, leading to salinity sensitivity, while OsWRKY72maternal specifically repressed OsAAT30, resulting in salinity tolerance. These results suggest that the OsWRKY72-OsAAT30/OsGSTU26 module may play an important role in heterosis for salt tolerance in an overdominant fashion in CY1000 hybrid rice, providing valuable clues to elucidate the mechanism of heterosis for salinity tolerance in hybrid rice.

Molecular regulation of ZmMs7 required for maize male fertility and development of a dominant male-sterility system in multiple species.

Understanding the molecular basis of male sterility and developing practical male-sterility systems are essential for heterosis utilization and commercial hybrid seed production in crops. Here, we report molecular regulation by genic male-sterility gene maize male sterility 7 (ZmMs7) and its application for developing a dominant male-sterility system in multiple species. ZmMs7 is specifically expressed in maize anthers, encodes a plant homeodomain (PHD) finger protein that functions as a transcriptional activator, and plays a key role in tapetal development and pollen exine formation. ZmMs7 can interact with maize nuclear factor Y (NF-Y) subunits to form ZmMs7-NF-YA6-YB2-YC9/12/15 protein complexes that activate target genes by directly binding to CCAAT box in their promoter regions. Premature expression of ZmMs7 in maize by an anther-specific promoter p5126 results in dominant and complete male sterility but normal vegetative growth and female fertility. Early expression of ZmMs7 downstream genes induced by prematurely expressed ZmMs7 leads to abnormal tapetal development and pollen exine formation in p5126-ZmMs7 maize lines. The p5126-ZmMs7 transgenic rice and Arabidopsis plants display similar dominant male sterility. Meanwhile, the mCherry gene coupled with p5126-ZmMs7 facilitates the sorting of dominant sterility seeds based on fluorescent selection. In addition, both the ms7-6007 recessive male-sterility line and p5126-ZmMs7M dominant male-sterility line are highly stable under different genetic germplasms and thus applicable for hybrid maize breeding. Together, our work provides insight into the mechanisms of anther and pollen development and a promising technology for hybrid seed production in crops.

An R2R3 MYB transcription factor confers brown planthopper resistance by regulating the phenylalanine ammonia-lyase pathway in rice.

Brown planthopper (BPH) is one of the most destructive insects affecting rice (Oryza sativa L.) production. Phenylalanine ammonia-lyase (PAL) is a key enzyme involved in plant defense against pathogens, but the role of PAL in insect resistance is still poorly understood. Here we show that expression of the majority of PALs in rice is significantly induced by BPH feeding. Knockdown of OsPALs significantly reduces BPH resistance, whereas overexpression of OsPAL8 in a susceptible rice cultivar significantly enhances its BPH resistance. We found that OsPALs mediate resistance to BPH by regulating the biosynthesis and accumulation of salicylic acid and lignin. Furthermore, we show that expression of OsPAL6 and OsPAL8 in response to BPH attack is directly up-regulated by OsMYB30, an R2R3 MYB transcription factor. Taken together, our results demonstrate that the phenylpropanoid pathway plays an important role in BPH resistance response, and provide valuable targets for genetic improvement of BPH resistance in rice.

Novel Salinity-Tolerant Third-Generation Hybrid Rice Developed via CRISPR/Cas9-Mediated Gene Editing.

Climate change has caused high salinity in many fields, particularly in the mud flats in coastal regions. The resulting salinity has become one of the most significant abiotic stresses affecting the world's rice crop productivity. Developing elite cultivars with novel salinity-tolerance traits is regarded as the most cost-effective and environmentally friendly approach for utilizing saline-alkali land. To develop a highly efficient green strategy and create novel rice germplasms for salt-tolerant rice breeding, this study aimed to improve rice salinity tolerance by combining targeted CRISPR/Cas9-mediated editing of the OsRR22 gene with heterosis utilization. The novel alleles of the genic male-sterility (GMS) and elite restorer line (733Srr22-T1447-1 and HZrr22-T1349-3) produced 110 and 1 bp deletions at the third exon of OsRR22 and conferred a high level of salinity tolerance. Homozygous transgene-free progeny were identified via segregation in the T2 generation, with osrr22 showing similar agronomic performance to wild-type (733S and HZ). Furthermore, these two osrr22 lines were used to develop a new promising third-generation hybrid rice line with novel salinity tolerance. Overall, the results demonstrate that combining CRISPR/Cas9 targeted gene editing with the "third-generation hybrid rice system" approach allows for the efficient development of novel hybrid rice varieties that exhibit a high level of salinity tolerance, thereby ensuring improved cultivar stability and enhanced rice productivity.

The OsEIL1-OsERF115-target gene regulatory module controls grain size and weight in rice.

Grain size is one of the essential determinants of rice yield. Our previous studies revealed that ethylene plays an important role in grain-size control; however, the precise mechanism remains to be determined. Here, we report that the ethylene response factor OsERF115 functions as a key downstream regulator for ethylene-mediated grain development. OsERF115 encodes an AP2/ERF-type transcriptional factor that is specifically expressed in young spikelets and developing caryopses. Overexpression of OsERF115 significantly increases grain length, width, thickness and weight by promoting longitudinal elongation and transverse division of spikelet hull cells, as well as enhancing grain-filling activity, whereas its knockout mutations lead to the opposite effects, suggesting that OsERF115 positively regulates grain size and weight. OsERF115 transcription is strongly induced by ethylene, and OsEIL1 directly binds to the promoter to activate its expression. OsERF115 acts as a transcriptional repressor to directly or indirectly modulate a set of grain-size genes during spikelet growth and endosperm development. Importantly, haplotype analysis reveals that the SNP variations in the EIN3-binding sites of OsERF115 promoter are significantly associated with the OsERF115 expression levels and grain weight, suggesting that natural variations in the OsERF115 promoter contribute to grain-size diversity. In addition, the OsERF115 orthologues are identified only in grass species, implying a conserved and unique role in the grain development of cereal crops. Our results provide insights into the molecular mechanism of ethylene-mediated grain-size control and a potential strategy based on the OsEIL1-OsERF115-target gene regulatory module for genetic improvement of rice yield.

Allelic Diversification of the Wx and ALK Loci in Indica Restorer Lines and Their Utilisation in Hybrid Rice Breeding in China over the Last 50 Years.

Hybrid rice technology has been used for more than 50 years, and eating and cooking quality (ECQ) has been a major focus throughout this period. Waxy (Wx) and alkaline denaturation (ALK) genes have received attention owing to their pivotal roles in determining rice characteristics. However, despite significant effort, the ECQ of restorer lines (RLs) has changed very little. By contrast, obvious changes have been seen in inbred rice varieties (IRVs), and the ECQ of IRVs is influenced by Wx, which reduces the proportion of Wxa and increases the proportion of Wxb, leading to a decrease in amylose content (AC) and an increase in ECQ. Meanwhile, ALK is not selected in the same way. We investigated Wx alleles and AC values of sterile lines of female parents with the main mating combinations in widely used areas. The results show that almost all sterile lines were Wxa-type with a high AC, which may explain the low ECQ of hybrid rice. Analysis of hybrid rice varieties and RLs in the last 5 years revealed serious homogenisation among hybrid rice varieties.

A novel strategy for creating a new system of third-generation hybrid rice technology using a cytoplasmic sterility gene and a genic male-sterile gene.

Heterosis utilization is the most effective way to improve rice yields. The cytoplasmic male-sterility (CMS) and photoperiod/thermosensitive genic male-sterility (PTGMS) systems have been widely used in rice production. However, the rate of resource utilization for the CMS system hybrid rice is low, and the hybrid seed production for the PTGMS system is affected by the environment. The technical limitations of these two breeding methods restrict the rapid development of hybrid rice. The advantages of the genic male-sterility (GMS) rice, such as stable sterility and free combination, can fill the gaps of the first two generations of hybrid rice technology. At present, the third-generation hybrid rice breeding technology is being used to realize the application of GMS materials in hybrid rice. This study aimed to use an artificial CMS gene as a pollen killer to create a smart sterile line for hybrid rice production. The clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) technology was used to successfully obtain a CYP703A3-deficient male-sterile mutant containing no genetically modified component in the genetic background of indica 9311. Through young ear callus transformation, this mutant was transformed with three sets of element-linked expression vectors, including pollen fertility restoration gene CYP703A3, pollen-lethality gene orfH79 and selection marker gene DsRed2. The maintainer 9311-3B with stable inheritance was obtained, which could realize the batch breeding of GMS materials. Further, the sterile line 9311-3A and restorer lines were used for hybridization, and a batch of superior combinations of hybrid rice was obtained.

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice.

Nitrogen (N) is a major driving force for crop yield improvement, but application of high levels of N delays flowering, prolonging maturation and thus increasing the risk of yield losses. Therefore, traits that enable utilization of high levels of N without delaying maturation will be highly desirable for crop breeding. Here, we show that OsNRT1.1A (OsNPF6.3), a member of the rice (Oryza sativa) nitrate transporter 1/peptide transporter family, is involved in regulating N utilization and flowering, providing a target to produce high yield and early maturation simultaneously. OsNRT.1A has functionally diverged from previously reported NRT1.1 genes in plants and functions in upregulating the expression of N utilization-related genes not only for nitrate but also for ammonium, as well as flowering-related genes. Relative to the wild type, osnrt1.1a mutants exhibited reduced N utilization and late flowering. By contrast, overexpression of OsNRT1.1A in rice greatly improved N utilization and grain yield, and maturation time was also significantly shortened. These effects were further confirmed in different rice backgrounds and also in Arabidopsis thaliana Our study paves a path for the use of a single gene to dramatically increase yield and shorten maturation time for crops, outcomes that promise to substantially increase world food security.

Transcription factor OsMADS25 improves rice tolerance to cold stress.

Cold stress is the limiting factor of rice growth and production, and it is important to clone cold stress tolerant genes and cultivate cold tolerance rice varieties. The MADS transcription factors play an important role in abiotic stress signaling in rice. This study showed that OsMADS25 was up-regulated by low temperature and abscisic acid (ABA), suggesting that OsMADS25 may be involved in ABA-dependent signaling. The OsMADS25 overexpression vector, pCambia1300-221-OsMADS25-Flag, was constructed and introduced into the rice variety Zhonghua 11 (ZH11) through Agrobacterium tumefacian-mediated genetic transformation. Two homozygous lines with high expression levels were selected for phenotypic identification. OsMADS25 overexpression lines show significantly improved cold stress tolerance and the sensitivity to ABA at the seedling stage of rice. Reactive oxygen species (ROS) was detected by diaminobenzidine (DAB) staining and nitroblue tetrazolium (NBT) staining. After treatment with cold stress, little ROS accumulation was observed in OsMADS25 overexpression lines compared to wild-type ZH11. In conclusion, OsMADS25 plays a role in scavenging reactive oxygen species (ROS) and could improve rice tolerance to cold stress involved in ABA-dependent pathway.

Days to heading 7, a major quantitative locus determining photoperiod sensitivity and regional adaptation in rice.

Success of modern agriculture relies heavily on breeding of crops with maximal regional adaptability and yield potentials. A major limiting factor for crop cultivation is their flowering time, which is strongly regulated by day length (photoperiod) and temperature. Here we report identification and characterization of Days to heading 7 (DTH7), a major genetic locus underlying photoperiod sensitivity and grain yield in rice. Map-based cloning reveals that DTH7 encodes a pseudo-response regulator protein and its expression is regulated by photoperiod. We show that in long days DTH7 acts downstream of the photoreceptor phytochrome B to repress the expression of Ehd1, an up-regulator of the "florigen" genes (Hd3a and RFT1), leading to delayed flowering. Further, we find that haplotype combinations of DTH7 with Grain number, plant height, and heading date 7 (Ghd7) and DTH8 correlate well with the heading date and grain yield of rice under different photoperiod conditions. Our data provide not only a macroscopic view of the genetic control of photoperiod sensitivity in rice but also a foundation for breeding of rice cultivars better adapted to the target environments using rational design.

Development of high-lysine rice via endosperm-specific expression of a foreign LYSINE RICH PROTEIN gene.

BACKGROUND: Lysine (Lys) is considered to be the first limiting essential amino acid in rice. Although there have been extensive efforts to improve the Lys content of rice through traditional breeding and genetic engineering, no satisfactory products have been achieved to date. RESULTS: We expressed a LYSINE-RICH PROTEIN gene (LRP) from Psophocarpus tetragonolobus (L.) DC using an endosperm-specific GLUTELIN1 promoter (GT1) in Peiai64S (PA64S), an elite photoperiod-thermo sensitive male sterility (PTSMS) line. The expression of the foreign LRP protein was confirmed by Western blot analysis. The Lys level in the transgenic rice seeds increased more than 30 %, the total amount of other amino acids also increased compared to wild-type. Persistent investigation of amino acids in 3 generations showed that the Lys content was significantly increased in seeds of transgenic rice. Furthermore, Lys content in the hybrid of the transgenic plants also had an approximate 20 % increase compared to hybrid control. At the grain-filling stage, we monitored the transcript abundance of many genes encoding key enzymes involved in amino acid metabolism, and the results suggested that reduced amino acid catabolism led to the accumulation of amino acids in the transgenic plants. The genetically engineered rice showed unfavorable grain phenotypes compared to wild-type, however, its hybrid displayed little negative effects on grain. CONCLUSIONS: Endosperm-specific expression of foreign LRP significantly increased the Lys content in the seeds of transgenic plant, and the the Lys increase was stably heritable with 3 generation investigation. The hybrid of the transgenic plants also showed significant increases of Lys content in the seeds. These results indicated that expression of LRP in rice seeds may have promising applications in improving Lys levels in rice.

Rice zinc finger protein DST enhances grain production through controlling Gn1a/OsCKX2 expression.

The phytohormone cytokinin (CK) positively regulates the activity and function of the shoot apical meristem (SAM), which is a major parameter determining seed production. The rice (Oryza sativa L.) Gn1a/OsCKX2 (Grain number 1a/Cytokinin oxidase 2) gene, which encodes a cytokinin oxidase, has been identified as a major quantitative trait locus contributing to grain number improvement in rice breeding practice. However, the molecular mechanism of how the expression of OsCKX2 is regulated in planta remains elusive. Here, we report that the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST) directly regulates OsCKX2 expression in the reproductive meristem. DST-directed expression of OsCKX2 regulates CK accumulation in the SAM and, therefore, controls the number of the reproductive organs. We identify that DST(reg1), a semidominant allele of the DST gene, perturbs DST-directed regulation of OsCKX2 expression and elevates CK levels in the reproductive SAM, leading to increased meristem activity, enhanced panicle branching, and a consequent increase of grain number. Importantly, the DST(reg1) allele provides an approach to pyramid the Gn1a-dependent and Gn1a-independent effects on grain production. Our study reveals that, as a unique regulator of reproductive meristem activity, DST may be explored to facilitate the genetic enhancement of grain production in rice and other small grain cereals.

To explore the best freeze-drying technology of Polygonatum sibiricum delar. Ex redoute and to establish the best HPLC method for the determination of main effective components of it from different areas in China.

To establish the freeze-drying process and the HPLC method to determine the main effective components and content of freeze-dried Polygonatum sibiricum of China. The results show that the freeze-drying process is a slice thickness at 3 mm, boiling time 2 min, pre-freezing temperature at -35 °C, low-temperature holding at -10 °C, low temperature sublimation at 10 °C and drying temperature at 35 °C. The optimum HPLC conditions of freeze-dried Polygonatum sibiricum was SepaxGp-C18 (4.6 × 250 mm, 5 µm), gradient elution of mobile phase A (acetonitrile)-B (ultra-pure water), detection wavelength of 254 nm, the flow rate of 1 mL/min, column temperature of at 30 °C and injection volume of 10 µL. Polygonatum sibiricum from different producing areas contains a variety of amino acids, diosgenin, sugars, and other active ingredients. The protein content of Polygonatum sibiricum from Yunnan Pu'er is the highest among them, and that of Shangluo in Shaanxi is the lowest. The total sugar content of Polygonatum sibiricum in Puer is the highest, while that of Polygonatum sibiricum in Wenshan is the lowest. And the diosgenin of Polygonatum sibiricum in Lijiang is the highest. In this study, the freeze-drying process of Polygonatum sibiricum was established, and the main effective components and contents of freeze-dried Polygonatum sibiricum was determined as the better HPLC method, provided theoretical and technical support for the rational development and utilization of Polygonatum sibiricum.

From Green Super Rice to green agriculture: Reaping the promise of functional genomics research.

Producing sufficient food with finite resources to feed the growing global population while having a smaller impact on the environment has always been a great challenge. Here, we review the concept and practices of Green Super Rice (GSR) that have led to a paradigm shift in goals for crop genetic improvement and models of food production for promoting sustainable agriculture. The momentous achievements and global deliveries of GSR have been fueled by the integration of abundant genetic resources, functional gene discoveries, and innovative breeding techniques with precise gene and whole-genome selection and efficient agronomic management to promote resource-saving, environmentally friendly crop production systems. We also provide perspectives on new horizons in genomic breeding technologies geared toward delivering green and nutritious crop varieties to further enhance the development of green agriculture and better nourish the world population.

Indication of the expression of transgene in rice plant based on hyperspectral remote sensing technique II--growth monitoring of samples in the contrast experiment.

Since the complication of monitoring and evaluating the problems about the transgenic expression and its impacts on the receptor in the transgenic crop breeding and other relevant evaluated works, the authors in the present work tried to assess the differences of spectral parameters of the transgenic rice in contrast with its parent group quantitatively and qualitatively, fulfilling the growth monitoring of the transgenic samples. The spectral parameters (spectral morphological characteristics and indices) chosen are highly related to internal or external stresses to the receipts, and thus could be applied as indicators of biophysical or biochemical processes changes of plant. By ASD portable field spectroradiometer with high-density probe, fine foliar spectra of 8 groups were obtained. By analyzing spectral angle and continuum removal, the spectral morphological differences and their locations of sample spectra were found which could be as auxiliary priori knowledge for quantitative analysis. By investigating spectral indices of the samples, the quantitative differences of spectra were revealed about foliar chlorophyll a+b and carotenoid content. In this study both the spectral differences between transgenic and parent groups and among transgenic groups were investigated. The results show that hyperspectral technique is promising and a helpful auxiliary tool in the study of monitoring the transgenic crop and other relevant researches. By this technique, quantitative and qualitative results of sample spectra could be provided as prior knowledge, as certain orientation, for laboratory professional advanced transgenic breeding study.

Senescence-Specific Expression of RAmy1A Accelerates Non-structural Carbohydrate Remobilization and Grain Filling in Rice (Oryza sativa L.).

Remobilization of pre-anthesis NSCs (non-structural carbohydrates) is significant for effective grain filling in rice (Oryza sativa L.). However, abundant starch particles as an important component of NSCs are still present in the leaf sheath and stem at the late stage of grain filling. There are no studies on how bioengineering techniques can be used to improve the efficiency of NSC remobilization. In this study, RAmy1A was expressed under the senescence-specific promoter of SAG12, which was designed to degrade starch in the leaf sheath and stem during grain filling. RAmy1A mRNA successfully accumulated in the leaf, stem, and sheath of transgenic plants after anthesis. At the same time, the starch and total soluble sugar content in the leaf, stem, and leaf sheath were obviously decreased during the grain-filling period. The photosynthetic rate of transgenic lines was higher than that of the wild types by an average of 4.0 and 9.9%, at 5 and 10 days after flowering, respectively. In addition, the grain-filling rate of transgenic lines was faster than that of the wild types by an average of 26.09%. These results indicate an enhanced transport efficiency of NSCs from source tissues in transgenic rice. Transgenic rice also displayed accelerated leaf senescence, which was hypothesized to contribute to decreased grain weight.

Exploring Natural Allelic Variations of the ß-Triketone Herbicide Resistance Gene HIS1 for Application in indica Rice and Particularly in Two-Line Hybrid Rice.

BACKGROUND: Benzobicyclon (BBC) is a ß-triketone herbicide (bTH) used in rice paddy fields. It has the advantages of high efficiency, low toxicity, high crop safety, and good environmental compatibility, and shows efficacy against paddy weeds resistant to other types of herbicides. However, as some important indica rice varieties are susceptible to BBC, BBC is currently only registered and applied in japonica rice cultivation areas. RESULTS: By analyzing haplotypes of the bTHs broad-spectrum resistance gene HIS1 and phenotypes for BBC in 493 major indica rice accessions in China, we identified a novel non-functional allelic variant of HIS1 in addition to the previously reported 28-bp deletion. Through detection with markers specific to the two non-functional mutations, it was clear that 25.4% of indica conventional varieties, 59.9% of fertility restorers, and 15.9% of sterile lines were susceptible to BBC. In addition, due to natural allelic variations of the HIS1 gene in the sterile and restorer lines, some two-line hybrid sterile lines were sensitive to bTHs, and the corresponding restorers were resistant. We showed the potential effectiveness of using bTHs to address the issue of two-line hybrid rice seed purity stemming from the self-crossing of sterile lines during hybrid rice seed production. Finally, allelic variations of the HIS1 gene may also play an important role in the mechanized seed production of hybrid rice. CONCLUSIONS: Our findings offer guidance for the application of BBC in indica rice areas and provide a non-transgenic approach to address the seed purity issue of two-line hybrid rice.