Dendrobium huoshanense Z. Z. Tang S. J. Cheng is a rare medicinal orchid characterized by slow growth and low propagation efficiency. This study aimed to establish a comprehensive and efficient in vitro propagation system for D. huoshanense by optimizing culture media via a response surface methodology (RSM). Key factors, including plant growth regulators and organic additives, were optimized for distinct developmental stages. The results indicated that the optimal conditions for protocorm proliferation were 0.2 mg·L⁻¹ naphthaleneacetic acid (NAA), 1.1 mg·L⁻¹ 6-benzylaminopurine (6-BA), and 97.0 g·L⁻¹ potato homogenate (PH), resulting in a proliferation coefficient of 59.3. The optimal conditions for protocorm differentiation were 1.1 mg·L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 1.1 mg·L⁻¹ 6-BA, and 85.0 g·L⁻¹ banana homogenate (BH), with an average of 6.4 buds per explant. For protocorm-like body (PLB) proliferation, the optimal conditions were 0.3 mg·L⁻¹ NAA, 2.0 mg·L⁻¹ kinetin (KT), and 150.0 g·L⁻¹ PH, resulting in a proliferation coefficient of 55.2. The optimal medium for PLB differentiation comprised 1.1 mg·L⁻¹ 2,4-D, 1.6 mg·L⁻¹ KT, and 140.0 g·L⁻¹ PH, yielding an average of 7.4 buds per explant. The optimal conditions for the proliferation of cluster shoots were 0.3 mg·L⁻¹ 2,4-D, 1.0 mg·L⁻¹ KT, 1.6 mg·L⁻¹ melatonin (MT), and 150.0 g·L⁻¹ BH, resulting in a proliferation coefficient of 6.2. For seedling rooting, the optimal medium was supplemented with 1.4 mg·L⁻¹ NAA, 0.7 mg·L⁻¹ indole-3-butyric acid (IBA), and 90.0 g·L⁻¹ BH. This study not only provides a practical protocol for its rapid propagation and conservation but also a valuable theoretical framework for tissue culture of other endangered orchid species. Response surface methodology enabled the optimization of culture conditions for Dendrobium huoshanense, achieving highly efficient regeneration and providing a theoretical basis for large-scale propagation and conservation of this endangered orchid.

The L-type amino acid transporter (LAT) family facilitates the cellular transport of amino acids and polyamines. However, the functions of LAT transporters in rice remain insufficiently characterized. In this study, we identified a significant negative association between OsLAT1 transcript levels and tiller number in rice. Transcriptional analysis revealed that OsLAT1 is predominantly expressed in leaves, basal tissues, and panicles. Subcellular localization assays showed that the OsLAT1 protein is localized to the endoplasmic reticulum and is strongly induced by Asp), Leu, spermidine (Spd), and spermine (Spm). Furthermore, under hydroponic conditions, moderate concentrations of arginine (Arg) and serine (Ser) partially promoted bud outgrowth and biomass in OsLAT1-overexpressing plants, whereas these effects diminished at higher Arg/Ser concentrations. In contrast, OsLAT1 facilitated the transport of spermidine (Spd) and spermine (Spm), thereby promoting axillary bud elongation and rice growth. These findings provide insights into amino acid transporter-mediated regulation of rice plant architecture and offer potential targets for yield improvement.

This study demonstrates that CRISPR-mediated cis-regulatory element editing (CRE editing) of the SD1 promoter effectively reduces plant height in Kam sweet rice, without compromising yield or grain quality, offering a precise strategy for crop improvement. Kam sweet rice, a unique aromatic variety, faces challenges with excessive plant height and suboptimal yield. This study explores a precision breeding approach by strengthening an endogenous TCP19–SD1 repression module through CRISPR-Cas9-mediated CRE editing to modulate the expression of the SD1 gene, a key regulator of gibberellin biosynthesis and stem elongation. By introducing an adenine insertion in the GGCCCCCC cis-regulatory element in the SD1 promoter, we enhanced the binding affinity of the transcription factor TCP19, resulting in down-regulated SD1 expression. This led to a reduction in gibberellin levels, shortening internodes, and reducing plant height. Phenotypic evaluations revealed that the edited lines exhibited significantly shorter plant height while maintaining grain yield and nitrogen utilization efficiency compared to wild-type plants. Microscopic analysis of the internodes confirmed that the reduced plant height correlated with decreased cell length. Transcriptomic studies indicated that CRE editing modulated a network of genes involved in both gibberellin and auxin signaling pathways, critical for plant growth. Importantly, the genetic modification did not adversely affect grain quality. This study demonstrates the potential of strengthening endogenous transcriptional repression via CRE editing as a precise alternative to conventional gene knockout techniques, offering a powerful strategy for optimizing complex agronomic traits in rice, with applications in modern crop breeding strategies.

Rice is the staple crop for more than half of the global population, and improving grain yield, grain quality, and stress resistance remain central goals of modern rice breeding. Among current precision breeding strategies, genome editing has created new opportunities for crop improvement, but its success depends heavily on the selection of effective target genes. In this context, negative regulators of agronomic traits are particularly valuable because their disruption or attenuation can relieve constraints on desirable phenotypes and generate beneficial variation. In this review, we summarize recent progress in the identification and functional characterization of negative regulatory genes associated with rice grain yield, grain quality and stress resistance. We further integrate the current knowledge of their molecular functions, regulatory mechanisms, and genetic networks and discuss their potential applications in genome editing-assisted breeding. This review provides a target-oriented framework for understanding negative regulation in rice and facilitating the development of improved varieties with increased productivity, quality and stress resistance.

Cymbidium goeringii Rchb. f. is an endangered orchid that is difficult to culture due to low rhizome proliferation, limited differentiation, and severe browning. An in vitro propagation system was established to culture this orchid by evaluating the effects of different basal media and decomposed animal-derived additives on rhizome proliferation, and the effects of anti-browning agents on rhizome differentiation and shoot rooting. Optimal medium for rhizome proliferation was determined to be 1/2 Murashige and Skoog (MS) supplemented with 0.5 mg·L−1 6-benzylaminopurine (6-BA), 1.0 mg·L−1 α-naphthaleneacetic acid (NAA), 7.5 g·L−1 activated charcoal, and 0.1 g·L−1 decomposed squid, resulting in a rhizome proliferation coefficient of 15.0 within 90 d. For rhizome differentiation, 1/2 MS supplemented with 1.0 mg·L−1 6-BA, 0.1 mg·L−1 NAA, and 0.25 g·L−1 succinic acid, significantly suppressed browning (9.5

Synergistically improving the yield and grain quality of rice remains a major breeding challenge. Amino acid transporters play key roles in regulating plant growth and development,but their mechanisms in synergistically regulating yield and quality remain unclear.

Nitrogen (N) fertilizers increase rice yield but it may also reduce rice quality, and the underlying mechanism is not elucidated. Here the impacts of different N levels on starch synthesis of two super hybrid indica rice cultivars were investigated. The apparent amylose content (AAC) showed a reduction of 15.70%-18.95% in response to N application from 0 to 400 kg N ha-1, while protein content increased by 35.73%-46.56%. More protein accumulation in endosperm at 400 kg N ha-1, affecting starch development and resulting in a higher proportion of fa (DP 6-12) chain and reduced fb2 (DP 13-24) and fb3 (DP ≥ 37) chains. Metabolomics and transcriptomics analyses indicated that under 200 kg N ha-1 treatment, transcription levels of starch synthase and starch branching enzymes were increased compared to 0 and 400 kg N ha-1, leading to an increase in AAC and changes in amylopectin chain length distribution. Nitrogen fertilizer increased the activity of nitrate reductase, glutamine synthetase (GS), and glutamate synthetase (GOGAT), promoting the GS/GOGAT cycle. Enhancement of nitrogen metabolism affected starch synthesis metabolism. The appropriate application of N fertilizer regulated the balance between carbon and nitrogen metabolism, improving processing and cooking qualities of super hybrid rice.

Tillering is a key determinant of rice yield and is regulated by both phytohormone signaling and nitrogen availability. However, how strigolactone-related pathways interface with nitrate-associated processes during tillering remains unclear. Here, we show that the TCP transcription factor OsFC1, a component of strigolactone- and brassinosteroid-associated pathways, exhibits natural promoter haplotypes that are associated with tillering-related variation. OsFC1 overexpression (OsFC1-OE) reduced the transcript abundance of the nitrate transporter gene OsNPF6.3, whereas OsFC1 knockdown (OsFC1-Ri) increased OsNPF6.3 expression. OsFC1 bound to the promoter region of OsNPF6.3 that harbours a TCP-binding motif (GTGGGCCC), and this binding correlated with reduced OsNPF6.3 transcript abundance. Overexpression of OsNPF6.3 in OsFC1-OE plants restored axillary bud elongation and increased tiller number, while knockout of OsNPF6.3 in OsFC1-Ri plants suppressed bud outgrowth and reduced tillering. Together, our findings reveal that OsFC1 acts as a critical molecular link integrating phytohormone signaling and nitrate transport to coordinate rice tillering via affecting OsNPF6.3 transcription.

Amino acids are crucial nutrients for growth in crops. In this study, we found an amino acid transporter-like 13 (OsATL13), that coordinately determined rice yield and quality. OsATL13 was primarily expressed in the root and panicle, its protein was localized on plasma membrane, and it principally transported phenylalanine and methionine. Overexpression (OE) of OsATL13 increased the tiller number by 31.4%, resulting in a 16.18% increase in grain yield compared to Zhonghua 11 (ZH11). It also decreased amylose content and increased protein content in OsATL13 OE lines compared to ZH11, whereas the OsATL13 mutant exhibited opposite effects. RNA-seq analysis revealed that upregulation of OsATL13 influenced the expression of genes associated with nitrogen and starch metabolism pathways. Notably, exogenous treatment with phenylalanine and methionine promoted axillary buds outgrowth, increased tiller number and rice yield, improved milled and head rice rates, and decreased chalky rice rate. Furthermore, rapid viscosity analysis supported the observation that phenylalanine and methionine treatments influenced rice eating and cooking quality. This research offers new perspectives on the synchronized enhancement of both rice yield and quality with amino acid transporter OsATL13.

Rice paddies are a major, persistent source of atmospheric methane (CH4), emission rates depend on the partitioning of photosynthate carbon between the rice plant and the rhizosphere microbiome. Although ratoon season rice (RR) is shown to emit far less CH4 than main-crop rice (MC), the mechanisms have remained unresolved. This work conducts a 2-year field experiment in which RR is compared with MC and with late rice (LR) synchronized to the RR heading stage. Relative to MC and LR, RR lowers daily CH4 flux by 91%, raises daily grain yield by 34%-57%, and increases net economic return by 90%-136%. Mechanistically, 13C-labelling reveals that RR diverted more newly fixed carbon to the grain and less to the rhizosphere, thereby restricting acetate availability for methanogens. Rhizosphere metagenomics show reduced abundance of Methanobacteriaceae and down-regulation of methanogenic genes in RR. This carbon-reallocation pattern is underpinned by an abscisic acid (ABA)-mediated interaction between OsCIPK2 and OsSWEET1A, which simultaneously curtailed carbon efflux from roots and enhanced grain filling. This study is the first to establish a comprehensive framework of "ABA regulation-carbon allocation-microbial function-emission reduction and efficiency enhancement." It provides targetable strategies for carbon allocation and microbial management within climate-smart rice farming systems.

Tartary buckwheat, known for its rapid growth, short growth cycles, and adaptability, is cultivated worldwide, particularly in East Asia and Eastern Europe. However, weed infestations severely impact yield and quality, and the absence of effective herbicides poses a considerable challenge to Tartary buckwheat production. To address this, we used ethyl methane sulfonate (EMS) mutagenesis to create a mutant population of Tartary buckwheat seeds. We applied a targeted screening process using the herbicide tribenuron-methyl (TM) to select mutants with reduced sensitivity to the herbicide. Integrative analysis of transcriptomic and metabolomic data indicated that TM primarily inhibited photosynthesis and amino acid biosynthesis pathways in sensitive plants, leading to toxicity. Conversely, resistant plants could reduce the toxic effects of TM on Tartary buckwheat by increasing antioxidant enzyme activity and enhancing secondary metabolic pathways such as flavonoid biosynthesis. TM also upregulated the expression of a gene encoding uridine diphosphate glucuronic acid transferase-like (FtUGT79L). Overexpression of FtUGT79L in Arabidopsis substantially increased total flavonoid content and improved the resistance of Arabidopsis to TM at the seedling and adult stages. The study provides insights into innovative approaches for breeding herbicide-resistant Tartary buckwheat germplasm and serves as an important reference for the development of herbicide-resistant varieties of other crops.

This review synthesizes how amino acid (AA) metabolism regulates rice stress tolerance, growth and quality through stress protection and growth-modulating pathways, bridging mechanisms to field applications. Under abiotic stresses, rice accumulates specific AAs—notably proline (Pro), γ-aminobutyric acid (GABA), and branched-chain AAs (BCAAs)—as osmoprotectants and antioxidants, correlating strongly with stress tolerance. Genetic evidence establishes causality: overexpression of biosynthetic genes (e.g., OsOAT for Pro, OsDIAT for BCAAs), while suppressing catabolism (e.g., OsProDH knockout) or engineering AA transporters (AATs) (e.g., ABA-induced OsANT1 for amino acids redistribution) enhances tolerance. Integrated AA biosynthetic, catabolic, and transport pathways collectively maintain cellular function under stress. These insights enable practical strategies: exogenous AA treatments (e.g., Pro, GABA) mitigate stress damage, while breeding/engineering (e.g., OsAAP3, OsAAP11, and OsProDH knockout) develops high-yield, high-quality, and stress-tolerant rice. Future work should translate molecular insights into field applications, addressing trade-offs between growth, nutrition, and tolerance to enhance climate-resilient rice production.

Black and red rice are known for their rich nutritional content, yet most varieties suffer from a firm texture and insufficient fragrance. In this study, we aimed to develop a fragrant and soft-textured black and red rice variety using the CRISPR/Cas9 technology to knock out the OsWx gene, which is associated with amylose content (AC), and the OsBADH2 gene, responsible for rice aroma. Our results showed that, compared to wild-type, CRISPR lines of XHZ, HM, NWZ, and PGZ targeting OsWx and OsBADH2 exhibited a reduction in AC content, altered gel consistency, and a more than 50% increase in gel consistency. Headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis revealed that the 2-acetyl-1-pyrroline (2-AP) content in the grains of xhz-cBADH2 Wx and hm-cBADH2 Wx reached 189.04 μg kg-1 and 309.03 μg kg-1, respectively. Furthermore, we observed a slight increase in anthocyanins and proanthocyanidins in these co-edited lines, without significant effects on their agronomic traits. Furthermore, to investigate the genes involved in the quality formation of black and red rice for the knockout of OsBADH2 and OsWx, we conducted RNA-seq analysis. The results indicated that knockout of OsBADH2 and OsWx affected the expression of genes involved in carotenoid biosynthesis, multiple amino acid metabolism genes, and endosperm starch and sucrose metabolic pathways. These findings suggest that the CRISPR/Cas9 technology can effectively target OsBADH2 and OsWx to develop high-quality black and red rice varieties with enhanced aroma and softer texture, providing a new strategy for the improvement of colored rice.

Rice tillering determines grain yield, yet the molecular regulatory network is still limited. In this study, we demonstrated that the transcription factor OsMADS60 promotes the expression of the auxin transporter OsPIN5b to affect auxin distribution and inhibit rice tillering and grain yield. Natural variation was observed in the promoter region of OsMADS60, with its expression level negatively correlated with tiller number and inducible by auxin. Overexpression of OsMADS60 resulted in reduced tillers and grain yield, whereas CRISPR-mediated knockouts of OsMADS60 led to increased tillering and yield. OsMADS60 was found to directly bind the CArG motif [CATTTAC] in the OsPIN5b promoter, thereby upregulating its expression. Moreover, we found that auxin content in various tissues of OsMADS60 and OsPIN5b overexpression lines increased relative to the wild-type ZH11, whereas the auxin levels in mutant lines showed the opposite trend. Genetic analysis further confirmed that OsPIN5b acted downstream of OsMADS60, coregulating the expression of genes involved in hormone pathways. Our findings reveal that OsMADS60 modulates auxin distribution by promoting OsPIN5b expression, thereby influencing rice tillering. This regulatory mechanism holds significant potential for the genetic improvement of rice architecture and grain yield.

Gama-aminobutyric acid (GABA) plays an important role in regulating plant growth and response to various stresses. Here, we studied the natural variation of GABA transporter gene OsGAT3 in rice germplasm and found four primary promoter haplotypes. OsGAT3 was highly expressed in roots, culms, and leaf blades and was strongly induced in roots by GABA, salt, drought, and low temperature. OsGAT3 expression level was positively correlated with tiller number and nitrogen utilization efficiency (NUtE). OsGAT3 was localized in the plasma membrane. Overexpression of OsGAT3 significantly promoted GABA accumulation in basal parts suggesting it boosted axillary bud formation, tillering, and grain yield. OsGAT3 overexpression also enhanced tolerance to drought and salt stress. Transcriptome analysis revealed enriched phenylpropanoid and pentose phosphate pathways, and MAPK signaling underlining tolerance to salt stress, whereas drought tolerance was attributed to enhanced photosynthesis, glyoxylate and dicarboxylate metabolism, and carotenoid biosynthesis. Taken together, our data support the potential application of the gene for increasing yield under abiotic tolerance.

Low -affinity nitrate transporter genes have been identified in subfamilies 4-8 of the rice nitrate transporter 1 (NRT1)/ peptide transporter family (NPF), but the OsNPF3 subfamily responsible for nitrate and phytohormone transport and rice growth and development remains unknown. In this study, we described OsNPF3.1 as an essential nitrate and phytohormone transporter gene for rice tillering and nitrogen utilization efficiency (NUtE). OsNPF3.1 possesses four major haplotypes of its promoter sequence in 517 cultivars, and its expression is positively associated with tiller number. Its expression was higher in the basal part, culm, and leaf blade than in other parts of the plant, and was strongly induced by nitrate, abscisic acid (ABA) and gibberellin 3 (GA3) in the root and shoot of rice. Electrophysiological experiments demonstrated that OsNPF3.1 is a pH -dependent low -affinity nitrate transporter, with rice protoplast uptake assays showing it to be an ABA and GA3 transporter. OsNPF3.1 overexpression significantly promoted ABA accumulation in the roots and GA accumulation in the basal part of the plant which inhibited axillary bud outgrowth and rice tillering, especially at high nitrate concentrations. The NUtE of OsNPF3.1-overexpressing plants was enhanced under low and medium nitrate concentrations, whereas the NUtE of OsNPF3.1 clustered regularly interspaced short palindromic repeats (CRISPR) plants was increased under high nitrate concentrations. The results indicate that OsNPF3.1 transports nitrate and phytohormones in different rice tissues under different nitrate concentrations. The altered OsNPF3.1 expression improves NUtE in the OsNPF3.1-overexpressing and CRISPR lines at low and high nitrate concentrations, respectively.

Pitaya (Hylocereus undatus; 2n=22) is an important fruit crop from the Cactaceae family, originally domesticated in Mexico and the USA, and is now widely cultivated for its nutritional benefits. It is characterized by its distinctive triangular-shaped stems and large, showy flowers, thriving in arid and semi-arid environments, particularly in hot, dry climates. However, systematic chromosomal studies, including chromosomal mapping of cytogenetic markers in pitaya, are limited, presenting challenges for its cytogenetic improvement. To address this issue, we designed oligo-barcodes specific to thirty-three chromosome regions based on the pitaya reference genome and applied them to both pitaya and cactus (Selenicerus grandifloras; 2n=22) for oligo-barcodes mapping, karyotyping, and chromosome identification. We utilized FISH technology, employing oligo, rDNA, and tandem repeat probes for chromosomal mapping, identification, and karyotyping of pitaya and related species. We successfully localized oligo-barcodes on eleven pairs of chromosomes in both pitaya and cactus, demonstrating the effectiveness of the synthesized oligo-barcodes. We used two ribosomal DNA (rDNA) probes (45S and 5S) and two tandem repeat probes (GTR11 and STR3) in pitaya (both diploid and tetraploid) and two other Cactaceae species (S. grandifloras and Opuntia humifusa; 2n=40) for chromosomal mapping. The analysis of rDNA distribution and CMA (Chromomycin A3) banding across different chromosomes in pitaya and cacti highlights the concept of conserved rDNA. This study provides fundamental insights into cytogenetic markers and their localization across different chromosomes in pitaya and other Cactaceae species.

Amino acids are necessary nutrients for the growth of Oryza sativa (rice), which can be mediated by amino acid transporter; however, our understanding of these transporters is still limited. This study found that the expression levels of amino acid permease gene OsAAP12 differed between indica and japonica rice. Altered expression of OsAAP12 negatively regulated tillering and yield in transgenic rice lines. Subcellular localization revealed that OsAAP12 was primarily localized to the plasma membrane. Moreover, it was indicated that OsAAP12 transported polar neutral amino acids asparagine (Asn), threonine (Thr), and serine (Ser) through experiments involving yeast heterologous complementation, fluorescence amino acid uptake, and amino acid content determination. Additionally, exogenous application of amino acids Asn, Thr, and Ser suppressed axillary buds outgrowth in OsAAP12 overexpression lines compared with wild-type ZH11. Conversely, the opposite trend was observed in CRISPR mutant lines. RNA-seq analysis showed that the expression patterns of genes involved in the nitrogen and cytokinin pathways were generally altered in OsAAP12 modified lines. Hormone assays indicated that OsAAP12 mutant lines accumulated cytokinins in the basal part of rice, whereas overexpression lines had the opposite effect. In summary, CRISPR mutant of OsAAP12 boosted rice tillering and grain yield by coordinating the content of amino acids and cytokinins, which has potential application value in high-yield rice breeding.

Kam sweet rice is a cultural treasure in Qiandongnan, Guizhou Province. However, the situation with low yield and economic value in Kam sweet rice urgently requires improved mechanistic understanding of tillering to increase its yield. In this study, we found that the rate of axillary bud elongation differed significantly among Kam sweet rice varieties, which was positively correlated with tiller number. Transcriptome analysis suggests that genes involved in nitrogen metabolism and plant hormone signaling pathways could be the main reasons for the differences in tillering among these varieties. The amino acid transporter OsAAP11 in the transcriptome was essential for bud outgrowth and rice tillering based on the phenotypic performance of its transgenic plants. Further results found that OsAAP11 was able to transport amino acids such as proline, glycine, and alanine in rice. Natural variations were found in the promoter region of this gene in different Kam sweet rice varieties, which may lead to differences in the transcription levels of OsAAP11. Overall, the results suggest that the natural variations of OsAAP11 in rice might lead to variations in its expression levels, further affecting bud outgrowth and tillering through regulating the transport and accumulation of amino acids.

Kam Sweet Rice is a high-quality local variety of Guizhou province in China, but most varieties have awns on lemma. In this study, we aimed to obtain awnless varieties of Kam Sweet Rice by blocking the awn development-related gene OsGAD1 using CRISPR/Cas9 technology. We determined that natural variations of the OsGAD1 triggered different lengths of awns of Kam Sweet Rice. We found that the awning rate of the CRISPR lines of OsGAD1 in Guxiangnuo, Goujingao and Gouhuanggang decreased by over 65%, and the number of grains per panicle and yield per plant increased by more than 17% and 20% compared to the wild-types. Furthermore, we indicated that blocking OsGAD1 resulted in an increase of over 2% in the brown rice rate and milled rice rate in these varieties. In addition, the analysis of the transcriptome revealed that the regulation of awn development and yield formation in CRISPR lines of OsGAD1 may involve genes associated with phytohormone and nitrogen pathways. These results suggest that blocking OsGAD1 in Kam Sweet Rice using CRISPR/Cas9 technology can be used for breeding programs seeking high yield and grain quality of Kam Sweet Rice.