Selection of pre-embryogenic callus from a core structure from mature seed-derived callus is the key for high-efficiency plant regeneration and transformation of switchgrass different cultivars. Switchgrass (Panicum virgatum L.) has been identified as a dedicated biofuel crop. For its trait improvement through biotechnological approaches, we have developed a highly efficient plant regeneration and genetic transformation protocol for both lowland and upland cultivars. We identified and separated a pre-embryogenic "core" structure from the seed-derived callus, which often leads to development of highly regenerative type II calluses. From the type II callus, plant regeneration rate of lowland cultivars Alamo and Performer reaches 95%, and upland cultivars Blackwell and Dacotah, 50 and 76%, respectively. The type II callus was also amenable for Agrobacterium-mediated transformation. Transformation efficiency of 72.8% was achieved for lowland cultivar Alamo, and 8.0% for upland cultivar Dacotah. PCR, Southern blot and GUS staining assays were performed to verify the transgenic events. High regenerative callus lines could be established in 3 months, and transgenic plants could be obtained in 2 months after Agrobacterium infection. To our knowledge, this is the first report on successful plant regeneration and recovery of transgenic plants from upland switchgrass cultivars by Agrobacterium-mediated transformation. The method presented here could be helpful in breaking through the bottleneck of regeneration and transformation of lowland and upland switchgrass cultivars and probably other recalcitrant grass crops.

Alfalfa (Medicago sativa L.) is an important and widely cultivated forage grass. The productivity and forage quality of alfalfa are severely affected by salt stress. Melatonin is a bioactive molecule with versatile physiological functions and plays important roles in response to various biotic and abiotic stresses. Melatonin has been proven efficient in improving alfalfa drought and waterlogging tolerance in recent studies. In our reports, we applied melatonin exogenously to explore the effects of melatonin on alfalfa growth and salt resistance. The results demonstrated that melatonin application promoted alfalfa seed germination and seedling growth, and reduced oxidative damage under salt stress. Further application research found that melatonin alleviated salt injury in alfalfa plants under salt stress. The electrolyte leakage, malondialdehyde (MDA) content and H 2 O 2 content were significantly reduced, and the activities of catalase (CAT), peroxidase (POD), and Cu/Zn superoxide dismutase (Cu/Zn-SOD) were increased with melatonin pretreatment compared to control plants under salt stress with the upregulation of genes related to melatonin and antioxidant enzymes biosynthesis. Melatonin was also involved in reducing Na + accumulation in alfalfa plants. Our study indicates that melatonin plays a primary role as an antioxidant in scavenging H 2 O 2 and enhancing activities of antioxidant enzymes to improve the salt tolerance of alfalfa plants.Plants 2020, 9, 220 2 of 17 world's irrigated land affected by saline or sodic globally [7,8]. Salinity has become one of the most important environmental stress factors impairing worldwide agricultural productivity. Under salinity conditions, the seed germination, growth and development processes of alfalfa are inhibited and they finally impair the herbage yield, as well as the forage quality [9]. Breeding new alfalfa cultivars with high salt tolerance is always needed. Genetic engineering and conventional breeding have been proven efficient for improving salt tolerance of various plant species [10][11][12][13]. However, they are time-consuming and complicated. Exogenous application of certain plant growth regulators such as phytohormone and other small molecules has been proven efficient at overcoming the harmful effects of salt stress on plants [14][15][16][17]. Moreover, plant growth regulators in low concentrations always play a role in plants, and are cost-effective. Foliar spraying of salicylic acid (SA) on faba bean inhibits Na + accumulation and lipid peroxidation, improving the antioxidant resistance and finally mitigating the damage caused by salinity [18]. Exogenously applied poly-γ-glutamic acid on wheat maintains the Na + and K + homeostasis and enhances antioxidant capacity by alleviating salinity damage under salt stress conditions [19]. Exogenous spermidine application to salt-stressed cucumber improves the photosynthetic capacity and the activity of key enzymes for CO 2 fixation by regulating the expression of related genes, and tolerance to salinity is thus...

The Drought and Salt Tolerance gene (DST) encodes a C2H2 zinc finger transcription factor, which negatively regulates salt tolerance in rice (Oryza sativa). Phylogenetic analysis of six homologues of DST genes in different plant species revealed that DST genes were conserved evolutionarily. Here, the rice DST gene was linked to an SRDX domain for gene expression repression based on the Chimeric REpressor gene-Silencing Technology (CRES-T) to make a chimeric gene (OsDST-SRDX) construct and introduced into perennial ryegrass by Agrobacterium-mediated transformation. Integration and expression of the OsDST-SRDX in transgenic plants were tested by PCR and RT-PCR, respectively. Transgenic lines overexpressing the OsDST-SRDX fusion gene showed obvious phenotypic differences and clear resistance to salt-shock and to continuous salt stresses compared to non-transgenic plants. Physiological analyses including relative leaf water content, electrolyte leakage, proline content, malondialdehyde (MDA) content, H2O2 content and sodium and potassium accumulation indicated that the OsDST-SRDX fusion gene enhanced salt tolerance in transgenic perennial ryegrass by altering a wide range of physiological responses. To our best knowledge this study is the first report of utilizing Chimeric Repressor gene-Silencing Technology (CRES-T) in turfgrass and forage species for salt-tolerance improvement.

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BackgroundIntracellular Na+ (K+)/H+ antiporters (NHXs) have pivotal functions in regulating plant growth, development, and resistance to a range of stresses. To gain insight into the molecular events underlying their actions in switchgrass (Panicum virgatum L.), we analyzed transcriptomic changes between PvNHX1-overexpression transgenic lines and wild-type (WT) plants using RNA sequencing (RNA-seq) technology.ResultsThe comparison of transcriptomic data from the WT and transgenic plants revealed a large number of differentially expressed genes (DEGs) in the latter. Gene ontology (GO) and KEGG pathway analyses showed that these DEGs were associated with a wide range of functions, and participated in many biological processes. For example, we found that PvNHX1 had an important role in plant growth through its regulation of photosynthetic activity and cell expansion. In addition, PvNHX1 regulated K+ homeostasis, cell expansion and pollen development, indicating that it has unique and specific roles in flower development. We also found that transgenic switchgrass exhibited a higher level of transcription of defense-related genes, especially those involved in disease resistance.ConclusionWe showed that PvNHX1 had an important role in plant growth and development through its regulation of photosynthetic activity, cell expansion, K+ homeostasis, and pollen development. Additionally, PvNHX1 overexpression activated a complex signal transduction network in response to various biotic and abiotic stresses. In relation to plant growth, development, and defense responses, PvNHX1 also had a vital regulatory role in the formation of a series of plant hormones and transcription factors (TFs). The reliability of the RNA-seq data was confirmed by quantitative real-time PCR. Our data provide a valuable foundation for further research into the molecular mechanisms and physiological roles of NHXs in plants.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1278-5) contains supplementary material, which is available to authorized users.

Transgenic switchgrass overexpressing Lolium perenne L. delta1-pyrroline 5-carboxylate synthase (LpP5CS) in group I (TG4 and TG6 line) and group II (TG1 and TG2 line) had significant P5CS and ProDH enzyme activities, with group I plants (TG4 and TG6) having higher P5CS and lower ProDH enzyme activity, while group II plants had higher ProDH and lower P5CS enzyme activity. We found group II transgenic plants showed stunted growth, and the changed proline content in overexpressing transgenic plants may influence the growth and development in switchgrass. RNA-seq analysis showed that KEGG enrichment included phenylpropanoid biosynthesis pathway among group I, group II and WT plants, and the expression levels of genes related to lignin biosynthesis were significantly up-regulated in group II. We also found that lignin content in group II transgenic plants was higher than that in group I and WT plants, suggesting that increased lignin content may suppress switchgrass growth and development. This study uncover that proline can appropriately reduce lignin biosynthesis to improve switchgrass growth and development. Therefore, appropriate reduction in lignin content and increase in biomass are important for bioenergy crop to lower processing costs for biomass fermentation-derived fuels.

Melatonin (N-acetyl-5-methoxytryptamine) is a pleiotropic signaling molecule that plays important roles in plant growth, development and stress responses. Alfalfa (Medicago sativa L.) is an important and widely cultivated leguminous forage crop with high biomass yield and rich nutritional value. The effects of exogenous melatonin content regulation on alfalfa stress tolerance have been investigated in recent years. Here, we isolated and introduced the MsASMT1 (N-acetylserotonin methyltransferase) gene into alfalfa, which significantly improved the endogenous melatonin content. Compared with wildtype (WT) plants, MsASMT1 overexpression (OE-MsASMT1) plants exhibited a series of phenotypic changes, including vigorous growth, increased plant height, enlarged leaves and robust stems with increased cell sizes, cell numbers and vascular bundles, as well as more branches. We also found that the flavonoid content and lignin composition of syringyl to guaiacyl ratio (S/G) were decreased and the cellulose content was increased in OE-MsASMT1 transgenic alfalfa. Further transcriptomic and metabolomic exploration revealed that a large group of genes in phenylalanine pathway related to flavonoids and lignin biosynthesis were significantly altered, accompanied by significantly reduced concentrations of the glycosides of quercetin, kaempferol, formononetin and biochanin in OE-MsASMT1 transgenic alfalfa. Our study first uncovers the effects of endogenous melatonin on alfalfa growth and metabolism. This report provides insights into the regulation effects of melatonin on plant growth and phenylalanine metabolism, especially flavonoids and lignin biosynthesis.

In plants, Δ1-pyrroline- 5-carboxylate synthase (P5CS) is the rate-limiting enzyme in proline biosynthesis. In this study, we introduced the LpP5C S ( Lolium perenne L.) gene into switchgrass by Agrobacterium -mediated transformation. The transgenic lines (TG) were classified into two groups based on their phenotypes and proline levels. The group I lines (TG4 and TG6) had relatively high proline levels and improved biomass yield. The group II lines (TG1 and TG2) showed low proline levels, severely delayed flowering, stunted growth and reduced biomass yield. Additionally, we used RNA-seq analysis to detect the most significant molecular changes, and we analyzed differentially expressed genes, such as flowering-related and CYP450 family genes. Moreover, the biomass yield, physiological parameters, and expression levels of reactive oxygen species scavenger-related genes under salt stress all indicated that the group I plants exhibited significantly increased salt tolerance compared with that of the control plants, in contrast to the group II plants. Thus, genetic improvement of switchgrass by overexpressing LpP5CS to increase proline levels is feasible for increasing plant stress tolerance.