Transgenic Breeding Research Articles

Molecular Cloning and Functional Characterization of Bisabolene Synthetase (SaBS) Promoter from Santalum album

Bisabolene-type sesquiterpenoids, which have multiple bioactivities, including anticancer activity, are one of the main groups of compounds in the essential oil extracted from Santalum album L. and other Santalum species. Bisabolene synthetase (SaBS) is a key enzyme for the synthesis of bisabolene in S. album, but the regulation of the SaBS gene’s expression is poorly understood. In this study, a 1390-bp promoter sequence of the SaBS gene was isolated from the leaves of six-year-old S. album. A bioinformatics analysis showed that certain environment stresses and phytohormone-activated cis-acting elements were distributed in different regions of the SaBS promoter (PSaBS). Transgenic Arabidopsis carrying full-length PSaBS had significantly higher β-glucuronidase (GUS) activity than the untreated control after treatment with salicylic acid (SA), suggesting that PSaBS is a SA-inducible promoter. Histochemical GUS staining and GUS fluorometric assays of transgenic Arabidopsis showed that the GUS activity directed by PSaBS was mainly expressed in stem tissue, followed by leaves and flowers. Moreover, different regions of PSaBS showed significantly different GUS activity. A 171-bp fragment upstream of the transcriptional initiation codon (ATG) is the core promoter region of PSaBS. Our results provide insight into and a greater understanding of the transcriptional regulation mechanism of the SaBS gene, which could serve as an alternative inducible promoter for transgenic plant breeding.

The conventional and transgenic breeding approaches for biofortification of rice grains with high fe & zn contents and enhancing their bioavailability to combat micronutrient malnutrition-a review

In the developing countries million of people are suffering from malnutrition.In these countries rice is a staple food in daily dietary system but among grains it is the poorest supplier of iron and zinc which leads to micronutrient malnutrition reducing adult productivity, causing anemia,retarding mental and physical growth. Alternative to improving malnutrition and balancing diet are supplementation chemical fortification and dietary diversification which failed to solve the problem due to high cost,limited infra structure for processing regulatory control, marketing problem of fortified food and poverty.Hence there is a need to adopt the cost effective micronutrient bio-fortification system. But Fe and Zn bio-fortification of rice grains will not be enough to satisfy the human micronutrient requirement due to the presence of the phytic acid,an antinutrient that can prevent the absorption of iron and Zinc decreasingtheir bioavailability. Although phytase is an enzyme capable of degrading phytic acid and improving bioavailability of micronutrients but its use by yeast fermentation as feed additive increases feeding cost.The improvement for iron, zinc and reduced phytic acid in rice grains will provide greater opportunity to eradicate malnutrition among poor families but greatly increasing iron levels in rice grains could result in decreased crop yield which is not acceptable to the farmers. Therefore,the identification of desirable donors containing high iron, zinc and low phytic acid and incorporating these traits into the back ground of high yielding cultivars through self sustainable and cost effective classical and transgenic breeding approach willalso provide further opportunity to develop high yielding cultivated rice varieties with high level of Fe,Zn and low phytic acid eradicating malnutrition. Agronomic management practices also influence micronutrient uptake and their accumulation in bio-fortified rice grains.

Enlightened oversight of genetically engineered crops for the next generation

AbstractRisk‐disproportionate regulatory oversight has hampered the use of genetic engineering to sustainably improve crops for the duration of a human generation (≈25 years). There is scientific consensus that transgenic breeding methods are safe. Current regulations are often driven by unfounded public fear and hamper crop improvement that is critical to meeting current environmental and nutritional needs and future food security. The public good requires progressive nations to enact policies that enlighten the oversight of modern breeding methods so that current nutritional needs can be met, future food crises can be averted, and agriculture can become more sustainable.

Major Achievements of Plant Biotechnology in Crop Improvements

Biotechnology is any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for a specific use. Agricultural biotechnology is the area of biotechnology involving applications to agriculture. Based on an understanding of DNA, scientists have developed solutions to increase agricultural productivity. Integrating recombinant techniques into conventional breeding programs could substantially enhance the efficiency of agricultural research and development. Breeding could be accelerated due to the more targeted transfer of desired genes into the crop. The major transgenic breeding objectives are; improving the agronomic traits and quality traits of different crops. Deliberate alteration of the genome of an organism by introduction of one or a few specific foreign genes is referred to as 'genetic engineering' or 'genetic transformation', and the modified organism is described as a 'transformed' or 'transgenic' organism. Achievements of biotechnology in crops were producing Bt crops, herbicide resistance crops, salinity tolerant crops, drought-tolerant crops and so on. Genetically engineered crop varieties that farmers deploy. Transgenic crops, especially those with resistance to biotic and abiotic stress factors, fit well into small-scale farming systems and can easily be integrated without adjusting traditional cropping practices. The comparatively low setup cost for adopting genetically engineered technologies at the farm level also makes this technology useful for semi-subsistence agriculture. Plant tissue culture also one of the applications of biotechnology used to improve crops and used to increase the speed or efficiency of the breeding process, to improve the accessibility of existing germplasm and to create new variation for crop improvement. Generally, the role of crop biotechnology for food security and poverty reduction should not be overrated. Many problems in low- and middle-income countries are not amenable to technological solutions.

Overexpression of BnaAOX1b Confers Tolerance to Osmotic and Salt Stress in Rapeseed.

Alternative oxidases (AOXs) are the terminal oxidase in the cyanide-resistant respiration pathway in plant mitochondria, which play an important role in abiotic stress and are proposed as a functional marker for high tolerant breeding. In this study, ten AOX genes (BnaAOXs) were identified, and CysI and CysII of AOX isoforms were highly conserved in rapeseed. Among them, Bna.AOX1b was mainly expressed in the ovule and displayed varying expression between rapeseed cultivars which showed different salt resistance in seed germination. We identified its mitochondrial localization of this gene. To investigate the function of BnaAOX1b in rapeseed, transgenic rapeseed lines with overexpressed BnaAOX1b were created and seed germination and seedling establishment assays were performed under osmotic, salt, and ABA treatment. The results indicated that overexpression of BnaAOX1b significantly improved seed germination under osmotic and salt stress and weakened ABA sensitivity. In addition, post-germination seedling growth was improved under high salt condition, but showed hypersensitivity to ABA. RNA-sequencing analysis indicated that the genes involved in electron transport or energy pathway were induced and a number of gene responses to salt stress and ABA were regulated in Bna.AOX1b overexpressing seeds. Taken together, our results imply that Bna.AOX1b confers tolerance to osmotic and salt stress in terms of seed germination and seedling establishment by regulating stress responsive genes and the response to ABA, and could be utilized as a candidate gene in transgenic breeding.

Construction of a high-resolution genetic map and identification of quantitative trait loci for salt tolerance in jute (Corchous spp.)

BackgroundJute (Corchorus spp.) is the most important natural fiber crop after cotton in terms of cultivation area and production. Salt stress greatly restricts plant development and growth. A high-density genetic linkage map is the basis of quantitative trait locus (QTLs) mapping. Several high-density genetic maps and QTLs mapping related to salt tolerance have been developed through next-generation sequencing in many crop species. However, such studies are rare for jute. Only several low-density genetic maps have been constructed and no salt tolerance-related QTL has been mapped in jute to date.ResultsWe developed a high-density genetic map with 4839 single nucleotide polymorphism markers spanning 1375.41 cM and an average distance of 0.28 cM between adjacent markers on seven linkage groups (LGs) using an F2 jute population, LGs ranged from LG2 with 299 markers spanning 113.66 cM to LG7 with 1542 markers spanning 350.18 cM. In addition, 99.57% of gaps between adjacent markers were less than 5 cM. Three obvious and 13 minor QTLs involved in salt tolerance were identified on four LGs explaining 0.58–19.61% of the phenotypic variance. The interval length of QTL mapping varied from 1.3 to 20.2 cM. The major QTL, qJST-1, was detected under two salt stress conditions that explained 11.81 and 19.61% of the phenotypic variation, respectively, and peaked at 19.3 cM on LG4.ConclusionsWe developed the first high-density and the most complete genetic map of jute to date using a genotyping-by-sequencing approach. The first QTL mapping related to salt tolerance was also carried out in jute. These results should provide useful resources for marker-assisted selection and transgenic breeding for salt tolerance at the germination stage in jute.

ZmPGIP3 Gene Encodes a Polygalacturonase-Inhibiting Protein that Enhances Resistance to Sheath Blight in Rice.

Plant polygalacturonase-inhibiting protein (PGIP) is a structural protein that can specifically recognize and bind to fungal polygalacturonase (PG). PGIP plays an important role in plant antifungal activity. In this study, a maize PGIP gene, namely ZmPGIP3, was cloned and characterized. Agarose diffusion assay suggested that ZmPGIP3 could inhibit the activity of PG. ZmPGIP3 expression was significantly induced by wounding, Rhizoctonia solani infection, jasmonate, and salicylic acid. ZmPGIP3 might be related to disease resistance. The gene encoding ZmPGIP3 was posed under the control of the ubiquitin promoter and constitutively expressed in transgenic rice. In an R. solani infection assay, ZmPGIP3 transgenic rice was more resistant to sheath blight than the wild-type rice regardless of the inoculated plant part (leaves or sheaths). Digital gene expression analysis indicated that the expression of some rice PGIP genes significantly increased in ZmPGIP3 transgenic rice, suggesting that ZmPGIP3 might activate the expression of some rice PGIP genes to resist sheath blight. Our investigation of the agronomic traits of ZmPGIP3 transgenic rice showed that ZmPGIP3 overexpression in rice did not show any detrimental phenotypic or agronomic effect. ZmPGIP3 is a promising candidate gene in the transgenic breeding for sheath blight resistance and crop improvement.

Functional Characterization of Resistance to Powdery Mildew of VvTIFY9 from Vitis vinifera.

Powdery mildew is a disease caused by fungal pathogens that harms grape leaves and fruits. The TIFY gene family is a plant-specific super-family involved in the process of plants’ development and their biotic and abiotic stress responses. This study aimed to learn the function of the VvTIFY9 gene to investigate molecular mechanisms of grape resistance to powdery mildew. A VvTIFY9 protein encoding a conserved motif (TIF[F/Y]XG) was characterized in grape (Vitis vinifera). Sequence analysis confirmed that VvTIFY9 contained this conserved motif (TIF[F/Y]XG). Quantitative PCR analysis of VvTIFY9 in various grape tissues demonstrated that the expression of VvTIFY9 was higher in grape leaves. VvTIFY9 was induced by salicylic acid (SA) and methyl jasmonate (MeJA) and it also quickly responded to infection with Erysiphe necator in grape. Analysis of the subcellular localization and transcriptional activation activity of VvTIFY9 showed that VvTIFY9 located to the nucleus and had transcriptional activity. Arabidopsis that overexpressed VvTIFY9 were more resistant to Golovinomyces cichoracearum, and quantitative PCR revealed that two defense-related genes, AtPR1 and AtPDF1.2, were up-regulated in the overexpressing lines. These results indicate that VvTIFY9 is intimately involved in SA-mediated resistance to grape powdery mildew. This study provides the basis for exploring the molecular mechanism of grape resistance to disease resistance and candidate genes for transgenic disease resistance breeding of grape plants.

ZmXa21-L gene encodes a plant receptor-like kinases (RLKs) protein that enhances resistance to bacterial blight in rice

Rice bacterial blight, which is caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious rice diseases worldwide. The rice Xa21 gene encodes plant receptor-like kinases (RLKs) that confer broad-spectrum resistance against Xoo. In this study, a full-length cDNA encoding a putative RLK protein with high sequence homology to the rice Xa21 gene was isolated from maize and designated as ZmXa21-L. The full-length cDNA of ZmXa21-L contained a 2796 bp open reading frame encoding a precursor protein of 932 amino acid residues. Comparative and bioinformatics analyses revealed that ZmXa21-L showed a high degree of homology with other Xa21-like proteins and contained the leucine-rich repeat domain, transmembrane region, and serine/threonine kinase domain. Subcellular localization revealed the presence of ZmXa21-L in the cell membrane. The expression level of ZmXa21-L was significantly induced by wounding, salicylic acid, and jasmonate treatments. ZmXa21-L was transformed into rice under the control of a ubiquitin promoter. After inoculation with Xoo strains, ZmXa21-L transgenic rice showed enhanced resistance to bacterial blight compared with wild-type rice. Moreover, investigation of the agronomic traits revealed that the ZmXa21-L expression increased the panicle number and grain yield per plant in the transgenic rice. Results demonstrate that ZmXa21-L is a promising candidate gene in transgenic breeding for bacterial blight resistance and crop improvement.

The Role of Soil Fungi in K+ Plant Nutrition

K+ is an essential cation and the most abundant in plant cells. After N, its corresponding element, K, is the nutrient required in the largest amounts by plants. Despite the numerous roles of K in crop production, improvements in the uptake and efficiency of use of K have not been major focuses in conventional or transgenic breeding studies in the past. In research on the mineral nutrition of plants in general, and K in particular, this nutrient has been shown to be essential to soil-dwelling-microorganisms (fungi, bacteria, protozoa, nematodes, etc.) that form mutualistic associations and that can influence the availability of mineral nutrients for plants. Therefore, this article aims to provide an overview of the role of soil microorganisms in supplying K+ to plants, considering both the potassium-solubilizing microorganisms and the potassium-facilitating microorganisms that are in close contact with the roots of plants. These microorganisms can influence the active transporter-mediated transfer of K+. Regarding the latter group of microorganisms, special focus is placed on the role of endophytic fungus. This review also includes a discussion on productivity through sustainable agriculture.

Ectopic expression of secretory peptide PdEPF3 in Arabidopsis confers drought tolerance with reduced stomatal density

Drought is one of the most prevalent environmental stresses that affect plant growth and development. Improvement in drought tolerance is associated with reduced stomatal density and higher water use efficiency (WUE). In this study, an epidermal patterning factor (EPF), PdEPF3, from a fast-growing poplar clone, NE-19 [<em>Populus nigra</em> × (<em>Populus deltoides</em> × <em>Populus nigra</em>)], was characterized. Quantitative reverse transcription polymerase chain reaction showed that the transcription of <em>PdEPF3</em> was induced by drought. We further found that the transgenic <em>Arabidopsis</em> overexpressing <em>PdEPF3</em> had an earlier seedling germination and longer primary roots under osmotic stress treatments, compared with the WT and mutant <em>epf1-1</em>. In addition, ectopic overexpression of <em>PdEPF3</em> in <em>Arabidopsis</em> plants was able to enhance drought tolerance. This observation was associated with the reduced stomatal density of transgenic plants, which would limit transpiration and reduce water loss, consequently improving the WUE of plants. Interestingly, the reduction of stomatal density in transgenic plants overexpressing <em>PdEPF3</em> did not affect their photosynthetic capacity. These results indicate that <em>PdEPF3</em> could be used in transgenic breeding to enhance plant drought tolerance.

Molecular cloning and expression analysis of a stress-responsive WRKY transcription factor gene, BnWRKY57, from Brassica napus

WRKY transcription factors play important roles in regulation of various plant biological processes, including response to abiotic stresses. WRKY genes might be potential targets for transgenic breeding to enhance stress tolerance in rapeseed (Brassica napus). The present study aimed to clone and characterize WRKY57 (BnWRKY57) gene derived from B. napus and to analyze patterns of BnWRKY57 expression under drought and salt stresses in two B. napus cultivars with different levels of tolerance to drought and salt. The full-length coding region of BnWRKY57 gene with 882bp long (GenBank Accession Number: MG699908) was cloned and sequenced. BnWRKY57 gene encodes a hydrophilic polypeptide of 293 amino acids. It shared high homology with other known WRKY57s from Brassicaceae family. The promoter of BnWRKY57 gene contained cis regulatory elements involved in response to phytohormones, light, biotic and abiotic stresses suggesting this gene may play a role to modulate different signaling pathways. Real time quantitative RT-PCR analysis revealed that BnWRKY57 expression was responsive to drought and salt stresses. BnWRKY57 gene showed different expression patterns between leaves and roots and also between the B. napus cultivars under stress conditions. Overall, the findings suggest that BnWRKY57 gene may contribute to adaptive responses to drought and salt stresses in B. napus.

Heterologous Expression of the DREB Transcription Factor AhDREB in Populus tomentosa Carrière Confers Tolerance to Salt without Growth Reduction under Greenhouse Conditions

The DREB transcription factors regulate multiple stress response genes, and are therefore useful for molecular plant breeding. AhDREB, a stress-inducible gene, was isolated from Atriplex hortensis L. and introduced into Populus tomentosa Carrière under the control of the CaMV35S promoter. Under salt stress, the chlorophyll content and net photosynthetic rate were higher in transgenic lines than in the wild type (WT). Moreover, the rate of electrolyte penetration (REC) was lower in the transgenic lines. Additional analyses revealed that the AhDREB transgenic plants generally displayed lower malondialdehyde (MDA) activity but higher superoxide dismutase (SOD) and peroxidase (POD) activities and proline content than the WT under salt stress. RNA sequencing indicated that AhDREB could enhance tolerance to salt by activating various downstream genes in the transgenic plants. Furthermore, no growth inhibition was detected in transgenic plants expressing AhDREB driven by the constitutive CaMV35S promoter. The transcriptome showed 165 and 52 differentially expressed genes in transgenic plants under stress and non-stress conditions, respectively, among which no significant metabolic pathway was enriched and no unintended effects have yet been identified. Together, these results suggest that AhDREB may be a good candidate gene for increasing salt tolerance in transgenic poplar breeding.

Effect of Abiotic Stresses on Brassica Species and Role of Transgenic Breeding for Adaptation

Abiotic stresses extremely affect crop productivity and agro-morphological and biochemical properties of all Brassica species. Due to these stresses, yield of many important Brassica species decreases by disturbing their normal growth rate. In this review, we have highlighted the latest reports about the impact of different abiotic stresses on different growth stages and other morpho-physiological processes of important Brassica species such as canola/rapeseed (Brassica napus), Indian mustard (Brassica juncea), Brassica oleracea and Brassica rapa. Many researchers reported that abiotic stresses influence the important morpho-biochemical characteristics such as root and shoot length, shoot fresh and dry weight, proline and relative water contents, chlorophyll amount, antioxidant enzymes activity of important Brassica species. Cell injury also occurs by disturbance in normal oxidative processes due to these stresses. Against these stresses genetic modification have been described for the development of transgenic plant. The present study will be useful to identify the best abiotic stress tolerant Brassica genotypes for further genetic engineering program and crop improvement programs.

Nucleus Accumbens Drd1-Expressing Neurons Control Aggression Self-Administration and Aggression Seeking in Mice

We recently developed a mouse model of appetitive operant aggression and reported that adult male outbred CD-1 mice lever-press for the opportunity to attack subordinate male mice and relapse to aggression seeking during abstinence. Here we studied the role of nucleus accumbens (NAc) dopamine receptor (Drd)1- and Drd2-expressing neurons in aggression self-administration and aggression seeking. We trained CD-1 mice to self-administer intruders (9 d, 12 trials/d) and tested them for aggression self-administration and aggression seeking on abstinence Day 1. We used immunohistochemistry and in situ hybridization to measure the neuronal activity marker Fos in the NAc, and cell-type-specific colocalization of Fos with Drd1- and Drd2-expressing neurons. To test the causal role of Drd1- and Drd2-expressing neurons, we validated a transgenic hybrid breeding strategy crossing inbred Drd1-Cre and Drd2-Cre transgenic mice with outbred CD-1 mice and used cell-type-specific Cre-DREADD (hM4Di) to inhibit NAc Drd1- and Drd2-expressing neuron activity. We found that aggression self-administration and aggression seeking induced higher Fos expression in NAc shell than in core, that Fos colocalized with Drd1 and Drd2 in both subregions, and that chemogenetic inhibition of Drd1-, but not Drd2-, expressing neurons decreased aggression self-administration and aggression seeking. Results indicate a cell-type-specific role of Drd1-expressing neurons that is critical for both aggression self-administration and aggression seeking. Our study also validates a simple breeding strategy between outbred CD-1 mice and inbred C57-based Cre lines that can be used to study cell-type and circuit mechanisms of aggression reward and relapse.SIGNIFICANCE STATEMENT Aggression is often comorbid with neuropsychiatric diseases, including drug addiction. One form, appetitive aggression, exhibits symptomatology that mimics that of drug addiction and is hypothesized to be due to dysregulation of addiction-related reward circuits. However, our mechanistic understanding of the circuitry modulating appetitive operant aggression is limited. Here we used a novel mouse model of aggression self-administration and relapse, in combination with immunohistochemistry, in situ hybridization, and chemogenetic manipulations to examine how cell types in the nucleus accumbens are recruited for, and control, operant aggression self-administration and aggression seeking on abstinence Day 1. We found that one population, dopamine receptor 1-expressing neurons, act as a critical modulator of operant aggression reward and aggression seeking.

Overexpression of an alfalfa glutathione S-transferase gene improved the saline-alkali tolerance of transgenic tobacco.

ABSTRACTAbiotic stresses restrict the productivity and quality of agricultural crops. Glutathione S-transferase (GST) utilizes glutathione to scavenge reactive oxygen species (ROS) that result from abiotic stresses. This study aimed to determine the expression pattern of the MsGSTU8 gene and its effects on saline-alkali tolerance. MsGSTU8, from alfalfa (Medicago sativa ‘Zhaodong’), was transformed into transgenic tobacco (Nicotiana tabacum) and overexpressed to determine its effects on saline-alkali tolerance. The gene products in alfalfa localized to the cytoplasm and the transcript levels were higher in the leaves than the roots and stems. Expression was strongly induced by cold, drought, salt and saline-alkali stresses as well as abscisic acid (ABA) treatments. The transgenic tobacco lines had significantly higher transcription levels of the abiotic stress-related genes and higher GST activity than the wild types. Transgenic tobacco lines with saline-alkali treatments maintained their chlorophyll content, showed improved antioxidant enzyme activity and soluble sugar levels, reduced ion leakage, O2.−, H2O2 accumulation and malondialdehyde content. Our results indicate that overexpression of MsGSTU8 could improve resistance to saline-alkali stresses by decreasing the accumulation of ROS and increasing the levels of antioxidant enzymes. Furthermore, they suggest that MsGSTU8 could be utilized for transgenic crop plant breeding.

Overexpression of the Wheat (Triticum aestivum L.) TaPEPKR2 Gene Enhances Heat and Dehydration Tolerance in Both Wheat and Arabidopsis.

Wheat (Triticum aestivum L.) yield and quality are adversely affected by heat, drought, or the combination of these two stresses in many regions of the world. A phosphoenolpyruvate carboxylase kinase-related kinase gene, TaPEPKR2, was identified from our previous heat stress-responsive transcriptome analysis of heat susceptible and tolerant wheat cultivars. Based on the wheat cultivar Chinese Spring genome sequence, TaPEPKR2 was mapped to chromosome 5B. Expression analysis revealed that TaPEPKR2 was induced by heat and polyethylene glycol treatment. To analyze the function of TaPEPKR2 in wheat, we transformed it into the wheat cultivar Liaochun10, and observed that the transgenic lines exhibited enhanced heat and dehydration stress tolerance. To examine whether TaPEPKR2 exhibits the same function in dicotyledonous plants, we transformed it into Arabidopsis, and found that its overexpression functionally enhanced tolerance to heat and dehydration stresses. Our results imply that TaPEPKR2 plays an important role in both heat and dehydration stress tolerance, and could be utilized as a candidate gene in transgenic breeding.

Characterization of a strong and constitutive promoter from the Arabidopsis serine carboxypeptidase-like gene AtSCPL30 as a potential tool for crop transgenic breeding

BackgroundTransgenic technology has become an important technique for crop genetic improvement. The application of well-characterized promoters is essential for developing a vector system for efficient genetic transformation. Therefore, isolation and functional validation of more alternative constitutive promoters to the CaMV35S promoter is highly desirable.ResultsIn this study, a 2093-bp sequence upstream of the translation initiation codon ATG of AtSCPL30 was isolated as the full-length promoter (PD1). To characterize the AtSCPL30 promoter (PD1) and eight 5′ deleted fragments (PD2-PD9) of different lengths were fused with GUS to produce the promoter::GUS plasmids and were translocated into Nicotiana benthamiana. PD1-PD9 could confer strong and constitutive expression of transgenes in almost all tissues and development stages in Nicotiana benthamiana transgenic plants. Additionally, PD2-PD7 drove transgene expression consistently over twofold higher than the well-used CaMV35S promoter under normal and stress conditions. Among them, PD7 was only 456 bp in length, and its transcriptional activity was comparable to that of PD2-PD6. Moreover, GUS transient assay in the leaves of Nicotiana benthamiana revealed that the 162-bp (− 456~ − 295 bp) and 111-bp (− 294~ − 184 bp) fragments from the AtSCPL30 promoter could increase the transcriptional activity of mini35S up to 16- and 18-fold, respectively.ConclusionsAs a small constitutive strong promoter of plant origin, PD7 has the advantage of biosafety and reduces the probability of transgene silencing compared to the virus-derived CaMV35S promoter. PD7 would also be an alternative constitutive promoter to the CaMV35S promoter when multigene transformation was performed in the same vector, thereby avoiding the overuse of the CaMV35S promoter and allowing for the successful application of transgenic technology. And, the 162- and 111-bp fragments will also be very useful for synthetic promoter design based on their high enhancer activities.

Comparative transcriptome study provides insights into acquisition of embryogenic ability in upland cotton during somatic embryogenesis

BackgroundThe conversion from non-embryogenic callus (NEC) to embryogenic callus (EC) is the key bottleneck step in regeneration of upland cotton (Gossypium hirsutum), and hinders the transgenic breeding of upland cotton. To investigate molecular mechanisms underlying acquisition of embryogenic potential during this process, comparation analysis of transcriptome dynamics between two upland cotton cultivars with different somatic embryogenesis abilities was conducted.ResultsDifferentially expressed genes involved in the transformation from NEC to EC were detected in the two different cultivars. Principal component analysis based on DEGs showed that the NEC tissues of the two cultivars were highly heterogeneous, whereas the derived EC tissues were similar, which suggested the homogeneousness of EC between different lines. In the highly embryogenic cultivar CCRI 24, more of these genes were down-regulated, whereas, in the recalcitrant cultivar CCRI 12, more were up-regulated. Bioinformatics analysis on these DEGs showed that the vast majority of differentially expressed genes were enriched in metabolism and secondary metabolites biosynthesis pathways. Flavonoid biosynthesis and phenylpropanoid biosynthesis pathways were enriched in both cultivars, and the associated genes were down-regulated more in CCRI 24 than in CCRI 12. We deduced that vigorous secondary metabolism in CCRI 12 may hinder primary metabolism, resulting in tardiness of cell differentiation. Interestingly, genes involved in the plant hormone signal transduction pathway were enriched in the recalcitrant cultivar CCRI 12, but not in CCRI 24, suggesting more radical regulation of hormone signal transduction in the recalcitrant cultivar. Signal transduction rather than biosynthesis of plant hormones is more likely to be the determining factor triggering NEC to EC transition in recalcitrant cotton lines. Transcription factor encoding genes showed differential regulation between two cultivars.ConclusionsOur study provides valuable information about the molecular mechanism of conversion from NEC to EC in cotton and allows for identification of novel genes involved. By comparing transcriptome changes in transformation from NEC to EC between the two cultivars, we identified 46 transcripts that may contribute to initiating embryogenic shift.

Pyramiding of nine transgenes in maize generates high-level resistance against necrotrophic maize pathogens.

Key message Nine transgenes from different categories, viz. plant defense response genes and anti-apoptosis genes, played combined roles in maize to inhibit the necrotrophic pathogens Rhizoctonia solani and Bipolaris maydis. Maize sheath blight and southern corn leaf blight are major global threats to maize production. The management of these necrotrophic pathogens has encountered limited success due to the characteristics of their lifestyle. Here, we presented a transgenic pyramiding breeding strategy to achieve nine different resistance genes integrated in one transgenic maize line to combat different aspects of necrotrophic pathogens. These nine genes, selected from two different categories, plant defense response genes (Chi, Glu, Ace-AMP1, Tlp, Rs-AFP2, ZmPROPEP1 and Pti4), and anti-apoptosis genes (Iap and p35), were successfully transferred into maize and further implicated in resistance against the necrotrophic pathogens Rhizoctonia solani and Bipolaris maydis. Furthermore, the transgenic maize line 910, with high expression levels of the nine integrated genes, was selected from 49 lines. Under greenhouse and field trial conditions, line 910 showed significant resistance against maize sheath blight and southern corn leaf blight diseases. Higher-level resistance was obtained after the pyramiding of more resistance transgenes from different categories that function via different mechanisms. The present study provides a successful strategy for the management of necrotrophic pathogens.