Stable Genetic Transformation Research Articles

Optimization of stable genetic transformation protocol in castor (Ricinus communis L. cv. TMV 5) using beta glucuronidase reporter gene for pioneer of desirable genes

The simple and stable protocol was standardised for castor (Ricinus communis L. cv. TMV 5) genetic transformation using Agrobacterium tumefaciens strain LBA4404 harbouring the binary plasmid pBAL2 (18.8 kb). Cotyledonary nodes from ten days old, in vivo seedlings were utilized as target cells for Agrobacterium mediated transformation. Explant pre-culture studies were carried out at 2, 4, 6, 8, 10 and 12 day intervals. The 4th day old explants cultivated on mMS medium (MS medium+B5 Vitamins) using plant growth regulators had the highest response percentage (50.6%). Kanamycin (0-175 mg/L) and Hygromycin (0-13 mg/L) sensitivity in well-developed shoots was investigated. Of the two antibiotics, Kanamycin 50 mg/L and Hygromycin 3 mg/L was found optimum. Different levels of acetosyringone (0-200 mg/L) were used in the co-cultivation medium to study the transformation efficiency of castor. Among the different concentrations, maximum number of explants showed GUS expression at 100 mg/L of acetosyringone in the co-cultivation medium at 2 days of co-cultivation period and the Cotyledonary node produced multiple shoots development and plantlet establishment in 0.3 mg/L TDZ, 0.6 mg/L PF-68, kanamycin 50 mg/L, 0.3 mg/L GA3, 1.5 mg/L IBA and 0.6 mg/L AgNO3. The rooted shoots were successfully acclimatized. Histochemical GUS assay was used to monitor T-DNA delivery into the target cells. PCR and Southern hybridization were used to confirm the transformants with the NPT II and GUS gene. A very high frequency (29.3%) of β-glucuronidase (GUS) gene expression was obtained through Agrobacterium-mediated gene transfer into cotyledonary node explants of Castor. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of Cator with desirable gene of agronomic importance.

A simple and efficient gene functional analysis method for studying the growth and development of peach seedlings.

Stable genetic transformation of peach [Prunus persica (L.) Batsch] still faces many technical challenges, and existing transient expression methods are limited by tissue type or developmental stage, making it difficult to conduct functional analysis of genes regulating shoot growth. To overcome this dilemma, we developed a three-step method for efficient analysis of gene functions during peach seedling growth and development. This method resulted in transformation frequencies ranging from 48 to 87%, depending on the gene. From transformation of germinating seeds to phenotyping of young saplings took just 1.5months and can be carried out any time of year. To test the applicability of this method, the function of three tree architecture-related genes, namely PpPDS, PpMAX4, and PpWEEP, and two lateral root-related genes, PpIAA14-1 and -2, were confirmed. Since functional redundancy can challenge gene functional analyses, tests were undertaken with the growth-repressor DELLA, which has three homologous genes, PpDGYLA (DG), PpDELLA1 (D1), and -2 (D2), in peach that are functionally redundant. Silencing using a triple-target vector (TRV2-DG-D1-D2) resulted in transgenic plants taller than those carrying just TRV2-DG or TRV2. Simultaneously silencing the three DELLA genes also attenuated the stature of two dwarf genotypes, 'FHSXT' and 'HSX', which normally accumulate DELLA proteins. Our study provides a method for the functional analysis of genes in peach and can be used for the study of root, stem, and leaf development. We believe this method can be replicated in other woody plants.

A protoplast-based transient gene expression assay for the identification of heat and oxidative stress-regulatory genes in perennial ryegrass

BackgroundWith the accumulating omics data, an efficient and time-saving transient assay to express target genes is desired. Mesophyll protoplasts, maintaining most stress-physiological responses and cellular activities as intact plants, offer an alternative transient assay to study target genes’ effects on heat and oxidative stress responses.ResultsIn this study, a perennial ryegrass (Loliumperenne L.) mesophyll protoplast-based assay was established to effectively over- or down-regulate target genes. The relative expression levels of the target genes could be quantified using RT-qPCR, and the effects of heat and H2O2-induced oxidative stress on protoplasts’ viability could be quantitatively measured. The practicality of the assay was demonstrated by identifying the potential thermos-sensor genes LpTT3.1/LpTT3.2 in ryegrass that over-expressing these genes significantly altered protoplasts’ viability rates after heat stress.ConclusionThis protoplast-based rapid stress regulatory gene identification assay was briefed as ‘PRIDA’ that will complement the stable genetic transformation studies to rapidly identify candidate stress-regulatory genes in perennial ryegrass and other grass species.

Setting up Agrobacterium tumefaciens-mediated transformation of the tropical legume Aeschynomene evenia, a powerful tool for studying gene function in Nod Factor-independent symbiosis.

Most legumes are able to develop a root nodule symbiosis in association with proteobacteria collectively called rhizobia. Among them, the tropical species Aeschynomene evenia has the remarkable property of being nodulated by photosynthetic Rhizobia without the intervention of Nod Factors (NodF). Thereby, A. evenia has emerged as a working model for investigating the NodF-independent symbiosis. Despite the availability of numerous resources and tools to study the molecular basis of this atypical symbiosis, the lack of a transformation system based on Agrobacterium tumefaciens significantly limits the range of functional approaches. In this report, we present the development of a stable genetic transformation procedure for A. evenia. We first assessed its regeneration capability and found that a combination of two growth regulators, NAA (= Naphthalene Acetic Acid) and BAP (= 6-BenzylAminoPurine) allows the induction of budding calli from epicotyls, hypocotyls and cotyledons with a high efficiency in media containing 0,5 μM NAA (up to 100% of calli with continuous stem proliferation). To optimize the generation of transgenic lines, we employed A. tumefaciens strain EHA105 harboring a binary vector carrying the hygromycin resistance gene and the mCherry fluorescent marker. Epicotyls and hypocotyls were used as the starting material for this process. We have found that one growth medium containing a combination of NAA (0,5 μM) and BAP (2,2 μM) was sufficient to induce callogenesis and A. tumefaciens strain EHA105 was sufficiently virulent to yield a high number of transformed calli. This simple and efficient method constitutes a valuable tool that will greatly facilitate the functional studies in NodF-independent symbiosis.

Development of a stable genetic transformation system in Erigeron breviscapus: a case study with EbYUC2 in relation to leaf number and flowering time.

A stable genetic transformation system for Erigeron breviscapus was developed. We cloned the EbYUC2 gene and genetically transformed it into Arabidopsis thaliana and E. breviscapus. The leaf number, YUC2 gene expression, and the endogenous auxin content in transgenic plants were significantly increased. Erigeron breviscapus is a prescription drug for the clinical treatment of cardiovascular and cerebrovascular diseases. The rosette leaves have the highest content of the major active compound scutellarin and are an important component in the yield of E. breviscapus. However, little is known about the genes related to the leaf number and flowering time of E. breviscapus. In our previous study, we identified three candidate genes related to the leaf number and flowering of E. breviscapus by combining resequencing data and genome-wide association study (GWAS). However, their specific functions remain to be characterized. In this study, we cloned and transformed the previously identified full-length EbYUC2 gene into Arabidopsis thaliana, developed the first stable genetic transformation system for E. breviscapus, and obtained the transgenic plants overexpressing EbYUC2. Compared with wild-type plants, the transgenic plants showed a significant increase in the number of leaves, which was correlated with the increased expression of EbYUC2. Consistently, the endogenous auxin content, particularly indole-3-acetic acid, in transgenic plants was also significantly increased. These results suggest that EbYUC2 may control the leaf number by regulating auxin biosynthesis, thereby laying a foundation for revealing the molecular mechanism governing the leaf number and flowering time of E. breviscapus.

Engineering Camelina sativa Seeds as a Green Bioreactor for the Production of Affordable Human Pro-insulin that Demonstrates Anti-diabetic Efficacy in Rats.

The current production of recombinant insulin via fermenter-based platforms (Escherichia coli and yeast) could not fulfill its fast-growing commercial demands, thus leading to a great interest in its sustainable large-scale production at low cost using a plant-based system. In the present study, Agrobacterium tumefaciens-mediated nuclear stable genetic transformation of an industrial oilseed crop, Camelina sativa, to express pro-insulin (with three furin endoprotease cleavage sites) fused with cholera toxin B subunit (CTB) in their seeds was successfully achieved for the first time. The bar gene was used as a selectable marker for selecting transformants and producing herbicide-resistant camelina plants. The transformation process involved the infiltration of camelina inflorescences (at flower buds with partially opened flowers) with A. tumefaciens and harvesting the seeds (T0) at maturity. The T0 seeds were raised into the putative T1 plants sprayed with Basta herbicide (0.03%, v/v), and the survived green transformed plants tested positive for pro-insulin and bar genes. A transformation frequency of 6.96% was obtained. The integration and copy number of the pro-insulin transgene and its expression at RNA and protein levels were confirmed in T1 plants using Southern hybridization, semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (sqPCR), and quantitative real-time Time PCR (qPCR) and western blot analysis, respectively. Enzyme-linked immunosorbent Assay (ELISA) quantified the amount of expressed pro-insulin protein, and its anti-diabetic efficacy was validated in diabetic rats on oral feeding. Transgenic plants integrated the pro-insulin gene into their genomes and produced a maximum of 197µg/100mg of pro-insulin (0.804% of TSP) that had anti-diabetic efficacy in rats.

ZjHXK5 and ZjHXK6 negatively regulate the sugar metabolism of Ziziphus jujuba Mill.

Hexokinase (HXK) plays a crucial role in plants, catalyzing the phosphorylation of hexose substances, which is one of the key steps in sugar metabolism and energy production. While HXK genes have been well-studied in model plants, the evolutionary and functional characteristics of HXK gene family in jujube is unknow. In this study, the HXK gene family members were identified by bioinformatics methods, the key members regulating glucose metabolism were identified by transcriptome data, and finally the function of the key genes was verified by instantaneous and stable genetic transformation. Our results showed that seven HXK genes were identified in the jujube genome, all of which were predict located in the chloroplast and contain Hexokinase-1 (PF00349) and Hexokinase-2 (PF03727) conserved domains. Most of HXK proteins were transmembrane protein with stable, lipid-soluble, hydrophilic. The secondary structure of ZjHXK proteins main α-helix, and contains two distinct tertiary structure. All ZjHXK genes contain nine exons and eight introns. Predictions of cis-regulatory elements indicate that the promoter region of ZjHXK contains a large number of MeJA responsive elements. Finally, combined with the analysis of the relationship between the expression and glucose metabolism, found that ZjHXK5 and ZjHXK6 may the key genes regulating sugar metabolism. Transient overexpression of ZjHXK5 and ZjHXK6 on jujube, or allogeneic overexpression of ZjHXK5 and ZjHXK6 on tomato would significantly reduce the content of total sugar and various sugar components. Transient silencing of ZjHXK5 and ZjHXK6 genes results in a significant increase in sucrose and total sugar content. Interestingly, the expression of ZjHXK5 and ZjHXK6 were also affected by methyl jasmonate.

Establishment of a genetic transformation system for cordycipitoid fungus Cordyceps chanhua.

Cordyceps chanhua is a well-known edible and medicinal mushroom with a long history of use in China, and it contains a variety of secondary metabolites with interesting bioactive ingredients. However, recent researches have mainly focused on cultivation conditions, secondary metabolite compositions and pharmacological activities of C. chanhua, the lack of an efficient and stable genetic transformation system has largely limited further research on the relationship between secondary metabolites and biosynthetic gene clusters in C. chanhua. In this study, single-factor experiments were used to compare the effects of different osmotic stabilizers, enzyme concentrations and enzyme digestion times on protoplast yield, and we found that the highest yield of 5.53 × 108 protoplasts/mL was obtained with 0.7 M mannitol, 6 mg/mL snail enzyme and 4 h of enzyme digestion time, and the regeneration rate of protoplasts was up to approximately 30% using 0.7 M mannitol as an osmotic stabilizer. On this basis, a PEG-mediated genetic transformation system of C. chanhua was successfully established for the first time, which lays the foundation for further genetic transformation of C. chanhua.

Cloning, Characterization, and Expression Pattern Analysis of the BBM Gene in Tree Peony (Paeonia ostii)

BABY BOOM (BBM) is one of the members of the plant-specific APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor superfamily. It acts as a key regulator of plant cell pluripotency, playing a significant role in promoting somatic embryogenesis. In this study, a BBM gene named PoBBM was screened, cloned, and identified from the third-generation full-length transcriptome data of Paeonia ostii. Its open reading frame was 2136 bp, encoding 711 amino acids. Sequence feature analysis revealed that it possessed two AP2 conserved domains and eight motifs, including bbm-1. The phylogenetic tree indicated that PoBBM clusters with AtBBM in the euANT group of the Arabidopsis AP2 family, which is most closely related to grape VvBBM and may have the same ancestry as grape. Subcellular localization demonstrated that the PoBBM protein was localized in the nucleus. Semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess the PoBBM transcript levels during ten developmental stages of somatic embryos and in five tissue types of peonies. The results indicate that PoBBM was highly expressed in the early stages of peony somatic embryo development. The expression on 0–15 d was the highest and decreased gradually with somatic embryogenesis. The gene is almost not expressed after 40 d since somatic embryo formation. PoBBM was expressed in roots, stems, leaves, seeds, and calli, with the highest levels in seeds, followed by leaves and calli. The PoBBM protein displayed transcriptional self-activation activity, which may facilitate further research on its relationships with other proteins. The above results provide a key gene PoBBM for somatic embryogenesis in peonies, which is significant for advancing the establishment of a stable and efficient regeneration and genetic transformation system for peonies.

Establishment of a Rapid and Effective Agrobacterium-Mediated Genetic Transformation System of Oxalis triangularis 'Purpurea'.

Oxalis triangularis 'Purpurea' has significant ornamental value in landscaping. There is a critical necessity to elucidate the gene functions of O. triangularis 'Purpurea' and dissect the molecular mechanisms governing key ornamental traits. However, a reliable genetic transformation method remains elusive. In this study, our investigation revealed that various transformation parameters, including recipient material (petioles), pre-culture time (2-5 days), acetosyringone (AS) concentration (100-400 μM), Agrobacterium concentrations (OD600 = 0.4-1.0), infection time (5-20 min), and co-culture time (2-5 days), significantly impacted the stable genetic transformation in O. triangular 'Purpurea'. Notably, the highest genetic transformation rate was achieved from the leaf discs pre-cultured for 3 days, treated with 200 μM AS infected with Agrobacterium for 11 min at OD600 of 0.6, and subsequently co-cultured for 3 days. This treatment resulted in a genetic transformation efficiency of 9.88%, and it only took 79 days to produce transgenic plants. Our transformation protocol offers advantages of speed, efficiency, and simplicity, which will greatly facilitate genetic transformation for O. triangular 'Purpurea' and gene function studies.

Recent Advances in Natural Deastringency and Genetic Improvement of Chinese PCNA Persimmon (Diospyros kaki)

Persimmon (Diospyros kaki) is a worldwide fruit cultivated mainly in the East Asia, Mediterranean, Caucasus, Latin America, and Oceania regions. This fruit contains abundant proanthocyanidins (PAs, also called condensed tannins), whose biosynthesis is the main cause of fruit astringency. As the original centre and top producing country, China has discovered a unique type with desirable natural deastringency, the Chinese pollination-constant non-astringent (C-PCNA) persimmon. Studies have revealed that the C-PCNA trait is controlled by a single and dominant locus, which differs from that of another type, the Japanese PCNA type, with recessive loci. In the C-PCNA type, accumulating evidence has shown that the astringency removal process involves two pathways (“dilution effect” and “coagulation effect”). Moreover, molecular marker-assisted selection (MAS) for the natural deastringency trait locus in C-PCNA has been used to test the non-astringency/astringency trait of hybrid offspring at the seedling stage. Importantly, persimmon can bear male flowers, female flowers, and perfect flowers, but sex-linked MAS has been developed for female-only persimmon. This sex-linked MAS, together with astringency-linked MAS and embryo rescue technology, may even shorten the conventional cross-breeding period of about 2–3 years. In addition, recently studies have established a stable genetic transformation system for persimmon transgenic improvement. Despite these efforts, how synthetic PAs and metabolism pathways lead to a deastringent trait remains unclear for persimmon. Thus, our review summarizes the latest research progress on the natural deastringency mechanism in C-PCNA, and we provide a new viewpoint for the genetic improvement of persimmon breeding in China.

Tracking organelle activities through efficient and stable root genetic transformation system in woody plants.

Due to the protracted transgenic timeline and low efficiency in stable genetic transformation of woody plants, there has been limited exploration of real-time organelle imaging within stable transgenic woody plant cells. Here, we established an efficient in vivo genetic transformation system for woody plants using an Agrobacterium rhizogenes-mediated approach. This system was successfully validated in multiple perennial woody species. Using citrus as a model, we introduced organelle-targeted fluorescent reporters via genetic transformation and investigated their subcellular localization and dynamics using advanced imaging techniques, such as confocal microscopy and live-cell imaging. Moreover, we subjected transgenic MT-GFP-labeled mitochondria in root cells to stress conditions simulating agricultural adversities faced by fruit crops. The stress-induced experiments revealed notable alterations in mitochondrial morphology. Our study contributes novel insights into membrane trafficking processes, protein localization dynamics, and cellular physiology in woody plants, while also providing stable and efficient genetic transformation methods for perennial woody species.

Development and application of virus-induced gene silencing (VIGS) for studying ApTT8 gene function in Agapanthus praecox ssp. orientalis

Agapanthus praecox ssp. orientalis is a famous summer-blooming perennial that produces hundreds of florets with blue or white tepal. In comparison, other colours are infrequent in the genus Agapanthus. Previous analysis revealed that delphinidin was the main but not the only anthocyanidin, petunidin and cyanidin also existed in the tepal. This indicated that the colouration and regulation mechanism in A. praecox was unique and worthy of study. However, the stable genetic transformation was low efficiency and time-consuming. A rapid and effective gene functional identification method was urgently needed. In this study, the first use of the VIGS system in A. praecox was described in detail. Tobacco rattle virus (TRV) caused systemic infection with mild symptoms in A. praecox, which was also confirmed by qRT-PCR. The reporter gene ApPDS (phytoene desaturase) was silenced using the injection and soaking method in basal plates of inflorescence, and the photo-bleaching symptoms were observed in tepal. The optimal Agrobacterium concentration was OD600 = 1.0. Then the VIGS system was used to silence a bHLH-like transcriptional factor, ApTT8, to identify its function in anthocyanin biosynthesis. The results showed that the total anthocyanin content was reduced to 12.21-fold compared with the empty control. Additionally, tepals were taken as recipients of VIGS using vacuum infiltration method. Total anthocyanin content was reduced to 3.44-fold in ApTT8-silenced tepal. The silencing efficiency of tepal was as high as 85.09 %. The structure genes, ApF3’H, ApF3’5’H, ApF3H, ApDFR, ApANS and ApUFGT were also significantly down-regulated. The successful silencing of the ApPDS and ApTT8 in this study indicated that the optimized TRV-mediated VIGS system could be used to target gene silencing in florets and tepal of A. praecox. This method will significantly shorten the time required for studying molecular mechanism of flower colouration and is of great significance for the research of A. praecox functional genomics.

Cloning, Characterization, and Functional Analysis of EuTIL1, a Gene-Encoding Temperature-Induced Lipocalin in Eucommia ulmoides Oliv

Eucommia ulmoides Oliver is a unique tertiary relict tree species in China belonging to the Eucommia family and genus. It is a traditional and precious Chinese medicinal herb with anti-tumor, antibacterial, antioxidant, anti-inflammatory, and bidirectional blood pressure regulation effects. Eucommia ulmoides mainly grows in temperate regions of China, but due to its sensitivity to low-temperatures, it is difficult to introduce into new regions. To study the role of Eucommia ulmoides lipocalin in plants. This investigation was conducted utilizing gene cloning, bioinformatics analysis, quantitative real-time polymerase chain reaction (qRT-PCR), subcellular localization, and stable genetic transformation to transfer EuTIL1 into Nicotiana tabacum Xanthi. The wild-type transgenic vector and EuTIL1 tobacco were cold-treated, and the corresponding protective enzyme activity and cold-induced gene expression levels were measured to analyze the functions of the genes. In this study, the full-length of the temperature-induced lipocalin gene (EuTIL1) cDNA was cloned from the leaves of Eucommia ulmoides using the rapid amplification of cDNA ends (RACE) method. The sequence analysis showed that the full-length cDNA of EuTIL1 was 917 bp and encodes a protein of 188 aa residues, which is a member of the Lipocalin-2 family. Subcellular localization analysis revealed that EuTIL1 was found in the plasma membrane. The transgenic tobacco lines expressing EuTIL1 under the control of the CaMV 35S promoter had increased tolerance to cold compared to wild-type (WT) plants. The average water loss rate of EuTIL1 transgenic plants was 12.4%, the average conductivity at 24 h was 55.11%, and the malondialdehyde content at 24 h was significantly lower than that of wild-type plants. The maximum soluble sugar (SS) content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, and catalase activity of EuTIL1 plants after low-temperature treatment were 22.03 mg/g, 726.87 U/g, 1283.94 U/g, and 356.84 U/g, respectively, which are significantly higher than those of the wild-type. Meanwhile, in the EuTIL1 transgenic tobacco plants, the expression of the NtDREB1, NtDREB2, NtDREB4, and NtCOR15a elevated under the low-temperature treatment condition. In conclusion, our study demonstrates that EuTIL1 is a gene involved in the cold-stress response and has the potential to enhance cold tolerance in plants, providing a potential molecular basis for the study of Eucommia ulmoides introduction and serving as a candidate gene for evaluating cold-tolerant plants.

Application and Expansion of Virus-Induced Gene Silencing for Functional Studies in Vegetables

Increased consumption of vegetables has been recommended worldwide as a part of a healthy diet; therefore, determining gene function among breeding materials is crucial for vegetable improvement to meet the sustainable development of new vegetable varieties. However, genetic transformation is time-consuming and laborious, which limits the exploration of gene function for various vegetable crops. Virus-Induced Gene Silencing (VIGS) can perform large-scale and rapid gene silencing in plants due to a reduction in the experimental period and its independence from the stable genetic transformation, providing an excellent opportunity for functional research. VIGS can accelerate model plant research and make it easier to analyze gene function and validation in vegetable crops. Moreover, with the advent of technologies such as virus-mediated heterologous protein expression and the development of CRISPR/Cas9 technology, virus-mediated genetic tools have ushered in a new era in genetics and crop improvement. This study summarizes recent achievements in VIGS and Virus-Induced Gene Editing (VIGE) in vegetables. We also identify several challenges in the current state of VIGS technology in vegetables, serving as a guide for future research.

Agrobacterium tumefaciens-mediated transformation of embryogenic callus and CRISPR/Cas9-mediated genome editing in ‘Feizixiao’ litchi

Litchi (Litchi chinensis Sonn.) is a type of commercially prevalent subtropical and tropical fruit. Since litchi has a highly heterozygous genetic background and a long reproductive cycle, conventional breeding methods (such as hybridization) have limited ability to nurture new litchi cultivars. Here, an efficient and stable Agrobacterium tumefaciens-mediated genetic transformation of embryogenic callus was established in ‘Feizixiao’ litchi. Transgenic materials were verified using polymerase chain reaction (PCR) analysis, β-glucuronidase (GUS) assay, and green fluorescent protein (GFP) assay. To implement the technology of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology in ‘Feizixiao’ litchi and verify the validity of these transformation systems, the litchi polyphenol oxidase gene (LcPPO, JF926153) was knocked out. Various categories of mutations, covering base insertions, deletions, and substitutions, were found in transgenic materials via sequence analysis. The transformation system achieved high feasibility and efficiency, and the system of CRISPR/Cas9 was successfully employed to edit genes in ‘Feizixiao’ litchi. This work provides an essential foundation for investigating the functions of genes and accelerating litchi genetic improvement.

Establishment of Stable Genetic Transformation Conditions for Novel Thermotolerant Scenedesmus sp. (Chlorophyta)

Microalgae became an attractive source of high value compounds such as fatty acids, carotenoids and other nutrients in recent years. In a previous study, the characterization of a novel thermo-tolerant freshwater microalga, Scenedesmus sp. ME02 with high phenolic content, particularly rich in flavonoids was done. Here, two different approaches are reported to increase the efficiency of the growth parameters of this strain. Firstly, the effects of phosphorus (P) depletion on biomass and lipid characteristics of Scenedesmus sp. ME02 were investigated. Although, P depletion resulted in a slight reduction in biomass productivity of microalgae and had a minimal effect on the lipid content and productivity; fatty acid methyl ester profile changed substantially. Particularly, percent oleic acid (18:1) increased by more than two-fold, whereas percent linolelaidic acid (C18:2n6t) and linolenic acid (C18:3n6) decreased significantly when cells were switched from P-replete (control) to P-deplete conditions. Overall, the polyunsaturated fatty acid content was significantly reduced. Secondly, stable genetic transformation conditions for Scenedesmus sp. ME02 via electroporation were optimized using two different transformation vectors. GFP gene expression was detected in colonies transformed with both vectors by RT-PCR. GFP protein expression was detected in the transgenic lines one year after initial transformation by confocal microscopy.

Enhancement of the anthocyanin contents of Caladium leaves and petioles via metabolic engineering with co-overexpression of AtPAP1 and ZmLc transcription factors.

Metabolic engineering of anthocyanin synthesis is an active research area for pigment breeding and remains a research hotspot involving AtPAP1 and ZmLc transcription factors. Caladium bicolor is a desirable anthocyanin metabolic engineering receptor, with its abundant leaf color and stable genetic transformation system. We transformed C. bicolor with AtPAP1 and ZmLc and successfully obtained transgenic plants. We then used a combination of metabolome, transcriptome, WGCNA and PPI co-expression analyses to identify differentially expressed anthocyanin components and transcripts between wild-type and transgenic lines. Cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside and peonidin-3-O-rutinoside are the main components of anthocyanins in the leaves and petioles of C. bicolor. Exogenous introduction of AtPAP1 and ZmLc resulted in significant changes in pelargonidins, particularly pelargonidin-3-O-glucoside and pelargonidin-3-O-rutinoside in C. bicolor. Furthermore, 5 MYB-TFs, 9 structural genes, and 5 transporters were found to be closely associated with anthocyanin synthesis and transport in C. bicolor. In this study, a network regulatory model of AtPAP1 and ZmLc in the regulation of anthocyanin biosynthesis and transport in C. bicolor was proposed, which provides insights into the color formation mechanisms of C. bicolor, and lays a foundation for the precise regulation of anthocyanin metabolism and biosynthesis for economic plant pigment breeding.

Optimization of the Genetic Transformation System of Lettuce

Lettuce (Lactuca sativa L.) is an annual vegetable crop of the family Asteraceae. It is the most consumed leaf vegetable in the world and is highly valued for its edible and medicinal value. Using transgenic technology, introducing functional genes into plants can shorten the breeding time and improve the quality of lettuce. However, in the genetic transformation of lettuce, the application of transgenic technology is limited by the low conversion rate. In this experiment, using ‘S39’ cotyledons as the test material, to establish a stable genetic transformation system. The results showed that when the leaf regeneration medium was 0.01 mg/L 6-BA and 0.4 mg/L NAA, the highest regeneration rate reached 97.9%, which was the best leaf regeneration hormone concentration. When the infection fluid was used in the OD600 value of 0.2, the infected leaves were in good condition and controllable Agrobacterium was the most suitable infection fluid concentration. When the infection time was 15 min, the infection effect was the best, the leaves grew well, and the resistant plants could grow. The screening in a medium containing 30 mg/L of Kana concentration and 250 mg/L of Cef was the suitable medium formulation. NAA 0.1 mg /L and 6-BA 0.2 mg /L were selected as the optimal concentrations in the rooting medium.

Effect of BBM gene on callus growth and ginsenoside content in Panax quinquefolius

Baby Boom(BBM) gene is a key regulatory factor in embryonic development and regeneration, cell proliferation, callus growth, and differentiation promotion. Since the genetic transformation system of Panax quinquefolius is unstable with low efficiency and long period, this study attempted to transfer BBM gene of Zea mays to P. quinquefolius callus by gene gunship to investigate its effect on the callus growth and ginsenoside content, laying a foundation for establishing efficient genetic transformation system of P. quinquefolius. Four transgenic callus of P. quinquefolius with different transformation events were obtained by screening for glufosinate ammonium resistance and molecular identification by PCR. The growth state and growth rate of wild-type and transgenic callus were compared in the same growth period. The content of ginsenoside in transgenic callus was determined by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-MS/MS). The results showed that transgenic callus growth rate was significantly higher than that of wild-type callus. In addition, the content of ginsenoside Rb_1, Rg_1, Ro, and Re was significantly higher than that in wild-type callus. The paper preliminarily proved the function of BBM gene in promoting growth rate and increasing ginsenoside content, which provided a scientific basis to establish a stable and efficient genetic transformation system for Panax plants in the future.