Genetic Transformation System Research Articles

Somatic Embryogenesis of Myrciaria dubia (Kunth.) McVaugh

Myrciaria dubia (caçari or camu-camu) is a species with great relevance because of its high levels of bioactive compounds and antioxidant activity. The species is propagated mainly by seed, which leads to high genetic diversity. Producing plants that maintain their characteristics on a large scale at a low cost is a challenge for fruit species, especially in the Myrtaceae family. Therefore, this study aimed at assessing the effects of different plant growth regulators at various concentrations for the induction of somatic embryogenesis from caçari nodal segments and leaf disk explants. Two independent experiments were performed using nodal segments and leaf disks from plants grown in the greenhouse. In the first experiment, the combined effect of auxin and cytokinin at different concentrations was evaluated: 2,4-dichlorophenoxyacetic acid at 0, 1, 2, and 4 mg⋅L–1; and benzylaminopurine at 0, 0.25, 0.5, and 1 mg⋅L–1). In the second experiment, the application of different plant growth regulators (benzylaminopurine, kinetin, thidiazuron, and isopentenyl adenine) and their concentrations (0, 2, 4, and 6 mg⋅L–1) were evaluated. In both experiments, the basic culture medium was woody plant medium. Callus formation via nodal segments and leaf disks occurred in the first 30 d from cultivation and proved to be responsive to induction by 2,4-dichlorophenoxyacetic acid and benzylaminopurine. Auxin proved to be essential for somatic embryogenesis induction in nodal segments using 1 and 4 mg⋅L–1 of 2,4-dichlorophenoxyacetic acid alone. The results of this experiment will help advancing protocols for regeneration of somatic embryos and elucidating the physiological, molecular, and genetic mechanisms involved in the process of somatic embryogenesis for M. dubia. The development of an efficient protocol for in vitro clonal propagation of this species also lays the groundwork for further optimization of the system for genetic transformation.

Ectopic Expression of PgF3′5′H in Commercial Gypsophila paniculata Cultivar through Optimized Agrobacterium-Mediated Transformation

Gypsophila paniculata is one of the most popular cut flowers in the world whose major cultivars are blooming white. As is well known, blue flowers could be generated via the overexpression of the gene encoding flavonoid 3′5′-hydroxylase (F3′5′H) in species that naturally lack it. In this study, we established the regeneration and a genetic transformation system for the commercial cultivar ‘YX4’ of G. paniculata and introduced the F3′5′H of Platycodon grandiflorus (PgF3′5′H) successfully into ‘YX4’ using the established protocol. A total of 281 hygromycin (Hyg)-resistant plantlets were obtained, and 38 of them were polymerase chain reaction (PCR) positive, indicating a 13.5% transformation efficiency. Shoot apex without meristem was more suitable for explant due to its high regeneration capacity, and the supplement of thidiazuron (TDZ) provided the most efficient promotion of adventitious bud induction, whereas the supplement of 6-Benzyladenine (6-BA) and 1-naphthaleneacetic acid (NAA) did not affect much. Additionally, the combination of 1 day (d) pre-culture, 5 d co-culture, 10 min infection, 30 mg·L−1 additional acetosyringone (AS) supplement, and 10 mg·L−1 Hyg selection formed the optimized system for ‘YX4’ transformation. This reliable and efficient agrobacterium-mediated transformation of the valuable commercial cultivar ‘YX4’ will contribute not only to the creation and improvement of G. paniculata cultivars, but also to the function research of genes associated with important ornamental traits.

Application of tissue culture technology of medicinal plants in sustainable development of Chinese medicinal resources

Chinese medicinal resources are the cornerstone of the sustainable development of traditional Chinese medicine industry. However, due to the fecundity of species, over-exploitation, and limitations of artificial cultivation, some medicinal plants are depleted and even endangered. Tissue culture, a breakthrough technology in the breeding of traditional Chinese medicinal materials, is not limited by time and space, and can allow the production on an annual basis, which plays an important role in the protection of Chinese medicinal resources. The present study reviewed the applications of tissue culture of medicinal plants in the field of Chinese medicinal resources, including rapid propagation of medicinal plant seedlings, breeding of novel high-yield and high-quality cultivars, construction of a genetic transformation system, and production of secondary metabolites. Meanwhile, the current challenges and suggestions for the future development of this field were also proposed.

Differentiating the pathogenicity of millet blast races and establishing a stable genetic transformation system

<p indent="0mm">The fungal pathogen <italic>Magnaporthe</italic> causes blast disease and destroys millet, rice, and wheat crops worldwide, which threatens global food production. Blast disease has spread in the main millet-producing area of China and is pathogenically differentiated in different ecological regions. Therefore, a study on the pathogenicity of millet blast races, the resistance spectrum of millet cultivars in the adult stage, and establishing a genetic transformation system for the blast fungus is of great significance for distributing local millet resources, controlling blast, and improving the yield and quality of millet. Herein, we collected blast races from six regions nationwide, isolated them from single spores, and successfully induced extensive conidial spores in tomato plants. Furthermore, we conducted artificial <italic>in-vitro</italic> inoculation experiments with different millet varieties to evaluate their resistance spectra at the seedling stage and lay a solid foundation to isolate disease-resistant genes. The results showed that the pathogenicity of the strains to different varieties was divergent; however, the virulence of the races from Hainan Province was significantly stronger than that from other regions. Thus, the HN-1 strain with the highest infectivity was inoculated into 17 different adult-stage cultivars in the field. Consequently, four cultivars with adult-plant resistance were screened out. We established a stable genetic transformation system in the HN-1 background to provide a technical foundation for future studies on millet blast virulent proteins.

&amp;lt;italic&amp;gt;De novo&amp;lt;/italic&amp;gt; domestication and preliminary cultivar development of sand rice (&amp;lt;italic&amp;gt;Agriophyllum squarrosum&amp;lt;/italic&amp;gt;), a sand dune pioneer species

<p indent="0mm">The comprehensive utilization of desert plants and sandy land resources is of far-reaching significance to the eco-civilization progress and food security in the arid region of northern China. The pioneer species <italic>Agriophyllum squarrosum</italic> is broadly distributed on the mobile sand dunes in the arid and semi-arid areas of northern China. Its seed, also called sand rice, has well-balanced nutritional values and has been consumed for more than <sc>1300 years.</sc> In 2010, our group first initiated the domestication research of sand rice by investigating the various historical literature and food consuming records of sand rice. Genetic modification and breeding are currently dependent on natural populations and artificial mutagenesis. A few modified lines with better agronomic traits have been initially isolated, laying a material foundation for further cultivation evaluation in different areas and cultivar declaration. However, the speed domestication of sand rice still requires systematically analyzing phenotypic variation among different germplasms, the establishment of a genetic transformation system, and isolating elite lines with a synchronous germination rate and strong seedling vigor.

First Report of Diaporthe cercidis and D. nobilis Causing Leaf Blotch on Populus davidiana × P. bolleana in China.

First Report of Diaporthe cercidis and D. nobilis Causing Leaf Blotch on Populus davidiana × P. bolleana in China.

Efficient disruption of CmHk1 using CRISPR/Cas9 ribonucleoprotein delivery in Cordyceps militaris.

Cordyceps militaris, an entomopathogenic ascomycete, produces edible medicinal mushrooms known to have medicinal and therapeutic functions. To develop the genetic transformation system in C. militaris, green fluorescent protein (GFP) mutants of C. militaris were generated by PEG-mediated protoplast transformation. The CRISPR/Cas9 ribonucleoprotein (RNP) targeting the class III histidine kinase of C. militaris (CmHk1) was then delivered into protoplasts of C. militaris through the transformation system. Mutations induced by the RNP in selected mutants were detected: 1nt deletion (6 mutants), 3 nt deletion with substitution of 1nt (1 mutant), insertion of 85 nts (1 mutant), 41 nts (2 mutants), and 35 nts (5 mutants). An in vitro sensitivity assay of the mutants indicated that knockout of CmHk1 reduced sensitivity to two fungicides, iprodione and fludioxonil, but increased sensitivity to osmotic stresses compared to the wild type. Summing up, the CRISPR/Cas9 RNP delivery system was successfully developed, and our results revealed that CmHk1 was involved in the fungicide resistance and osmotic stress in C. militaris.

The Establishment of a Genetic Transformation System and the Acquisition of Transgenic Plants of Oriental Hybrid Lily (Lilium L.).

Lily (Lilium spp.) has elegant flowers and beautiful colors, which makes it popular among people. However, the poor stress resistance and self-propagation ability of lily limit its application in landscaping to a great extent. In addition, transgenic technology is an important means to improve plant characteristics, but the lack of a stable and efficient genetic transformation system is still an important factor restricting the development of lily transgenic technology. Therefore, this study established a good lily regeneration system by screening different explants and plant growth regulators of different concentrations. Then, the genetic transformation system of lily was optimized by screening the critical concentration of antibiotics, the concentration of bacterial solution, and the infection time. Finally, the homologous lily cold resistance gene LlNAC2 and bulblet generation gene LaKNOX1 were successfully transferred to 'Siberia' and 'Sorbonne' to obtain lily transgenic lines. The results showed that when the stem axis was used as explant in 'Siberia', the induction rate was as high as 87%. The induction rate of 'Sorbonne' was as high as 91.7% when the filaments were used as explants. At the same time, in the optimized genetic transformation system, the transformation rate of 'Siberia' and 'Sorbonne' was up to 60%. In conclusion, this study provides the theoretical basis and technical support for improving the resistance and reproductive ability of Oriental lily and the molecular breeding of lily.

Establishing a high-efficiency &lt;i&gt;in vitro&lt;/i&gt; regeneration system for &lt;i&gt;Agrobacterium&lt;/i&gt;-mediated transformation in &lt;i&gt;Fragaria nilgerrensis&lt;/i&gt;

<italic>Fragaria nilgerrensis</italic> Schlechtendal ex J. Gay, a diploid wild strawberry, has many excellent characteristics such as fruit with white color and peach fragrance, and strong disease resistance. However, the lack of an efficient and stable regeneration and genetic transformation system for <italic>F. nilgerrensis</italic> has largely limited the functional studies of related genes governing excellent traits. In this study, a regeneration system for <italic>F. nilgerrensis</italic> leaf discs was developed by optimizing factors such as different hormone combinations, dark culture times and casein hydrolysate (CH) concentrations, with an average regeneration rate of 97.3% at 45 d of culture. By paraffin section observation of callus with different colors induced from the leaf disc regeneration, light yellow callus was determined to be embryogenic and the regenerative pathway was identified as indirect organogenesis. Based on this, an average transformation percentage of 8.67% was achieved by screening kanamycin concentration and referring to transformation procedures described by predecessors. PCR-positive transformants were obtained within 4-5 months by confirmation of PCR and histochemical GUS, and transgene integration was identified by transformants regeneration. The establishment of efficient regeneration system provided a feasible platform for genetic transformation, which laid the foundation for further gene functional studies in <italic>F. nilgerrensis</italic>.

Agrobacterium‐mediated transformation via establishment of stable tissue culture system in Zoysia matrella (L.) Merrill ‘Wakaba’

AbstractZoysia matrella (L.) Merrill is a perennial C4 warm‐season turfgrass grown for landscapes, golf courses, sports fields and recreation parks. To create a new breeding method by genome editing, an efficient genetic transformation system is essential. In this study, we report the efficient protocol of Agrobacterium‐mediated transformation through the establishment of a stable tissue culture system for Z. matrella ‘Wakaba’. The embryogenic callus was induced from shoot apices of nodal segments incubated in Murashige and Skoog (MS) medium containing 2 mg/l 2,4‐dichlorophnoxyacetic acid (2,4‐D), 0.1 mg/l 6‐benzylaminopurine (BAP) and 5 μM CuSO4 (MS‐DBC). Repeated subculture of compact high‐quality callus in MS‐DBC medium produced a highly regenerative callus with dense pre‐embryogenic clusters, and it was used as a transformation target. Agrobacterium strain EHA105 harboring pANIC8B vector containing the β‐glucuronidase gene (GUS) and hygromycin B phosphotransferase gene was used. Agrobacterium‐infected calli were cocultured for 5 days with 100 μM acetosyringone and then subjected to selection pressure of 50 mg/l hygromycin. This optimized protocol yielded transformation efficiencies of up to 6.6%. Southern blot analysis verified one to three copies of the GUS gene in different independent transgenic plants. All transgenic plants were morphologically normal, and the GUS expressions were stable. Our optimized in vitro and transgenic system will facilitate the new breeding technology of genome editing in zoysiagrass.

Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation.

Paeonia lactiflora Pall. is an important ornamental plant with high economic and medicinal value, which has considerable development prospects worldwide. The lack of efficient tissue culture techniques and genetic transformation systems has become a master obstacle for P. lactiflora research. The purpose of the present study focuses on obtaining an efficient and stable genetic transformation method using callus as the receptor and exploring an efficient protocol for callus induction and proliferation associated with P. lactiflora. Callus induction and proliferation were performed using MS medium with various concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (6-BA) and thidiazuron (TDZ). The sensitivity of callus to kanamycin and cefotaxime was determined. Several parameters such as Agrobacterium cell density, infection time and co-culture duration were studied to optimize transformation efficiency. Agrobacterium strains EHA105 and pBI121 binary vector harboring the β-glucuronidase (GUS) gene were used for transformation. Expression of the GUS reporter gene was detected by GUS assay, polymerase chain reaction (PCR) and Quantitative Real-time PCR (RT-qPCR). The MS medium containing 1.0 mg·L-1 NAA, 0.5 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was optimal for callus induction and MS medium containing 0.5 mg·L-1 NAA, 1.0 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was the best for callus proliferation. The concentrations of kanamycin and cefotaxime used for screening positive callus were 125 mg·L-1 and 200 mg·L-1, respectively. Among various combinations analyzed, the best transformation result was obtained via the 25 min of infection of Agrobacterium at 0.6 OD600 and 3 d of co-culture. Overall, this study provided technical support and theoretical guidance for improving the callus induction and proliferation efficiency and the study of gene function in P. lactiflora.

CRISPR-Cas technology opens a new era for the creation of novel maize germplasms.

Maize (Zea mays) is one of the most important food crops in the world with the greatest global production, and contributes to satiating the demands for human food, animal feed, and biofuels. With population growth and deteriorating environment, efficient and innovative breeding strategies to develop maize varieties with high yield and stress resistance are urgently needed to augment global food security and sustainable agriculture. CRISPR-Cas-mediated genome-editing technology (clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated)) has emerged as an effective and powerful tool for plant science and crop improvement, and is likely to accelerate crop breeding in ways dissimilar to crossbreeding and transgenic technologies. In this review, we summarize the current applications and prospects of CRISPR-Cas technology in maize gene-function studies and the generation of new germplasm for increased yield, specialty corns, plant architecture, stress response, haploid induction, and male sterility. Optimization of gene editing and genetic transformation systems for maize is also briefly reviewed. Lastly, the challenges and new opportunities that arise with the use of the CRISPR-Cas technology for maize genetic improvement are discussed.

Establishment of an efficient regeneration and Agrobacterium transformation system in mature embryos of calla lily (Zantedeschia spp.).

Calla lily (Zantedeschia spp.) have great aesthetic value due to their spathe-like appearance and richness of coloration. However, embryonic callus regeneration is absent from its current regeneration mechanism. As a result, constructing an adequate and stable genetic transformation system is hampered, severely hindering breeding efforts. In this research, the callus induction effectiveness of calla lily seed embryos of various maturities was evaluated. The findings indicated that mature seed embryos were more suitable for in vitro regeneration. Using orthogonal design experiments, the primary elements influencing in vitro regeneration, such as plant growth regulators, genotypes, and nanoscale materials, which was emergent uses for in vitro regeneration, were investigated. The findings indicated that MS supplemented with 6-BA 2mg/L and NAA 0.1mg/L was the optimal medium for callus induction (CIM); the germination medium (GM) was MS supplemented with 6-BA 2mg/L NAA 0.2mg/L and 1mg/L CNTs, and the rooting medium (RM) was MS supplemented with 6-BA 2mg/L NAA 0.7mg/L and 2mg/L CNTs. This allowed us to verify, in principle, that the Agrobacterium tumefaciens-mediated genetic transformation system operates under optimal circumstances using the GUS reporter gene. Here, we developed a seed embryo-based genetic transformation regeneration system, which set the stage for future attempts to create new calla lily varieties.

Commercial scale genetic transformation of mature seed embryo explants in maize.

A novel, efficient maize genetic transformation system was developed using Agrobacterium-mediated transformation of embryo explants from mature seeds. Seeds from field grown plants were sterilized and crushed to isolate embryo explants consisting of the coleoptile, leaf primordia, and shoot apical meristem which were then purified from the ground seed bulk preparation. The infection of relevant tissues of seed embryo explants (SEEs) by Agrobacterium was improved by the centrifugation of the explants. Transgenic plants were obtained by multiple bud induction on high cytokinin media, followed by plant regeneration on hormone-free medium. Three different selectable markers (cp4 epsps, aadA, and nptII) were successfully used for producing transgenic plants. Stable integration of transgenes in the maize genome was demonstrated by molecular analyses and germline transmission of the inserted transgenes to the next generation was confirmed by pollen segregation and progeny analysis. Phenotypic evidence for chimeric transgenic tissue was frequently observed in initial experiments but was significantly reduced by including a second bud induction step with optimized cytokinin concentration. Additional improvements, including culturing explants at an elevated temperature during bud induction led to the development of a revolutionary system for efficient transgenic plant production and genome editing. To our knowledge, this is the first report of successful transgenic plant regeneration through Agrobacterium-mediated transformation of maize mature SEEs. This system starts with mature seed that can be produced in large volumes and the SEEs explants are storable. It has significant advantages in terms of scalability and flexibility over methods that rely on immature explants.

Genetics, Genomics, and Breeding in Melon

Melon is an important horticultural crop worldwide. The high diversity of melon makes it a model plant for various different properties. Some quantitative trait loci or candidates have been discovered, but few were verified as limiting genetic transformation and genome editing systems. Identifying new genetic resources with resistance and special fruit quality traits is imperative to develop effective and useful breeding technologies in melon. This review describes the advances in genetics, genomics, and the breeding of melon and puts forward some recommendations in these areas.

Understanding the biosynthesis and regulatory mechanisms of bioactive compounds in Taraxacum species (dandelions), a model system for natural rubber, food, and medicinal plant biology

Plants that belong to the genus Taraxacum are commonly referred to as dandelions; these are some of the most evolved plants in the Asteraceae, with more than 2000 species distributed worldwide. Dandelions not only include medicinal plants but also rubber-producing plants. Recent advances in biotechnology and the growing demand for dandelions have increased research attention toward dandelions. Owing to the availability of the whole genome sequence of Taraxacum kok-saghyz, and Taraxacum mongolicum (accession no. GWHBCHF00000000 and GWHAAAA00000000), and large transcriptome and metabolome databases for T. kok-saghyz, Taraxacum officinale, T. mongolicum, and Taraxacum antungense (accession no. PRJNA378120, PRJEB48186, PRJNA897666, and PRJNA578773), significant progress has been made in understanding the biosynthesis and regulatory mechanisms of phenolic acids, flavonoids, terpenoids, rubber, and other bioactive compounds. This review focuses on Taraxacum spp.; specifically, We describe the following: (1) the recent results of genetic transformation systems, (2) biosynthetic pathways of bioactive compounds and natural rubber, (3) cross-talk in the biosynthesis of different secondary metabolites, (4) the relevance of the environment and the reproductive mode of dandelions in secondary metabolite biosynthesis, and (5) future research directives for dandelions. This review also lays the foundation for further research on other medicinal and edible plants.

On the role of cell surface associated, mucin-like glycoproteins in the pennate diatom Craspedostauros australis (Bacillariophyceae).

Diatoms are single-celled microalgae with silica-based cell walls (frustules) that are abundantly present in aquatic habitats, and form the basis of the food chain in many ecosystems. Many benthic diatoms have the remarkable ability to glide on all natural or man-made underwater surfaces using a carbohydrate- and protein-based adhesive to generate traction. Previously, three glycoproteins, termed FACs (Frustule Associated Components), have been identified from the common fouling diatom Craspedostauros australis and were implicated in surface adhesion through inhibition studies with a glycan-specific antibody. The polypeptide sequences of FACs remained unknown, and it was unresolved whether the FAC glycoproteins are indeed involved in adhesion, or whether this is achieved by different components sharing the same glycan epitope with FACs. Here we have determined the polypeptide sequences of FACs using peptide mapping by LC-MS/MS. Unexpectedly, FACs share the same polypeptide backbone (termed CaFAP1), which has a domain structure of alternating Cys-rich and Pro-Thr/Ser-rich regions reminiscent of the gel-forming mucins. By developing a genetic transformation system for C. australis, we were able to directly investigate the function of CaFAP1-based glycoproteins invivo. GFP-tagging of CaFAP1 revealed that it constitutes a coat around all parts of the frustule and is not an integral component of the adhesive. CaFAP1-GFP producing transformants exhibited the same properties as wild type cells regarding surface adhesion and motility speed. Our results demonstrate that FAC glycoproteins are not involved in adhesion and motility, but might rather act as a lubricant to prevent fouling of the diatom surface.

CRISPR/Cas9-mediated genome editing of RsGL1a and RsGL1b in radish (Raphanus sativus L.).

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool widely used for genome editing in various organisms, including plants. It introduces and facilitates the study of rare genetic mutations in a short time and is a potent tool to assist in plant molecular breeding. Radish (Raphanus sativus L.) is an important Brassicaceae vegetable cultivated and consumed worldwide. However, the application of the CRISPR/Cas9 system is limited by the absence of an efficient transformation system in radish. This study aimed to establish a CRISPR/Cas9 system in radish employing the Agrobacterium-mediated genetic transformation system reported recently. For this purpose, we performed genome editing using the CRISPR/Cas9 system targeting the GLABRA1 (GL1) orthologs, RsGL1a and RsGL1b, that induces leaf trichome formation in radish. A Cas9/single guide RNA (sgRNA) vector with a common sgRNA corresponding to RsGL1a and RsGL1b was transferred. A total of eight T0 plants were analyzed, of which six (editing efficiency 75%) had a mutated RsGL1a, five (62.5%) had a mutated RsGL1b, and five showed mutations in both RsGL1a and RsGL1b. Most mutations in T0 plants were short (<3 bp) deletions or insertions, causing frameshift mutations that might produce non-functional proteins. Chimeric mutations were detected in several T0 generation plants. In the T1 generation, the hairless phenotype was observed only in plants with knockout mutations in both RsGL1a and RsGL1b. The majority of mutant alleles in T0 plants, with the exception of the chimeric mutant plants detected, were stably inherited in the T1 generation. In conclusion, we successfully knocked out RsGL1a and RsGL1b using the CRISPR/Cas9 system and demonstrated that both RsGL1a and RsGL1b independently contribute to the induction of leaf trichome formation in radish. In this study, genome-edited plants without T-DNA, which are useful as breeding material, were obtained. The findings prove the feasibility of genome editing in radish using a CRISPR/Cas9 system that could accelerate its molecular breeding to improve agronomically desirable traits.

An efficient Agrobacterium-mediated transient transformation system and its application in gene function elucidation in Paeonia lactiflora Pall.

Paeonia lactiflora Pall. is known as the king of herbaceous flowers with high ornamental and precious medicinal value. However, the lack of a stable genetic transformation system has greatly affected the research of gene function in P. lactiflora. The Agrobacterium-mediated transient gene expression is a powerful tool for the characterization of gene function in plants. In this study, the seedlings of P. lactiflora were used as the transformation receptor materials, and the efficient transient transformation system with a GUS reporter gene was successfully established by Agrobacterium harboring pCAMBIA1301. To optimize the system, we investigated the effects of germination time, Agrobacterium cell density, infection time, acetosyringone (AS) concentration, co-culture time, negative pressure intensity, Tween-20 concentration and different receptor materials on the transient transformation efficiency of P. lactiflora. The results showed that the highest transient transformation efficiency (93.3%) could be obtained when seedlings in 2-3 cm bud length were subjected to 12 h infection of resuspension solution comprising 1.2 OD600 Agrobacterium, 200 μM AS and 0.01% Tween-20 under 10 of negative pressure intensity followed by 3 days of co-culture in darkness condition. This method is more suitable for the study of gene function in P. lactiflora. Subsequently, stress resistance genes PlGPAT, PlDHN2 and PlHD-Zip were used to verify the effectiveness of this transformation system. These results can provide critical information for identification of key genes in non-model plants, such as P. lactiflora, and promote the development of molecular biology research for P. lactiflora.

Over-Expression of Phosphoserine Aminotransferase-Encoding Gene (AtPSAT1) Prompts Starch Accumulation in L. turionifera under Nitrogen Starvation.

It has been demonstrated that the phosphorylation pathway of L-serine (Ser) biosynthesis (PPSB) is very important in plant growth and development, but whether and how PPSB affects nitrogen metabolism and starch accumulation has not been fully elucidated. In this study, we took the energy plant duckweed (strain Lemna turionifera 5511) as the research object and used a stable genetic transformation system to heterologously over-expressing Arabidopsis AtPSAT1 (the gene encoding phosphoserine aminotransferase, the second enzyme of PPSB). Our results showed that, under nitrogen starvation, the transgenic plants grew faster, with higher values of Fv/Fm, rETR, and Y(II), as well as fresh and dry weight, than the wild-type. More promisingly, the accumulation of starch was also found to be significantly improved when over-expressing AtPSAT1 in the transgenic plants. qRT-PCR analysis results showed that the expression of genes related to nitrogen assimilation, carbon metabolism, and starch biosynthesis was up-regulated, while the expression of starch degradation-related genes was down-regulated by AtPSAT1 over-expression. We propose that the increased starch accumulation caused by AtPSAT1 over-expression may result from both elevated photosynthetic capacity and nitrogen utilization efficiency. This research sheds new light on the mechanism underlying the ability of PPSB to coordinate nitrogen and carbon metabolism, and provides a feasible way to improve starch production, that is, through engineering PPSB in crops.