Detection of soybean transgenic event GTS-40-3-2 using electric field-induced release and measurement (EFIRM).

Polymerase chain reaction (PCR) technology has become a standard technique for the detection of genetically modified organisms (GMOs). However, this method requires a PCR amplification process which is both expensive and time-consuming. Herein, we propose electric field-induced release and measurement (EFIRM) technology as an alternative method for GMO screening. The specificity and sensitivity of the EFIRM assay were proven to be comparable to those of the real-time PCR method for detecting genetically modified soybeans. After all the parameters had been evaluated, the actual evaluation of soybean samples from soybean cargoes was performed. An actual EFIRM screening was performed on 157 soybean cargo samples, which had 102 transgenic soybean samples containing the GTS-40-3-2 gene, through a blind trial at the Dalian port of China. Our results showed that 101 transgenic soybean samples were correctly detected, with only one false-negative case, and 55 non-transgenic soybean samples were detected as negative; this demonstrates that the EFIRM assay is an effective, accurate, simple, and economical novel method for detecting transgenic products, which may have a positive impact on the development of rapid on-site GMO monitoring platforms.

Insights into the DNA methylation of sea cucumber Apostichopus japonicus in response to skin ulceration syndrome infection.

DNA methylation is an important epigenetic modification that regulates gene expression in many biological processes, including immune response. In this study, whole-genome bisulfite sequencing (WGBS) was carried out on healthy body wall (HB) and skin ulceration syndrome (SUS) infected body wall (SFB) to gain insights into the epigenetic regulatory mechanism in sea cucumber Apostichopus japonicus. After comparison, a total of 116,522 differentially methylated regions (DMRs) were obtained including 67,269 hyper-methylated and 49,253 hypo-methylated DMRs (p < 0.05, FDR < 0.001). GO enrichment analysis indicated that regulation of DNA-templated transcription (GO: 0006355), where DNA methylation occurred, was the most significant term in the biology process. The integration of methylome and transcriptome analysis revealed that 10,499 DMRs were negatively correlated with 496 differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these DEGs were enriched in the phosphoinositide 3-kinase-protein kinase B (PI3K/Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Interestingly, two serine/threonine-protein kinases, nemo-like kinase (NLK) and mTOR, were highlighted after functional analysis. The variations of methylation in these two genes were associated with SUS infection and immune regulation. They regulated gene expression at different levels and showed interaction during response process. The validation of methylation sites showed high consistency between pyrosequencing and WGBS. WGBS analysis not only revealed the changes of DNA methylation, but also presented important information about the regulation of key genes after SUS infection in A. japonicus.

In-depth profiling of miRNA regulation in the body wall of sea cucumber Apostichopus japonicus during skin ulceration syndrome progression.

MicroRNAs (miRNAs) are small non-coding RNAs that mediate mRNA degradation or translation repression. Previous study showed that the expression of miRNAs was significantly changed in the body wall of sea cucumber Apostichopus japonicus after skin ulceration syndrome (SUS) infection, which is a dynamic process. However, the critical miRNAs from body wall that involved in different infection stages of SUS remain unknown. In this study, four cDNA libraries were constructed with the body wall from healthy and three SUS-infected stages of A. japonicus. A total of 248 conserved miRNAs and five novel miRNAs were identified through Illumina HiSeq 2000 platform. Compared to the control, 238 miRNAs showed significant differential expression at three stages of SUS progression. Totally, 3149 miRNA-mRNA pairs were identified by target prediction and 314 miRNA-mRNA pairs showed negative correlation. It is noteworthy that 15 miRNAs and four mRNAs were located at the crucial positions of the network built with the anti-correlated miRNA-mRNA pairs. GO and KEGG enrichment analysis indicated that the predicted targets were involved in many immune-related processes. Deep analysis of miR-31c-5p, miR-29b-3p, NF-kB, mucin 2 and titin showed that they may play important roles in the pathogens attachment and recognition, signaling transduction and lesions repair of A. japonicus after SUS infection. These results would be useful for further investigating the potential roles of critical miRNAs and mRNAs in A. japonicus immune regulation.

Comparative study of three phenoloxidases in the sea cucumber Apostichopus japonicus.

In order to preliminarily illustrate the functional differences of phenoloxidases (POs) in Apostichopus japonicus, the full-length cDNAs of two POs (named as AjPOⅡ and AjPOⅢ, respectively) were cloned from the coelomocytes of A. japonicus using 3'- and 5'-rapid amplification of cDNA ends method, and combined with the previously acquired full-length cDNA of a laccase-type PO from A. japonicus (Accession No. KF040052, named as AjPOⅠ), the sequence structure and phylogenic status of POs from A. japonicus (AjPOs) were comparatively analyzed, and the transcriptional expression of AjPOs in different tissues, at different developmental stages and after different bacterial challenges was determined with quantitative real-time PCR method. Sequence analysis indicated AjPOⅡ and AjPOⅢ were both laccase-type POs, coincident to the results of phylogenic analysis. Sequence analysis also showed that AjPOⅠ had a transmembrane domain (J. Jiang et al., 2014), AjPOⅡ contained a signal peptide, and AjPOⅢ possessed a signal peptide and a transmembrane domain, implying that three AjPOs might play different roles in immune and physiological processes. Transcriptional expression analysis showed that AjPOⅡ and AjPOⅢ were most abundant in tube feet, while AjPOⅠhad the highest expression level in coelomocytes (J. Jiang et al., 2014), suggesting that AjPOⅠ may be mainly involved in immune response, while AjPOⅡ and AjPOⅢ are probably responsible for other physiological processes in addition to immune response. Besides, three AjPOs were determined to have different expression patterns during organism development and different spectrums of response against bacteria, which further indicated that there might be immune and physiological functional differentiation among three AjPOs.

Comparative analysis of immunocompetence between females and males in the sea cucumber Apostichopus japonicus.

In order to preliminarily understand the immune difference between females and males in the sea cucumber Apostichopus japonicus, the activities assay of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), phenoloxidase (PO), acid phosphatase (ACP) and alkaline phosphatase (ALP) with biochemical methods, the detection of PO isozymes with native-PAGE and catechol staining, and the test of antibacterial activities with bacterial growth curve determination method were performed in this study using cell-free coelomic fluid (CCF) and coelomocyte lysate supernatant (CLS) from females and males as the samples. The PO activities were not detected in the CLS and showed no significant difference between the CCF from females and males. However, totally five PO isozyme bands were detected in the CLS of females while only four were detected in the CLS of males after zymogram analysis. These results implied that the PO isozymes in the coelomocytes of viripotent A. japonicus were inactive under natural condition and may be activated by some certain treatments during native-PAGE, and PO might play different immune and physiological roles between females and males. In addition, the activities of SOD, CAT, POD and ALP in the CCF and the activities of CAT, POD, ACP and ALP in the CLS from males were all significantly higher than those from females. The results collectively suggested that in viripotent A. japonicus, the gender had a remarkable effect on the immunity, and the immunocompetence of males might have an advantage over that of females. Furthermore, the activities of all determined enzymes except PO and the number of detected PO isozymes showed higher values in CLS than in CCF, implying that in viripotent A. japonicus, the coelomocytes might take more immune responsibility in comparison with CCF.

RNA sequencing analysis to capture the transcriptome landscape during skin ulceration syndrome progression in sea cucumber Apostichopus japonicus.

BACKGROUND: Sea cucumber Apostichopus japonicus is an important economic species in China, which is affected by various diseases; skin ulceration syndrome (SUS) is the most serious. In this study, we characterized the transcriptomes in A. japonicus challenged with Vibrio splendidus to elucidate the changes in gene expression throughout the three stages of SUS progression. RESULTS: RNA sequencing of 21 cDNA libraries from various tissues and developmental stages of SUS-affected A. japonicus yielded 553 million raw reads, of which 542 million high-quality reads were generated by deep-sequencing using the Illumina HiSeq™ 2000 platform. The reference transcriptome comprised a combination of the Illumina reads, 454 sequencing data and Sanger sequences obtained from the public database to generate 93,163 unigenes (average length, 1,052 bp; N50 = 1,575 bp); 33,860 were annotated. Transcriptome comparisons between healthy and SUS-affected A. japonicus revealed greater differences in gene expression profiles in the body walls (BW) than in the intestines (Int), respiratory trees (RT) and coelomocytes (C). Clustering of expression models revealed stable up-regulation as the main pattern occurring in the BW throughout the three stages of SUS progression. Significantly affected pathways were associated with signal transduction, immune system, cellular processes, development and metabolism. Ninety-two differentially expressed genes (DEGs) were divided into four functional categories: attachment/pathogen recognition (17), inflammatory reactions (38), oxidative stress response (7) and apoptosis (30). Using quantitative real-time PCR, twenty representative DEGs were selected to validate the sequencing results. The Pearson's correlation coefficient (R) of the 20 DEGs ranged from 0.811 to 0.999, which confirmed the consistency and accuracy between these two approaches. CONCLUSIONS: Dynamic changes in global gene expression occur during SUS progression in A. japonicus. Elucidation of these changes is important in clarifying the molecular mechanisms associated with the development of SUS in sea cucumber.

Expression analysis of microRNAs related to the skin ulceration syndrome of sea cucumber Apostichopus japonicus.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes. To investigate the miRNAs related to skin ulceration syndrome (SUS) of Apostichopus japonicus, small RNA libraries of body wall, intestine, respiratory tree and coelomocytes from healthy and diseased A. japonicus were sequenced on Illumina Hiseq 2000 platform. A total of 247 conserved and 10 novel miRNAs were identified across all libraries. After pair-wise comparisons, 215 miRNAs in body wall, 36 in intestine, 2 in respiratory tree and 38 in coelomocytes showed significant expression differences. Further analyses were conducted on some tissue-specific differentially expressed miRNAs: miR-8 and miR-486-5p in body wall, miR-200-3p, let-7-5p and miR-125 in intestine, miR-278a-3p and bantam in respiratory, miR-10a and miR-184 in coelomocytes. Notably, these miRNAs in some species were reported to function in various physiological or pathological processes associated with immune regulations. Using stem-loop quantitative real time PCR, six representative miRNAs in four tissues were selected to validate the sequencing results. The Pearson's correlation coefficient (R) of the six miRNAs ranged from 0.777 to 0.948, which confirmed the consistency and accuracy between these two approaches. This study provides comprehensive expression and regulation patterns of functional miRNAs in different tissues and gives insights into the tissue-specific immune response mechanisms in SUS-infected A. japonicus.

Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning, expression and substrate specificity analysis.

Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses.

In vitro antibacterial analysis of phenoloxidase reaction products from the sea cucumber Apostichopus japonicus.

Three phenoloxidases (POs) of Apostichopus japonicus, AjPOs (AjPO1, AjPO2 and AjPO3), were partially purified from the coelomocytes with an electrophoretic method, and then employed for the in vitro antibacterial analysis. Using L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate, AjPO1 and AjPO2-derived compounds inhibited the growth of Vibrio splendidus and Staphylococcus aureus, while AjPO3-derived compounds only inhibited the growth of V. splendidus. When dopamine was used as a substrate, AjPO1 and AjPO3-derived compounds inhibited the growth of V. splendidus and Vibrio harveyi, while AjPO2-derived compounds only inhibited the growth of V. splendidus. Moreover, AjPO1-derived compounds showed stronger inhibition in V. harveyi than AjPO3-derived compounds did. However, all of the three AjPO reaction products showed no inhibitions on the growth of Pseudoalteromonas nigrifaciens, Shewanella baltica, Micrococcus lysodeikticus, Streptococcus dysgalactiae and Nocardiopsis sp. with L-DOPA or dopamine as a substrate. Scanning electron microscope (SEM) observation of V. harveyi treated by AjPOs and dopamine showed that AjPO1-derived compounds resulted in massive bacteriolysis, AjPO2-derived compounds caused no obvious alteration on bacterial morphology, and AjPO3-derived compounds increased the ratio of spheroidal bacteria. All these results suggested that AjPO reaction products derived by L-DOPA and dopamine had different but limited antibacterial spectrum, and the different antibacterial effects observed among three AjPOs resulted from the different reaction products generated by AjPOs with the same substrate.

Expression analysis of immune related genes identified from the coelomocytes of sea cucumber (Apostichopus japonicus) in response to LPS challenge.

The sea cucumber (Apostichopus japonicus) occupies a basal position during the evolution of deuterostomes and is also an important aquaculture species. In order to identify more immune effectors, transcriptome sequencing of A. japonicus coelomocytes in response to lipopolysaccharide (LPS) challenge was performed using the Illumina HiSeq™ 2000 platform. One hundred and seven differentially expressed genes were selected and divided into four functional categories including pathogen recognition (25 genes), reorganization of cytoskeleton (27 genes), inflammation (41 genes) and apoptosis (14 genes). They were analyzed to elucidate the mechanisms of host-pathogen interactions and downstream signaling transduction. Quantitative real-time polymerase chain reactions (qRT-PCRs) of 10 representative genes validated the accuracy and reliability of RNA sequencing results with the correlation coefficients from 0.88 to 0.98 and p-value <0.05. Expression analysis of immune-related genes after LPS challenge will be useful in understanding the immune response mechanisms of A. japonicus against pathogen invasion and developing strategies for resistant markers selection.

Identification and expression analysis of two Toll-like receptor genes from sea cucumber (Apostichopus japonicus).

Toll-like receptors (TLRs) are a family of type I integral membrane glycoproteins which play pivotal roles in innate immunity. In this study, two TLRs named AjTLR3 and AjToll were cloned from sea cucumber (Apostichopus japonicus). The full-length cDNA sequences of AjTLR3 and AjToll are 3484 bp and 4211 bp, with an open reading frame (ORF) of 2679 bp and 2853 bp, encoding 892 and 950 amino acids, respectively. Both AjTLR3 and AjToll are composed of a leucine-rich repeat (LRR) domain, a transmembrane (TM) domain and an intracellular Toll/interleukin-1 receptor (TIR) domain. Evolution analysis revealed that AjTLR3 and AjToll were clustered with the vertebrate-like TLRs (V-TLRs) and the protostome-like TLRs (P-TLRs), respectively. These two genes were widely expressed in all five tested tissues (body wall, coelomocytes, tube feet, intestine and respiratory tree), but showed different expression patterns. The significantly up-regulated expressions of AjTLR3 and AjToll after peptidoglycan (PGN), lipopolysaccharides (LPS), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C) challenges suggested that they were functionally involved in the immune responses to the Cram-positive bacteria, Gram-negative bacteria, fungi and double-stranded RNA (dsRNA) viruses, respectively.

Molecular characterization and expression analysis of heat shock cognate 70 after heat stress and lipopolysaccharide challenge in sea cucumber (Apostichopus japonicus).

A gene encoding hsc70 was cloned from the sea cucumber Apostichopus japonicus and named AjHsc70. The full-length cDNA sequence was 2,508 bp, containing a 5'-UTR of 77 bp, an ORF of 2,010 bp encoding 670 amino acids, and a 3'-UTR of 421 bp. Quantitative RT-PCR analysis revealed that AjHsc70 was expressed constitutively in all of the tested tissues with respiratory tree tissue showing the highest expression level. AjHsc70 expression was significantly induced by lipopolysaccharide, and the expression levels peaked at different sampling times in the body wall (24 h), coelomocytes (12 h), and intestine and respiratory tree tissues (6 h). After heat stress, AjHsc70 expression in intestine, coelomocytes, and body wall decreased acutely at first and then increased slightly. AjHsc70 expression patterns indicated that hsc70 plays an important role in mediating the responses of A. japonicus to bacterial challenge and heat stress.

Precise Authenticity of Quinoa, Coix Seed, Wild Rice and Chickpea Components Using Optimized TaqMan Real-Time PCR.

Functional food such as, quinoa, coix seed, wild rice and chickpea have experienced rapidly increasing demand globally and exhibit high economic values. Nevertheless, a method for rapid yet accurate detection of these source components is absent, making it difficult to identify commercially available food with labels indicating the presence of relevant components. In this study, we constructed a real-time quantitative polymerase chain reaction (qPCR) method for rapid detection of quinoa, coix seed, wild rice and chickpea in food to identify the authenticity of such food. Specific primers and probes were designed with 2S albumin genes of quinoa, SAD genes of coix seed, ITS genes of wild rice and CIA-2 genes of chickpea as the target genes. The qPCR method could specifically identify the four wild rice strains, yielding, LODs of 0.96, 1.14, 1.04 and 0.97 pg/µL quinoa, coix seed, wild rice and chickpea source components, respectively. Particularly, the method allowed the identification of the target component with content below 0.01%. A total of 24 commercially available food samples of different types were detected by using the method and the results indicate that the developed method is applicable to the detection of different food matrices, as well as authenticity verification in deeply processed food.

Molecular characterization and expression analysis of a complement component 3 in the sea cucumber (Apostichopus japonicus).

The complement system has been discovered in invertebrates and vertebrates, and plays a crucial role in the innate defense against common pathogens. As a central component in the complement system, complement component 3 (C3) is an intermediary between innate and adaptive immune system. In this study, a new isoform of C3 in the sea cucumber Apostichopus japonicus, termed AjC3-2 was identified. Its open reading frame (ORF) is 5085 bp and encodes for 1695 amino acids with a putative signal peptide of 20 amino acid residues. The mature protein molecular weight of AjC3-2 was 187.72 kDa. It has a conserved thioester site and a linker R(689)RRR(692) where AjC3-2 is splitted into ß and α chain during posttranslational modification. The expression patterns of two distinct sea cucumber C3 genes, AjC3-2 and AjC3, were similar. During the different development stages from unfertilized egg to juvenile of the sea cucumber, the highest expression levels of AjC3-2 and AjC3 genes were both found in late auricularia. In the adult, the highest expression of these two genes was observed in the coelomocytes and followed by the body wall. AjC3-2 and AjC3 genes expression increased significantly at 6 h after the LPS challenge. These results indicated that these two C3 genes play a pivotal role in immune responses to the bacterial infection in sea cucumber.

Expression of immune-related genes in embryos and larvae of sea cucumber Apostichopus japonicus.

The echinoderm immunity system has been extensively investigated in adults in several classes such as echinoid and holothuroidea. However, the defense mechanism in embryos and larvae remains largely unexplored. To profile the immune-related genes expression in embryos and larvae and to monitor the stimulation of the innate immune response by lipopolysaccharides (LPS) challenge, we investigated the expression patterns of nine immune-related genes in embryos and larvae of sea cucumber (Apostichopus japonicus) at eleven developmental stages using quantitative real-time PCR (qRT-PCR). The expression of six encoding proteins including heat shock protein70 (Hsp70), Hsp90, Hsp gp96, thymosin-beta, ferritin and DD104 protein was detected at all eleven development stages according to mRNA expression data. However, the expression of mannan-binding C-type lectin (MBCL) was detected at early auricularia to juvenile stages, while lysozyme and serine proteinase inhibitor (SPI) were detected only at juvenile stage. Out of these nine genes, three (MBCL, lysozyme and SPI) were found to be up-regulated in mRNA expression upon LPS challenge, whereas the other six showed no significant change. Our study presents a first preliminary view into the expression patterns of immune-related genes at different developmental stages of sea cucumber, which increases the available information on echinoderm immunity.

Bibliometric Profile of Global Microplastics Research from 2004 to 2019.

Microplastics (MPs) have generated worldwide attention due to their global distribution in the environment, and their potential harmful effects on human and animal health. To analyze MPs-related scientific publications from a global point of view, we created a bibliometric profile, by searching the Web of Science Core Collection database for the topic "microplastic* or (micro near/1 plastic*)", in publications dated from 2004 to 2019. The results revealed an increasing trend in publication output, and identified contributions of different countries and their collaborations, as well as influential authors and productive journals in the field of MPs research. Using co-citation network analysis in VOSviewer, we mined cited references for knowledge bases about analytical methods, potential sources and spatial distributions of MPs, the impacts of MPs on organisms, and the interaction of MPs with contaminants, as well as microorganisms. We also identified four global hotspots for MPs related research, using author keywords co-occurrence network analysis of all extracted publications, as well as Essential Science Indicators highly cited papers from Clarivate Analytics. Results of this study provide a valuable reference for ongoing MPs-related research, which may be of intrigue and awesome noteworthiness for relevant researchers.

Rapid PCR-free meat species mitochondrial DNA identification using Electric Field Induced Release and Measurement (EFIRM®).

This work details the usage of EFIRM® (Electric Field Induced Release and Measurement) for PCR-free rapid electrochemical detection of mitochondrial DNA. EFIRM® was able to perform highly sensitive detection of animal species for meat contamination testing without multistep sample lysis, DNA extraction, or PCR amplification steps, demonstrating the capability to detect the presence of foreign meat species that only constituted 0.1% of the total mass of a food sample (achieving sensitivity equivalent to that of PCR). The EFIRM® strategy utilizes surface immobilized nucleic acid probes that complement to mitochondrial sequence of Ovis Aries, Sus Scrofa, and Bos Taurus and are immobilized in a polypyrrole matrix on a 96-electrode array. Quantification was performed through amperometric measurement of oxidation-reduction reactions on a streptavidin-peroxidase enzyme chain that completes the nucleic acid complex. All electrochemical procedures were performed using a high-throughput potentiostat system that allows parallelized electrochemical measurement and interfacing to the 96-electrode array.

Ovary-drip transformation: a simple method for directly generating vector- and marker-free transgenic maize (Zea mays L.) with a linear GFP cassette transformation.

The presence of selectable marker genes and vector backbone sequences has affected the safe assessment of transgenic plants. In this study, the ovary-drip method for directly generating vector- and selectable marker-free transgenic plants was described, by which maize was transformed with a linear GFP cassette (Ubi-GFP-nos). The key features of this method center on the complete removal of the styles and the subsequent application of a DNA solution directly to the ovaries. The movement of the exogenous DNA was monitored using fluorescein isothiocyanate-labeled DNA, which showed that the time taken by the exogenous DNA to enter the ovaries was shortened compared to that of the pollen-tube pathway. This led to an improved transformation frequency of 3.38% compared to 0.86% for the pollen-tube pathway as determined by PCR analysis. The use of 0.05% surfactant Silwet L-77 + 5% sucrose as a transformation solution further increased the transformation frequency to 6.47%. Southern blot analysis showed that the transgenic plants had low transgene copy number and simple integration pattern. Green fluorescence was observed in roots and immature embryos of transgenic plants by fluorescence microscopy. Progeny analysis showed that GFP insertions were inherited in T(1) generation. The ovary-drip method would become a favorable choice for directly generating vector- and marker-free transgenic maize expressing functional genes of agronomic interest.

Detection of vector- and selectable marker-free transgenic maize with a linear GFP cassette transformation via the pollen-tube pathway.

The pollen-tube pathway is feasible to transform vector- and selectable marker-free linear gene cassettes into plants to address the biosafety issues. However, its transformation frequency is low and the screening of selectable marker-free transformants by PCR analysis is time-consuming and expensive. In this study, a linear GFP cassette (Ubi-GFP-nos) flanked by 25bp T-DNA borders was transformed into maize via the pollen-tube pathway. The forepart of each maize ear was divided into five segments (segments I-V) at an interval of two rows of kernels. The segments that were most likely to contain transgenic kernels were identified by monitoring GFP expression in the immature embryos. A total of 21 ears were transformed with the linear GFP cassette. Seven out of 19 ears exhibited positive GFP expression in the immature embryos. Transgenic kernels were primarily identified in segments III and IV. A total of 121 plants derived from kernels located within segments III and IV of the remaining two ears were screened by PCR analysis. Six plants (4.96%) showed the presence of the GFP cassette. Southern blot analysis showed that the transgenic plants had simple integration patterns. The identification of transgenic kernels would facilitate PCR screening for marker-free transgenic plants.

Transfer of a minimal linear marker-free and vector-free smGFP cassette into soybean via ovary-drip transformation.

A tissue culture-independent plant transformation method, called ovary-drip transformation, was established in which a minimal linear gene cassette [35S CaMV promoter, open reading frame of soluble modified green fluorescent protein (smGFP), and NOS terminator] was transformed into soybean. The method is characterized by directly dripping a DNA solution, which is supplemented with a surfactant, onto the ovary wound 6-8 h after self-pollination. The growth of the pollen tube was measured after self-pollination. The movement of smGFP across the passageway toward the embryo sac was monitored using fluorescein isothiocyanate-labeled DNA. The transformation frequency reached 3.2% by PCR analysis. Southern analysis of the primary transformants denoted the integration of a single site smGFP. The transgenic plants exhibited a high level of smGFP expression which was visible in the immature embryos of the transgenic soybean.