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Eight new green fluorescent protein GFP binary vectors were developed by inserting gfp reporter gene cassettes into pGreen vectors. We chose one of them, pG52KF, with the npt II selection and gfp reporter gene and one recombinant construct, pG52KFp, for a preliminary evaluation in citrus using Agrobacterium -mediated transformation.
High-transformation efficiency was observed, whereas green fluorescence greatly facilitated the early in vivo screening and categorizing of the transformants. These pGreen-derived GFP binary vectors, freely available on request, provide more and flexible options for genetic transformation in citrus and other woody plants. They allow early selection by kanamycin and later verification of transformed plants by GUS assay. This will obviously result in damage to or removal of the plantlet if an early stage of screening is required.
Since the green fluorescent protein GFP gene gfp was cloned and used as a visual marker for gene expression study Chalfie et al. Another nonbinary GFP vector was also used in citrus protoplast transformation Fleming et al. These vectors usually are developed for specific use. Generally, it is very time-consuming to modify them to develop new constructs for other applications. The pGreen is considered to be a new concept binary Ti vector Hellens et al.
High-transformation efficiencies have been demonstrated in tomato and rice using some original pGreen vectors Hellens et al. The pGreen consists of a plasmid backbone plus a Bgl II-flanked T-DNA region containing two unique blunt restriction sites, Hpa I and Stu I, located internally to the left and right border sequences LB and RB , respectively, which facilitate the cloning of any blunt selection and reporter gene cassettes.
Two Bgl II sites are useful in cloning and posttransformation analysis. These special features allow new binary vectors to be developed optimally and quickly to meet user's different demands. Moreover, pGreen contains 18 unique cloning restriction sites within the pBlueScript polylinker maximizing the options to clone an alien gene for construct development.
Compared with other binary vectors, the size of the vector is small because the origin of the replication element required by Agrobacterium is in a helper plasmid, pSoup, thus making the manipulation relatively easy.
In this article, we report the development of eight new pGreen-derived GFP vectors and preliminary studies to evaluate one of the vectors using citrus Agrobacterium -mediated transformation.
Phil Mullineaux's laboratory Hellens et al. The gfp used here originated from the jellyfish Aequorea victoria but was modified Haseloff et al. A motif encoding a transit peptide has been attached to drive the GFP to endoplasmic reticulum ER where it can be stably accumulated, and codons also modified to favor recognition by, and expression in, plant genomes that can enhance fluorescence.
Such one-step reaction can be performed only when the inserted fragment does not have the Stu I site. Twelve clones from each transformation were randomly selected and cultured for plasmid minipreparation. Eight new green fluorescent protein vectors derived from three pGreen plasmids. Epicotyl segments cut from 4-week-old etiolated seedlings of grapefruit Citrus paradisi Macf.
Duncan were used as explants in this preliminary evaluation. The transformation and regeneration procedure was as described by Yu et al. The gfp expression was observed and photographed under a fluorescent microscope Zeiss SV6 4 to 6 weeks after initial selection culture Ghorbel et al.
Regenerated shoots were cut off from 6-week explants on the selection medium and micrografted onto Carrizo rootstocks. Grafted plantlets were grown on a filter paper bridge in culture tubes containing liquid MS medium for 4 weeks before transplanting into autoclaved soil Yu et al. After 4 weeks in soil, a leaf disc of 0.
The expected sizes of PCR products would be bp and bp, respectively. PCR products were resolved on 1. The number of gfp- and npt II-amplified plantlets divided by the total number of cocultured segments was used as transformation efficiency in percentage form. The clone numbers with different insert directions were listed behind each combination of ligation. These vectors can be used in different applications, including protoplast transformation or AMT.
The lane numbers here matched those in column 1 in Table 1. Both white arrows indicated the common bp plasmid backbone fragment. NS, Biolabs, Beverly, Mass. Four-week explants with new shoots were chosen to observe the gfp expression under a fluorescent microscope.
A total of 12 and 13 shoots were regenerated from pG52KF and pG52KFp cocultured segments, and green fluorescence was observed on six and five of them, respectively, at different levels and patterns Fig.
PCR detection using the leaves of micrografted plantlets revealed that all 11 green fluorescent plantlets harbored both npt II and gfp genes Fig. Observed green fluorescence and amplified gene fragments strongly suggested that these plantlets were transgenic.
Both genes were also amplified from 10 shoots five from the vector and five from pG52KFp without green fluorescence. The transformation efficiency of pG52KF was found to be 7. The insert size appeared as a factor related to transformation efficiency. Of the remaining four plantlets without fluorescence, three had only npt II amplified one of them in lane 10 in Fig. There was no plantlet with only the gfp amplified. The new shoots with no and strong fluorescence were observed simultaneously to be regenerated from the same explants Fig.
It was unclear if the phenomenon resulted from possible chimerism or the nature of the integrated gene expression. A Nontransformed control with red fluorescence; B—L transformed shoots from the individual explants with different patterns and levels of green fluorescence, co-occurring with nontransformed shoots at the same explants B, C, and D , weak E and F , partial G and H , strong with localized reduction or absence I, J, and K , or very strong L.
Polymerase chain reaction detection of npt II A and gfp B genes in regenerated plantlets with 1—7 or without 8—13 fluorescence observed. Both sizes of npt II and gfp amplicons were as expected, bp and bp, respectively. The gfp was not detected in lane 10 from a sample that did not show fluorescence. It also occurred on some tobacco regenerants, although Agrobacterium was mostly detected on regenerated roots rather than on regenerated leaves Matzk et al.
Moreover, citrus species are not natural hosts of A. The gene fragment from Poncirus was also amplified from the gfp- expressed plantlets from pG52KFp data not shown.
The visual green fluorescence in these fragile transformants will directly ensure the early identification in vivo and categorization of transgenic plants without any damage. This preliminary evaluation and resulting transformation efficiency suggested the pG52KF may be a promising vector for citrus transformation; it is being continuously used in our laboratory as well as being evaluated on apple AMT Zhu, personal communication, 27 Jan.
These new pGreen-derived GFP vectors, with all special features from pGreen and added gfp reporter gene for in vivo screening of transformants, will allow more and flexible options for transformation and genetic engineering in citrus and many other recalcitrant horticultural crops. Mullineaux for providing pGreen vectors and other plasmids harboring gfp cassettes, Dr.
This research was supported in part under Project No. User Name Password Sign In. Previous Section Next Section. In this window In a new window. Fluorescence observation and polymerase chain reaction verification.
Development and verification of pGreen-derived binary Ti vectors. CrossRef Medline Google Scholar. Jr Agrobacterium -mediated transformation of citrus using two binary vectors. Plant Cell Tissue Organ Cult. This Article HortScience February vol. Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Download to citation manager.
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