The Selection and Analysis of Transformants
Using either Agrobacterium or direct gene transfer systems, it is now possible to introduce DNA into virtually any regenerable plant cell type. However, only a minor fraction of the treated cells become transgenic while the majority of the cells remain untransformed. It is therefore essential to detect or select transformed cells among a large excess of untransformed cells, and to establish regeneration conditions allowing recovery of intact plants derived from single transformed cells.
Selectable marker genes are essential for the introduction of agronomically important genes into important crop plants. The agronomic gene(s) of interest are invariably co-introduced with selectable marker genes and only cells that contain and express the selectable marker gene will survive the selective pressure imposed in the laboratory. Plants regenerated from the surviving cells will contain the selectable marker joined to the agronomic gene of interest. The selection of transgenic plant cells has traditionally been accomplished by the introduction of an antibiotic or herbicide-resistant gene, enabling the transgenic cells to be selected on media containing the corresponding toxic compound. The antibiotics and herbicides selective agents are used only in the laboratory in the initial stages of the genetic modificationprocess to select individual cells containing genes coding for agronomic traits of interest. The selective agents are not applied after the regeneration of whole plants from those cells nor during the subsequent growth of the crop in the field. Therefore, these plants and all subsequent plants and its products will neither have been exposed to, nor contains the selective agent. By far, the most widely used selectable gene is the neomycin phosphotransferase II (NPTII) gene (Fraley, 1986) which confers resistance to the aminoglycoside antibiotics kanamycin, neomycin, paromomycin and G-418 (Bevan et al., 1983; Guerche et al., 1987). Another widely used selectable marker is the hptII gene. The HPTII gene derived from E. coli is an aminocyclitol antibiotic that interferes with protein synthesis. The bacterial hpt gene was modified for expression in plants (Waldron et al., 1985) and has then been widely used as selectable marker in plant transformation. Hygromycin is generally more toxic to cells than kanamycin and quick as well as effective in killing of sensitive cells. Transformants are selected by applying hygromycin concentrations ranging from 20-200mg/l. It has been employed to transform diverse plant species, especially grasses in which nptII is ineffective (Miki and McHugh, 2004).
A number of other selective systems have been developed based on resistance to bleomycin (Hille et al., 1986), bromoxynil, chloramphenicol (Fraley, 1983).
Reporter genes are ‘scoreable’ markers which are useful for screening and labeling of transformed cells as well as for the investigation of transcriptional regulation of gene expression. Furthermore, reporter genes provide valuable tools to identify genetic modifications. They do not facilitate survival of transformed cells under particular laboratory conditions but rather, they identify or tagtransformed cells. They are particularly important where the genetically modified plants cannot be regenerated from single cells and direct selection is not feasible or effective. They can also be important in quantifying both transformation efficiency and gene expression in transformants. The reporter gene should show low background activity in plants, should not have any detrimental effects on plant metabolism and should come with an assay system that is quantitative, sensitive, versatile, simple to carry out and inexpensive. A main use of reporter genes is promoter characterization. They are transcriptionally fused to the promoter of interest and assayed to determine the expression conferred by the promoter. They are also used in gene silencing approaches, in studying transposon activity, as markers for specific cellular compartments and in selection of transformed cells and tissues (Rosellini, 2012). Arrays of reporter genes have been reported so far of which the most widely used include uidA (GUS), lacZ (β-galactosidase), GFP (Green fluorescent protein) and Luc (Luciferase). In addition, several anthocyanin pigmentation genes are also used as RGs due to their stability and easy detection.The gene encoding for the enzyme-glucuronidase, GUS, has been developed as a reporter system for the transformation of plants (Jefferson et al., 1986;Vancanney et al., 1990).The ß- glucuronidase enzyme is a hydrolase that catalyzes the cleavage of a wide variety of ß -glucuronides, many of which are available commercially as spectrophotometric, fluorometric and histochemical substrates. There are several useful features of GUS which make it a superior reporter gene for plant studies. Firstly, many plants assayed to date lack detectable GUS activity, providing a null background in which to assay chimaeric gene expression. Secondly, glucuronidase is easily, sensitively and cheaply assayed both in vitro and in situ in gels and is robust enough to withstand fixation, enabling histochemical localization in cells and tissue sections. Thirdly, the enzyme tolerates large amino-terminal additions, enabling the construction of translational fusions. The gene encoding firefly luciferase has proven to be highly effective as a reporter because the assay of enzyme activity is extremely sensitive, rapid, easy to perform and relatively inexpensive. Light production by luciferase has the highest quantum efficiency known of any chemiluminescent reaction. Additionally, luciferase is a monomeric protein that does not require posttranslational processing for enzymatic activity (De Wet et al., 1985).
The use of green fluorescent protein (GFP) from the jellyfish Aequorea victoria to label plant cells has become an important reportermolecule for monitoring gene expression in vivo, in situ and in real time. GFP emits green light when excited with UV light. Unlike other reporters, GFP does not require any other proteins, substrates or cofactors. GFP is stable, species-independent and can be monitored noninvasively in living cells. It allows direct imaging of the fluorescent gene product in living cells without the need for prolonged and lethal histochemical staining procedures. In addition, GFP expression can be scored easily using a long-wave UV lamp if high levels of fluorescence intensity can be maintained in transformed plants. Another advantage of GFP is that it is relatively small (26 kDa) and can tolerate both N- and C-terminal protein fusions, lending itself to studies of protein localization and intracellular protein trafficking (Kaether and Gerdes, 1995). It has been reported that high levels of GFP expression could be toxic to plant growth and development (Rouwendal et al., 1997). Solution to this problem comes from the utilization of GFP mutant genes. Among the various GFP mutations, the S65T (replacement of the serine in position 65 with a threonine) is one of the brightest chromophores characterized by its faster formation and greater resistance to photo bleaching than wild-type GFP photo bleaching. Furthermore, this mutant is characterized by having a single excitation peak ideal for fluorescin isothiocyanate filtersets (Heim et al., 1995) and also by its harmless action to the plant cell (Niwa et al., 1999).
Alternative selectable markers for plants fall into two categories. Some markers confer resistance to chemicals other than antibiotics that kill plant cells such as herbicides and lethal concentrations of the amino acids lysine and threonine. The enzyme that confers resistance to high concentrations of lysine and threonine can interfere with amino acid biosynthesis and if expressed at high levels cause abnormal plant development. The relevant genes are therefore not suitable as marker systems. Other alternative marker systems rely on the growth of plant cells in the presence of unusual nutrients, including cytokinin, glucuronides, xylose or mannose, which will not allow non-transformed plant cells to grow. For example, plant cells usually do not use mannose as a source of sugar. The delivery of a gene allowing mannose to be metabolised in plant cells and the subsequent cultivation of those cells in a medium containing mannose as the sole source of sugar would allow only those cells which have taken up the gene to grow. When these systems, which are still in their development phase, work reliably on a large scale in a wide range of different environments risk assessments will have to be conducted to assess the potential ecological impacts of plants that can grow on a new substrate, the impact on the overall plant metabolism and the consequences on human or animal diet from increased levels of metabolites in these crops that might not be present in the conventional counterpart.
It is not possible to remove marker genes once they are integrated into a plant genome unless a particular mechanism forremoval is incorporated along with the marker gene and the gene of interest at the time of the transformation. As was mentioned above, it is possible to avoid introducing into plant cells antibiotic resistant marker genes which are only used for the assembly and amplification of the DNA constructs in bacteria, and therefore are not necessary during the plant step of the transformation procedure. The removal prior to commercialisation of marker genes which are driven by plant promoters and are used for selection of plant cells has become the aim of both consumers and industry. Extensive research with this aim is being carried out both by industry and academic institutions. Among the technologies being assessed are:
1. The use of meganucleases (e.g.: Cre/lox system). These are enzymes which can specifically recognise long DNA sequences. These recognition sequences are introduced on both sides of the antibiotic resistant marker gene to be introduced into the plant cell. Once the transformed cells have been selected on the corresponding antibiotic, the meganuclease is introduced into the plant cell, and will allow the excision of the antibiotic resistant marker gene. This technology has proven to be very efficient in certain plants, but difficult to handle in others possibly because the meganuclease recognises sites in the plant genome itself.
2. The presence of homologous DNA sequences on both side of the antibiotic resistant marker gene may allow for random recombination and elimination of the gene. This process of homologous recombination occurs at low frequency and may be plant specific.
It is possible to introduce the trait of interest and the antibiotic resistant marker on different DNA constructs. Following transformation, each molecule integrates on a differentchromosome. In this case it is possible to segregate the trait of interest from the marker gene at the next generation. Frequencies of integration on separate chromosomes can be quite low when compared to integration at the same locus.
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