Plant Transformation and Genetic Markers

Progress in plant genetic engineering has been spectacular since the recovery of the first transformed plants in the early 1980s. Molecular techniques have been applied to an array of species, resulting in the generation of numerous transgenic plants. These plants were initially transformed with marker genes (genetic markers), but subsequently with commercially important genes including those enabling agronomic improvement, easier processing and alternative uses.

Early experiments involving gene transfer used Agrobacterium tumefaciens for the introduction into tobacco of genes conveying antibiotic resistance. The development of sophisticated methods to regenerate callus, and in some cases intact plants, from protoplasts, has opened the way for an alternative procedure for engineering plants using direct DNA transfer. The protocols employed included chemical treatments and electroporation. Following the development of particle bombardment methods, the transformation of most crop species has been rapidly achieved using various modifications of the technique

The genetic markers developed for use in plant cells in general have been derived from either bacterial or plant sources and can be divided into two types: selectable and screenable markers.

Selectable markers are those which allow the selection of transformed cells, or tissue explants, by their ability to grow in the presence of an antibiotic or a herbicide. The most frequently used selectable markers are kanamycin and hygromycin. In addition to selecting for transformants, such markers can be used to follow the inheritance of a foreign gene in a segregating population of plants.

Screenable markers encode gene products whose enzyme activity can be easily assayed, allowing not only the detection of transformants but also an estimation of the levels of foreign gene expression in transgenic tissue. Markers such as b-glucuronidase (GUS), luciferase or b-galactosidase allow screening for enzyme activity by histochemical staining or fluorimetric assay of individual cells and can be used to study cell-specific as well as developmentally regulated gene expression.
Routinely, the marker genes used have been linked to promoters derived from plant pathogens, such as the T-DNA of Agrobacterium tumefeciens or plant viruses. In this way it is to be expected that such promoters function well in all plant cells, although increasingly it has become clear that the levels of expression directed by such promoters may be modulated to a certain extent by developmental factors operating in the intact plant.

Selectable/screenable marker genes for the identification of transformed plant cells

Selectable marker genes
Two main aspects of the marker gene have to be considered. Firstly, its structure (nucleic acid sequence), which will determine factors such as regulation of transcription (constitutive, environmental or developmental expression), rate of transcription, transcript stability and efficiency of translation. Secondly, the gene product itself, which is obviously responsible for the dominant expression of a suitable selective phenotype. The selectable functions on most general transformation vectors are prokaryotic antibiotic-resistance enzymes which have been engineered to be expressed constitutively in plant cells. In some experiments, enzymes affording protection against specific herbicides have also been used successfully as dominant marker genes. The enzyme coding sequence is normally fused to promoters isolated from T-DNA or the CaMV genome at the 5’ end, and a polyadenylation signal, often again from a T-DNA gene, at the 3’ end. Besides genes conferring resistance to antibiotics, genes giving resistance to herbicides such as glyphosate have also been used recently as dominant markers for transformed cells. The selective agent concerned must be able to exert stringent selection pressure on the plant tissue concerned and kanamycin resistance has proved a very useful transformation marker.

Screenable marker genes
Screenable marker genes are included on many transformation vectors for two reasons. Firstly, to allow independent verification of the transformed status of tissues growing on media containing selective antibiotics or herbicides. Secondly, as a principal means of identifying transformants in conditions where transformation frequencies are high. The most commonly used screenable markers are, chloramphenicol acetyl transferase (CAT), b-glucuronidase (GUS), b-galactosidase and luciferase. A specific use of a screenable marker is as a reporter gene; both in the development of transformation systems using transient expression assays to monitor success, or to test out DNA sequences which may be able to regulate gene expression in stably transformed tissues.
One of the most direct approaches for studying the expression of foreign genes in transformed plant tissue is to measure the abundance, or activity, of the gene products encoded by the transferred genes. Many promoters, and putative gene regulatory sequences have been analysed by making fusions with reporter genes. These are usually the coding sequences of bacterial enzymes for which convenient and sensitive assays are available and whose activities are not normally found in higher plant tissues. Among the most common are octopine and nopaline synthase, CAT, NPT-II and GUS. In situations where the gene product under investigation has no enzymatic activity, gene expression may be screened by western blotting or other standard immunological methods. In addition to this, molecular analysis like polymerase chain reaction, Southern analysis and Northern analysis are carried out to check for the integration of the transgenes.

The enzymatic assays for the most commonly used reporter genes viz., nptII and b-glucuronidase (gus) are outlined below.

Neomycin phosphotransferase assay (NPT-II)

Neomycin phosphotransferase-II (NPT-II) is a small (25 kd) bacterial enzyme which catalyses the ortho-phosphorylation of a number of aminoglycoside antibiotics including neomycin and kanamycin. The most commonly used radioactive assay is explained. The reaction involves transfer of the g-phosphate group of ATP to the antibiotic molecule, which detoxifies the antibiotic by preventing its interaction with the target site-the ribosome. This transfer reaction has been exploited to develop a sensitive solid phase assay for the enzyme. The total proteins are first extracted from the tissues to be analysed for the presence of the transgenes. The total proteins are later separated on a 10% non-denaturing polyacrylamide at 4 0C. Both kanamycin and g 32P ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzyme reaction (30 min at room temperature), the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper (for 3h) and the radiolabeled kanamycin is visualised by autoradiography (Fig. 3). With this method, 1 ng of active enzyme can be easily detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.

Analysis of GUS expression
The E.coli b-glucuronidase gene has been developed as a reporter gene system for the transformation of plants. b-glucuronidase, encoded by the uidA locus, is a hydrolase that catalyses the cleavage of a wide variety of b-glucuronides, many of which are available commercially as spectrophotometric, fluorometric and histochemical substrates. There are several useful features of the GUS gene which make it a superior reporter gene for plant studies. Firstly, many plants assayed to date lack detectable glucuronidase activity, providing a null background in plants. Secondly, glucuronidase is easily, sensitively and cheaply assayed both in vitro, in situ in gels and is robust enough to withstand fixation, enabling histochemical localization in cells and tissue sections (Fig. 1). The preferred histochemical substrate for tissue localization of GUS is 5-bromo-4 chloro-3-indolyl-b-D-glucuronide (X-gluc). The advantage of these substrates is that the indoxyl group produced upon enzymatic cleavage dimerizes to indigo which is virtually insoluble in an aqueous environment. The histochemical assay for GUS consists of soaking tissue in substrate solution and watching for blue colour to appear. The basic steps of this staining/fixation method are as follows. Tissue to be stained is placed in the well of an ELISA plate and freshly prepared staining solution is added. If the tissue floats or will not wet well, Triton-X-100 is added to the staining solution to a final concentration of 0.1%. The plates wrapped in plastic film to prevent drying are incubated at 37 0C overnight. After staining, the tissue is examined under a stereomicroscope for the presence of blue sectors. The tissue can be cleared by removing the staining solution and soaking the tissue in 95% ethanol with no loss of indigo.

Analysis for the transgenes at the molecular level is mainly carried out by Polymerase Chain Reaction and genomic Southern analysis. Polymerase chain reaction shows the presence of the transgene (Fig. 2) where as stable integration of the transgene is confirmed by genomic Southern analysis (Fig. 4). To analyze DNA where Agrobacterium vectors are used for transformation, it is important to prepare plant DNA from sterile tissue, as contamination with A.tumefaciens DNA will interfere with the interpretation of the results.

The polymerase chain reaction is used to amplify a segment of DNA that lies between two regions of a known sequence. A DNA polymerase uses two oligonucleotides as primers for a series of synthetic reactions that are catalysed by a DNA polymerase. These oligonucleotides typically have different sequences and are complementary to sequences that lie on the opposite strands of the template DNA and flank the segment of DNA that is to be amplified. The template DNA is first denatured by heating in the presence of a large molar excess of each of the two oligonucleotides and the four dNTPs. The reaction mixture is then cooled to a temperature that allows the oligonucleotide primers to anneal to their target sequences, after which the annealed sequences are extended with DNA polymerase. The cycle of denaturation, annealing and DNA synthesis is then repeated many times. Because the products of one round of amplification serve as templates for the next, each successive cycle essentially doubles the amount of the desired DNA product.

In some instances testing for the presence of foreign gene sequences by DNA ‘dot blotting’ may be used to verify the transformed nature of a tissue. Genomic Southern analysis yields information on the copy number of the integrated DNA sequences, whether any multiple inserts are tandemly linked or dispersed, and on the stability of this DNA in the F1 progeny of the transformed plants.

Southern analysis is used to determine the presence and organization of particular sequences of DNA in gel-fractionated nucleic acid. It is based on two principles. First, the transfer of fractionated DNA to a support membrane by capillary action so that the relative position of the DNA remains unchanged. Second, the hybridization of the immobilized DNA to radioactively labeled hybridization probes (complementary DNA) for the detection of specific DNA sequences. The process involves:

Restriction enzyme digestion of high molecular weight plant DNA.
Separation of the digested fragments on a 0.7% agarose gels by electrophoresis
The depurination, denaturation and neutralization of the DNA within the gel
Transfer and immobolization of the nucleic acid to a support membrane
The incubation of the membrane with a single stranded, radioactive labelled DNA hybridization probe and after washing, the visualization of hybridizing homologous DNA restriction fragments by autoradiography using X-ray film.

GUS fusions are now widely used to study plant-pathogen and plant-symbiont interactions. They can be used both to study expression of particular genes and to mark and monitor populations of microorganisms in soil or in association with plants. GUS offers a promising route for transformation, monitoring and molecular genetic analysis of the numerous important epi- and endophytic organisms that are not readily cultured, such as oomycetes and mycorrhizal fungi. In addition GUS fusions can be used in many laboratory model systems lacking endogenous activity. Unlike lac Z-encoded b-galactosidase, GUS can readily traverse membranes when an appropriate transit or signal sequence is fused to its amino terminus. GUS fusions have been used to study targeting and transport of chimeric proteins into chloroplasts, mitochondria and the endoplasmic reticulum (ER).

The first transgenic plants were recovered in 1983. It is remarkable that in just over a decade the tools of recombinant DNA technology and cell biology are at the disposal of plant breeders. Important practical issues can now be addressed and agricultural productivity should be the direct beneficiary of advances in this field. -----------------------------------------------------------------------------

Dr. K. Sankara Rao and Dr. V. K. Rohini are at the Department of Biochemistry, Indian Institute of Science, Bangalore – 560 012