Genetic Problems and Gene Technology

Neumann, Karl-Hermann; Imani, Jafargholi; Kumar, Ashwani

doi:10.1007/978-3-540-93883-5_13

Karl-Hermann Neumann²,
Jafargholi Imani³ &
Ashwani Kumar⁴

Part of the book series: Principles and Practice ((PRINCIPLES))

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Abstract

In addition to aneuploidy, long-term cultures commonly show cells with ploidy levels higher than haploidy or diploidy. Such ploidy instability observed in originally haploid or diploid cultures is actually a general characteristic of cell cultures. Cytogenetic stability, however, is a prerequisite for genetic manipulations, as well as for the use of such cultures for breeding purposes. Still, genetic instability also sometimes offers a chance to isolate genotypes with properties important for practical applications, but again the problem of genetic stability of the offspring arises again.

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Cloning, General

Genetic Modification (GMOs), General

Mutations and Experimental Mutagenesis

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Somaclonal Variations

Ploidy Stability

In addition to aneuploidy, long-term cultures commonly show cells with ploidy levels higher than haploidy or diploidy. Such ploidy instability observed in originally haploid or diploid cultures is actually a general characteristic of cell cultures. Cytogenetic stability, however, is a prerequisite for genetic manipulations, as well as for the use of such cultures for breeding purposes. Still, genetic instability also sometimes offers a chance to isolate genotypes with properties important for practical applications, but again the problem of genetic stability of the offspring arises again.

Here epigenetic variations come into play. Following Chaleff, these are defined as reactions of cultures maintained after removal of the stimulus that caused these reactions. By contrast, “normal” physiological reactions cease operation after removal of the stimulus.

The classical method to determine the ploidy level is to count chromosomes at the metaphase of mitosis. Cells in active divisions are mostly ploidy stable, whereas a broad scattering of DNA levels can be observed by cytophotometry in older cell cultures with a low division rate. In this case, reliable chromosome counts would reflect a higher genetic stability and homogeneity than really exist. This can be seen in Fig. 13.1 for a Datura cell suspension at the stationary growth phase, in which the amount of DNA was determined by means of cytophotometric measurements (see Chap. 6). Such inhomogeneities occur particularly in cultures of haploid origin. Cytophotometric methods, however, are usually not sensitive enough to detect aneuploidies, or the loss of sections of chromosomes. For this, chromosome analysis is still recommended.

As mentioned before for studies assessing cell-specific DNA contents, the  so-called C-value is often used. 1C represents the DNA content of the haploid genome of the species in the G1-phase of the cell cycle, and 2C that of the G2-phase. The 1C-value can be easily obtained by cytophotometric determination of DNA content in the tetrads for standardization of the method. In carrots, for example, measurable (albeit small) differences in DNA content can be observed between varieties, and therefore it is recommended to perform such measurements for each variety separately.

In the G1-phase of the cell cycle, the DNA content of diploid cells is defined as 2C, and in the G2-phase cells as 4C. In the S-phase, intermediate values are found. Values higher than those defined for the diploid genome can be also observed in cells of intact plants raised from seeds. Moreover, and despite some data scattering particularly for stem cells, comparing the profiles of DNA content of cells of intact plants, and of cell culture systems clearly demonstrates more uniformity in DNA content for the former.

In barley plants raised from microspores, a distinct inhomogeneity can be observed within a plant, with different ploidy levels for the leaves (Fig. 13.2). Under the conditions employed, direct somatic embryogenesis could not be induced. The plants were obtained from a callus produced by anther culture via separate shoot and root differentiation. The root tip of these plants consists of cell lines of different ploidy levels (Neumann 1995). This is not the case for plants raised by the “bulbosum” method. The same scattering of ploidy levels can be detected in the callus from which the plant was obtained. Apparently, the root primordia developed from a group of cells within the callus that contained cells of different C-values. In the shoots of these plants, variations in C-values were recorded in the leaves, and infertile flowers were observed. If the initiation of shoot primordia were analogous to that of the root primordia considered above, then these leaf and flower trends would be explained.

These results have important implications. As we know today, plants derived from somatic embryogenesis develop from a single cell (see Sect. 7.3). Therefore, in experiments on the genome, or for use in breeding programs and the like, these plants would be by far preferable to those obtained from callus cultures with separate differentiation of roots and shoots. For some plant species, even nowadays the term recalcitrant is used with respect to the induction of somatic embryogenesis. For these species, the following results could be of help in producing ploidy homogenous material (Figs. 13.3, 13.4).

The protocol is based on two assumptions: first, cells with the lowest ploidy level have the shortest cell cycle duration; and second, in cell suspensions highest cell division activity takes place in small cell aggregates, comparable to the meristematic nests described for callus cultures. High cell division activity is supported by a kinetin supplement, and these cell aggregates are isolated by means of some sieving technique. The end result are ploidy stable Datura suspensions maintained for 3–4 weeks (Kibler and Neumann 1980; Neumann 1995). The application of this protocol to barley suspensions of haploid origin with a broad scattering of C-values in several successive subcultures resulted in ploidy homogenous cell material (Fig. 13.4 Neumann 1995).

Some More Somaclonal Variations

Following Larkin and Scowcroft (1981), scattering of ploidy levels (as described above), and some other epigenetic variations of cultured cells can be categorized as somaclonal variations. These include mutations, chromosomal rearrangements, changes in chromosome structure, gene amplification, gene methylations, activation of transposons, exchange of sections of chromosomes, and others.

For a number of plant species (rice, wheat, maize, lettuce, tobacco, tomato, and rapeseed), so-called point mutations have been detected in plants raised from cell cultures. An early review on this topic was published by Scowcroft et al., already in 1987. Plants with such mutations were obtained from the same callus as others free of these, and it was speculated that these mutations were produced during cell culture. This, however, can not be regarded as a conclusive proof. Usually, an original explant consists of 10,000 to 15,000 cells and more, and one can not exclude the “accidental” existence of cells already mutated in the mother plant, which are later propagated in culture. Here, protoplast cultures and somatic embryogenesis should be a more suitable system.

In higher plants, the fraction of repeated DNA sequences amounts to about 40–60% of the total genome, and some sequences can occur in up to million copies. The fraction “repetitive sequences” with a moderate number of copies includes genes for rRNA. In tissue culture-derived individual plants of Triticale, for a ratio of four fragments of an rRNA sequence to each other obtained by application of the restriction enzyme Tag 1, quantitative variations were detected by Brown and Lörz (1986). These changes were stable through meiosis. For interpretation of such results, it has to be considered that changes in the number of copies of genes can be also induced by phytohormones, or herbicides (Widholm 1987). It remains unclear to which extent such changes are heritable. Notably, an amplification of some DNA stretches induced by GA3 applications to carrot plants, which resulted in a reduction of the diameter of the taproot, was not inherited.

Already Larkin et al. (1985) described tissue culture-derived plants of 14 species in which changes in chromosome structure were observed. These included deletions, exchange of chromosome sections, isochromosome formations, inversions, DNA amplifications, and others. Furthermore, such changes were observed in 17 of 551 tissue culture-derived hexaploid wheat plants. In 14 plants, aneuploidy of a chromosome was detected, and four plants were euploid. These variations were interpreted as being due to the formation of isochromosomes, or translocations within the genome.

The question has to be raised which are the causes of somaclonal variations, and what makes plants derived from tissue cultures particularly prone to such changes.

The simple conventional understanding of genetic regulation of cellular life can be summarized as: DNA is transcribed into RNA, which acts as template to synthesize proteins, these being responsible for essentially all processes occurring within the cell, or its reactions to the environment. As DNA nucleotide sequences become increasingly known, however, unexplainable inconsistencies with this central dogma appear. This raises questions like why apparently genetically identical twins are not really identical in some ways. Epigenetic factors seem to be responsible for these anomalies, and these may be associated with the so-called junk DNA (see above). It has to be kept in mind that only a few percent of our DNA codes for proteins through mRNA. For a long time, this junk DNA was considered as a byproduct of millions of years of evolution. This could still be true to some extent, but here at least part of the information for epigenetic factors could be localized. Somaclonal variations, often observed in cultured cell material, or embryos derived thereof, can be defined as epigenetic factors, and this may be a valuable system for studying the broad spectrum of epigenetics as such, and its regulation of cells.

During recent years, ever more evidence points to an epigenetic system of regulation of growth and development that seems to control gene activity. This includes DNA methylation, DNA amplification, histone acetylation, and others without changes in the nucleotide sequence. Changes in this system are also heritable. It remains to be seen whether these are part of a regulatory system above that of the classical DNA/RNA/protein system, or rather largely independent factors related to the classical schema.

As mentioned elsewhere, during callus formation in vitro two of these epigenetic factors, i.e., DNA methylation and DNA amplification, were characterized in carrot cultures. These proved to be transient. During the logarithmic growth phase, DNA amplification was decreased and DNA methylation was promoted. This was an indication of a rearrangement of epigenetic factors (or systems) at transfer from the original, rather quiescent root cells, to proliferating callus cells. At the stationary phase often associated with rhizogenesis, methylation was reduced, and the formation of amplified DNA sequences was increased again. Here, a presumably qualitatively different, new epigenetic system would have been established. Changes in methylation can also be induced by growth regulators, notably auxins (Loschiavo et al. 1989; Arnholdt-Schmitt 1993). Repeated elements are known to be preferably methylated (Arnholdt-Schmitt et al. 1995).

During such rearrangements, errors may occur in the reorganization of the epigenetic system, expressed as somaclonal variations. Such somaclonal variations are frequent in transgenic material. Transgenic plants are usually derived from tissue culture systems, and since such epigenetic changes can be heritable, the genome of transgenic plants is often rather unstable. This could contribute to answering the second part of the question posed above.

As answers to the first part of this question, DNA amplifications, transposons, and somatic reorganizations of the genome can be considered. The latter seems to involve mainly changes in polygenic traits. Furthermore, somatic crossing over, exchange of material of sister chromatides, variations in the methylation pattern of DNA, activation or inactivation of genes due to mutations of DNA sequences originally not coding, though associated with coding DNA stretches, and other aspects can be discussed. To date, however, there is insufficient experimental evidence published in the literature pointing to any one mechanism as the cause of somaclonal variations.

The situation is not much different in responding to the second part of the question posed above. As early as the 1970s, D’Amato discussed the high plasticity of the genome of higher plants in tissue culture. Based on the availability of sufficient data already at that time, several mechanisms seemed to be possible, related mainly to the original material from which the explants were obtained. This may contain cells with DNA replications without concurrent division of the nucleus, or eventually the cell. If such cells occur in an explant at the initiation of cell division, then when these cells divide, polyploidy will result. Such DNA extrareplications have been recorded in more than 80% of angiosperms screened. As already described, these can be more or less tissue-specific, and seem to be related to differentiation. In meristematic tissue, they are almost absent. In mature tissue like the Phaseolus suspensor, an amplification of DNA without concurrent division of the chromatids results in giant chromosomes (Nagl 1970). Consequently, ploidy homogeneity of cell cultures would depend at least partly on the developmental status of the tissue used to obtain explants for culture. The use of explants from meristematic areas should yield culture systems showing higher ploidy homogeneity.

Important are also reactions of the nucleus during callus inductions. Here, endoreduplications are often observed, and also unequal nucleus fragmentation due to multipolar spindle formation. As a result, cells with more than one nucleus are produced (D’Amato et al. 1980). Later, cell divisions will be associated with the loss of one or the other chromosome, eventually leading to aneuploidy. As already mentioned elsewhere, phytohormones seem to play a central role in these processes, particularly the auxin/cytokinin ratio.

In general, the variability of the genome increases with the age of cultures. This can be observed mainly for the occurrence of endoreplications without concurrent division of the nucleus, as well as of the cell. The extent of this influence depends on the hormonal supplement to the nutrient medium, but also on nitrogen form and concentration, the osmotic pressure of the medium, and other factors. Thus, which cell type will find optimum conditions under which it can dominate will depend on the composition of the nutrient medium. An example is the influence of kinetin to enrich haploid cells in a suspension originally showing a broad scattering of C-values (see above).

A similar significance can be assigned to changes of the nutrient medium to induce differentiation. Thus, by changing the culture conditions, it is possible to selectively promote growth of certain genomes. By implication, careful genetic characterization, particularly of plant material used for plant breeding, or gene technology is recommended here.

As discussed earlier, somaclonal variation can cause problems for cell culture systems preserving germplasm for long duration through subculturing. These problems can be circumvented by use of various methods of cryopreservation (Sect. 3.6).

As mentioned before, somaclonal variation can be exploited to select germ lines with properties beneficial for one or the other application. Here again the problem of stability of such traits arises. Although some changes in the genome were shown to be heritable, these changes were conferred mostly to areas of low significance for the growth and development of intact plants derived from cell suspensions. Similar to mutations induced by chemical treatments, or by X-rays, somaclonal variations are rather accidental. To improve the yield of crop plants, or the resistance to environmental stresses and attacks of pests, a controlled change of the genome is required. Success depends on understanding the physiological or biochemical basis of the process in the plant to be altered, and its dependence on genetic factors. As will be described later, here gene technology comes into play.

An important aspect in tracking genomic changes induced either naturally as somaclonal variations, or by artificial means such as X-rays is the method of selection. Generally, a positive method is applied through which plants with altered genomes are not, or at least less affected in an environment hostile to the unchanged wild type (Figs. 13.5, 13.6). An example is a stepwise increase of a toxin. Callus cultures, cell suspensions, or meristem cultures are suitable for an induction of mutations. In callus cultures, as well as in meristem cultures, the cells are not all exposed equally neither to the mutagen, nor to the same selection pressure. Consequently, a variable number of cells escape either the one, and/or the other. Plants obtained from such cultures through regeneration of shoots and roots may be chimeras, as described for “ploidy chimeras” above. An alternative could be the use of protoplasts, which can be plated and selected after initiation of culture to be used to produce plants following somatic embryogenesis.

Plating entails the transfer of protoplasts onto agar not yet fully hardened, and through further solidification of the agar, their position is subsequently fixed. The position of individual protoplasts on the plate can be marked, and their further development can be easily observed. Cultures of a given size (100s of cells) can be exposed to the selection pressure. The problems of selections will be addressed again after the discussion of gene technology. A summary of work done on mutations before the advent of gene technology is given by Jacobs et al. (1987).

The discovery of many biochemical pathways in microbes was based on the use of deficiency mutants. Meanwhile, mutants of a few master plant systems, like Arabidopsis or rice, are employed for studying the metabolism of higher plants (metabolomics). Details on this are found in the sections dealing with metabolism, and gene technology in this book.

Gene Technology

Gene technology is a term that encompasses a wide range of techniques for genetic analyses based on the direct manipulation of DNA, and the transfer of genes between different species. The increased use of gene technology in biotechnology for numerous categories of common products and other important aspects (pharmaceuticals, food quality, agricultural chemicals, disease resistance, functional food, plant nutrition, etc.), as well as for basic investigations to understand gene regulation has an ever increasing impact on our society. In the medical/pharmaceutical field, biotechnology signifies a dramatic change in the approach to drug discovery, research and development, diagnosis, and disease management. Examples of biopharmaceuticals, i.e., enzymes, or regulators of enzyme activity, hormones, or hormone-like growth factors, cytokines, vaccines, monoclonal antibodies, and gene transfer in humans can be discussed (Ritschel and Forusz 1994).

Farmers, breeders, and scientists have been improving existing species of plants and animals by selective crossbreeding for thousands of years. Gene technology is the latest addition to these breeding techniques. It provides a means of introducing new characteristics into a species not possible by conventional breeding. Because the building blocks of genetic material are the same in all living systems, and many organisms share genes, gene technology increases the pool from which breeders can select beneficial traits from species unrelated to the crop they wish to improve.

Generally, plants have around 30,000 genes, and animals up to 50,000 genes. A gene is a segment of a long chemical polymer called deoxyribonucleic acid (DNA). Because the chemical units of DNA are the same in all living systems, it is possible to transfer a gene, and the physical characteristics it controls, from one organism to another without altering other characteristics of that organism, not usually the case when using conventional breeding methods. A transgenic organism carries, in  all its cells, the foreign gene that was inserted by laboratory techniques. Each transgenic organism is produced by introducing cloned genes, composed of deoxy ribonucleic acid (DNA) from microbes, animals, or plants, into plant and animal cells. Transgenic technology consists of methods that also enable the transfer of genes between different species. Introducing a new gene into a crop using gene technology is difficult and time-consuming. However, once that gene exists in one variety, it can be transferred to other, related varieties with crossbreeding techniques that have been used by plant breeders for centuries.

Transferring a gene from one source (plants, bacteria, fungi, insects) to another, especially across kingdoms (from an animal to a plant, for example), requires a laboratory step that is often called gene splicing, or genetic modification (GM). The integration of a desired gene into a target organism at present generally is random.

Transformation Techniques

There are two common ways of introducing DNA into plant cells—indirect, and direct. In the former approach, scientists make use of microbes that commonly infect plants, e.g., Agrobacterium tumefaciens, or a virus. The new piece of spliced DNA is placed into a bacterium or a virus, which acts as a courier to carry the DNA into the plant cell. The new DNA is then incorporated into the recipient cell’s own DNA.

The latter approach of introducing a new gene into a plant involves, for example, particle bombardment (gene gun), polyethylene Glycol (PEG), and microinjection. In both approaches, the cells containing the new gene are grown under tissue culture conditions, and after somatic embryogenesis (see Sect. 7.3.) can develop into a fully functional plant, complete with the new, desirable characteristic.

Direct Gene Transfer

To the Agrobacterium system, alternative direct transformation methods have been developed (Shillito et al. 1985; Potrykus 1991) such as polyethyleneglycol-mediated transfer (Uchimiya et al. 1986), microinjection (de la Pena et al. 1987), protoplast and intact cell electroporation (Fromm et al. 1985, 1986; Lörz et al. 1985; Arencibia et al. 1995), and gene gun technology (Sanford 1988). However, Agrobacterium-mediated transformation has remarkable advantages over these direct transformation methods. It reduces the copy number of the transgene, potentially leading to fewer problems with transgene co-suppression and instability (Koncz et al. 1994; Hansen et al. 1997). In addition, it is a single-cell transformation system, and usually does not form chimeric plants, which are more frequent when direct transformation is used (Enríquez-Obregón et al. 1997, 1998). Two barley transformation systems—Agrobacterium-mediated, and particle bombardment—were compared in terms of transformation efficiency, transgene copy number, expression, inheritance, and physical structure of the transgenic loci, using fluorescence in situ hybridization (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. Whereas 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable, and inherited as a simple Mendelian trait. By contrast, transgene silencing was frequently observed in the T1 populations of the bombardment-derived lines (Travella et al. 2005). Because of genotype-dependent transformation by A. tumefaciens, however, the use of ballistic methods for recalcitrant genotypes is sometimes essential.

Still, as mentioned above, the advantages of Agrobacterium-mediated transformation of plant tissue are generally a low transgene copy number, minimal rearrangements, and higher transformation efficiency than for the direct DNA delivery techniques (Gelvin 1988; Pawlowski and Somers 1996), as described below.

Agrobacterium-Mediated Gene Transformation

Since the application of Agrobacterium-mediated transformation to monocotyledonous species such as rice, maize, barley, sugarcane, and wheat, and also to animal cells, as has been recently reported also here the use of Agrobacterium is still in central focus. In summary, the main characteristics of the Agrobacterium system in these, as well as dicotyledonous species are:

•
a rather high frequency of transformation,
•
proper integration of the foreign gene into the host genome,
•
low copy number of the gene inserted.

This results in most cases in a correct expression of the transgene itself. Because of its significance, it is necessary to give some details of this transformation process.

The first reliable method for plant transformation was based on a pathogen that attacks plants, and causes crown gall disease—formation of galls at the “crown” of a plant. The organism that causes this disease is Agrobacterium tumefaciens, a soil-borne plant pathogenic bacterium. The galls are produced at the site of infection, and consist of a mass of undifferentiated cells, also known as tumors. Agrobacterium produces these tumors by transferring a piece of its DNA (T-DNA, transferred DNA) into the plant. This is a natural transfer of DNA from a prokaryote into an eukaryote. Plant transformation mediated by A. tumefaciens has become the most commonly used method for the introduction of foreign genes into plant cells, and the subsequent regeneration of transgenic plants.

The first evidence indicating this bacterium as the causative agent of crown gall goes back 100 years (Smith and Townsend 1907). Since that time, a large number of researchers have focused on the study of this neoplastic disease, and its causative pathogen, and this for various reasons. During the first and most extensive period, scientific effort was devoted to disclosing the mechanisms of crown gall tumor induction. Then, in the 1970s, pathogenicity transferred between bacteria via conjugation, and evidence of plasmid involvement was investigated, and finally in the 1970–1980s the Ti (tumor-inducing) plasmid was characterized. It was apparent to researchers working with A. tumefaciens that this gram negative soil bacterium co-opted normal plant cell metabolism by leaving a small portion (the transfer DNA, or T-DNA) of its Ti-plasmid in the genome of an infected plant cell. Several research groups realized that if the genes normally present within the bacterium T-DNA were to be replaced with other genes, then one could obtain expression of the new genes in plant cells. However, the bacterium infected only one, or a few cells, and so not all cells of a plant harbored the new genes flanked by the T-DNA. Consequently, it was necessary to include a selectable marker in the T-DNA, so that only those cells that had taken up the engineered T-DNA, including the selectable marker, could be identified and allowed to grow.

When interacting with susceptible dicotyledonous plant cells, virulent strains of A. tumefaciens and Agrobacterium rhizogenes, another pathogen, have the exceptional ability to transfer a particular part (T-DNA) of their large plasmid, the Ti-plasmid (Fig. 13.7, >250 kb), into the nucleus of the infected cell, where it is then stably integrated into the host genome, and transcribed. This induces the diseases known as crown gall (A. tumefaciens) and hairy roots (A. rhizogenes). Two types of genes are responsible for tumor formation: the oncogenic genes, encoding for enzymes involved in the synthesis of auxins and cytokinins, and additionally T-DNA contains genes encoding for the synthesis of opines. These compounds, produced by the condensation of amino acids and sugars, are synthesized and excreted by the crown gall cells, and consumed by A. tumefaciens as carbon and nitrogen sources. The synthesis of the opine form is dependent on the bacterial strain; e.g., for plasmids of C58 chromosomal background, it is the nopaline synthase that is responsible for nopaline formation from arginine, and  it is agropine for strain EHA 105 with TiAch5 chromosomal background (Roger et al. 2000).

Mechanisms of gene transfer mediated by A. tumefaciens

The process of gene transfer from Agrobacterium tumefaciens into plant cells can be considered in terms of several steps, as follows:

•
Infestation of bacteria
•
Induction of the bacterial virulence genes
•
Generation of the T-DNA transfer complex
•
Integration of the T-DNA complex into the plant genome.

An essential, and also the earliest step in tumor induction is the infestation of plant cells by A. tumefaciens after its attachment to the plant cell surface (Matthysse 1986). Non-attaching mutants show a loss of the tumor-inducing capacity (Cangelosi et al. 1987; Thomashow et al. 1987; Bradley et al. 1997).

The polysaccharides of the A. tumefaciens cell surface are proposed to play an important role in the colonization of, and also during the interaction with the host plant. The gene responsible for successful bacterium attachment to the plant cell is located at the chromosomal 20 kb locus.

Lipopolysaccharides (LPS) are an integral part of the outer membrane, and include the lipid membrane anchor, and the antigen polysaccharide. A. tumefaciens, like other plant-associated Rhizobiaceae bacteria, produces also capsular polysaccharides (antigens) lacking lipid anchor, of strong anionic nature and tightly associated with  the cell. There is some evidence indicating that capsular polysaccharides may play  a specific role during the interaction with the host plant. In the particular case of A. tumefaciens, a direct attachment of wild type bacteria to plant cells was observed.

This attachment region is composed of at least six essential operons (virA, virB, virC, virD, virE, virG), and two nonessential ones (virF, virH). The number of genes per operon differs Tzfira & Citovsky (2008).

VirA is a transmembrane dimeric sensor protein that detects signal molecules, mainly small phenolic compounds, released from wounded plants (Pan et al. 2003). The signals for VirA activation include acidic pH, phenolic compounds, such as acetosyringone (Winans 1992), and certain classes of monosaccharides that act synergistically with phenolic compounds (Ankenbauer and Nester 1990; Cangelosi et al. 1990; Shimoda et al. 1990; Doty et al. 1996). VirA protein serves as periplasmic, or input domain (important for monosaccharide detection), and two transmembrane domains act as a transmitter (signaling) and receiver (sensor; Chang and Winans 1992; Parkinson 1993). One of these transmembrane domains corresponds to the kinase domain, and plays a crucial role in the activation of VirA, phosphorylating itself (Huang et al. 1990; Jin et al. 1990a, b) in response to signaling molecules from wounded plant sites.

VirG functions as a transcriptional factor regulating the expression of vir genes when it is phosphorylated by VirA (Jin et al. 1990a, b). The C-terminal region is responsible for the DNA binding activity, while the N-terminal is the phosphorylation domain, and shows homology with the VirA receiver (sensor) domain.

The activation of vir systems also depends on external factors like temperature and pH. Virulence capacity is reduced by high temperature (exceeding 32°C), because of a conformational change in the folding of VirA (Jin et al. 1993; Fullner et al. 1996). This indicates that the temperature for co-culture is crucial for genetic transformation. Actually, it should be between 21 and 28°C.

The activation of vir genes results in the generation of single-stranded (ss) molecules representing the copy of the bottom T-DNA strand. Any DNA placed between T-DNA borders will be transferred to the plant cell as single-stranded DNA, and integrated into the plant genome. The proteins VirD1 and VirD2 play a key role in this step, recognizing the T-DNA border sequences and nicking (cf. endonuclease activity) the bottom strand at each border. After endonucleotidic digesting, the rest of the VirD2 protein covalently attaches to the 5′ end of the ssT strand. This association prevents exonucleolytic attack to the 5′ end of the ssT strand (Dürrenberger  et al. 1989), and distinguishes the 5′ end as the leading end of the T-DNA transfer complex. T-DNA strand synthesis is initiated at the right border, and it proceeds in the 5′ to 3′ direction until the termination process takes place. The left border may also act as a starting site for ssT strand synthesis, but the efficiency is much lower (Filichkin and Gelvin 1993). Inside the plant cell, the ssT-DNA complex is targeted to the plant nucleus after passing through the cell- and nuclear membranes. For the export of VirE2 to the plant cell, VirE1 is essential. VirE2 contains two plant nuclear location signals (NLS), and VirD2 one (Bravo Angel et al. 1998). This indicates that both proteins probably play important roles once the complex is in the plant cell, mediating the complex uptake into the nucleus (Herrera-Estrella et al. 1990; Rossi et al. 1993; Tinland et al. 1995; Zupan et al. 1996). VirD2 and VirE2 also ensure that the DNA is efficiently transported mammalian into the nuclei.

The final step of T-DNA transfer is its integration into the plant genome. The mechanism involved in the T-DNA integration has not yet been fully characterized. It has to be considered that integration occurs by some illegitimate recombination (Gheysen et al. 1989; Lehman et al. 1994; Puchta 1998). According to this model, pairing of a few bases, known as microhomologies, are required for a pre-annealing step between the T-DNA strand coupled with VirD2, and plant DNA. These homologies are very low, and provide only a minimum specificity for the recombination process by positioning VirD2 for the ligation. The 3′ end, or adjacent sequences of T-DNA find some low homologies with plant DNA, resulting in the first contact (synapses) between the T strand and plant DNA, and forming a gap in the 3′–5′ strand of plant DNA. Displaced plant DNA is subsequently cut at the 3′-end position of the gap by endonucleases, and the first nucleotide of the 5′ attaches to VirD2 pairs with a nucleotide in the sense (5′–3′) plant DNA strand. The 3′ overhanging part of T-DNA, together with the displaced plant DNA are digested either by endonucleases, or by 3′–5′ exonucleases. Then, the 5′ attached to the VirD2 end, and the other 3′ end of the T strand (paired with plant DNA during the first step of integration process) join the nicks in the anti sense plant DNA strand. Once the introduction of the T strand into the 3′–5′ strand of the plant DNA is completed, a torsion followed by a nick into the opposite plant DNA strand is produced. This situation activates the repair mechanism of the plant cell, and the complementary strand is synthesized using the earlier inserted T-DNA strand as template (Tinland et al. 1995).

The import of DNA into mammalian nuclei is generally inefficient by this system. The Agrobacterium virulence proteins VirD2 and VirE2 perform important functions for the transport into nuclei. The reconstituted complexes consisting of the bacterial VirD2, VirE2, and single-stranded DNA (ssDNA) in use in vitro for import into HeLa cell nuclei (Ziemienowicz et al. 1999) are inefficient. The import of ssDNA requires both VirD2 and VirE2 proteins, and a VirD2 mutant lacking its C-terminus nuclear localization signal is inefficient.

The system described above was efficiently performed by plant cells. Here it was shown that VirE2 can be a protein without ssDNA, to be able to be imported into cell nuclei. The smaller ssDNA required only VirD, whereas the import of longer ssDNA additionally required VirE2. RecA, another ssDNA binding protein, could substitute for VirE2 in the nuclear import of T-DNA, but not in earlier events of T-DNA transfer to plant cells (Ziemienowicz et al. 2001).

The first records on transgenic tobacco plant expressing foreign genes appeared at the beginning of the last decade, although many of the molecular characteristics of this process were unknown at that time (Herrera-Estrella 1983; Potrykus 1991). Since that crucial turn in the development of plant science, a great progress in understanding of the Agrobacterium-mediated gene transfer to plant cells has been achieved. However, A. tumefaciens naturally infects only dicotyledonous plants, and many economically important plants are monocots, including cereals, and remained inaccessible for genetic manipulation for a long time.

Agrobacterium-mediated gene transfer into monocotyledonous plants was not possible until recently, when reproducible and efficient methodologies were established for rice (Cheng et al. 1998), banana, corn (Ishida et al. 1996), wheat (Cheng et al. 1997; Rasci-Gaunt et al. 2001; Wu et al. 2003), sugarcane (Enríquez-Obregón et al. 1997, 1998; Arencibia et al. 1998), and barley (Tingay et al. 1997; M.B. Wang et al. 1998, 2001; Murray et al. 2001).

Many factors, including plant genotype, explant type, Agrobacterium strain, and binary vector, influence the Agrobacterium-mediated transformation of monocotyledonous plants. Inoculation and co-culture conditions are very important for the transformation of monocots. For example, antinecrotic treatments using antioxidants and bactericides, osmotic treatments, desiccation of explants before or after Agrobacterium infection, and inoculation and co-culture medium compositions had influences on the ability to recover transgenic monocots. The plant selectable markers used, and the promoters driving these marker genes have also been recognized as important factors influencing stable transformation frequency. Extension of transformation protocols to elite genotypes, and to more readily available explants in agronomically important crop species will be the challenge of the future. Further evaluation of genes stimulating plant cell division or T-DNA integration, and of genes increasing the competency of plant cells to Agrobacterium may improve transformation efficiency in various systems (Cheng et al. 2004).

In this book, we present updated information about the mechanisms of gene transfer mediated by A. tumefaciens, and assessments for the application of this method to the transformation of monocotyledonous and dicotyledonous plants, as examples using barley and carrots, respectively, and as practiced in our own research program (see later).

Transformation is currently used for the genetic manipulation of more than 120 species of at least 35 families, including the major economic crops, vegetables, fruit trees, as well as ornamental, medicinal, and pasture plants (Birch 1997), based on Agrobacterium-mediated, or direct transformation methods. The number of GM plant species increases continuously. The argument that some species cannot accept the integration of foreign DNA in their genome, and lack the capacity to be transformed can not be accepted, in view of the increasing number of species that have already been transformed; however, the establishment of an efficient tissue culture system still forms the basis for genetic manipulation.

As mentioned above, efficient methodologies for Agrobacterium-mediated gene transfer have been established mainly for dicotyledonous plants. To extend these to monocotyledonous plant species, it is important to account for critical aspects in the Agrobacterium tumefaciens–plant interaction, and the cellular and tissue culture methodologies developed for these species. The suitable genetic material (bacterial strains, binary vectors, reporter, marker genes, and promoters), and the molecular biology techniques available in the laboratory are necessary considerations for selection of the DNA to be introduced. This DNA must be able to be expressed in the plants, enabling the identification of transformed plants in a selectable medium, and using molecular biology techniques to test and characterize the transformation events (for a review, see Birch 1997).

The optimization of A. tumefaciens–plant interaction is probably the most important aspect to be considered. It includes the integrity of the bacterial strain, its correct manipulation, and the study of reactions in wounded plant tissue, which may develop into a necrotic process in the wounded tissue, or affect the interaction and release of inducers or repressors of the Agrobacterium virulence system. The type of explants is also important, and must be suitable for regeneration, enabling the recovery of whole transgenic plants. The successful establishment of a method for the efficient regeneration of one particular species is crucial for its transformation.

It is recommended to work firstly on the establishment of optimal conditions for gene transfer, through preliminary experiments on transient gene expression using reporter genes (Jefferson 1987), like the “green fluorescent protein” (GFP; Tsien 1998; Chalfie and Kain 1998), DSRED Lux, and Bax. The proapoptotic protein Bax can serve as rapid gene answer by fast-killing of plant cells (Eichmann et al. 2006; Technologie-Lizenz-Buero (TLB) der Baden-Wuertingenschen Hochschule GmbH, Germany). This is supported by the fact that Agrobacterium-mediated gene transfer is a complex process, and many aspects of the mechanisms involved still remain unknown. Transient expression experiments help to identify also the explants that may be used as targets for gene transfer, providing definitive evidence of successful transformation events, and correct expression of the transgene.

Preliminary studies also include the use of histologically defined tissues of different explants, and regeneration of whole plants, preferably by somatic embryogenesis (Sect. 7.3). Transient expression experiments may be directed to the regenerable tissue and cell. Optimization of transient activity is a futile exercise if experiments are conducted on non-regenerable tissues, or under conditions inhibiting regeneration, or altering the molecular integrity of the transformed cell. As mentioned before, Agrobacterium-mediated transfer introduces a smaller number of copies of foreign DNA per cell than is the case for particle bombardment or electroporation, but high efficiencies of stable transformation may be obtained even from cells without positive results in transient expression assays.

These aspects are important in establishing an appropriate transformation procedure for any plant, particularly for those species categorized as recalcitrant. Cereals, legumes, and woody plants, which are difficult to transform or still remain untransformed today, can be included in this category. Many species originally considered for this category have nevertheless been transformed in recent years.

Selectable Marker Genes

Production of transgenic plants usually requires the use of selection marker genes, which enables the selection of genetically modified cells, and their regeneration into whole plants. For this reason, genes coding for antibiotic resistance are frequently used. These genes have no intentional function in the genetically modified organism, and no agronomic or other value in agriculture.

Current methods of generating transgenic plants employ a “selectable marker gene”, which is transferred together with any other gene of interest usually on the T-DNA between its borders. The presence of a suitable marker is necessary to facilitate the detection of genetically modified plant tissue during its development.

There are two types of marker genes:

1.
Selectable markers to protect the organism against a selective agent, based on antibiotic resistance; herbicide tolerance, and metabolic marker genes.
2.
Markers for screening. A marker for screening will make cells containing the gene “look” different.

The most frequently used marker genes in GM plants are the kanamycin resistance gene (neomycin phosphotransferase II, NPT II) from the bacterial transposable element Tn5, and the hygromycin resistance gene associated with hygromycin phosphotransferase (hpt). Hygromycin B and kanamycin are inhibitors of RNA translation.

Herbicide-tolerant marker genes are screenable selectable markers, which makes the identification of transformed progeny efficient and fast. The Bar and pat genes have been widely used as selectable markers for plant transformation, and are ideal for large- or small-scale screening to identify transformants that can easily be done in the greenhouse or in the field. Also herbicide screening has been used successfully to identify transformants. Transgenic cells and plants expressing the pat (phosphinotricin acetyltransferase isolated from soil bacteria) gene are able to degrade the herbicide agent phospinotricin (glufosinate). This gene is resistant to the herbicides Basta, Bialaphos, and Ignite.

Metabolic marker genes are:

•
2-DOG system

2-deoxyglucose is a plant growth inhibitor. Transgenes (2-deoxyglucose-6-phosphate-phosphatase) can grow on medium containing 2-Dog.

•
Palatinose system

Usually, plant cells are not able to assimilate palatinose. Therefore, if this is the carbohydrate source in the medium, no growth occurs. On the other hand, the transgenes containing palatinase can grow on medium containing palatinose, by converting it into fructose+glucose.

•
Mannose system

Mannose can not be metabolized in plants. Transgenes with mannose-6-phosphate-isomerase can grow on medium containing mannose.

Reporter Genes

In order to identify transformed cells or plants that have been growing on a selective medium, it is necessary to have an easily assayable reporter gene. The most useful reporter genes encode an enzyme activity not found in the organism being studied. A number of these genes are currently being used (cf. uidA, GFP, DsRED, Luc, LuxA/LuxB).

Green fluorescent protein (GFP)

In addition to fluorescent stains, also fluorescent proteins are used. The most prominent of these is the green fluorescent protein (GFP; Chalfie et al. 1994; Tsien 1998; Chalfie and Kain 1998). Chalfie and Tsien received the 2008 Noble Prize in Chemistry for this discovery. A general overview of this topic is given by Tsien (1998). This rather unusual molecule was detected in the 1960s in the luminescent jellyfish Aequorea victoria. The protein emits a green fluorescence in UV light. In modified forms, it has been used to make biosensors, and many animals have been created that express GFP as a proof of concept that a foreign gene can be expressed throughout a given organism. Reporter plants carrying GFP are used to analyze cytological events, or to localize selected proteins. GFP is coupled to the gene to be analyzed. In most cases, the target protein is not disturbed, and GFP can be used as a chemical “flash light”. The position of the protein to be investigated can be localized by use of a fluorescent microscope. This provides the possibility not only to localize the protein, but also to gain a fair estimation of its concentration. Additionally, it is possible to analyze new “switches” for genes, i.e., promoters, and their activation in an organism. These very interesting properties have been used to analyze many physiological and biochemical problems. Meanwhile, beside GFP, some other fluorescence proteins, like DsRed of corals, have become known and are being used in molecular biology.

Variants of GFP

As mentioned above, the green fluorescent protein (GFP) has become an invaluable tool for pure and applied biological research. The inert nature of the protein, and the many potential uses of GFP, including the possibility to observe the behavior of proteins in living cells, were quickly recognized, and triggered the development of novel GFP variants with improved characteristics. An intensive search was initiated for other autofluorescent proteins, which fluoresce at different wavelengths than that of GFP. Mutagenesis of the wild type gene yielded improved variants such as enhanced GFP (EGFP), as well as color variants such as the cyan (CFP) and yellow (YFP) fluorescent proteins. A recent paper has described a family of fluorescent proteins related to GFP. The most useful of these newly discovered proteins is DsRed, which is derived from the coral Discosoma. DsRed has an orange–red fluorescence with an emission maximum at 583 nm. It has a high quantum yield, and is photostable. These characteristics make DsRed an ideal candidate for fluorescence imaging, particularly for multicolor experiments involving GFP and its variants. A codon-optimized version of DsRed is now available under the name DsRed1. The red fluorescent protein DrFP583 (DsRed) is the first true monomer, mRFP1, derived from the Discosoma sp. fluorescent protein (Geoffrey et al. 2000; Bevis and Glick 2002).

Dendra

The first representative of a new class of photoactivatable fluorescent proteins that is capable of pronounced light-induced spectral changes is the monomeric variant Dendra from octocorals (Dendronephthya sp., red activatable), suitable for protein labeling. Dendra is capable of 1,000- to 4,500-fold photoconversion from the green to the red fluorescent states in response to either blue or UV light. It demonstrates high photostability of the activated state, and can be photoactivated by a common, marginally phototoxic, 488-nm laser line. The suitability of Dendra for protein labeling, and to quantitatively study the dynamics of fibrillarin and vimentin in mammalian cells is recommended (Gurskaya et al. 2002).

Proapoptotic protein Bax

For promoter studies based on rapid gene answer by the fast killing of the plant cell, the proapoptotic protein Bax also seems suitable (Eichmann et al. 2006; Technologie-Lizenz-Buero der Baden-Wuertembergischen Hochschule GmbH, Germany). Figure 13.8 shows collapsed barley epidermal cells expressing Bax protein. Here, the leaves were transformed, via ballistic delivery of expression vectors, into single epidermal cells of barley leaf segments, according to a transient transformation protocol originally developed for wheat (Schweizer et al. 1999). Both GFP and the Bax gene were under control of the CaMV35S promoter.

β-Glucuronidase (GUS)

The E. coli β-glucuronidase (GUS) is one of the most popular reporter gene currently in use. The protein has a molecular weight of 68,200, and appears to function as a tetramer. It is very stable, and will tolerate many detergents, widely varying ionic conditions, and general stress. It is most active in the presence of thiol-reducing agents such as β-mercaptoethanol, or DTT. It may be assayed at any physiological pH, with an optimum between 5.2 and 8.0. The GUS gene, like the GFP, can be used in gene fusion. This means that the GUS coding sequence is under the direction of the controlling sequence of another gene.

Agrobacterium containing some GUS plasmids show significant GUS activity. This seems to be due in part to read-through transcription from the gene in which the GUS coding region might be located. Agrobacterium without these constructs shows little, if any detectable GUS activity. In order to solve this problem, one laboratory has constructed GUS genes carrying an intron, which has to be processed before expression takes place. This totally eliminates expression in any untransformed system (Vancanneyt et al. 1990).

Procedures for Assay of GUS Gene Expression

Histochemical assay

The best substrate currently available for the histochemical localization of β-glucuronidase activity in tissues and cells is 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc; see Table 13.1). The substrate works very well, giving a blue precipitate by incubation at 37°C for 8–16 h at the site of enzyme activity. There are numerous variables that affect the quality of histochemical localization, including all aspects of tissue preparation and fixation, as well as the reaction itself.

Table 13.1 Compositions of X-Gluc buffer for GUS assay  (10 ml; store at –20°C after filter (0.2 µ) sterilizing)

Full size table

The product of glucuronidase action on X-Gluc is not colored. Indeed, the indoxyl derivative produced must undergo an oxidative dimerization to form the insoluble, highly colored indigo dye. This dimerization is stimulated by atmospheric oxygen, and can be enhanced by using an oxidation catalyst such as a K⁺ ferricyanide/ferrocyanide mixture. Without a catalyst, the results are often very good, but one has to consider the possibility that nearby peroxidases may enhance the apparent localization of glucuronidase.

Fixation conditions will vary with the tissue, and its permeability to the fixative. Glutaraldehyde can be used; it does not easily penetrate leaf cuticle, but is immediately available to stem cross sections. Formaldehyde seems to be a more gentle fixative than glutaraldehyde, and can be used for longer periods of time.

Whole tissues, callus, suspension culture cells and protoplasts, or whole plants or plant organs can be stained, but the survival of stained cells is not certain. After staining, clearing the tissue with 70% ethanol seems to improve the contrast in many cases.

Fluorometric assay

Fluorometry is preferred over spectrophotometry because of its greatly increased sensitivity, and wide dynamic range. Although spectrophotometric substrates for GUS are available, GUS activity in solution is usually measured with the fluorometric substrate 4-methylumbelliferyl-b-D-glucuronide (MUG). The assay is highly reliable, and simple to use.

Antibiotics Resistance Genes

(ABR genes, restricted use recommended)

On 2 April 2004, a group of experts of the European Union (GMO panel of the European Nutritional Office EFSA) responsible for security problems in “green technology” accepted a report on the use of antibiotics-resistant marker genes in gene technologically transformed plants. These experts declined a general prohibition, and recommended a differential and cautious use. The use of some antibiotics-resistant markers would be prohibited, and the use of some others would be restricted. For the nptII marker gene employed in most transformed plants, no changes were recommended.

Horizontal gene transfer from a transformed plant into microorganisms is very unlikely; still, the experts considered this a possibility. The frame of considerations was set by accepting the notion, as basis, that even in the case of such an unlikely horizontal transfer, human and environmental health would not be negatively influenced. The following criteria were applied:

•
The medical significance of an antibiotic, its present use in human and veterinary medicine, and its efficiency to control some infectious diseases
•
The natural distribution of antibiotics resistances in microorganisms of soil and water, but also in the digestive systems of humans and other mammals.

The experts recommended a release of transformed organisms into the environment only if they contain ABR marker genes occurring at large under natural conditions, and inhibiting the action of only those antibiotics not in use in medicine.

As any other group of experts of EFSA, the GMO panel consists of independent and highly esteemed scientists. This group of scientists will usually be consulted in granting permission to grow transformed plants in the EU, and present scientific recommendations. Decisions, however, are made by the political institutions—EU Commission,  EU Counsel, and EU Parliament. Not all antibiotics resistance genes are similar. The GMO panel of EFSA categorizes antibiotics-resistant genes into three groups.

Group 1

These are ABR genes occurring at large in natural microbial communities. These antibiotics have either no, or very limited significance for human or veterinary medicine (nptII gene for kanamycin resistance, or hph gene for hygromycin resistance).

Example: nptII gene: this gene was abundantly used for many years to label transgenic plants. It was originally isolated from a transposon (jumping gene). It transmits resistance to several antibiotics, e.g., kanamycin, or neomycin. These are used only on patients not able to tolerate any other antibiotics. Kanamycin can have strong side effects.

For marker genes of this group, it is assumed that their use in transgenic plants will have no influence on the group’s already existing distribution in the environment. The EFSA experts recognized no arguments to restrict the use of these ABR genes. It was recommended to permit unlimited use of GM plants carrying these ABR marker genes for field experiments, as well as for commercial farming.

Group II

It is assumed that marker genes of this group used in transgenic plants would hardly have an influence on their present distribution. If an influence on the health of humans and animals exists, it would only be small. The EFSA experts recommended using these markers only in field experiments, but not in crops for the market. ABR genes are widely distributed in natural populations of microorganisms. Antibiotics are still prescribed to control specific diseases. To these belong the ampr gene (resistance to ampicillin), the aadA gene (resistance to streptomycin), and the Cmr gene (resistance to chloramphenicol).

Example: ampr gene: this gene transmits resistance to the antibiotic ampicillin. It originates from E. coli bacteria, and is used in approved transgenic plants (Bt 176 corn). Generally, ampicillin is only rarely prescribed, to cure certain infectious diseases.

Group III

These are ABR genes transmitting resistance to antibiotics of great significance in controlling diseases in humans. Even if the effect of these antibiotics is not impaired by use in GM plants, this is not approved as a precaution, and the use of these genes should be avoided. The EFSA panel recommended that GM plants with such marker genes should be used neither for experimental purposes, nor for commercial production.

Example: Npt gene: this gene transmits resistance to the antibiotic amikacin, an important storage antibiotic effective in controlling a number of infectious diseases.

In the near future, a prohibition to use antibiotics in transgenic plants can be expected.

One alternative could be the use of genes of D-amino acid-oxidase (dao1; Erikson et al. 2004) as selection markers:

I.
D-Alanine and D-serine where β-NADH is β-nicotinamide adenine dinucleotide, reduced form, and β-NAD is β-nicotinamide adenine dinucleotide, oxidized form.
II.
D-Valine and D-isoleucine do not affect plants, but are metabolized by dao1 into keto acids, which inhibit plant growth.

Elimination of Marker Genes

Production of transgenic plants usually requires the use of selection marker genes, which enables the selection of genetically modified cells, and their regeneration into whole plants. For this purpose, as discussed above, genes coding for antibiotic resistance are frequently used. However, there is no agronomic, or other value of the selection marker genes for the use of transgenic plants in agriculture. The presence of antibiotics resistance markers in transgenic plants intended for human or animal consumption may also be a cause of concern. Fears have been expressed that such genes may be transferred horizontally to microorganisms of the gut flora of man or animals, leading to the spread of antibiotics resistances in pathogenic microorganisms. Though extensive studies have failed to detect a measurable risk of this occurrence, many biotechnologists view the negative publicity related to the presence of unnecessary marker genes as sufficient reason to warrant their removal.

We present some common methods for the elimination of undesirable marker genes, which have already been successfully used in plant and animal cells.

Cre-lox Recombination-Based Systems

A group of site-specific recombinase enzymes that catalyze recombination at the specific target sequences have received considerable attention for the manipulation of heterologous genomes in vivo. Of these, the recombinase protein from bacteriophage P1 (Cre is a 38 kDa) mediates intramolecular (excisive or inversional), and intermolecular (integrative) site-specific recombination between loxP sites (locus of X-ing over); it consists of two 13-bp inverted repeats separated by an 8-bp asymmetric spacer region (see review by Sauer 1993).

One molecule of Cre binds per inverted repeat, or two Cre molecules line up at one loxP site. The recombination occurs in the asymmetric spacer region. Those eight bases are also responsible for the directionality of the site. Two loxP sequences, in opposite orientation to each other, invert the intervening piece of DNA, and two sites of same orientation dictate excision of the intervening DNA between the sites, leaving one loxP site behind. This precise removal of DNA can be used for the elimination of an endogenous gene, or activation of a transgene (Fig. 13.9).

The advantage of this system is the automatic elimination during seed production—for example, when a seed-specific promoter is used. The following generation should therefore be marker gene-free. Homologous recombination is efficient in chloroplasts (Corneille et al. 2001).

Ac/Ds System

The Nobel Prize winner Barbara McClintock in 1949 challenged the thesis that genes remain fixed at particular sites in a chromosome. She showed that genes can change their position, and can move to other chromosomes. These “jumping genes”, or “transposons”, occur in many organisms. This phenomenon plays an important role in biology, because it contributes to the genetic variability of organisms. The transposons that have been most comprehensively characterized are those of the “Ac/Ds family”. A transposon contains a gene for a particular enzyme (Ac transposase, activator element), which recognizes certain signals (Ds sequences, dissociation element) in the DNA, cuts pieces out of the DNA at these points, and reintegrates these into the genetic material at a different, unpredictable site. This ability can be exploited to remove undesirable marker genes from genetically modified plants (Fig. 13.10).

The marker-free GMP can be then selected by progeny segregation. The advantage of this system is not only to unlink the marker gene, but also to create a series of plants with different transgene loci from one original transformant, which is particularly highly valued if recalcitrant plants have to be transformed. This repositioning enables expression of the transgene at different genomic positions, and consequently at different levels. However, as segregation of the transgene and marker are required, and transposons tend to jump into linked positions, this approach is definitely more time-consuming than Cre-lox recombination-based systems.

Double Cassette System

The selectable marker in the transgenes also prevents retransformation with additional genes with the same selection procedure. Two co-transformation approaches are comparable: one Agrobacterium strain with one plasmid bearing two T-DNAs (double cassette), and two Agrobacterium strains each with one plasmid (one with the target gene, and one with the selection gene). It is thus desirable to create marker-free transgenic plants. This was successfully achieved in tobacco; rice, and barley with binary “double cassette” vectors, which carry two separate cassettes on the same plasmid, each bracketed by a left and a right T-DNA border sequence (Fig. 13.11; Komari et al. 1996; Slafer et al. 2002). One cassette contains the gene encoding β-glucoronidase (uidA), and the other a hygromycin or kanamycin resistance gene.

Both T-DNA segments were co-transferred with a frequency of about 50% in the plants mentioned above. As the two cassettes were frequently inserted into different chromosomes, or chromosome arms, the resistance gene segregated independently from the marker gene in the T2 generation.

Concluding remarks

Agrobacterium tumefaciens is more than only the causative agent of crown gall disease affecting dicotyledonous plants. It now is also the natural system for the introduction of foreign genes into plants, enabling its genetic manipulation. Similarities have been found between T-DNA and conjugal transfer systems. They are evolutionally related, and apparently evolved from a common ancestor.

Although the gene transfer mechanisms remain largely unknown, great progress has been made in the practical implementation of transformation protocols for both dicotyledonous and monocotyledonous plants. Particularly important is the extension of this single-cell transformation methodology to monocotyledonous plants. This advance has biological and practical implications. Firstly, there are advantages of A. tumefaciens-mediated gene transfer over the direct transformation methods, which originally were the only available for genetic manipulation of economically important crops such as cereals and legumes. Secondly, it has been demonstrated that T-DNA is transferred to dicotyledonous and monocotyledonous plants by an identical molecular mechanism. This confirmation implies that potentially any plant species can be transformed by this method, if a suitable transformation protocol is established.

For better understanding of gene technology, a few examples of our own research program will be described.

Agrobacterium-Mediated Transformation  in Dicotyledonous Plants

The Agrobacterium-mediated transformation protocols differ from one plant species to the other, and within species, from one cultivar to the other. Therefore, the optimization of Agrobacterium-mediated transformation methodologies requires the consideration of several factors that can be determined in the successful transformation of one species. The optimization of Agrobacterium-plant interaction in competent cells from different tissues, and the development of a suitable method for regeneration from transformed cells are needed.

In this book, we present the carrot as model for genetic manipulation in a successful establishment of a routine protocol for Agrobacterium-mediated transformation of dicotyledons. Here, we have optimized the integration rate of a desired gene into the plant genome.

The basis for a successful production of transgenic carrot plants, as an example for dicots, has been formed by lengthy experience in the field of somatic embryogenesis and cell cycle synchronization, as well as extensive work on the differentiation and DNA organization of this species. One example will be the integration of a surface protein of hepatitis B (SHBs), another the integration of a second gene for phosphoenolpyruvate carboxylase (PEPCase; Sect. 9.2.2), Hordeum vulgare Bax inhibitor 1 (HvBI-1), and some attempts to target foreign genes (ROL genes; see Sect. 7.3) for integration into the genome and gene studies related to plant pathogen resistance. For monocots, the transformation of barley will be described.

Transgenic Carrot: Potential Source of Edible Vaccines

Transgenic plants have a high scientific and economical potential for the production of foreign proteins of biomedical importance. Since the early 1990s, it has been shown that transgenic plants could express viral and bacterial antigens, with preservation of their immunogenic properties. Such plants could therefore serve as an inexpensive vaccine, because it would not be necessary to make a large capital investment into a production facility for a vaccine. These vaccines stimulate the immune response at the mucosal level, and thus would be particularly effective against diseases. For example, enough antigens for one dose of hepatitis B vaccine could be produced in unprocessed plant material at a cost of US$ 0.005. This advantage of plant-derived vaccines is important, because it can lead to a much less complicated vaccine development decision-making process. This represents an alternative for the production of vaccines against infectious diseases of worldwide importance, e.g., hepatitis B. Moreover, transgenic plants expressing sufficient levels of antigens bear the potential of oral delivery of antigenic proteins as edible vaccines, if the antigen is resistant against the gastric passage.

The hepatitis B virus (HBV) is distributed worldwide, with an estimated 350 million persistent carriers (Maynard et al. 1986). HBV infections are responsible for a high proportion of the world cases of cirrhosis, are the cause of up to 80% of all cases of hepatocellular carcinoma (HCC), and are directly responsible for more than one million deaths each year (report of WHO meeting, 1993). Major pathways for transmission of HBV include parental exposure to blood or other infective body fluids, transmission from mother to infant, as well as sexual transmission. In order to minimize HBV infections, large-scale immunization is required (Chen et al. 1988, 1996). Current hepatitis B vaccines contain the major, or small hepatitis B virus surface protein (SHBs), and are produced in transgenic yeast. Vaccines may also be produced in genetically engineered higher plants. There are several reports on transgenic plants producing biomedically relevant molecules, such as antibodies in tobacco plants (Mason et al. 1992, 1996; Tsuda et al. 1998), and potatoes (Ehsani et al. 1997). Furthermore, a plant-derived edible vaccine against HBV expressed in lettuce and lupine has been reported (Kapusta et al. 1999). More recently, successful clinical tests on humans, using raw potatoes after incorporation of SHBS, have been carried out (Thanavala et al. 1995). For vaccination purposes, however, HBV surface proteins should preferably be produced in an edible plant that is easy to store and to transport, and that can be consumed without cooking. Thus, carrots (Daucus carota L., ssp. sativus) seem to be very suitable for the production of HBV vaccines.

For production of major hepatitis B virus surface protein, we linked the MAS promoter to SHBs. In carrot cell suspensions, as well as in roots of mature transgenic carrot plants, the hepatitis B virus surface protein was produced (Imani et al. 2002; Figs. 13.12, 13.13).

Mannopine synthase (mas) promoter of Agrobacterium tumefaciens is a strong inducible promoter, active in roots and leaves, as well as in callus of carrot (Imani et al. 2002) and tobacco, and induced by wounding, auxins, and cytokinins (Langridge et al. 1989). Because of its inducibility by exogenous application of auxins, we tested its suitability for engineering HBsAg in carrot. The HBV concentration depends on the form of application of NAA to enhance the activity of the MAS promoter, and the production of HBsAg. The following experiments using callus cultures are encouraging.

As control for the function of an additional application of an auxin in the production of the viral protein, callus cultures were initiated in explants of the secondary phloem of the taproots of transgenic and of untransformed carrot plants of the same developmental stage in a liquid medium (Steward et al. 1952; Neumann 1995). The cultured explants were bathed in the nutrient medium containing IAA; consequently, the effect of the auxin on the production of the viral protein is much higher than after an application to intact plants growing in soil (Fig. 13.13).

Production levels of SHBs antigen in carrot, tobacco, tomato, banana, and other plants were compared. Expression of SHBs was reported to be rather low, at  25 ng/g f. wt. in carrot (Kumar et al. 2007).

In order to reach high transformation rates as a basis for strain selection, an effective method to transform carrot suspension cells is required. The percentage of cells successfully transformed by Agrobacterium is usually very low. In this report, we show that in carrot (Daucus carota L., ssp. sativus) cell suspension transformation efficiency was strongly improved by using cell cycle synchronized cells (Chap. 12). Fluorodeoxy-uridine (FDU, Sigma, Germany) was added for 24 h to inhibit thymidine synthesis. This blocked the cell cycle at the transition from the Gl- to the S-phase. Then, the block was released by applying thymidine. A high rate of transformation was obtained when A. tumefaciens was added concurrently with thymidine. As examples of efficient and long-term foreign gene expression in transgenic cells, the reporter enzyme β-glucuronidase (GUS) was used as model. The GUS gene was linked to an inducible mannopine synthase promoter (MAS) from the Ti-plasmid of A. tumefaciens. In carrot cell suspensions containing the gus gene, the corresponding GUS protein was produced. In roots of mature transgenic carrot plants generated via somatic embryogenesis and raised in soil, as well as in callus cultures derived thereof, the GUS protein was also produced (Imani et al. 2002).

Here, a synchronization of the cell cycle strongly promoted transformation. From the cell suspension, we regenerated transgenic carrot plants following somatic embryogenesis. Carrot cell suspensions were first transformed with a MAS::GUS construct, which was used as control for the functioning of the system. Based on the experience gained in this model system, the SHBs gene was successfully introduced.

Cell Culture and Transformation Procedures

The B5 medium (Gamborg et al. 1968; Sect. 3.4) for carrot cell suspensions originally derived from cultured petiole explants was supplemented with 2.26 µM 2.4D (dichlorophenoxy acetic acid) to promote cell division. For synchronization of the cell cycle, an FDU/thymidine system was employed, as described earlier (see Sect. 7.3, and Fig. 12.1; Blaschke 1977; Blaschke et al. 1978; Neumann 1995; Froese and Neumann 1997). By these means, 80–90% of the dividing cultured carrot cells can be synchronized. In short, to arrest cycling cells at the transition from Gl- to the S-phase, fluorodesoxyuridine (FDU, 10^–6M) was added to the nutrient solution for 24 h to inhibit the synthesis of thymidine, and consequently of DNA replication. Thereafter, the cells were transferred to a fresh nutrient medium supplemented with 10^–5M thymidine to initiate the transition of the synchronized cultures from the late G1-phase into the S-phase for DNA replication.

In order to make use of an advantageous preferential integration of foreign DNA into replicating DNA, a suspension of A. tumefaciens containing the plasmid pGE2 with the GUS construct (Fig. 13.14) was simultaneously supplied to the synchronized carrot cell suspension, together with thymidine. After 48 h of co-culture, cefotaxime (500 µg/ml) was added to remove excess Agrobacterium (Fig. 13.15).

After transformation, the cells were either subcultured in a B5 medium, again with 2.26 µM 2.4D for proliferation, or transferred into a hormone-free medium to enable the development of somatic embryos (Li and Neumann 1985). The germinated embryos were raised to intact plants growing in soil. Integration of foreign genes into the carrot genome was confirmed by genomic southern blotting (Imani et al. 2002).

The integration efficiency for GUS was determined by the X-Gluc reaction, resulting in the production of a blue color proportional to the number and expression activity of transformed cells. Comparing the intensity of the color in the cultures shown in Fig. 13.16, it is evident that the integration of the MAS::GUS constructs is greatly enhanced by adding A. tumefaciens simultaneously with thymidine to initiate DNA replication.

Adding the bacteria 24 h after the application of thymidine results in a markedly reduced reaction, indicating a lower level of transformation, probably due to loss of cell cycle synchronization, and a reduced number of cells passing through the S-phase. Quantification of transgene expression with densitometric techniques shows a fourfold increase in gene expression of synchronized cell cultures, compared to unsynchronized cultures (Fig. 13.16). In such transformed cell suspensions, the development of somatic embryos was initiated (see Sect. 7.3). The embryos were then raised to intact plantlets, and transferred to soil in the greenhouse. After approximately 3 months of culture in soil, in various parts of the plants a blue color evidently indicated the long-lasting expression of β-glucuronidase.

Optimization of transformation and protein extraction techniques in our laboratory, followed by the screening of several transgenic cell lines, revealed significant differences in expression levels of proteins of interest. This helped to increase the concentration of SHBs protein to 15 µg/g in carrots (Ph.D. Thesis of H. Lorenz, 2006). Thus, transgenic carrots may have a high potential for the production of various oral vaccines.

Based on this method, Cyclamen persicum Mill. embryogenic cell suspension cultures were transformed. A high rate of transformation was obtained when A. tumefaciens was added concurrently with thymidine (Imani et al. 2007). The regeneration of transformed cyclamen plants via somatic embryogenesis has been described by Winkelmann et al. (2000).

Uses of Transgenes to Increase Host Plant Resistance to Plant Pathogens

As is known, an infection by plant pathogens leads to plant cell death as self-protection (programmed cell death, PCD), which should inhibit the spreading of fungi within the plant. PCD is a mechanism to remove aged, unwanted, damaged, or infected cells from multicellular organisms. It is under genetic control, and must be tightly regulated to avoid false ultimate decisions and diseases. In the interaction of plants with pathogenic microbes, PCD appears to play different roles depending on the lifestyle of the disease-inducing agent. Among cell death regulator proteins, only a few are structurally and functionally conserved across eukaryotic kingdoms. One of these PCD regulator proteins is the BAX inhibitor-1 (BI-1), an endoplasmic reticulum membrane protein that can suppress diverse kinds of PCD. BI-1 controls heterologous BAX-induced cell death, hypersensitive reaction, and abiotic stress-induced cell death in plants (reviewed by Hückelhoven 2004).

Grey mold caused by Botrytis cinerea is a severe disease for many dicotyledonous crop plants, and it also occurs as post-harvest disease in carrots. Owing to its necrotrophic lifestyle, B. cinerea destroys plant tissue by secretion of hydrolytic enzymes, host-nonspecific toxins, and reactive oxygen species (ROS; Von Tiedemann 1997; Gronover et al. 2001; Tenberge et al. 2002; Siewers et al. 2005). The species is difficult to control by chemical means, because pesticide resistance occurs rapidly in fungal populations (Staub 1991). B. cinerea might be a target for genetically engineered resistance.

To restrict pathogenesis of the necrotroph Botrytis cinerea (carrot root pathogen), we generated the carrot (Daucus carota ssp. sativus) cultivar Rotin expressing HvBI-1 protein under control of constitutive CaMV35S and inducible MAS promoter (Imani et al. 2006). From the corresponding lines, we regenerated plants by tissue culturing and somatic embryogenesis, under selective conditions according to Imani et al. (2002). For pathogen challenge, excised leaves from 7–8 week old plants were inoculated with agar blocks containing B. cinerea (strain 7890).

Symptom development on excised leaves was examined in three independent experiments. Symptom development progressed slowly until 4 days after inoculation. However, runaway spreading of leaflet necrosis, and overgrowth by grey  mold started on wild type leaves as of the 5th day after inoculation. Symptom development progressed until 4 weeks after inoculation, when the leaves were necrotic, and overgrown by the fungus (Fig. 13.17).

Together, HvBI-1 can delay, or prevent diseases caused by hemibiotrophic and necrotrophic fungal pathogens in carrots. This supports the assumption that factors promoting biotrophic fungal growth concurrently cause inhibition of fungi with a necrotrophic lifestyle phase. To address the problem that overexpression of PCD inhibitors in plants might confer undesired side effects on biotrophs, one strategy could be expression of BI-1 under control of a necrotrophy-specific and/or a tissue-specific promoter.

Agrobacterium-Mediated Transformation  in Monocotyledonous Plants

Generation of Transgenic Barley Plants

Recent work on Agrobacterium-mediated genetic transformation of monocotyledonous plant species has focused on the use of the so-called super-binary vector systems, i.e., binary vectors carrying a DNA fragment from the A. tumefaciens virulence region (Komari et al. 1996; Torisky et al. 1997).

Using highly virulent strains of Agrobacterium, and improved vectors, much progress in the transformation efficiency of cereals has been made (Tingay et al. 1997; M.B. Wang et al. 1998, 2001; Murray et al. 2001; Rasci-Gaunt et al. 2001; Wu et al. 2003). Nowadays, quite routinely applied transformations of cereals are still rather time-consuming and cost-intensive. For transformation, the strongly constitutive ubiquitin and CaMV35S promoters are used. At present, a shortage exists of suitable regulatable promoters for the expression of defense-associated genes (Stuiver and Custers 2001).

For constitutive overexpression and for tagging expression, we cloned a functional cDNA fusion of the green fluorescing protein (GFP) and HvBI-1 into the binary vector pLH6000 (http://www.dna-cloning-service.de/lh-vectors.htm; DNA Cloning Service, Hamburg, Germany), maintaining the original cauliflower mosaic virus 35S promoter (CaMV35S). For barley transformation, pLH6000 CaMV35S::GFP, as well as pLH6000 CaMV35S::GFP-HvBI-I were then introduced into A. tumefaciens strain AGL1 (Lazo et al. 1991). GFP-BI-1 fusion protein accumulates in nuclear membranes (Matthews et al. 2001; Deshmukh et al. 2006).

The barley cultivar Golden Promise was grown in a growth chamber, or in the greenhouse at 22°C, 60% relative humidity, and a photoperiod of 16 h (150 µmol/s per m² photon flux density). Stable genetic transformation of barley was performed as described by Tingay et al. (1997; see flow sheet in Fig. 13.18, and Table 13.2).

Table 13.2 Different culture media used for barley transformation

Full size table

Twelve to 14 days after anthesis, immature kernels were surface sterilized for  3 min with 70% ethanol, and 20 min with a sodium hypochlorite solution containing 3% active chlorine, followed by rinsing 3 times with sterile distilled water. Excised immature embryos were then infected with A. tumefaciens. The callus induced grew on Murashige and Skoog medium containing 50 mg hygromycin B/l (Roche, Germany). Established calli were then subcultured on regeneration medium supplemented with 25 mg hygromycin B/l, until rooted plantlets could be transferred to soil. Timentin (150 mg/l) was applied until tests for the presence of bacteria proved negative. The GFP reporter was visualized with either a standard fluorescence microscope, or the confocal laser scanning microscope TCS SP2 AOBS (excitation: laser line 488 nm, emission: 500–540 nm; Leica Microsystems, Bensheim, Germany; Schultheiss et al. 2005; Deshmukh et al. 2006; Fig. 13.19).

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Justus-Liebig-Universität Giessen, Institut für Pflanzenernährung, Heinrich-Buff-Ring 26-32, Giessen, 35392, Germany
Prof. Dr. Karl-Hermann Neumann
Justus-Liebig-Universität Giessen, Institut für Phytopathologie und Angewandte Zoologie, Heinrich-Buff-Ring 26-32, Giessen, 35392, Germany
Dr. Jafargholi Imani
Department of Botany, University of Rajasthan, Jaipur, 302003, India
Prof. Dr. Ashwani Kumar

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Prof. Dr. Karl-Hermann Neumann
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Dr. Jafargholi Imani
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Prof. Dr. Ashwani Kumar
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Neumann, KH., Imani, J., Kumar, A. (2009). Genetic Problems and Gene Technology. In: Plant Cell and Tissue Culture - A Tool in Biotechnology. Principles and Practice. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-540-93883-5_13

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DOI: https://doi.org/10.1007/978-3-540-93883-5_13
Published: 29 January 2009
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Genetic Problems and Gene Technology

Abstract

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Cloning, General

Genetic Modification (GMOs), General

Mutations and Experimental Mutagenesis

Keywords

Somaclonal Variations

Ploidy Stability

Some More Somaclonal Variations

Gene Technology

Transformation Techniques

Direct Gene Transfer

Agrobacterium-Mediated Gene Transformation

Selectable Marker Genes

Reporter Genes

Variants of GFP

β-Glucuronidase (GUS)

Procedures for Assay of GUS Gene Expression

Antibiotics Resistance Genes

Elimination of Marker Genes

Cre-lox Recombination-Based Systems

Ac/Ds System

Double Cassette System

Agrobacterium-Mediated Transformation  in Dicotyledonous Plants

Transgenic Carrot: Potential Source of Edible Vaccines

Cell Culture and Transformation Procedures

Uses of Transgenes to Increase Host Plant Resistance to Plant Pathogens

Agrobacterium-Mediated Transformation  in Monocotyledonous Plants

Generation of Transgenic Barley Plants

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About this chapter

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Genetic Problems and Gene Technology

Abstract

Similar content being viewed by others

Cloning, General

Genetic Modification (GMOs), General

Mutations and Experimental Mutagenesis

Keywords

Somaclonal Variations

Ploidy Stability

Some More Somaclonal Variations

Gene Technology

Transformation Techniques

Direct Gene Transfer

Agrobacterium-Mediated Gene Transformation

Selectable Marker Genes

Reporter Genes

Variants of GFP

β-Glucuronidase (GUS)

Procedures for Assay of GUS Gene Expression

Antibiotics Resistance Genes

Elimination of Marker Genes

Cre-lox Recombination-Based Systems

Ac/Ds System

Double Cassette System

Agrobacterium-Mediated Transformation in Dicotyledonous Plants

Transgenic Carrot: Potential Source of Edible Vaccines

Cell Culture and Transformation Procedures

Uses of Transgenes to Increase Host Plant Resistance to Plant Pathogens

Agrobacterium-Mediated Transformation in Monocotyledonous Plants

Generation of Transgenic Barley Plants

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Agrobacterium-Mediated Transformation  in Dicotyledonous Plants

Agrobacterium-Mediated Transformation  in Monocotyledonous Plants