In this article we will discuss about:- 1. General Principles of Gene Transfer 2. Regeneration of Plants from Transformed Cells 3. Experimental and Commercial Transgenic Crop Plants 4. Preparing Genes for Introduction into Plants 5. Limitations of Transformation Techniques.
General Principles of Gene Transfer:
The ultimate objective of modern plant breeding is to improve a top variety in one single additional character in a predictable and precise manner without disturbing the rest of the genome. Today this is being realised through examples of successful transfer of specific traits into higher plants by gene transfer.
Techniques that open up to the plant breeder the possibility of transferring in a planned manner characters from one organism to another have been developed in microbial genetics. It should be stressed right at the outset that the expression “gene” has different meanings in agriculture and in molecular biology.
Since Mendel’s experiments in the late nineteenth century, agriculture has described a gene the expression was introduced in 1909 by Johannsen – as a segregating character, and has visualised different crossing- over rates as different distances on the Morgan gene map.
In plant breeding, a gene is a mathematical expression; nevertheless, linkage groups and crossing-over rates can be symbolized on chromosomes to give a rather concrete picture. In molecular genetics, a gene is known physically as a sequence of nucleotides linked up in the DNA macromolecule.
It is known that a gene or, more precisely, a structural gene, consists of a longer or shorter linear sequence of nucleotides as a part of the huge double-stranded DNA molecule.
Together with its promotor – an activating point for an enzyme that transcribes the DNA into RNA, and perhaps some other regulatory mechanisms like DNA-binding proteins (in bacteria we know specific operators) — one or more structural genes form an operon. Such an operon is transcribed into single-stranded messenger RNA (mRNA).
At the ribosomes, the mRNA is translated into peptides by linking amino acids, which are delivered from transfer RNAs (tRNA). In all processes, further modifications take place, e.g., processing until the protein(s) forms the final enzyme.
The enzymes are responsible for the total pathways of the plant. With the exploitation of this causality, the connection between genotype and phenotype is successfully established in molecular genetics. This path can be copied to a large extent in vitro in an artificial system.
It is possible to synthesise DNA sequences; it is also possible to transcribe and translate DNA and RNA in vitro and to obtain finally, the corresponding protein. The genetic code (the sequence of nucleotides, three of which code for one amino acid) is unveiled; we do understand the letters of life.
But, what is even more exciting, this path can increasingly be followed in the reverse direction. If, for a given phenotype, sufficient quantities of a responsible protein can be isolated, the pathway from the phenotype to the gene can be followed as well (Fig. 9.5).
To use gene transfer techniques, the following prerequisites must be fulfilled:
1. The gene must be available as a fragment of DNA.
2. This DNA should be clonable.
3. The gene has to be manageable in a transfer system.
4. Integration of the DNA in the cellular genome must be achievable.
5. The transformed cell must be regenerate into a functional plant.
6. The plant has to express the transferred trait.
In principle, all prerequisites are already fulfilled today.
The first structural gene (ribulose bisphosphate carboxylase, rubisco. a part of the fraction- I protein) was isolated in 1977 by Coen Fifty per cent of the soluble leaf protein consists of this enzyme, the large subunit protein of which is coded from the DNA of the chloroplasts. Thus, it was an ideal candidate for isolation and identification of its gene.
It was followed by the gene identification for storage proteins such as zein, hordein, and phaseolin. In all these cases, for a limited period of time most of the RNA present is responsible for the production of that particular protein.
The highest RNA peak should correspond to that protein, thus the RNA was identified. This analytical step from the protein to the RNA is not reversible due to energy reasons and due to its very high complexity; from RNA to DNA, however, the enzyme reverse transcriptase can produce single-stranded copy DNA (cDNA), which can either be enzymatically converted into genomic DNA or, by DNA hybridization techniques, can be probed to cleaved DNA pieces.
The cleavage is done again, enzymatically, by using restriction enzymes, of which about 150 are known today, after the first ones were detected by Arber and Dussiox in 1962. The detection of these enzymes was probably one of the key events of present genetics, which was honoured with the Nobel prize in 1978.
Each restriction enzyme cleaves the DNA at different specific points, with or without sticky ends. The small pieces can be analysed for their nucleotides and, like a puzzle, several pieces can be linked to end up in a nucleotide sequence of a part of a gene or of a complete gene.
Once a specific DNA has been identified, this DNA has to be propagated, it has to be identically, cloned to result in enough DNA for transfer experiments. For cloning, bacteria are used. First, the DNA is linked to a plasmid, cosmid, or similar vectors that are able to replicate.
Such circular DNA is quite common and is known by abbreviations (often the initials of the responsible scientist or laboratory) such as pBR 322, one plasmid that was used in a number of fundamental transformation experiments.
Once the plasmid with its additional information is in a bacterium, it will be multiplied; normally, some additional sequences, such as an antibiotic resistance, are added that allow the easy detection of bacteria which multiply the engineered plasmid with its foreign piece of DNA. Today, cloning of a gene is no longer a problem.
For the subsequent gene transfer, following methods are available:
1. Microinjection
2. Insertion via micro-projectiles
3. Direct transfer
4. Vector-mediated gene transfer
Based on the positive results of microinjection in animal systems, this technique was tried in plants. Although under applied aspects such a laborious method will only be of very limited importance, one procedure will be discussed here. Neuhaus (1987) microinjected gene constructs into multicellular structures of Brassica napus, which have a high competence for plant regeneration through embryogenesis.
Microspore-derived embryoids of B. napus were individually, microinjected with the neomycin phosphotransferase II (NPT II) gene, under control of the cauliflower mosaic virus promoter 35S and the transcription terminator of a modified pDH 51. Transformation efficiencies between 27 and 51% were determined in primary regenerants.
Stable integration of full-length, microinjected genes into high-molecular-weight DNA was proven by Southern blot analysis of genomic DNA isolated from regenerated plants. As the starting material was multicellular, chimeras were produced. The chimeric nature of such primary regenerants was overcome by in vitro segregation through secondary embryogenesis, resulting in wild-type and pure transformants.
The basic principle of the direct gene transfer method has been summarised by Potrykus (1987). In this procedure, foreign DNA is linked to a strong promoter and to marker sequences, and is incubated together with protoplasts.
Under the influence of 8% PEG, heat shock (5 min at 45°C) and/or electric shock (electroporation, 1400 V, 10, µs), the DNA is taken up and may be integrated into the nuclear DNA. It could be demonstrated that using direct gene transfer, a number of monocots could be transformed. These include wheat, maize, rice, etc.
Vectors are most commonly used for gene transfer, particularly the Ti-plasmid of Agrobacterium tumefaciens. Details are available in the review of Horsch (1987). For A. tumefaciens-based gene transfer, the infection of a plant cell by the bacterium is a prerequisite. This occurs easily in nature only with dicots, while monocots are no host.
Thus, this transfer technique is hampered by host-range limitations, which are, especially disturbing when aiming at gene transfer into cereals. The host range poses an even more severe problem in the other biological vector system, plant DNA viruses. Despite its restriction in host range, Agrobacterium-mediated transfer has been commonly used.
Historically, most progress has been obtained in incorporating specific tolerance to herbicides. One reason for the rapid advances in this field is the fact that the biochemical pathways of modern herbicides are known; they are quite often based on just one or a few enzymes, allowing for a straightforward strategy.
A specific tolerance to one of the most commonly used herbicides, glyphosate, has been transferred first to petunia and subsequently to tobacco and tomato. The strategy used was an overexpression of 5-enolpyruvylshikimate-e-phosphate synthase (EPSPS), which is blocked at normal concentrations by the herbicide.
Overproducing mutants were first screened for in-cell suspension cultures of petunia by sequential step-up from 0.1 mM glyphosate to 20 mM glyphosate. In the tolerant cultures, about 20 times more EPSPS was produced.
The pure protein was then partially sequenced. From there, the mRNA could be used to isolate a cDNA, which was finally transferred via Agrobacterium to petunia cells. Transformed callus proved to be tolerant to high glyphosate concentrations.
The objective of genetically engineering plants with herbicide tolerance is philosophically different from that of disease resistance: one is intended to permit the continued use of pesticides and the other to use less pesticides.
Unfortunately, our knowledge of biochemical mechanisms of disease resistance is very limited, and a straightforward strategy comparable to the approach in herbicide resistance is not easily available.
Nevertheless, substantial progress has been made in inducing virus resistance via gene technology. This strategy, however, is quite different from the previous one. It is known that a virus causes cross-protection, which means that a related virus cannot infect the same plant. The coat protein of the virus is responsible for this protection.
The gene responsible for the coat protein production of TMV via Agrobacterium has been transferred to tobacco. It was expressed and prevented symptom formation in tobacco plants after TMV inoculation. The same strategy was successfully used to protect potatoes and other crops against viruses.
Another promising approach is the incorporation of the Bacillus thuringiensis toxin gene into host plants to protect against insects. The toxin, a protein, is produced very specifically, in Bacillus thuringiensis, a natural antagonist to caterpillars, diptera, and beetles. The gene for the toxic protein has been isolated, cloned, and transferred to tobacco cotton and some crop plants.
In tobacco, it has been demonstrated that the gene is expressed and the correct gene product is formed. After some amplifications, the concentration of the toxin is high enough to protect the plant against attack by caterpillars.
Regeneration of Plants from Transformed Cells:
Once a plant cell has incorporated the introduced DNA in a stable manner, the second task to be done by the plant genetic engineer is to regenerate an entire fertile transgenic plant from this single transformed cell. This is purely a cell biological problem.
The generated plants are usually, obtained from explants. It should be kept in mind that for application of this technique on commercial scale, the frequency of transformation should be high and reproducible. Further, the method should be applicable to a wide range of genotypes.
Two regeneration pathways are available. One is organogenesis, the regeneration via adventitious shoots, induced either on callus tissue or directly on a cultured explant such as a leaf, hypocotyl or cotyledon fragment. This is the ‘classical’ regeneration applied in leaf disc transformation and is very efficient for some plant species like those belonging to the solanaceae family.
The adventitious shoots grow out and can be rooted to produce a complete plant (a transgenic plant). The second regeneration pathway is somatic embryogenesis, in which undifferentiated cells develop into embryo like structures through a sequence of events like the development of zygotic embryos. These somatic embryos can germinate and grow into a mature plant.
In general, regeneration from protoplast is more difficult than from explants or callus tissue. For details on these aspects the reader should refer Dale (1993), Fisk and Dandekar (1993), Wijbrandi and De Both (1993), and Smith and Hood (1995). Table 9.6 lists the species that have been transformed and regenerated into complete transgenic plants.
Table 9.7 shows principal transformation methods in current use and examples of the crop species they have been successful with. Table 9.8 shows the vegetable crops for which genetic transformation has been reported. These examples demonstrate the wider application of technique.
Experimental and Commercial Transgenic Crop Plants:
With advances in recombinant DNA technology and transformation procedures, it has become possible to transfer genes into crop plants from unrelated sources like plants, microbes and animals. Most of these modifications are for disease and pest resistance, product quality and tolerance to environmental stress.
There are also opportunities to modify crop plants to give specialised products for industrial or pharmaceutical uses. A few important types and origins of genes inserted into crop plants as summarised by Dale (1993) are given in Table 9.9.
At, Agrobacterium tumefaciens; Ar, Agrobacterium rhizogenes; PB, particle bombardment; EP, electroporation; PG, polyethylene glycol mediated; MI, microinjection; IR, injection of reproductive organs; LP, liposome mediated; US, ultra sonication.
The genetic engineering of plants provides an opportunity to change the properties or performance of plants in order to improve their usefulness. Genetic engineering may be used to modify the expression of genes already present in the plant or to introduce new/novel genes from species with which the target plant cannot be crossed.
This opens the scope for introducing synthetic genes as well. This technology extends the possibilities for plant breeding to include totally new plant traits. Single gene may be added to the target genotypes without the alteration of any other features of the genotype which may happen with conventional back-crossing or even with marker assisted backcrossing.
Preparing Genes for Introduction into Plants:
For successful expression of foreign genes in plants, suitable gene construct has to be prepared. The name “construct” is used because the sequences normally do not exist and this combination must be put together (constructed). In gene construct, in addition to sequences coding for gene protein, the promoter and termination sequences are added at the 5′ and 3′ ends respectively.
The promoter requires a site for initiation of transcription with regulatory sequences to ensure the desired expression of gene in the desired tissues. The correct 5′ sequence alone will not necessarily ensure effective expression. Other features of gene may prevent expression.
Termination sequences are required to ensure termination of transcription and thereby gene expression. The coding region requires a translation initiation and termination codon.. It must be ensured that the codon remains in the frame, in further refinement, the codon usage may be needed to be adjusted to optimise plant expression.
Introns are also used to enhance expression. The presence of introns may have different effects in different plants. For example, there are reports that insertion of an intron between the CaMV 35S promoter and GUS increased expression 15-fold in rice and had no effect in tobacco protoplasts. A variety of promoter sequences have been used to drive genes in plant cells.
Common ones include:
nos: Agrobacterium nopaiine synthase gene, moderate activity, constitutive
ocs: Agrobacterium octopine synthase gene
mas: Agrobacterium mannopine synthase gene
35S: Cauliflower mosaic virus (CaMV) 35S RNA gene, most commonly used promoter both in dicots and monocots, 10-40 fold more efficient than nos promoter
Adh1: Promoter of alcohol dehydrogenase gene in maize
Enhancer and silencer sequences are also used to regulate the activity of promoters. An enhancer may be defined as a DNA sequence which increases the activity of promoter of a gene while DNA sequences suppressing promoter activity are known as silencers.
Limitations of Transformation Techniques:
A large and continuously growing number of plant species have been successfully transformed. However, still there are limitations of transformation techniques. Many of the direct DNA transfer methods are highly genotype dependent. Development and improvement of genotype- independent cell culture and plant regeneration techniques are required to allow more general application of plant transformation techniques.
Protocols have been standardised, which are specific to species and the genotypes. Gene expression may not be stable in some systems because of gene silencing, especially of homologous genes. Techniques for reliable gene targeting in plants may be useful in overcoming this problem.
Ideally, transformation techniques that can routinely insert genes at precise loci and with control of flanking sequences are needed. Genes inserted into chloroplasts are mainly incorporated by homologous recombination but nuclear genes may also be targeted by using homologous recombination.
Loss of expression may be due to methylation and differential methylation may explain differences in expression of transgenes. Gene replacement by homologous recombination would allow the elimination of undesirable genes by direct replacement or the modification of gene function.
Application of transformation may have restricted use in certain cases depending upon suitable promoters. Very precise control of the tissues and the developmental stages at which the gene is expressed is needed to have the desired transgenic plant.
Another problem encountered with transformation relates to controlling the number of copies of the gene to be inserted. Direct DNA transfer methods do not allow such control and hence a large number of transformants are to be raised to select the ideal one. Agrobacterium mediated transformations result in a high frequency of single copy transformants.
Production of transgenic plants usually involves co-transformation with a selectable marker gene. This gene is in most cases an antibiotic resistance gene or herbicide resistance gene.
This is a necessary evil as this gene may not be desirable in the transgenic plant but is required to select the transformed cell by growing all the cells on a medium containing antibiotic or herbicide, where only the transformed cells may survive and the non-transformed ones will die.
Non-selectable marker genes or reporter genes may be used to optimise transformation, but these genes do not allow selection of transformed cells on a medium containing a selection agent.
A few selectable marker genes for use in plant transformation are as follows:
Reporter genes that have been used in plant transformation are 3-glucuronidase, Luciferase, Anthyocyanin regulators and Green fluorescence jellyfish.