2. Methods of Genetic Transformation: Electroporation and Polyethylene Glycol Treatment

RAY SHILLITO Agrevo USA Co., 703 NOR-AM Road, P. O. Box 538, Pikeville, NC 27863, USA. E-mail: shillir@agrgold.hcc.com

ABSTRACT. Methods for direct gene transfer into protoplasts via polyethylene glycol (PEG) treatment and electroporation were developed in early 1980's. Genetic transformation of protoplasts led to the production of the first transgenic cereals. This paper describes events leading up to the stable transformation of protoplasts via these techniques, the possible mechanisms involved, the improvement of the methods, and their application to the mole­cular improvement of cereals. While no longer the predominant method for transforming cereal crops, protoplast transformation still plays an important role in basic studies of gene regulation and function, understanding of the transformation process, and in the production of transgenic crops, particularly of many grass species.

Introduction

The challenge facing scientists in the early 1980' s was of introducing cloned genes into plants to study their function, and to confer new and useful traits. By 1982, this had been achieved for bacteria (Avery et aI., 1944, Cosloy and Oishi, 1973, Klebe et aI., 1983), and animal cells (Colbere-Garapin et aI., 1981). In plants, Agrobacterium tumefaciens had been shown to transfer DN A into cells of a limited number of species. By 1983 it was successfully used to transfer T-DNA genes into plant tissue cultures (Fraley et aI., 1983) and into regenerating protoplast cultures of tobacco and petunia (Marton et aI., 1979). However, the method had limitations because of its dependence on a unique biological association. Many species, particularly the important gramineous crops, were not known host plants for Agrobacterium (de Cleene and de Ley, 1979). The method by which Agrobacterium transferred the DNA, and its integration into the genome, were a black box. In addition, it was not known whether Agrobacterium-derived elements were required for integration into the genome. Even today there are steps in the process which are poorly understood. At the time, there was also discussion over whether gramineous plants were inherently incapable of transformation (Potrykus, 1990).

The advent of methods to introduce DNA directly into plant protoplasts

I.K. Vasil (ed.), Molecular Improvement a/Cereal Crops, 9-20

© 1999 Kluwer Academic Publishers.

10 Ray Shillito

via polyethylene glycol (or other polycation) treatment and electroporation - methods not dependent on Agrobacterium - gave new optimism to those trying to transform monocots. They were quickly adapted and used to transform cell cultures of Lolium, Triticum and Zea mays (Potrykus et aI., 1985, Lorz et al., 1985, Fromm et al., 1986), and eventually to introduce genes into protoplasts capable of forming fertile plants. With the advent of biolistic transformation techniques, the use of polyethylene glycol (PEG) and electroporation for stable transformation has declined, but it still remains an important tool in studying transient gene expression, and a method of choice for rice and many grass species.

Mechanism

Polyethylene Glycol (and polycation)-Mediated Direct Gene Transfer

Transfer of DNA across membranes by treatment with polycations such as PEG is not a fully understood process. The method is similar to fusion of protoplasts and involves the incubation of DNA (circular or linear) with protoplasts, addition of a polycation together with a divalent ion such as calcium or magnesium, and the subsequent dilution of the solution with a buffer which also contains divalent ions. Protoplasts are unstable in the absence of divalent cations, and the choice of calcium or magnesium is governed by the goal of the experiment. In general, calcium promotes better transient expression, whereas magnesium gives better transformation rates (Shillito, unpublished, Negrutiu et al., 1987). The conformation of DNA is strongly affected by PEG (Salianov et al., 1978, Matsuzawa and Yoshikawa, 1993), and particularly by PEG of a molecular weight above 600 which is normally used for direct gene transfer. Thus it is generally believed that the polycation compacts the DNA and also allows it to associate with the membrane, due to neutralisation of charges on the DNA and the membrane. The polycation solution has a high osmotic pressure, and withdraws water from the protoplast, as well as promoting adhesion of protoplasts to each other. It is supposed that DNA uptake takes place during the dilution phase, but the mechanism is unknown. Also unclear is how the DNA makes its way to the nucleus (or chloroplast) through the cell. Loyter et al., (1982) studied the behaviour of DNA/calcium co-precipitates in animal cells. However, co­precipitates are not generally used in plant cells; there are few comparable studies (Gisel et al., 1996) and the mechanism may be different.

Electroporation-Mediated Direct Gene Transfer

Electroporation is more clearly understood than polycation-mediated DNA uptake. A polycation such as PEG may be used to potentiate uptake and/or

Electroporation and Polyethylene Glycol Treatment 11

integration (Shillito et al., 1985), but is not required. The uptake of DNA into the protoplast (or cell) is induced by pore fonnation in the cell membrane. A voltage applied across the membrane promotes and/or increases the stability of micropores which continually fonn in a resting membrane. The likelyhood of a pore opening is related to the voltage and the time for which it is applied. It is supposed that the open pore allows DNA to cross the membrane, and the DNA then finds its way to the nucleus. There have been some suggestions that the DNA may be induced to cross the membrane by the electric field inherent in electroporation. Pores are inherently unstable, and are kept open by the presence of the electric field. They must close in order for the protoplasts to remain viable. During electroporation animal cells must be incubated on ice for a period of time in order to allow the pores to close (Neumann et al., 1982), and in spite of extensive research (Chang et al., 1992) the mechanisms remain unknown (Maccarrone et al., 1995). The mechanism has not been investigated closely in plant cells but pore closing appears to be a rapid process (Shillito, unpub­lished). This may be due to the less structural nature of plant cell membranes.

DNA Integration

The way in which DNA enters the nucleus and is expressed and/or inte­grated is also not fully understood. It is clear that DNA may be acted on by recombination mechanisms before integration (Bates et al., 1990), leading to complex inserts (Takano et al., 1997). Recombination before integration was first suggested by the fact that cotranfonnation led to integration of a non-selected gene together with the gene of interest (Schocher et al., 1986). Furthennore, DNA introduced by either of these methods is integrated into the genome at one locus about 75% of the time, although many copies may be integrated at each locus. Experiments showing reconstitution of genes from fragments confinned that recombination occurs at a high rate, both when double stranded (Baur et al., 1990) and single stranded DNA (Bilang et al., 1992) are introduced.

Development of PEG-mediated Transformation and Electroporation

The further development of transfonnation methods depended on a number of factors. These were effective selectable markers, totipotent tissue cul­tures, and a method that did not require Agrobacterium. Before the first successful demonstration of non-Agrobacterium-mediated protoplast transfonnation, there were a large number of publications that attempted to show that DNA was taken up and/or incorporated into protoplasts. However, it was, and still is, very difficult to prove physical uptake of DNA into protoplasts via PEG treatment. It was known as early as the late 1970's

12 Ray Shillito

that DNA could adsorb to cell components, and particularly to the outer membrane of protoplasts and to nuclear membranes (Ohyama et aI., 1977, Lurquin et al., 1979). Plasmid DNA can remain associated with carrot protoplasts after PEG treatment and subsequent DNAase treament. When the protoplasts are lysed, this DNA can become bound to the nucleus, giving rise to misleading results. The large number of equivocal reports raised the barrier for proof of stable transformation. The lack of suitable markers for transformation was also a block to progress, as early workers had to rely on showing DNA uptake and the gene constructs they used were not optimised for expression in plant cells.

Thus hormone independence conferred by the Ti plasmid of Agrobacterium tumefaciens was the first marker that was convincingly used as a selectable marker in plant cells. This allowed DNA uptake into proto­plasts to be demonstrated by Davey et al., (1980) and Krens et al., (1982) using Ti plasmids that were taken up and gave rise to tumorous cells. The advent of 'disarmed' Ti plasmids and binary vectors (Hoekema et al., 1983, Bevan, 1984) simplified the use of the method and the advent of good markers such as neomycin phosphotransferase (NPT; De Block et al., 1984) allowed the tumor genes to be discarded as markers. However, the only real test of whether Agrobacterium-derived elements were required for transformation was to use DNA that did not contain the critical vir genes and border elements of T-DNA.

The first demonstration of non-Agrobacterium-mediated transformation of tobacco protoplasts, with a simple E.coli plasmid, was published by Paszkowski. et al., (1984). The proofs for transformation were not only molecular (Southems, border fragments), but also segregation in micro­spores and transmission of a phenotype to progeny, linked to the segregation of the same integrated DNA fragment. Once published, this method, which came to be known as 'direct gene transfer', was used by a number of laboratories and spurred a renewed interest in cereal protoplast culture.

Shortly afterwards, the stable transformation of plant protoplasts via electroporation was also demonstrated (Shillito et al., 1985). Around the same time, electroporation was used to show transient expression of genes in maize protoplasts (Howard et al., 1985, Fromm et al., 1985). This was followed by stable transformation of non-regenerable protoplasts of Lolium multiflorum and Triticum monococcum using PEG (Potrykus et aI., 1985, Lorz et aI., 1985), and non-regenerable protoplasts of maize by electro­poration (Fromm et al., 1986).

Studies of transient expression tend to be dominated by electroporation with a low voltage pulse, rather than by PEG transformation. Low voltage electroporation has been used in a very large number of species to optimize conditions in order to carry out stable transformation, but most extensively to explore promoter function (e.g. Yang, 1985, Hauptmann et al., 1987, Junker et al., 1987). Great care must be used in using transient results to

Electroporation and Polyethylene Glycol Treatment 13

optimize a system for stable transformation. The best conditions for transient expression are not necessarily those that give the best yield of stable transformants.

Improvement of the Techniques

Most stable protoplast transformation has been carried out using PEG, prob­ably because of the simplicity of the technique. Advances, such as the use of magnesium in the medium (Negrutiu et aI., 1987), have increased efficiencies in some systems to the 1% range, close to that which can be achieved using a combination of electroporation and PEG (Shillito et al., 1985). There have been few reports of optimisation of protocols for PEG­mediated transformation (Maas and Werr, 1989, Armstrong et aI., 1990). An efficiency of 1-3% of regenerable colonies can often be achieved, a level at which colonies can be screened for the gene of interest, and the selectnl"e marker can be eliminated from the process. Typically 80 x 106 protopl~"lS can be used per experiment leading to 105 to 106 tranformants. These numbers are not approachable via other methods.

Over the years, it has been suggested that the transfer of DNA into protoplasts was due to impurities in the PEG (better results are obtained with particular sources or batches of PEG), or is potentiated by use of other polycationic agents. In addition, other treatments such as radiation (Kohler et al., 1989), and synchronisation of cultures have been suggested. However, the method remains little changed from that first used -application of between 8 and 20% PEG of a molecular weight of 4000-8000 to protoplasts, followed by slow dilution of the PEG with a buffer containing divalent cations (Johnson et al., 1989). The main criterion for choosing the concentration of PEG is simply to use the amount that will not kill too many (more than 50%) of the protoplasts (Vasil et al., 1988).

An important development is the use of PEG transformation to introduce DNA into the chloroplast genome. This technique was pioneered by Maliga and co-workers (Carrer et al., 1993, Bock et aI., 1994) and shows great promise for the engineering of crops which outcross into wild populations. However, it is inherently difficult and inefficient, and requires transforma­tion efficiencies of green tissue that are not yet available with gramineous species.

In the late 1980's there were two main schools of electroporation - those that used a short, high voltage pulse (e.g. Shillito et al., 1985, Schocher et al., 1986, Riggs and Bates, 1986) and those using a long, lower voltage pulse (Fromm et aI., 1986, Guerche et aI., 1987). This second approach also led to the use of square-wave pulses (Lindsey and Jones, 1987). In general, the longer pulse seems to lead to better transient expression, but the data is not clear on which approach gives the best stable transformation. The

14 Ray Shillito

highest transformation efficiencies (% of surviving colonies which give rise to a selectable transformed colony) have been achieved using high voltage electroporation in the presence of PEG (Shillito et aI., 1985) although simple electroporation with a long pulse can lead to comparable efficiencies in some cases (Guerche et aI., 1987).

There have been a number of studies that compared the penetration of DNA and or dyes due to pulsing with different machines and voltages (Rathus and Birch, 1992, Penmetsa and Ha, 1994, Bates, 1994). However, there is little or no clear direction to be gained from the literature. Most protocols tend to be developed by trial and error. The best approach is to tailor the voltage of the applied pulse to the protoplast diameter. For high voltage electroporation, the voltage required is inversely proportional to the diameter of the protoplast. For example, a tobacco protoplast with an average diameter of 50 pm will transform optimally at about 1500 V/cm, with a t~ (exponential decay constant: time for the voltage to decay to one half of its original level) of 10 j.ls, whereas a maize or similar protoplast derived from an embryogenic cell culture typically has a diameter of 25 j.lm

and therefore will require 3000 V /cm for the same pulse length. Pulse length is controlled by adjusting the resistance of the medium, and/or changing the size of capacitor discharged across the cell containing the protoplasts. For low voltage (- 400 V) systems, the parameters do not appear to be as clearly defined, and most protocol development requires a review of the literature on similar protoplast systems and some optimisation experiments to tune the system.

Electroporation has only rarely been applied to regenerable cereals, but grasses such as Dactylis (Hom et al., 1988) and Agrostis (Lee et al., 1996) have been transformed, as have rice (Chamberlain et al., 1994, Xu and Li, 1994, Rao et al., 1995) and barley (Salmenkallio et al., 1995). A novel approach, that of tissue electroporation, described by D'Halluin et aI., (1992) has seen limited use, as has electroporation of partially digested tissues (Yang et al., 1993).

Application to Cereals

At the time when protoplast transformation was being developed, all the monocot protoplast systems available were non-regenerable, and this remained a major hurdle on the road to transformed maize and other grami­neous plants (Vasil and Vasil, 1984). Rice was the first to be regenerated successfully from protoplasts (Fujimura et al., 1985), as was expected due to its excellent tissue culture characteristics. However, others remained recalcitrant. The first non-rice plants regenerated from protoplasts were those of Dactylis glomerata (Hom et a1.1988) and other grasses (Dalton, 1988), wheat (Vasil et al., 1990), and maize (Rhodes et aI., 1988). In 1989

Electroporation and Polyethylene Glycol Treatment 15

the first fertile maize plants (Prioli and Sondahl, 1989, Shillito et al., 1989) were reported. Regeneration of fertile plants from these recalcitrant species opened them up to transformation by PEG and electroporation. Eventually, protoplast cultures were used to produce fertile transgenic plants from Japonica, Javanica (Toriyama et al., 1988, Shimamoto et aI., 1989, Li et al., 1992) and Indica (Datta et al., 1990) rice and maize (Golovkin et aI., 1993), as well as grasses such as fescue (Wang et aI., 1992).

Conclusions

Over the years, many different approaches have been tried to introduce DNA into plants. Zhou et aI., (1988) claimed to have transformed cotton via injection into ovules, and De La Pena et aI., (1987) described transformation of rye plants by injection into immature inflorescences. However, transmis­sion to progeny was not demonstrated, and these methods have yet to be confirmed by independent laboratories. Sonication is another technique that has been used to introduce DNA into protoplasts (Joersbo and Brunstedt, 1990), but this has found little application.

In 1995, Jahne et aI., stated that 'to date only three methods have been found to be suitable for obtaining transgenic cereals: transformation of toti­potent protoplasts, particle bombardment of regenerable tissues and, more recently, tissue electroporation.' With the exception of Agrobacterium­mediated transformation of embryos and embryogenic callus (see chapter by Komari and Kubo, this volume), this statement remains essentially true today. The advent of direct gene transfer and electroporation gave new hope to those who wished to engineer cereals. This was a method which was independent of Agrobacterium, and it was quickly applied to cereal cultures. If an efficient totipotent protoplast culture could be produced, it promised an easy route to transformed crops (e.g., rice; Shimamoto et aI., 1989, and maize, Morocz et al., 1990). While totipotent cell culture systems were developed fairly early for a number of dicotyledons, the monocots, and particularly the cereals, proved difficult to bring into culture and then retrieve as fertile plants (Vasil and Vasil, 1992). A concerted effort was therefore made by a number of groups to develop embryogenic suspension cultures which were, and to-date remain, the only source of totipotent grass protoplasts. Such cultures provided the first transgenic cereals, and have been most useful in rice and several forage grass species (Vasil, 1994).

Stable transformation via electroporation and PEG-mediated direct gene transfer has now been eclipsed in the most part by other means of transforming cereals, particularly the biolistics procedure (see Chapter 3). However, direct gene transfer to protoplasts has remained popular for trans­forming some grasses, and rice. The method remains particularly useful where large numbers of transformants are needed, where a particularly

16 Ray Shillito

regenerable protoplast culture is available (e.g., rice) or where intellectual property issues dictate the approaches available. The method can also be used for screening gene banks where a selectable phenotype expressed in culture is available. An example would be the direct transfer of a herbicide or antibiotic resistance (Gallois et al., 1992). In addition, protoplast transfor­mation, and particularly electroporation, can be used to deliver proteins and protein complexes to plant cells (Ashraf et al., 1993). The use of these techniques will remain vital in dissecting processes such as Agrobacterium­mediated transformation (Hansen et al., 1997), and the use of recombinases (Albert et al., 1995, Lyznik et al., 1996) and other approaches to control integration of genes into the genome.

While not the predominant method for transforming cereal crops, transformation of protoplasts via PEG and electroporation still remains an important weapon in our scientific armory.

Acknowledgements

Special thanks to Martha Wright (Novartis Seeds) for her help and encour­agement witht his article. Thanks are also due my many colleagues in tissue culture and transformation research with whom I have worked or discussed these topics over the years.

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