WO2000062601A1 - Agrobacterium-mediated genetic transformation of multicellular marine algae, resultant strains and their products - Google Patents

Abstract

Methods are provided for transforming multicellular marine algae utilizing, e.g., Agrobacterium tumefaciens as a gene delivery system. In particular, methods are described for wounding multicellular marine algae and, by incorporating an inoculation method that minimizes the exposure of the algae to a non-salt water medium, inoculating the same with Agrobacterium tumefaciens carrying one or more genes for introduction to the recipient algal cell. The methods may be used to transform multicellular marine algae for the purpose of producing new products, modifying existing traits or introducing new traits.

Description

TITLE OF THE INVENTION

Agrobacterium-mediated Genetic Transformation of Multicellular Marine Algae, Resultant Strains and their

Products

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 60/129,490, filed April 15, 1999, the whole of which is hereby incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

Part of the work leading to this invention was carried out with United States Government support provided under a grant from the National Science Foundation, Grant No. MCB-9987302. Therefore, the U.S.

Government has certain rights in this invention.

BACKGROUND OF THE INVENTION Marine algae are a large and diverse group of photosynthetic organisms that live in ocean and brackish waters. They are distinct from land plants in several ways, including phylogenetically, biochemically and morphologically. They are morphologically diverse, ranging in size from tiny microscopic, unicellular forms less than 50 μm in length to large macroscopic, multicellular forms (commonly called seaweeds) over ten meters in length. They are generally of simple construction with little cell differentiation and no true vascular tissue. Furthermore, they are biochemically diverse, and include organisms with a broad array of pigment compositions, cell wall compositions and biosynthetic pathways, many of which are not found in land plants. Marine algae are also diverse in their forms of reproduction, which are very different from those of land plants. Marine algae lack flowering structures that characterize land plants. Furthermore, all marine algae, even those that grow attached to the shoreline, produce planktonic unicellular stages at some point of their life cycle. Evolutionarily, marine algae are more ancient than land plants and have evolved to become the largest group of photosynthetic eukaryotic organisms in our oceans and embayments. As defined here, marine algae include non- angiosperm, photosynthetic, eukaryotic organisms that live in ocean or saline water. Marine algae have considerable commercial value

(e.g., Hannisak, 1998; Radmer, 1996). Probably the best known commercially valuable marine algae are the multicellular macroalgae, or seaweeds. Seaweeds have been traditionally exploited for two principal commercial uses: 1) they are eaten as food items, and 2) they are sources of polysaccharide extractives called phycocolloids . Phycocolloids are used in the food industry as gelling, thickening and emulsifying agents and include carrageenans, agars and alginates. Examples of seaweeds that are used as food include the red alga Porphyra , commonly called nori and eaten as sushi, which has a worldwide production valued at over $1.5 billion dollars annually, and the brown alga Laminaria , which is eaten as kombu and has a worldwide production valued at over $2.8 billion dollars annually. The phycocolloid industry, based upon several species of red and brown algae, is valued at over $580 million dollars annually.

Also of commercial value are a variety of agrichemicals and specialty compounds, such as fatty acids and pigments (e.g., beta carotene, phycoerythrin) , which come from a variety of microscopic and macroscopic marine algae.

Marine algae are becoming increasingly attractive as a potential source of new products as the search for commercially valuable new compounds extends into the seas. Marine algae could, for example, serve as a source of valuable pharmaceutical proteins and medicinal compounds through a technology referred to as "bioengineering," or the manipulation of an organism so that it produces a product not ordinarily produced.

This technology is currently being applied to land plants. In fact, because of the high protein content of some marine algae, their biosynthetic characteristics and the ease and low cost of growing them for biomass, marine algae may provide an advantage over some land plant systems. However, new technologies such as bioengineering require the ability to introduce foreign genes into, or to genetically engineer or transform, the organism of choice. Therefore, the development of new commercial applications for marine algae would benefit greatly from the ability to apply genetic transformation technology to these organisms and then produce new strains with novel traits.

Marine algal strain improvement efforts have traditionally relied upon classical plant breeding techniques, with the single most commonly used method being strain selection of wild individuals or of genetic variants produced by mutagenesis. Sexual hybridization has not had nearly the impact in marine algal genetic improvement as in land plant improvement because it has been much more difficult to perform realiably. Similarly, protoplast fusion to produce somatic hybrids has had little impact to date on marine algal genetic improvement because of difficulties in producing viable protoplasts capable of regeneration. Recently, a new method of protoplast fusion was developed for certain seaweeds, but even this method requires reproductive structures or spores (Cheney et al., 1995) . In addition, protoplast fusion is limited in what it can accomplish. Protoplast fusion cannot, for example, produce a new strain with a completely new trait obtained from an unrelated organism. This can be accomplished only through genetic transformation. Thus, using current genetic modification/improvement methods, there is very limited capacity to produce new strains of marine algae with novel traits.

Over the past decade, genetic transformation has become the principal strain improvement method for land plants. Today, genetic transformation is being used to replace traditional plant breeding because of the latter' s slower rate and more limited scope in what can be accomplished. Genetic transformation has become commonplace in land plants and has been reported in over 120 species in 35 families including nearly every agriculturally-important crop species (see, e.g., Dale, 1995) . The annual market value of genetically transformed (or transgenic) agricultural crops is estimated to be $500 million today and is expected to increase to over $7 billion by 2005 (Vasil, 1998) . Examples of what has been accomplished through genetic transformation in agricultural crop species include: delayed-softening and delayed-ripening tomatoes; corn and soybeans with improved (e.g., healthier) amino acid contents; potatoes with increased starch content; and corn, cotton and soybean species carrying genes conferring resistance to viral, fungal and insect pathogens to reduce fungicide and insecticide use (see, e.g. , Christou, 1996) .

A crucial step in developing a genetic transformation system for any new group of organisms is to have an efficient method of gene transfer, that is, a method for introducing foreign genes into the target organism that is both reliable and reproducible. The three most widely used methods for gene transfer in land plants consist of: 1) Agrobacterium-mediated gene transfer; 2) biolistics or microparticle bombardment; and 3) electroporation. Λgrojacteriurn-mediated gene transfer was the first method successfully used for transforming land plants. It is commonly used for genetic transformation in dicotyledonous agricultural crops, in particular, in species of the Solanaceae such as tobacco, potato and tomato. It has been less successfully applied to monocotyledonous species. The Agrobacterium species are land plant pathogenic soil bacteria that infect wounded plants and transfer genes to the host cells. Two species of Agrobacterium have been used for gene transfer: A. tumefaciens, which causes tumorous growths, or crown galls, by infecting leaf or shoot tissue, and A. rhizogenes, which causes hairy root disease by infecting root tissue. In the case of A. tumefaciens, genetic transformation is accomplished by the transfer of a small piece of DNA, called T-DNA, from the bacterium into the plant cell, where it becomes integrated into the nuclear genome. The T-DNA region and other genes involved in gene transfer and virulence (the vir-region) are located on a plasmid, called the tumor inducing (Ti) plasmid in Agrobacterium tumefaciens . Transformation of land plant cells by infection with A . tumefa ciens and subsequent transfer of the T-DNA into the host cell have been well documented (e.g., Hooykass et al., 1992). Biolistics or microparticle bombardment is the other most commonly used method of gene transfer in land plants today (see review by Christou, 1996; Birch, 1997). It is especially widely used in those species where Agrojacterium-mediated gene transfer has been shown to be ineffective. The method involves coating the gene (or DNA) to be introduced onto tungsten or gold microparticles one μm or less in diameter and "shooting" these particles at host cells or tissue at a great rate of speed (Sanford et al., 1990) . The microparticles are able to penetrate cell walls and deliver genes into the cytoplasm of cells, where they can eventually become integrated into the host cell genome.

Electroporation (see Christou, 1996) , less commonly used today than the other two methods for transforming land plants, requires the use of protoplasts, which are subjected to short pulses of electric current to cause the per eabilization of the cell plasma membrane. The electrical breakdown of cell membranes allows the sequestering of substances (such as foreign DNA) through the membrane and into the cell. In contrast to its broad success and applications in land plants, genetic transformation is not an established and broadly applicable technology in marine algae. Stable, inheritable genetic transformation of a foreign gene has been reported in only a very small number of marine algae, all of which are microscopic, unicellular forms. There is no evidence that inheritable genetic transformation has ever been successfully carried out in the larger, macroscopic, multicellular forms of marine algae (eg., Stevens et al., 1997 and Minocha, 1999) . To date, genetic transformation has been reported in six species of unicellular marine algae, all of which used either microparticle bombardment or electroporation. This includes: three diatom species - Cycol tella cryptica , Navicula saprophila , and Phaeodactylum tricornutum (Apt et al., 1996; Dunahay et al., 1995); two dinoflagellate species - Amphidinium sp . and Symbiodinium microadriaticum (Lohuis and Miller, 1998); and one green algal species, the high salinity tolerant Dunaliella salina (Porath et al., 1997). In the case of two dinoflagellate species (Lohuis and Miller, 1998), the expression of the foreign gene apparently was not stable, but rather decreased over time. There are also reports of stable genetic transformation in several unicellular fresh water algae; in particular for the fresh water unicellular green alga Chlamydomonas reinhardtii (reviewed in Stevens and Purton, 1997) and the fresh water colonial green alga Volvox carteri (see, e.g., Schiedlmeier et al., 1994). Finally, there is a brief report of transient gene expression in an additional diatom species, Skeletonema costa tum, following electroporation (Smith and Alberte, 1995) .

There is no evidence that inheritable genetic transformation has been successfully achieved with macroscopic, multicellular marine algae. There are three reports of transient (or unstable) gene expression for such algae (reviewed in Stevens and Purton, 1997 and Minocha, 1999) . Transient expression of a foreign gene in multicellular algae has been accomplished, using microparticle bombardment or electroporation, in two species of red macroalgae, Kappaphycus alvarezii (previously called Eucheuma cottonii) (Kurtzman and Cheney, 1991) and Porphyra inia ta (Kubler et al., 1994), and the green macroalga Ulva lactuca (Huang et al . , 1996). These three studies reported the expression of the beta-glucuronidase or GUS reporter gene in transformed cells but were unable to demonstrate stable incorporation and expression of the foreign gene into the genome of the algae tested and the subsequent production of transformed progeny from such cells. In addition, Qin et al. (1998, 1999) have reported gene transfer by microparticle bombardment and the expression of the lacZ gene in sporophytes of the brown macroalga Laminaria japonica regenerated parthenogenetically from female gametophytes . However, the regenerated sporophytes are haploid and cannot reproduce. Thus, there is a need for an efficient method of inheritable gene transfer that can be applied to a broad range of multicellular marine algal species.

BRIEF SUMMARY OF THE INVENTION In one aspect, the invention is directed to a method for introducing foreign genes into or causing genetic transformation of multicellular marine algae. The method of the invention comprises, as a first step, wounding an alga in a manner that is sufficient to penetrate at least the cuticle, or outer cell wall layer in order to facilitate access of T-DNA from an Agroba cterium species, e.g., A . tumefa ciens . In the second step, cells from at least one transformation- competent Agrobacterium species are applied to the wounded algal tissue to transform the marine alga, preferably in a manner that minimizes the exposure of the alga to be transformed to a non-salt water environment, e.g., by spraying or swabbing the surface of the alga with the bacterial suspension. The chosen transformation-competent Agrobacterium species contains at least one plasmid to effect transformation of the alga. Furthermore, the plasmid preferably is genetically engineered such that it can cause insertion of a commercially desirable gene into the DNA of the transformed algae and expression of the inserted gene. The plasmid preferably also contains a promoter gene and at least one second gene selected from the group consisting of genes which code for at least one screenable marker, genes which code for selection agent resistance, or combinations thereof.

In another aspect, the invention is directed to a stable transgenic multicellular marine alga comprising and expressing a DNA sequence coding for a gene foreign to said alga. Preferably, the alga is a species from the genus Porphyra , Chondrus or Laminaria . The invention is also directed to a transgenic alga comprising and expressing a DNA sequence coding for an antigen of a pathogenic microorganism or an antigenic determinant thereof, said antigen or antigenic determinant thereof eliciting a secretory immune response in a human or other animal upon oral administration of cellular material from said alga. The expressed protein (antigen or antigenic determinant thereof) can be in the form of a fusion protein.

Thus, the invention is directed to the use of the first technology for genetically transforming macroscopic marine algae or seaweeds. This new technology now permits foreign genes to be introduced into and expressed in a seaweed and, thereby, provides the means by which seaweeds can be utilized for completely new purposes, namely as production systems for therapeutic proteins. With this new technology, transgenic seaweeds, such as the widely-eaten and broadly-cultivated red seaweed Porphyra, can be inexpensively cultivated in the ocean or on land to provide an attractive alternative to mammalian cell culture for the production of biopharmaceutical proteins, including antibodies and vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims, taken in conjunction with the accompanying drawings, in which: Fig. 1 is a diagram showing steps in the method of the invention, wherein:

Fig. la shows spreading of Porphyra blades on top of a sterile filter paper disk on a solidified agar plate, just prior to the wounding step in the method of the invention;

Fig. lb shows the use of a biolistic instrument to wound the Porphyra blades;

Fig. lc shows inoculation of wounded Porphyra blades with Agrobacterium bacteria using a fine mist sprayer;

Fig. Id shows turning over the filter paper disk so that the wounded surface of the Porphyra blades come into direct contact with the agar plate; Fig. le shows removal of the Agrobacterium bacteria from the Porphyra blades following co-culture by repeated washing steps; and

Fig. If shows culture of the transformed Porphyra blades in an aerated flask.

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention extends the potential of Agrojbacterium-mediated gene transfer to the marine environment, and specifically to multicellular marine algae, by incorporating a wounding and an inoculation method that minimizes the exposure of the algae to a non-salt water medium. Preferably, the wounding method comprises microparticle bombardment or biolistics; however, any method of wounding that is sufficient to penetrate at least the cuticle algal layer or outer cell wall layer may be used in the method of the invention.

The preferred inoculation step is to spray wounded algal tissue or cells with a fine mist containing the designated Agrobacterium culture. The inoculated algae are then co-cultured with the Agrobacterium cells on a medium that is also hospitable, e.g., solid agar containing 50% seawater, to ensure survival of the transformed algal cells.

The red alga Porphyra was selected as a model system because of its commercial value and the wealth of knowledge available about its field and laboratory culture and its ability to produce large numbers of regenerative cells. Porphyra is a member of the Rhodophyta, or red algae, and is not related to land plants. It grows as a one or two cell thick blade or sheet. The species described here is one cell thick. Porphyra , or nori as it is commonly called, is one of the two most widely cultivated and commercially valuable macroalgae or seaweed in the world and would benefit greatly from the application of genetic engineering technology.

In preliminary transformation experiments with Porphyra , microparticle bombardment or biolistics was used alone to introduce the reporter gene GUS into Porphyra blades. This method failed to produce reliable evidence of even transient gene expression after four separate attempts. The Agrojacterium-mediated gene transfer method described herein, however, comprising both wounding and inoculation steps, was successful in producing stable genetic transformants of Porphyra . The preferred method of the invention consists of the following steps:

Step la. Culturing of a transformation-competent Agrobacterium species Choice and culture of a suitable Agrobacterium strain is crucial. The strain should be virulent and contain a plasmid with a promoter gene and a reporter or selectable marker gene plus the gene of interest. Appropriate strains are from terrestrial Agrobacterium species, such as A. tumefaciens or A. rhizogenes, or from plasmid-containing marine representatives of the genus Agrobacterium .

The bacteria need to be grown in liquid prior to inoculation. The medium of preference and methods of culture are well established for Agrobacterium culture.

In the method of the invention, the bacterial cultures were subcultured for two or more days, such that they were maintained at or near exponential growth phase prior to inoculation. A typical OD₆₂o (or OD₆6o) determination value before dilution was around 1.0 1.8. The bacteria were kept on a shaker until use to prevent clumping. Just before use, the bacteria were spun down and concentrated by centrifugation and then diluted such that the final bacterial concentration had an ODδ₂o of between 0.6-1.0.

Step lb. Preparation of algal samples

Prior to the transformation experiment, the algal sample, e.g. the Porphyra blades, are maintained in a growth chamber at a light intensity, temperature, and growth medium suitable for optimal growth. Just prior to wounding by biolistics, the samples are spread out on top of a sterile filter paper disk that lies on top of a plate of solidified agar (Fig. la) . A typical agar plate composition is 0.6% phytoagar in 50% seawater with LB medium, plus e.g., kanamycin when appropriate, i.e., when the Agrobacterium bacterium contains a kanamycin resisitance gene.

Step 2. Wounding of host tissue The preferred method of wounding in the present invention utilizes biolistics or microparticle bombardment. This is not the usual method of wounding in Agrobacterium transformation in land plants. In this procedure, "naked" or non-DNA coated tungsten particles are shot by a helium-powered microparticle bombardment instrument, e.g., made by BioRad Corp., to accomplish wounding of the Porphyra blades (see Fig. lb) . Microparticle bombardment was selected because it allows for thousands of extremely small particles (approximately 1 μm in diameter or less) to be shot at and wound the host tissue simultaneously and in a manner in which the particle projectile force can be varied in a reproducible manner. It appears that these particles penetrate the cuticle layer of the blades and even the walls of the cells without causing much cell death. The specific steps involved in using the microparticle bombardment device are well described (Sanford et al . , 1990) . The agar plates containing Porphyra blades were positioned in the biolistic chamber at an appropriate distance from the nozzle according to the settings of the instrument, e.g., at approximately 8 or 11 cm from the nozzle.

Step 3. Treatment of bacteria and/or host tissue with virulence-inducing compound Prior to bacterial inoculation of the host tissue, the Agrobacterium culture can be treated with the virulence-inducing compound acetosyringone . The host tissue can also be treated with acetosyringone as well. The bacteria were treated with acetosyringone at a concentration of 100 μM, typically for 0.75-2 hrs prior to inoculation.

Step 4. Inoculation of host tissue with Agrobacterium bacteria In order to avoid as much contact as possible of the Agrojbacterium bacteria with seawater or the Porphyra blades with fresh water, the preferred method of inoculating the host tissue is to spray a suspension of the bacteria onto the tissue to be inoculated as a fine mist (see Fig. lc) . The sprayer can be attached to a source of pressurized air so that the bacteria are sprayed at the Porphyra blades under an adjustable amount of force. The spray method described here allows for control of the application pressure so as to cause little if any damage to the Porphyra blades. In general, the wounding and inoculation procedures are selected so as not to interfere with the growth habits or reproductive capacity of the algae.

Step 5. Co-culture of Agrobacteri um bacteria on host tissue

After the host tissue has been inoculated with Agrobacterium bacteria, the two are co-cultured for a period of time to permit bacterial infection and DNA transfer to occur (see Fig. Id). The length of time for co-culture can vary but is typically 2-3 days. During this time, the Porphyra blades were maintained in a growth chamber at low light and at a temperature suitable for Porphyra growth and for Agroba cteri um activity, for example, at a temperature of 21-22°C.

Step 6. Removal of Agrobacterium bacteria and selection of transformants

At the end of the co-culture phase, the Agrobacterium bacteria are removed from the surface of the blades by repeated washing of the blades in seawater in a beaker until the seawater is clear and there is no cloudiness left caused by the bacteria (see Fig. le) . This typically takes 4-5 transfers (100 ml each) per plate of blades. Following such washing, blades were transferred to 50 ml centrifuge tubes filled with seawater and gently shaken back and forth to further remove bacteria from the surface of the blades. This procedure was again repeated several times until the seawater appeared clear. After the bacteria are removed from the surface of the blades by the washing step, the Porphyra blades are placed into culture flasks and grown under optimal conditions, in a culture medium in which other antibiotics are added to kill any remaining Agrobacterium bacteria (see Fig. If). Blades are cultured thus for a short period of time to allow for recovery, after which they are either transferred to a selection medium containing antibiotics that permit selection for transformed (i.e., selectable-marker gene containing) cells or are examined for the presence of the cells containing the reporter gene. The transformed cells can then, by asexual reproduction, be made to produce monospores which grow into stable transformed blade progeny (Tl generation) . These blades also can pass on the foreign gene to their progeny (the T2 generation) in a similar fashion.

The following examples are presented to illustrate the advantages of the present invention and to assist one of ordinary skill in making and using the same. These examples are not intended in any way otherwise to limit the scope of the disclosure.

EXAMPLE I

Transformation of Porphyra yezoensis, or nori, using Agrobacterium tumefaciens for gene delivery

This example describes the transformation of Porphyra yezoensis, or nori , using Agrobacterium tumefaciens for gene delivery. The foreign genes for neomycin phosphotransferase-II (NPT-II) and beta-glucuronidase (GUS) were expressed in Porphyra .

Young blades of Porphyra yezoensis belonging to existing strain U-51 and new strain #9-13 were grown from monospore cultures until they reached a size of approximately 1-3 cm long. These small blades were cultured in an enriched seawater medium in an aerated flask. The blades were growing at a high rate and had a high rate of cell division at the time of wounding. Prior to wounding, approximately 10-20 Porphyra blades were spread out onto a sterile filter disc which was placed on top of an agar plate consisting of LB medium plus kanamycin (at 50 mg/1) made up using 50% seawater and solidified with 0.6% phytoagar.

The Porphyra blades were wounded by "shooting" them with naked (i.e., non-coated) tungsten microparticles shot from a BioRad Corp. helium-powered biolistic instrument. The tunsgten particles were prepared following the manufacturer's directions. Typically, two 450 psi rupture discs were used, giving a force of approximately 850-900 psi. The plate carrying the Porphyra blades was typically placed approximately 8 cm (or 11 cm) from the nozzle, although other distances were also shown to produce good results.

After the Porphyra blades were wounded, they were inoculated with the Agrobacterium tumefaciens bacteria described below. Both immediate inoculation after wounding and inoculation 1-2 days after wounding were tested and found to work, although the preferred method is immediate inoculation.

The Porphyra blades were inoculated with LBA4404 strains of Agrobacterium tumefaciens containing either the plasmid pBI121 (Jefferson et al., 1987) or the plasmid pBI141 (May et al., 1995). Plasmid pBI121 contains the GUS gene under the control of a CaMV 35s promoter and the NPT-II gene under the control of a nos promoter. The plasmid pBI141 contains the GUS gene under the control of a rice actin 1 (Act 1) promoter and the NPT-II gene under the control of a nos promoter. Prior to inoculation, the Agrobacterium strains were grown in liquid LB culture medium plus kanamycin sulfate (50 mg/1) on a shaker until there was approximately

10-20 ml of bacterial culture at an OD6₂o of around 1.8.

This volume was then centrifuged to concentrate the bacteria. Resuspension was in fresh LB media plus kanamycin to make up a final volume of around 20-30 ml in a small flask (at an OU6₂o of around 0.8) . This bacterial suspension was then treated with the virulence-inducing compound acetosyringone (at a concentration of 100 μM) approximately 0.75-2 hours prior to inoculation of the Porphyra blades. During treatment with acetosyringone, the bacterial culture was maintained on a shaker to prevent clumping.

The Porphyra blades were inoculated with the Agrobacterium strains described above by spraying a portion of the bacteria (approx. 2.5-5 ml per plate) onto the microparticle-wounded surface of the blades, using a glass paper-chromatography sprayer. This sprayer has the benefit that it can be easily attached by a hose to a source of pressurized air and the bacteria can be sprayed on to the blades as a very fine mist under adjustable pressure. In between inoculations, the sprayer can be washed and sterilized as necessary. During the bacterial inoculation process, the plates containing the Porphyra blades are set at an angle of around 60° to allow excess bacteria culture medium to collect at the bottom of the plates. This excess culture medium was collected by micropipet and removed from the plates as it appeared.

After the Porphyra blades were sprayed with the bacteria, there was an initial short period of co-culture for approximately 0.5-0.75 hrs in a growth chamber at a low light intensity and at a temperature suitable for both Porphyra growth and Agrobacterium activity, typically around 21-22°C. Next, the filter paper holding the blades was flipped over onto a new agar plate, so that the side of the blades that had been sprayed was now in contact with the surface of the new plate. The new agar plate also consisted of LB media plus kanamycin in 50% seawater and solidified with 0.6 % phytoagar. After the blade surfaces were inverted, the filter paper was carefully removed, making sure that the blades remained in contact with the surface of the agar. Co-culture then was continued on the new plates for 2-3 days .

At the end of the 2-3 day period of co-culture, the bacteria were removed by washing the Porphyra blades repeatedly in seawater. Washing was carried out by placing the blades from one plate into a beaker of 100 ml of seawater, swirling them around for several minutes, and then transferring them to a new beaker of seawater. The seawater appeared very cloudy. This procedure was repeated 4-5 times until the seawater appeared clear. Then the blades were transferred to 50 ml plastic centrifuge tubes filled with seawater and gently shaken back and forth for a couple of minutes. This washing step was repeated several times until the seawater again went from being cloudy to clear. The blades were then placed into gently-aerated or standing culture flasks and grown at the same temperature described above or lower, in a culture medium containing AgroJacterium-killing antibiotics . After several days of recovery, samples of blades from each plasmid treatment were assayed for the presence of the beta-glucuronidase (GUS) gene by staining them with X-Gluc stain. Both plasmid treatments produced blades with blue cells expressing the GUS gene. Control plants that were not innoculated with Agrobacterium organisms did not exhibit GUS expression.

Furthermore, in order to make certain that the expression of the GUS gene was not due to the Agrobacterium itself or to the presence of some other bacteria, a separate experiment was conducted as above.

This time, however, the strain of Agrobacterium used contained the plasmid p35s GUS INT (Vancanneyt et al.,

1990) . The plasmid p35s GUS INT contains a GUS gene with an intron inserted into it, preventing its expression by Agrobacterium or any other bacteria. The GUS gene is under the control of a CaMV 35s promoter, and the plasmid also contains a NPT-II gene under the control of a nos promoter. Porphyra blades treated with the p35s GUS INT plasmid also produced blue cells expressing the GUS gene.

After approximately 2-3 weeks in culture, the blades from all three plasmid treatments were transferred to new culture media in 6 well culture plates, one plant per well. Following another 2-4 weeks of incubation, blades from each treatment started to release monospores, which in turn grew into new blades.

Monospores are a product of vegetative reproduction and are produced by mitosis. Some blades produced by these monospores (i.e., the "Tl progeny" of the original transformants) were transferred to a selection medium containing the antibiotic geneticin at a concentration of 125 μg/ml. This concentration of geneticin had been shown in preliminary experiments to be toxic to very young non-transformed Porphyra blades in just 7-10 days. Blades that survive this selection treatment are putative transformants and were shown to possess blue cells indicating the presence of the GUS gene. Other blades produced by the released monospores of the original transformant were transferred to normal, i.e., non-selective, media, cultured and shown to produce progeny that were also capable of expressing the GUS gene. These Tl progeny in turn, in non-selective media, released monospores that produced new blades (i.e., T2 generation) , which were also capable of expressing the GUS gene.

Additional transformation experiments have been performed on Porphyra yezoensis using the same methods as described above but with a different Agrobacterium strain and different plasmid vectors. For example, one of the new vectors used had been developed for Agrobacterium-mediated gene transfer by the CAMBIA institute in Australia, pCAMBIA1304. This vector was carried by Agro-bacterium tumefaciens strain GV3101 and contained reporter genes for both GUS ( gush) and the green fluorescent protein ( gfp) under the control of a CaMV 35s promoter. Using the protocol described above, we have obtained transformants and progeny that express both the GUS and GFP genes. The advantage of using GFP as a reporter gene is that transformed cells can be identified and subsequently cultured without having to use a toxic stain as is neccassary with GUS identification.

Additionally, transformation experiments have been conducted in which the vector pCAMBIAl304 has been modified such that its CaMV 35s promoter is replaced with a homologous promoter. The new vector, pCPYl, contains an homologous promoter from Porphyra yezoensis isolated from the gene encoding the largest subunit of DNA-dependent RNA poly erase II, RPB1. (Dr. John Stiller, University of Washington, private communication) . Using the same transformation protocol described above, we have produced transformants with pCPYl that express both the GUS and GFP genes. Thus, we have observed expression of both the GUS and GFP genes using different Agrobacterium tumefaciens strains and different vectors carrying two different promoters.

EXAMPLE 2

Transformation of Chondrus crispus using Agrobacterium tumefaciensfor gene delivery

For the transformation of a Chondrus species, the steps given above in EXAMPLE 1 are repeated except that Porphyra yezoensis is substituted for by Chondrus crispus . Chondrus crispus, another macroscopic red alga, is taxonomically different from, and anatomically much more complex than, Porphyra yezoensis . Whereas Porphyra yezoensis consists of a one-cell thick blade and belongs to the most primitive group of red algae (the Bangiophyceae) , Chondrus crispus consists of a branched frond composed of a 6-7 cell thick cortical layer plus a several-cell thick central medulla (e.g., Fredericq et al., 1992) and belongs to the most advanced group of red algae, the Florideophyceae . Chondrus crispus is commercially valuable as a source of the phycocolloid kappa carrageenan, although this species actually produces three different types of carrageenan: kappa, lambda and (in smaller amounts) iota. Historically, Chondrus crispus was the single most important source of carrageenan in the world until relatively recently.

Other Embodiments Other macroscopic marine algae are susceptible to transformation according to the method of the invention, as long as the exterior, or cuticle, layer can be penetrated by a wounding method, e.g., by biolistics. These algae include, but are not limited to, commercially valuable brown seaweeds belonging to the group known as kelps (e.g., species of Laminaria ) , which are eaten as human and animal food and used to produce a variety of industrial products, and the green seaweeds Ulva and Enteromorpha , which have been used for bioremediation and food, respectively. Other promoter and/or reporter or selection gene systems are also appropriate in the method of the invention.

A preferred form of the host marine algae is partially flattened thalli which are reproductive or can be induced to reproduce. The advantage of a flattened thallus is that it provides a larger surface area for wounding by biolistics. Some examples of macroscopic marine algae besides Porphyra that have largely flattened thalli and, therefore, would be very applicable to the transformation techniques of the invention are species of the brown alga Laminaria ( kelps), the green alga Ulva and the red algae Chondrus and Mazzaella .

USE

An unlimited variety of foreign genes, which code for polypeptides non-native to a marine algal species, can be introduced and transcribed in the chosen marine algae by the method of the invention. This includes the production of important proteins or other products of commercial value, such as pigments, antibodies, hormones, pharmaceuticals, vaccines and the like. The choice of the particular gene or genes to be delivered into the alga depend on the purpose of the transformation. Some specific examples of the application of the invention are described below.

1) The production of a transgenic strain of marine algae that provides oral immunization against hepatitis, diarrheal or other infectious diseases, thus acting as an edible "vaccine," e.g., as in Richter et al . , 1996: For diarrheal diseases, this would be accomplished in Porphyra, for example, by transforming the alga with Agroba cterium tumefaciens harboring a plasmid containing the gene encoding LT-B or an LT-B fusion protein (Haq et al., 1995) or the Norwalk virus capsid protein (NVCP) . Methods of preparing oral vaccines in land plants are described, e.g., in Curtis, III, et al . , U.S. Patent Nos. 5,654,184; 5,679,880; and 5,686,079, the whole of which are hereby incorporated by reference herein. 2) The production of a transgenic strain of marine algae with enhanced disease resistance to marine fungi:

Resistance to the marine fungus Pythium, for example, would be accomplished in Porphyra by transforming the alga with an Agrobacterium tumefaciens strain harboring a plasmid that contains a gene(s) coding for anti-fungal proteins, e.g., anti-fungal cell wall proteins, and/or marine fungal recognition and resistance genes. Disease-resistant strains of multicellular macroalgae seaweeds like Porphyra , which are cultivated in a multi-billion dollar a year mariculture industry, would are highly desirable.

3) The production of a transgenic strain of marine algae with an enhanced level of a nutritionally valuable compound, such as the carotenoids, the polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) : This would be accomplished in Porphyra , for example, by transforming the alga with an Agroba cterium tumefaciensstrain harboring a plasmid containing a gene for either increasing the overall lipid content (by, for example, introducing the acetyl-CoA carboxylase (ACCase) gene (Dunahay et al., 1996)), or converting EPA to DHA with a desaturase gene. Additional details for genetically modifying lipid content in algae are found in Dunahay et al., 1997.

4) The production of a transgenic strain of marine algae capable of expressing therapeutic human antibodies: Analogous to the use of transgenic land plants (Russell, D., 1999), transgenic multicellular marine algae can an alternative source or bioreactor for the production of therapeutic antibodies.

References

Apt, K. , P. Kroth-Pancic and A. Grossman, 1996. Stable nuclear transformation of the diatom Phaeoda ctyl um tri cornutum . Mol. Gen. Genet. 252: 572-579.

Birch, R. , 1997. Plant transformation: problems and strategies for practical application. Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:297-326.

Cheney, D., and C. Duke, 1995. Methods for producing improved strains of seaweed by fusion of spore- protoplasts, and resultant seaweeds and phycolloids, U.S. Patent No. 5,426,040. Christou, P., 1996. Transformation technology. Trends in Plant Science 1:423-431.

Dale, P., 1995. R & D regulation and field trialing of transgenic crops. Trends in Biotech. 13: 398-403.

Dunahay, T., E. Jarvis and P. Roessler, 1995. Genetic transformation of the diatoms Cycol tella cryptica and Navicula saprophila . J. Phycology 31: 1004-1012.

Dunahay, T., E. Jarvis, S. Dais and P. Roessler, 1996. Manipulattion of microalgal lipid production using genetic engineering. Applied Biochem. and Biotechnology 57/58: 223-231.

Dunahay, T., et al., 1997. Method to transform algae, material therefore, and products produced thereby. U.S. Patent No. 5, 661,017. Fredericq, S., J. Brodie, and M. Hommersand, 1992. Developmental morphology of Chondrus crispus

(Gigartinales, Rhodophyta). Phycologia 31: 542-563.

Haq, T., H. Mason, J. Clements, and C. Arntzen, 1995. Oral immunization with a recominant bacterial antigen produced in transgenic plants. Science, 268: 714-716.

Hooykass, P. and R. Schilperpoort , 1992. Agrobacterium and plant genetic engineering. Pit. Mol Biol. 19: 15-38.

Huang, X., J. Weber, T. Hinson, A. Mathieson and S. Minocha, 1996. Transient expression of the GUS reporter gene in the protoplasts and partially digested cells of Ulva lactuca (Chlorophyta) . Bot . Marina 39: 467-474.

Jefferson, R. , T. Kavanagh, and M. Bevan, 1987. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901- 3907.

Kubler, J. , S. Minocha and A. Mathieson, 1994. Transient expression of the GUS reporter gene in protoplasts of Porphyra (Rhodophyta). J. Mar. Biotechnol. 1: 165-169. Kurtzman, A. and D. Cheney, 1991. Direct gene transfer and transient expression in a marine red alga using the biolistic method. J. Phycol. 27 (Suppl) : pg 42.

Lohuis, M. and D. Miller, 1998. Genetic transformation of dinoflagellates (Amphidinum and Symbiodinium) : expression of GUS in microalgae using heterologous promoter constructs. Plant J. 13:427-435.

May, G., R. Afza, H. Mason, A. Wiecko, F. Novak, and C. Arntzen, 1995. Generation of transgic banan {Musca acumina ta ) plants via Agrobacterium-mediated transformation. Biotechnoloogy 13: 486-492. Minocha, S. Genetic engineering of marine macroalgae: current status and future perspectives. World Aquaculture 30: 29 - 30, 57.

Porath, J. , I. Gokhman, A. Lers, A. Levy, and A. Zamir, 1997. Developing a transformation system in Dunaliella . Phycologia 36 (Suppl) : pg 89.

Qin, Song, Peng Jiang, Xin-Ping Li, Xi-Hua Wang and Cheng-Kui Zeng, 1998. A transformation model for Laminaria Japonica (Phaeophyta, Laminariales) . Chin. J. Oceanol. Limnol . 16 (Suppl): 50-55.

Qin, Song, Guo-Qiong Sun, Peng Jiang, Li-Hong Zou, Yun Wu, and Cheng-Kui Zeng, 1999. Review of genetic engineering of Laminaria Japonica (Phaeophyta,

Laminariales) in China. Hydrobiologia 398/399: 469-472.

Radmer, R. , 1996. Algal diversity and commercial algal products. Bioscience 46: 263-270.

Richter, L., H. Mason, and C. Arntzen, 1996. Transgenic plants created for oral immunization against diarrheal diseases. J. Travel Med. 3: 52-56. Russell D., 1999. Feasibility of antibody production in plants for human therapeutic use. In: Current Topics Microbiology, vol 240, Hammond, J. (eds) , Plant Biotechnology, ppll9-138. Sanford, J. , et al . , 1990. Method for transporting substances into living cells and tissue and apparatus therefore. U.S. Patent No. 4,945,050.

Schiedlmeier, B., et al., 1994. Nuclear transfer of Volvox carter! . Proc. Natl. Acad. Sci. USA 91:5080-5084.

Stevens, D. and S. Purton, 1997. Genetic engineering of eukaryotic algae: progress and prospects. J. Phycology,

33:713-722

Vancanneyt, G., R. Schmidt, A. O'Connor-Sanchez, L. Willmitzer and M. Rocha-Sosa, 1990. Construction of an intron-containing marker gene: splicing of the intron in trangenic plants and its use in monitoring early events in Agrobacteriuffi-mediated plant transformation. Mol. Gen Genet. 220: 245-250. Vasil, I. 1998. Biotechnology and food supply for the 21st century: a real-world perspective. Nature Biotechnology 16:399-400.

While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein.

Claims

CLAIMSWhat is claimed is:

1. A method for causing genetic transformation of multicellular marine algae, said method comprising: culturing cells of a transformation-competent Agrobacterium species, said cells containing a Ti plasmid that contains a gene of interest; wounding a multicellular marine alga to be transformed in a manner that is sufficient to penetrate at least the cuticle, or outer cell wall layer of said alga; applying cells of said transformation-competent Agroba cterium species to wounded cells of said alga; and co-culturing said applied cells of said Agrobacterium species with said wounded algal cells for a time sufficient to effect transformation of some of said algal cells.

2. The method of claim 1, further comprising isolating transformed marine algal cells.

3. The method of claim 1, wherein the method of wounding in said wounding step comprises the use of bolistics or microparticle bombardment.

4. The method of claim 1, further comprising, prior to said applying step, treating said cells of said transformation-competent Agroba cterium species or said multicellular marine alga to be transformed with a virulence-inducing compound.

5. The method of claim 4, wherein said virulence- inducing compound is acetosyringone.

6. The method of claim 1, wherein, in said applying step, said cells of said transformation-competent Agrobacterium species are applied to wounded cells of said alga in a manner that minimizes exposure of said alga to be transformed to a non-salt water environment.

7. The method of claim 6, wherein said cells of said Agrobacterium species are applied to wounded cells of said alga by spraying or swabbing the surface of the alga with a suspension of cells of said Agrobacterium species .

8. The method of claim 1, wherein, in said co- culturing step, said applied cells of said Agrobacterium species are co-cultured with said wounded algal cells under conditions of low light exposure.

9. The method of claim 1, wherein, in said wounding step, said wounded algal cells are cells of blade tissue.

10. The method of claim 9, wherein said cells of blade tissue are not totally flattened.

11. The method of claim 9, further comprising, following said co-culturing step, the step of removing said applied cells of said Agrobacterium species from said cells of blade tissue.

12. The method of claim 11, wherein said removing step comprises repeated washing of the surfaces of said blade tissue .

13. The method of claim 2, further comprising, following said isolating step, culturing said transformed marine algal cells and isolating progeny of said cells, wherein said progeny cells are capable of expressing said gene of interest.

14. The method of claim 1, wherein cells of more than one transformation-competent Agroba cterium species are cultured and subsequently applied to wounded cells of said alga.

15. The method of claim 1, wherein said Ti plasmid is genetically engineered such that it is capable of causing insertion of a commercially desirable gene into the DNA of wounded marine algal cells and expression of the inserted gene.

16. The method of claim 15, wherein said Ti plasmid further comprises a promoter gene and at least one selectable gene, wherein said selectable gene is chosen from the group consisting of genes which code for at least one screenable marker, genes which code for selection agent resistance, and combinations thereof.

17. The method of claim 1, wherein said multicellular marine alga is a species from a genus selected from the group consisting of Porphyra , Chondrus and Laminaria .

18. The method of claim 1, wherein said transformation- competent Agrobacterium species are from terrestrial Agrobacterium species or from plasmid-containing marine representatives of the genus Agrobacterium .

19. The method of claim 1, wherein said transformation- competent terrestrial Agrobacterium species are A. tumefaciens or A. rhizogenes.

20. A stable transgenic multicellular marine alga, said alga comprising a DNA sequence coding for a gene foreign to said alga, wherein said alga is further capable of expressing said DNA sequence and of transferring said expressible DNA sequence to progeny of said alga.

21. The stable transgenic multicellular marine alga of claim 20, wherein said alga is a species from a genus selected from the group consisting of Porphyra , Chondrus and Laminaria .

22. A transgenic strain of marine algae comprising and capable of expressing a DNA sequence coding for an antigen of a pathogenic microorganism or an antigenic determinant thereof, wherein said antigen or antigenic determinant thereof is capable of eliciting a secretory immune response in a human or other animal upon oral administration of cellular material from said algae.

23. A transgenic strain of marine algae with enhanced disease resistance to marine fungi compared to a non- transformed said strain.

24. A transgenic strain of marine algae capable of producing a compound having an enhanced health benefit compared to a non-transformed said strain.

25. The transgenic strain of claim 24, wherein said compound having an enhanced health benefit is an antibody having therapeutic activity.

26. A method for eliciting a secretory immune response in a human or other animal, said method comprising: orally administering an effective amount of a composition comprising transgenic marine algae, or tissue thereof, wherein said transgenic algae, or algal tissue, comprise and are capable of expressing a DNA sequence coding for an antigen of a pathogenic microorganism or an antigenic determinant thereof, wherein said antigen or antigenic determinant thereof is capable of eliciting a secretory immune response in a human or other animal upon oral administration of cellular material from said algae.

27. The method of claim 26, wherein said transgenic algae, or algal tissue, comprise and are capable of expressing a DNA sequence comprising any gene, combination of genes, gene fragment or combination of gene fragments coding for said antigen.

28. The method of claim 26, wherein said transgenic algae, or algal tissue, comprise and are capable of expressing a synthetic DNA sequence coding for said antigen.

29. The method of claim 26, wherein said transgenic algae, or algal tissue, comprise and are capable of expressing two or more DNA sequences coding for two or more antigens that can be the same or different.