Multicellular living organisms and unmodified parts thereof and – Method of producing a plant or plant part using somatic cell...
Reexamination Certificate
2000-06-12
2003-03-11
Campell, Bruce R. (Department: 1661)
Multicellular living organisms and unmodified parts thereof and
Method of producing a plant or plant part using somatic cell...
C800S295000, C800S296000, C800S292000, C435S421000, C435S420000, C435S419000, C435S430000, C435S410000, C435S440000, C435S449000, C435S450000, C435S453000, C435S454000
Reexamination Certificate
active
06531646
ABSTRACT:
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 National Sea Grant Enhancement Grant entitled “Developing a commmercially viable seaweed aquaculture industry in New England.” Therefore, the U.S. government has certain rights in this invention.
BACKGROUND OF THE INVENTION
The red alga Porphyra, or nori as it is commonly called, is the most widely eaten and commercially valuable seaweed in the world. Porphyra's main use is as the purple-black wrapping around the delicacy known as “sushi.” Nori is commercially grown in Japan, China, Korea and Taiwan, and over 45,000 dry metric tons of nori are produced annually, worth over $2 billion US dollars. Because of its high protein and vitamin content, nori is considered to be a valuable health food. The market for nori sheets in the US alone is estimated to be worth at least $50 million dollars annually and is growing at a rate of over 17% per year. In addition, certain species of Porphyra also serve as important commercial sources of the red pigment r-phycoerythrin, which is utilized as a fluorescent “tag” for immunofluorescent studies and can cost as much as $360 per mg.
There are thought to be approximately 70 species of Porphyra worldwide, the majority of which are found in the North Pacific Ocean; approximately 33 species of Porphyra occur in Japan alone. Nori cultivation is a well developed industry, particularly in Japan where it has undergone significant technical improvements since the 1960's. Improvements made to the technical aspects of nori cultivation include the development of techniques for controlled culturing of its conchocelis stage in shells and for artificial seeding of spores produced by the conchocelis onto cultivation nets which can be stored until placed in the ocean.
As has been demonstrated repeatedly with agricultural crops and other types of cultivation, genetic improvement of cultured species is generally crucial for maximizing yield and developing cost-effective cultivation programs. Seaweeds, including Porphyra, are no exception. However, unlike land plants, seaweed strain improvement techniques have generally been restricted to classical breeding methods, particularly strain selection.
As a result of strain selection efforts, today there are several dozen cultivars of two Porphyra species,
P. yezoensis
and
P. tenera
, farmed in Japan. These cultivars were developed primarily as a result of the intensive strain selection program in Japan. Over many years of repeated selection, improvements were made in increasing the average length of fronds, as well as the length of the growing seasons of these two species (see Patwary and van der Meer, 1992). By comparison, efforts to develop new Porphyra strains through sexual hybridization have been far less successful. Intra- and interspecific crosses have been attempted in Porphyra but have contributed little (Suto, 1963). Often the products of sexual crosses have exhibited abnormal growth or chimeric (i.e., sectored) blades. In addition, because most commercially valuable species are monoecious, and thus easily self-fertilized, it is difficult in Poryhrya to produce F1 progeny of specific parents by sexual hybridization.
Thus, the most successful method of producing new strains of Porphyra to date has been through repeated strain selection. However, this approach has a number of disadvantages and limitations. In particular, repeated strain selection usually requires many years of intensive effort and is very labor intensive. In addition, the existing genetic variability in one or more populations of interest may not be sufficient for strain selection purposes. Furthermore, desirable and agronomically-beneficial traits that may be found in other species can not be taken advantage of by applying the methods that have been used in Porphyra in the past. Future improvements in the production of nori, both within and outside of the United States, will therefore most likely depend on the production of new strains that will have to be developed by new strain improvement methods.
One method of strain improvement that permits the rapid development of new strains and the transfer of genes and traits between species is somatic hybridization via protoplast fusion. Protoplast fusion is a well-developed technique in land plants and is just beginning to be successfully applied to seaweeds. In this technique, protoplasts are produced by enzymatically removing the cell walls that surround plant cells, and the protoplasts are then fused together to form a hybrid or a cybrid (i.e., cytoplasmic hybrid). Protoplast fusion can also be used to produce a polyploid or aneuploid. Like sexual hybrids, somatic hybrids generally exhibit combinations of traits found in the two parental plants hybridized. One major advantage of protoplast fusion is that it provides the opportunity to produce unique genomic combinations which are impossible or impractical by sexual hybridization, such as hybridizing individuals of different species and producing cybrids. Protoplast fusion has been reported in only a small number of seaweeds to date, including the green algae Ulva and Enteromorpha (Reddy et al. 1992) and the red algae Gracilaria (Cheney, 1990; Cheney and Duke, 1995), and Porphyra (e.g., Fujita and Migita, 1987; Fujita and Saito, 1990).
Efforts at protoplast fusion in Porphyra date back to 1986, when Saga et al. attempted to fuse protoplasts of a Porphyra species with those of the green alga Enteromorpha without success. Later, Fujita and Migita (1987) reported successfully fusing protoplasts of a wild type strain and a green mutant strain of
P. yezoensis
using the chemical fusagen polyethylene glycol (PEG). However, although they observed fusion and heterokaryon formation, they ultimately were able to produce only chimeric fronds which in turn produced greenish conchocelis that gave rise to green F1 fronds. In 1990, Fujita and Saito used both PEG and electrofusion techniques in fusion efforts with protoplasts from several Porphyra species. Similarly, Araki and Morishita (1990) attempted to fuse protoplasts between
P. yezoensis
and
P. tenera
. Mizukami et al. (1995) used electrofusion to fuse protoplasts between
P. yezoensis
and
P. suborbiculate
and report producing “hybrid-like” thalli. However, none of the fusion studies to date on Porphyra have resulted in a consistent reproducible technique that produces Porphyra strains with stable, improved properties. Effective and practical protoplast fusion methods that can be used for strain improvement in Porphyra would, therefore, be highly desirable.
BRIEF SUMMARY OF THE INVENTION
This invention is directed to a new method for producing wall-less cells, or protoplasts, from the commercially valuable edible seaweed Porphyra (also known as nori) and the use of protoplast fusion techniques for the production of new and improved strains of the same.
The method of the invention features the use of conchoporangial branch conchocelis for at least one of the sources of protoplasts for protoplast fusion. Protoplasts produced from conchosporangial branch conchocelis of one Porphyra species may be mixed with protoplasts produced from blade material, conchocelis or conchospores of a second Porphyra species and fused using, e.g., a chemical fusing agent like polyethylene glycol (PEG) or electrofusion. Other possible fusogens include sodium nitrate, dextran, high pH-high calcium containing solutions or combinations thereof. After fusion has occurred, fusion products are isolated, cultured to multicellular material and regenerated to whole plants. Alternatively, the multicellular material can be used as an undifferentiated cell mass.
Hybrids produced by the method of the invention are expected to possess combinations of the genetic material found in their respective parental species, and, therefore, are expected to exhibit combinations of their traits. Polyploids and aneuploids produced by the method of invention are ex
Cheney Donald P.
Roberts Kathryn M.
Watson Katherine L.
Campell Bruce R.
Grunberg Anne Marie
Northeastern University
Weingarten Schurgin, Gagnebin & Lebovici LLP
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