Multicellular living organisms and unmodified parts thereof and – Method of using a plant or plant part in a breeding process... – Method of breeding using interspecific crosses
Reexamination Certificate
1999-07-15
2002-02-05
LeGuyader, John L. (Department: 1635)
Multicellular living organisms and unmodified parts thereof and
Method of using a plant or plant part in a breeding process...
Method of breeding using interspecific crosses
C800S298000, C435S006120, C435S410000, C435S468000
Reexamination Certificate
active
06344597
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to novel interspecific
Nicotiana excelsior×N. benthamiana
hybrid seeds and plants and to a method of producing interspecific Nicotiana hybrids having enhanced properties for biomass and the production of recombinant proteins using a viral vector system.
The difference between intra- and interspecific hybrids can be visualized by the Mendelian behavior of intraspecific hybrids and the essentially non-Mendelian behavior of interspecific hybrids. This difference results from the complete chromosome homology in hybrids between plants of the same species (intraspecific) and the partial or nonchromosome homology that characterizes hybrids between plants of different species (interspecific). The intermediate or gray area between the extremes among interspecific hybrids is seen in the behavior of those involving closely related species. Such species will have the same chromosome number, and the extent of chromosome homologies may be very high. Hybrids of this kind produce seed when self-pollinated and show evidence of Mendelian patterns of segregation for some traits.
Cytological studies of meiosis in some F
1
hybrids may show evidence of chromosome irregularities that reflect the chromosomal differences that mark the parents as different species. A hybrid between distantly related species may show reduced pairing between chromosomes of the different genomes. Meiosis in such hybrids may exhibit the typical chromosome behavior characteristic of monogenomic haploids. Most interspecific F
1
hybrid combinations in the genus Nicotiana fall into this latter category. Their chromosome doubled counterparts are called amphidiploids, or simply allopolyploids, when a parent of the hybrid may itself be a combination of different ancestral genomes.
N. tabacum
represents a typical example.
The relative difficulty of producing interspecific F
1
hybrids increases in proportion to the decrease in taxonomic relations between their parents. Although some F
1
hybrids are stable when converted to fertile polyploids, even hybrids between distantly related parents share some degree of chromosome homology. There are advantages to maintaining seed stocks of allopolyploids by self-pollination, particularly if the hybrid is difficult to obtain by conventional cross-pollination.
Interspecific hybridization and introgression in natural populations of plants and animals is a known source of genetic variation and adaptation. The term introgression, or introgressive transfer, of genetic traits is used in a narrower sense with short-term breeding projects. The goal of experimental interspecific hybridization is usually the introgressive transfer of a specific trait from one species (the nonrecurrent parent) into the genome of another (the recurrent parent). The trait must be expressed with reasonable phenotypic fidelity as a dominant or partial dominant in the F
1
hybrid and all subsequent backcross generations, when nonrecurrent chromosomes are lost at random, otherwise the proposed interspecific transfer could not be made. Foreign genes from one species can become unstable when translocated into the germplasm of another.
Chromosome pairing between the foreign genomes in an interspecific hybrid may be extensive or minimal although the F
1
hybrid is usually sterile. Fertility may be restored by treating germinating seed of the hybrid in 0.4 percent aqueous colchicine for about 4 hours to induce chromosome doubling. Immersion of the seeds in 0.1 percent colchicine for 24 hours may also be effective. Thereafter, the seed is rinsed in sterile distilled water and planted by sowing on the surface of pasteurized soil in glass or plastic preparation dishes. The seedlings later are transplanted to pots of soil in the greenhouse and mature plants that show good pollen development are self pollinated or backcrossed to the recurrent parent.
Three conventional approaches to interspecific hybridization for the purpose of alien transfer and incorporation of germplasm into stable diploid lines are (a) diploid×diploid then doubling to produce the allopolyploid and backcrossing to produce the sesquidiploid, (b) autotetraploid×autotetraploid to produce the allopolyploid directly, and © autotetraploid×diploid to produce the sesquidiploid directly as described in Technical Bulletin 1586, U.S. Department of Agriculture, 1979.
Gene transfer mediated by
Agrobacterium tumefaciens
vectors has become routine in tobacco (DeBlock et al., 1987; Grierson, et al., 1990; Hilder, et al., 1990; Lindbo and Dougherty, 1992). Commercial use of genetic transformation in agriculture depends on the incorporation of foreign genes into high-yielding germplasm. Plant breeders may want to combine several foreign genes into a single elite germplasm source. In order to accomplish this goal, designer chromosome construction is desirable.
Designer, or artificial, chromosomes have been produced in yeast. In plants, the scaffold for designer chromosome construction can be found in a breeding line that possesses the full complement of chromosomes from its own species plus an additional chromosome from a related species, Campbell, et al., Construction of a designer chromosome in tobacco,
Theor Appl Genet
(1994) 87:837-842. Additional chromosomes are often meiotically stable as homozygotes, and because recombination between additional chromosomes and the rest of the plant genome is rare (Gerstel 1945) the integrity of a foreign-gene linkage package can be preserved.
The placement of this linkage package on an additional chromosome will also minimize disturbance to the rest of the plant genome. The genetic structure of high-yielding germ plasm can be disturbed either through insertional mutagenesis or the disturbance of beneficial linkage blocks. Insertional mutagenesis, caused by the integration of foreign genes into plant coding sequences, can occur frequently. Konez, et al. (1989) estimated that at least 30% of all T-DNA insertions occur in transcribed regions of the Arabidopsis and Nicotiana genomes. Beneficial linkage blocks are formed through intermating and recombination followed by selection.
The desired traits for a plant may not be present in the germplasm of the species of interest. In such a case, traditional breeding within the species may not give acceptable results. Introducing a desired trait from one species into a related one by interspecific hybridization is followed by introgression into the recurrent parent.
Methods for expressing genes in plants has been described in U.S. Pat. Nos. 5,316,931; 5,589,367; 5,811,653 and 5,866,785, all of which are incorporated herein. Certain species have either an acceptable biomass or an acceptable expression of a sequence inserted into a plant viral vector, but no current species have both high levels of biomass and high levels of viral vector performance. There is a need for a method to develop in one species both the characteristics of increased biomass and improved viral vector performance.
SUMMARY OF THE INVENTION
The present invention is directed to a method of crossing two different species of Nicotiana to produce an interspecific hybrid which results in an increased biomass and improved host susceptibility to a viral vector.
In accordance with this invention, interspecific Nicotiana hybrids have been produced that are characterized with having a biomass of greater than that usually obtained from tobacco with CP-fusion constructs and a virion yield of greater than 1.0 mg. virion/gram fresh weight.
In one embodiment of the invention, there is provided a novel Nicotiana interspecific hybrid. This invention thus relates to the seeds of Nicotiana interspecific hybrid, to the plants of Nicotiana interspecific hybrid and to methods for producing a Nicotiana interspecific hybrid plant produced by crossing the Nicotiana interspecific hybrid with itself or another Nicotiana interspecific hybrid line.
Thus, any such methods using the Nicotiana interspecific hybrid are part of this invention: selfing, backcrosses, hybrid pro
Large Scale Biology Corporation
LeGuyader John L.
Rothwell Figg Ernst & Manbeck
Zara Jane
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