Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-07-11
2004-08-10
Johannsen, Diana B. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S019000, C435S091200, C435S410000
Reexamination Certificate
active
06773889
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to the isolation of molecular markers of genetic mutation in plants by representational difference analysis (RDA). In particular, the invention relates to the preparation of genetic probes for early identification of plants that are not true-to-type. More particularly, the invention relates to the use of such genetic probes as a diagnostic and quality control tool for monitoring the development of genetic polymorphisms arising during tissue culture regeneration of plants.
The large-scale production of commercially elite plants by in vitro micropropagation is technically available for a number of species. A common problem encountered when growing plants by tissue culture is the development of tissue culture-induced genomic polymorphisms in which genetic changes in the nuclear, mitochondrial and/or chloroplast genomes result in a lack of homogeneity among the regenerants and the production of inferior plants that are not true-to-type (i.e., “off-type” plants) with little commercial value. The development of genetic polymorphisms in tissue culture is termed “somaclonal variation”. As used herein, the term “off-type” refers to a plant that exhibits a phenotypic difference from a normal plant.
Investment in plants that are later discovered to be off-types can have severe financial implications for both growers and plant producers, who may only discover that the plants are off-types after the plants have grown for some period of time in the field. Traditionally, an experienced examiner is required to identify off-type plants by a morphological description and visual monitoring of plant characteristics. However, many characteristics are only expressed in a more mature stage of plant development and not in in vitro plants or very young plants. For example, in banana plants, off-types such as dwarfs, mosaics (which have irregular bright yellow spots or stripes on the leaf) and masadas (which have abnormal foliage showing depressions and thicker leaves) are difficult to identify at both the tissue culture and nursery stages [Israeli et al. (1991)
Scientia Hortic.
48, 71-88]. Therefore, a method for early monitoring and identification of off-type plants, while still at the tissue culture or nursery stages, would be a very advantageous tool to enable the selection of desired true-to-type plants for further propagation.
Attention was first drawn to somaclonal variation by Larkin and Scowcroft [
Theoret. Appl. Genet.
60,197-24 (1981)] and a great deal of literature on the subject has accumulated [see reviews by M. Lee and R. L. Phillips,
Ann. Rev. Plant Physiol. Mol. Biol.
39, 413-437 (1988); R. L. Phillips et al., in Progress in Plant Cellular and Molecular Biology, Kluwer Academic Publishers, Netherlands, pp. 136 (1990); C. A. Cullis, The Molecular Biology of Plant Cells and Cultures in Comprehensive Biotechnology, M. W. Fowler and G. S. Warren, eds., Pergarmmon Press, NY (1991); A. Karp,
Oxford Surveys Plant Mol. Cell. Biol
7, 1-58 (1991)]. A large number of variants in morphological and biochemical characteristics, as well as in changes at the DNA level, have been characterized. At the genomic level, changes in ploidy level, chromosomal rearrangements, activation of transposable elements, gene amplification, single gene mutations and variation in quantitative traits have been reported. Variations in mitochondrial and chloroplast genomes in regenerants, particularly in cereals, have also been reported [M. D. Morere-Le Pavan et al.
Theoret. Appl. Genet.
85, 1-19 (1992)], thus illustrating that all compartments of the genetic information of the plant cell are susceptible to the phenomenon of somaclonal variation. In addition to the alterations in genomic DNA sequence and organization, stable changes in nucleic acid methylation patterns have been implicated as a major factor in epigenetic changes [S. M. Kaeppler and R. L. Phillips,
Proc. Natl Acad. Sci. USA
90, 8773-8776 (1993); M. J. M. Smulders et al.,
Theoret. Appl. Genet
91, 1257-1264 (1995); B. Arnholdt-Schmitt et al.,
Theoret. Appl. Genet.
91, 809-815 (1995); P. Bogani et al.,
Genome
38, 901-912 (1995)]. The question of how and when during the tissue culture process variations occur has also been addressed. A study in wheat indicated that the variation in these cultures originated during the callus phase and that the extent of variation could be affected by manipulation of the culture medium [B. F. Carver and B. B. Johnson,
Theoret. Appl. Genet.
78, 405-418 (1989)].
A series of PCR (polymerase chain reaction) based technologies have been used by investigators to compare and highlight the differences between DNAs isolated from related sources. For example, restriction fragment length polymorphisms (RFLP) have been used, particularly by plant breeders, as genetic markers in developing genetic linkage maps in which chromosomal regions associated with desirable phenotypic traits may be identified and tracked during subsequent selective breeding to produce improved plant lines. RFLPs are genetic differences detectable by DNA fragment lengths, typically revealed by agarose gel electrophoresis after restriction endonuclease digestion of DNA. There are large numbers of restriction endonucleases available, characterized by their nucleotide cleavage sites and their source, e.g., the bacteria
E. coli
. Variations in RFLPs result from nucleotide base pair differences which alter the cleavage sites of the restriction endonucleases, or by insertions, yielding different sized fragments. Other point mutations in the genome usually go undetected. Thus, RFLP differences often are difficult to identify. Although RFLP has advantages in detecting genetic variation, it is labor intensive.
Sequence tagged sites (STS) of DNA polymorphisms have been developed by the use of RFLP. However, the development of STS requires an already identified difference which can be tested for in the unknown cell lines. Thus, STS is not a useful approach to isolate differences between uncharacterized cultivars.
Random amplified polymorphic DNAs (RAPD) and amplified fragment length polymorphisms (AFLP) are two further techniques that simply compare the DNA from any number of different samples and can be used to detect the level of difference between them. The RAPD method employs DNA amplification by PCR using short primers of arbitrary sequence (random amplified polymorphic DNA). Differences as small as single nucleotides between genomes can affect the RAPD primer's binding/target site, and a PCR product may be generated from one genome but not from another. RAPD detection of genetic polymorphisms represents an advance over RFLP in that it is less time consuming, more informative, and readily adaptable to automation. However, RAPD is limited in that only dominant polymorphisms can be detected (i.e., this method does not offer the ability to examine simultaneously all the alleles at a locus in a population). However, because of its sensitivity for the detection of polymorphisms, RAPD method has been widely used for analyzing genetic variation within species or closely related genera, both in the animal and plant kingdoms. In particular, RAPD has been used by several groups for off-type plant detection and, recently, a potential RAPD marker has been identified for a dwarf banana off-type [O. Damasco et al.,
Plant Cell Rep.
16, 118-122 (1996)]. However, this use of the RAPD technique was restricted to attempting to generate a marker for dwarfism, with no reference made to attempting to find generalized markers of polymorphism. Both RFLP and RAPD have been used to distinguish between regenerants from embryogenic carrot cell lines [L. Georgetti et al.,
Mol. Gen. Genet.
246, 657-662 (1995)]. The RAPD technique however, generally has several disadvantages, including a lack of reproducibility of results and the necessity of using of a large number of different primers to detect variation in only a small portion of the genome.
AFL
Cullis Christopher A.
Kunert Karl
Rademan Samantha
Case Western Reserve University
Day Jones
Johannsen Diana B.
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