The importance of genetic transformation lies in the fact that this technique allows for the insertion of a single or a few genes into an established genotype, avoiding the random assortment of genes produced through meiosis. The mixing of genetic material of both parents that results from hybridization provides genetic diversity, but it also generally requires generations of continued hybridization and selection to produce progeny carrying the desired traits of both parents. Transformation has the potential to produce a genotype that is essentially unchanged except for the improvement of a particular trait. Genetic transformation is not without its own inherent difficulties. The regeneration of many tree fruit species and/or cultivars is problematic and one of the most serious hindrances to the application of gene transfer technologies to perennial tree fruit crops. In those species that can be reliably transformed, the technology is generally only successful with a few genotypes and, in some cases, these genotypes are not commercially important. In some species, transformation has been obtained only from seedling material. This reduces the usefulness of the technology, since hybridization using transgenic genotypes as parents would then generally be required for cultivar development in order to combine the trait improved through transformation with the host of additional traits necessary for a commercial cultivar. It is precisely the process of hybridization that genetic trans formation seeks to bypass. The difficulties of developing transgenic tree fruit are clearly indicated by the fact that of the 8,906 field releases of transgenic plants in the United States from 1987 to July 2002, only 54 were temperate tree fruit species.
The process of producing transgenic plants of tree fruit species can vary from months to years. Following the initial confirmation of transformation, the processes of rooting and propagation add significant time to the process. Once confirmed transgenic lines are obtained, the requirements for evaluating their performance are the same as those for conventional cultivar development. Transgenic plants require careful and exhaustive testing not only to evaluate the effect of the transgene but also to confirm the trueness-to-type and stability of the characteristics of the original cultivar.
Difficulties notwithstanding, there are several inherent advantages in the use of gene transfer for tree fruit improvement. Once a useful transformant is isolated, assuming stability of transgene expression (and this assumption has yet to be adequately tested for tree fruit), vegetative propagation—the normal route of multiplying tree fruit— provides for virtually unlimited production of the desired transgenic line. Fixation through the sexual cycle is not required. Also, while the dominance of a few major cultivars in many tree fruit crops such as pear, apple, and sour cherry may be a hindrance to the acceptance of new cultivars produced through hybridization, it can maximize the impact of a major cultivar improved through transformation.
Genetic Marker-Assisted Selection and Gene Identification
Genomics research is aimed at identifying functional genes and understanding the action and interactions of gene products in controlling plant growth and development. For plant breeders, perhaps the two most important aspects of genomics research are the development of genetic markers and gene identification (and isolation).
Molecular markers are segments of deoxyribonucleic acid (DNA) that reveal unique sequence patterns diagnosed by one or more restriction enzymes in a given cultivar, line, or species. These DNA segments are associated with genes that control plant characteristics. If the marker is found in DNA isolated from a particular plant line or cultivar, the character with which it is associated will likely be expressed in that plant. The closer the marker DNA segment is to the segment of DNA, or gene, controlling the character of interest, the stronger the association between the marker and the character. If the marker DNA is contained in the gene of interest, the association will be 100 percent. To develop markers, trees are rated for the expression of characteristics such as resistance to a particular disease, fruit size, sugar content, etc. This is a critical step in marker development. The value of a marker in predicting a trait or phenotype is a function of the accuracy of the evaluation of the trait in the mapping population. A marker cannot be accurately correlated with a characteristic if the characteristic cannot be accurately rated or quantified. DNA is extracted from the evaluated plants, and then any one of a number of molecular marker systems is used to identify polymorphisms that are preferentially present in those trees that carry the trait(s) under consideration. For a discussion of the various marker systems, see Abbott in Khachatourians et al. (2002). Once the presence of a marker or markers is associated with a trait, and particularly if this association is consistent across populations with varying genetic backgrounds, the marker can be used to predict the presence of the trait in hybrid progeny. In programs using molecular markers, the presence of marked or mapped traits can be evaluated by sampling a small amount of leaf material from young seedlings in the greenhouse, rather than waiting for seedlings to fruit or display tree characteristics in field plantings.
Molecular markers have the potential for speeding the breeding process and reducing costs. The prospect of selecting promising seedlings at an early stage of growth in the greenhouse by using a saturated genetic map to tag single gene traits as well as multigenic traits, and planting only these promising genotypes in the field, is an attractive prospect. But molecular markers are not a panacea for fruit breeding. Considerable work is involved in the development of molecular markers, particularly the initial trait evaluation in mapping populations. Also, the fact remains that hybridization will produce random gene assortment, and it is likely that several generations or more will be necessary for cultivar development (especially if genes are introduced from unimproved germplasm). Using molecular markers, seedlings with little likelihood of value can be rapidly discarded, leaving field space for more promising material. The seedlings that remain, however, must be subjected to the same rather lengthy evaluation process as previously described. For a more detailed discussion of marker-assisted selection of tree fruit, see Luby and Shaw (2001).
Gene identification utilizes the close linkage between a molecular marker and a functional gene to allow for "chromosome walking" and, ultimately, the isolation of that gene. The marker that is linked to a gene of interest can serve as an anchorage for the initiation of map-based positional cloning, from which a physical map can be established. Narrowing a gene of interest down to a limited region of a chromosome requires further fine mapping analysis to ensure the gene is located between two known molecular markers. The genomic region identified as carrying the gene is then subjected to genome sequencing, verification of complementary DNAs, identification of sequence change or rearrangement, and functional analysis (e.g., functional complementation or reverse genetics such as transfer DNA insertion or gene silencing). These analyses ensure that the gene isolated dictates the observed phenotype. The map-based approach has been successfully used for gene isolation in herbaceous species including Arabidopsis thaliana, maize, and tomato.
Microarray technology is used to isolate genes based on the analysis of gene expression in a particular cell type of an organism, at a particular time, under particular conditions. For instance, microarrays allow comparison of gene expression between healthy and diseased cells. Microarrays are based on the binding of complementary single-stranded nucleic acid sequences. Microarrays typically employ glass slides, onto which DNA molecules are attached at fixed locations. Ribonucleic acid (RNA) species isolated from different sources or treatments are labeled by attaching specific fluorescent dyes that are visible under a microscope. The labeled RNAs are hybridized to a microscope slide where DNA molecules representing many genes have been placed in discrete spots. By analyzing the scanned images, the change of expression patterns of a gene or group of genes, in response to different treatments or developmental stages, can be identified. The identified genes serve as potential candidates for further functional characterization.
The value of gene identification, whether through the use of molecular markers and chromosome walking, microarrays, or other technologies, is the availability of these plant-based genes for genetic transformation and targeted plant improvement. Currently, most genes available for plant transformation have been isolated from microorganisms, and only a few from plant species. The isolation and use of plant genes, particularly from woody species, for transformation will be an important step in the genetic improvement of temperate tree fruit.
The current revolution in genetics will have a dramatic effect on plant breeding. The new genetic technologies are nowhere more needed than in temperate tree fruit breeding, a process that is relatively slow and inefficient. At the same time, the relative lack of genetic information and the difficulties of regenerating most temperate tree fruit make application of the new biotechnologies most difficult for these species. The close collaboration of molecular biologists and fruit breeders will be critical to progress both in basic research on fruit genetics and the application of new knowledge and technologies to the development of new improved temperate tree fruit cultivars.
Related Topics: CULTIVAR SELECTION; PROPAGATION; ROOTSTOCK SELECTION
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