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Chromosome Transfer in Plants






Partial nuclear genome transfer in plants has generally been performed by asymmet-


ric hybridization; however, in most cases, the amount of donor chromosomes elimi- nated in hybrids tends to be low. It is even more diffi cult to eliminate chromosomes if the species involved are closely related. In plants, little progress has been made in the development of new methods for the trans- fer of a limited number of chromosomes from one species to another. Szabados et al. (1981) established a procedure for mass iso- lation of chromosomes from wheat (Triticum monococcum) and parsley (Petroselinum hortense), and showed evi- dence of introduction into parsley, maize or wheat protoplasts. Unfortunately, the cul- ture period for hybrid protoplast was too short to verify the feasibility of this proce- dure as a partial genome transfer method. Later, de Laat and Blaas (1987) microin- jected metaphase chromosomes isolated from kanamycin-resistant Nicotiana plumbaginifolia suspension cells into pro- toplasts of wild-type N. plumbaginifolia; however, only visual observation was made on the transfer process. Griesbach (1987) microinjected Petunia alpicola chromo- somes into protoplasts of P. hybrida and demonstrated biochemically that enzymes from the donor could be detected in the calli formed; however, no cytological analy- sis was performed, and no plants were obtained. One of the most promising methodologies, which proved to be an excellent tool in mammalian cells is micro- cell-mediated chromosome transfer, which for plants is called microprotoplast-medi- ated chromosome transfer (MMCT). This technique is a combination of several important steps, some of which were devel- oped more than a decade ago. For example, the fi rst step in MMCT is to induce donor chromosomes to be scattered in small num- bers throughout the cytoplasm to form micronucleated cells. This phenomenon is induced by microtubule (MT)-depolymeriz- ing agents, which prevent formation of a mitotic spindle, arresting the cells in metaphase. Micronucleation has been known in mammalian cells for many years (Phillips and Phillips, 1969); however, for a long time the lack of an efficient and


 


 

reversible anti-MT drug in plants hampered the development of MMCT. In 1987, de Laat et al. (1987) demonstrated that Amiprophos- methyl (APM) induced a high degree of chromosome metaphase arrest in cells of N. plumbaginifolia and, after a prolonged exposure, the chromosome decondensed and formed micronuclei. Falconer and Seagull (1987) observed that APM is a rapid and reversible anti-MT agent for plant cells. APM was reported to be a highly effi cient mitosis-arresting agent that induced forma- tion of a high frequency of micronuclei in N. plumbaginifolia (Ramulu et al., 1988a) and in Solanum tuberosum, Daucus carota and Haplopappus gracilis cells (Ramulu et al., 1988b). They also observed that a large per- centage of the micronuclei contained 1–3 chromosomes and suggested the application of their data to initiate a new method to pro- duce microcell hybrids in plants, which would be suitable for genetic manipulation and gene mapping. Ramulu et al. (1994) reported that the herbicide Cremart® (butamiphos) was also very effi cient as an anti-MT agent to induce mitosis arrest and the formation of micronuclei in plants.

Micronucleation is only the fi rst step in the development of a chromosome transfer procedure, because individual micronuclei must to be isolated from the cells by an effi - cient enucleation process. Lorz et al. (1981) enucleated protoplasts of Hyoscyamus muticus, Nicotiana tabacum and Zea mays using a density gradient of percoll, calcium chloride and mannitol to produce cyto- plasts and miniprotoplasts. Later, Lesney et al. (1986) enucleated Solanum nigrum L. protoplasts using a mannitol/sucrose gradi- ent. In mammals, the most effi cient enucle- ation process, which has been used for MMCT since the fi rst establishment as a partial genome transfer procedure (Fournier and Ruddle, 1977), has been CB, which is a fungal metabolite that interferes with the microfi lament attachment to the cell mem- brane (Carter, 1967) inducing the extrusion of the nuclei from the cell. Combination of this treatment with centrifugation proved to be very effi cient for enucleation in mam- malian cells (Fournier and Ruddle, 1977).


 

Wallin et al. (1978) observed that CB plus centrifugation was effi cient for enucleation of plant protoplasts; however, Lorz et al. (1981) reported reduction of viability and plating effi ciency of miniprotoplasts pro- duced using this chemical in concentra- tions from 1 to 200 mg/ml and incubation times of up to 24 h. Verhoeven and Ramulu (1991) demonstrated that a continuous iso- osmotic gradient of mannitol and percoll in association with CB was very effi cient to produce of microprotoplasts of N. plumbaginifolia. Additionally, the negative effect of CB on regeneration of subproto- plasts was compensated by a better frac- tionation and by higher yields of evacuolated, intact subprotoplasts. Ramulu et al. (1993) observed that the most impor- tant parameters for production of a large number of small microprotoplasts were the synchronization of the suspension cell cycle with hydroxyurea or aphidicolin, the presence of CB during protoplast isolation and ultracentrifugation, and passage of microprotoplasts through sieves of 48, 20, 15 and 5 mm. With these parameters, these authors were able to isolate a fraction with approximately 80% of microprotoplasts containing 1–4 chromosomes. The culmina- tion in the establishment of MMCT for plants occurred when Ramulu et al. (1995) reported the application of this method for transgenic S. tuberosum and N. plumbaginifolia as donors, and Lycopersicon peruvianum or N. tabacum as recipients. They successfully produced hybrid plants containing 24 or 48 L. peru- vianum chromosomes and one S. tubero- sum chromosome. Additionally, they observed several plants with phenotypes between that of wild tomato and potato; one of these plants had 71 tomato chromo- somes, fi ve potato chromosomes and two chromosomes with interchanged or recipro- cally translocated parts of the wild tomato and potato chromosomes. For the S. tubero- sum + N. tabacum combination, all plants resembled the recipient parent, N. tabacum; however, they expressed donor characteristics such as kanamycin resist- ance and b-glucuronidase (GUS) activity.


 


Later, Ramulu et al. (1996a) using the anti- MT agent cremart and transgenic potato suspension cells as chromosome donor, were able to produce several hybrid plants containing one chromosome of potato, car- rying a single copy of the nptll and gus genes, and a complete set of tobacco or wild tomato. In the fi rst backcross progeny of these hybrids, they recovered monosomic and disomic additions, and introgression plants showing integration of gus and nptll genes. This demonstrates that the trans- ferred potato chromosomes had a normal function in the wild tomato genome back- ground. In all the above microprotoplast hybrids, the chromosome donor parents were transgenic and the hybrids were regenerated under selection pressure. If there is a need for a donor parent to carry a selectable marker for MMCT to be applied, the broad use of this procedure would be limited. Binsfeld et al. (2000) using non- transgenic donors obtained microprotoplast hybrid plants of Helianthus annuus con- taining 2–8 chromosomes of H. giganteus or

H. maximiliani. Further analysis of 12 microprotoplast hybrids indicated that despite some meiotic abnormality (bridges, laggard chromosomes, univalent or multi- valent pairing), most of the hybrids pre- sented regular chromosome pairing (Binsfeld et al., 2001). In addition, the hybrids produced highly viable pollen per- mitting sexual transmission of the trans- ferred chromosomes to their progeny. Chromosome elimination or rearrangement also seems to occur in the progeny. These results clearly indicate that there is no need for selection pressure to maintain an alien chromosome in the recipient parent.

 

 






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