Hannu Ahokas
Plant Breeding Section, ARC, FIN-31600 Jokioinen, Finland. "R"
Most selection methods on transgenic plant cells, tissues or seedlings are based on chemical resistance caused by a selectable marker gene, usually of strange origin. This selectable sequence is commonly also stably integrated in the transformed genome, at least with an inducible expression. An avoidance of using such strange, sometimes potentially harmful selectable markers, here the use of some gene mutants, is described, their complementation could serve as a selectable marker using linked or unlinked gene constructs for the transgenosis. The method can be used at selection of transgenics principally in all cormophytes, the adjoining paper (Ahokas 1997) describes a potentially useful mutant for selection in barley.
Several reports of transgenic plants have shown high incidences of co-transgenosis even with unlinked plasmids with frequencies varying from 18 to 100% (Schocher et al. 1986, Damm et al. 1989, Christou & Swain 1990, Spencer et al. 1990, Herve et al. 1993, Altpeter et al. 1996, Aragao et al. 1996, Gath Jensen et al. 1996, Zhong et al. 1996).
The mutated gene sequence responsible for the marker deficiency must be known and its wild type sequence cloned adequately, or the wild type sequence made by DNA synthesis. Conditional lethals or chlorotics are maintained and multiplied under optimal conditions. The adjoining paper (Ahokas 1997) describes the barley mutant msm1-SCS which is productive at a growth temperature above 18oC, but does not survive at temperatures below 10oC. The detection of the deficient gene in msm1-SCS may be easier since apparently confined to the plastid or mitochondrial genome. On the other hand, co-transgenosis of the nuclear and organelle genome simultaneously probably confers a mosaicism in the plastom, requiring a backcross with the genetic mosaic as a pollen parent, to save only the nuclear transgenic genome.
Limits may be set for the transgenosis methods by the fact that all kinds of mutants are not prone to all methods of producing transgenics. For barley grains, the electrophoretic transfection (Ahokas 1989a, b) might be reasonable, and does not suffer from regeneration problems. Stable transgenics have been obtained with electrophoretic applications also in other species (Griesbach & Hammond 1993, Yang et al. 1993, Songstad et al. 1995).
The treated mutant plants are grown under optimal conditions. If so desired, their seeds are multiplied with another generation, or are directly subjected to selective lethalizing conditions where transgenics and possible backmutations are assumed to grow adequately or to survive. Considering the msm1-SCS mutant, a magnitude of 107 seedlings can be screened on germination trays of a few square meters during a year in a chilled and illuminated chamber without using e.g. harmful and expensive antibiotics. The viable seedlings are saved and studied as possible double transgenics and used as pollen parents to cross the nuclear transgene.
Selection of wild type transgenics could be extended to viable mutants like recessive dwarf and shrunken endosperm which show response to wild type genes in trans. With recessive dwarfs, the screening can be done under less demanding conditions. The heterozygotes of dwarfs mutants of barley, are usually distinct after a few days of growth, permitting handling and screening of great seed lots originating from descendants of transgenic treatments using the normalizing allele of the dwarf gene.
References:
Ahokas, H. 1989a: Transfection of germinating barley seed electrophoretically with exogeneous DNA. - Theor. Appl. Genet. 77: 469-472.
Ahokas, H. 1989b: Electrophoretic transfection of cereal grains with exogenous nucleic acid. - Biotieteen Päivät 1989 (Abstracts), Soc. Biochem. Biophys. Microbiol. Fenn., Techn. Univ. of Helsinki, Espoo, 2 p.
Ahokas, H. 1997: Cytoplasmic male sterility in barley. XVI. Conditional lethal albino in msm1, a double cytoplasmic mutant. - Barley Genet. Newslett. (submitted).
Altpeter, F., Vasil, V., Srivastava, V. & Vasil. I. K. 1996: Integration and expression of the high-molecular-weight glutenin subunit 1Ax1 gene into wheat. - Nat. Biotechnol. 14: 1155-1159.
Aragao, F. J. L., Barros, L. M. G., Brasileiro, A. C. M., Ribeiro, S. G., Smith, F. D., Sanford, J. C., Faria, J. C. & Rech, E. L. 1996: Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment. - Theor. Appl. Genet. 93: 142-150.
Christou, P. & Swain, W. F. 1990: Cotransformation frequencies of foreign genes in soybean cell cultures. - Theor. Appl. Genet. 79: 337-341.
Damm, B., Schmidt, R. & Willmitzer, L. 1989: Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts. - Mol. Gen. Genet. 217: 6-12.
Gath Jensen, L., Olsen, O., Kops, O., Wolf, N., Thomsen, K. K. & Wettstein, D. v. 1996: Trangenic barley expressing a protein-engineered, thermostable (1,3-1,4)-B-glucanase during germination. - Proc. Natl. Acad. Sci. USA 93: 3487-3491.
Griesbach, R. J. & Hammond, J. 1993: Incorporation of the GUS gene into orchids through embryo electrophoresis. - Acta Hort. 336: 165-169.
Herve, C., Rouan, D., Guerche, P., Montane, M.-H. & Yot, P. 1993: Molecular analysis of transgenic rapeseed plants obtained by direct transfer of two separate plasmids containing, respectively, the cauliflower mosaic virus coat protein gene and a selectable marker gene. - Plant Sci. 91: 181-193.
Schocher, R. J., Shillito, R. D., Saul, M. W., Paszkowski, J. & Potrykus, I. 1986: Co-transformation of unlinked foreign genes into plants by direct gen transfer. - Biotechnology 4: 1093-1096.
Songstad, D. D., Somers, D. A. & Griesbach, R. J. 1995: Advances in alternative DNA delivery techniques. - Plant Cell Tiss. Org. Cult. 40: 1-15.
Spencer, T. M., Gordon-Kamm, W. J., Daines, R. J., Start, W. G. & Lemaux, P. G. 1990: Bialaphos selection of stable transformants from maize cell culture. - Theor. Appl. Genet. 79: 625-631.
Yang, J., Ge, K., Wang, Y., Wang, B. & Tan, C. C. 1993: Highly efficient transfer and stable integration of foreign DNA into partially digested rice cells using a pulsed electrophoretic drive. - Transgen. Res. 2: 245-251.
Zhong, H., Sun, B., Warkentin, D., Zhang, S. Wu, R., Wu, T. & Sticklen, M. B. 1996: The competence of maize shoot meristems for integrative transformation and inherited expression of transgenes. - Plant Physiol. 110: 1097-1107.