Genetic methods of producing monoploids in maize can be combined with current tissue culture techniques. The indeterminate gametophyte (ig) method of generating monoploids (Kermicle, 1966, Science 166:1422-1424) is particularly convenient because parental monoploids are produced in a useful frequency. This allows the inbred A188, or any genotype which readily produces tissue cultures capable of plant regeneration, to be used as the male parent in crosses with W23 ig. The resulting parental monoploids have the nuclear genome of the male parent and should readily culture.
The R-nj marker system (Nanda and Chase, 1966, Crop Science 6:213-215) of identifying monoploids by mature seed anthocyanin pigmentation has been used extensively. Although previous work has shown that it is possible to identify and recover monoploid cultures from immature embryos using R-nj as a marker, the frequency was lower than expected (Green and Donovan, 1978, Intl. Congress Plant Tissue and Cell Culture, Calgary, Canada, p. 157). This may have been due to variability in R-nj pigmentation in immature embryos isolated at a size suitable for producing cultures capable of plant regeneration. Experiments varying the growth media components, both organic and inorganic, have not improved the pigmentation response of immature embryos. Several R alleles, R-sc, R-scm:2, and R-scm:3, have been tested and produce scutellar pigmentation in immature embryos more reliably than R-nj. Crosses have been made to combine ig with each of these three alternate R alleles. The homozygous ig R (R-sc, R-scm:2, R-scm:3 plants will be crossed by A188 and embryos isolated and cultured to compare the pigmentation response of the R alleles, and to establish haploid cultures capable of plant regeneration.
A second method of obtaining monoploid maize cultures is being tested. Isolated shoot apices of fourteen-day-old seedlings can be used as explant tissue for initiating cultures capable of plant regeneration (Rice et al., 1978 Symp. Propagation of Higher Plants Through Tissue Culture, Knoxville, Tenn.). This allows a seedling marker, such as purple coleorhiza, glossy, or yellow-green, to be used in the identification of monoploids. For example, seed from crosses of W23 R-nj ig x A188 gl can be screened for scutellum color and the monoploids tentatively selected. After sterilization and germination, the remaining hybrid diploid seedlings can be discarded and the parental monoploids (glossy seedlings) sectioned for callus initiation. Root tips are easily obtained at this point for cytological confirmation of monoploidy.
The seedling method potentially offers several advantages over the immature embryo technique. First, it permits the use of both seed and seedling markers, for more efficient selection of monoploids. Secondly, no alteration in normal gene expression is necessary, contrary to the early pigmentation needed with the R alleles in immature embryos. Thirdly, only the putative monoploids are handled under sterile conditions. In addition, putative monoploid seeds can be accumulated and germinated as needed to initiate the desired monoploid cultures. Lastly, cytological confirmation of monoploidy coincides with callus initiation.
Carol A. Rhodes and C. E. Green
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