Our objective was to develop a reliable system of obtaining monoploid maize tissue cultures using the ig system to generate paternal monoploids. Immature embryos of certain genotypes will readily initiate cultures capable of regenerating plants (C. E. Green and R. L. Phillips, 1975, Crop Sci. 15:417). The R-sc locus scutellar pigmentation system and Adh1 isozymes were chosen as genetic markers to detect haploidy.
This scheme has several improvements over other systems of obtaining monoploid cultures (C. E. Green and C. M. Donovan, 1979, Abstract 1714, 3rd Intl. Cong. PTCC, Calgary; H. S. Dhaliwal and P. J. King, 1979, TAG 55:257). The substitution of R-sc for the R-nj allele resulted in a consistently high color response which was easier to score because of the pigment location. The male parent's r-r allele was replaced with r-g, which eliminated pigmentation that could confuse the scoring of scutellum color. A second genetic marker (Adh1) was incorporated, which increased the screening efficiency for monoploids. Adh1-F and -S isozymes do not affect culture initiation and analysis requires only a small amount of tissue. Even if paternal monoploid tissue has spontaneously doubled prior to the cytogenetic analysis, the Adh1 pattern reveals the initial monoploid level. Maternal monoploidy versus accidental self-pollination cannot be differentiated.
Two major attempts were made to initiate monoploid cultures. Ears from crosses of W23 R-sc/R-sc Adh1-F/Adh1-F ig/_ x [A188/Hayes White] x r-g/r-g Adh1-S/Adh1-S were harvested 12-18 days post-pollination. Embryos were isolated and placed sequentially on standard media (0.5 mg/l 2,4-D MS). After three days of light exposure (95W/m2), pigment response was scored and the colorless embryos were transferred for a three-week period of culture initiation. Tissue samples of these cultures were assayed for Adh1 isozymes.
A consistently high proportion (93.5%) of the embryos expressed scutellar pigment. Six paternal monoploids were identified out of 7241 embryos, which was much fewer than expected. Several factors could have contributed to this discrepancy: (a) twenty-two of the colorless embryos did not grow after isolation and could not be analyzed for Adh1 isozymes; (b) embryos could not be readily found in about 6% of the normally developing kernels; (c) heterozygosity for ig in the maternal parent would have reduced the expected frequency of paternal monoploids; (d) the paternal genotype also might have influenced this frequency.
Variations of genetic and environmental factors of the basic embryo isolation procedure were tested in an attempt to improve the percent of embryos which expressed the scutellum pigment marker. Immature R embryos were placed on MS media, exposed to light and scored daily for pigmentation. After three days, the explanted embryos were either frozen and later quantitatively assayed for anthocyanins or incubated for 2-3 weeks for culture initiation.
A comparison of different R alleles showed that R-nj embryos gave a more variable response than either R-scm:2 or R-scm:3 embryos at similar stages of development. Pigment appeared on the upper surface of the scutellum with R-sc, R-scm:2 and R-scm:3, which was more easily seen than the R-nj pattern. Established diploid cultures with the R-sc allele often produced darkly pigmented tissues that resembled scutella. Cultures of the paternal genotype (both haploid and diploid) produced similar structures that were never pigmented. This apparent expression of R-sc in established cultures increases its effectiveness as a genetic marker in this system.
Increased irradiance did not alter the final percentage of pigmented embryos, but it did result in more anthocyanin per embryo. Color response increased with longer periods of light exposure although culture initiation decreased. Embryos with a colorless genotype showed the same decline in frequency of culture initiation under the same light treatments; thus the reduced culture initiation frequency was not an effect of pigmentation.
The cytokinins BAP, zeatin, and 2iP at 10-6, 10-7, and 10-8 M in the media did not affect the final color response of the embryos, although BAP increased coleoptilar anthocyanin content. All three cytokinins promoted growth of the coleoptile, which usually indicates poor culture initiation. There were no differences in color response among 2,4-D treatments of 0, 0.5, or 2.0 mg/l.
Abscisic acid (ABA) at concentrations of 1.0 mg/l or more resulted in a higher proportion of pigmented embryos, and more pigment per embryo, as well as inhibiting culture initiation. Lower doses of ABA (0.1, 0.25, 0.5 mg/l) did not promote pigmentation as much and still reduced culture initiation. Transfer of the embryos to media without ABA after 6, 12, or 24 hours avoided some of this inhibitory ABA effect, but the transfer procedure itself appeared to depress culture initiation. There was no correlation between pigmentation and culture initiation.
The combination of R-sc scutellar pigmentation with a second marker for Adh1 isozymes allowed identification of monoploid cultures from immature maize embryos under standard culture initiation conditions. Two of the six paternal monoploid cultures were capable of regenerating plants. The optimum size for culture initiation from monoploid embryos might differ from diploids because of their differences in ploidy and heterozygosity.
Carol Rhodes and C. E. Green
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