The genetic behavior of unstable alleles controlling aleurone layer color following pollen treatment with exogenous DNA
--M.K Haritonova, N.V. Krivov and V.N. Lysikov

Instability of the heterochronous mutation Cg2, like that of any other locus within the maize genome, is most probably due to the activity of a genetic factor whose presence can be inferred from the Cg2 -----> Cg2+ and Cg2+ ----> Cg2 mutational changes (Krivov, MNL66:53-54, 1992). One possible way of detecting such a factor is to transfer it to a homogeneous genetic background by treating pollen of the appropriate recipient with exogenous DNA isolated from maize plants carrying the Cg2 alleles. We suspected regulatory sequences to be integrated into pollen grains far more frequently as compared with structural genes, and expected the appearance of plants with the Cg2 phenotype or of unstable mutations at some genomic locations.

Therefore, in order to test the assumption of a genetic factor controlling the cg2 locus instability, pollen of the marker line wx sh was treated with exogenous DNA isolated from plants of genotype zb*-8 lg g12 v4 Ch/+++++ Cg2 and applied to stigmata of the same marker line wx sh.

The pollen was collected in the morning, separately from each tassel, by shaking manually, and immediately mixed with a buffer solution containing donor DNA. For a better penetration of the exogenous DNA into pollen grains, the DNA preparation was diluted with a mixture of 0.3M sucrose and TE buffer consisting of 10ml 0.01M nhbc HCl, pH 8.0 and 1ml 0.001M EDTA. The DNA was diluted in this buffer until its concentration reached 100 mg/ml, and pollen was admixed to it with energetic stirring to produce a paste-like mixture. Then the resulting paste was applied to the stigmata of the previously prepared ears of the recipient plants. The control plants received pollen mixed with the buffer containing no DNA.

Grain content of the control ears was lower (out of 10 ears, only one set 6 seeds) than that of DNA-treated ears (8 ears out of 10 set 2, 3, 3, 5, 6, 7, 9 and 16 seeds). This supports the assumption (Mishra et al., 1987) that the introduction of high molecular weight DNA molecules into pollen is possible without significantly reducing pollen viability.

Pollen treatment in the M1 resulted in 8 plants forming ears, two of which carried mutant kernels. These were visually detectable as colored dots in the aleurone layer against the colorless background of the kernel. The dot color and pattern are very similar to those of Dt on 9S. The Dt:colorless ratio in these ears was 10 Dt:3 colorless and 5 Dt:2 colorless, respectively. In the M2, one more plant was detected carrying an identical mutation, as indicated by the fact that its ear exhibited the same pattern of colored dots, at a ratio of 7 Dt:16 colorless.

It can be seen from Table 1 that, on the whole, the progeny of five plants grown from Dt and colorless kernels largely consists of either Dt kernels (4/6, 4/7, 4/8) or colorless ones (1/1, 1/2, 2/2, 3/2, 3/4, 4/3, 4/4). However, upon selfing the plants grown from colorless kernels, a large proportion of the progeny of two of these (3/3 and 3/5) had Dt kernels, and upon selfing the plants grown from Dt kernels, significant deviations from the theoretically expected 3:1 ratio were observed, either due to an excess (2/3, 3/1, 3/9, 4/2, and 5/2) or deficit (3/6, 3/7, 4/1, 4/5, and 5/3) of colorless kernels.

Table 1. Individual tests for the progeny of Dt-like and colorless kernels in the M2.
Color distribution in progeny
Plant No. Parental Kernel Color Colorless Dt Self-colored Total x2
1/1 colorless 225 225
1/2 Dt 6 6
2/1 Dt 26 16 42 30.5
2/2 colorless 44 44
2/3 Dt 58 114 172 7.0
3/1 Dt 21 6 27 40.1
3/2 colorless 10 10
3/3 colorless 19 9 28
3/4 colorless 14 14
3/5 colorless 9 13 22
3/6 Dt 2 37 1 40 8.5
3/7 Dt 2 30 32 6.0
3/8 Dt 3 13 16 0.33
3/9 Dt 36 14 1 51 56.5
4/1 Dt 1 38 39
4/2 Dt 61 11 72 137.0
4/3 colorless 38 38
4/4 colorless 111 111
4/5 Dt 2 18 3 23 3.3
4/6 Dt 22 22
4/7 Dt 51 51
4/8 Dt 10 1 11
5/1 Dt 1 5 6 0.2
5/2 Dt 10 8 18 9.0
5/3 Dt 3 43 46 8.4

It is quite evident that the isolated alleles mutate at a high rate in somatic and generative cells. These mutation events are most likely to be due to genetic element transpositions within the maize genome. However, we will refrain from a detailed genetic analysis of induced instability since the scale of the testcrosses performed is still not large enough. Also, it is necessary to test this new mutator system for interaction with the existing, well-studied mutator systems, as well as to try to determine the class of the genetic element (receptor or regulator) integrated into the marker line wx sh genome.

The unstable mutation induction could most probably have occurred by integration of a regulator resembling the Dt element into the recipient genome, the allele for anthocyanin synthesis already carrying a receptor element responding to the Dt-like element. This resulted in some cells acquiring violet color against the colorless aleurone background as early as the M1. The receptor element is less likely to be integrated into the allele for anthocyanin synthesis in the recipient, since its integration into the allele fails to produce the dotted-like pattern observed by us in some of the recipient ears. The dominant allele expression in some aleurone cells is always associated with the receptor excision, resulting in a reversion to the wild-type allele. A simultaneous integration into the recipient genome of both the regulator and the receptor appears unlikely. Currently, all three assumptions are being tested. However, even now it is clear that the Dt-like element is dominant, which is indicated by the 3/8 plant segregation and by the colorless x Dt testcrosses performed, where a 1:1 (8 Dt:12 colorless) segregation ratio was observed in F1. 

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