FREISING, GERMANY
Bavarian State Institute for Agronomy
CHISINAU, MOLDOVA
Institute of Genetics
Aneuploidy as a possible cause of haploid-induction in maize --Chalyk, S, Baumann, A, Daniel, G, Eder, J The technology based on utilization of maternal haploid plants induced by special haploid-inducing lines (Eder, Chalyk, TAG 104:703-708, 2002) is used increasingly in practical maize breeding. The mechanism of haploid-inducing capacity of certain lines has not been understood until now. Previously it was supposed that, in the genotypes inducing haploids, two sperms are developed with different speed (Bylich, Chalyk, MNL 70:33, 1996). As a result, one of the sperm develops to a state ready for fertilization, but the other one does not. The existence of only a single normal sperm in a pollen grain could be the reason for a broken doubled fertilization and the development of kernels with haploid embryos (Enaleeva et al., Dokl. Biol. Sci. 353:225-226, 1996). But in contrast to these results, Mahendru and Sarkar (Indian J. Genet. Plant Breed. 60:37-43, 2000) could not find any difference between the two sperms in pollen of a haploid inducing line. Investigation of pollen grains has brought inconsistent results. So, further research has to be initiated to understand what processes make an egg cell develop an embryo without fertilization.

While working with maternal haploids we often observed among them some plants showing an unusual phenotype. These plants did not look like haploids or hybrids, and they expressed the dominant anthocyanin marker genes of the paternal inducer-line. As a rule, these plants developed slowly, were later and often sterile. In different years we observed such plants with a different frequency, usually in a range from 0% to 1%. Root tip-analysis of these plants showed that most of them were aneuploid.

Since the plants were the result of a cross between different breeding populations and a haploid-inducing line it could be supposed that the source of the aneuploid gametes was the haploid-inducing line. When maternal haploids are produced, the haploid-inducing line is used as a paternal parent. Consequently we can suppose that a portion of the pollen grains in haploid-inducing lines has aneuploid sperms.

To test this supposition, chromosome-numbers in microsporocytes of two haploid-inducing lines, MHI and M471H, were analyzed. The cells were analyzed in meiosis at the stage of diakinesis. For comparison, the same analysis was conducted for two normal lines, A619 and MC3. During analysis a number of aneuploid microsporocytes were observed. The results are presented in Table 1. In the line MHI, which was grown in the greenhouse, the frequency of aneuploid cells was 14.7%. When the line was grown in the field the frequency of aneuploid microsporocytes was somewhat lower, 11.0%. In plants from the greenhouse the number of bivalents ranged from 8 to 12. The highest frequency was found for cells with 11 bivalents. Their frequency was 9.7%. There were 4.3% cells with 9 bivalents. From a total of 278 cells in which we determined the number of bivalents, there was only one cell with 8 bivalents and one cell with 12 bivalents. When the line MHI was grown in the field, on analyzing a total of 299 cells we observed 6.4% microsporocytes with 9 bivalents and 4.7% microsporocytes with 11 bivalents.

Another haploid-inducing line, M741H, was grown in the field only. A high frequency of aneuploid microsporocytes was observed in this line as well. In 5.7% of the cells, we observed 9 bivalents, and in 3.6% of the cells, 11 bivalents. Just one cell with 7 bivalents was observed out of 192 microsporocytes.

In the normal lines A619 and MC3, the frequency of aneuploid cells was much lower than in the haploid-inducing lines. One aneuploid cell was observed in line A619 out of 108 cells. In line MC3, four aneuploid microsporocytes were observed out of 253 cells. Among them, three cells possessed 9 bivalents and one cell had 11 bivalents. This result shows that some aneuploid microsporocytes can be found in normal lines as well. This quite agrees with the fact that in normal maize, haploid plants sometimes appear spontaneously. According to Randolph (MNL 12:12, 1938; MNL 14:23-24, 1940), the frequency of spontaneous maternal haploids normally ranges from one to two haploids per one thousand plants.

The facts that the frequency of aneuploid microsporocytes was much higher for haploid inducers than for normal maize, and that among haploids, aneuploid plants appear, let us suppose that haploid-inducing lines do produce some portion of gametes with aneuploid sperms. Aneuploid sperms break doubled fertilization and this can make an egg cell develop into a haploid embryo without being fertilized.

On the basis of the results obtained, it is possible to build a hypothesis that haploid-inducing lines can have a genetic factor, or factors, that cause abnormal division of chromosomes during microsporocyte formation. The factor or factors lead to development of aneuploid sperm. Aneuploid gametes can break doubled fertilization and stimulate egg cell division without fertilization. As a result of this process a haploid embryo can be formed from an unfertilized egg cell.

This hypothesis is supported by several experimental results obtained in Germany and in Moldova. Nevertheless, for its complete proof some additional investigations are needed. We would be greatly interested in cooperation with researchers that can make a contribution to the cytological analysis of the mechanism of haploid induction in maize.

Table 1. Frequency of aneuploid microsporocytes in meiosis of two haploid-inducing lines, MHI and M741H, and two normal lines, A619 and MC3.
 
Line  Conditions 
Total cells analyzed, no.
Total aneuploid cells
Cells with following number of bivalents
         
7
8
9
11
12
     
No.
%
No.
%
No.
%
No.
%
No.
%
No.
%
A619 field
108
1
0.9
           
1
0.9
   
MC3 field
253
4
1.6
       
3
1.2
1
0.4
   
MHI greenhouse
278
41
14.7
   
1
0.4
12
4.3
27
9.7
1
0.4
MHI field
299
33
11.0
       
19
6.4
14
4.7
   
M741H field
192
19
9.8
1
0.5
   
11
5.7
7
3.6
   

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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