Maize Genetics Cooperation Newsletter vol 81 2007
Institute of Genetics and Physiology
Large-scale production of haploids in maize became possible when the ability to induce maternal haploids was revealed in the Stock 6 line. Stock 6 and its derivatives have a wide distribution among maize breeders, who, besides using haploids for breeding work, try to improve the haploid-inducing ability and explain the nature of this phenomenon. Producing haploids every year by applying the same inducer, MHI (Chalyk, MNL 73, 1999), we have noticed that their frequency significantly varies: depending on the method of pollination, artificial or natural (Rotarenco, MNL 76, 2002), in different maternal genotypes and even within one ear (Chalyk, MNL 73, 1999). It is known from the literature that delayed pollination increases the frequency of haploids (Randolph, 1946; Seaney, 1954; Chase, 1969). However, the opposite results have been obtained in our experiments. The main purpose of our work was to estimate the influence of delayed pollination on the percentage of kernels with haploid embryos when a haploid-inducing line was used.
A number of hybrids and inbred lines were crossed with the MHI inducer line by hand pollination. Plants were divided into four groups within one maternal genotype--with two-day, four-day, seven-day and ten-day ears. The start day of the ear-age recording was the day of silk emergence, and such ears were considered one-day ears. When the ears reached the desired age, they were pollinated with the inducer.
After harvesting, those genotypes that had rather good seed set and the best expression of the R1-nj gene (a marker gene allowing kernels with haploid embryos to be identified) were selected. Thus, four inbred lines and four hybrids were used for the experiment (Table 1). The number of ears in each ear-age group varied from 10 to 15. Therefore, the total number of ears analyzed for each genotype exceeded 40. In the lines MK01 and Мо17, a total number of plants of less than 40 were divided into three and two groups, respectively. In the Mo17xB73 hybrid there were 37 plants and they were divided into two groups.
Table 1.
Frequencies of haploid induction (%) in the four
groups of ears and in general for each genotype, coefficients of correlation.
|
Maternal genotype |
Day of
pollination after silk emergence |
General
average |
Coefficient
of correlation, r |
|||
|
Inbred
lines |
2 days |
4 days |
7 days |
10 days |
||
|
A464 |
7.3 |
5.6 |
3.9 |
4.6 |
5.4 |
-0.45** |
|
A619 |
7.1 |
6.1 |
4.8 |
5.3 |
5.8 |
-0.34* |
|
MK01 |
10.3 |
6.2 |
6.2 |
- |
7.6 |
-0.56** |
|
Mo17 |
6.82 |
5.4 |
- |
- |
6.11 |
-0.27 |
|
On average for lines |
|
6.2*** |
|
|||
|
Hybrids |
|
|
|
|||
|
Modavian450 |
6.2 |
4.4 |
4.0 |
3.8 |
4.5 |
-0.39* |
|
Porumbeni295 |
4.3 |
2.9 |
4.5 |
3.3 |
3.7 |
-0.15 |
|
Porumbeni359 |
7.0 |
4.2 |
2.6 |
2.9 |
4.1 |
-0.61** |
|
Mo17xB73 |
5.5 |
4.2 |
- |
- |
4.8 |
-0.43* |
|
On average for hybrids |
|
4.3 |
|
|||
*, **, *** significant
at 5%, 1% and 0.1% level, respectively.
In all genotypes the delay of pollination caused a decrease of frequency of kernels with haploid embryos (Table 1). According to the coefficients of correlation, this decrease was statistically significant for most genotypes. Additionally, a significant difference was revealed between thee averaged percent of haploids in the inbred lines (6.2) and the hybrids (4.3). The average number of haploid kernels per ear did not change significantly, except for the MK01 line (Table 2).
Silk
is known to appear gradually in maize.
In our experiment, the frequencies of haploid kernels on the bottom and
top half of ears were estimated (Table 3). The coefficients of correlation had negative values and in
most cases were statistically significant. This kind of estimation at a greater degree showed the
decrease of haploid-kernel frequency due to the delayed pollination. The highest percentage of kernels with
haploid embryos was on the top half of the two-day ears, 11.3 on average for
the lines, and 9.2 for the
Table 2.
Average
number of haploid kernels per ear in the four groups of ears and in general for
each genotype, coefficients of correlation.
|
Maternal genotype |
Day of pollination after silk emergence |
General average |
Coefficient of correlation, r |
|||
|
Inbred lines |
2 days |
4 days |
7 days |
10 days |
||
|
A464 |
7.8 |
7.2 |
7.8 |
5.1 |
7.0 |
-0.18 |
|
A619 |
10.4 |
13.8 |
10.1 |
8.2 |
10.6 |
-0.17 |
|
MK01 |
16.1 |
9.4 |
10.7 |
- |
12.1 |
-0.37* |
|
Mo17 |
11.2 |
7.5 |
- |
- |
9.3 |
-0.31 |
|
On average for lines |
|
9.7 |
|
|||
|
Hybrids |
|
|
|
|||
|
Modavian450 |
9.4 |
8.8 |
10.0 |
9.6 |
9.4 |
0.01 |
|
Porumbeni295 |
11.4 |
10.6 |
14.5 |
9.8 |
11.6 |
-0.04 |
|
Porumbeni359 |
11.4 |
11.2 |
6.3 |
6.4 |
8.8 |
-0.36 |
|
Mo17xB73 |
14.7 |
15.0 |
- |
- |
14.8 |
0.02 |
|
On average for hybrids |
|
11.1 |
|
|||
* significant at 5% level.
hybrids. Thus, the highest frequency of haploid kernels was in ovules/silks that at the time of pollination were the youngest.
We hypothesize that the reason for the different influence of delayed pollination on the frequency of haploid kernels in our work and in the common opinion (delayed pollination increases haploid frequency) is connected with the unique way of haploid-kernel occurrence caused by inducers. Sarkar and Coe (1966), working with the Stock 6 inducer, found a higher frequency of haploid kernels at the top half of the ears. A spontaneous frequency of haploid induction in maize is 0.1% (Chase, 1951), whereas using inducers allows haploids with frequencies from 2.3% (Coe, 1959) up to 6% (Sarkar et. al, 1994; Shatskaya et. al, 1994; Chalyk, 1999) to be produced. In our experiment, several two-day ears at the top half had frequencies that exceeded 20%. Such essential distinction between the frequencies of spontaneous and induced occurrence of haploids and the contradiction of the influence of delayed pollination can be connected with the different causes of haploidy in these two cases.
Each year among haploids we find plants that have expression of marker genes which belong to the inducer; however, the plants do not differ from other haploids by their phenotype. Probably, these results of gene transformation have some causal reasons, but there is an opinion that it might be a product of the haploid induction. In other words, instead of one normal sperm there are some fragments of its DNA molecule in the embryo sac, and one of these fragments fertilizes the ovule which provokes its development. If we take this as a fact, then the plants produced by the inducers are not real haploids.
The assumption above needs to be proved experimentally. Now, we would like to discuss the possible reasons for the decrease of haploid-kernel frequency caused by delayed pollination in our experiment. The reason that might have an influence on the frequency of haploids is heterofertilization. It was found earlier that the frequency of heterofertilization in the MHI inducer is much higher than in a genotype without the haploid-inducing ability (Rotarenco and Eder, MNL 77, 2003). Additionally, this year, an experiment with the goal of revealing the influence of delayed pollination on the frequency of heterofertilization was carried out.
Two groups of plants of a heterogeneous population with two-day (21 ears) and ten-day (11 ears) ears were pollinated by a pollen mixture made (50/50) of the pollen collected from two lines, X28C (possessing the R1-nj gene) and 092 (no marker genes); neither line was a haploid inducer. The frequency of heterofertilization in the two-day-ear group was 0.48 %, and in the ten-day ears 1.97 %. The difference between the groups was significant at the 0.1% level. The average number of kernels per ear was 250. This establishes that the delay of pollination influenced the increase of heterofertilization frequency. Most likely, it is connected with an increase in the number of pollen tubes that penetrate into an embryo sac in older silk. Probably, this occurs because of an increase in the silk diameter during the plant vegetation. Therefore, in the case of single fertilization after pollination with a haploid-inducer in older silk, the opportunity of compensation of missing sperm from another pollen tube is high in comparison with young silk. This might be the reason for the decrease in haploid-kernel frequency.
Probably, the significant difference in the haploid frequency between the lines and the hybrids in our experiment is connected with heterofertilization (Table 1). Theoretically, the silk diameter in hybrids is bigger than in inbred lines, and consequently, the frequency of heterofertilization might be higher in hybrids, resulting in a negative influence on haploid induction.
Some additional experiments are needed to before reaching a final conclusion on this problem, but these results might be useful, especially for improving haploid inducers.
Table 3.
Frequencies of haploid induction (%) at bottom and top
half of ears in the four ear groups, coefficients of correlation.
|
Maternal genotype |
Day of pollination after silk emergence |
Coefficient of correlation, r |
||||||||
|
2 days |
4 days |
7 days |
10 days |
|||||||
|
bottom |
top |
bottom |
top |
bottom |
top |
bottom |
top |
bottom |
top |
|
|
A464 |
4.2 |
8.1 |
4.8 |
4.9 |
3.3 |
5.2 |
1.1 |
4.2 |
-0.53* |
-0.37* |
|
A619 |
4.9 |
10.4 |
3.3 |
9.1 |
1.9 |
7.6 |
1.8 |
7.3 |
-0.54** |
-0.37* |
|
MK01 |
7.7 |
16.2 |
4.0 |
11.9 |
4.1 |
11.2 |
- |
- |
-0.54** |
-0.45* |
|
Mo17 |
4.0 |
10.4 |
2.7 |
8.9 |
- |
- |
- |
- |
-0.37* |
-0.1 |
|
Modavian450 |
4.0 |
9.0 |
3.0 |
6.8 |
2.9 |
5.3 |
3.1 |
5.0 |
-0.19 |
-0.46* |
|
Porumbeni295 |
2.3 |
7.4 |
1.7 |
4.4 |
2.5 |
7.1 |
1.7 |
5.5 |
-0.1 |
-0.17 |
|
Porumbeni359 |
4.3 |
11.2 |
1.6 |
7.3 |
0.9 |
4.6 |
2.3 |
3.9 |
-0.47* |
-0.61** |
|
Mo17xB73 |
3.5 |
9.1 |
2.2 |
6.8 |
- |
- |
- |
- |
-0.4* |
-0.37* |
*, ** significant
at 5% and 1% level, respectively
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