Existence of pollen grains with a pair of morphologically different sperm nuclei as a possible cause of the haploid- inducing capacity in ZMS line
--Bylich, VG and Chalyk, ST

Maternal haploids in maize can be obtained when haploid-inducer lines are used as pollen parent. It is quite logical to assume that pollen contains a factor or factors determining the haploid-inducing capacity.

Enaleeva et al. (XI Intern Symp, Leningrad: 29-30, 1990) studied the events which occur in embryo sacs after pollination with pollen of the haploid-inducer line PEMS-2. This line induces nearly 8% of maternal haploids when it is used as a male parent. The development of either the embryo or central cell was established in some embryo sacs. Some embryo sacs have been discovered where development of the embryo lags behind that of the endosperm. These events are explained by failure in double fertilization. Single fertilization of an egg or of a central cell occurs. The authors supposed that the developing triploid endosperm can stimulate the unfertilized egg to divide and to develop into a haploid embryo.

In our work the pollen of the ZMS haploid-inducer line has been studied. This line induces up to 3 and more percent of maternal haploids. Pollen from MK01 line has been used as the control. Study of the pollen grains has been carried out using an automatic system which includes a light scanning microscope and a computer complex. Fresh mature pollen has been fixed in a mixture of ethanol and acetic acid. Staining has been done after hydrolysis in HCl. A sample containing 3165 pollen grains was analyzed.

It has been established that the pollen grains of the ZMS line can be divided into five types. This division has been carried out according to morphological traits of sperm nuclei. The types are as follows.

1. The pollen grains with two normal well developed sperm nuclei (NN) belong to the first type. The percentage of such pollen grains is 93.50%.

2. The pollen grains with sperm nuclei, still incompletely structured (GG), are included in the second type. Such sperm nuclei differ from the normal ones in their larger size and round shape. Perhaps, they have not undergone complete development and are not ready for fertilization. The percentage of such pollen grains is 0.09%.

3. The third type of pollen grains is characterized by the presence of two sperm nuclei smaller than normal, with configuration nonspecific for maize and increased chromatin density (gg). The presence of such sperm nuclei might result from pollen grain senescence or effects of unfavourable enviroments. Their percentage is 0.09%.

4. The fourth type of pollen grains differs from the above mentioned ones in the presence of two morphologically different sperm nuclei (NG). One sperm nucleus is quite normal for its morphological traits. The other one differs in its larger size and an uncertain round shape that corresponds to the sperm nuclei of the second type of pollen grains. 176 pollen grains belonging to the fourth type have been found, for a percentage of 5.56%.

5. The fifth type embraces the pollen grains which, like ones of the fourth type, have two sperm nuclei differing from each other. One sperm nucleus is quite normal and the other one is significantly smaller in its size and possesses increased chromatin density (Ng). The second sperm nucleus corresponds to those observed in pollen grains belonging to the third type. 24 pollen grains have been discovered and studied belonging to the fifth type, for a percentage of 0.76%.

The fourth and fifth types of pollen grains are of certain interest. We have not observed pollen grains with two different sperm nuclei in the MK01 control line. It may be assumed that the presence of a single normal sperm and a single sperm incapable of fertilization causes induction of maternal haploids. It is still difficult to judge if the pollen grains belonging to both types, NG and Ng, can serve as the haploid-inducing factor or the pollen grains of only one type possess the ability to induce haploids. In any case the total percentage of the pollen grains included in the fourth and fifth types is 6.32% of the pollen from the ZMS line. It exceeded approximately two-fold the maximal percentage of the maternal haploids which the ZMS is capable of inducing. Bearing in mind that Enaleeva et al. (1990) have observed single development either of the embryo or of the endosperm it may be assumed that a normal sperm nucleus of the fourth and fifth types (NG and Ng) can fertilize an egg, or a central cell. This can explain why the frequency of the maternal haploids induced is approximately two-fold less than that of the pollen grains with a single normal sperm nucleus.

We assume that the presence of two morphologically different sperm nuclei may result from their different speed of development. The presence in a pollen grain of one normally developed sperm nucleus and a second sperm nucleus which is either insufficiently well developed or has lost its ability for fertilization because of senescence may be the main cause for induction of maternal haploids in ZMS line. 

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