Comparative characteristic of microsporogenesis polyploidy forms of an apple-tree

Cytoembryological control at all stages of a selection process is an integral part of selection programs using polyploidy; and the complex work of plant breeders and cytologists promotes increase of its efficiency.

At the first stages, while selecting parent material for selection, cytoembryological researches give an opportunity to establish peculiarities of male and female gametophyte development and, as a result, to define the quality of formed gametes of polyploid forms, to pick up the most rational combinations of crossover, in some way to predict and to estimate the results of these crossovers. For many years since 1970 [11] apple-tree selection at polyploid level has been one of ARRIHCS’s research directions. The Cytoembryological laboratory scientific team systematically studies the generative sphere of tetraploid forms of apple-trees used in the selection at polyploid level.

Materials and methods

The following forms were chosen as the object of this study: the forms one part of which consisted of diploidic and tetraploid chimeras of the I; Antoknovka ploskaya(2-4-4-4х), Giant Spy (2-4-4-4х), Papirovka (2-4-4-4х), Welsi -F (2-4-4-4x) , Welsi-M (2-4-4-4х), and another part of which consisted of homogeneous tetraploids: Macintosh (4х), Melba (4х), Spartak (4х) and the new homogeneous tetraploid forms obtained in the process of implementation of the program of apple selection at polyploid level: 13-6-106 (plantlet of Suvorovets cultivar), 25-35-120,25-35-121,25-35-144 – bred from the crossover of two tetraploid Welsi-F forms (4х) x Papirovka (4х), 25-37-40, 25-37-33, 20-9-27 – bred from the crossover of the tetraploid form of Welsi with diploid cultivar Antonovka . In this case it is likely that unreduced gametes of male parent of Antonovka cultivar took part in the fertilization. Forms 25-37-47 (SR0523, 2х x Antonovka ploskaya, 4х), 25-37-45 (Orlovskaya girlanda, 2х x Welsi-F, 4х) and 30-47-88 (Liberty, 2х x 13-6-106, 4х) - bred in progeny by crossover of the types 2х x 4х. Probably, unreduced gametes of diploidic female parent took part in formation of hybrid germs. The form 25-37-35 is bred in progeny by the method of crossover of two tetraploid forms of the Welsi cultivar under open pollination.

In some cases microsporogenesis of polyploid forms was compared with their diploidic analogs.

Miosis at microsporogenesis was studied using temporary, squash [13], and on permanent [10] preparations. Advantage of the latter method is that the structure of the generative sphere remains undisturbed and it is possible to note those features, which escape notice of a scientist when using temporary squash preparations.

Results

Study of bud cuts on permanent preparations showed that the asynchronous miosis course at microsporogenesis is typical for both diploidic and tetraploid forms of an apple-tree. Asynchrony has a general basipetal tendency and is manifested at the level of different flowers of one inflorescence: the first miosis begins in the central flower of the inflorescence, then - in the flowers appeared in the inflorescence later. Further asynchrony is fixed within one flower: the top, more developed anthers might demonstrate the end of the miosis, whereas the bottom and less developed manifest only its beginning. Moreover, asynchrony took place within different pollen sacs and within the same anther. On the longitudinal cut of one pollen sac its miosis passes as though wavy: at the same time it is possible to observe 3... 4 consecutive stages of miosis. The diploidic cultivars of apple-trees manifested similar phenomenon repeatedly as it was noted in the course of the research [1, 2, 5, 6, 7, 9, 15, etc.]. Among cornel (Cornus mas L. ) this phenomenon under the formation of microspores occurs even more often [3]. Besides, it strengthens, if the corresponding stages of organogenesis occur in poor conditions [4].

Thus, it is obvious that this phenomenon is typical of all member of Malus cultivar, and also of other horticultural crops.

Miosis of all studied polyploid forms in the course of microspores forming progressed with significant number of abnormalities at all subsequent stages. The percent of microsporocytes with abnormal way of mitosis may vary in wide ranges (from 1,7 to 96,7 %), depending on the stage of miosis and form. The total of 19 tetraploid forms have been studied. Abnormalities occurring in the course of miosis can be divided into three groups: the first group - forms with the most correct course of miosis, their quantity at all stages is ranging from 10 to 20% on the average. These are forms 25-37-45 (4х), Papiroska (2-4-4-4х), 25-35-121 (4х), 25-35-144 (4х), and 20-9-27 (4х). The second group is the most numerous. It includes 11 forms, the number of abnormalities of which makes from 20 to 40%. And the third group includes only three forms: Welsi-F (4х), 30-47-88 (4х) and Macintosh (4х). On the average, their number of abnormalities is more than 40%. The correct course of miosis at microsporogenesis is noted in the form 25-37-45 (4х). Its number of abnormalities is several times less, than that of other forms (figure 1), and microsporocytes with a normal way of fission ranges at different stages from 80 to 96%. The most abnormal course of miosis is characteristic for a tetraploid form of Macintosh cultivar.

The heterotypical division of miosis of the majority of tetraploid forms is characterized by greater number of abnormalities, than homeotypic one. The maximum of abnormalities is accounted for metaphase-I stage, then, during anaphase-I and telophase -I, it considerably decreases. At the stage metaphase -P and anaphase -P the number of abnormalities again increases a little, and at the end of miosis (the stage telophase -P, tetrads) decreases. The only exception is the course of miosis at microsporogenesis of Macintosh cultivar (4х). Heterotypical division at this form proceeds according to general for the majority of tetraploids scheme, and from the metaphase -P stage the number of abnormalities sharply increases and reaches the maximum (96,7 %) by the moment the miosis ends. The quantity of normal tetrads at this form totals only 3, 3%. Such distinctions in the course of miosis of different tetraploid forms can be explained to a certain degree. For this purpose we have to better understand the substance of abnormalities morphology. It should be noted that the number of abnormalities and a variety of morphological types of disturbances are interrelated. The forms with more correct course of miosis usually demonstrate not so broad variety of anomalies. For example, Papirovka tetraploid has more regular miosis at microsporogenesis; it has the minimum number of abnormalities. The same is true for the new tetraploid form 25-37-45. On the contrary, Welsi-F forms (4х), 30-47-88 (4х) and Mekintosh (4х) have the maximum percent of abnormalities in the course of miosis and also have the maximum variety of morphological types of abnormalities.

Comparing different separate forms we can see that the majority of them have morphological types of abnormalities, and also that certain abnormality types at the corresponding stages show a certain parallelism. In this regard the tetraploid form of Macintosh cultivar differs significantly. Its microsporocytes, except the types of the abnormalities typical for all other forms, often manifest formation of tripolar spindles. This type of abnormalities and its consequences of Macintosh (4х) are traced throughout all subsequent stages of miosis and, as a result, lead to mass formation of polyades. The number of these polyades containing from 5 to 9 microspores in one sporadic group, totals 94.2% of the total number of checked microsporocytes. This explains low viability of pollen of this form, and also the high rate of yield of diploidic seedlings in their hybrid progeny.

The phenomenon of formation of tripolar spindles was observed on miosis of the remote hybrids [14]. It is considered that this abnormality is supervised genetically. From our point of view, this statement has certain basis. IT is confirmed by the presence of tripolar spindles in miosis and of a diploidic form of Macintosh cultivar. But in case of diploidic form such microsporocytes occur with the frequency of 0,5, and in case of tetraploid form — with the frequency of 17,6%. Probably, it can proove the existence of the gene dose effect: the increase of the number of homologous chromosomes leads to change of manifestation degree of a feature

The presence in the tetraploid set of chromosomes of, at least, four homologous chromosomes is the reason for which the conjugation of homologs in miosis and the divergence of chromosomes are often abnormal, because in parallel with bivalents, they form uni - tri - and quadrivalent. Association with a valence higher than four occur rarely.

The prevailing type of abnormalities at the stage of M_I and M_II demonstrated by the majority of tetraploid forms is premature forwarding of 1... 4, rarely 6... 8 of chromosomes prior to anaphase movement in the main group. At various forms it averages between 35 to 98% of total number of abnormalities. This phenomenon probably is explained by both presence of univalents and low density of chromosomes conjugation (frequency chiasma), which is often manifested in casautopolyploids of other cultivars and in remote hybrids [12, 14].

In stages A_I and A_II the most frequent (45... 98% of total number of disturbances) abnormality is retarded fission in some significant part of associations which remain in the center of a maturation spindle of division while the main groups of chromosomes already departed to the poles of the spindle. Sometimes chromosome bridges are noted - a consequence of formation of dicentric chromosomes. Such phenomenon was observed in gamma mutants of currants [8].

It is also worth mentioning that such anomalies, as blow-out of separate chromosomes or their groups outside the boundaries of the achromatic spindle can also be often observed. Very often (from 15 to 45% of the total number of abnormalities manifested by different forms) they occur during the second division of miosis. Such abnormality leads to the formation of supernumerary figures of division in the subsequent stages and, as a result of it, formation of additional kernels and microkernels at telophase I and II stages, supernumerary microspores at the stage of tetrads.

Comparative studying of miosis of polyploidy forms and their diploidic analogs (Antonovka ploskaya, 4х - Antonovka, 2х; Papirovka, 4х - Papirovka, 2х; Macintosh, 4х — Macintosh, 2х, etc.) allowed establishing that usually the same types of abnormalities occur in tetraploid forms. Their frequency is the only thing that differs. As a rule, abnormalities at the correspondent stages of tetraploid forms occur considerably more often than at their diploidic analogs. Besides, in certain cases deviations are special for each cultivar. As a result of certain deviations in the course of miosis forms, which apparently ought to form diploidic gametes, act differently. Aside from the significant amount of the defective pollen which is forming as a result of abnormalities of numerical distribution of chromosomes, some of forms form a certain amount of haploid pollen instead of expected diploidic forms that makes essential corrections to results of selection work. In this regard, hybridization of diploidic cultivars with such forms sometimes has a considerable amount of diploidic descendants (for example, Welsi-F and Welsi-M) is formed. This circumstance should be considered when performing the crossover.

Detailed study of the course of miosis at microsporogenesis allows to conclude that despite the significant amount of abnormalities, miosis of the majority of examined tetraploid forms ended with the formation of the sufficient amount of normal diploidic pollen. The characteristic of miosis is confirmed by the results of the analysis of the ploidy of the hybrid progeny bred with participation of these forms (see the Table). Basing on the research the majority of examined tetraploid forms it can be recommended for the hybridization with the purpose of mass production of triploid seedlings. To achieve the desirable results it is necessary only to correct the volume of selection works with regard to the characteristic of concrete tetraploid forms.

Efficiency of the complex cooperation of plant breeders and cytologists is already proven in use. At ARRIHCS goal-directed crossovers of different chromosomal forms (2х x 4х, 4х x 2х) gave 36 thousand hybrid seedlings, 18 elite forms were separated, 10 new triploid cultivars, 8 of which are included in National Measurement Assurance System, 2 are released.