c. NECESIDADES DE PROFESORADO Y OTROS RRHH

A sunflower ovule is initiated in the base of an ovary, about 7-10 days before flowering.

The ovule primordium is formed by periclinal divisions of cell in the subepidermal placental layer (Figure 1a, b, c).

Complimentary Contributor Copy

First stages of ovule development in the sunflower.

Figure 1a, b, c – tubular flower at the stage of ovule primordium initiation.

Figure 2a, b – the ovule primordium with initial cell in subepidermal layer.

Figure 3a, b – the initial cell elongates and starts mitotic division.

Complimentary Contributor Copy

First stages of ovule development in the sunflower (Continued).

Figure 4a, b, c – the initial cell divided into archesporial cell and basal cell, integument initiates in epidermal layer of ovule, the ovule starts being curved.

Figure 5a, b – the archesporial cell elongates, the integument actively develops, the vascular band initiates in ovule.

Complimentary Contributor Copy

First stages of ovule development in sunflower (Continued).

Figure 6a, b, c – tubular flower at the stage of megasporocyte formation, the ovule curves in anatropous position.

Complimentary Contributor Copy

First stages of ovule development in sunflower (Continued).

Figure 7a, b – megasporocyte before meiosis: the first stage of integumentary tapetum formation – two cell in epidermal layer start flatten.

Figure 8 – megasporocyte before meiosis, integumentary tapetum is formed around megasporocyte.

Complimentary Contributor Copy

Embryo sac formation and anomalies in ovule development in sunflower. 9 – ovule with chalazal megaspore, three micropilar megaspore degenerated, walls of integumentary tapetum cell are thickened, 10 – mature embryo sac, 11 – mature embryo sac and aposporous embryo sac with egg apparatus, antipodal cells and two polar nuclei, 12 –ovule with a cell complex instead of a embryo sac, 13 – ovary with mature main embryo sac and aposporous embryo sac, 14 – mature embryo sac, integumentary tapetum forms derivates around the embryo sac, 15– ovule without main embryo, 16 – ovule without main embryo sac and with aposporous embryo sac, 17a,b,c – ovule with integumentary embryo in degenerated embryo sac: a,b – neighboring section of one ovule, c - integumentary embryo, 18 – ovule with embryo and endosperm.

Complimentary Contributor Copy

1-4 – CMS line VIR 114 A, 5-8, 18 – cv. Peredovik, 9-17 – CMS line VIR 116 A;

1-13, 17-18 – longitudinal slides were stained with Heidenhain's hematoxylin and alcyan blue, 14-16 – total ovule (phase contrast).

ac – archesporial cell, aes – aposporous embryo sac, anc – antipodal cell, an – anther, bc – basal cell, bn – basal area of nucellus, cc – cell complex, ccn – central cell nucleus, ch - chalaza, cln – cell of lateral area of nucellus, dea - degenerating egg apparatus, ec – egg cell, em – embryo, en – endosperm, es – embryo sac, ic - initial cell, ie – integumentary embryo, ii – initial of integument, hc – hypostase cell, in – integument, it – integumentary tapetum, izi – inner zone of integument, mc – micropile, msc – microsporocyte, ms – microspore, ozi – outer zone of integument, ov – ovary, pn – polar nuclei, pt – pistil, sec – subepidermal cell, tc – table-like cell, vb – vascular bands.

Scale bars: 1b, 2a, 3a, 4b, 5a, 6b, 7b, 8, 9, 10, 11, 12, 17a,b,c, – 30 mkm, 1a, 4a, 6a – 150 mkm, 13 – 300 mkm.

Complimentary Contributor Copy

Previous studies suggested that usually 1-2 (for wild species up to 3-4) archesporial cells were formed in subepidermal layer of the primordium ovule, which then develops into megasporocyte directly without any mitotic divisions (Ustinova, 1964; Dzyubenko, 1959;

Newcomb, 1973a; Toderich, 1988, etc.). In this study, a different order of structures formation were observed. One to two (rarely 3) subepidermal cells (sec) divided mitotically and gave rise to initial (ic) and first basal cells (bc1) located under initial cell in the ovule primordium of cultivated sunflower. That results in couples of cells located around future long axis of an ovule (Figure 2a,b). The ic cell of the central couple divided once again periclinally and formed an archesporial cell (ac) on the top and second basal cell (bc2) on the bottom (Figure 3a,b, 4a,b,c). Both basal cells (bc1+ bc2) became a part of basal area of nucellus (bn). Other pair of cells (analog ic+bc1 cells) located around the archesporial cell formed a lateral area of nucellus (cln) (Figure 4c, 5b, 6c, 8, 9). The archesporial cell represented an apical area of nucellus.

Thus, the apical, lateral, and basal areas of nucellus were identified on the early stage of sunflower ovule development. About the division the nucellus onto different areas see Shamrov, 1998, 2008. Therefore, the ovule of a sunflower can be considered not as tenuinucellar, but rather as medionucellar, with sindermalny variation and single-layer sub-variation, according to Shamrov's classification (2008). According to Endress‘s classification (2011), it would be an incompletely tenuinucellar ovule, that is without a hypodermal cell layer between the megasporocyte and nucellus apex, but with hypodermal tissue at the nucellus flanks and below megasporocyte.

The single integument (in) was developed in epidermal placental layer at the same time as the formation of an archesporial cell (Figure 4b,c, 5a,b). The layer of hypostases cells (hc) developed at the level of initial cells of integument and on top of them, the basal cells (bc1) differentiated. An axial row was formed as ac+bc2+bc1+hc (Figure 4c). The basal cells bc1 increased in size and became strongly vacuolated, with enlarged and poorly stained nucleus.

Because two to three such cells were present in an ovule, they formed a peculiar light zone transparent in the center of the ovule. The basal cell located under the archesporial cell was larger than the latter.

Archesporial cell (ac) elongated and gave rise to megasporocyte (msc). At the same time the internal epidermal cells of integument located nearby also underwent changes. They started differentiating into integumentary tapetum (it) which was well expressed already at the stage of mononuclear embryo sac and consisted of one layer of dark-stained, 1-2-nuclear, flattened cell of table-like form with thickened walls (Figure 7a,b, 8, 9). Later, the integumentary tapetum became a two to three layers deep and formed derivates around the mature embryo sac (Figure 10, 11, 14), as typical for Asteraceae (Musial et al., 2012, 2013;

Bencivenga et al., 2011).

During differentiation of archesporial cell to megasporocyte, the cell of basal area of the nucellus underwent one more mitotic division and formed two cells of table-like form (tc1 and tc2). An axial row was formed as msc+tc1+tc2+bc1+hc (Figure 6c). The table-like cells and basal cells together formed the basal area of nucellus. The cells of the basal area of nucellus remained for a long time and formed a pathway from an archesporial cell (or megasporocytes and megaspore) to hypostasis. Formation of rows of table-like cells, the cells of hypostases, in a broad sense, under a megasporocyte is a characteristic for a number of floral plants, in particular for cereals, such as wheat and corn (Batygina, 1974, 1987, Voronova et al., 2003). It was noted that the archesporial cell of the wheat is terminating an

Complimentary Contributor Copy

already existing number of the cells located along a polar axis of nucellus (Batygina, 1974).

As one can see, in sunflower the differentiation of archesporial cell begins with the formation of a special cell which will further form a basal area of nucellus. But unlike cereals, where rows of table-like cells remained unchanged up to seed maturation, in sunflower only two table-like cells got formed. Soon these cells got elongated and formed a narrow row together with the underlying cells of basal area of nucellus (Figure 9, 10).

Formation of apospory embryo sac took place within the row (Figure 11, 13). Apospory embryo sacs, as a rule, had the improper shape and was formed from the integumentary tapetum behind the main embryo sac, but was not adjoined to it.

Initial cells of aposporous embryo sac were positioned near the basal area of nucellus and, probably, originated from the same subepidermal cell (sec) of ovule primordium, like an archesporial cell (Table 1). Thus, the initial cells of aposporous embryo sacs arose from the cells of the nucellus, and not from the integument as proposed by Ustinova (1970). And, probably, they can be related to the cells that Dzyubenko (1959) called sporogenous cells without considering their origin.

Cells of lateral area of nucellus (cln = ic+bc1) exist only at early stages of ovule development. Initially located around an archesporial cell, they are forced out downwards during the process of growth (Figure 4c, 5b, 6c, 7a). These cells continued to put pressure on the cells of the lateral area of nucellus and shifted them down towards the chalaza with the development of megasporocyte and then megaspore. The cells of the lateral area of nucellus were compressed, but remained distinct up to the stage of mononuclear embryo sac (Figure 8, 9).

The young ovule of sunflower is ortotropuos (Figure 1a,b, 2a, 3a), but one side of the integument got curved because of the more intensive growth (Figure 4a, 5a, 6a,b). The mature ovule of sunflower is anatropous (Figure 13), unitegmic, with integumentary tapetum or endothelium, like in other Asteraceae.

Table 1. The sequence of divisions at an early stage of sunflower ovule development

Complimentary Contributor Copy

The ovule showed a zonal differentiation, that is particularly visible in the central part.

The cells of the outer zones were elongated with thin cell wall, whereas the cells of the inner zone were disintegrated and had thick cell wall intensively stained with alcian blue (Figure 9, 12, 13, 17a,b, 18). The vascular bundle passed through chalaza and penetrated integument almost reaching the micropyle (Figure 5b, 6c, 7a), like in some other Asteraceae species (Musial et al., 2013).

The megasporocyte developed directly from archesporial cell without any mitotical division (Figure 5a,b, 6b,c, 7a,b, 8). The meiotic divisions of megasporocyte produced a linear tetrad of haploid megaspores. Three of them degenerated and Polygonum-type embryo sac was formed from one chalazal megaspore (Figure 9, 10).

The embryo sac was completely developed by the time of pollination. The mature embryo sac consisted of the three-cellular egg apparatus, the central cell with big, fused, secondary nucleus, and antipodal cells (Figure 10). Egg apparatus consisted of three pear-shaped cells: two synergids and the egg. The egg cell nucleus was rather large, with obvious nucleolus, and positioned in the apical part of the cell. The synergid nuclei were barely distinguishable and positioned in the center of cells. A filiform apparatus with hamiform evagination was found in the basal part of synergids. The bulk of cytoplasm of the central cell was located near the egg apparatus and thin bundles between large vacuoles. The large central cell nucleus was located near the apical end of the egg cell. Antipodes were linear. Antipodal cells were strongly vacuolated, with the large, polyploid nuclei. Usually, there are only two antipodes in the mature embryo sac. Antipodal cell were adjoined to the central cell, usually larger, underlying, and can increase 2-3 times in size. The remains of the third antipode were sometimes observed in the chalazal end of embryo sac. The antipodal complex as a whole took half of the general linear size of the embryo sac.

An additional embryo sacs were found in the ovule of the plants pollinated by perennial wild species. They were found at different times after pollination, from two hours to nine days, and in different combination of pollinations (see Table 2).

The relative position of additional and main embryo sacs confirmed that the additional embryo sac was aposporous. Besides, the groups of big megaspore-like cells were found around chalazal end of main embryo sacs at earlier stages of development (Voronova, Gavrilova, 2007). These are the cells of the basal area of the nucellus that have originated from the subepidermal cells of the ovule (see Table 1).

The main embryo sac in all cases had a normal structure that is characteristic for sunflower. Aposporous embryo sac included the same elements as the main one: the egg cell, the synergids, the central cell with polar nuclei or secondary nucleus, and antipodes. All elements of additional embryo sac were similar to those of the main, but differed in sizes and by the level of differentiation. There were more than three antipodal cells (Figure 11).

Aposporous embryo sacs lied below the level of integumentary tapetum. Absence of integumentary tapetum around an additional embryo sac leaded to a variety of shapes and sizes of this embryo sac. Apparently, the integumentary tapetum plays a role of a bearing element and gives definite form to the whole structure of the main embryo sac.

The asynchrony in development of the main and additional embryo sac was noted. The additional embryo sacs, as a rule, delayed in development. For example, the main embryo sac could be fully formed while the polar nuclei of additional embryo sac did not fuse yet (Figure 11).

Complimentary Contributor Copy

Table 2.

Anomalies in ovule development after pollination of CMS was caused by disorders in the preceding meiotic divisions (Pustovoit et al, 1976). Also they noted that in some tetraploid sunflowers the ovule is underdeveloped in general and lacking the embryo sac (Efremov, 1967). In this investigation, all ovule structures were formed normally and phases of their development and relative positioning of elements corresponded to the stages of flower development. Two small and 1-3 large cells with light, strongly vacuolated cytoplasm and small nuclei were found in a cavity surrounding integumentary tapetum, instead of an embryo sac (Figure 12). Possibly, these cells are derivatives of nucellus. Usually, nucellus cells appear squeezed by the developing megasporocyte and degenerate by the time of formation of an embryo sac. It is possible to assume that the transport of nutrients in this zone remains in the absence of embryo sac, and, nucellus cells remain viable for a long time in the absence of effect from outside megasporocyte.

Lack of the main embryo sac and formation of additional embryo sacs could be observed at the same time in the same ovule (Figure 16). In case of formation of seeds from similar ovule, the embryo in them could be formed by apomixes only.

Integumentary embryos were formed in some ovules of CMS line VIR 116 (Figure 17a,b,c). after parent plants were pollinated by H. occidentalis pollen. Though pollination was carried out, fertilization was not observed even 7-9 days after the pollination. Normally, both, the embryo and endosperm were observed in the ovule at that time. But in some plants of CMS line VIR 116 A, the embryo sac still remained in an 8-shaped form at this stage. It

Complimentary Contributor Copy

was still possible to discern the egg apparatus, the central cell with the secondary nucleus, and the antipodes (Figure 17b). Sometimes the egg cell was significantly increased in size, while synergids were compressed and darkened. The egg nucleus could be larger than the secondary nucleus of the central cell. The cytoplasm of the central cell occupied wall position with a huge vacuole in the center. The secondary nucleus was located near the egg cell.

The integumentary tapetum became multilayered and folded, and its cells were transparent with small and poorly visible nuclei. The integumentary tissues near the chalazal pole of the embryo sac were destroyed, the cells were disintegrated and cavities were formed.

Integumentary embryos were located in the chalazal region of the integumentary tapetum with their apical poles turned to the micropyle, which is distinct from the sexual embryo (Figure 17a,c). The embryos consisted of 1-2 suspensor cells and 7-8 embryonic cells. Cells were dark-colored and had large nuclei. The lighter nucleoli with dark-colored center were visible in the nucleus (Figure 17a). These embryos corresponded to Asterad-type Senecio – variation (Voronova, 2010).

In control pollination of VIR 116 A line with the pollen of the fertile analogue of VIR 116 B and of cv. Peredovik the formed embryos and endosperm at the similar stage of ovule development were observed (Figure 18); later the seeds were fully formed. Unfortunately, the development of fully formed seeds after pollination of the CMS line with pollen of wild sunflower species were not observed. The seeds formed in the head did not contain embryos.

It is known that interspecific hybridization in sunflower is possible, but the number of seeds formed is very insignificant, such as 1-10 for a head (Gavrilova and Anisimova, 2003).

C

ONCLUSION

Three types of deviations in the reproductive system of CMS lines were found during this study: absence of an embryo sac; apospory; and integumentary embryony. The last two represent different forms of apomixis.

It was noted that apomixes phenomenon is connected with hybridization in different systematic groups (Carman, 1995). CMS lines obtained a sterile cytoplasm from wild sunflower species (Leclerq, 1969) and, therefore, have an interspecific hybridization in their pedigrees (Voronova, Gavrilova, 2007). It is possible, that the reproductive system of CMS lines is less stable than in cultivated species and cultivars. Therefore, the alternative ways of development, such as apospory and integumentary embryony, are rare for the cultivated sunflower but typical for other representatives of the Asteraceae family (Noyes, 2007) and work under stress conditions, for example pollination with an alien pollen, delay or the full absence of the fertilization.

Based on the earlier publications (Molchan, 1973; Petrov, 1988; Bencivenga et al, 2011),, it could be assumed that the pollination of cultivated sunflower with the pollen of wild Helianthus species and the absence of normal fertilization can stimulate appearance of different anomalies in the morphogenesis of ovule structures. The disturbance in fertilization can lead to interruption of normal ―cross talk between the sporophyte and megagametophyte‖

(Bencivenga et al, 2011) resulting in the alternative way of development of reproductive system in sunflower lines.

Complimentary Contributor Copy

The interspecific hybridization method was used during the breeding work on sunflower and the pollination process was changed repeatedly. At first, there was a search of self-fertile forms and then, among them, the sterile lines were located. CMS lines were maintained using so called "B lines" or fertile analogs. For production of heterosis industrial hybrids, the process of pollination was changed again and CMS lines were cross-pollinated. The balance existing in reproductive system of stable cross-pollinated cultivars was apparently disrupted in lines and hybrids.

Integumentary embryony, as one of apomixes forms, represented indubitable theoretical and practical interest because it implies embryo formation from cells of somatic tissue of ovule. By origin, integumentary embryos represent clones of a parent organism; their formation can result in genetic heterogeneity of seed (Batygina, Vinogradova, 2007). This means that in the progeny of a single plant some of the seeds may have a paternal heredity and part of the maternal. Furthermore, both parents can participate in the endosperm formation. For this reason, possibility of integumentary embryos formation in cultural sunflower perhaps may explain the deviations from expected result of crossings, so-called partial hybridization and lack of splitting in hybrids. These were noted by different authors (Lyashchenko 1940, 1948; Gavrilova et al., 1997; Faure et al., 2002; Gavrilova and Anisimova, 2003). When sunflower was pollinated by wild species under interspecies hybridization, part of progeny tended to resemble a cultivated sunflower, whereas when wild species was pollinated by sunflower, the progeny looked like wild species. It is possible that genes of the endosperm inherited from both parents would be included in the work at the early stages and affected the development of the embryo.

It was observed in other plants that the initial cells of integumentary embryos are formed at a certain stage of ovule development, corresponding to the time when the normal embryo should start to develop (Naumova, 1993, 2008). In this study, a similar picture was observed.

The alternative (aposporic) embryo sacs were formed if the violation in the development of basic reproductive structures occurred at an earlier stage. The integumentary (somatic) embryos were developed if the violation has occurred at a stage when zygotic embryo must be formed. Apospory embryo sac and integumentary embryo were formed at the determined stage of ovule development, the first during the formation of female gametophyte, and the second during the development of zygotic embryo. Formation of the alternative and additional structures depends on general stage of the ovule development and on the time that passed after pollination.

A

CKNOWLEDGMENTS

I am grateful to Dr. V. A. Gavrilova (N.I.Vavilov Research Institute of Plant industry) and Dr. V. T. Rozhkova and Dr. T. T. Tolstaja (the Kuban experimental station) for their help in obtaining the experimental material and Dr. T. Lobova for her help in editing the

I am grateful to Dr. V. A. Gavrilova (N.I.Vavilov Research Institute of Plant industry) and Dr. V. T. Rozhkova and Dr. T. T. Tolstaja (the Kuban experimental station) for their help in obtaining the experimental material and Dr. T. Lobova for her help in editing the

In document IMPRESO SOLICITUD PARA MODIFICACIÓN DE TÍTULOS OFICIALES 1. DATOS DE LA UNIVERSIDAD, CENTRO Y TÍTULO QUE PRESENTA LA SOLICITUD (página 114-117)