Ботанический журнал, 2021, T. 106, № 7, стр. 658-664

Development of the male gametophyte

The study of the introduction of plants should primarily be aimed at identifying the features of the generative sphere development, including the development of the male gametophyte. The first sampling of male buds, carried out on May 6, 2012, showed that the microsporocytes in the microsporangia were isolated, i.e., meiosis had already begun in them (Table 2). Moreover, in P. abies (an indigenous species), this process was at a more advanced stage. Further observations revealed that the general pattern of meiosis in all studied Picea species is basically the same.

The calendar dates of individual stages of microsporogenesis and microgametogenesis in the studied species in 2012 are shown in Table 2. It was found that in microsporocytes of a separate sporangium, meiosis begins almost synchronously, but in its course, deviations in the rate of passage of individual phases are observed in different cells. Therefore, the percentage of cells in one or another phase was given in the table to characterise these deviations.

The time period from the beginning of the active phases of meiosis (diakinesis and further to the stage of microspore tetrads) in P. pungens is 6 days, and in P. abies – 4 days. At the stage of microspore tetrads in all studied species, synchronization of cell development occurs, i.e. ‘lagging’ microsporocytes ‘catch up’ in development with the others at this stage.

The study showed that the studied Picea species differ quite noticeably in the timing of the meiosis in microsporocytes. In 2012, the stage of microspore tetrads in P. abies was recorded on May, 8, in P. canadensis – on May, 13, in P. pungens – on May, 19. Thus, the indigenous species considerably advanced (by 5–11 days) the introduced species by the time of meiosis onset in microsporocytes.

As a result of microsporogenesis, microspores are formed. Their nuclei are divided twice according to the type of mitosis, which leads to male gametophytes formation. There are four cell divisions from the microsporocyte stage to the mature pollen stage. Mature pollen consists of the remains of two protallial, siphonogenic, spermogenic, and sterile cells. In all studied species, the development of the male gametophyte (up to the stage of mature pollen) lasted about 10 days. In 2012, pollen dispersion in P. abies and P. canadensis began on May, 29, and in P. pungens – only on June 5. The pollen dispersion lasted 2–3 days.

The repeated observations of the course of meiosis and the development of pollen, carried out in 2013, showed that on April 29, microsporocytes in the microsporangia of P. abies, P. canadensis, and P. pungens were still in the prophase of meiosis I. But already on May 5, meiosis in P. abies and P. canadensis ended, and tetrads of microspores were formed in the microsporangia. Meanwhile, the completion of meiosis in P. pungens was recorded much later – only on May, 11. Thus, microsporogenesis in 2013 in the studied species ended 3–8 days earlier than that in 2012. In 2013, mature pollen in the anthers of P. abies and P. canadensis was recorded already on May, 17, but pollen dispersion itself, due to the cooling, began only on May, 25. In P. pungens, this phenomenon began on June 3. Thus, for two years of observation, the pollen dispersion of the studied species took place almost at the same time, differing by no more than 1–4 days.

While investigating the process of microsporogenesis in Picea obovata, Yu.F. Rozhdestvensky (1981) also observed a high sensitivity of microsporocytes during prophase I to the effects of unfavorable weather factors, in particular, to fluctuations in air temperature. The author noted that a decrease in temperature caused an increase in the number of abnormal deviations in the developing gametophyte.

No investigations on the development of pollen grains in P. pungens was found. For P. canadensis, one can compare the results of our studies with others (Owens, Molder, 1979). According to this author, under the conditions of British Columbia (Canada), prophase in the mother cells of pollen of this species in 1975 began on April 14, and the appearance of mature pollen was recorded on May 26. In 1976, these stages were recorded on April 12 and May 21, respectively, i.e. almost at the same time. Interestingly, in the same area, but 500 m higher, in 1975, the prophase of meiosis and mature pollen in P. canadensis were observed much later – on May 26 and June 9, respectively. Unfortunately, this comparison gives little to explain such significant differences and understand the adaptation mechanism, since the authors did not provide data on the air temperature regime.

Thus, the studies showed that the duration and timing of meiosis phases are determined both by the biological characteristics of the species and by the specific conditions of the growing season. Most researchers consider the air temperature regime to be the decisive factor for microsporogenesis (Mergen et al., 1965; Luomatoki, 1977; Rozhdestvensky, 1981; Cram, 1984; Williams, 2009; Vorobiev, Tebenkova, 2013). Moreover, each phenological phase occurs at a certain sum of positive temperatures (Sarvas, 1970; Cram, 1984). This sum of temperatures always represents almost the same percentage of the annual sum of temperatures. This fact was determined by Professor Lensser, the director of the Pulkovo Observatory, back in 1867 (Sarvas, 1970).

According to our data, under the conditions of Karelia, the sum of effective temperatures for the full development of pollen by the time of pollen dispersion in P. abies and P. canadensis reaches 250°C, which is 14% of the sum of effective temperatures for the growing season, whereas in P. pungens – 360°C and 20%, respectively. It is known that introduced species, which start growth processes relatively early and finish them early, are most adapted to new conditions (Vstovskaya, 1983; Plotnikova, 1988; Shkutko, 1991; Popova et al., 2016). Whereas, the least adapted species are those with late start and end of vegetation. Consequently, in our study of the introduced species P. сanadensis is characterised by a greater degree of adaptation than P. pungens. Having studied these introduced species in the Nizhnii Novgorod region, R.A. Vorobiev and D.N. Tebenkova (2013) came to a similar conclusion.

Development of the female gametophyte and embryogenesis

In the development of the female gametophyte of conifers, there are two well distinguished stages: free-nuclear and cellular. The first fixations in 2012 and 2013, carried out on May 16–17, respectively, showed that the female gametophyte in all three studied species of the genus Picea is at the free-nuclear stage (Table 3). In the middle of the ovule nucellus, there is a cavity lined with a layer of secretory tissue cells, the so-called nucellar tapetum.

On the preparations recorded on June 1 (2012) and June 5 (2013), one can see that the initiation of archegonia has already occurred, and the gametophyte has a cellular structure. At this time, the central cell of the archegonia is surrounded by a single-row layer of sheath cells. The archegonia maturation in the studied species continued until early July in 2013, whereas, in 2012, this process ended in June. Several archegonia are usually formed in one female gametophyte. Introduced species often have signs of archegonian degeneration (Kishchenko et al., 1994). According to scientific literature data, the cause of archegonia degeneration in ovules of conifers is most often under-pollination or pollination with sterile pollen (Mergen et al., 1965; Khromova, 1985; Surso, 2017). Another form of degeneration of the female gametophyte in the introduced species is observed at a later stage, namely, at the stage of early embryogenesis. In this case, the egg cell and the sheath cells are no longer distinguishable, only the cell membranes are partially preserved. No traces of proembryo or embryo were found. The embryonic cavity in the developing seed is empty. Endosperm is present, but its cells are lighter than those of normal seeds. A similar pattern was observed in Larix sibirica (Trenin, 1986) and P. sibirica (Tretyakova, 1990).

The initial development stages of the coniferous plants embryo occur in the archegonia. On the preparations of the P. abies and P. canadensis seeds recorded at the end of July, 2013, the embryo was at the final stage of early embryogenesis. In P. pungens, a similar stage occurred only on August 9 (Table 2). A similar dependence but 1–2 weeks earlier was found in 2012. The embryo at this stage already has an elongated-cylindrical shape characteristic of conifers. The primordia of the cotyledons and the plume, or the embryonic apex of the shoot, can be seen at the distal end of the embryo. There is no sharp boundary between the distal and proximal poles of the embryo. Its cells are poorly differentiated and characterized by a meristematic organization. The growth of the embryo ends quickly and already in the second ten-day period of August in the seeds of the studied species of the genus Picea, it reaches normal size and consists of leaf-like formations, or cotyledons, and the stem part, or hypocotyl, located below the cotyledons. In addition to the rudimentary apex of the shoot, in the embryo, one can find a root rudiment, consisting of a meristem and a massive root cap. After this stage, visible structural changes in the embryo no longer occur (Kishchenko et al., 1994). The stage of late embryogenesis was previously recorded in P. abies August 1–9, and most recently in P. pungens f. glauca (August 11–20).

The embryo in the seeds of the genus Picea, like other conifers, is surrounded by endosperm and protected by the seed coat. The endosperm in coniferous seeds is transformed from the cells of the female gametophyte. The transformation of female gametophyte cells into storage tissue cells occurs during fertilization and in the phase of proembryogenesis. Two zones are distinguished by the type of storage substances in the endosperm. The cells of the inner zone, adjoining the developing embryo, contain mainly starch. The inner zone of the endosperm is completely resorbed during embryogenesis. In a mature seed, the embryo is surrounded by an endosperm containing lipids and proteins. These storage substances are utilised during seed germination.

It is worth noting that from 37 to 51% of the introduced seeds lack an embryo, but they sometimes contain endosperm (Table 2). The same phenomenon in Pinus sibirica under conditions of introduction was observed by I.N. Tretyakova et al. (2004) and V.S. Svintsova and I.N. Tretyakova (2014). Such deviations in indigenous species are much less common (Bazhina et al., 2018). The true nature of the anomaly remains unclear. There is a hypothesis about the existence of a so-called hidden genetic load (Koski, 1973). According to the author, most of this load remains unexpressed in the course of cross-pollination. However, even with a slight self-pollination, the effect of recessive genes quickly becomes visible. Most of the species of conifers are known to be devoid of mechanisms preventing self-fertilization after self-pollination. The relatively small number of individuals of the studied species and the distance between them prevent cross-pollination. Therefore, the hypothesis about the genetic load leading to the formation of empty seeds seems highly probable. According to I.I. Kharova (1989) and M.F. Surso (2017), degenerative processes in non-pollinated ovules and blocking the development of the female gametophyte in P. sylvestris also occur under conditions of free self-pollination. According to M.F. Surso (2017), pollination, absence of pollination, and pollination with non-viable pollen lead to a destructive type of ovule development at the early stages in most coniferous species, which is one of the reasons of underdeveloped seeds formation. The author showed that low growth energy of pollen tubes, sterility of male gametophyte, as well as death of embryos in early embryogenesis cause ovules necrosis at the later stages of their development, resulting in empty seeds formation. Consequences of self-pollination and self-fertilization in Pinaceae begin to appear in pro-embryogenesis and early embryogenesis due to the physiological incompatibility of the tissues of the embryo and the female gametophyte (Mergen et al., 1965; Forshell, 1974; Williams, 2009). These disturbances lead to an increase in the yield of empty seeds (Zavada, Taylor, 1986; Kormuták, Lindgren, 1996; Slobodník, 2002). All this leads to a sharp decrease in seed germination of the introduced species of coniferous plants (Sedaeva et al., 2008; Tretyakova, Voroshilova, 2014). Such deviations in indigenous species are much less common (Bazhina et al., 2018; Surso, 2015).

Thus, of all studied species of the genus Picea, the maximum delay in the passage of all main stages of morphogenesis, as well as the highest percentage of empty seeds, was observed in P. pungens, which indicates the least degree of adaptation of the generative sphere of this introduced species to new conditions.