Pollen development in Douglas lk (Pseudotsuga menziesii)'

JOHN N. OWENS AND MARJE MOLDER Department of Biology, University of Victoria, Victoria, British Cobrinbia

Received January 12, 1971

OWENS, J. N., and M. MOLDER. 1971. Pollen development in Douglas fir (Pseudotsuga ~nerrziesii). Can. J. Bot. 49: 1263-1266.

The normal sequence of pollen development in Douglas fir is described and is essentially the same as that for Pinus. Mature pollen grains normally consist of five cells: two lens-shaped prothallial cells, a stalk cell, a body cell, and a large tube cell. The normal sequence of development is described as well as the structure and histochernistry of the spore wall. Possible reasons are given for earlier misinterpreta- tions of pollen development and spore wall structure in Douglas fir.

Introduction

Most standard texts describe the development of the conifer microspore into the mature pollen grain using Pinus as the example. Pinus is typical of most conifers but we should be aware that considerable variation exists within the conifers. Conifer pollen always has two wall layers, the exine and intine, but only about one-third of the genera have wings or bladders of any sort on the pollen grain. The formation of a five-celled, ma- ture pollen grain consisting of two prothallial cells, a stalk cell, a body cell, and a tube cell (formed in that order) is the prevalent course of development in conifers. However, considerable variation exists in the number of prothallial cells formed, the number of cells present when the pollen is shed, and the number and structure of gametes formed (Chamberlain 1957).

Recent studies of pollen formation have em- phasized meiosis for the purpose of determining chromosome number since pollen mother cells are ideal for this purpose. Studies of pollen growth in vitro to follow pollen development after pollination have also been given consideration. Such studies have occasionally included partial descriptions of Douglas fir (Pseudotsuga men- ziesii (Mirb.) Franco) pollen development but the fragmentary observations have resulted in confusion regarding the normal sequence of pol- len development. Barner and Christiansen (1962) stated that the pattern of pollen development in Douglas fir was uncertain since the entire se- quence of nuclear divisions had never been ob- served. In a subsequent paper (Christiansen 1969), a sequence of pollen development quite different from that of Pinus was suggested. Both

1Research supported by National Research Council of Canada grant 1982.

tube nucleus and stalk cell were suggested to have arisen during meiosis and the generative cell was considered absent during any stage of development. Consequently, an incorrect se- quence of pollen development emerged.

I t is very easy to misinterpret pollen develop- ment if all stages are not observed. Failure to observe all stages is not surprising when only whole mounts or squash preparations are used for study. From the single-celled microspore stage onward, the wall of the developing pollen grain is thick and finely sculptured. As a result, staining of nuclei is difficult and observation through the thick wall obscures details. In addi- tion, microspores become densely packed with large starch grains which remain throughout pol- len development. The combination of poor stain- ability, a thick cell wall, and abundant starch grains makes nuclear divisions difficult to see. Paraffin-embedded, sectioned, and stained pollen cones during pollen development, however, clearly show all stages without the difficulties described above.

Methods and Materials Pollen cones were collected from early March, just

after meiosis is completed, to early April when pollina- tion occurs. Cones were collected from several trees near Corvallis, Oregon in 1961 and 1962 as part of a study of cone development. These were fixed in Randolph's modified Navashin solution (Johansen 1940). Cones were also collected in 1969 near Victoria, British Colum- bia, and fixed in neutral formalin or FAA. All material was dehydrated in a tertiary-butyl alcohol series and embedded in tissuemat or paraplast according to the method of Johansen (1940). Cones were sectioned serially at 10 p and stained with safranin and hematoxylin. Histochemical tests for starch (IKI), cellulose (IKI- HzS04 method), lignin (phloroglucinol test), and pectin (hydroxylamine -ferric chloride reaction) were made on embedded material to distinguish the wall layers clearly and to describe them more fully (Jensen 1962).

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1264 CANADIAN JOURNAL OF BOTANY. VOL. 49, 1971

Observations

Meiosis in Douglas fir begins in October and passes through the early prophase stages to pachy- tene. Pollen mother cells then pass into a diffuse diplotene stage in which they remain arrested until late February when meiosis resumes. Meio- sis is usually completed by the end of February at lower elevations. Each microsporangium is then filled with several hundred tetrads of hap- loid, thin-walled microspores borne in a fluid (Fig. 1). Each microspore contains a single hap- loid nucleus and a little starch (Fig. 1). During the first 3 weeks after meiosis. no cell divisions occur within the microspores. Microspore cell walls rapidly thicken eq~ially on all surfaces. Microspores enlarge slightly but remain together within the microspore mother cell wall. Rapid accumulation of starch occurs until the cyto- plasm of each microspore is densely packed with large starch grains (Figs. 2, 9). The outer wall of the developing pollen grain (the exine) thickens to about 2 p which is equal to that of the mature pollen grain. By the last week of March micro- spores have enlarged sufficiently and rounded out enough to rupture the thin microspore mother cell wall, the fragments of which appear scattered throughout the microsporangium. After separation, microspores retain an angular ap- pearance for a short time (Figs. 2, 3). The micro- spore nucleus then divides. One daughter nucleus remains in the center of the pollen grain and the other presses against the cell wall. A new cell wall forms, dividing the cytoplasm unequally. The nucleus and a small amount of cytoplasm adjacent to the wall form the first lens-shaped prothallial cell (Fig. 3). The wall that now forms inside the exine and also arouild the first prothal- lial cell is the intine. It appears lighter in color

than the exine and is separated from the exiile by a distinct thin, dark line. The microspore nucleus in the center of the developing pollen grain di- vides a second time forming a second prothallial cell which is pressed tightly against the first. The cytoplasm is again divided unequally but the second prothallial cell, though still lens-shaped, is thicker (Fig. 4). A wall, contin~ious with the intine, forms around the second prothallial cell. The intine at this three-celled stage becomes al- ., most equal in thickness to the exine with the wall around the second prothallial cell remaining slightly thinner (Fig. 4). The microspore nucleus divides a third time forming a generative nucleus adjacent to the second prothallial cell (Figs. 5, 6). Cell wall formation again unequally divides the cytoplasm. The other daughter nucleus remains in the center of the developing pollen grain and is termed the tube nucleus. No special wall forms around the tube nucleus. The wall of the develop- ing pollen grain, consisting of exine and intine, represents the wall of the tube cell. No thick cell wall forms between the generative and tube cells, only a thin membrane. The portion of the generative cell adjacent to the second prothallial cell does form a rudimentary cell wall indistin- guishable from the intine. This wall extends very slightly out around the generative cell and forms a shallow, cup-shaped wall projecting from the wall of the second prothallial cell into the center of the developing pollen grain (Figs. 6, 8). The thin membrane enclosing the generative cell is continuous with this cup-shaped cell wall. The generative cell then divides equally forming the stalk cell adiacent to the second ~rothallial cell and the body cell adjacent to the tube nucleus (Figs. 7, 8). No thick cell wall forms around these cells; they remain enclosed within only a thin membrane. Whether this "membrane"

FIGS. 1-6. Pollen grain developnlent as seen in sectioned pollen cones during March. X 600. Fig. 1. Tetrads of microspores as they appear early in March just after meiosis is complete. Each microspore con- tains a single haploid nucleus and little starch. Fig. 2. Separate microspores after the pollen mother cell wall has ruptured. Microspores rapidly accumulate starch, shown here as small white discs, the exine (E) thickens, and the microspore nucleus (M) divides to form the first prothallial cell. Fig. 3. Division of the cytoplasm is unequal in the formation of the first prothallial cell (PI). The rnicrospore nucleus remains in the center of the cell and divides a second time forming the second prothallial cell. Fig. 4. The second prothallial cell (P2) is lens-shaped but thicker than the first. By this time, late in March, the pollen grain has enlarged, rounded out somewhat and the intine (I) becomes almost equal in thickness (2 p) to the exine. Note that the intine forms around both prothallial cells. Fig. 5. The microspore nucleus divides a third time. The abundant, large starch grains are quite visible at this stage. Fig. 6. The four-celled stage of the pollen grain. The micro- spore nucleus has divided to form the generative cell (G) adjacent to the second prothallial cell and the tube nucleus (T). The portion of the generative cell adjacent to the second prothalIia1 forms a rudimentary cell wall (W) which extends very slightly out around the generative cell.

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FIG. 7. The generative cell divides equally to form the stalk cell and body cell. X 600. Frc;. 8. The mature five-cellcd pollen grain as seen at pollination, consisting of two lens-shaped, prothallial cells enclosed by the intine: a stalk cell (S) whose base IS partially enclosed by the CLIP-shaped Intine (W); the body cell ( B ) cn- closed only by a thin membrane (Mb) ; and the large tube n ~ l c l e ~ ~ s (T). Exine, intine, and starch grains arc clearly evident. X 600. FIG. 9. Starch accum~~lates rapidly so that at the three-celled stage the developing pollen grain is filled with large starch grains. X 780. FIG. 10. Abundant cellulose is present i n the intinc. Thc swollen intinc is a r e s ~ ~ l l o f the IK I -H2S04 test for cellulose. X 780. FIG. 11. The exine appears irreg~llarly laniinated with irregular dark lines between lighter layers. X 2000.

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OWENS AND MOLDER: DOUGLAS-FIR POLLEN 1265

consists only of the cell membrane or also in- cludes an extremely thin cell wall could not be determined. The mature pollen grain of Douglas fir at the time of pollination, therefore, usually consists of five cells: two small lens-shaped pro- thallial cells, a stalk cell, a body cell, and a large tube cell (Fig. 8).

Allen (1943) reported that only about one pol- len grain in each 100 examined was at the five- celled stage while the rest were at the four-celled, generative cell stage (Fig. 6). In our material, cones showed, just before pollen release, a few pollen grains still a t the three-celled stage, many at the four-celled stage, but most at the five-celled stage. Since pollination usually occurs over a 2-week period on any tree, the pollen when shed is no doubt quite variable in stage of develop- ment, being somewhere between the three- and five-celled stages. No pollen grains were observed in which the body cell had divided to form the two sperm.

The structure of the pollen grain wall or sporoderm is similar to that of other conifers (Chamberlain 1957). Wings or bladders and con- spicuous pores or furrows are absent. The outer surface of the wall has a very fine granular sculp- turing. The detailed structure of the wall is sim- ilar to that described for other pollen grains by Clowes and Juniper (1968). The outer layer or exine is about 2 p thick (Fig. 8). Histochemical tests indicate the presence, in-the exine, of a small amount of lignin which is probably in the form of a complex polymer of lipid, lignin, and car- bohydrate called sporopollenin. The exine ap- pears irregularly laminated with thin dark lines between lighter layers (Figs. 9, 11). The dark areas have been interpreted in pollen of other plants as thin cavities occupied by cytoplasm left by the tapetal cells (Clowes and Juniper 1968). The intine is also about 2 p thick and continuous within the exine and around the two prothallial cells (Fig. 8). Histochemical tests indicate the presence of both cellulose (Fig. 10) and a small amount of pectin. No tests for callose were made. The pectin is more abundant at the border of the intine and exine and is probably the layer which appears as the dark line in histological preparations (Fig. 8). The inner sur- face of the intine is rough with many tiny canals which extend out toward the exine and resemble plasmodesmata (Fig. 10).

Discussion Several points are clarified by this description

of Douglas-fir pollen development and by the accompanying photographs. Pollen grain devel- opment is similar to that described for Pitzus (Chamberlain 1957). The pollen grain at pollina- tion normally consists of either four or five cells. It is difficult to estimate what proportion of pol- len grains are at the four- or five-celled stage, however, since pollination on a tree normally extends over a 2-week period and pollen cones vary this amount in their stage of development. Allen (1943) estimated that only 176 of the pollen had reached the five-celled stage. The present study covering pollen for 3 different years indi- cates that about 50% are at the five-celled stage or are dividing to form the five-celled stage. This type of development was suggested by Lawson (1909) and Allen (1943) but was never confirmed by a study at all stages. Generative cell formation and the cup-shaped partial wall enclosing it are of interest in Douglas fir since confusion exists in the literature regarding their presence and interpretation. It has been suggested that the generative cell does not occur in Douglas fir (Christiansen 1969). The present study, however, shows that it is present as it is in other conifers. Moreover, a pore has been suggested to exist at the proximal pole of the pollen grain, adjacent to the prothallia1 cells (Barner and Christiansen 1962). Their interpretation was based on the study of mature pollen. They described an aper- ture at the proximal pole surrounded by a thick- ening of the intine. Upon squashing, two small discs, a flat one and a concave one, were ob- served near the proximal pole which were inter- preted as degenerated prothallial cells or mem- branes covering a pore. The pore was interpreted as the orifice of a membrane enclosing the body and stalk cells. In fact the two discs were no doubt prothallial cells extruded when pressure was applied and the wall separating the second prothallial cell and the stalk cell probably broke open and was interpreted as a pore. Moreover the "pore," which appears as a ring in whole mounts, is probably the cup-shaped outer partial wall of the stalk cell formed adjacent to the sec- ond prothallial cell, as seen in polar view.

The use of paraffin-embedded and sectioned cones containing developing pollen has provided a means of observing all developmental stages

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1266 CANADIAN JOURNAL OF BOTANY. VOL. 49, 1971

and eliminates the problems of interpreting cel- lular detail through a very thick, finely sculptured spore wall. Further studies of pollen develop- ment, in conifers at least, should make use of the parafk technique in conjunction with whole mounts to arrive at a correct interpretation of all stages.

ALLEN, G. S. 1943. The embryogeny of Pseudotsuga taxi- folia (Lamb.) Britt. Arner. J. Bot. 30: 655-661.

BARNER, H., and H. CHRISTIANSEN. 1962. The formation of pollen, the pollination mechanism, and the deter- mination of the most favourable time for controlled pollination in Pseudotsuga menziesii. Silvae Genet. ll(4): 89-102.

CHA~ERLAIN, C. J. 1957. Gymnosperms structure and evolution. Re~rinted ed. Johnson Re~r in t Comora- .- tion, New YO;~.

CHRISTIANSEN, H. 1969. On the pollen grain and the fer- tilization mechanism of Pseudotsu~a menziesii (Mirbel) Franco var. Viridis Schwer. ~ i lvae Genet. 18(4): 971 104.

C ~ o m s , F. A. L., and B. E. JUNIPER. 1968. Plant cells. Blackwell Scientific Publications, Ltd., Oxford.

JENSEN, W. A. 1962. Botanical histochernistry. W. H. Freeman and Co., San Francisco.

JOHANSEN. D. A. 1940. Plant microtechniaue. McGraw- Hill, N ~ W York.

LAWSON, A. A. 1909. The garnetophytes and embryo of Pseudotsuga Donglasii. Ann. Bot. (London), 23(90): 163-180.

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