Factors affecting seed set in Douglas-fir (Pseudotsuga menziesii)

JOHN N. OWENS', ANNA M. COLANGELI, AND SHEILA J. MORRIS Department of Biology, University of Victoria, Victoria, B.C., Canada V8W 2Y2

Received May 7, 1990

OWENS, J . N . , COLANGELI, A. M., and MORRIS, S. J . 1991. Factors affecting seed set in Douglas-fir (Pseudotsuga menziesii). Can. J . Bot. 69: 229-238.

Cone and seed development in Douglas-fir were studied from pollination until seed release in 1986. Cone abortion at, and shortly after, pollination was high, resulting from a combination of low temperatures and possibly high moisture and pop- ulations of microorganisms on cones. Seed potential averaged about 75 seeds per cone with 31 filled seed per cone, giving an average seed efficiency of 39%. The major loss of seed resulted from insufficient pollen in the ovules. Other causes were ovule and embryo abortion at various stages of development. The effects of prezygotic and postzygotic events on seed set are discussed with respect to the reproductive success of Douglas-fir.

Key words: Douglas-fir, seed set, cone, ovule, development, abortion.

OWENS, J . N. , COLANGELI, A. M., et MORRIS, S. J . 1991. Factors affecting seed set in Douglas-fir (Pseudotsuga menziesii). Can. J . Bot. 69 : 229-238.

Le dCveloppement des c6nes et des graines du sapin Douglas de la pollinisation jusqu'au relhchement des graines, en 1986, a CtC CtudiC. L'avortement des c6nes au moment de, et peu aprks, la pollinisation a CtC ClevC, causC par une combinaison de basses tempCratures et possiblement d'humiditC ClevCe et de populations abondantes de microorganismes dans les c6nes. Le potentiel sCminal Ctait de 75 graines par c6ne dont 31 graines pleines par the, soit une efficacitt stminale de 39%. La plus forte perte de graines provient d'une insuffisance de pollen dans les ovules. D'autres causes incluent I'avortement des ovules et des embryons B divers stades du dCveloppement. Les effets des CvCnements prCzygotiques et postzygotiques sur la mise en graine en relation avec le succks reproductif du sapin Douglas sont discutCs.

Mots clks : sapin Douglas, mise en graine, cane, ovule, dCveloppement, avortement. [Traduit par la rkdaction]

Introduction (Sorensen 1969). Manv studies of conifer-seed set consider

Seed set in Douglas-fir is often low and quite variable in wild stands (Owston and Stein 1974) and seed orchards (Reynolds and El-Kassaby 1989; McAuley 1990), especially when trees are naturally wind pollinated. In a survey of four coastal Douglas-fir seed orchards, over 4 years, the average seed potential per cone was 70, but filled seed averaged only 2i per cone, or about 29% (McAuley 1990). Low seed set is a major concern in conifers, but the causes have not been thor- oughly investigated. Generally, it is thought to be caused by the prezygotic event of low pollination success (Owens and Blake 1985) and the postzygotic event of low self-fertility (genetic load) (Sorensen 1969). Conifer-seed set may be increased by using control pollination (Owens et al. 1981) or supplemental pollinations (Bridgwater 198 1).

In flowering plants, several prezygotic events reduce seed set, including pollen-pistil interactions (Knox 1984), self- incompatibility (Cornish et al. 1988), gametophytic competi- tion (Mulcahy 1978), and selection at fertilization. Postzygotic events can also reduce seed set in flowering plants, and these include developmental selection during embryo development (Weins et al. 1987), female choice (Stephenson and Bertin 1983), plus several others (in Willson and Burley 1983). In conifers, prezygotic events other than low pollination success are thought to be of little importance, but there have been few investigations to substantiate this belief (in Owens and Blake 1985). In contrast, postzygotic events are known to be very important, especially genetic load which leads to the abortion of embryos at various st?.ges of development. This is thought to be caused by the increased frequency of deleterious and lethal genes through selfing in normally outcrossing conifers

'Author to whom all correspondence should be addressed.

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only genetic factors ( ~ k - ~ w i n ~ 1965; Sorensen 1969; Fowler 1965), whereas others have considered developmental factors (Om-Ewing 1957; Plym Forshell 1953; Dogra 1967; Colangeli 1989; Owens et al. 1990). None have considered specific gene control of development.

To fully understand the factors influencing seed set in con- ifers, all stages of the reproductive cycle must be studied in detail to determine quantitatively when cone, ovule, embryo, or seed losses occur. The relative importance of the causes may vary between trees as well as within a tree in different years. Therefore, any study will only be a small sample of a large population, but owing to the conservative nature of .con- ifer reproductive cycles, it should accurately indicate when most seed losses occur for that species. Once this has been established, subsequent studies can focus on the possible phys- iological or molecular controls of these critical stages. The objective of this study was to determine at which stages of cone and seed development most seed losses occurred in Douglas-fir.

Materials and methods Five 15-year-old trees bearing abundant seed-cone buds were

selected in the spring of 1986 in the Dewdney Seed Orchard (British Columbia Ministry of Forests), 15 km north of Victoria, B.C. The five trees were grown from open-pollinated seed collected from five different parent trees at 760-1070 m elevations from the coast-inte- rior transition zone and south coast mainland seed orchard planning zones in B.C. (Hanson 1985). The trees were growing within a 30-m radius in three adjacent rows in the seed orchard. Meteorolog- ical data were obtained from the Environment Canada Atmospheric Environment Services station, 6 km north of the seed orchard.

From 48 to 61 cone buds per tree were enclosed in windowed paper pollination bags several days before seed cones emerged and became

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receptive. Four to eight pollination bags per tree were placed on branches bearing 4 to 19 cones each in the lower two-thirds of the crown. Cone buds were monitored daily until they became receptive. On April 1 1 and again on April 14, 0.9 mL of a mix of fresh pollen from other unrelated trees within the seed orchard was blown into each pollination bag so that all receptive cones were pollinated. On May 12, pollination bags were removed, the number of aborted cones determined, and pollination bags replaced with nylon-mesh insect bags.

On each collection date observations were recorded on the stage of cone development and the incidence of cone abortion. Two cones were randomly selected from bagged branches on each tree every 2 weeks from April 25 through August 14. Twenty ovules were ran- domly sampled from along the length of each cone. In April and May the ovules and a small portion of the ovuliferous scale were sliced longitudinally along both sides and fixed. After early June the seed coats had become hard and ovules readily separated from the ovuli- ferous scales. From June 4 through July 3 seed coats were removed from the ovules, the megagametophytes were sliced longitudinally along both sides, and the central portion was fixed. During this time megagametophytes that had obviously aborted and shrivelled were counted in the sample of 20 ovules but were not fixed.

On the subsequent collection dates of July 16 and 30 and August 14, the 20 ovules per cone were first sorted into flat (empty) and round (full). 'The round ovules were dissected, and those con- taining shrivelled megagametophytes were counted as having aborted about the time of fertilization. Developing embryos were clearly vis- ible in most of the remaining ovules. These were classed according to stage of embryo development. Round ovules, which contained no obvious embryos but had not aborted, were fixed and sectioned to anatomically determine their stage of development. All specimens were placed in Navashin's fixative, dehydrated in a tert-butyl alcohol series (Johansen 1940), and embedded in Tissueprep. Embedded specimens were serially sectioned longitudinally at 6 p,m and stained with safranin and hematoxylin for microscopic observations.

On most collection dates, samples of ovules and megagameto-

1 phytes were sliced into thin (1 mm) median longitudinal segments and the chalaza1 end discarded. These specimens were fixed in 4% ~ glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 2 h at room ~ temperature. Tissue was rinsed in buffer, postfixed in 1% buffered osmium tetroxide for 1 h, rinsed in buffer, and dehydrated in an ethanol series. Tissues were then rinsed in propylene oxide and embedded in Spurn's resin (Spurn 1969) at 60°C for 18 h. These spec- imens were sectioned at 700 nm, mounted on glass slides, and stained (Richardson et al. 1960).

All seed cones have, at their base and tip, several rudimentary or sterile ovuliferous scales. These, and the number of fertile scales between, were determined for each mature cone. Seed potential (SP) per cone was calculated as two times the number of fertile ovuliferous scales. Seed efficiency (SEF) per cone was the number of filled seed divided by the SP times 100.

Sectioned ovules were studied anatolnically to determine the pol- lination success (PS, the percentage of fertile ovules containing pollen in the micropyle), fertilization success (FS, the percentage of ovules in which at least one egg was fertilized), and the frequency of ovule or embryo abortion during each major developmental stage. These microscopic observations were combined with observations obtained by the dissection of ovules during later development to determine the rate of development and stages at which ovule or embryo abortion occurred. Only four trees were used for the complete analysis. The fifth tree had such a high rate of cone abortion that too few cones remained for complete anatomical or SEF studies.

On Atlgust 14 when cones began to dry and open, 10 of the cones remaining in the bags were sampled from each tree. Each cone was placed distal end up in an envelope to prevent seed release and air dried until cones opened. Seeds were then carefully removed sepa- rately from each cone and classed as round or flat Dissection dem- onstrated that flat seeds had aborted. All round seeds per cone were

X-rayed for 2.5 min at 15 kV using a Faxitron 804 X-ray unit, and the number of filled (FS) and empty seed determined for each cone.

On August 27, 10 unbagged primary branches were observed in the same region of the crown from which cones were collected. All cones were counted, and the number of aborted and developed cones recorded. The approximate time of cone abortion was determined by the size and morphology of the aborted cones, all of which remained attached to the branches (Fig. 1).

PS and mean number of pollen grains per micropyle were deter- mined from counts of serial longitudinal sections of 214 ovules col- lected from the four sample trees on April 25, May 8 and 22, and June 4.

Ovule abortion was determined from anatomical observations of ovules during early development (April and May collections) and counts of flat seeds in later colections (June through August). FS was calculated as the proportion of 284 mature ovules that contained developing proembryos, early embryos, or aborted embryos sampled on June 18 and July 2 and 16. This calculation was adjusted for the variable pollination success of cones sampled earlier from each tree. The proportion of ovules pollinated but not fertilized could not be accurately determined because of the difficulty in identifying pollen tubes in nucelli. Therefore, this was calculated as the difference between the sum of unpollinated and early aborted ovules and the fertilized ovules. The latter was the sum of the SEF and the number of ovules containing degenerated embryos. The frequency of embryo abortion was detemined from anatomical observations of 136 ovules during early embryo development on July 16 and included only ovules in which all embryos had aborted rather than the proportion of embryos that aborted in the multiarchegoniate megagametophytes. Analysis of variance and Student-Newman-Keuls tests were used to determine the clonal effect on cone and seed characteristics at the 95% probability level.

Results

Cone abortion Cone abortion, at or soon after pollination, was high

throughout the seed orchard in 1986. In the five sample trees, abortion of unbagged cones was 20.2, 29.5, 60.3, 64.2, and loo%, with a mean of 54.8%. This was much higher than abortion of cones within bags, which ranged from 4.6 to 55.5%, with a mean of 28.2%. Precise comparisons of these values are not possible because cones within the bags were being destructively sampled throughout the study and some early cones may have aborted if they had not been collected. Tree 5 had the highest abortion rate of bagged (55.5%) and unbagged (100%) cones.

Bagged cones dissected during early stages of cone abor- tion, during or soon after pollination, showed no external signs of abortion. Softening and browning occurred first in the cone axis and spread outward, with the large bracts the last to turn brown. Many cones aborted while still upright and receptive. Others aborted while bending downward or soon after becom- ing pendant (Fig. 1) early in May. Few cones aborted after this time, but some outside the bag showed abnormalities because of insect infestations. Cones that touched the inside of pollination bags often aborted, but the first sign of damage was at the point of contact with the bag rather than in the cone axis.

Weather records show only 2 days of below-freezing tem- peratures ( - 0.6 and - 0.2"C) and 14 days when temperatures were near freezing (0.6 to 2.0°C) between April 10 and May 15 when cone abortion occurred. Temperatures below 2°C caused the automatic water-misting system to operate. Precipitation was also recorded on 25 of the 31 days between April 10 and

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OWENS ET AL.

TABLE 1. Mean number and standard error (SE) of sterile and fertile ovuliferous scales, seed potential, number of filled seed, and seed efficiency based on 10 cones from bagged branches collected on August 14 from each

of four Douglas-fir trees

Sterile scales Seed No. filled Seed Fertile potential seed efficiency

Tree Basal Distal scales (sp) (FS) @Em

1 9.5(0.6)ab 2.4(0.2)b 37.1(1.7)b 74.2(3.3)b 42.8(5.5)a 57.4(6.3)a 2 9.3(0.6)ab 3.3(0.3)a 41.7(0.9)a 83.4(1.9)a 33.8(3.8)a 40.3(4.1)b 3 10.9(0.7)a 2.5(0.2)b 32.2(0.8)c 64.4(1.6)c 10.8(1.5)h 16.8(2.3)c 4 7.8(0.5)b 2.0(0.2)b 39.2(0.9)ab 78.4(2.0)ab 31.7(3.7)a 40.5(4.8)b

Mean 9.4f0.3) 2.6f0.1) 37.6f0.8) 75.0(1.6) 29.8f2.7) 38.8f3.2)

NOTE: Means followed by the same letter are not significantly different ( p < 0.05), as determined by Student-Newman-Keuls test.

TABLE 2. The percentage of ovules with pollen (pollination success, PS), number of ovules in which at least one egg was fertilized (fertilization success, FS), and the proportion of pollinated

ovules in which at least one egg was fertilized (adjusted fertilization success, AFS)

PS

Ovules FS AFS -- Total with Total Fertilized Pollinated Fertilized

Tree ovules pollen % ovules ovules % ovules ovules %

1 50 45 90 72 50 69 65 50 77 2 54 40 74 72 34 47 53 34 64 3 48 33 69 69 10 15 48 10 21 4 62 56 90 72 51 72 64 51 80

Total 214 174 285 145 230 145 Mean 8 1 5 1 63

May 15. Between the natural rainfall and the misting system, trees received some "precipitation" on 30 of the 31 days.

Reproductive potential of cones Significant tree variation ( p < 0.01) was found for all cone

and seed characteristics, including number of basal and distal sterile scales, fertile scales, SP, FS, and SEF. Tree 3 was con- sistently lower than the other trees in the last four parameters, based on the Student-Newman-Keuls test (Table 1). Trees varied in their SP, ranging from 64.4 to 83.4, with a mean of 75.0. This was determined primarily by the total number of ovuliferous scales present rather than variation in the propor- tions of proximal and distal rudimentary or sterile scales. The number of FS per cone ranged from 10.8 to 42.8, with a mean of 29.8 for the four trees. The SEF ranged from 16.8 to 57.4, with a mean of 38.8 (Table 1).

Pollination and fertilization success The PS ranged from 69 to 90% of ovules, with a mean of

81 % (Table 2). The number of pollen grains per micropyle ranged from 0 to 11, with a mean of 3.19. Most pollen remained attached to or near the stigmatic hairs after being taken into the micropyle (Fig. 2). Within 2 weeks from the time pollen was taken into the micropyle, it began to swell and germinate. The exine usually remained attached or near the stigmatic surface (Fig. 2). A high proportion of pollen ger- minated, but the exact proportion could not be determined because some pollen was lost during sectioning and staining. Pollen elongated down the micropylar canal and many had reached the nucellus by the June 4 collection. Pollen tubes then formed and several usually penetrated the nucellus (Fig. 6). Their pathway through the nucellus was often irreg- ular and it was not possible using embedded specimens to determine if growth of some pollen tubes was slowed or

arrested. Fertilization occurred in 5 1 % of the ,285 ovules sec- tioned (FS). However, the total number of pollinated ovules that was fertilized (the adjusted fertilization success, AFS) was 63% and ranged from 2 to 80% in the four trees (Table 2). In many of these, pollen tubes may have become arrested during penetration of the nucellus.

Ovule development and causes for reduced SEF On the first collection date (April 25) cones were still erect

or starting to bend downward, but ovules were no longer receptive. The stigmatic tips which entrap the pollen had grown inward, engulfing the pollen. This occurred by elongation of outer cells on the stigmatic surface, causing stigmatic hairs and attached pollen to be carried into the micropyle (Fig. 2).

Early ovule development showed few abnormalities or abor- tions. On April 25 the megagametophyte was at the early to mid free nuclear stage, with a distinct megaspore wall. In par- affin-embedded specimens the megagametophyte shrank and pulled away from the surrounding tapetum, which usually became dislodged from adjacent nucellar tissues (Fig. 2). Dur- ing the next 2 weeks the sac-like megagametophyte increased slightly in size and the nucellus grew down the micropylar canal (Fig. 3). No ovule abortion was observed at the free nuclear stage nor was there evidence of abortion at meiotic and the functional megaspore stages.

Cell wall formation began about mid-May. On May 22, 27 of the 65 ovules sampled were undergoing cell wall formation (Fig. 4), and a few ovules contained aborting megagameto- phytes (Fig. 5). Several archegonial initials were evident as enlarged cells at the micropylar end of the megagametophyte (Fig. 4). Cell wall formation began at the periphery of the megagametophyte and progressed inward, eventually filling the central vacuole.

By early June, the ovule was almost fully enlarged and rounded, and the integument had begun to differentiate into

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OWENS

the three layers of the seed coat. Cell wall formation was com- plete in the megagametophyte, and prothallial cells continued to divide. Archegonial initials divided to form the central cell and the primary neck cell, which in turn divided to form a single layer of neck cells. The central cell enlarged many times and took on its characteristic frothy, vacuolate appearance. Archegonial iacket cells were evident around each central cell. " The nucellar tip bore many empty and vacuolate cells, indi- cating secretory activity (Owens and Monis 1990). Pollen formed narrow pollen tubes that penetrated the nucellus (Fig. 6). Few abortions or other abnormalities occurred at the central cell stage. Any aborted ovules present at this time appeared to have aborted during cell wall formation before ovules fully enlarged. Several ovules appeared to have been arrested during the free nuclear stage. This resulted in what is commonly called "flat seeds." In these, the integument dif- ferentiated into a thin seed coat, but the ovule did not expand and "round UD." The nucellus remained thin and the mega- u

gametophyte degenerated, leaving a flat, empty nucellus (Fig. 7) or one that contained only remnants of the megaga- metophyte. This type of megagametophytic abortion occurred whether in the presence or absence of pollen in the micropyle. Another abnormality observed at this stage was disorientation of the nucellar tip.-Rather than elongating down the narrow micropylar canal, it bent laterally (Fig. 8). This greatly increased the distance Dollen would have to grow to contact the nucellar tip, which'had become oriented &ward one side of the ovule.

By mid-June the mature megagametophyte had developed. The central cell had divided to form the small ventral canal cell and a large egg cell. The prothallial cells of the mega- gametophyte overgrew the neck cells to form a small arche- gonial chamber. The entire megagametophyte was enclosed by a megaspore wall. In the 121 ovules sampled from the four trees on June 4 and 18, the mean number of archegonia was 3.9, with a range of 1-7. Only 1.6% of the ovules had 1, 2, or 7 archegonia, whereas 23.7% had 3 ,22% had 5 , and 44.1 % had 4 as shown in Fig. 8.

On June 18 there was considerable variation in stage of development of the 248 archegonia observed in the 5 9 sec- tioned ovules. Only 5% of the ovules had megagametophytes arrested at the free nuclear stage, 6% had aborted at some earlier stage of development but had degenerated to the point that the precise stage was impossible to determine, and 76% had mature megagametophytes (Fig. 9). Another 8% were at one of the proembryo stages (Figs. 9-1 l ) , and 5% were at an early embryo stage (Figs. 12-14). At this time many of the eggs in mature megagametophytes had recently been fertilized (Fig. 9), but it was not possible to determine the proportion of ovules remaining unfertilized (Fig. 9). This had to be deter- mined from ovules in subsequent collections (Fig. 10) because eggs within a megagametophyte can be fertilized over several

ET AL. 233

days. Consequently, an ovule might have some fertilized and unfertilized eggs (Fig. 10) for several days, but all may even- tually become fertilized. The cytoplasm of fertilized eggs becomes more vacuolate and granular (Figs. 10, 11) than that of unfertilized eggs (Fig. 10). However, the cytoplasm of both degenerates within about 1 week after which fertilized and unfertilized archegonia appear the same. Too few samples were resin embedded in this study to adequately determine the per- centage of eggs that were fertilized.

Megagametophytes in which no eggs were fertilized began to degenerate, dry, and shrivel within 2-4 weeks. Initially, this could not be detected by dissection but was evident in specimens prepared for anatomical study.

On July 2 about 5% of the ovules sampled had aborted ear- lier, producing flat seeds. These were not fixed or sectioned. The same proportion of early aborted ovules occurred in sub- sequent collections. Of the 62 ovules sectioned on July 2 , 45% were unfertilized but had not yet aborted. All remaining ovules contained early embryos (Figs. 12- 14). There was an average of 1.4 early embryos per ovule (Fig. 14), and only four of the 46 early embryos observed were aborting. This was the earliest stage when embryo abortion became common.

Starting on July 16, the 20 ovules from each cone were first separated into early aborted (flat) ovules and developing ovules. Dissected developing ovules, in which the megaga- metophyte had begun to dry, were counted as aborted. From anatomical observations it was concluded that most aborted because they were not fertilized. However, some of these megagametophytes could have aborted during proembryo or very early embryo development.

On July 16, 47% of the dissected ovules contained shriv- elled megagametophytes (Fig. 15) that had recently aborted, apparently because they were not fertilized or because all embryos had aborted. Embryos were developing on 48% of the ovules, with 36% at the cotyledon stage and 12% at the club-shaped stage. Another 12% contained aborted embryos.

By July 30, seeds that contained well-developed embryos generally could be determined by their external appearance. Filled seeds had a darkened region at the micropylar end. This resulted from the thick grey nucellus at the micropylar end which can be seen through the immature seed coat. As the seed coat matured it darkened, and this distinction was no longer possible. On July 30, 33% of the fully enlarged seed had embryos, all at the cotyledon stage, and 67% had shriv- elled megagametophytes (Fig. 15). Again, it could not be determined if this resulted from no fertilization or from abor- tion of all early embryos. On August 14 mature cones were starting to dry, turn brown, and open. Mature seeds (Fig. 17) showed nearly the same percentage of filled (36%) (Fig. 16) and degenerated megagametophytes (64%) (Fig. 15) as on July 30. These seeds could not be distinguished externally

FIG. 1. Branch collected in late May, showing a large developing seed cone and a small seed cone (arrow), which aborted soon after becoming pendant early in May. FIGS. 2-4. Median longitudinal sections of paraffin-embedded ovules attached to ovuliferous scales (0s) showing integument (i), nucellus (n), tapetum (t), and megagametophyte (m). Fig. 2. Ovule collected on April 25 during mid free nuclear stage of the rnegagametophyte. Ungerminated pollen @) is in rnicropylar canal (mc), inside closed micropyle with collapsed stigmatic hairs (sh). Fig. 3. Ovule collected on May 22 during the late free nuclear stage of the rnegagametophyte enclosed by the megaspore wall (mw). Elongated pollen is in the micropyle. Fig. 4. Ovule collected on May 22 at onset of cell wall formation. Archegonial initials (ai) are evident. FIG. 5. Ovule collected on May 22 showing aborted megagametophyte. FIG. 6. Near-median longitudinal section of a resin-embedded ovule collected June 10 showing elongated pollen and pollen tubes @t) penetrating the nucellus. a, archegonia. FIG. 7. Median longitudinal section of a paraffin-embedded "flat" ovule collected on June 4 showing empty nucellus (en). FIG. 8. Longitudinal section of a paraffin-embedded abnormal ovule with nucellus and four archegonia (a) oriented laterally.

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OWENS ET AL. 235

100 90 80 70 60 50 40 30 20 10 0

FIG. 18. Summary of the seed efficiency and the causes of seed losses in four Douglas-fir trees in 1986. "Other prezygotic factors" is an estimate based on measures of all other factors.

(Fig. 17). Embryos were well developed and filled the cor- rosion cavity (Fig. 16).

NO POLLEN

19%

Discussion

OTHER PREZYGOTlC

FACTORS 11%

I

OVULE ABORTION

11%

In the Douglas-fir trees selected for this study, both seed and seed-cone losses significantly reduced seed production. The 39% SEF obtained here is typical for many conifers (Schopmeyer 1974; Owens and Blake 1985) and for outcross- ing flowering species (Weins et al. 1987). However, the rel- ative importance of the different causes of seed loss may be quite different in conifers than in flowering plants (Sedgley and Griffin 1989). A summary of the observed and estimated losses that occurred in Douglas-fir is given in Fig. 18. Observed losses include prezygotic ones resulting from no pol- lination and ovule abortion, whereas observed postzygotic losses result from embryo abortion. Other prezygotic losses such as pollen-nucellar interactions can only be estimated because the observed losses never account for all the seed loss.

I I I I I

It is assumed that conifers have a low frequency of prezygotic selection but a well-developed postzygotic selection mecha-

EMBRYO DEGENERATION

20%

. -

nism through polyembryony. In most flowering plants, how- ever, there are highly developed prezygotic mechanisms (Willson and Burley 1983; Sedgley and Griffin 1989). Our

I

SEED EFFICIENCY

39%

results are similar to observations made in four coastal Doug- las-fir seed orchards over 4 years (McAuley 1990). In that study, seed efficiency varied from 16 to 45%-over the 4 years and averaged about 29%. Causes of seed losses were estimated as 15-30% because of ovule abortion, 25-35% resulting from inadequate pollination and late embryo abortion, and 2-44% as a result of insect damage. Seed efficiency and causes of seed losses were quite variable from year to year and from one seed orchard to another.

Cone loss at about the time of pollination is a common phe- nomenon in many conifers and can usually be attributed tolow temperatures at that time (Owens and Blake 1985; Colangeli

et al. 1990). Cone loss in the trees sampled averaged about 55% outside the pollination bags, which was similar to other trees in the orchard, but only about 28% inside the bags. The paper pollination bags increase temperatures inside, and this enhances cone development (Owens et al. 1981) and reduces frost damage. Heavy cone loss occurred even though ambient temperatures only went below freezing on 2 days during the study period. Another factor contributing to the high cone loss outside the pollination bags, at or following pollination, was the water-misting system. Although it raised the air tempera- ture around the cones, it kept them wet for prolonged periods. This along with the frequent rains enhanced the growth of microorganisms on foliage and cones (Colangeli et al. 1990) which may be harmful to cones, especially if concentrated around the succulent cone axis. A concurrent study of unbagged cones and foliage from the same five trees showed significantly higher bacterial populations in 1986 when cone abortion averaged 55% than in 1987 when cone abortion aver- aged I 1 %. One bacterium has been tentatively identified as an ice-nucleating Pseudomonas (Colangeli et al. 1990). They are endemic epiphytes on a wide variety of plant species, includ- ing conifers. These ice-nucleating bacteria cause ice formation at temperatures just above freezing, which occurred frequently during the pollination period. The bacterial population may have been lower inside the pollination bags, but this was not determined.

Cone drop not related to temperature is common in Pinus (Sweet 1973), in which it is a function of "female choice" as used by Weins et al. (1987) and Stephenson and Winsor (1986). In Pinus, pollinated ovules are thought to produce growth regulators which affect nutrient transport to developing cones. If too few ovules are pollinated, the cone aborts (Sweet 1973). Sarvas (1962) estimated that when more than 20% of the ovules in a Pinus sylvestris cone aborted (because of a lack of pollen), the cone aborted and about 80% of cone drop resulted from low levels of pollen. In certain other pines, seed-

-

FIGS. 9-14. Median longitudinal sections of resin-embedded ovules showing nucellus (n) and megagametophyte (m). Fig. 9. Two archegonia in mid-June. The lower one has just been fertilized as evidenced by vacuolate (v) egg cytoplasm (ec). Egg nucleus (en), neck cells (nc), and ventral canal cell (vc) are still visible. Upper archegonium shows early free nuclear (fn) proembryo and second male gamete ( g ) in neocytoplasm (nc). Pathway of pollen tube (pt) is evident. Fig. 10. Three archegonia in mid-June. The upper one has not been fertilized as evidenced by nongranular cytoplasm. The lower two have been fertilized as evidenced by vacuolate granular cytoplasm. The middle archegonium shows three of four proembryo-free nuclei. Fig. 11. A 12-cell proembryo showing apical (at), suspensor (st), and open (ot) tiers. Note vacuolate granular egg cytoplasm and small archegonial jacket (aj) cells. Fig. 12. Suspensor- and apical-tier elongation causing collapse of archegonial jacket cells (arrow) and start of corrosion cavity formation and early embryo development. Fig. 13. Three archegonia in which fertilization has occurred, showing early embryos with long suspensor tier in lower two. Fig. 14. Three early embryos (ee) with suspensors (s) from three separate archegonia. Note collapsed megagametophyte cells (arrow) around the early embryos. FIG. 15. Dissected seed showing seed coat (sc) and shrivelled nucellus and megagametophyte (arrow). FIG. 16. Dissected mature filled seed showing megagametophyte and embryos (e) with cotyledons (c). FIG. 17. Mature Douglas-fir seed with wings removed showing dark upper and light lower surfaces and integument tip (it).

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236 CAN. J. BOT. VOL. 69, 1991

cone development and vegetative shoot elongation coincide and compete for nutrients, often resulting in high proportions of cone drop (Sweet and Bollman 1970). In other conifers, e.g., Picea (Dogra 1967; Owens and Blake 1984) and Thuja (Owens et al. 1990), unpollinated ovules abort before they mature, which may reduce cone size but does not cause cone abortion. In Douglas-fir and many conifers (Owens and Blake 1985) ovules develop normally and megagametophytes differentiate fully in the absence of pollen. Megagametophytes then abort if fertilization does not occur. This does not adversely affect seed-cone development or cone size and often seed size, since cone and seed enlargement are nearly completed by the time of fertilization.

Although all conifers are anemophilous and most produce prodigious pollen that is widely dispersed, many ovules are not pollinated. This is a major cause of low seed set (Owens and Blake 1985). In the present study, this accounted for a 19% reduction in seed set (Fig. 18). About 81% of ovules contained pollen, and ovules averaged 3.19 pollen grains per micropyle. This was about the same number of pollen grains found in the micropyles of Douglas-fir in two earlier studies in which pollen was applied in different quantities and at dif- ferent times (Owens et al. 1981; Owens and Simpson 1982). In those studies, although up to 15 pollen grains reached the stigmatic tip, only 3 or 4 were taken into the micropyle. This roughly corresponds to the average number of archegonia (3.9) per ovule found in this study.

Failure of ovules to be pollinated may be traced to the pol- lination mechanism in Douglas-fir, which like in Larix (Owens and Molder 1979; Owens et al. 1981), involves a stigmatic tip that consists of two lobes bearing long stigmatic hairs on either side of the micropyle. Pollen attaches to the hairs over several days, then the outer cells around the micropyle elongate and hairs with the attached pollen are engulfed into the micropyle. Once this has occurred, late-arriving pollen cannot enter the sealed micropyle. Therefore, as in most other conifers, indi- vidual ovules are receptive for only a few days, but each cone may be receptive for several days longer, and pollen is usually shed over an even longer time (Owens and Blake 1985). Despite the efficient pollination mechanisms and long recep- tive periods, unpollinated ovules are a major cause of low seed set in most conifers studied thus far (Owens and Blake 1985; Colangeli and Owens 1990). This commonly results because not enough pollen is available or it is filtered out of the air by rain. Even in bagged cones, which were pollinated twice in this study, 19% of the ovules had no pollen. This can result from unequal distribution of pollen within the bags, varying receptivity of ovules along the cone axis, and portions of receptive cones being covered by foliage restricting entrance of pollen.

The complex interactions that exist between pollen and the stigma and style of angiosperms (Dumas and Knox 1983) have not been clearly demonstrated between the pollen and the nucellus of conifers. Nevertheless, when all other factors have been accounted for in the few conifers that have been studied, there are significant propcrtions of seed losses that may be attributed to this prezygotic stage of development. This stage accounted for about 11% of seed loss in Douglas-fir (Fig. 18). In Tsuga heterophylla this stage accounted for 14% of seed loss in self-pollination and only 2% of seed loss in cross- pollination, suggesting a prezygotic self-incompatibility mech- anism (Colangeli 1989). Pollen tubes became arrested in nucelli of selfed and outcrossed Pinus peuce (Hagman and

Mikkola 1963) and in interspecific crosses of Picea (Mikkola 1969) and Pinus (Hagman and Mikkola 1963). Pettitt (1982) detected proteins and glycoproteins in the exine and pollen tube (intine) wall of Cycas and, more recently, the release of enzymes from pollen tubes of Pinus, Picea, Abies, and Cedrus pollen grown in vitro (Pettitt 1985). These observations sug- gest that there are significant, but perhaps not highly evolved, prezygotic selection mechanisms operating in conifers and that they may be greater in selfed than in outcrosses. However, no detailed studies have been made of conifer pollen-tube devel- opment within the nucellus that demonstrate the interactions which may occur between the gametophytic pollen tube and sporophytic nucellus.

Douglas-fir cones in this study had an SP of 75, which is typical for Douglas-fir in seed orchards (McAuley 1990). The SP of Douglas-fir is about midway in the range of SP found within the Pinaceae but is generally higher than the SP in most other conifer families (Schopmeyer 1974). The high SP in many conifers would appear to allow considerable opportunity for developmental selection as occurs in many angiosperms through competition among ovules with genetically diverse embryos (Wiens et al. 1987; Sedgley and Griffin 1989). How- ever, this does not occur in Douglas-fir or many other conifers (Owens and Blake 1985) because, at fertilization, ovules are almost fully enlarged, seed coats are well developed, and ovules no longer have a vascular connection to the seed cone. Ovules are held in place by the ovuliferousa scales, and further development relies on reserves already in the megagameto- phyte. Therefore, the parent cone has little control over post- zygotic seed development. In some conifers such as Pinus (Sweet 1973), Picea (Dogra 1967), and Thuja (Owens et al. 1990), which do have small vascular connections to the ovules, unpollinated ovules abort before fertilization. Abortion in these cases results from pollen-ovule interactions rather than from developmental selection (Sweet 1973). This may allow the remaining pollinated ovules to develop more vigorously from the reserves available from the cone axis. There may be no conifers in which there is maternal control over fixed abortion systems (Weins et al. 1987). An exception may be Cephalo- t a u s in which the number of maturing ovules is reduced to one.

Because most conifers have multiarchegoniate ovules, com- petition between genetically diverse embryos occurs within ovules rather than between ovules. The latter is true in angio- sperms (Weins et al. 1987). This developmental selection (Bucholz 1922) between embryos within one megagameto- phyte has been described in many conifers (in Singh 1978), but its physiology is poorly understood. Cohabiting proem- bryos, whether resulting from outcrossing as in this study or from selfing as shown in Tsuga heterophylla (Colangeli 1989), rarely show abnormalities. Embryo abortions become more frequent when young embryos are thrust into the corrosion cavity and have more intimate contact with the megagameto- phyte and one another. This could mean that any gene control from the megagametophyte is more important in later embry- ogeny than during early embryogeny or, alternatively, that the zygotic genome is not fully activated until later embryogeny when losses caused by lethal genes occur. At the present time we know nothing about gene control of conifer embryogenesis. More is known for only a few species of angiosperms (Goldberg et al. 1989).

Selfing in most conifers results in abortion of embryos during early development (Orr-Ewing 1957; Dogra 1967;

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OWENS ET AL. 237

Sorensen 1969; Colangeli 1989). This is commonly called self- COLANGELI, A. M., MCAULEY, L., and OWENS, J. N.. 1990. The inviability in conifers and occurs at cleavage (if present) or potential role of ice-nucleating bacteria in inducing frost damage

during subsequent early embryo development and is thought in Douglas-fir conelets. New For. 4: 55-61.

to be a result of the accumulation of deleterious recessive CORNISH, E. C., ANDERSON, M. A,, and CLARKE, A. E. 1988. Molecular aspects of fertilization in flowering plants. Annu. Rev.

genes. Self-embryo survival is variable and was reported as Cell Biol. 4: 209-228. 12% in ~ o u ~ l a s - f i r (Orr-Ewing 1957), 25% in Pinus sylvestris DOGRA, P. D. 1967. Seed sterility and disturbances in embryogeny (Sarvas 1962), and 29% in Thuja plicata (Owens et al. 1990). in conifers with particular reference to seed testing and tree However, most conifer genetic studies are based only on self- breeding in Pinaceae. Stud. For. Suec. 45: 3-97. seed survival and not self-embryo survival (Sorensen 1969). DUMAS, C., and KNOX, R. B. 1983. Callose and determination of In our study of Douglas-fir, embryo abortion under outcrossing pistil viability and incompatibility. Theor. Appl. Genet. 67: conditions was about 20%; in Tsuga heterophylla (Colangeli 1-10,

and Owens 1989) and Thuja plicata it was 19% (Owens et al. FOWLER, D. P. 1965. Effects of inbreeding in red pine, Pinus resi- tlosa Ait. 11. Pollination Studies. Silvae Genet. 14: 12-23.

1990). Therefore, GOLDBERG, R, B., BARKER, S, J . , and PEREZ-GRAU, L, 1989, Reg- self-inviability, and where only seed set is used to determine ulation of gene expression during plant embryogenesis. Cell, selfing, the effects of selfing may be overestimated. Some 56: 149-160. embryo abortion will occur in all crosses, but the frequency HAGMAN, M., and MIKKOLA, L. 1963. Observations on cross-, has not been determined for most species. Not all conifers have self-, and interspecific pollinations in Pinuspeuce Griseb. Silvae high levels of self-inviability. In Thuja plicata embryo abor- Genet. 12: 73-79. tion was no higher and SEF no lower in selfed than in out- HANSON, P. 1985. Seed orchards of British Columbia. British

crossed trees (Owens et al. 1990), indicating an unusually low Columbia Ministry of Forests, Victoria.

level of self-inviability. This may be common in conifers JOHANSEN, D. A. 1940. Plant microtechnique. McGraw-Hill Publi- cations, New York.

whose strategies have under levels KNox, R. B, 1984, Pollen-pistil interactions, Itl Encyclopedia of of self-pollination. plant physiology (NS). Intercellular interactions. Edited by H. F.

In Douglas-fir, as in most conifers, the most important Linskens and J. Heslop-Harrison. Springer-Verlag, Berlin. deterrent to reproductive success is failure to initiate cones pp. 508-608. (Owens and Blake 1985). This is followed by several prezy- MCAULEY, L. 1990. Coastal Douglas fir cone analysis results. British gotic and postzygotic factors. Failure of ovules to be polli- Columbia Ministry of Forests, Silviculture Branch, Internal nated, low pollination success, was the second most important Report 8653K. factor in Douglas-fir as it is in most conifers. Other prezygotic MIKKOLA, L. 1969. Observations on interspecific sterility in Picea.

factors including failure of pollinated ovules to be fertilized Ann. Bot. Fenn. 6: 285-339.

and ovule abortion played lesser roles, Events at fertilization MULCAHY, D. L. 1978. Further evidence that gametophytic selection

showed no obvious adverse effects, but percentage of fertilized modifies the genetic quality of the sporophyte. Actual. Bot. 1-2: 57-60.

eggs was not precisely determined in this ORR-EW~NG, A, L, 1957. Further inbreeding studies with Douglas degeneration was the only postzygotic factor detected other fir. For. Chron. 33: 3 18-332. than losses caused by insects. More detailed ultrastructural - 1965. Inbreeding and single crossing in Douglas-fir. For. Sci. studies may reveal more subtle effects causing losses during 11: 279-290. pollen tube penetration of the nucellus, fertilization, and OWENS, J. N., and BLAKE, M. D. 1984. The pollination mechanism proembryo and early embryo development. of Sitka spruce (Picea sircherzsis). Can. J. Bot. 62: 1 136-1 148.

1985. Forest tree seed production. A review of literature and recommendations for future research. Can. For. Serv. Petawawa

Acknowledgements Natl. For. Inst. Inf. Rep. P1-X-53. OWENS, J . N., and MOLDER, M. 1979. Sexual reproduction of Larix

This research was supported by a Canada - British occidentalis. Can. J. Bot. 57: 2673-2690. Columbia Forst Resource Development Agreement contract OWENS, J. N., and MORRIS, S. J . 1990. The cytological basis for and Natural Sciences and Engineering Research Council of cytoplasmic inheritance in Pseudotsuga menziesii: I. Pollen tube Canada grant A1982 to Dr. J. N. Owens. We wish to thank and archegonial development. Am. J . Bot. 77: 433-445.

~~j~ ~ ~ l , j ~ ~ for her critical review of the manuscript. we OWENS, J . N., and SIMPSON, S. J. 1982. Further observations on the

wish to acknowledge the photographic assistance of Mr. T. pollination mechanism and seed production of Douglas-fir. Can. J. For. Res. 12: 43 1-434. Gore and the cooperation of the B.C. Ministry of Forests for OWENS, J. N., SIMPSON, S, J., and MOLDER, M. 1981. The

allowing us to use trees within their seed orchard and Forestry pollination mechanism and the optimum time of pollination in Canada for use of their seed X-ray unit. Douglas-fir (Pseudotsuga menziesii). Can. J. For. Res. 11:

36-50. BRIDGWATER, F. E., and TREW, I. F. 1981. Supplemental mass pol- OWENS, J. N., COLANGELI, A. M., and MORRIS, S. J. 1990. The

lination. In Pollen management handbook. Edited by E. C. effect of self, cross and no pollination on ovule, embryo, seed, Franklin. U.S. Dep. Agric. Agric. Handb. 587. pp. 52-57. and cone development in western redcedar (Thuja plicata Donn).

BUCHOLZ, J. T. 1922. Developmental selection in vascular plants. Can. J. For. Res. 20: 66-75. Bot. Gaz. 73: 249-286. OWSTON, P. W. , and STEIN, W. I. 1974. Pseudotsuga Carr. Douglas

COLANGELI, A. M. 1989. Pollination and pollen and seed develop- fir. p. 674-685. In Seeds of woody plants of the United States. ment in western hemlock. Ph.D. thesis, University of Victoria, Edited by C. S. Schopmeyer. U.S. Dep. Agric. For. Serv. Victoria, B.C. Handb. 450. pp. 674-685.

COLANGELI, A. M., and OWENS, J. N. 1989. Postdormancy seed- PET~ITT, J . M. 1982. Ultrastructural and immunocytochemical dem- cone development and the pollination mechanism in western onstration of gametophytic proteins in the pollen tube wall of the hemlock (Tsuga heterophylla). Can. J. For. Res. 19: 44-53. primitive gymnosperm Cycas. J. Cell Sci. 57: 189-213.

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