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Viability and longevity of pollen of

Nothofagus species in South Chile
a

Paola Bez , Magaly Riveros & Carlos Lehnebach

Instituto de Botnica, Facultad de Ciencias , Universidad Austral

de Chile , Casilla 567, Valdivia, Chile
b

Instituto de Botnica, Facultad de Ciencias , Universidad Austral

de Chile , Casilla 567, Valdivia, Chile E-mail:
Published online: 17 Mar 2010.

To cite this article: Paola Bez , Magaly Riveros & Carlos Lehnebach (2002) Viability and longevity
of pollen of Nothofagus species in South Chile, New Zealand Journal of Botany, 40:4, 671-678, DOI:
10.1080/0028825X.2002.9512822
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New Zealand Journal of Botany, 2002, Vol. 40: 671-678

0028-825X/02/4004-0671
$7.00 The Royal Society of New Zealand 2002

671

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Viability and longevity of pollen of Nothofagus species in south Chile

PAOLA BEZ
MAGALY RIVEROS*
CARLOS LEHNEBACH
Instituto de Botnica
Facultad de Ciencias
Universidad Austral de Chile
Casilla 567
Valdivia, Chile

breeding programmes and pollination studies is

discussed.
Keywords pollen; pollen longevity; viability; in
vivo germination; Nothofagus; Chile

INTRODUCTION

Species of the genus Nothofagus are important

components of the temperate forests of the Southern
Abstract The reproductive success of some Hemisphere, notably in Chile, Argentina, and New
Nothofagus species from South Chile is poor. Zealand (Wardle 1984; Donoso 1987; Poole 1987).
Although self-incompatibility is known to occur, In Chile, Nothofagus forests extend from Central
seed set by out-crossing pollination is still low. Chile (33S) to Tierra del Fuego (56S) (Rodrguez
Therefore, in this study we focused on the pollen et al. 1983; Veblen et al. 1995,1996). Owing to the
physiology of these species. Using direct and indirect excellent quality of their wood, species have been
methods, the viability and longevity of pollen grains extensively exploited with consequent deforestation
from young and mature individuals of the species N. of the Nothofagus environment (Armesto et al.
antarctica, N. obliqua, N. dombeyi, and N. betuloides 1992). Although attempts are being made to replant
were assessed. Pollen germination in vivo was areas which have been denuded of trees, a problem
evaluated in hand-pollinated and naturally pollinated frustrating these efforts is the low rate of viable seed
receptive stigmas of both categories. To assess production (Riveros et al. 1995a). This low
pollen viability indirectly, in vitro germination on a reproductive success is exacerbated by the fact that
sucrose-agar culture medium was performed. trees do not flower regularly and in some years
Alexander's procedure (an indirect method) was widespread non-flowering occurs (Riveros et al.
used to evaluate viability immediately after pollen 1995b, 1998). This leads to intermittent production
collection and longevity after 3, 6, and 12 months of large seed crops, a phenomenon known as masting
of storage. Pollen germination in vivo ranged from (Kelly 1994; Ogden et al. 1996).
52 to 74% in young individuals and 68 to 84% in
Several studies dealing with the breeding system
mature trees. In vitro germination ranged from 46
of
this genus have shown that Nothofagus species
to 71 % in young individuals and 49 to 85 % in mature
possess
a highly self-incompatible reproductive
trees. Fresh pollen dyed with Alexander's stain
system;
thus,
seed set results mainly from crossreached 91-100% viability. Similar values were
pollination
(Riveros
et al. 1995a, 1998; Palma et al.
obtained after three months of storage. After 6
1996).
Although
fruit
development in Nothofagus is
months, pollen viability reduced to 66% and after
one year of storage, viability had dropped to 21 to often near 100% (e.g., each flower results in a fruit),
50%, with mature individuals showing the highest the majority of fruits lack viable seeds (Riveros et
loss of viability. The relevance of these findings to al. 1995a). Riveros et al. (1995b) suggested that
incompatible pollen (from the same individual) as
well as compatible but "weak" pollen from different
individuals may trigger fruit development but only
occasionally will it lead to embryo formation.
*Author for correspondence. Email: mrivero@uach.cl
Moreover,
since Nothofagus
species are
B01035; Published 28 November 2002
anemophilous,
pollination
and
reproductive
success
Received 18 July 2001; accepted 2 July 2002

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672
are constrained to some extent by the climatic
conditions during flowering (Riveros et al. 1995b),
e.g., anthesis and pollen release are sensitive to
temperature and humidity (Palma et al. 1996).
After shedding, pollen grains of these
anemophilous species are exposed to a hostile
environment, such as dry conditions, and yet they
have to reach a receptive stigma while still viable.
In some Nothofagus species, the percentage of viable
pollen that actually reaches the stigmas fluctuates
between 47 and 93% (Palma et al. 1996). This
difference in the viability may be a result of several
abiotic (e.g., temperature and relative humidity),
genetic, and physiological factors (Shivanna & Johri
1985; Shivanna & Rangaswamy 1992; Kearns &
Inouye 1993; Aizen & Rovere 1995). Tree age and
size have also been suggested to increase the
proportion of non-viable pollen (Aizen & Rovere
1995). Furthermore, pollen longevity, considered as
the period in which pollen remains able to germinate
on an appropriate (receptive and compatible) stigma
(Dafni & Firmage 2000), varies significantly from
species to species, ranging from minutes after
shedding to months under laboratory conditions
(Shivanna & Johri 1985). The importance of studies
dealing with pollen viability and longevity has been
largely recognised in pollination biology as a priority
for helping to understand species reproductive
performance and for the successful implementation
of breeding programmes (Dafni & Firmage 2000).
In this study we investigated the viability and
longevity of pollen of four Nothofagus species from
south Chile and whether pollen viability is somehow
reduced when trees of these species get older.
Using direct and indirect viability tests, we assessed
pollen performance from young and mature
individuals and pollen longevity after several months
of storage.

MATERIAL AND METHODS

This study involved four species of the genus
Nothofagus, N. antarctica, N. betuloides, N.
dombeyi, and N. obliqua var. obliqua, growing in
four localities of the X Region of Chile (Botanical
Gardens of the Universidad Austral de Chile,
3948'S; Punahue, County of Riihue, 3940'S;
Puyehue National Park, 4044'S; Vicente Perez
Rosales National Park, 4102'S.). During the
flowering period (September to December), five
young and five mature flowering trees of each
species were chosen and used as pollen donors.

New Zealand Journal of Botany, 2002, Vol. 40

Diameter at breast height (DBH) and historical
records were used to estimate the ages of the
individuals sampled; trees c. 15 years old, or in the
second flowering of their life span, were considered
young and those with a DBH over 80-100 cm,
usually over 100 years old, as mature. In each
species, two anthers from each of 20 randomly
selected male flowers per tree were collected. In
total, 200 anthers from young and 200 from mature
trees were collected from each species. Anthers were
placed in aluminum envelopes (Rotafoil) and
stored at 4C in the refrigerator until examined. Fresh
pollen was also collected and stored in small plastic
vials until used for hand-pollinations. Direct and
indirect techniques were used to assess pollen
viability of young and mature individuals of each
species.
Direct pollen viability assessment
Germination in vivo
The in vivo germination test measures the
germinability of pollen grains on a conspecific
stigma: stigmatic germinability (Dafni & Firmage
2000). Following Riveros et al. (1995a), flowering
branches of young and mature individuals were
bagged before anthesis. To ensure that stigmas were
virgin, male flowers were emasculated at the bud
stage and female flowers were covered with paper
bags prior to the stigmas becoming receptive.
Stigmatic receptivity was determined by eye and
with reference to records of female flower life-span
reported elsewhere (Riveros et al. 1995b).
Conspecific fresh pollen from another tree was used
for hand-pollination treatments and then the
branches were bagged again to prevent any other
pollen deposition.
After seven days, to allow pollen germination and
pollen tube development, branches were unbagged
and female flowers were collected and fixed in
Carnoy's solution (Sass 1958). Following Shivanna
& Rangaswamy (1992), stigmas were stained with
aniline blue, gently squashed, and placed on a
microscopic slide for scoring of germination. Except
for the emasculation and bagging of the flowering
branches, the same protocol was followed to score
germination on naturally pollinated stigmas.
Germination was scored under the microscope in 360
hand-pollinated flowers and 360 naturally pollinated
flowers.
To determine the average number of pollen grains
germinating per stigma of each species, the stigmatic
surfaces of 10 flowers of each category were

Bez et al.Viability and longevity of Nothofagus pollen

measured with an ocular micrometer. Stigmatic
surface was expressed in mm2. Nothofagus species
studied have two tricarpelate (3 stigmas each) and
one bicarpelate flower (2 stigmas) per inflorescence,
in total 8 stigmas per inflorescence; 160 stigmas
were measured per species.

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Indirect pollen viability assessment and pollen

longevity
Germination in vitro
A culture medium of 10% sucrose, 1% agar, and 100
ml of distilled water was used to evaluate
germination. After 24 hours of incubation under
laboratory conditions at 25C, pollen cultures
were scored according to Shivanna & Rangaswamy
(1992). Pollen grains were considered germinated
when the length of the tube was more than the
diameter of the pollen grain (Shivanna & Rangaswamy 1992).
Alexander's procedure
Alexander's (1980) stain is a dye containing
malachite green and acid fuchsin, which differentially stains aborted and viable pollen; malachite
green stains cellulose in pollen grains, and acid
fuchsin stains the protoplasm. Thus, aborted grains
appear green while grains with protoplasm appear
pink. The number of viable pollen grains from stored
samples was determined using a haemocytometer.
Pollen longevity was assessed immediately after
collection, and 3, 6, and 12 months after collection.
Data analysis
ANOVA was used to analyse all data using
Statgraphic plus (Statistical Graphics Corp. 19941996). When variances were not homogeneous,
Kruskal-Wallis non-parametric tests were applied.

Table 1 Total stigmatic surface in young and mature

individuals. n = 160. Different letters indicate statistical
difference; F = 306.2; d.f., 159; P = 0.00018.

Species
N. antarctica
N. obliqua
N. dombeyi
N. betuloides

673

RESULTS
Pollen grain germination on the stigmatic
surface
The total stigmatic surface was highest for young
trees of N. antarctica (0.29 0.1 mm2). Smaller
surfaces were recorded in N. dombeyi, N. obliqua,
and N. betuloides, in decreasing order. Total
stigmatic surface was larger in young than in mature
trees, except for N. betuloides (Table 1).
The number of germinated pollen grains per
stigmatic surface was higher on stigmas of young
trees under natural pollination than in the mature
ones, contrasting with the values obtained through
hand-pollination treatments (Table 2). The highest
germination rate was observed on stigmas of young
trees ofN. obliqua. After hand-pollination of female
flowers, young trees had lower germination rates per
stigmatic surface than older ones, except for N.
dombeyi (9.40 in young and 8.38 in mature trees)
(Table 3). Young trees of N. obliqua and N.
betuloides showed the highest germination rates,
10.8 and 10.6 germinating grains per stigma,
respectively. On the other hand, mature trees of N.
antarctica had the highest average of germination
(15.53). Usually, mature trees had a greater number
of germinated pollen grains per stigmatic surface.

Table 2 Average number of pollen grains germinated

on naturally pollinated stigmas of young and mature
individuals. Different letters indicate statistical difference.
F = 290.97 4; d.f., 149; P = 0.000.

Species
N. antarctica
N. obliqua
N. dombeyi
N. betuloides

Young

Mature

xSD

xSD

6.70 1.1 a
8.85 2.2 a
1.49 0.7 b
5.80 2.1 a

2.93 0.1b
2.90 0.1b
4.20 3.4 a
5.30 3.5 b

Table 3 Average number of pollen grains germinated

on hand-pollinated stigmas of young and mature
individuals. Different letters indicate statistical difference;
F = 223.079; d.f., 149; P = 0.0035.

Young

Mature

Surface mm2

Surface mm2

Species

0.29 0.1 a
0.190.1 a
0.20 0.1 a
0.120.1b

0.180.1b
0.17 0.2 b
0.17 0.3 b
0.20 0.1 a

N. antarctica
N. obliqua
N. dombeyi
N. betuloides

Young

Mature

xSD

xSD

6.81 0.7 a
10.80 0.9 a
9.40 3.3 a
10.60 3.8 b

15.53 3.0 b
11.101.0b
8.38 1.2 b
11.00 4.0 a

New Zealand Journal of Botany, 2002, Vol. 40

674
100 n

Fig. 1 Evaluation of pollen

viability from young (open bars)
and mature (solid bars) trees of
Nothofagus antarctica, N. obliqua,
N. dombeyi, and N. betuloides
using in vitro germination.

80-

60 H

40 H

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20-

N. antarctica

N. obliqua

N. dombeyi

N. belutoides

100 -

Fig. 2 Evaluation of pollen

viability from young (open bars)
and mature (solid bars) trees of
Nothofagus antarctica, N. obliqua,
N. dombeyi, and N. betuloides
using Alexander's stain.

80 -

60-

40 20 H

0
N. antarctica

N. obliqua

N. dombeyi

Evaluation of pollen viability using in vitro

germination
Of the responses of cultured pollen grains among the
young trees, the deciduous species N. antarctica
showed the highest germination rate (71.25%) and
N. obliqua the lowest (45.34%) (Fig. 1). Germination
of pollen was generally higher from mature than
from young trees, e.g., N. betuloides reached 84.78%
germination. Of mature trees, N. obliqua showed the
lowest germination percentage (48.71). Comparing
germination percentages from young and mature
trees, and excluding N. antarctica, a weak trend

N. belutoides

towards higher germination with the increased age

of individuals was observed (Fig. 1).
Evaluation of pollen viability using
Alexander's stain
In general, pollen viability was high in all species
and categories as assessed by Alexander's stain.
Only young and mature individuals of N. betuloides
showed the lowest viability (91.40 and 90.96%,
respectively). Also, a slight trend towards increased
viability from young to mature individuals was
registered in this assessment (Fig. 2).

Bez et al.Viability and longevity of Nothofagus pollen

Fig. 3 Percent of viability in
pollen grains from young trees of
the four species of Nothofagus
immediately after collection and
following 3, 6, and 12 months of
storage.

675
N. antarctica

100 i

N. obiiqua
N. dombeyi
N. belutoides
60 -

Xl

40 -

>

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20 -

12

Time of storage (months)

Fig. 4 Percent of viability in

pollen grains from mature trees of
the four species of Nothofagus
immediately after collection and
following 3, 6, and 12 months of
storage.

N. antarctica

100

N. obiiqua
/V. dombeyi

80

belutoides
60
4020
0
3

12

Time of storage (months)

Pollen longevity
There was a loss of viability in young and mature
individuals with increasing storage time (Fig. 3,4).
The viability of pollen from young trees began to
decline after three months of storage. The most
significant loss of viability occurred between 6 and
12 months of storage, affecting all species. Viability
ofN. betuloides pollen decreased from 91.43% at the
start to 28.93%. The loss of viability in pollen from
mature individuals showed a rather similar trend to
that of young trees. Between 6 and 12 months,
viability fell to almost half of the initial level in all

the species (Fig. 4). Overall, the loss of viability was

higher in mature than in young trees, with the
exception of N. obliqua.

DISCUSSION
In general, the germination success of pollen of the
Nothofagus species studied here was high when
assessed by in vivo and in vitro methods (50-70%).
There was higher pollen germination in handpollinated stigmas than in naturally pollinated ones.

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676
This high germination is understandable partly due
to the origin of the pollen, from a different donor to
avoid auto-incompatibility reactions. On the other
hand, the low germination observed on naturally
pollinated stigmas might have been limited by
dehydration during dispersal (Shivanna & Johri
1985), a consequence of the environmental
conditions and/or nature of the stigmas (in
Nothofagus they are dry). Also, it is important to note
that under natural pollination conditions the amount
of foreign pollen reaching the stigmatic surface is
high (see values in Riveros et al. 1995b), and this
may interfere with pollen germination. In fact, under
natural pollination conditions, only between 5% and
16% of the fruit contain an embryo (Riveros et al.
1998).
It is interesting to note that young individuals
recorded low stigmatic germinability after hand
pollination compared with natural pollination.
Reasons for this fluctuation are not clear; but it could
be attributed to stigma receptivity during the
pollination or changes in the pollen physiology
during collection and handling. It has been reported,
for instance, that pollen viability is so labile that it
may differ when pollen is collected at different times
of the day. In fact, pollen collected from flowers in
anthesis for one hour show decreased germination
(Shivanna & Rangaswamy 1992). Also, it is
important to note that pollen germination and
viability have a genetic component; consequently,
results will be different depending on the genetic
variability of the individuals used as donors (Vasil
1987). For some Nothofagus species (N. dombeyi,
N. betuloides, and N. nitida), Premoli (1996)
reported a high genetic variation within individuals
of populations but low variation between
populations.
The effect of environmental conditions when
pollination and fertilisation took place should be kept
in mind. Eisikowitch & Woodell (1974) noted that
pollen of species in humid environments has slower
dehydration rates than those of dry areas. In Chile,
for instance, springtime flowering species have
managed to delay pollen dispersal until late in the
afternoon, 46 p.m., since relative humidity is high
during the morning, when it is sometimes misty,
conditions that preclude anther opening and pollen
dispersal (Riveros et al. 1995b).
Although the in vivo germination test has the
advantage that it simulates natural pollination (Dafni
& Firmage 2000) and it is more valid than in vitro
germination tests (Shivanna & Johri 1985), the latter
are faster and relatively simpler. However, the main

New Zealand Journal of Botany, 2002, Vol. 40

limitation of this test is the difficulty in achieving
satisfactory germination in many species (Shivanna
& Rangaswamy 1992). In fact, although some tests
were carried out in this study using nutritious media
such as Brewbacker & Kwak's and Robert's
medium, results were negative. After several futile
attempts using calcium and boron for achieving
germination, a culture medium of 10% sucrose and
1% agar was found to allow germination. Mulugeta
et al. (1994) observed a similar situation when
attempting to germinate pollen from the
anemophilous species Kochia scoparia in culture
media enriched with calcium. Dafni (1992)
suggested that many pollen grains can germinate in
water or in aqueous solutions of sucrose with no
additives, especially binucleated ones, as is the case
in Nothofagus.
Besides a carbohydrate source, boron and calcium
play important roles in pollen germination and pollen
tube growth (Brewbacker & Kwak 1963). Lack of
boron and/or calcium usually affects pollen
germination in several plant species; pollen tube
growth is markedly affected, often showing
abnormalities such as coiling and swelling of the tip,
or growing tubes may even burst (Shivanna &
Rangaswamy 1992). Nevertheless, in our study such
abnormalities were never observed. This may
indicate that Nothofagus pollen requires only a
carbohydrate source and suitable conditions of
environmental temperature and relative humidity to
successfully germinate. Pollen germination has been
observed when pollen is still in the anthers (M.
Riveros pers. obs.), and this is consistent with the
suggestion that Nothofagus pollen has few
requirements for germination. The germination
results obtained in the present work are reasonably
high compared with those from pollen germinated
in nutritious media in cultivars of avocado (Sahar &
Spiegel-Roy 1984) or Capsella bursa-pastoris
(47%) (Leduc et al. in Kearns & Inouye 1993).
Alexander's stain, which is meant to distinguish
aborted from non-aborted pollen (Alexander 1980),
revealed a very high rate of stainability, reaching
100% in some species (e.g., N. dombeyi and N.
obliqua). Pollen grains stored at 4C under low
humidity conditions maintained high stainability
even after six months of storage. Non-vital stains,
however, may provide only rough estimates of
viability and caution may be needed in their
interpretation. Marcellan & Camadro (1996)
assessed the viability of asparagus pollen grains after
storage at low temperatures by staining and by in
vitro germination; the stain did not show changes in

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Bez et al.Viability and longevity of Nothofagus pollen

pollen viability during storage but pollen
germination in vitro decreased 70% after 14 days of
storage. The use of Alexander's procedure in the
present work may have led to overestimation of
pollen viability, since staining capacity depends not
on the viability but on the content of the pollen
grains. Hence, such a measure of pollen stainability
used here may depart considerably from the real
value of pollen viability (Dafni 1992). Recently,
Rodriguez-Riano & Dafni (2000) have developed a
new procedure to assess pollen viability and also
recommended the use of some type of control (such
as killed pollen) to check the potential of the dye
before using it.
Pollen from mature trees of Nothofagus did not
show lower viability as observed by Aizen & Rovere
(1995) in Austrocedrus chilensis, which aborts 0.5
7.8% of the pollen produced with the percentage
increasing considerably with tree age and size. The
lack of pollen abortion as trees get older is significant
considering the longevity of some Nothofagus.
Furthermore, this means that pollination experiments
conducted on young trees are as reliable as those
conducted on mature and taller trees; the latter
usually have less accessible flowers.
The pollen produced by the Nothofagus species
studied here is viable and vigorous, but, after
dispersal, germinability seems to be affected by
environmental conditions. Appropriate management
of pollen physiological requirements will be
advantageous in breeding programmes aiming to
improve the quality of the seeds set after handpollination treatments. Moreover, the appropriate
management of pollen longevity in these species is
valuable. Under natural conditions pollen is usually
released 10-15 days before the female flowers are
receptive (Riveros et al. 1995b), a feature that
constrains the success of pollination programmes
within and between genotypes of forestry interest
or between species with different flowering
seasons.

ACKNOWLEDGMENTS
The authors are grateful to the Direccin de Investigacin
de la Universidad Austral de Chile (DIDUACH Grant S98-37) for the financial support to undertake this research;
also to Enrique Rico, Alastair Robertson, and Jill Rapson
for valuable comments and suggestions on an earlier draft
of this paper.

677

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