Annals of Botany 86: 133±148, 2000
doi:10.1006/anbo.2000.1170, available online at http://www.idealibrary.com on
Floral Structure, Stigma Receptivity and Pollen Viability in Relation to Protandry
and Self-incompatibility in Silky Oak (Grevillea robusta A. Cunn.)
A . K A L IN G A N IR E *{, C . E . H A R WO O D{, M . U . SL E E { and A . J . SI M O N S }
{Department of Forestry, Australian National University, ACT 0200, Australia, {CSIRO Forestry and Forest
Products, PO Box E4008 Kingston, ACT 2604, Australia and }International Centre for Research in Agroforestry,
PO Box 30677 Nairobi, Kenya
Received: 24 December 1999 Returned for revision: 11 February 2000
Accepted: 24 March 2000
The reproductive biology of Grevillea robusta growing under exotic conditions in Kenya and Australia is reported.
The species showed both protandry and a self-incompatibility mechanism. The stigma was wet and papillate with a
distinct groove in the middle. The anthers dehisced prior to anthesis, when the perianth opened. Stigmatic receptivity
began 1 d after anthesis, with the greatest pollen germination rates and longest pollen tubes obtained 2 d after
anthesis. Nectar secretion commenced with pollen dehiscence and was abundant at anthesis. Most stigmatic grooves
opened widely 1±2 d after anthesis and stigmas showed taller papillae and abundant secretion. Controlled pollinations gave a greater fruit set from cross-pollination (5.9 % in April and 17.5 % in July) than open-pollination (0.1 %
in April and 3.3 % in July). No fruit set from self-pollination was obtained in April, and very few fruit set for
geitonogamous (two out of 1622; 0.1 %) or for autogamous (one out of 2707 ¯owers; 0.04 %) pollination treatments
in July. Following self-pollination, growth of pollen tubes was poorer than in other treatments, and was generally
arrested in the upper style. Cross-pollinated ¯owers produced normal and straight pollen tubes, while self-pollen
tubes had growth abnormalities. Most of the open-pollinated ¯owers were found without pollen or with only selfpollen on their stigmas indicating that the amount of cross-pollen reaching the stigma under open-pollination may be
a factor limiting seed production. Flowers shed soon after the fertilization phase were those with ungerminated pollen
or no pollen. Although a very low rate of sel®ng may occur, G. robusta presents a self-incompatibility system and
# 2000 Annals of Botany Company
allogamy is its primary breeding behaviour.
Key words: Grevillea robusta, silky oak, Proteaceae, protandry, controlled pollinations, receptivity, pollen-tube
growth, self-incompatibility, pollination.
I N T RO D U C T I O N
Grevillea robusta A. Cunn. ex R. Br. (silky oak) is a tree
belonging to the dicotyledonous plant family Proteaceae,
subfamily Grevilleoideae in the tribe Grevilleeae (McGillivray, 1993). The species has a restricted natural range on
the east coast of Australia from latitude 228500 S to 308100 S
(Harwood, 1992; McGillivray, 1993). Grevillea robusta has
been introduced to many countries in south and central
America, south Asia, and in the highlands of eastern and
central Africa (Harwood, 1989) where it is very common
and popular for farm plantings. It is grown as a shade tree
for tea and co€ee plantations, in agroforestry plantings for
fuel wood and timber and also as an ornamental tree.
Detailed studies of the reproductive biology of the
Proteaceae have been mainly limited to species of the
genus Banksia L.f. (e.g. Carthew, 1993; Sedgley et al., 1993,
1994; Goldingay and Carthew, 1998) and Macadamia
Muell. (e.g. Sedgley et al., 1985; Wallace et al., 1996) and
recently Dryandra (Matthews and Sedgley, 1998). Grevillea
R. Br. ex Knight, the largest genus in the family Proteaceae,
has received less attention and existing information has
* For correspondence at: CSIRO Forestry and Forest Products,
PO Box E4008, Kingston ACT 2604, Australia. Fax ‡61 2 6281 8266,
e-mail Antoine.Kalinganire@€p.csiro.au
0305-7364/00/070133+16 $35.00/00
focused mainly on levels of self-incompatibility (Herscovitch and Martin, 1990; Harriss and Whelan, 1993; Ayre
et al., 1994; Hermanutz et al., 1998; Hogbin et al., 1998).
The most comprehensive work on the biology of G. robusta
is that of Brough (1933), who reported in detail its
life history, ¯oral morphology and seed development,
and that of McGillivray (1993) who reported its taxonomy.
Harwood (1992) and Kalinganire et al. (1996) have
reported the ¯owering pattern and pollen vectors for
G. robusta.
Proteaceous species are generally considered protandrous
(see Johnson and Briggs, 1975; Collins and Rebelo, 1987;
Ladd et al., 1998). The protandrous behaviour of G. robusta
has been previously reported by Brough (1933) for planted
trees in Sydney and by Venkata Rao (1971) for landraces in
India; anthers dehisce prior to stigma receptivity and pollen
is deposited onto the pollen presenter. In contrast, the study
of Owuor and Oduol (1992) concluded that in Kenya the
species is protogynous. There have been no reported studies
of stigma development and pollen tube growth and viability
before, during and after anthesis in G. robusta.
Although most species of Proteaceae are largely selfincompatible, there is some indication that several selfcompatible species preferentially out cross (Goldingay and
Carthew, 1998). Grevillea shows highly variable breeding
# 2000 Annals of Botany Company
134
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
systems (Harriss and Whelan, 1993; Ayre et al., 1994;
Hermanutz et al., 1998), although McGillivray (1993)
stated that self-compatibility is probably predominant in
the genus. For G. robusta, Brough (1933) noted allogamy
appeared to be its predominant behaviour, although some
self-fertilization did occur. However, Owuor and Oduol
(1992) found it to have an autogamous breeding system in
exotic stands in Kenya with 63 % fruit set from selfpollination and 69 % from open-pollination. Using isozyme
studies, Harwood et al. (1992) reported G. robusta to be
predominantly outcrossing in two natural populations. The
contradictory results reported for the species to date suggest
a need for more research to elucidate its breeding system.
The availability of seed is often a limiting factor for
planting programmes in most East African countries and an
understanding of the factors a€ecting seed production has
important practical applications, for both genetic improvement and operational seed production. This study investigates important aspects of the reproductive biology of
G. robusta: protandrous behaviour including ¯ower structure and development, and the self-incompatibility mechanism dealing with pollen tube growth and fruit set
following self-, outcross- and open-pollination. Moreover,
the study attempts to de®ne the temporal sequence of
stigma receptivity, the relationship of stigma receptivity to
pollen release and the degree of pollen viability.
M AT E R I A L S A N D M E T H O D S
Plant material and study site
The study was conducted at two sites: Malava, western
Kenya, from January 1996 to April 1997; and Canberra,
southern Australia, in December 1997.
The Malava site (08280 N, 348510 E; 1600 m a.s.l.) is part
of a ®eld trial site maintained by the International Centre
for Research in Agroforestry (ICRAF) and Kenya Forestry
Research Institute. The site has a slope of around 5 % with
a south-westerly aspect. The adjacent vegetation is mainly
natural evergreen forest with some cleared farmland nearby.
The mean annual rainfall (years 1990 to 1995) is 2413 mm
with a bimodal distribution. Rain falls in every month of
the year, with May and September the wettest months.
During the study period, the monthly means of daily
temperature maxima ranged from 278C (August) to 338C
(March) and corresponding minima from 148C (July) to
168C (November). The monthly means of daily relative
humidity at 0700 h ranged from 76 % (November) to 97 %
(July). The soil is classi®ed as very-®ne, kaolinitic, isohyperthermic, udic kandic ustalf, with a pH of 6.2 (Matungulu,
1994).
A trial of 90 open-pollinated G. robusta families from
19 Australian provenances was planted on the site in April
1991, in a design which maintained both family and
provenance identity. Provenance details (Table 1) spanned
the range of the species' natural distribution. The trial,
within 1.1 ha, used a randomized complete block design
with seven complete replicates and single-tree plots. Spacing
between trees was 4 m 4 m. At the time of the study,
mean height of trees was around 14 m, mean diameter at
breast height 18 cm, and overall survival exceeded 95 %.
The plant material used for the timing of stigmatic
exudates, measurements of groove and papillae size and for
an in-vitro pollen germination test, as part of the ¯oral
receptive study, was collected on planted trees growing in
the Australian National Botanic Gardens (ANBG), at
Canberra. Preliminary observations had indicated the
duration of ¯owering in Canberra was very similar to that
in Kenya and the ambient temperature in the two locations
at ¯owering was also similar. Thus the Canberra observations should be applicable to Kenya. At Canberra
the average daily maximum temperature for December,
the ¯owering and study period, was 26.28C and the
T A B L E 1. Details of the natural provenances of Grevillea robusta planted in the provenance±progeny trial at Malava
(western Kenya)
Seedlot no.
CSIRO
seedlot no.
No. of families
included
Provenance name
Alt
(m)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
17694
15872
17693
17610
17699
15873
17612
17185
17613
17611
17614
17616
15615
17617
17618
17620
17619
17621
17622
6
4
8
4
5
4
5
1
3
4
4
2
3
4
4
10
4
4
11
Porters Gap
Linville
Bunya Mountains
Mudgereeba
Albert River
Emu Vale
Nimbin
Woodenbong
Grevillea
Tyalgum
Duck Creek
Paddys Flat
Bottle Creek
Mummulgum
Rappville
Mann River
Fine Flower
McPhersons
Boyd River
650
140
1000
20
280
550
50
200
180
80
160
180
200
100
40
60
60
40
200
Lat
(8S)
26
26
26
28
28
28
28
28
28
28
28
28
28
28
29
29
29
29
29
45
29
54
5
16
14
38
26
26
22
43
44
48
50
7
24
33
48
53
Long
(8E)
151
152
151
153
153
152
153
152
152
153
152
152
152
152
152
152
152
152
152
30
16
37
22
6
17
13
45
47
11
33
26
39
49
58
29
29
54
27
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
Floral structure and development
In¯orescences of G. robusta are terminal and may form
up to six branches each bearing hermaphroditic ¯owers
grouped into racemes (Fig. 1) (Johnson and Briggs, 1975;
McGillivray, 1993). In this study, each individual branch is
referred to as a raceme. Floral development in G. robusta
has been described by Brough (1933) and the main stages
are illustrated in Fig. 2. Each ¯ower of G. robusta contains
two ovules (McGillivray, 1993; Hermanutz et al., 1998).
Flower structure and development were studied at the
Malava site. In¯orescence length, number of racemes per
in¯orescence and raceme length were recorded. Daily
observations began on 25 Mar. 1996 and ended on 26
Jul. 1996. Basic stages of ¯ower development were de®ned
by detailed examination of ®ve trees. Flower parameters
were measured on 43 trees ( four in¯orescences per tree and
four racemes per in¯orescence sampled) representing 16
families in six provenances (Emu Vale, Mudgereeba, Albert
River, Nimbin, Rappville, Mann River; details in Table 1)
distributed across the species' natural range. Style length
was measured on ®ve ¯owers selected at random around
each raceme. Only the length of the style was recorded as
the pedicel does not change signi®cantly during ¯oral
development. The number of ¯owers per raceme were
recorded at all stages. Open ¯owers were counted and
recorded as were the number of ¯owers with nectar.
Time (no. of days from 0d):
Stage:
10
(i)
20
(ii)
31
(iii)
conflorescence
or raceme
main
raceme
Length of inflorescence
corresponding average daily minimum was 11.28C. The
average daily relative humidity recorded during the study
period at 0700 h was 62 %. The trees were 35-years-old and
¯owering heavily at the time of the study. The natural
provenance origin of the trees is uncertain; they originated
from seeds collected from one or more mother trees in a
planted stand in Western Australia.
len
gt
h
of
ra
ce
135
m
e
primary
peduncle
F I G . 1. A four-branched in¯orescence of Grevillea robusta shows
di€erent con¯orescences or racemes.
Furthermore, observations on capped ¯owers were made
on 12 trees (three in¯orescences per tree and two racemes
per in¯orescence sampled). Capped ¯owers are individual
¯owers with the staminal structure remaining in place over
the stigma holding the ¯ower's pollen and excluding other
pollen.
Fruit set at di€erent stages of stigmatic development
Timing of stigma receptivity was investigated by assessment of fruit set following controlled pollination at Malava.
Preliminary observations showed that cross-pollination
between unrelated trees produced fruit with viable seeds.
Consequently, crosses were made between unrelated trees,
i.e. from di€erent families, for the experiments. Flowers
33
(iv)
35
(v)
38
(vi)
39
(vii)
54
(viii)
F I G . 2. Individual ¯ower development stages of Grevillea robusta and the mean number of days to reach each ¯oral stage from ¯ower macroscopic
initiation. The main stages represented in sequence are from left to right: (i) ¯ower bud macroscopic initiation; (ii) early buds; (iii) late buds;
(iv and v) two stages of early looping; (vi) advanced looping; (vii) pollen presentation (anthesis); and (viii) fruit set.
136
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
were emasculated to remove self-pollen and isolated, then
hand pollinated.
The emasculation and isolation technique used was a
modi®cation of the procedure described by Owuor and
Oduol (1992). Emasculation was carried out at the late
looping stage (Fig. 2; stage vi), approximately 48 h before
the style elongated and was freed from the perianths. At this
stage pollen presenters are easily pulled out by hand or
using a pin, thus being separated from the perianths enclosing the anthers. The undehisced anthers were then removed.
All other ¯owers were trimmed o€ using ®ne scissors. All
groups were emasculated at the same time.
In¯orescences were isolated from pollinators by the use
of muslin bags. Crosses were carried out by applying
pollen-laden stigmas to stigmas of emasculated ¯owers.
Bags were retained until the end of the receptive phase, i.e.
the fall of the perianths, about 20 d after cross-pollination.
For this experiment, three trees from three di€erent
families were used. Flowers from the main raceme for one
in¯orescence per tree and per treatment were tagged. Flower
stages and the corresponding number of ¯owers (given in
brackets) assessed were: ÿ2 d (170); ÿ1 d (141) before
anthesis; at 0 d (132) or anthesis; and 1 d (159); 2 d (127);
3 d (156); 4 d (132); and 5 d (149) after anthesis. The
number of ¯owers used for each treatment ranged from 21 to
64 ¯owers per tree. Di€erent sources of pollen were applied
on the three trees, thus avoiding any chance of incompatibility. Fruit set was recorded 60 d after pollination.
Pollen germination at di€erent stages of stigmatic
development
To determine the ¯oral stage which corresponds to
optimum stigmatic receptivity, pollen germination on
stigmas and subsequent pollen tube growth at various
¯oral stages was assessed. Two 35-year-old trees growing in
the ANBG, Canberra, were used as mother trees for this
trial. One raceme per tree was tagged for each stigmatic
development stage. Flower stages assessed were: ÿ2 d prior
to anthesis; ÿ1 d prior to anthesis; at 0 d or anthesis; and
1 d; 2 d; 3 d; and 4 d after anthesis. In this study anthesis is
de®ned as the time when the perianth opens and pollen is
presented to ¯oral visitors. Flowers were emasculated
approx. 2 d before anthesis (Fig. 2; stage vi) and isolated
from visitors using polyester pollination bags (bag type PBS
10-1; PBS International, UK). Flowers for ÿ2 d, ÿ1 d
prior to anthesis and on the day of anthesis (0 d) had their
self-pollen removed and were pollinated immediately with
cross-pollen from two di€erent trees, whose relatedness is
uncertain. For other stages the pollination time was referred
to as the emasculation day, which corresponds approximately to 2 d before anthesis, and ¯owers were cross-pollinated
as above. Emasculated ¯owers were cross-pollinated at the
appropriate stage using pollen of fresh 0 d ¯owers from
di€erent trees as above. The isolation bags were replaced
immediately after pollination. Pollen could be seen on
stigmas after application.
At least 25 ¯owers per stage and per tree were harvested
24 h after pollination and styles processed for ¯uorescence
microscopy for pollen germination and tube growth as
follows. Immediately after harvesting pistils were ®xed in
`Carnoy' ®xative (absolute ethanol : chloroform : acetic acid;
6 : 3 : 1) for at least 2 h and then transferred to 70 % ethanol
for storage. Pistils were later cleared and softened with
sodium hydroxide (0.8 M NaOH) for up to 1.5 h at 608C,
rinsed in distilled water and then stained overnight with
water-soluble aniline blue in potassium phosphate (K3PO4)
bu€er. This method causes callose in the pollen grains and
tubes to be stained and to ¯uoresce brightly under a shortwave (UV) light. Pistils were then placed on a microscope
slide with a drop of 80 % glycerol and squashed beneath a
coverslip. Pollen tubes were examined by ¯uorescence
microscopy and photographed or stored in the dark for
further viewing. Samples were viewed through a Nikon
Optiphot epi¯uorescence microcope with a ¯uorescene
source.
Twenty-®ve ¯owers for each treatment on each tree were
examined microscopically. A ¯ower with at least one
germinated pollen grain was scored as receptive. A pollen
grain was considered germinated when the length of its tube
was more than the diameter of the pollen grain (Shivanna
and Rangaswamy, 1992). Fluorescing pollen grains without
any pollen tubes were scored as ungerminated.
Timing of stigmatic exudates, and groove and papillae size
The stigmatic features of unmanipulated ¯owers at
di€erent stages were assessed and recorded. Flower stages
assessed were: ÿ2 d; ÿ1 d before anthesis; at 0 d or
anthesis; and 1 d; 2 d; 3 d; and 4 d after anthesis. One
35-year-old tree growing in the ANBG, Canberra, was used
for this experiment. One in¯orescence per treatment was
tagged and bagged as above. For stages ÿ2 d, ÿ1 d and
0 d, ¯owers were identi®ed, tagged and immediately
collected for scanning electron microscopy (SEM). For
treatments 1 d, 2 d, 3 d, 4 d and 5 d after anthesis, on the
day of anthesis (0 d), all ¯owers at looping stage were
clipped o€ the raceme using ®ne scissors. The open ¯owers
remained untouched for collection at the appropriate stage.
Five fresh pistils were collected per stage for examination.
All ¯owers from di€erent stages used in this experiment
were collected at the same time.
The timing of stigmatic exudation and associated groove
and papillae size and adhesion of pollen on the stigma were
determined using SEM. Samples for SEM were prepared
following a cryo-SEM method (Craig and Beaton, 1996;
R. Heady, pers. comm., 1998). This method allowed fresh
stigmatic exudates to be checked, which would have dried
out using other methods. Fresh sample material was
attached to a mounting plate using tissue freezing medium
and rapidly frozen by plunging into liquid nitrogen slush at
ÿ2308C. The plate with attached sample was then inserted
into the preparation chamber of an Oxford CT1500 Cryo
Preparation System and slowly warmed to ÿ808C in order
to remove ice crystals on the surface of the specimen. The
frozen sample was then coated with a 10 nm layer of gold
and transferred to a cryo stage at ÿ1858C ®tted inside the
chamber of a Cambridge Instruments S360 SEM. The
electron optics system of the SEM was optimized for high
resolution, but with sucient depth-of-®eld to enable the
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
entire selected image to be focused. This required a 30 mm
diameter ®nal aperture, a working distance of approx.
18 mm, electron beam current of 80 pA and an accelerating
voltage of 15 kV. The sample was maintained at ÿ1858C
throughout the SEM viewing operation.
The papillae length, the pollen size and the width of the
stigmatic groove opening at its widest point, half way along
its length, were estimated from micrographs of ®ve di€erent
stigmas per stage using the `NIH Image Program' version
1.57 (Public Domain Image Processing and Analysis
Program, National Institute of Health, USA). However,
groove and papillae size could not be measured with
precision as pollen and stigmatic secretions obstructed the
reading for most stages. Estimates for groove width were
recorded for up to 2 d after anthesis, while other stages
were only checked for stigmatic ¯uids. Five stigmas were
observed and at least 30 pollen grains were measured per
¯oral stage.
In order to check for any e€ect of emasculation on the
stigmatic receptivity, ®ve ¯owers were emasculated at
approx. 1 d before anthesis and ®ve other ¯owers on the
same in¯orescence were left unmanipulated. The pistils of
these ten ¯owers were collected 24 h later and examined by
SEM for stigmatic exudates, a probable indication of
stigmatic receptivity.
Duration of viability of pollen grains on the stigma
Pollen grains of G. robusta were tested for their capacity
to germinate and produce a normal pollen tube after
exposure on the pollen presenter and stigmatic region under
®eld conditions. Pollen was collected from ¯owers of the
same stage (age) from three trees at the ANBG, Canberra,
at ÿ2 d, ÿ1 d, at 0 d (anthesis), and 1 d, 2 d, 3 d and 4 d
after anthesis.
Racemes of ¯owers at di€erent stages were identi®ed,
tagged and isolated using polyester bags to avoid any pollen
contamination by visitors until the pollen was collected for
viability testing. On a tagged raceme, as di€erent ¯ower
stages are present on a single raceme, only ¯owers of the
same stage following a given treatment were left and others
were removed. Flowers at stages ÿ2 d, ÿ1 d and 0 d were
tagged and pollen collected immediately. Pollen from
¯owers at the same stage was mixed for a germination test.
For other stages, ¯owers were tagged on the day of
anthesis and then collected at the appropriate time
depending on the treatment. At least 100 ¯owers per stage
and per tree were collected and pollen was removed from
the stigmas using a ®ne sterile pin. The fresh pollen was
mixed in a vial and tested immediately for germination. A
fresh pin was used for each stage.
Preliminary studies indicated that media containing only
sucrose might not result in optimum pollen germination. In
the study 100 ml of aqueous nutrient agar solution was
made up using 20 g sucrose, 1 g agar, 0.01 g boric acid,
0.03 g calcium nitrate, 0.02 g magnesium sulphate, and
0.01 g potassium nitrate. Two drops of the medium were
placed on each end of a microscope slide. As pollen grains
of many species exhibit a population e€ect (e.g. Mulcahy
et al., 1992) the density of pollen grains in the culture
137
medium may be critical (Shivanna and Rangaswamy,
1992). Consequently when the medium had set, a suitable
amount of pollen (not too small or too large; about 100
pollen grains) was added over each drop and mixed
thoroughly with a needle to obtain homogenous pollen
distribution. The microscope slide was then placed in a
Petri dish on two glass rods placed parallel at about 50 mm
apart on moist ®lter paper and then incubated at 228C.
After 24 h the microscope slide was viewed under the
microscope at 160 magni®cation. A coverglass was
lowered on the preparation prior to the microscopic
examination. The number of pollen grains was counted
and germination assessed.
Germination was scored and recorded following Shivanna and Rangaswamy (1992). Pollen grains in ten
randomly selected microscope ®elds were observed. To
avoid repeated scoring of the same group of pollen, the
preparation was moved under the microscope to view
adjacent but non-overlapping ®elds. From each ®eld, the
total number of pollen grains and the number of germinated grains were recorded, and the length of pollen tubes
measured with an ocular micrometer. Five individual
straight pollen tubes were measured in each ®eld.
Assessment of self-compatibility and barriers to sel®ng
A combination of ®eld work and ¯uorescence microscopy
was used to investigate incompatibility mechanisms and to
determine barriers to sel®ng. Data from ®eld observation
and plant material for microscopy were collected
between March and September 1996 at Malava and
between November 1997 and January 1998 at Canberra.
A controlled pollination technique was used in order to
investigate the e€ect of di€erent pollination treatments on
fruit set. This involved 27 trees, selected from nine di€erent
families (i.e. three trees per family) from three di€erent
natural provenances (Bunya Mountains, Boyd River and
Duck Creek; details in Table 1). The above provenances
and families were selected randomly from the heavily
¯owering families in the stand. The same trees were used
in April and July to check for seasonal variation.
Four di€erent pollination treatments were applied to
either one or two in¯orescences on each tree. A minimum of
30 ¯owers per in¯orescence was used. In¯orescences were
tagged and randomly allocated to one of four experimental
treatments: (1) no experimental manipulation (left for openpollination): in¯orescences were left uncovered to allow
visits by pollen vectors; (2) xenogamy (cross-pollination):
self-pollen removed (by emasculation as described above)
and in¯orescences bagged, and pollen from trees of a
di€erent provenance applied to stigmas 48 h after emasculation. Pollen was collected randomly from ¯owering trees,
maintaining the same source of pollen for the trees of each
family at one period, but not necessarily being the same
trees for both experimental periods (April and July); (3)
autogamy (natural self-pollination; spontaneous sel®ng):
in¯orescences were bagged and left unmanipulated; (4)
geitonogamy (induced self-pollination): self-pollen removed
and in¯orescences bagged; pollen taken from other in¯orescences on the same tree, just after pollen dehisces from
138
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
the anthers onto the pollen presenter but prior to anthesis,
was transferred to stigmatic areas.
For treatments (2) and (4), ¯owers were emasculated,
isolated and pollinated as described earlier. Fruit set was
evaluated 60 d after anthesis. The presence or absence of
fruit developed from each ¯ower was recorded.
The level of incompatibility among families was assessed
by controlled cross-pollination. At Malava, ®ve families
each from Boyd River (BR) (only three families are
reported) and Bunya Mountains (BM) provenances were
used in this study. The two populations are widely
separated, being at opposite ends of the natural distribution
of the species. The choice for cross mating presumably
avoided any possibility of recent common ancestry. One to
®ve trees per family were crossed with pollen from one to
®ve other families in the provenance and/or crossed with
one to ®ve families in the other provenance.
The e€ectiveness of di€erent pollination treatments was
assessed by detection of pollen tubes in treated ¯owers. The
experiment involved eight trees of di€erent families (one
tree per family) in July 1996. In¯orescences with at least 120
¯owers were identi®ed and tagged. These were randomly
allocated to one of the four experimental treatments (open,
xenogamy, autogamy and geitonogamy) as described
above. The isolation, emasculation and hand-pollination
procedures were the same as described under fruit set.
Samples of the ®ve ¯owers were randomly collected from
over the whole raceme for each treatment of each tree
(40 ¯owers per treatment) 48 h after pollination. Preliminary observations showed growth of the pollen to take
between 24 and 48 h from pollen germination to fertilization. In the case of the autogamy treatment, ¯owers were
sampled 72 h after even-aged ¯owers had their pollen
presented, giving self-pollen a chance to germinate. For the
open-pollination treatment (1), ¯owers were sampled after
they had started to fade and the stigmas to turn brown
(i.e. more than 72 h after anthesis). The fading of the
¯owers was known to have no e€ect on the visibility of
pollen tubes in other Proteaceae (e.g. Goldingay et al.,
1991).
Sampled ¯owers were ®xed and processed for ¯uorescence microscopy as described above. Forty pistils per
treatment were processed and examined microscopically
using aniline blue as a callose stain. Pollen tubes were
counted close to the stigmatic region, where individual
pollen grains and attached tubes could be observed. Additional observations were made in the transmission tissue,
further down the style, to check for continued growth of
pollen tubes. It was dicult to observe pollen tubes in the
lower style, thus observations for the lower style could not
be scored with precision and were discarded from analysis.
However, cross-pollen tubes were occasionally observed
entering the ovary, and self-pollen tubes did not appear to
enter the lower style region. Counts were made of the
number of pollen grains per ¯ower, pollen grains germinated on the stigma and the number of grains with pollen
tubes in the upper style. Fluorescing pollen grains without
any pollen tubes were scored ungerminated. Fluorescing
pollen tubes were scored as normal tubes if they appeared
straight, or scored abnormal if they had either bulbous
swellings or directionless growth.
Flowers which fell 5 to 10 d after anthesis were checked
for pollen germination and pollen tube growth. These were
collected fresh under two di€erent trees at Malava (Kenya)
and 56 single ¯owers were examined for pollen germination
on the stigmas. At Canberra, dropping ¯owers were
sampled in the crown of three trees in the ANBG. A total
of 720 ¯owers were collected fresh from three hanging
plastic dishes per tree on four di€erent occasions; they were
®xed immediately and later processed for ¯uorescence
microscopy.
Statistical analysis
Most data are categoral and were approximated by a
binomial distribution. A logistic regression model with a
link function (logit) was ®tted to analyse data on fruit set
under di€erent pollination treatments and the proportion
of pollen tubes ( presence or absence of tubes) in the style.
For pollen viability on stigmas, the signi®cance of treatment di€erences in the proportion of germinated pollen
grains and pollen tube length and the size of the stigmatic
groove and pollen on di€erent stigmatic phases was
assessed by analysis of variance (ANOVA) (McCullagh
and Nelder, 1989; Mead et al., 1993). Log transformations
of the original data were applied to the number of pollen
grains per stigma and the number of germinated grains
(compatibility experiment and dropping ¯owers), and
square-root transformations to the proportion of pollen
germination ( pollen germination on di€erent stigmatic
phases) before ANOVA was carried out.
For traits observed for ¯oral structure and development,
the signi®cance of provenance and family di€erences were
tested by analysis of variance (GLM procedure; Mead et al.,
1993).
R E S U LT S
Flower structure and development
In¯orescence length averaged 151 mm (range 50 to 264;
Table 2). Signi®cant (P 5 0.05) provenance and family
variation was evident. Mudgereeba and Mann River had
longer in¯orescences than other observed provenances with
an average of 168 mm and 167 mm, respectively. The
overall mean number of racemes per in¯orescence was 3.9,
with provenances di€ering signi®cantly in number. There
was signi®cant variation in in¯orescence length at the
provenance and family level. The total number of ¯owers
per raceme was the only trait with signi®cant tree-to-tree
variation within families.
Racemes were on average 105 mm (range 10 to 175 mm;
Table 2) in length when fully developed, bearing 84 (range
15 to 159 ¯owers) early ¯oral green buds and 40 (range 0 to
134) golden-yellow or orange coloured ¯owers at anthesis.
The open-pollinated ¯owers set few fruits with a low
fruit : ¯ower ratio (0.1 %; range 0 to 18.2 %).
Flowers developed over an average period of 39 d from
bud initiation to anthesis, and a further 60 d was required
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
139
T A B L E 2. Family means for ¯oral parameters at the time of anthesis for Grevillea robusta
No. of trees
In¯or. length
(mm)
Emu Vale1
Emu Vale2
4
2
135
136
4.1
3.2
92
107
Mudgereeba1
Mudgereeba2
2
1
169
167
3.6
4.8
Albert River1
Albert River2
Albert River3
4
3
4
148
149
164
Nimbin1
Nimbin2
Nimbin3
4
2
3
Rappville1
Rappville2
Rappville3
Mann River1
Mann River2
Mann River3
Family name{
Mean
Range
F-values:
Provenance
Family
Tree in family
Final length
of style (mm)
Loss of ¯owers to
anthesis{ ( %)
22
55
21
22
70
41
118
95
50
40
25
24
68
52
4.1
4.7
4.1
109
82
123
44
31
57
24
22
22
49
64
48
194
145
117
4.5
4.3
4.0
126
71
92
59
32
29
22
21
21
43
72
58
3
3
2
141
130
126
3.0
3.2
2.8
95
85
79
26
26
27
25
23
24
58
75
49
2
1
3
183
156
161
3.8
3.8
3.9
107
110
119
32
58
38
24
20
26
48
52
61
(43)
151
50±264
3.9
1±6
105
10±175
40
0±134
23
13±30
56
3±100
3.79**
2.89*
0.36ns
No. of racemes Raceme length No. of ¯owers
per in¯or.
(mm)
per raceme
5.99**
1.64ns
0.55ns
1.14ns
2.82*
0.28ns
1.83ns
0.06ns
3.14*
6.11***
1.05ns
0.74ns
0.60ns
0.37ns
2.05ns
*** P 5 0.001; ** P 5 0.01; * P 5 0.05; ns, non-signi®cant.
{ Details for families are given in Table 1.
{ Di€erence between the number of ¯owers at the early bud stage and the number of ¯owers at anthesis, as a percentage of the number of
¯owers observed at early bud stage.
for fruits to reach maturity. The mean number of days from
¯ower bud initiation to di€erent ¯ower stages, including the
number of days between stages, is given in Table 5 and
illustrated in Fig. 2. As the ¯ower matures the style elongates nearly three-fold from 8.6 + 0.5 mm at early bud, to
23 + 3.1 mm (up to 30 mm; Table 2). The pollen presenter
of the mature ¯ower is thus about 25 mm away from the
nectar (the main attractant for pollinators), which is
produced near the base of the ¯ower. Flower development
is accompanied by a gradual change in style colour from
light green at initiation, orange at anthesis to green/brown
at fruit maturity.
An average of 99 % of the ¯owers in a raceme may abort
(range 100 to 82 %). Many were lost between initiation and
early looping (mean of 42 % of ¯owers initiated). More
¯owers were lost between early looping and anthesis
(overall 14 %) and another 43 % from fruit initiation to
fruit maturity. There was no signi®cant correlation between
the number of ¯owers at anthesis and the number of fruit
set.
The ¯owers opened acropetally, mostly towards dawn
(Kalinganire, unpubl. res., 1996) with opening lasting 3 to
5 d for each whole raceme. Generally, nectar ¯ow started
within 24 h of anthesis of the ®rst ¯ower to present pollen
on a raceme and the ¯ow lasted for about 5 d for the raceme
(range 4±7 d; Table 3), until about 24 h after the last ¯ower
had presented its pollen. Occasional ¯owers were observed
open without any nectar until 1 d after opening.
For each individual ¯ower, observations indicated that
nectar secretion occurred within 24 h of the stigma being
freed from the perianth and continued for 2 d. Within a day
after anthesis, nectar secretion occurred together with the
production of stigmatic exudates, the stigma appearing
shiny and wet. However, from these observations it was not
possible to establish when the exudates started ¯owing from
the stigmas.
Fruit set at di€erent stages of stigmatic development
Results of the controlled pollination experiment (Fig. 3)
indicated that fruit set was possible for the period between
1 d before and 2 d after anthesis. No fruits were set on
¯owers pollinated 2 d before anthesis and 3 to 5 d after
anthesis, suggesting non-receptivity of the stigma at these
periods. However, it was not possible to determine the
timing of stigma receptivity from this study as deposited
pollen may remain viable to germinate when the stigma
becomes receptive. For example stigmas might not be
receptive 1 d before anthesis although fruit set was
obtained.
Pollen germination at di€erent stages of stigmatic
development
The percentage of receptive stigmas, de®ned by the
germination of pollen, at di€erent stigmatic stages was
140
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
T A B L E 3. The main ¯owering stages de®ned for Grevillea robusta at Malava
Stage
Main stages
Day*
s.d.
Description
Start of formation of ¯ower bud; green
Individual buds visible; purple/green
Style elongates with a small loop; purple/orange
Style elongates with a very large loop and gradual emergence between
two perianth segments; orange
Freeing of the stigmatic region, stigma shiny; orange
Perianth segments bearing the dehisced anthers fall; orange
Embryo starts to swell; orange/green
Fruit from green to brown
1
2
3
4
Initiation
Early bud
Early looping
Advanced looping
0
10
31
35
±
0.1
0.4
0.4
5
6
7
8
Anthesis/nectar
Fertilization
Fruit development
Fruit maturity
39
44
54
99
0.5
0.6
0.4
11.0
* The start (mean day) of a given stage and the end (mean day) of the preceding stage.
signi®cantly di€erent among treatments (P 5 0.05). There
was no pollen germination on stigmas observed 2 d or 1 d
before anthesis (Fig. 4).
On the day of anthesis the few pollen grains germinated
had very short pollen tubes in the styles. Peak stigma
receptivity in G. robusta was recorded 2 d post-anthesis with
98 % of observed stigmas being receptive and pollen grains
having long tubes. Stigmas observed 3 and 4 d postanthesis, showed high pollen germination but with short
14
Fruit set (%)
12
10
8
6
4
2
0
−2
−1
0
1
2
3
4
Time of pollination
Percentage of receptive stigmas
F I G . 3. Fruit set from cross-controlled pollinations carried out at
varying times [days before (ÿ) and after (‡) anthesis] on emasculated
¯owers at Malava. Bars show s.e.
100
90
80
70
60
50
40
30
20
Timing of stigmatic exudates, and groove and papillae size
Stigma development over a 7 d period and receptivity
features of individual G. robusta ¯oral periods are given in
Table 4. Groove width was at a maximum 1 d after anthesis
(419 + 36 mm; range 379 to 491 mm). There was no pollen
adhering to the stigma 2 d before anthesis (Fig. 5); thereafter all stages examined had stigmas covered by pollen.
Overall pollen size was 40.8 + 2.7mm. Pollen sizes at
di€erent stigmatic stages were not signi®cantly di€erent.
One day and 2 d before anthesis, papillae were visible and
grooves apparently open (Figs 5 and 6) but no stigmatic
exudates were observed. Anther dehiscence was 1 d before
anthesis and pollen was observed adhering to the stigma
(Fig. 6). At anthesis (Fig. 7), papillae are taller than at the
previous stages, but no stigmatic ¯uids were observed. All
stigmas examined 1 d after anthesis had their pollen and
papillae immersed in heavy droplets of exudates (Fig. 8),
and the width of the stigmatic groove increased to its
maximum at 1 d post-anthesis. Exudates ran freely (Fig. 8)
out of the stigmatic groove at the early period of this stage.
Stigmas observed 3 and 4 d after anthesis were characterized
by shorter papillae than in other stages (Fig. 9), a closing
groove and drying out of stigmatic ¯uids. Few stigmas
had remnants of exudates at these stages, which were hardly
ever seen 5 d after anthesis, and had faded and wrinkled
papillae (Fig. 10). Under ®eld conditions the stigma looks
dry during these stages, with collapsed brown papillae.
SEM examination revealed early stigmatic secretions on
all 20 emasculated ¯owers 24 h after emasculation, and no
exudates for the untreated ¯owers (nine out of ten ¯owers)
for the same period. These results suggest that emasculation
slightly enhances an early stigma receptivity.
Duration of viability of pollen grains on the stigma
10
0
pollen tubes and a messy aspect, i.e. pollen tubes which
lacked clarity of appearance and direction of growth.
−2
−1
0
1
2
3
4
5
Days before (−) and after (+) anthesis
F I G . 4. Percentage of receptive stigmas (de®ned by the germination of
pollen) following compatible pollen germination on stigmas at di€erent
¯oral stages at Canberra. Bars show s.e.
Treatment means for pollen germination and the corresponding pollen tube growth are given in Fig. 11. This study
found that pollen collected about 2 d before anthesis is
not mature as no germination was obtained. Signi®cant
di€erences (P 5 0.001) were obtained among treatments
(treatment ÿ2 d excluded from analysis) for pollen germin-
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
141
T A B L E 4. Flower development at various times during the stigma receptivity period of Grevillea robusta
Groove size (mm)
Pollen size (mm)
Days before (ÿ) or
after (‡) anthesis{
Mean + s.e.
Range
Mean + s.e.
Range
ÿ2
ÿ1
ÿ0
‡1
‡2
‡3
‡4
290 + 20
274 + 32
369 + 47
419 + 36
268 + 13
n.a.
n.a.
232±338
218±330
259±491
379±491
265±271
No pollen
37.9 + 5.2
43.2 + 2.6
40.3 + 3.3
40.5 + 2.5
31±48
35±50
34±45
38±43
F(4,13) ˆ 2.81ns
ANOVA
Exudate (yes/no)
N
N
N
Y
Y/N
Y/N
N
F(3,9) ˆ 0.63ns
{ Five ¯owers were observed at each time and the total number of stigmas measured were three for 1 d before anthesis (on 1 and 2 d after
anthesis) and ®ve stigmas for other stages. At least 30 pollen grains were measured each time.
n.a., Data not available as stigmas looked dry with few or no pollen grains at these stages.
T A B L E 5. Pollination success parameters following pollen grain germination and pollen tube growth for di€erent pollination
treatments in Grevillea robusta
Treatment
Empty ¯owers{ ( %)
Open-pollination
Autogamy
Geitonogamy
Cross-pollination
55
27
25
25
ANOVA
Logistic regression model
10.4***
Pollen grains per ¯ower
Germinated grains per ¯ower
Tubes in style per ¯ower{
7.1
6.7
11.6
19.7
2.1
3.2
2.6
9
0.6
0.2
0.5
4.9
7.2***
7.4***
31.81***
***P 5 0.001.
n ˆ 40 ¯owers from eight trees.
{ Empty ¯owers have stigmas without pollen grains as observed under ¯uorescence microscopy.
{ Pollen tubes were counted from the upper part of the style.
ated on stigmas of various phases and their corresponding
pollen tube growth under ®eld conditions. For pollen
collected 1 d before anthesis, 52 % of pollen grains germinated with short (17.6 mm) but normal pollen tubes. On the
day of anthesis 49 % of the pollen germinated with a mean
pollen tube length of 31.4 mm. The adhesion of pollen on
the presenter 1 d before anthesis and pollen germination
con®rm that pollen is mature 1 d before ¯ower opening.
The best results were obtained with pollen collected 1 d
after anthesis, when 95 % of pollen germinated and had
longer tubes (mean length 46.2 mm). Later pollen grain
collections (mostly 3 d after anthesis) showed poor
germination and shorter tubes with a messy appearance.
Assessment of self-compatibility and barriers to sel®ng
The e€ectiveness of natural open-pollination (open), self(autogamy and geitonogamy) and cross-pollination was
examined by scoring pollen germinated on the stigma,
pollen growth in the style and subsequent fruit set. The
di€erent types of pollination resulted in signi®cantly di€erent numbers of pollen grains on the stigma (P 5 0.001;
Tables 5 and 6 for ranges) with 7.1 (range 0 to 100), 6.7
(range 0 to 28), 9.2 (range 0 to 47), and 19.7 (range 0 to 81),
pollen grains per ¯ower respectively for open, autogamy,
geitonogamy and cross-pollination treatments. Nearly
T A B L E 6. Frequency distribution of pollen deposition on
stigmas for Grevillea robusta at Malava, under di€erent
types of pollination
Number of
pollen grains
on stigma
Type of pollination ( ¯owers %)
Open
Autogamy
Geitonogamy
Cross
0±1
2±10
11±20
21±30
31±60
60±100
62.2
24.4
4.4
4.4
2.2
2.2
40.9
22.8
31.8
4.5
0
0
48.1
31.9
6.5
9.5
4.0
0
34.9
30.1
4.7
14.0
14.0
2.3
two-thirds (65 %) of cross-pollinated ¯owers had more
than two pollen grains per ¯ower compared with only 38 %
of the ¯owers which were naturally open-pollinated
(Table 6). The number of ¯owers observed to have no
pollen on stigmas di€ered signi®cantly between treatments
(P 5 0.001; Table 5); open-pollinated ¯owers had the
highest proportion of bare stigmas (55 %).
There were signi®cant (P 5 0.001) di€erences between
the numbers of pollen grains germinated on the stigma for
all treatments. Although some (25±55 %) of the ¯owers had
no pollen germinated on their stigma, cross-pollinated
142
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
F I G S 5±10. Scanning electron micrographs (SEM) of Grevillea robusta stigmas. Fig. 5. Papillae ( p) 2 d before anthesis. Note the stigmatic groove
(g) free of pollen grains and exudates. Fig. 6. Pollen presenter ( pp) and papillae ( p) covered with pollen grains 1 d before anthesis. Fig. 7. Stigma
showing papillae ( p) and pollen grains ( po) deposited over the stigmatic groove on the day of anthesis. No stigmatic secretion observed. Fig. 8.
Receptive stigma of Grevillea robusta 1 d after anthesis showing pollen grains ( po) submerged by the stigmatic secretion (ss) running from the
stigmatic groove. Fig. 9. Stigma showing fading papillae with little remains of stigmatic ¯uids (ss) 4 d after anthesis. Fig. 10. Dry stigma and
collapsed papillae 5 d after anthesis. Remnants of exudates were not seen at this stage.
¯owers had the most germinated pollen grains (nine pollen
grains per ¯ower) and open-pollinated ¯owers the fewest
(2.1 pollen grains per ¯ower) (Table 5). For autogamous
and geitonogamous treatments, pollen grains germinated,
but most were unable to penetrate the stigma, and few
pollen tubes grew down the style towards the ovary. Crosspollinated ¯owers had signi®cantly more pollen tubes than
other treatments (P 5 0.001). No signi®cant di€erences
Pollen germination (%) and
pollen tube length (m)
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
100
90
80
70
60
50
40
30
20
10
0
germination
tube length
−2
−1
0
1
2
3
4
5
Days before (−) and after (+) anthesis
F I G . 11. Pollen germination and pollen tube growth in vitro for
Grevillea robusta pollen exposed to ®eld conditions for various lengths
of time. Pollen was tested on a nutrient agar. Bars show s.e.
between trees or families were obtained for these observations (P 5 0.05).
Pollen for cross-pollinated ¯owers germinated well with
apparently healthy and straight tube growth (Fig. 12). Tube
lengths in control cross-pollinations were similar and
uniform. However, pollen tubes that grew into the styles
following self-pollination were short and did not progress
past the upper style just next to the stigmatic region. Most
pollen for self- and open-pollinated ¯owers either did not
germinate or showed some pollen tube abnormalities.
Tubes were either distorted, presenting a messy growth,
with irregular or spiralling tubes (Fig. 13) or with a bulbous
appearance at the tube end.
Microscopic examination of dropped ¯owers collected
soon after the fertilization stage at Malava showed 75 %
(42 ¯owers out of 56 collected) had no pollen germinated on
their stigmas; 21 % had bare stigmas and 4 % had germinated pollen. This result suggested that dropping ¯owers
are generally not fertilized. A study on ¯owers collected in
the ANBG con®rmed the Kenyan results. A total of 490
dropped ¯owers was observed. Although 90 % of the ¯owers
had pollen grains on their stigmas (mean 13.7 + 1.0 pollen
grains), pollen grains germinated on only 35, or 7 %, of the
observed ¯owers (mean 4.5 + 1.9 %). Only 1.4 % (seven out
of 490 ¯owers) had pollen tubes growing in the upper style.
In the main controlled pollination experiment there were
signi®cant (P 5 0.001) di€erences in the probability of fruit
143
set between pollination treatments in both the April and
July experimental periods (Table 7). The results indicated
family di€erences in April (P 5 0.05) but not in July, and
provenance di€erences in both April (P 5 0.01) and July
(P 5 0.05).
In July, very low fruit set was obtained for autogamous
sel®ng (0.04 %; one fruit set out of 2707 ¯owers; 27 trees);
and for geitonogamous sel®ng (mean 0.1 %; two fruits out of
1622 ¯owers obtained from only one tree; 27 trees). No fruit
was set from either autogamy or geitonogamy treatments
in April. The overall fruit set observed was dependent on the
time of the pollination period (P 5 0.001), with higher fruit
set in July than in April. The fruit set following openpollination was considerably lower (mean 0.1 % in April
and 3.3 % in July; range 0±27.1 %) than that resulting from
hand cross-pollination (mean 5.9 %; range 0±21.4 in April
and 17.5 %, range 0±50 in July).
There was strong evidence of family in¯uence, for both
male and female parents, on fruit set (Table 8). The average
success rate of the best ®ve crosses was 45 % (range 35±55).
Thus, some pollen sources promoted higher fruit set. Moreover, there was a high overall number of inter-family crosses
without fruit set (27 %). No fruits were set following selfpollination of any of the 18 trees in the crossing programme
(Table 8).
DISCUSSION
Fruit set in relation to ¯oral development
Grevillea robusta trees bear approx. 50 000 hermaphroditic
¯owers, opening acropetally. The period from ¯oral
initiation to fruit maturity is approx. 3 months. Depending
on the size of an in¯orescence, it may take from 3 to 10 d
for all the ¯owers on an in¯orescence to open and in
most in¯orescences nectar ¯ows continuously from one or
more of the racemes over this period. As anthesis is accompanied by nectar secretion, at least within 24 h of
presentation, this strategy encourages ¯oral visitors and
hence self-pollen removal and pollen deposition from other
¯ower sources, thus increasing the chances of crosspollination.
F I G S 12 and 13. Fluorescence micrographs of Grevillea robusta upper style. Fig. 12. Style showing compatible controlled cross-pollination
displaying abundant pollen germination and growth of normal and straight pollen tubes 48 h after pollination. Fig. 13. Style with controlled selfpollen showing inhibition of pollen tube with a directionless and messy aspect 48 h after pollination.
144
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
T A B L E 7. Fruit set of Grevillea robusta from di€erent pollination treatments at Malava
April{
July{
July
Family code
Cross-pollination
( % fruit set)
Cross-pollination
( % fruit set)
Open-pollination
( % fruit set)
Boyd River (17622)
CEH 89
CEH 91
CEH 980
2.8
3.0
5.2
0
6.9
0.4
Duck Creek (17614)
CEH 26
CEH 28
CEH 29
16.7
9.2
10.8
10.7
16.8
15.3
31.9
32.4
13.3
Provenance (CSIRO no.)
Bunya Mountains (17693)
T5
T3
T2
Overall mean (no. ¯owers observed)
Variation (logistic regression model):
Among treatments
Inter-provenance
Inter-family
Fruit set over periods
7.7
2.8
6.0
12.8
6.8
5.7
5.9 (2112)
78.48***
5.93**
2.86*
56.41***
17.5 (2416)
0
4.8
9.1
0
4.0
4.2
3.3 (2718)
62.83***
3.91*
0.85ns
*** P 5 0.001; ** P 5 0.01; * P 5 0.05; ns, non-signi®cant.
{ April, No fruit set for self-pollination treatments and very low fruit set for open-pollination (two out of 2078 ¯owers; 0.1 %).
{ July, For geitonogamy (two out of 1622; 0.1 %) and for autogamy (one out of 2707 ¯owers; 0.04 %).
On the day of anthesis the pollen presenter, a specialized
structure, serves as both a pollen-reception zone and a
presenter of pollen for transfer to pollinating agents
(Collins and Rebelo, 1987). Pollen can be seen on the
presenters of individual ¯owers for up to 5 d. The peak of
¯ower opening on a raceme level coincides with the peak of
nectar production. This presumably maximizes the chances
of ¯ower visitation and cross-pollination. Also, a long
period of nectar secretion should encourage more visits per
raceme, leading to higher pollen removal and transfer.
However, the process does not exclude the possibility of
geitonogamy (the transfer of self-pollen to other ¯owers on
the same individual; Snow et al., 1996) or the existence of a
self-incompatibility mechanism.
Many ¯owers were aborted from initiation to early bud
stage and less than 44 % reached anthesis. Five to 10 d after
anthesis, ¯owers abort en masse (43 % of the total number
of ¯owers), with very few remaining until fruit maturity.
This ¯ower drop might be a result of competition for
assimilates. The heavy losses after anthesis suggest unsuccessful fertilization. After anthesis the ovary starts swelling
if fertilization takes place.
It is not clear from the above observations whether crosspollen or other resources are factors limiting fruit set for
natural open-pollinated ¯owers of G. robusta. This study
found that dropping ¯owers are not fertilized, and that
there was no fruit set from capped ¯owers. This is contrary
to the report by Brough (1933) who found fruit development on capped ¯owers. The absence of fruit set on
dropping and capped ¯owers indicates the presence of
incompatible, probably self-pollen on stigmas. The above
result, together with the large variation in the fruit : ¯ower
ratio, indicates poor fertilization, most probably related to
the lack of suitable pollinators. The very low fruit set (5.9 to
17.5 %) from controlled hand-pollination is due in part to
failure of some individual crosses to set any fruit. Other
crosses displayed higher levels of fruit set. As the pollen
parents used were successful in at least some crosses
(Table 8), this points to incompatibility in some crosses.
The clustering of ¯owers on the ends of branches,
brush-type presentation, bright colour and copious nectar
production suggest G. robusta is pollinated by animal
vectors. The structure of the in¯orescences and the nectar
reward suggest the species is most probably bird- or
mammal-pollinated, similar to other proteaceous species
(Collins and Rebelo, 1987). Individual ¯owers are adapted
to early-morning presentation for pollination, as suggested
by the timing of anthesis and nectar ¯ow.
The protandrous behaviour of Grevillea robusta
The study showed G. robusta to have a wet papillate
stigma, contrary to the dry stigma reported for Grevillea
species by Heslop-Harrison and Shivanna (1977). Some
other members of the Proteaceae have been reported to
have wet stigmas at the time of stigma receptivity; for
example Banksia (Fuss and Sedgley, 1991), Macadamia
(Sedgley et al., 1985), Dryandra (Matthews and Sedgley,
1998) and Grevillea leucopteris (Lamont, 1982). The SEM
examination revealed that stigmas were dry at anthesis, but
had copious secretion commencing 1 d thereafter for a
period of 3 d. The pollen germination studies indicated that
stigmatic receptivity of G. robusta began to develop 1 d
post-anthesis, but the greatest pollen germination rates on
stigmas and longest pollen tubes were observed 2 d postanthesis, and individual stigmas remained receptive for
approx. 3 d post-anthesis.
In the studies reported here, G. robusta pollen was not
viable until 1 d before its presentation to ¯oral visitors.
Pollen viability was retained for 4 d, providing a good
Bunya Mountains
Female
families
Bunya Mountains
Boyd River
BM11
BM12
BM13
BM21
BM23
BM31
BM41
BM52
BM53
BM54
BR11
BR12
BR13
BR15
BR21
BR23
BR51
BR52
Boyd River
Male families
BM11
BM12
BM13
0
0
6
0
0
5
11
0
BM21
0
6
BM23
0
BM31
0
5
0
BM41
BM52
BM53
BM54
BR11
BR12
10
10
0
43
10
0
0
4
0
8
0
3
5
45
0
5
19
41
0
6
11
7
0
34
0
28
0
5
0
35
BR13
BR15
BR21
BR23
BR51
28
4
12
2
3
0
12
0
6
10
4
2
0
1
11
0
14
7
3
10
0
0
32
0
0
0
0
47
27
0
0
18
0
BR52
7
0
2
0
{ Number of ¯owers pollinated per family cross varied from 108 to 782. Number of individual trees per family ranged from one to ®ve. Results for each family cross are shown and bold ®gures
indicate selfs. BM, Bunya Mountains; BR, Boyd River, e.g. BR11 ˆ Tree 1 of family 1 in Boyd River.
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
T A B L E 8. Fruit set ( %) from a range of selfs and inter-family crosses{ for Grevillea robusta at Malava
145
146
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
opportunity for its removal and deposition on di€erent
¯owers. Pollen collected on stigmas 3 d after anthesis and
germinated in-vitro had a messy appearance and shorter
tubes. This poor vigour casts doubt on its ability to grow
and fertilize ovules. The results conform with Thomson and
Thomson's (1989) statement that pollen may lose viability
over time, such that pollen removed late in the ¯ower's life
has less chance of fertilizing ovules than pollen removed
earlier. At anthesis, the stigma is probably not receptive.
Results obtained at Malava (Fig. 3) indicated that pollen
deposited before the period of receptivity remained viable
long enough to germinate when the stigma became receptive. Under ®eld conditions the stigma appears shiny and
wet 1 d after anthesis. Moreover, there is evidence of poor
pollen germination and pollen tube growth for pollen
collected more than 2 d after anthesis. Also, there is less
likelihood of collection taking place, as the ¯ower ceases
nectar production, thus becoming less attractive to visitors.
To maximize the likelihood of pollen germination, and
subsequent seed set, hand-pollinations should be made on
the day of peak stigma receptivity. This peak was 1±2 d after
anthesis when the highest rates of pollen grain germination
with normal pollen tubes in the styles were obtained. This
period was accompanied by morphological changes to the
stigma including longer papillae and the production of
stigmatic exudate. In Grevillea wilsonii (Collins and Grey,
1988) and G. barklyana (Vaughton, 1996), the stigmatic
papillae have also been shown to enlarge as receptivity
increases. The current study showed that no fruit set
occurred on stigmas hand-pollinated 4 and 5 d after
anthesis, indicating loss of stigma receptivity at this stage.
The stigmatic groove was apparently open and wider
than the diameter of the pollen grain before anthesis, but no
successful pollination was e€ected. Thus, groove width did
not limit pollen deposition within the groove, but the
stigma was not yet receptive. Therefore, the timing of
groove opening in G. robusta is not sucient to determine
stigma receptivity.
Within a ¯ower, the anthers of G. robusta dehisce prior to
anthesis and stigma receptivity. One day before, and on the
day of anthesis, self-pollen completely covers the pollen
presenter and the stigmatic groove. Thus, successful natural
cross-pollination may depend upon the removal of selfpollen before the stigma becomes receptive within 1 d of
anthesis. Consequently, protandry makes it possible for
most pollen to be dispersed before the stigma becomes most
receptive. Therefore, the protandrous ¯owering habit of
G. robusta makes individual ¯owers well adapted to outbreeding. But geitonogamy is always possible within the
same raceme, the same in¯orescence or the same tree as they
contain both male and female phase ¯owers at a range of
developmental stages.
The study has shown the existence of protandry in
G. robusta, with less than 2 d separation between male and
female maturity phases for individual ¯owers. This
protandrous mechanism was reported by Brough (1933)
and Venkata Rao (1971) but disputed by Owuor and Oduol
(1992). The latter reported G. robusta to have simultaneous
anther dehiscence and stigma receptivity. Protandry in
G. robusta allows some time for self-pollen removal. If self-
pollen is not removed by ¯oral visitors, self-pollination may
occur autogamously. Since viable self-pollen will still be
present on ¯owers when the stigma becomes receptiveÐ
unless removed by pollinators or rainÐ protandry will not
prevent autogamy in G. robusta.
Fertilization and sel®ng barriers in Grevillea robusta
In this study, the stigmatic surface was the ®rst site of
pollen grain inhibition for self-pollinated ¯owers. The
second site of inhibition was the upper part of the style:
most self-pollen tubes (Table 5) failed to penetrate to the
lower part of the style. Few self-pollen tubes grew normally
towards the ovary. Cross-pollen tubes were occasionally
seen penetrating the ovules, but no self-pollen could be
detected doing this. However, although there was no fruit
set following self-pollination, self-pollen tubes were occasionally seen extending beyond the upper style; and because
of diculties in observing fully the lower style it could not
be determined whether self-incompatibility occurs in the
ovary as well. Further investigation of the cytology
( pollen±ovule interaction) of fertilization might clarify
this question.
Fewer pollen grains were observed on open- and selfpollinated ¯owers than on cross-pollinated stigmas, and
open-pollinated ¯owers had a higher incidence of empty
¯owers (Tables 6 and 7). Together with the poor pollen
germination obtained on self- and natural open-pollinated
¯owers at Malava, these observations suggest self-pollen
fails to adhere and germinate on stigmas before it is washed
o€ by frequent heavy rains (see Lamont, 1982). Most crosspollinated stigmas presented more pollen grains than the
ovule number (two per ¯ower), giving a better chance of
successful fertilization.
Grevillea robusta set few fruit by self-pollination
(50.1 %), while cross-pollinated ¯owers set 5.9 % and
17.5 %, respectively, in April and July (Table 7). Most selfpollen tubes failed to fertilize the ovule after exhibiting
distorted growth patterns (Fig. 13), suggesting that this
failure resulted from self-incompatibility reactions. The
results demonstrated that G. robusta possesses strong
barriers to sel®ng with a likely gametophytic selfincompatibility system (de Nettancourt, 1977). This kind
of inhibition in the upper style has been reported in other
members of the Proteaceae (Herscovitch and Martin, 1990;
Fuss and Sedgley, 1991; Goldingay and Whelan, 1993).
The natural fruit set following open-pollination was low
(0.1 to 3.3 %), and was considerably lower than that which
resulted from controlled cross-pollination. Brough (1933)
observed variable fruit production (meagre to high) in an
exotic population at Sydney, Australia depending on the
weather conditions prevailing at the time of pollination,
while Owuor and Oduol (1992) reported very high fruit set
(69 %) with G. robusta landraces at Maseno in western
Kenya. The present results are consistent with those for
other members of the Proteaceae (e.g. Vaughton, 1991;
Harriss and Whelan, 1993) in general, and with Grevillea
species (0.01 to 0.09 fruit set; Hermanutz et al., 1998) in
particular.
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
In this study, fruit set in April was much lower than
in July for both open-pollination and cross-pollination.
This variation is probably due to heavy rains which fell
in April±May, which may have had an impact on fertilization of the ¯owers. Such conditions, i.e. rainy and cloudy
weather, naturally restrict pollinator activity and in general
present an unfavourable environment for pollination, and
consequently result in poor fruit production. Moreover,
pollination is adversely a€ected following the displacement
of pollen which is washed o€ the stigmas [see Boland and
Owuor (1996) for Calliandra calothyrsus under similar
environmental conditions], and probably by an increased
percentage of sterile pollen which occurs as a result of
repeated wetting and drying (Brough, 1933). The above
results emphasize the e€ects of climatic conditions at the
time of fertilization.
A signi®cant variation between trees in the rate of fruit
set for both cross- and open-pollinated ¯owers is evident
from this study. For natural open-pollinated ¯owers, such
variation may be due in part to di€erences in nectar
concentration and production, and hence energy rewards
(Heinrich and Raven, 1972), or to other factors such as the
number of in¯orescences displayed per tree or the colour of
the ¯owers. This may a€ect movement of pollinators
amongst trees and hence the rate of pollen transfer. Di€erences seen between cross-pollinated ¯owers within families
may also arise from di€ering compatibilities of di€erent
male parents for di€erent individual crosses. Furthermore,
this study revealed that few of the ¯owers that dropped
soon after natural fertilization had germinated pollen and
very few had pollen-tubes in their styles. The germination
failure is probably due to a self-incompatibility mechanism,
indicating that the ¯ower's own pollen was probably not
removed by ¯oral visitors. This was observed in other
Australian Proteaceae where self- and outcross-pollen are
often transported by pollinators but self-pollen is expected
to predominate on stigmas (Goldingay et al., 1991;
Carthew, 1993; Ayre et al., 1994). Thus, the unfertilized
¯owers, probably bearing self-pollen, dry up and drop o€
the raceme soon after the fertilization phase.
The poor fruit set following natural open-pollination is
due mainly to the absence of outcrossed pollen. This is
probably due to low visitation rates by e€ective pollinators
(Kalinganire et al., 1996). Flowers of G. robusta contain two
ovules and it was established (Kalinganire, unpubl. res.,
1996) that whenever there is fruit set the follicle always
contains two seeds, suggesting a low abortion rate once a
¯ower receives adequate pollen. As found in the Grevillea
species studied (Herscovitch and Martin, 1990; Hermanutz
et al., 1998), only two or three pollen tubes penetrate the
stylar transmission tissue and these have access to the two
ovules.
Fruit production per in¯orescence showed no relationship to the number of ¯owers remaining after anthesis and
appeared to be primarily dependent on the proportion of
¯owers with compatible pollen. This suggests a weak e€ect
of other resources limiting fruit set. Inadequate receipt of
cross-pollen on open-pollinated ¯owers contributed considerably to the low fruit set. If pollination was not a
limiting factor, higher rates of fruit set might be expected on
147
open-pollinated ¯owers than on cross-pollinated ¯owers
because of the stress experienced during emasculation. Low
rates of cross-pollination appear to be a primary cause of
low fecundity in perennial plants, which are often selfincompatible (Burd, 1994). Therefore a lack of cross-pollen
was mainly responsible for a low fruit set in G. robusta.
However, post-anthesis loss in cross-pollinated ¯owers
suggests resource limitation should not be excluded. As
suggested by Vaughton (1991) and Campbell and Halama
(1993), pollen and nutrients may interact in determining
levels of fruit set, suggesting that fecundity may depend on
the availability of both factors. It is unlikely that there is a
strict separation between the e€ects of pollen and resource
allocation on the reproductive output. Yet, as reported by
Bierzychudek (1981), if hand-pollinated plants produce
more seeds than naturally pollinated controls, then
reproduction is being limited by pollinator activity.
The present study has clearly shown the natural low fruit
set to be largely due to a lack of cross-pollen on stigmas and
a self-incompatibility mechanism. Cross-pollen transfer
seems likely to be a factor limiting fruit production because
of a lack of suitable pollinators at Malava (Kalinganire
et al., 1996) and probably high levels of incompatibility
among families. Misty weather conditions might a€ect the
overall fruit production at Malava, by cross- or natural
pollination, and it is suggested that breeding work should
be undertaken during the drier parts of the year even if
¯owering is poor. Therefore, it is likely that the low
fruit : ¯ower ratio is due to complex genetic factors, including self-incompatibility, as well as being in¯uenced by
environmental conditions, pollinator abundance and
possibly to a lesser extent by the availability of resources.
The study shows G. robusta to be highly self-incompatible
and outcrossing. The species might be capable of a very low
rate of sel®ng, but fruit set results indicate very high rates of
outcrossing for all genotypes studied. This is consistent with
the high outcrossing rates obtained by Harwood et al. (1992)
from isozyme studies of two natural populations. Allogamy
is con®rmed to be the primary breeding behaviour of
G. robusta. Self-incompatibility may help to explain why the
species has not shown any substantial genetic deterioration
as an exotic, especially in the eastern and central African
highlands. Very few selfed plants are produced in progeny;
therefore inbreeding does not develop as rapidly as in, for
example, eucalypts (Eldridge et al., 1993).
AC K N OW L E D G E M E N T S
This study comprised part of the PhD research undertaken
by Antoine Kalinganire and was funded by Intercooperation Suisse. The Australian National University (ANU)
Department of Forestry, CSIRO and ICRAF also provided
research support. The Australian National Botanic
Gardens (Canberra) supplied some of the plant material
for this study under permit number 20. Thanks to Sally
Stowe and Roger Heady of the ANU Electron Microscopy
Unit; Ross Cunningham and Christine Donnelly of the
Statistical Consulting Unit of the Graduate School (ANU)
and David Boshier (University of Oxford) for their
assistance, and Peter Kanowski (ANU), Mike Moncur,
148
Kalinganire et al.ÐProtandry and Self-incompatibility of Grevillea robusta
Alan Brown and Doug Boland (CSIRO) for helpful
comments. Thanks to Stephen Ruigu, Agnes Yobterik
and Amadou Niang (ICRAF Maseno) for their support,
and to Walter Adongo, Joseph Njeri and their team for
technical assistance during the ®eld work at Malava.
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