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The genus Schlumbergera Lemaire (Cactaceae, subtribe
not affect seed germination.
J. AMER. SOC. HORT. SCI. 120(2):313-317. 1995.
Pollen Germination, Pollen Tube Growth, Fruit Set,
and Seed Development in Schlumbergera truncata
and
S. ×buckleyi
(Cactaceae)
Thomas H. Boyle1, Renate Karle2, and Susan S. Han3
Department of Plant and Soil Sciences, French Hall, University of Massachusetts, Amherst, MA 01003
Additional index words. electron microscopy, flowering potted plants, ornamental plants
Abstract. The reproductive biology of Schlumbergera truncata (Haworth) Moran and S. xbuckleyi (T. Moore) Tjaden was
examined in a series of experiments. At anthesis, pollen grains are spherical, 54 to 62 µm in diameter, and tricellular. The
receptive surface of the stigma is densely covered with elongated papillae and is devoid of exudate during the period of
flower opening. When compatible pollen was applied to mature stigmas, germination occurred between 20 and 30 minutes
after pollination and pollen tubes penetrated the stigma surface between 30 and 40 minutes after pollination. Pollen tubes
exhibited a nonlinear pattern of growth in the upper two-thirds of the style, and the maximum rate of growth (»1.9
mm·h-1) occurred between 12 and 18 hours after pollination. Full seed set was attained between 32 and 48 hours after
pollination. Genotypic variation in the time required to achieve full seed set was partly attributable to differences in stylar
length. Seeds were fully mature 6 months after pollination, but delaying fruit harvest until 8 months after pollination did
Rhipsalidinac) is comprised of about five species of epiphytic
shrubs that are indigenous to southeastern Brazil (Barthlott, 1987;
Hunt, 1969). Schlumbergera truncata, also known as zygocactus
or crab cactus, is the most commonly cultivated species and is an
economically important floricultural crop in northern Europe and
North America (Cobia, 1992). The other four Sch/umber-gera
species are rare in cultivation but have been utilized for breeding.
Hybridization between S. truncata and S. russelliana (Hooker)
Britton & Rose has yielded many cultivars, including the well-
known Christmas cactus (Hunt, 1981). The collective name for
hybrids of S. truncata x S. russelliana parentage is S. ×buckleyi (T.
Moore) Tjaden (Tjaden, 1966). Cultivars of S. truncata and S.
×buckleyi are distinguished primarily by the morphology of their
flowers and phylloclades (stem segments) and their flowering time
under natural photoperiods (Barthlott and Rauh, 1977; Hunt, 1969,
1981). Schlumbergera truncata has also been crossed with S.
opuntioides (Loefgren & Dusen) Hunt (Barthlott and Rauh, 1977)
and S. orssichiana Barthlott & McMillan (Horobin and McMillan,
1985), but few cultivars have been produced from the resultant
progeny. Hence, nearly all of today’s commercial cultivars remain
either S. truncata or S. ×buckleyi (Cobia, 1992; McMillan, 1985).
The reproductive biology of Schlumbergia is not well charac-
terized. All Schlumbergia species except for S. obtusangula
(Schumann) Hunt are reported to be self-incompatible (Ganders,
1976: McMillan. 1991). Flowers of Schlumbergia are hermaph-
roditic and exhibit many features that are clearly adoptive to bird
pollination, i.e., a long flower tube, a staminal nectar chamber,
exserted stamens and style, and tepals in varying shades of red
(Barthlott and Rauh, 1975; Buxbaum, 1953; Hunt, 1969). Abendroth
(1969) observed hummingbirds (Trochilidac) visiting flowers of
S. truncata. Several reports suggest that fruit of S. truncata
J. AMER. SOC. HORT. SCI. 120(2):313-317. 1995.
requires 12 months or longer to reach maturity (Abendroth, 1969;
Barthlott and Rauh, 1975: Horobin, 1981). although Kölli (1988)
found that fruit matured 6 months after pollination. The mature
fruit is an indehiscent, fleshy berry (Barthlott and Rauh, 1975;
Buxbaum, 1955).
To optimize breeding efforts. it would be desirable to gain more
information about the reproductive biology of Schlumbergia. In
the present article we report on pollen germination, tube growth,
fruit set, and seed development in S. truncata and S. ×buckleyi.
Materials and Methods
General procedures. The cultivars that were used for study are
listed in Table 1. All cultivars were propagated vegetatively from
stock plants maintained at the Univ. of Massachusetts, Amherst (lat.
42°22.5'N). Plants were grown in glasshouses with temperature
setpoints of 18/22C (heat/vent). Actual glasshouse temperatures
ranged from 16C minimum to a maximum of » 40C for short
durations in the summer. Shading compound (Kool Ray, Continental
Products Co., Euclid, Ohio) was applied to the glass to maintain
photosynthetic photon flux (PPF) below ≈650 µmol·m–2·s–1.
Pollination was performed by rubbing recently dehisced an-
thers on the stigmatic lobes. Each flower was pollinated once using
pollen collected from two or more flowers. Four experiments were
conducted.
Effect of timing of harvest on seed maturity (Expt. 1).
Seventy-five flowers of ‘88-50’ were pollinated on the day of
Table 1. The Schlumbergera cultivars used for study and their taxonomic
statas and source.
313
anthesis with compatible (‘Dark Marie’) pollen. All pollinations
were performed within a 10-day interval. Fruit were harvested at
4,5,6,7, and 8 months after Pollination. Seeds that were shriveled
Table 2. Influence of timing of fruit harvest on seed set and the germina-
tion percentage for the cross female Schlumbergera ×buckleyi‘88–50’
x male S. truncata ‘Dark Marie’.
or collapsed were discarded, and the remaining seeds were counted
and germinated in covered petri dishes on top of blotter paper that
was moistened with deionized water. Seeds were germinated in a
controlled-environment chamber (model I-35LVL; Percival
Scientific, Boone, Iowa) kept at 20 ± 1 C and providing 52 ± 8
µmol·s–1·m–2 PPF for 12 h daily from cool-white fluorescent lamps
(Bachtaler, 1989).
Data were collected on seed set (number of seeds at harvest
minus shriveled or collapsed seeds) and number of seeds germi-
nated at 10, 20, and 30 days after sowing. The number of seeds
germinated at 40 days after sowing was also recorded for fruit
harvested 4 months after pollination. A seed was considered
germinated upon emergence of the radicle. The experiment con-
sisted of 10 replications (fruit) per harvest period. Percent seed
germination was calculated for each treatment and the data was
arcsin-transformed before analysis. All variables were analyzed
by SAS Institute’s (1985) General Linear Model (GLM) proce-
dure.
Morphology of the stigmatic surface and mature pollen grain
(Expt. 2). Five styles of ‘Dark Marie’ were collected at anthesis,
fixed in 2% (w/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7),
dehydrated through an ethanol series, critical-point dried in CO2,
and coated with 200 to 300 A of gold–palladium. Stigmas were
viewed on a scanning electron microscope (JSM-25S; JEOL,
Peabody, Mass.).
Fresh stigmas of ‘Dark Marie’ were examined under a dissect-
ing microscope (×20 to ×50) to determine the presence or absence
of stigmatic exudate. Styles were collected at four developmental
stages between anthesis and senescence (Scott et al., 1994). Five
stigmas were examined per developmental stage.
Pollen of ‘Buckleyi’ and ‘Dark Marie’ was collected from »10
flowers on the day of anthesis and was fixed in 3 ethanol: 1 glacial
acetic acid (v/v) for >24 h. Fixed pollen was dispersed on a
microscope slide and stained for »24 h with the DNA-specific
fluorochrome mithramycin A [0.50 µg.ml–1 in McIlvaine–Lillie
buffer at pH 7.0 (Coleman and Goff, 1985)]. Slides were examined
with a Zeiss epifluorescence microscope equipped with a 100-W
high-pressure Hg lamp and a filter set for ultraviolet–violet
waveband excitation (exciter filter BP 395-425, dichromatic beam
splitter FT 425, and barrier filter LP 450). A minimum of 500
grains were observed per cultivar.
Kinetics of pollen adhesion and germination (Expt. 3). Whole
flowers of ‘Eva’ and ‘88-29’ were collected on the day of anthesis
and placed in covered petri dishes containing blotter paper moist-
ened with deionized water. Flowers were transferred to the labo-
ratory, and compatible pollen (’Dark Marie’) was applied to the
stigmatic surface of each flower with a fine artist’s brush. A brush
was used for pollinations in order to maximize the number of
pollen grains in direct contact with the stigmatic surface and
minimize the variation in germination time due to pollen clumping
(Thomson, 1989). The covers were replaced on the dishes after
pollination, and the flowers were transferred to an incubator with
the same temperature, irradiance, and photoperiod conditions
described in Expt. 1. About 7 min elapsed between the flower
collection and placement in the incubator.
At 10, 20, 30, 40, 50, and 60 min after pollination, stigmas were
excised and placed in scintillation vials containing ≈5 ml of 3
ethanol : 1 glacial acetic acid (v/v). Immediately after placing the
stigmas in fixative, the vials were shaken twice with a vortexer set
at high speed (3 sec on/3 sec off/3 sec on) in order to dislodge grains
Harvest time Seed set Germination (%)
(months after (no. seeds/ Days after sowing
Pollination) fruit)
10
20
30
40
4215.1 1 5 9 12
5254.1 53 68 73 ---
6229.9 73 82 85 ---
7237.5 72 81 84 ---
8238.5 73 81 83 ---
F test NS *** *** *** ---
Contrasts
4 vs. 8 months ---
*** *** ***
---
5 vs. 8 months ---
*** *** ***
---
6 vs. 8 months --- NS
NS
NS
---
7 vs. 8 months ---
NS
NS NS ---
NS*,**,***Nonsignificant or significant at 0.05
≥ α
> 0.01, 0.01
≥ α
> 0.001,
or α ≤ 0.001, respectively, according to F test of contrast between
treatments.
that were not strongly adherent. Stigmas were fixed for ≥ 24 h,
stained for ≈30 min with 0.05% (w/v) aniline blue in lactophenol
(Arlington and La Cour, 1942), and mounted in pure lactic acid.
Dislodged pollen grains settled to the bottom of the vials and were
taken up in a drop of fixative with a Pasteur pipette and placed on
a microscope slide. Pollen was stained with aniline blue in
lactophenol and viewed 24 h later. Stigmas and pollen were
examined using visible light microscopy. A minimum of five
stigmas were examined for each time interval.
Kinetics of pollen tube growth and fruit set (Expt. 4). Twelve
plants of ‘88-50’ with mature flower buds (≈1 to 2 days before
anthesis) were transferred from the glasshouse to a controlled-
environment chamber set at 20 ± 1C and 65% ± 5% relative
humidity, and providing 52 ± 8 µmol·s–1·m–2 PPF for 12 h daily.
Flowers were pollinated on the day of anthesis with compatible
(’88-68’) pollen. Entire styles were excised at the point of attach-
ment to ovaries at either 6, 12, 18, 24, 28, 32, 36, 40, 44, 48, 60, or
72 h after pollination. Styles remained intact on control flowers.
Ten flowers were used for each treatment, and the treatments were
randomized over all plants. Plants were returned to the glasshouse
following upon completion of the last stylar excision (72 h).
Excised styles were fixed in 3 ethanol :1 glacial acetic acid (v/
v) >24 h, washed in deionized water, softened for 20 h in 4 N NaOH,
stained for ≈24 h in 0.1 % (w/v) decolonized aniline blue in 0.1 M
K3PO4 (Martin, 1959), and examined with an epifluorescence
microscope fitted with the same lamp and filter set described in
Expt. 2. The longest pollen tube in each style was measured to the
nearest millimeter.
Six months after pollination, the number of set fruit was
counted and fruit were harvested. The procedures used for extract-
ing and germinating seeds were identical to those described
previously. The experiment consisted of five replications (fruit)
per harvest period. The number of germinated seed was recorded
30 days after sowing, and the data was analyzed by SAS Institute’s
(1985) GLM procedure. Percent fruit set was calculated for each
treatment [(no. fruit set ÷ no. flowers pollinated) x 100] and the
data was evaluated by chi-square analysis.
A second study was performed for the cross female ‘Dark
Marie’ x male ‘Linda’. Eight plants of ‘Dark Marie’ were trans-
ferred from the glasshouse to a controlled-environment chamber
providing the same temperature, relative humidity, PPF, and
314 J. AMER. SOC. HORT. SCI. 120(2):313–317. 1995.
Fig. 1.
(A)
A scanning electron micrograph of the stigmatic surface of Schlumbergera
truncata ‘Dark Marie’. A portion of a stylar lobe is shown, illustrating the density
and arrangement of stigmatic papillae. Bar = 20 pm. (B) Ungerminated pollen of
Schlumbergera ×buckleyi ‘Buckleyi’. Fixed pollen was stained with mithramycin
and viewed using epifluorescence microscopy. The vegetative nucleus is flanked
by two smaller sperm nuclei (s). Bar = 50 µm.
photoperiod described previously. Flowers were pollinated on the
day of anthesis using freshly dehisced pollen. Entire styles were
excised at the point of attachment to ovaries at either 12, 24, 36, 48,
60, or 72 h after pollination, or the styles were left intact (controls).
The experiment consisted of seven treatments with five replica-
tions (fruit) per harvest period. The experimental protocol and
methods used for data collection and statistical analysis were
identical to those described for the cross female ‘88-50’ x male
‘88-68’.
Experiment 1. There were no significant differences between
fruit harvest dates with regard to the number of seeds per fruit
(Table 2). Date of fruit harvest, however, markedly influenced the
percentage of seeds germinated 10, 20, and 30 days after sowing.
Percent seed germination increased as fruit maturity increased
from 4 to 6 months after pollination. No increase or decrease in
percent seed germination was obtained by delaying fruit harvest
from 6 to 8 months after pollination.
J. AMER. SOC. HORT. SCI. 120(2):313-317. 1995.
Fig. 2. In viva pollen tube growth for the-cross female Schlumbergera ×buckleyi ‘88-50’
x male S. ×buckleyi ‘88-68’. Each data point is the mean of 10 styles ± 1 SE.
Experiment 2. The mature stigma of S. truncata and S. ×buckleyi
consists of five or six stigmatic lobes that are erect, connivent, and
≈3 mm in length. The receptive surface of the stigma is densely
covered with elongate papillae that are ≈25 µm wide and -120 to
150 µm long (Fig. 1A). No exudate was present on the papillate
surface during the period from anthesis to senescence. Pollen
grains collected at anthesis were spherical, 54 to 62 pm in diam-
eter, and tricellular (Fig. 1B). Each grain contained a large vegeta-
tive nucleus and two smaller sperm nuclei.
Experiment 3. Pollen adhesion commenced between 10 and 20
min after pollination, and the numbers of grains per style increased
substantially during the first hour after pollination. Pollen loads
averaged only 22 ± 10 grains per style at 20 min after pollination,
but increased to 148 ± 28 grains per style at 30 min, 300 ± 103
grains per style at 40 min, and -500 grains per style at 50 min.
Most of the pollen germinated between 20 and 30 min after
pollination, with stigmatic penetration occurring between 30 and
40 min after pollination. By 60 min after pollination, pollen tubes
had reached the transmitting tissue in the stigmatic lobes, turned
-90” and started growing towards the ovary.
Experiment 4. Pollen tubes exhibited a nonlinear pattern of
growth in vivo (Fig. 2). The rate of pollen tube growth was -0.8
mm·h–1 between 6 and 12 h after pollination but increased to -1.9
mm·h–1 between 12 and 18 h after pollination and then decreased
to ≈1.1 mm·h–1 between 18 and 24 h after pollination. The average
stylar length for ‘88-50’ (from the tip of the stigma to the entrance
of the ovary cavity) was 49.3 ± 0.4 mm (n = 10). Based on these
measurements, pollen tubes traversed -17% of the style within 6
h, 27% of the style within 12 h, 50% of the style within 18 h, and
63% of the style within 24 h after pollination (Fig. 2).
For the cross female ‘88-50’ x male ‘88-68’, fruit set did not occur
when styles were excised at or before 28 h after pollination (Table 3).
Excision of styles at 32 h resulted in only 20% fruit set and seed yields
were lower than for flowers with intact styles (controls). When styles
were removed at 36 h or later, fruit set was 100% and seed yields were
similar to the controls. For the cross female ‘Dark Marie’ x male
‘Linda’, fruit set was prevented when styles were excised at or before
36 h after pollination (Table 4). However, removal of styles 48 h after
pollination or later resulted in >80% fruit set and seed yields that were
comparable to the controls.
315
Table 3. Effect of timing of stylar excision after pollination on fruit set and Table 4. Effect of timing of stylar excision after pollination on fruit set and
number of viable seeds per fruit for the cross female Schlumbergera number of viable seeds per fruit for the cross female Schlumbergera
×buckleyi ‘88-50’ x male S. ×buckleyi ‘88-68’. truncata ‘Dark Marie’ x male S. truncata ‘Linda’.
zTreatment significantly different from control by chi-square test, P =
0.05; chi-square = 3.841 for 1 degree of freedom.
yF test performed on data from 32, 36, 40, 44, 48, 60, and 72 h and control
treatments.
NS,***Nonsignificant or significant at a ≤ 0.001, respectively.
zTreatment significantly different from control by chi-square test, P =
0.05; chi-square = 3.841 for 1 degree of freedom.
yF test performed on data from 48, 60, and 72 h and control treatments.
NSNonsignificant (P > 0.05).
important to plant breeders, who need to maintain and assess pollen
viability and maximize seed set. Investigations are needed to deter-
mine the optimum storage conditions and maximum longevity for
Schlumbergera pollen. Schlumbergera species vary considerably in
their natural flowering time (Barthlott and Rauh, 1977), and the
development of reliable pollen storage techniques would aid in
broadening the genetic base by interspecific hybridization.
Discussion
In vivo pollen tube growth typically proceeds at a linear rate in
species with tricellular pollen and at a nonlinear rate in species with
bicellular pollen (Mulcahy and Mulcahy, 1983; Willemse and
Franssen-Verheijen, 1988). Nonlinear pollen tube growth was
observed in the tricellular species Plumbago zeylanica L. and was
correlated with changes in the architecture of the transmitting
tissue (Russell, 1986). Our results demonstrate the existence of
another species with tricellular pollen and nonlinear pollen tube
growth in vivo. Additional research is needed to identify the causes
of nonlinear pollen tube growth in Schlumbergera.
The timing of seed maturation is of significant interest to plant
breeders who desire to minimize the time from pollination to seed
collection and maximize the yield of viable seed. Our results
indicate that Schlumbergera seed attains physiological maturity as
early as 6 months after pollination, thus corroborating the findings
of Kölli (1988). Kölli (1988) suggested that seed maturation
periods longer than 6 months probably result from growing plants
at suboptimal temperatures. Relatively low temperatures, as well
as high soil moisture levels and high relative humidity, can delay
seed ripening (George, 1985). It cannot be concluded with cer-
tainty that suboptimal temperatures were responsible for longer
seed maturation periods in the studies of Abendroth (1969),
Barthlott and Rauh (1975), and Horobin (1981) because the
authors did not report the temperature regimes under which fruit-
ing plants were grown.
Seed that is harvested prematurely typically exhibits poor
germination and is short-lived (Austin, 1972; Cochran, 1943; Kerr,
1963). Harvest and extraction of seed before the attainment of
physiological maturity was also detrimental to germination in
Schlumbergera. This is readily apparent from comparing the
germination percentages for seeds from 4-month-old fruit at 40
days after sowing (12%) vs. seeds from 5-month-old fruit at 10
days after sowing (53%) (Table 2). These two groups of seeds were
harvested and extracted on different dates but were similar in
physiological age, i.e., the interval from pollination until data
collection.
The Cactaceae belongs to the centrospermous order
(Chenopodiales), and all taxa within this order have tricellular
pollen (Gibson and Nobel, 1986). Generally, tricellular pollen
tends to lose its viability quickly, exhibits poor germination in
vitro, and is difficult to store for extended periods (Brewbaker,
1967; Hoekstra, 1973). These attributes of tricellular pollen are
Excision of styles at 36 h after pollination resulted in 100% fruit
set and high seed yields for ‘88-50’, but 0% fruit set for ‘Dark
Marie’ (Tables 3 and 4). The lack of conformity in fruit set for these
two clones may be due to differences in stylar length. The average
length of ‘Dark Marie’ styles (from the tip of the stigma to the
entrance of the ovary cavity) was 67.7 ± 0.6 mm (n = 10), i.e.,
≈40%
longer than ‘88-50’ styles (49.3 ± 0.4 mm). Thus, genotypic
variation in the time required to achieve full seed set is partly
attributable to differences in stylar length.
Self-incompatibility (SI) occurs in Schlumbergera (Ganders,
1976; McMillan, 1991) and in many other genera of the Cactaceae
(Boyle et al., 1994; Breckenridge and Miller, 1982; Ganders, 1976;
Ross, 1981; Taylor, 1976). However, little is known about the
physiology or genetic control of SI in this family. Several traits that
occur in Schlumbergera are typically associated with sporophytic
SI systems, i.e., tricellular pollen and dry, papillate stigmas
(Brewbaker, 1957; Heslop-Harrison et al., 1975). The related
genus Rhipsalidopsis also exhibits these traits, along with two
others that are associated with gametophytic SI systems: inhibition
of incompatible pollen tubes in the style and absence of reciprocal
differences in outcrosses (Boyle et al., 1994).
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