Reproductive Biology of Avocado (Persea americana Mill.) in Southern
Spain
M.L. Alcaraz and J.I. Hormaza
Estación Experimental la Mayora-CSIC. 29750 Algarrobo-Costa, Málaga, Spain
Keywords: Hass, pollen germination, stigmatic receptivity, temperature, relative
humidity,
Abstract
Spain is the only European country with a commercial production of
avocados, mainly in the coasts of Malaga and Granada. Only a very small fraction of
the flowers produced at anthesis in an avocado tree (less than 1%) are able to set
fruits and the production is very dependent of the environmental conditions.
Consequently, knowledge of avocado reproductive biology is important to increase
yield and to design appropriate breeding programs in this species. Avocado is a
tropical and subtropical fruit tree that shows protogynous dichogamy. Although the
flowers are bisexual, each flower opens twice: first functionally as female (stigmas
are receptive), then the flower closes and reopens the next day functionally as male
(dehiscence of anthers). The different avocado cultivars are classified by their floral
behaviour in two groups: A and B. In type A cultivars the flowers open in the
morning as females and in the afternoon of the following day as males whereas in
type B cultivars the flowers open in the afternoon as females and the following
morning as males. The main objective of this work was to determine the changes in
the effective pollination period (EPP) due to environmental factors, mainly
temperature and relative humidity. Thus, we analyzed the progamic phase, from
pollination to fertilization under the environmental conditions of Southern Spain:
pollen deposition on the stigmas, pollen germination, pollen tube growth and pollen
tube penetration into the ovule. The response of pollen germination to temperature
and humidity has also been evaluated for different genotypes both in vivo and in
vitro. The results are discussed in terms of the implications of the environmental
conditions on reproductive success and fruit set in avocado to elucidate the limiting
factors in production of this species and to design appropriate crosses.
INTRODUCTION
Avocado is an evergreen tree native to Central America and México. It is a
member of the Lauraceae, a mostly subtropical or tropical family included in the basal
Angiosperm clade Magnoliid within the order Laurales. Three horticultural races of
avocado have traditionally been recognized: Mexican, Guatemalan and West-Indian
described, respectively as subtropical, semitropical and tropical. These races are
distinguishable on the basis of morphological, physiological, and horticultural traits. No
sterility barriers have been reported between or among the three races. The avocado
shows a particular flowering behaviour: protogynous dichogamy. The different avocado
cultivars are classified by its floral behaviour in two groups: A and B (Stout, 1923); in the
type A cultivars the flowers open in the morning functionally as females, close at midday
and reopen in the afternoon of the following day functionally as males. The flowers of the
type B cultivars open as females during the afternoon, close overnight and reopen the
following morning in the male stage. Thus, the two groups are complementary. Due to its
protogynous dichogamy, avocado is considered to be an out-crossing species and,
consequently, planting complementary cultivars in orchards has been recommended to
achieve an adequate pollination and fruit set. In areas where avocados are grown
commercially, honey bees (Apis mellifera L.) are used for avocado pollination.
A correct understanding of the floral biology of this species is necessary not only
to optimize yield but also to perform appropriate crosses in avocado breeding programs.
MATERIALS AND METHODS
The experiments were carried out during two years in an experimental orchard
located at the Estación Experimental “La Mayora”, Malaga (Spain).
In vitro pollen germination was evaluated in different avocado cultivars: ‘Hass’
(hybrid), ‘Fuerte’ (hybrid), ‘Anaheim’ (Guatemalan), ‘Topa Topa’ (Mexican), ‘Maoz’
(West Indian) and ‘Gvar 13’ (unknown). Pollen was prehydrated for one hour
(Loupassaki et al, 1997) and germinated in a liquid medium containing 23%PEG, 10%
sucrose and a mixture of mineral salts [100mg/L KNO3, 100 mg/L H3BO3, 200mg/L
MgSO47H2O and 250mg/L Ca(NO3)4H2O]. Germination was evaluated after 24 hours.
Pollen grains were considered germinated when the pollen tube was longer than the
diameter of pollen grain.
To estimate stigmatic receptivity, approximately 60 flowers per treatment were
collected, and maintained in wet florist's foam at different temperatures (20ºC, 25ºC and
ambient temperature -26ºC- in the orchard) and relative humidities (50%, 75%, 95%).
The flowers of cultivar ‘Hass’ were hand-pollinated at the female (using anthers of
‘Fuerte’) and male (using anthers of ‘Hass’) stages. The flowers were collected during
three days after pollination and were fixed in FAA (formalin: acetic acid: 70% ethanol,
1:1:18 v/v, Johansen, 1940). The pistils were washed three times with distilled water and
maintained in sodium sulphite overnight at 4 ºC. The next day they were autoclaved for
10 min at 1 kg/cm2 in 5% sodium sulphite (Jefferies and Belcher, 1974) and mounted in
squash preparations with 0.1 aniline blue in 0.1N K3PO4 . Preparations were viewed under
a microscope with UV epifluorescense. Stigmatic receptivity was evaluated as the
capacity of stigmas to support pollen hydration, germination, and pollen tube growth in
the transmitting tissue of the style.
RESULTS AND DISCUSSION
Significant differences for in vitro pollen germination were recorded between the
different genotypes studied. The highest germination rate was observed in ‘Gvar 13’,
‘Anaheim’ and ‘Hass’ and the lowest in ‘Fuerte’, ‘Topa Topa’ and ‘Maoz’. The
percentage of germination of cultivars ‘Topa Topa’, ‘Maoz’ and ‘Fuerte’ are similar and
different to ‘Gvar 13’ and ‘Hass’. The germination percentage ranged from 7.09
(‘Fuerte’) to 34.34 (‘Hass’). These differences and the lack of differences in pollen
germination in vivo suggest that the germination medium should be optimized for each
cultivar (Figure 1).
No significant effects of temperature were found in the behaviour of these
cultivars except for ‘Maoz’ (a West Indian cultivar) that shows an increase in germination
when the temperature is increased. This could be due to the adaptation of the West Indian
race to warmer conditions.
Stigmatic receptivity has been evaluated on the basis of pollen germination and
penetration to the transmitting tissue in the female and the male stages. At 20ºC no
significant differences (p>0,005) were observed between both stages. However, at higher
temperatures (25ºC or natural conditions in the orchard, 26ºC) we find lower pollen
germination on the male phase resulting in significant differences in pollen germination
between both stages at 25ºC (Figure 2).
The effect of relative humidity on stigmatic receptivity was also evaluated on the
male and female stage (Figure 3) but no differences between both stages were found at
75% and 95%, although a significant decreases was observed at 50% (p=0,001). No
significant differences were obtained in the number of pollen tubes at the base of style
between the male and the female stages 24 hours after pollination on the different
treatments. In the upper part of the style the number of tubes ranged from 15 to 1, with an
average of 6, in the middle of the style the average number of pollen tubes was of 2 and
generally only one single tube was recorded at the base of style, although in some cases
two tubes could be recorded.
The results indicate that the capacity of the stigma to support pollen germination is
maintained on the male stage. This could explain the self pollination rate and the yield
obtained in solid blocks of single cultivars and could also facilitate crosses in avocado
breeding programs allowing the use of pollen from cultivars of the same floral group (A
or B).
ACKNOWLEDGEMENTS
Financial support for this work was provided by the Spanish Ministry of
Education (Project Grant AGL2004-02290/AGR). M.L.A. was supported by an I3P
predoctoral grant of CSIC.
Literature cited
Stout, A.B. 1923. A study in cross-pollination of avocado in southern California.
California Avocado Association Annual Report 8: 29-45.
Johansen, D. A. (1940). Plant microtechniques. New York: McGraw-Hill.
Jefferies, C. J. and Belcher, A. R. (1974). A fluorescent brightener used for pollen tube
identification in vivo. Stain Technology 49, 199–202.
Loupassaki, M., Vasilakakis, M. and I. Androulakis (1997). Effect of pre-incubation
humidity and temperature treatment on the in vitro germination of pollen grains.
Euphytica 94: 247-251.
Figures
Germination (%)
50
40
30
20
10
0
Hass
Fuerte
T. Topa
Gvar 13
Anaheim
Maoz
Cultivars
Room Temp
20ºC
30ºC
Fig. 1. In vitro pollen germination of 6 avocado cultivars at different temperatures.
Germination (%)
60
50
40
30
20
10
0
20ºC
25ºC
26ºC (Orchard)
Tem perature
Female Stage
Male Stage
Fig. 2. Percentage of avocado pollen germination at different maximum temperatures in
vivo in female and male stages.
Germiantion (%)
70
60
50
40
30
20
10
0
50%
75%
95%
Relative Humidity
Female Stage
Male Stage
Fig. 3. Percentage of germination at three different relative humidity percentages.