|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,
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.
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).
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.
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.
Fig. 1. In vitro pollen germination of 6 avocado cultivars at different temperatures.
Fig. 2. Percentage of avocado pollen germination at different maximum temperatures in vivo in female and male stages.
Fig. 3. Percentage of germination at three different relative humidity percentages.