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CROP ECOLOGY, MANAGEMENT & QUALITY

Pollination Requirements of Pigmented Grapefruit (Citrus paradisi Macf.) from Northwestern Argentina

^a Lab. de Investigaciones Ecológicas de las Yungas, CC 34, 4107, Yerba Buena, Tucumán, Argentina
^b Lab. Ecotono-CRUB, Univ. Nacional del Comahue, Quintral 1250, 8400 Bariloche, Río Negro, Argentina

^* Corresponding author (nchacoff{at}lab.cricyt.edu.ar).

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
The consequence of a proposed pollinator decline for agriculture is a subject of much ongoing debate. However, pollination requirements of many cultivated plants remain unknown. Citrus is complex in terms of pollination needs because of great variation in breeding systems among and within species, and even among and within cultivars. The objective of this study was to evaluate pollinator dependence of three cultivars of grapefruit (Citrus paradisi Macf.) planted in northwestern Argentina. Bagged flowering branches were assigned to different pollination treatments: emasculation, spontaneous self-pollination, hand self-pollination, and hand cross-pollination, and the results compared with those from open-pollinated flowers. We counted the number of pollen grains and pollen tubes in the style, fruit set, and seed production. We also assessed differences in germination rates of self- vs. cross-pollen grains. We found that hand- and open-pollinated flowers set about six times more fruit than emasculated and bagged (insect excluded) flowers. In addition, cross-pollen performed better in terms of grain germination and tube growth than self-pollen. Although being fully self-compatible, apomixis and wind pollination are not important factors for grapefruit reproductive success. Thus, insect pollinators represent a critical and potential limiting resource for seedless grapefruits from northwestern Argentina.

Abbreviations: FAA, formalin–acetic acid–ethyl alcohol.

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
STUDIES ON CROP pollination by native and nonnative insects are becoming increasingly critical because of a perceived global decline in pollinator stocks, a subject of much current debate with great economical and conservation consequences (Ghazoul, 2005a, 2005b; Steffan-Dewenter et al., 2005). Insect pollination is determinant or beneficial for agriculture since more than 65% of the 1500 cultivated species are pollinated by animals (Roubik, 1995). However, detailed studies of crop pollination systems are incomplete or out of date (Klein et al., 2006). To adequately evaluate the importance of animal pollination for food production and the impact of pollinator losses due to different anthropogenic disturbances, one of the first steps is to determine the pollination needs of different cultivated plants (Klein et al., 2006). Even when some highly productive crops may benefit from insect pollination, generalizations on pollinator requirements are usually based on results obtained from one or a few cultivars (Crane and Walker, 1984; Free, 1993; Roubik, 1995), a practice that can lead to misleading conclusions and mismanagement with great potential economical losses (Steffan-Dewenter et al., 2005; Westerkamp and Gottsberger, 2000).

The extensively cultivated genus Citrus, which includes such economically important crops as orange (Citrus sinensis), tangerine (C. reticulata), grapefruit (C. paradisi Macf.), and lemon (C. limon), exhibits tremendous variation in pollination requirements, making generalizations of little value. In Citrus, pollinator dependence and breeding systems vary among and within species, and even within cultivars of the same species (Cameron and Frost, 1968; Crane and Walker, 1984; Free, 1993; McGregor, 1976; Roubik, 1995). For instance, whereas ‘Clementine’ tangerine and some sweet oranges are completely or partially self-incompatible (Cameron and Frost, 1968; McGregor, 1976; Sanford, 1992)—thus requiring insects for pollination—spontaneous self-pollination prevails among ‘Eureka’ and ‘Lisbon’ lemons (Webber, 1946). This genus also exhibits large variation in the incidence of parthenocarpy (McGregor, 1976), and thus, the sexual process is not always necessary for fruit development. On the other hand, many cultivars of Citrus are known to be self-incompatible and therefore an appropriate supply of cross pollen and pollinating insects are needed for high fruit yields (Sanford, 1992).

In particular, the pollination requirements and breeding system of grapefruit are poorly understood, and the available information is confusing. Some studies state that cross-pollination is not necessary for fruit set (Krezdorn, 1986a; Roubik, 1995), whereas others demonstrate that the transfer of cross-pollen mediated by insects increases fruit production (Burger, 1985b). In some cultivars, most seeds are produced by asexual means, but this information does not necessarily indicate the absence of benefits derived from insect pollination (McGregor, 1976). In ‘Marsh’ grapefruit, for example, open-pollinated flowers set about twice as many seeds and four times as many fruits as spontaneously, self-pollinated flowers (Wright, 1937, in McGregor, 1976). ‘Star Ruby’ grapefruit (a seedless deep red, fleshed-fruit cultivar) produced a reduced number of functional pollen grains, and few fruits developed if flowers were not hand-pollinated (Burger, 1985b). Crane and Walker (1984) also stated that pollen transfer by insects increases fruit set in grapefruit.

In Argentina, citriculture is practiced mostly in the northwestern and northeastern regions of the country. In particular, grapefruit was brought from Jamaica to northern Argentina in 1911 (Dansa, 2001), and an increasing number of important cultivars have now been selected and propagated locally from naturally occurring mutations (Dansa, 2001). Feral Africanized honeybees (Apis mellifera) and other stingless and solitary bees (e.g., Trigona argentina and Augochlorpsis cupreola) visit grapefruit flowers in northwestern Argentina (Chacoff and Aizen, 2006). However, there is a lack of studies on the reproductive biology and relevance of insect pollinators for fruit production, both for the local cultivars or for others cultivated in the region.

The objective of this study was to assess experimentally the pollination requirements of three pigmented (red seedless) cultivars of grapefruit planted in northwestern Argentina to evaluate the possible effects of insect and cross-pollination on fruit production. Two are regional cultivars of great economic importance. We also examined differences in pollen grain germination and tube attrition of self- vs. cross-pollen, to evaluate whether postpollination events may favor outcrossing.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
The Tree Species
Grapefruit originated in the Caribbean (Gmitter, 1995; León, 2000) by natural hybridization between pummelo (C. grandis Osbeck) and sweet orange (C. sinensis Osbeck) (Moore, 2001; Nicolosi et al., 2000). Variation among grapefruit cultivars has arisen both naturally and through induced somatic mutations, which were propagated asexually (Gmitter, 1995). Cultivated Citrus are mostly shrubs or small trees with dense foliage and flowers, which are either solitary or grouped in small clusters. The conspicuous white corolla is composed of five distinct petals and alternating sepals. The flowers produce a strong scent when mature. The androecium consists of 20 to 40 stamens with partially fused filaments and yellow anthers. A nectariferous disc inside a ring of stamens secretes abundant nectar (Schneider, 1968). A globose, yellowish stigma terminates the style, which connects to a superior ovary containing 8 to 15 fused carpels, each enclosing two rows of ovules. Flowers are simultaneously hermaphroditic, releasing pollen when the stigma is receptive. Flowers open early during the morning and remain receptive until dusk of the following day.

In the study region, grapefruit trees bloom for approximately 45 d, between the end of August (late winter) and the end of September (midspring). Fruits start ripening during the austral fall and can remain on the tree for several months after maturation. Therefore, grapefruit harvest starts in March and continues until mid-August.

We recorded 50 species of insects visiting grapefruit flowers. However, about 95% of the visits were made by the alien feral, Africanized honeybees, with species of stingless (e.g., Trigona argentina and Tetragonisca angustula) and solitary bees (Xylocopa eximia and Megachile sp.) being the next most common visitors. Flowers received an average of 1.4 visits h^–1 during daytime (Chacoff and Aizen, 2006).

Study Area
We studied grapefruit cultivars planted in the Upper Bermejo River Basin, near Orán city, Salta province, northwestern Argentina (23°28' S, 64°24' W), in a zone corresponding to the premontane lowland forests of the Yungas (400–600 m above sea level) (Cabrera, 1976). Annual rainfall in this region averages 733 mm and varies between 280 and 1224 mm (Brown et al., 2001). Citrus plantations occupy 13500 ha in the Upper Bermejo River Basin, producing mostly grapefruit (57%) and sweet oranges (32%) (Peralta, 1999). Grapefruit from this region represents more than 30% of the Argentine production, and most of it (> 80%) is exported (Dansa, 2001; Federcitrus, 2003). Six red and very red seedless cultivars (Henninger Ruby, Río Red, Rouge La Toma, Flame, Foster Seedless, and Star Ruby) are the most commonly cultivated (Peralta, 1999).

We studied the pollination requirements of three seedless, pigmented cultivars in four commercial plantations. These cultivars included (i) Rouge La Toma, a very red cultivar that was selected and propagated from a naturally mutated Henninger's Ruby tree at La Toma plantation (Salta Province, northwestern Argentina); (ii) Río Red, a very red cultivar generated in 1963 by irradiation of buds of Ruby Red seedlings in Texas (Gmitter, 1995); and (iii) Foster Seedless, a red cultivar that arose from a natural mutation in a Foster tree at the INTA experimental station at Yuto (Ing. Palacios, personal communication, 2003; Jujuy Province, northwestern Argentina) and is now cultivated widely in the Upper Bermejo River Basin (Peralta, 1999). We selected these cultivars because of their relatively high economic importance and abundance in the study area.

Field Sampling
Pollination Experiments
We studied 6 to 12 trees in each plantation to assess their dependence on cross-pollination and their degree of self-compatibility. For Rouge La Toma, we selected 10 trees in the La Toma plantation during 2000 and 9 trees in the Produnor plantation during 2001; for Río Red, we studied 12 trees in the Citrusalta plantation during 2001 and 2002. For this last cultivar, six trees were surveyed during 2001; the same trees were used in the next year, except for one tree that did not flower profusely the second year and thus was replaced. We also added six new Río Red trees to our study during 2002. Foster Seedless was represented by six trees in the Peña Colorada plantation during 2002. Trees in each plantation were selected randomly. More details on the plantations and cultivars are provided in Table 1.

One week after pollination, we collected styles from flowers in all five treatments just before they started falling naturally to estimate pollen receipt and pollen tube growth. Styles were fixed and stored in individual microcentrifuge tubes containing FAA (formalin–acetic acid–ethyl alcohol, 5:5:90). In the laboratory, styles were cleared in a 10 mL L^–1 NaOH solution for 48 h and stained with 0.1% aniline blue in 0.1 mol L^–1 K₃PO₄ (Martin, 1959). Squashed preparations were examined with an epifluorescence microscope at 100x. For each style, we counted the number of pollen grains germinating on the stigma and the number of pollen tubes at the base of the style.

We estimated reproductive success from each treatment as final fruit set and seed set. Fruits were collected when ripe, in March, and developed seeds were counted. From these data, we determined fruit set (number of fruits/number of flowers) and seed set (number of seeds fruit^–1). Sample sizes for each variable, treatment, and cultivar are indicated in the Appendix.

Pollen Grain Germination Rates
To compare pollen germination rates of self- vs. cross-grains, we enclosed flower buds from branches of the Rouge La Toma cultivar (in La Toma plantation) during the flowering season of 2002. As buds opened, we manually pollinated virgin flowers with either cross- or self-pollen. Cross-pollen came from three different trees within the same plantation and cultivar. Flowers were rebagged after pollination. We collected and fixed styles in FAA at different times after pollination (2, 4, 8, and 24 h). Styles were stained as above and pollen grains that had germinated on the stigma counted with an epifluorescence microscope. In this particular experiment, we could not estimate rates of pollen-tube growth because no pollen tubes reached the base of the style within 24 h. In grapefruit, the period from pollination to fertilization has been reported to be from 2 to 12 d (Eti and Stosser, 1992; Ngo et al., 2001). In total, 223 styles were collected, which represented a mean of 28 styles treatment^–1 (pollen origin and time combination).

Data Analysis
We analyzed data using two-way mixed model ANOVA with two fixed factors, cultivar and pollination treatment. The tree factor was nested within cultivar and thus considered random. Year was also considered as a random factor (Bennington and Thayne, 1994). We performed orthogonal contrasts to assess significant differences among treatments. The contrasts were (i) emasculation vs. spontaneous self-pollination (Treatments 1 vs. 2), (ii) emasculation and spontaneous self-pollination vs. hand and open pollination (Treatments 1 + 2 vs. 3 + 4 + 5), (iii) hand (self and cross) vs. open pollination (Treatments 3 + 4 vs. 5), and (iv) hand cross- vs. hand self-pollination (Treatments 3 vs. 4) (Table 2).

We analyzed germination rates of self- vs. cross-pollen also using a general linear mixed model. Here, we considered time since pollination and pollen type (self vs. cross) as crossed fixed factors, and tree and its interactions with time and pollen type as random effects.

All the analyses were performed in SAS. We used the MIXED and GLIMMIX procedures for the general and generalized models, respectively (SAS Institute, 2004).

	RESULTS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
Flowers of the study cultivars of grapefruit received large pollen loads under natural conditions. Open-pollinated flowers had [mean ± (1 SE)] 56.2 (± 6.9) germinated grains on the stigma and 9.8 (± 1.1) pollen tubes reaching the ovary. Only 21 out of 642 open-pollinated flowers developed fruits; these fruits contained an average of 3.1 (± 0.9) seeds.

Pollination Experiments
Pollen loads on the stigmas differed greatly among pollination treatments, but they did not differ among cultivars (Table 2). Open- and hand-pollinated flowers had on average four times more pollen grains germinating on their stigmas than spontaneously self-pollinated flowers and eight times more than emasculated flowers (Fig. 1a). The number of germinated pollen grains on stigmas of emasculated flowers did not differ from that of self-pollinated flowers (emasculation vs. spontaneous self-pollination contrast, Table 2). Although emasculation was performed immediately before flower anthesis, a few pollen grains had accidentally fallen onto the stigmas when we removed anthers (see Appendix). Stigmas from open-pollinated flowers had 21% fewer germinating pollen grains than those from hand-pollinated flowers (cross- and self-pollination vs. open-pollination contrast, Table 2), whereas the number of pollen grains found in hand self-pollinated flowers did not differ significantly from the number found in hand cross-pollinated flowers (Fig. 1a). The differences among pollination treatment in number of germinated grains were relatively consistent among cultivars (nonsignificant treatment x cultivar interaction, Table 2; see also Appendix).

View larger version (24K): [in this window] [in a new window]   Figure 1. Least-square means of number of pollen grains: (a) pollen tubes, (b) final fruit set, (c) seed set, and (d) (± 1 SE). Different uppercase letters indicate significant differences among treatments (P < 0.05) according to the scheme of orthogonal contrasts (see "Data Analysis" and Table 2).

Emasculated and spontaneously self-pollinated flowers rarely set fruit (0.14%), and their fruit set was three to six times lower than those of open- and hand-pollinated flowers (Fig. 1c). Fruit and seed production of hand-pollinated flowers (self- and cross-) did not differ significantly from those of open-pollinated flowers (Fig. 1c–d). Hand-pollination with self- or cross-pollen resulted in equivalent fruit set, despite a trend for cross-pollinated flowers to produce more fruit than self-pollinated flowers (Fig. 1c). Fruit set and seed production did not differ significantly among cultivars, and all cultivars responded similarly to the different pollination treatments (nonsignificant treatment x cultivar interaction; Table 2 and Appendix). However, open-pollinated flowers from the Río Red cultivar did not produce more fruit than spontaneously self-pollinated flowers (Appendix).

Pollen Grain Germination Rates
Cross-pollen germinated faster than self-pollen. Differences in the number of germinated pollen grains between self- and cross-pollinated flowers developed over time (F_3,35.5 = 43.44, P < 0.001, Fig. 2). However, differences between treatments became apparent 24 h after the beginning of the experiment, when we found a significantly larger number of germinated cross- than self-pollen grains (F_1,45.3 = 5.99, P < 0.05, Fig. 2).

View larger version (11K): [in this window] [in a new window]   Figure 2. Mean (±1 SE) number of germinated pollen grains on the stigma over time (hours) after self- and cross-pollinations. Significant statistical differences (p < 0.05) for specific times are indicated by the symbol *. Closed circles (•) correspond to cross-pollen; open circles (°) correspond to self-pollen.

	DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

Pollination, pollen tube growth, fertilization, and subsequent seed development are the steps involved in the sexual reproduction of the vast majority of flowering plants (Richards, 1986). However, there are species in which fruit development occurs without sexual reproduction, that is, parthenocarpy (Richards, 1986). These fruits, consequently, are seedless or have seeds whose embryos develop apomictically (Gmitter, 1995). For instance, the Red Blush grapefruit cultivar is highly parthenocarpic (Krezdorn, 1986a). However, there are varying degrees of parthenocarpy in Citrus, and some parthenocarpic cultivars require at least the stimulus from pollen tube growth or even sexual fertilization and subsequent seed abortion to develop fruit (Krezdorn, 1986a). Although we did not conduct an embryological analysis to determine the origin of the seeds (zygotic or apomictic), we can conclude that animal pollination and pollen tube development are important factors for fruit production in all three study cultivars.

Moreover, the arrival of pollen grains to the stigma is also important for seed formation. "Seedless" Citrus cultivars, as the majority of commercial grapefruits, have between zero and six seeds (Palacios, 1978). In some cultivars, seedlessness actually results from pollen sterility (nonfunctional pollen) and/or low levels of ovule fertility. A slow pollen tube growth in those cultivars producing viable pollen is apparently induced by inhibitors in the style, causing pollen tubes to abort before reaching the ovary, thus precluding sexual fertilization (Krezdorn, 1986a). Since high levels of ovule (female) sterility is common in all commercially seedless grapefruits (e.g., Marsh and Red Blush), there is no way to increase seediness appreciably (Krezdorn, 1986b). This may explain why fruits from the different pollination treatments contained a similar low number of seeds (Table 2, Fig. 1d). In addition, improved pollination did not enhance the number of seeds per fruit, even though it increased fruit set.

The few pollen grains found on the stigmas of flowers isolated with mesh bags demonstrate a limited capacity for spontaneous self-pollination and suggest that grapefruit pollen cannot be windborne. Citrus pollen is heavy and sticky and thus cannot be transported by wind (Krezdorn, 1986a; Sanford, 1992; Schneider, 1968). Therefore, the presence of pollinating insects becomes critical for high fruit yield in the study cultivars. The exception may be Río Red, the nonlocal cultivar, for which levels of fruit set resulting from spontaneous self-pollination did not differ from those of open-pollinated flowers. In this cultivar, animal pollination seems not to be a prerequisite for fruit production.

Hand-pollinated flowers with pollen from the same or other trees of the same cultivar exhibited a slight increase in pollen tube growth and fruit production with respect to open-pollinated flowers, indicating that these grapefruit cultivars may be limited by inadequate pollination. For some grapefruit cultivars, a high proportion of sterile pollen has been reported (Burger, 1982; Frost and Soost, 1968); however, for the cultivars studied here, we observed pollen germinating on the stigma and the development of pollen tubes that reached the base of the style. Our results on pollen germination rates and pollen tube growth show the existence of differences in pollination quality according to pollen origin. We found more cross- than self-pollen germinating 24 h after pollination. Furthermore, more cross-pollen tubes reached the ovary at flower senescence. Higher performance of cross- vs. self-pollen is common in many species (Aizen et al., 1990; Cruzan, 1989; Snow and Spira, 1991). Thus, under natural conditions of mixed pollination, cross-grains could outcompete self-grains in siring seeds and/or eliciting initial ovary swelling and fruit development. Although Citrus blooms profusely, mature fruits are produced from a very small percentage of flowers (0.1–3.5% in Bustan and Goldschmidt, 1998). Yet, the presence of abundant insect pollinators is essential to guarantee maximum fruit set in our and other cultivars studied (Burger, 1985a; McGregor, 1976). For instance, trees from Star Ruby grapefruit that were exposed to bees inside cages produced more fruit than caged trees not exposed to bees (Burger, 1985a). More generally, fruit production, even in fully self-compatible plant species, may be strongly pollinator-limited (Klein et al., 2003a; Richards, 2001; Roubik, 2002a). Regrettably, most research in Citrus has been concentrated in breeding programs aimed at the search of adequate pollen donors in self-incompatible cultivars (Eti and Stosser, 1992; Ngo et al., 2001), or of plants resistant to different nutritional stresses, particularly to N and Mg deficiencies, or insect herbivory (e.g., Argov et al., 1999; Krezdorn, 1986b; Peralta, 1999). Thus, the degree of yield improvement that could be reached by encouraging efficient pollination and improving the pollination environment remains uncertain. Beekeepers usually perceive Citrus plantations as good nectar sources for honey production, whereas citriculturists are not much concerned about pollination requirements at all (Free, 1993; Sanford, 1992). In northwestern Argentina, most grapefruit pollination is conducted by feral Africanized honeybees (Chacoff and Aizen, 2006). Although these and other bees provide a valuable service, their abundance depends to some degree on the conservation of nearby forest remnants, where they nest and find nectar, and other resources throughout the year (Allen-Wardell et al., 1998; Kearns and Inouye, 1997; Klein et al., 2002).

The service that pollinators provide to agriculture is increasingly being taken into account in the economic cost structure associated to different crops (Constanza et al., 1997; Kenmore and Krell, 1998; Richards, 2001; Southwick and Southwick, 1992; Westerkamp and Gottsberger, 2000). Recent studies of pollination of coffee (Coffea arabica, C. canephora, and C. robusta) in Indonesia, Costa Rica, Brazil, and Panama reveal the importance of insect pollination and the conservation of the nearby forest for the fruit production of this self-compatible crops (De Marco and Monteiro Coelho, 2004; Klein et al., 2003b; Ricketts et al., 2004; Roubik, 2002b). Studies of native pollinators and the pollination requirements of crops, including some economically important ones, like coffee and Citrus, are still being developed. Conclusions drawn from these studies on the pollination requirements of crops could be linked to the conservation of habitat mosaics in which many pollinators thrive. The results reported in this study suggest that an adequate pollination service of these grapefruit cultivars provided by bees may be important to maintain and improve fruit production.

	APPENDIX

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 

	ACKNOWLEDGMENTS

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Received for publication September 15, 2006.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 

• Aizen, M.A., K.B. Searcy, and D.L. Mulcahy. 1990. Among and within flower comparisons of pollen tube growth following self- and cross pollinations in Dianthus chilensis (Caryophyllaceae). Am. J. Bot. 77:671–676.[CrossRef][ISI]
• Allen-Wardell, G., P. Bernhardt, R. Bitner, A. Burquez, S. Buchmann, J. Cane, A. Cox, V. Dalton, P. Feinsinger, M. Ingram, D. Inouye, C.E. Jones, K. Kennedy, P. Kevan, H. Koopowitz, R. Medellin, S. Medellin-Morales, G.P. Nabhan, B. Pavlik, V. Tepedino, P. Torchio, and S. Walker. 1998. The potential consequences of pollinator declines on the conservation of biodiversity and stability of food crops yields. Conserv. Biol. 12:8–17.[CrossRef]
• Argov, Y., Y. Rossler, H. Voet, and D. Rosen. 1999. Spatial dispersion and sampling of citrus whitefly, Dialeurodes citri, for control decisions in a citrus orchard. Agric. For. Entomol. 1:305–318.[CrossRef]
• Bennington, C.C., and W.V. Thayne. 1994. Use and misuse of mixed model analysis of variance in ecological studies. Ecology 75:717–722.[CrossRef][ISI]
• Brown, A.D., H.R. Grau, L. Malizia, and A. Grau. 2001. Argentina. p. 623–659. In M. Kappelle and A. D. Brown (ed.) Bosques Nublados del Neotrópico. InBio, Santo Domingo de Heredia, Costa Rica.
• Burger, D.W. 1982. Pollination and fruit set of Star Ruby grapefruit. J. Rio Grande Valley Hortic. Soc. 35:163–166.
• Burger, D.W. 1985a. Fruit production of caged ‘Star Ruby’ grapefruit trees. J. Rio Grande Valley Hortic. Soc. 38:15–18.
• Burger, D.W. 1985b. Pollination effects on fruit production of ‘Star Ruby’ grapefruit (Citrus paradisi Macf.). Sci. Hortic. (Amsterdam) 25:71–76.[CrossRef]
• Bustan, A., and E.E. Goldschmidt. 1998. Estimating the cost of flowering in a grapefruit tree. Plant Cell Environ. 21:217–224.[Medline]
• Cabrera, A.L. 1976. Regiones fitogeográficas Argentinas. Enciclopedia Argentina de Agricultura y Jardinería. Vol. 2. 2nd ed. Editorial Acme, Buenos Aires, Argentina.
• Cameron, J.W., and H.B. Frost. 1968. Genetics, breeding, and nucellar embryony. p. 325–366. In W. Reuther (ed.) The citrus industry. Vol. 2. Univ. of California Press, Berkeley.
• Chacoff, N.P., and M.A. Aizen. 2006. Edge effects on flower-visiting insects in grapefruit plantations bordering premontane subtropical forest. J. Appl. Ecol. 43:18–27.[CrossRef]
• Constanza, R., R. d'Arge, R.D. Groot, S. Farber, M. Grasso, B. Hannon, K. Limburg, R.V. O'Neill, J. Paruelo, R.G. Raskin, P. Sutton, and M. van der Belt. 1997. The value of the world's ecosystem services and natural capital. Nature 387:253–260.[CrossRef]
• Crane, E., and P. Walker. 1984. Pollination directory for world crops. Int. Bee Res. Assoc., Bucks, UK.
• Cruzan, M.B. 1989. Pollen tube attrition in Erythronium grandiflorum. Am. J. Bot. 76:562–570.[CrossRef][ISI]
• Dansa, A.M. 2001. Perfil de mercado de Cítricos. Secretaria de Agricultura, Ganadería, Pesca y Alimentos, Buenos Aires, Argentina.
• De Marco, P.J., and F. Monteiro Coelho. 2004. Services performed by the ecosystem: Forest remnants influence agricultural cultures' pollination and production. Biodivers. Conserv. 13:1245–1255.[CrossRef]
• Eti, S., and R. Stosser. 1992. Pollen tube growth and development of ovules in relation to fruit set in Mandarine, cv. ‘Clementine’ (Citrus reticulata blanco). Acta Hortic. 321:621–625.
• Federcitrus. 2003. Estadísticas 2003: Ranking de Empresas Exportadoras. Available at http://www.atcitrus.com (verified 30 Jan. 2007). Tucuman Citrus Assoc., Tucuman, Argentina.
• Free, J.B. 1993. Insect pollination of crops. 2nd. ed. Academic Press, New York.
• Frost, H.B. 1946. Seed reproduction: Development of gametes and embryos. p. 767–815. In H. J. Webber and L. D. Batchelor (ed.) The citrus industry. Vol. 1. Univ. of California Press, Berkeley.
• Frost, H.B., and R.K. Soost. 1968. Seed reproduction: Development of gametes and embryos. p. 290–320. In W. Reuther (ed.) The citrus industry. Vol. 2. Univ. of California Press, Berkeley.
• Ghazoul, J. 2005a. Buzziness as usual? Questioning the global pollination crisis. Trends Ecol. Evol. 20:367.
• Ghazoul, J. 2005b. Response to Steffan-Dewenter et al.: Questioning the global pollination crisis. Trends Ecol. Evol. 20:652.
• Gmitter, F.G.J. 1995. Origin, evolution, and breeding of the grapefruit. Plant Breed. Rev. 13:345–363.
• Kearns, C., and D. Inouye. 1993. Techniques for pollination biologists. Univ. Press of Colorado, Boulder.
• Kearns, C., and D. Inouye. 1997. Pollinators, flowering plants, and conservation biology. Bioscience 47:297–307.[CrossRef][ISI]
• Kenmore, P., and R. Krell. 1998. Global perspectives on pollination on agriculture and agroecosystem management. In Proc. Int. Workshop on the Conserv. and Sustainable Use of Pollinators in Agriculture, with Emphasis on Bees, Sao Paulo, Brazil. 7–9 Oct. 1998.
• Klein, A.M., I. Steffan-Dewenter, D. Buchori, and T. Tscharntke. 2002. Effects of land-use intensity in tropical agroforestry systems on coffee flower-visiting and trap-nesting bees and wasps. Conserv. Biol. 16:1003–1014.[CrossRef]
• Klein, A.M., I. Steffan-Dewenter, and T. Tscharntke. 2003a. Bee pollination and fruit set of Coffea arabica and C. canephora (Rubiaceae). Am. J. Bot. 90:153–157.[Abstract/Free Full Text]
• Klein, A.M., I. Steffan-Dewenter, and T. Tscharntke. 2003b. Fruit set of highland coffee increases with the diversity of pollinating bees. Proc. R. Soc. Lond. B. Biol. Sci. 270:955–961.[Medline]
• Klein, A., B. Vaissiere, J. Cane, I. Steffan-Dewenter, S. Cunningham, C. Kremen, and T. Tscharntke. 2006. Importance of pollinators in changing landscapes for world crops. Proc. Royal Soc. London B. doi:10.1098/rspb.2006.3721.
• Krezdorn, A.H. 1986a. Citrus flowering, fruit set and development. Citrus Research and Education Center, Lake Alfred, FL.
• Krezdorn, A.H. 1986b. Pollination and related factors affecting fruit quality. Citrus Research and Education Center, Lake Alfred, FL.
• León, J. 2000. Botánica de los cultivos tropicales. 3rd ed. Editorial Agroamericana, Instituto Interamericano de Cooperación para la Agricultura, San José, Costa Rica.
• Littell, R.C., G.A. Milliken, W.W. Stroup, and R. Wolfinger. 1996. SAS system for mixed models. SAS Inst., Cary, NC.
• Martin, F.W. 1959. Staining and observing pollen tubes in the style by means of fluorescence. Stain Technol. 34:125–128.[ISI][Medline]
• McGregor, S.E. 1976. Citrus. p. 149–157. In Insect pollination of cultivated crop plants. Agric. Handb. 496. U.S. Gov. Print. Office, Washington, DC.
• Moore, G.A. 2001. Oranges and lemons: Clues to the taxonomy of Citrus from molecular markers. Trends Gen. 17:536–540.[CrossRef][ISI][Medline]
• Ngo, B.X., A. Wakana, S.M. Park, Y. Nada, and I. Fukudome. 2001. Pollen tube behaviors in self-incompatible and self-compatible Citrus cultivars. J. Fac. Agr. Kyushu Univ. 45:443–457.
• Nicolosi, E., Z.N. Deng, A. Gentile, and S. La Malfa. 2000. Citrus phylogeny and genetic origin of important species as investigated by molecular markers. Theor. Appl. Genet. 100:1155–1166.[CrossRef][ISI]
• Palacios, J. 1978. Citricultura moderna. Hemisferio Sur, Buenos Aires, Argentina.
• Peralta, C. 1999. Informes citrícolas de la provincia de Salta. INTA ORAN, Salta, Argentina.
• Richards, A.J. 1986. Plant breeding systems. Paston Press, London.
• Richards, A.J. 2001. Does low biodiversity resulting from modern agricultural practice affect crop pollination and yield? Ann. Bot. (Lond.) 88:165–172.[Abstract/Free Full Text]
• Ricketts, T.H., G.C. Daily, P.R. Ehrlich, and C.D. Michener. 2004. Economic value of tropical forest to coffee production. Proc. Natl. Acad. Sci. USA 10:12579–12582.
• Roubik, D.W. 1995. Pollination of cultivated plants in the tropics. FAO Agric. Serv. Bull., FAO, Rome.
• Roubik, D.W. 2002a. Feral african bees augment neotropical coffee yield. p. 255–266. In P. Kevan and V. Imperatriz-Fonseca (ed.) Pollinating bees: The conservation link between agriculture and nature. Ministry of Environment, Secretariat for Biodiversity and Forests, Brasilia, Brazil.
• Roubik, D.W. 2002b. The values of bees to the coffee harvest. Nature 417:708.
• Sanford, M.T. 1992. Pollination of citrus by honey bees. RF-AA092. Florida Coop. Ext. Serv., Inst. of Food and Agric. Sci., Univ. of Florida.
• SAS Institute. 2004. The Glimmix procedure (experimental). Release SAS 9.1. SAS Inst., Cary, NC.
• Schneider, H. 1968. The anatomy of citrus. p. 1–23. In W. Reuther (ed.) The citrus industry. Vol. 2. Univ. of California Press, Berkeley.
• Snow, A.A., and T.P. Spira. 1991. Differential pollen tube growth rate and non random fertilization in Hibiscus moscheutos (Malvaceae). Am. J. Bot. 78:1419–1426.[CrossRef][ISI]
• Sokal, R.R., and F.J. Rohlf. 1995. Biometry. 3rd. ed. W.H. Freeman, New York.
• Southwick, E., and L. Southwick. 1992. Estimating the economic value of honey bees (Hymenoptera: Apidae) as agricultural pollinators in the United States. J. Econ. Entomol. 85:621–633.[ISI]
• Steffan-Dewenter, I., S.G. Potts, and L. Packer. 2005. Pollinator diversity and crop pollination services are at risk. Trends Ecol. Evol. 20:651–652.[CrossRef][Medline]
• Webber, H.J. 1946. Cultivated varieties of citrus. p. 569–584. In H.J. Webber and L.D. Batchelor (ed.) The citrus industry. Vol. 1. Univ. California Press, Berkeley.
• Westerkamp, C., and G. Gottsberger. 2000. Diversity pays in crop pollination. Crop Sci. 40:1209–1222.[Abstract/Free Full Text]

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