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2 Department of Life Sciences, Institutes for Applied Research, Ben-Gurion University of the Negev,P.O. Box 653, Beer-Sheva 84105, Israel;and 3 The Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences,Department of Field Crops, Vegetables and Genetics, Rehovot 76100, Israel
Received for publication July 22, 1999. Accepted for publication September 16, 1999.
ABSTRACT
Chromosome numbers and meiotic behavior are reported for the climbing cacti species Hylocereus undatus, Hylocereus polyrhizus, and Selenicereus megalanthus. The Hylocereus spp. are diploid (2n = 22), while S. megalanthus is a tetraploid (2n = 44). Irregular chromosome disjunction at anaphase I in pollen mother cells of S. megalanthus is probably the major cause of its reduced pollen viability and may contribute to low seed set, low number of viable seeds and, consequently, low fruit mass. A pollination study confirmed self-incompatibility in H. polyrhizus and a weakened incompatibility reaction in H. undatus and S. megalanthus. Major crossability barriers do not exist between the Hylocereus spp. investigated. Reciprocal intergeneric crosses were successful between Hylocereus spp. and S. megalanthus, suggesting that an Hylocereus sp. might be one of the diploid progenitors of the tetraploid S. megalanthus. The implications of the results on cacti nomenclature and systematics are briefly discussed.
Key Words: cacti • polyploidy • Hylocereus • Selenicereus • self-incompatibility • semi-sterility • systematics
Night-blooming climbing cacti of the genera Hylocereus (Berger) Br. & R. and Selenicereus (Berger) Br. & R. have received increased attention during the last decade for their potential as new exotic fruit crops. The species of these genera have been studied mainly from the physiological aspect, and a review of their reproductive biology has recently been published (Nerd and Mizrahi, 1997
).
These genera belong to the Cactaceae subfamily Cactoideae, tribe Hylocereeae (Br. & R.) Buxbaum (Barthlott and Hunt, 1993
). About 16 Hylocereus species are dispersed along Central America and Northern South America. The genus Selenicereus comprises 20 species distributed through tropical America and the Caribbean region (Barthlott and Hunt, 1993
).
Commercial plantations of H. undatus (Harworth) Br. & R. exist in Colombia (Cacioppo, 1990
), Nicaragua (INRA, 1994
), and Vietnam (Mizrahi, Nerd, and Nobel, 1997
); and of S. megalanthus (Schum. ex Vaupel) M. in Colombia (Cacioppo, 1990
; Mizrahi, Nerd, and Nobel, 1997
). Small-scale production of these species and of H. polyrhizus (Weber) Br. & R. is emerging in Israel.
Fruit size in these genera determines their economic value. Fruit of S. megalanthus (80–300 g) are much lighter than Hylocereus spp. (200–800 g). Weiss, Nerd, and Mizrahi (1994)
found a positive correlation between fruit fresh mass and total seed number, and a positive relationship seems to exist between the seed set data and pollen viability in these species. Pollen viability is high (>90% stainability) in the Hylocereus group but low in S. megalanthus, 25% for a Colombian clone and 40% for an Ecuadorian clone (Weiss, Nerd, and Mizrahi, 1994
). Lichtenzveig (1996)
observed that the reduced pollen stainability values of Colombian S. megalanthus clones occurred only at the early- and late-season flower buds as compared to 70–78% stainability during flowering peak. A high rate of ovule failure was reported in S. megalanthus (>77%) as compared to only 10% for H. undatus (Weiss, Nerd, and Mizrahi, 1994
).
Studies on Cactaceae cytology deal with chromosome numbers but not with structural chromosome aberrations (Beard, 1937
; Banerji and Sen, 1955
; Spencer, 1955
; Ross, 1981
). To the best of our knowledge there are no reports relating pollen viability to chromosomal aberrations in the Cactaceae.
Both self-fruitful and self-unfruitful (i.e., self-incompatible) species have been observed in Hylocereus. Hylocereus undatus was reported as self-compatible, but with partial self-fruitfulness (50–80% fruit set), while H. polyrhizus, which bears attractive fruit of high commercial potential, was reported as self-incompatible (Weiss, Nerd, and Mizrahi, 1994
). The largest fruits of each of the Hylocereus spp. were obtained by interspecific crossings. Fruit set occurs both after self- and cross-pollination (interclonal) in S. megalanthus (Weiss, Nerd, and Mizrahi, 1994
).
In the framework of a developmental project to introduce climbing cacti as crops for the Negev Desert of Israel, we studied the reproductive biology and cytology of H. undatus, H. polyrhizus, and S. megalanthus. The objective of the study reported here was to determine whether the reduced pollen viability and high rate of ovule failure of S. megalanthus occur due to chromosomal aberrations and to identify the factor(s) that prevent fruit set following self-pollination in H. polyrhizus.
MATERIALS AND METHODS
Plant materials and growing conditions
Three species were selected for this study: H. undatus (one clone), H. polyrhizus (two clones), and S. megalanthus (five clones) as shown in Table 1. The clones were originally introduced as cuttings to Israel either from the Huntington Botanical Garden in California or from three different commercial plantations in Colombia, or as seeds from the jungles in Ecuador (Table 1). The study was carried out at the Ben-Gurion University of the Negev campus in Beer-Sheva, during 1995 and 1996. The experiments were performed on 3–4 yr-old-plants grown on a trellis in a net-house. The plants were irrigated once a week with 2 L per plant during the cold wet season (November–April) and twice a week with 2.5 L per plant during the hot season (May–October); water contained 70 ppm N, 9 ppm P, and 70 ppm K. The average max/min greenhouse temperatures were 28°/6°C in the coldest month (January) and 35°/18°C in the hottest month (August). The maximum temperature was 45°C during the summer, and the minimum temperature was 2°C during the winter.
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Pollination techniques, seed set determination, and fruit mass
Two Hylocereus species (one H. undatus clone and two H. polyrhizus clones) and S. megalanthus were self-pollinated and intercrossed during the night. A full diallel was not achieved because the species did not flower synchronously. In selfing, pollen from the same flower was applied to the stigma; in interclonal, interspecific, or intergeneric crosses, pollen from two or more flowers was combined. The stigma was covered with parafilm 1–2 h before and immediately after hand-pollination to avoid contamination. For all pollinations, fresh pollen was collected from flowers at anthesis. Pollen abortion was evaluated under a light microscope throughout the flowering season using Alexander's stain (Alexander, 1969
). The percentage of pollen abortion was scored from a minimum of 400 grains pooled from at least two flowers per month for each species or clone.
Fruits were harvested at maturity, based on skin color, at seed maturation. Fruit mass and seed number were determined. Brown- and black-coated seeds were observed in some of the fruits. Brown-coated seeds were found nonviable. Seed viability was determined by germinating the black-coated seeds on moist filter paper, in covered petri dishes at 20° ± 2°C under permanent cool-white fluorescent lamps of 40 W. Germination percentage was determined 21 d after seed imbibition.
Pollen germination and pollen tube growth in vivo
Hylocereus polyrhizus clone 89–028 was either selfed or crossed reciprocally with H. undatus clone 89–024. Reciprocal crosses were made between H. undatus clone 89–024 and S. megalanthus clones as well as selfing of parental lines. From each described combination three to five flowers were pollinated. Samples of stigmatic lobes were removed 10–12 h after pollination, while pistils were separated from the flowers 2–4 d after pollination (DAP) and were immediately fixed with a 1:8:1 formalin:80% ethanol:acetic acid solution and stored. The pistils were cut into six sections and a cross slice was sampled from each section for fluorescence microscopy. Both stigmatic lobes and pistil samples were soaked in a 1% sodium carbonate solution for 1 h, washed with a 0.1% solution of aniline blue in 0.1 mol/L K3PO4 for at least 4 h. Squash preparations of the samples were observed under an epifluorescence Zeiss microscope equipped for UV excitation. Percentage of pollen germination was calculated from counts of at least 100 pollen grains in each sample; a pollen grain was considered germinated when the pollen tube length exceeded the grain diameter.
RESULTS AND DISCUSSION
Polyploidy in Hylocereeae
Chromosome numbers and meiotic behavior are reported for the first time for S. megalanthus and H. polyrhizus, and previously published chromosome counts are confirmed for H. undatus. According to the base number of x = 11 (Beard, 1937
; Ross, 1981
), both diploids and tetraploids were observed. In pollen mother cells (PMCs) of H. undatus and H. polyrhizus 2n = 22 chromosomes were observed. In S. megalanthus, chromosome counts in PMCs of three clones from Columbia (90–001, 90–002, and 90–003), as well as in two clones from Ecuador (88–023 and 96–666) were 2n = 44, indicating that S. megalanthus is a tetraploid. Meiotic chromosomes in the seven clones studied were of similar size and morphologically indistinguishable from one another.
Chromosome number variation, especially polyploidy, is believed to be one of the major phylogenetic processes in Cactaceae evolution (Beard, 1937
; Pinkava and McLeod, 1971
; Ross, 1981
; Cota and Philbrick, 1994
). Polyploidy is more common in the subfamilies Opuntiodieae, e.g., Opuntia (Pinkava and McLeod, 1971
; Pinkava, McGill, and Brown, 1973
; Baker and Pinkava, 1987
), and Cactoideae, e.g., Mammillaria and Echinocereus (Ross, 1981
; Cota and Philbrick, 1994
). Cytological studies on Hylocereeae have been reported for four genera: seven Hylocereus spp. (Beard, 1937
; Spencer, 1955
), five Selenicereus spp. (Beard, 1937
; Spencer, 1955
), one Weberocereus Br. & R. sp. (Beard, 1937
), and one Mediocactus Br. & R. sp. (Beard, 1937
). Except for Mediocactus coccineus (Salm-Dyck) Br. & R., thus far the only tetraploid (2n = 44) known in this tribe, all Hylocereeae species were reported as diploids (2n = 22).
The genus Mediocactus is no longer considered an independent taxon, but rather a synonym of the genus Selenicereus (Barthlott and Hunt, 1993
; Weiss, Scheinvar, and Mizrahi, 1995
). Two species were included in this genus: M. coccineus and M. megalanthus syn.: S. megalanthus (Weiss, Scheinvar, and Mizrahi, 1995
). Mediocactus was first classified by Britton and Rose (1963)
and described as follows, "in habit and flowers this plant much resembles Hylocereus, but differs from it in its tubercular ovary and in the felted and spine-bearing areoles of the fruit, which resemble those of Selenicereus ... its name implies intermediate characters ...." These morphological features might imply that the polyploidy observed in S. megalanthus originated from an intergeneric hybridization between diploid species of the genera Hylocereus and Selenicereus. If this is the case, S. megalanthus should be considered an allopolyploid.
Meiotic chromosomes behavior of Hylocereus spp. and Selenicereus megalanthus
Multivalent pairing was observed in most PMCs tested in S. megalanthus clones (Fig. 1A, B). Frequencies of the different meiotic configurations observed in the two clones are provided in Table 2. Bivalent formation and regular disjunction were observed during the first metaphase of meiosis in PMCs from the species H. undatus and H. polyrhizus (Fig. 1C, D).
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). In S. megalanthus the presence of multivalents and univalents was typical of metaphase I. An example of an univalent in "mitotic disjunction" at anaphase I in S. megalanthus 90–003 is presented in Fig. 1B. Occasional multivalents in an organism with regular bivalent pairing may be a consequence of heterozygosity of translocations. In perennials with vegetative propagation, such translocations can easily be fixed and maintained for many generations. In such cases one would expect ~50% pollen viability in clones heterozygous for a single translocation and further reduction in viability with more than one translocation. This, however, is not the case in the S. megalanthus clones tested. That is, despite the observed frequencies of multivalent pairing (3.8 per cell) (Table 2), pollen viability in S. megalanthus was above 70% during peak flowering. Thus, reciprocal translocations were ruled out as the cause for these multivalent associations in metaphase I.
In polyploids with multivalent pairing, chiasmata failure may result in unbalanced anaphase I disjunction due to presence of unpaired univalents. Having observed univalents in metaphase I cells, we were interested in determining the degree of unbalanced anaphase I disjunctions. Three different anaphase separations were observed for the S. megalanthus clones (90–003 and 88–023) at anaphase I; 22–22, 23–21, and 24–20 (Table 3). Some degree of aneuploidy could be tolerated, both under the assumption of autotetraploidy or allotetraploidy (with some degree of homoeology between parental genomes). For example, we have counted 2n = 28 in root tip cells of a hybrid resulting from the cross H. polyrhizus x S. megalanthus. The deviation from the expected triploid number (2n = 3x = 33) may be explained by fusion of a normal haploid gamete (n = 11) with an unbalanced, but still viable, n = 17 (2x - 5) gamete from the tetraploid pollen parent. Chromosome deficiency for both copies of a particular chromosome, in an autotetraploid, or of both partial homologues (homoeologues) in an allotetraploid, is likely to result in a nonviable gamete. Alternatively, a gamete deficient for one homologue from each of several nonhomologous chromosomes might still be viable. It is impossible to determine what portion of the 20-chromosome situations in anaphase I (Table 3) represents such a case. Clearly, the chromosome disjunction data at anaphase I (Table 3) explain the reduced pollen viability counts (70–78%) of S. megalanthus. While it is possible that some of the aneuploid pollen might be viable, it is likely that pollen grains from 24 to 20 disjunction events would be nonviable, hence the reduced stainability counts.
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; Ross, 1981
). The exceptions are Rebutia spegazziana, Rebutia cv. Nivea, Mammillaria compressa, and M. prolifera, each of which form three to five quadrivalents (Ross, 1981
). The frequency of multivalent pairing in S. megalanthus may assist in determining its status as an autotetrapolyploid or as an allotetraploid. Exclusive bivalent formation in metaphase I could serve as an indication for allopolyploidy. Quadrivalent formation, in certain frequencies, could indicate an autoploid origin (Jackson and Casey, 1982
). The observed frequencies of quadrivalent + trivalent in PMCs of both studied S. megalanthus clones are by far below the expected frequencies predicted by the model suggested by Jackson and Casey (1982)
. Therefore, it is unlikely that S. megalanthus is an autotetraploid species. If this is the case, the occurrence of an average number of 3.8 multivalents per PMC could reflect some chromosome homoeology. In such a case S. megalanthus might be a segmental allopolyploid indicating the homoeology between some of the chromosomes of its parental complements.
Possible cause of low fruit mass in S. megalanthus
Weiss, Nerd, and Mizrahi (1994)
reported a positive correlation between fruit fresh mass and total seed number in S. megalanthus (r = 0.46, which differ significantly from zero at P < 0.05). We observed a higher correlation between fruit mass and viable seed number; the number of viable seeds per fruit accounted for 42% of the variation in fruit mass (r = 0.65, which differ significantly from zero at P < 0.05). Selenicereus megalanthus ovaries contain in average 1969 ± 176 ovules (Weiss, Nerd, and Mizrahi, 1994
). A low percentage of these ovules developed into viable seeds: 8.7% after self-pollinations, 5.4 and 9.7% after intergeneric crosses with H. undatus and H. polyrhizus, respectively, and 17.9% after interclonal crosses, where clones 90–001, 90–002, and 90–003 were the female parent and clone 88–023 was the male (Table 4). Undeveloped brown seeds and viable black seeds were also observed in fruits of the columnar cactus Cereus peruvianus (L.) Mill. (Weiss, 1995
), which have similar characteristics to fruit of Selenicereus. Cereus peruvianus fruit have white juicy pulp, which develops from the feniculi, but the pulp originates only from the feniculi of the black seeds (Weiss, 1995
). Such might be the situation in S. megalanthus fruits and could be the reason for the correlation between fruit mass and viable seeds.
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Mating systems of the genera Hylocereus and Selenicereus
In the self-fruitful Hylocereus clones, fruits obtained by self-pollination were significantly smaller than those obtained by interspecific cross-pollination (Table 5). These results are in line with data reported by Weiss, Nerd, and Mizrahi (1994)
. Self-pollinations in H. polyrhizus resulted in inhibition of pollen tube growth at the ovary (Fig. 2A–D) and low percentage of light mass fruit set (Table 5). Hylocereus polyrhizus clones were reciprocally cross-compatible; the percentage of fruit set and the fruit mass were almost as high as after interspecific pollination (Table 5).
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)—are cross-compatible with each other despite their classification as independent botanical taxons. This means that the above species may be considered as members of the same gene pool (biological species), after Mayr (1970
).
The experimental results confirm that self-incompatibility (SI) occurs in H. polyrhizus. All species tested in this study possess hollow styles that, according to de Nettancourt (1977)
, probably restrain the contact between pollen tubes and the stylar tissue. Inhibition of incompatible pollen tubes was not confined to a particular stylar region for any of the pollination types, hence, inhibition of self-pollen tubes probably occurs at the ovary before fertilization. The term "late-acting self-incompatibility" (Sedgley, 1994
) seems appropriate to describe the SI mechanism in H. polyrhizus. The pollen tube growth behavior suggests that H. polyrhizus presents a gametophytic SI system, however, genetic studies are required to ascertain whether the gametophyte or sporophyte determines the incompatible phenotype of the pollen. Further observations are needed to determine the incompatibility reaction site in H. polyrhizus ovary; although postzygotic embryo abortion seems less possible, it cannot be ruled out yet.
The modern European (fresh) fruit markets are the main targets of the emerging Israeli climbing cacti industry. These markets have certain demands in terms of desired fruit mass as well as stable product delivery during the season. In Israel, climbing cacti cropping is a high input investment, mainly due to the required trellis, irrigation, and shedding facilities and labor cost. Hence, high fruit set of marketable size (mass) is a prerequisite for a lucrative and viable industry. In the absence of natural pollinators (native to Latin America), optimizing artificial pollination techniques including pollen storage protocols are of significant economic implications. In addition, development of self-compatible types with valuable fruit sizes is an important long-term goal in climbing cacti breeding. Such types will require less manual labor for pollen collection and pollination, thereby reducing farming costs.
FOOTNOTES
1 The authors thank Mrs. Hadassa van Oss (The Hebrew University of Jerusalem) for her skillful technical assistance and Prof. J. Janick for invaluable remarks on the manuscript. 
4 Current address: The Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, Department of Field Crops, Vegetables and Genetics, Rehovot 76100, Israel.
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Mayr, E. 1970 Populations, species and evolution—an abridgment of animal species and evolution. Belknap Press of Harvard University Press, Cambridge, Massachusetts, USA.
Mizrahi, Y., A. Nerd, and P. S. Nobel. 1997 Cacti as crops. In J. Janick [ed.], Horticultural Reviews, vol. 18, 291–320. John Wiley & Sons, New York, New York, USA.
Nerd, A., and Y. Mizrahi. 1997 Reproductive biology of cactus fruit crops. In J. Janick [ed.], Horticultural Reviews, vol. 18, 321–346. John Wiley & Sons, New York, New York, USA.
de Nettancourt, D. 1977 Incompatibility in angiosperms. Springer-Verlag, New York, New York, USA.
Pinkava, D. J., and M. G. Mcleod. 1971 Chromosome numbers in some cacti of Western North America. Brittonia 23: 171–176.
———, ———, L. A. McGill, and R. C. Brown. 1973 Chromosome numbers in some cacti of Western North America—II. Brittonia 25: 2–9.[CrossRef][ISI]
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Sedgley, M. 1994 Self-incompatibility in woody horticultural species. In E. Williams, A. Clarke, and R. Knox [eds.], Genetic control of self-incompatibility and reproductive development in flowering plants, 141-163. Kluwer Academic, London, UK.
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Weiss, J. 1995 The biology of flowering, pollination and fruit development in night blooming and day-blooming cacti. Ph.D. dissertation, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
———, A. Nerd, and Y. Mizrahi. 1994 Flowering behavior and pollination requirements in climbing cacti with fruit crop potential. HortScience 29: 1487–1492.
———, L. Scheinvar, and Y. Mizrahi. 1995 Selenicereus megalanthus (the yellow pitaya), a climbing cactus from Colombia and Peru. Cactus and Succulent Journal (U.S.) 67: 280–283.
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