Notes on Citrullus Spp.: Pollen Morphology, C Values, and Interspecific Hybridizations with the Gemsbok Cucumber

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Published March 3, 2017

RESEARCH Notes on Citrullus spp.: Pollen Morphology, C Values, and Interspecific Hybridizations with the Gemsbok Cucumber

Robert L. Jarret,* Gary R. Bauchan, W. Wyatt Oswald, Kathiravetpillai Arumuganathan, and John P. Shields

R.L. Jarret, USDA-ARS, Plant Genetic Resources Unit, 1109 Experiment ABSTRACT St., Griffin, GA 30223; G.R. Bauchan, USDA-ARS, Electron and Scanning electron and light microscopy Confocal Microscopy Unit, Bldg. 12 BARC-West, 10300 Baltimore were used to examine pollen of the currently Ave., Beltsville, MD 20705; W.W. Oswald, Emerson College, Institute recognized species (and forms) within the genus for Liberal Arts and Interdisciplinary Studies, 120 Boylston St., Boston, Citrullus (Cucurbitaceae). Materials examined MA 02116; K. Arumuganathan, Benaroya Research Institute, 1201 9th included: C. lanatus (Thunb.) Matsum. & Nakai, Ave., Seattle, WA 98101-2795; J.P. Shields, Dep. of Cellular Biology, 724 the citron (C. amarus Schrad.), the egusi Biological Sciences Building, Univ. of Georgia, Athens, GA 30602-2607. [C. mucosospermus (Fursa) Fursa], C. Received 30 Aug. 2016. Accepted 21 Nov. 2016. *Corresponding author colocynthis (L.) Schrad., C. rehmii De Winter, ([email protected]). Assigned to Associate Editor Yiqun Weng. C. ecirrhosus Cogn., and the gemsbok Abbreviations: P/E, polar/equatorial ratio; SEM, scanning electron cucumber [C. naudinianus (Sond.) Hook. f.]— microscopy. also referred to in the literature as Acanthosicyos naudinianus (Sond.) C. Jeffrey. Pollen of all species were similar in shape differing slightly atermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] in their polar (P) and equatorial (E) axes and Wis cultivated globally on a massive scale for the sweet flesh P/E. In general, all were characterized as of its fruits and, to a lesser extent, for its edible seeds (Minsart et prolate and tricolpate with a small polar area al., 2011; FAOSTAT, 2016; Watermelon Board, 2016). The current and reticulate ornamentation. Flow cytometry revealed C values for the taxa examined as literature (Chomicki and Renner, 2015) recognizes seven Citrul- ranging from 0.8 to 1.04 pg DNA per 2C nuclei. lus spp. that includes the gemsbok cucumber [Citrullus naudinianus A mutant affecting pollen ornamentation was (Sond.) Hook. f.]. Additional taxa have been described and/or pro- identified in PI 482261, a C. amarus citron from posed (Bailey, 1930; Mansfeld, 1959; Fursa, 1972b, 1983; Nesom, Zimbabwe. Pollen ornamentation on PI 482261 2011). Each recognized taxon possesses a unique combination of was rugulate. An examination of 15 additional distinguishing morphological characteristics. These include the accessions from neighboring areas in Zimbabwe absence of tendrils (C. ecirrhosus Cogn.), the presence of a caudex revealed a second rugulate type in PI 482312, [C. colocynthis (L.) Schrad. and C. ecirrhosus], storage root formation while the remaining 14 C. amarus accessions [C. naudinianus (Sond.) Hook. f.], a distinctive fruit pericarp texture were reticulate. Successful hybridizations were and coloration (C. rehmii De Winter), seed type [C. mucosospermus made between C. colocynthis (PI 652554) and (Fursa) Fursa], a dioecious (C. naudinianus) versus a monoecious C. naudinianus (GRIF 14032), and between (all other currently recognized Citrullus spp.) flowering habit, and C. rehmii (PI 670011) and C. naudinianus a perennial (C. naudinianus, C. ecirrhosus, C. colocynthis) versus an (GRIF 14032). The F1 hybrids of both crosses were extremely vigorous and exhibited some annual [C. lanatus (Thunb.) Matsum. & Nakai, C. amarus Schrad., characteristics (i.e., branching and storage root C. mucosospermus, C. rehmii] growth habit, among others. While formation) associated with the male parent. the potential contribution of one or more of these taxa to the Other characteristics such as tendril length and shape, and leaf size and shape, were Published in Crop Sci. 57:856–864 (2017). intermediate between the parents. To date, the doi: 10.2135/cropsci2016.08.0717

856 www.crops.org crop science, vol. 57, march–april 2017 improvement of cultivated C. lanatus or C. amarus remains Pseudocucumis. Jeffrey (1962) transferred Citrullus naudinia- to be determined, it is clear that, as a group, they potentially nus (Sond.) Hook. f. to Acanthosicyos based on biochemical possess traits of immediate or high agronomic significance. and seed coat characters, its spiny ovary and fruit, tendrils Understanding the relationships among these materials can reduced to spines, and the presence of cucurbitacin B as be useful in predicting the likelihood of successfully using aglycone (E being absent). Jeffrey (1964), however, recog- their genetic material for the improvement of the cultivated nized Acanthosicyos as being closer to Citrullus and noted crop species. that pollen of A. naudinianus bore no resemblance to that Palynology has been used in efforts to define or clarify of Cucumis and its allied genera. Jeffrey (1964) also noted relationships among members of the Cucurbitaceae (Mar- that Acanthosicyos was not a natural group. Acanthosicyos ticorena, 1963; Stafford and Doyle, 1994; Barth et al., 2005; naudinianus was placed in the tribe Benincaseae, subtribe Pruesapan and Van der Ham, 2005; Perveen and Qaiser, Benincasinae (with Citrullus), on the basis of its seed coat 2008; Lima and Miotto, 2011). Dieterle (1974), for exam- characteristics (Jeffrey, 2005). Jeffrey’s transfer of C. nau- ple, used scanning electron microscopy (SEM) to compare dinianus to Acanthosicyos was not supported by Fernandes similarities in the pollen morphology of certain species et al. (1986), Fursa (1972a, 1972b), Schaefer and Renner of Echinopepeon and that of a new genus from Mexico (2011), and most recently by the molecular data of Cho- (Apatzingania Dieterle). In her description of Apatzingania, micki and Renner (2015). Dieterle (1974) noted that, given the extreme diversity Although previous studies have examined pollen in pollen structure in the Cucurbitaceae, similarities in morphology in one or more species of Citrullus (Marti- pollen morphology may indicate a degree of taxonomic corena, 1963; Jeffrey, 1964; Wen’ge et al., 2003; Perveen relationship. Jeffrey (1964) examined pollen slides of vari- and Qaiser, 2008), none of these have presented images or ous taxa to evaluate natural groupings within the family data on pollen of C. rehmii or C. ecirrhosus, or attributed Cucurbitaceae. He found a high degree of correspondence data acquired on pollen of C. lanatus to one of its previ- between groups based on general plant morphology and ously recognized forms (Bailey, 1930; Fursa, 1972b, 1983). pollen morphology. Information on attempts to cross C. naudinianus with The taxonomic status and the relationship of Citrullus other members of the genus Citrullus appears to be lacking naudinianus Sond. [syn. Acanthosicyos naudinianus (Sond.) in the literature, with one exception (Shimotsuma, 1963). Jeffrey] toCitrullus has frequently been questioned (Jeffrey C values are available only for C. lanatus. This study was 1962; Meeuse, 1962). Recent molecular genetic data sup- conducted to examine the pollen morphology of all cur- port its inclusion within Citrullus (Chomicki and Renner, rently recognized Citrullus spp. (including C. naudinianus) 2015). Described as Cucumis naudinianus Sond. (Harvey in an attempt to document material not previously exam- and Sonder, 1862), Citrullus naudinianus (Sond.) Hook. f. ined and to confirm previously published descriptions, to (Hooker, 1871), Colocynthis naudinianus (Sond.) Kuntze establish C values for Citrullus spp., and to evaluate the (Kuntze, 1891), and Pseudocucumis naudinianus (Sond.) C. feasibility of hybridizing C. colocynthis and C. rehmii with Jeffrey, Citrullus naudinianus is also referred to as gemsbok C. naudinianus. cucumber. This plant is endemic to Botswana, Namibia, Mozambique, South Africa, Zambia, and Zimbabwe, MATERIALS AND METHODS where it is found in woodlands, grasslands, and wooded Plant Material grasslands in areas of sandy soil. The plant bears pale green All plant materials were acquired directly from the S-9 fruits that somewhat resemble those of Acanthosicyos horri- genebank in Griffin, GA (Jarret et al., 1990). The citron (or dus Wewl. ex Hook. f. (Bosch, 2004) and certain Cucumis preserving melon) and the egusi melon are referred to as spp. The fruits and seeds of C. naudinianus are eaten raw C. amarus and C. mucosospermus, respectively, per Chomicki and or roasted and provide an important source of water in the Renner (2015), recognizing that universal agreement on their Kalihari (Bosch, 2004). Extracts of its storage root have nomenclature has yet to be achieved. Unless noted otherwise, antimicrobial activity (Hedimbi et al., 2012). Its chromo- seed of all plant introductions (PI) and GRIFFIN (GRIF) num- some number (n) is 11 (Shimotsuma, 1963; Ojo, 2016). bers, including PI 665007 (C. lanatus ‘Sugar Baby’); PI 306782 According to Meeuse (1962), the simple spine-like (C. mucusospermus); PI 652554 (C. colocynthis); GRIF 14032 and PI 596694 (C. naudinianus); C. amarus PI numbers 482261, tendrils, tuberculate fruits, and the chemical composi- 482257, 482259, 482265, 482276, 482283, 482298, 482309, tion of the bitter substances in the fruit (which have a 482311, 482312, 482315, 482316, 482322, 482355, 482361, composition similar to those found in Cucumis) of Citrullus and 532624; PI 381824 [Lagenaria siceraria (Molina) Standl.]; naudinianus suggested a relationship to Cucumis. How- GRIF 5611 (Luffa acutangula (L.) Roxb.]; PI 512102 [Cucurbita ever, its floral characters indicated that it was a species of okeechobeensis subsp. martinezii (L.H. Bailey) T.C. Andres & Citrullus. Hence, Meeuse (1962) recommended the reten- G.P. Nabhan ex T.W. Walters & D.S. Decke]; and PI 618817 tion of the true species of Citrullus as the section Citrullus (Cucumis spp.), were sown directly into peat pots containing a and the inclusion of C. naudinianus in a separate section, greenhouse potting mix (Fafard 3B, Conrad Fafard). All plants

crop science, vol. 57, march–april 2017 www.crops.org 857 were subsequently transplanted into 3.78-L plastic pots and MX-Macro lens, MXG-5040RZ, and MXG-10C with a 300´ maintained in the greenhouse without supplemental lighting. objective. The digital microscope has a motorized stage, which In the summer of 2015, male flowers of GRIF 14032 were used allows several images at varying degrees of focus to be com- to pollinate three flowers of PI 652554 C.( colocynthis) and PI pressed together to develop an image where all fields of view 670011 (C. rehmii). Fruit were allowed to mature for 45 d. The are in focus. seeds were then extracted, washed, and stored at −20°C in an For comparative purposes, some samples were sput- airtight foil pouch until use. ter-coated (Structure Probe) for 60 sec. (~15 nm thick) and examined with a Zeiss 1450EP SEM at 10 kV with a probe of In Vitro Seed Germination 400 nA at the Georgia Electron Microscopy Facility, Univer- Seeds of C. ecirrhosus (PI 673135), C. rehmii (PI 670011), and sity of Georgia, Athens. Measurements of pollen dimensions hybrid seed of C. colocynthis (PI 652554) ´ C. naudinianus obtained on both instruments were comparable. (GRIF 14032) and C. rehmii ´ GRIF 14032 were germinated in vitro. Seed were soaked for 1 h in water and then transferred Light Microscopy to a solution containing 10% (v/v) household bleach (0.5% Light microscopy was used to score the pollen of the parents, sodium hypochlorite) and stirred for 10 min. In a laminar flow F1, F2, and BC1 progeny of crosses between PI 482261 (mutant) hood, the seeds were rinsed three times with sterile water and and PI 482309 (normal). Pollen from freshly harvested flowers placed in open Petri dishes to dry. A pair of sterile vise grips was was shaken onto a glass slide, covered with a cover slip, and used to crack the seed coat. Seeds were then placed in 25-mm immediately observed using an Olympus model BX51 micro- −1 ´ 150-mm culture tubes (1 seed tube ) containing 20 mL of scope. Images were taken with an Omax model A35100U a media composed of one-half Murashige and Skoog basal salts digital camera. (Murashige and Skoog, 1962), 30 g L−1 of sucrose, 8 g L−1 of Difco Bacto agar, and 0.05% (v/v) Plant Preservative Mixture Flow Cytometry (Caisson Laboratories). The pH of all media were adjusted to The procedure used to analyze nuclear DNA content in plant 5.8. Cultures were incubated at 28°C on a 16-h photoperiod cells was modified from Arumuganathan and Earle (1991a). under cool-white fluorescent lights. Briefly, the procedure consisted of preparing suspensions of intact When the cotyledons of the plants were fully expanded nuclei by chopping 50 mg of plant leaf in MgSO4 buffer mixed and roots longer than 3 cm had developed, plants were removed with DNA standards and stained with propidium iodide in a from the culture tubes, the media were washed off with running solution containing DNase-free RNase. Fluorescence intensities water, and the plantlets were transferred to peat pots containing of the stained nuclei were measured by a flow cytometer. Values the potting mix noted previously, amended to contain ~15% for nuclear DNA content were estimated by comparing fluores- (v/v) washed coarse sand. Plantlets were covered with a clear cence intensities of the nuclei of the test population with those plastic bag and placed in the shade for 7 d to acclimate. The of an appropriate internal DNA standard that was included with plastic bag was removed after the acclimation period and the the tissue being tested. Nuclei from Oryza sativa ‘Nipponbare’ plants (in peat pots) were transplanted into plastic pots contain- (0.96 pg per 2C were used as the internal standard). The pellet ing the same soil mixture. was suspended by vortexing vigorously in 0.5 mL of solution

containing 10 mM MgSO4×7H2O, 50 mM KCl, 5 mM Hepes Scanning Electron Microscopy (SEM) (pH 8.0), 3 mM dithiothreitol, 0.1 mg mL−1 propidium iodide, Flowers were harvested in Griffin in the early morning 1.5 mg mL−1 DNase-free RNase (Rhoche), and 0.25% Triton (<8:00 AM), placed in 50-mL conical centrifuge tubes with X-100. The suspended nuclei were withdrawn using a pipettor, a 4-cm ´ 4-cm piece of moist paper towel, and shipped on filtered through 30-mm nylon mesh, and incubated at 37°C for ice via next-day courier transport to Beltsville, MD, for obser- 30 min before flow cytometric analysis. Suspensions of sample vation immediately after delivery. For comparative purposes, nuclei were spiked with a suspension of standard nuclei (prepared flowers were also harvested, packed as noted previously, and in the above solution) and analyzed with a FACScalibur flow transported directly to Athens, GA, for observation. cytometer (Becton Dickinson). For each measurement, the prop- Anthers were dissected out of freshly cut flowers and placed idium iodide fluorescence area signals (FL2-A) from 1000 nuclei on a 25-mm black double-sided sticky tab (PELCO Carbon Con- were collected and analyzed by CellQuest software (Becton ductive tabs, Ted Pella) which was covering a 25-mm (diameter) Dickinson, 1999) on a Macintosh computer. The mean position SEM stub (AMRAY aluminum stub, Ted Pella). Anthers and of the G0/G1 (nuclei) peak of the sample and the internal stan- pollen were observed using a Hitachi Variable Pressure TM3030 dard were determined by CellQuest software. The mean nuclear tabletop SEM with a Deben Cool Stage (TM-3000, Woolpit). DNA content of each plant sample, measured in picograms, was Samples were not pretreated before imaging. The Deben Cool based on 1000 scanned nuclei. Stage was cooled to −25°C, which allowed for a longer imaging period (1–1.5 h) before tissue deterioration. Images were cap- RESULTS AND DISCUSSION tured using the Hitachi TM3030 software. No differences in size, shape, or ornamentation were Color Images observed between pollen samples analyzed ~24 to 30 h Color images were obtained of the flowers, anthers, pistils, after harvest when packed on ice and shipped to Beltsville and pollen using a Hirox KH-7700 Digital Microscope with a (as described) for observation and those carried (on ice) to

858 www.crops.org crop science, vol. 57, march–april 2017 Athens for observation within 3 h of harvest. Measure- and shape within and among Citrullus spp. has not been ments (SEM) taken on these samples were not significantly determined. The lack of available plant material of several different from those taken on samples examined locally, taxa precluded a broader survey in this study. However, we immediately (<30 min) after harvest, via light microscopy. suggest that similarities in gross pollen morphology among Pollen grains of all taxa examined were similar in size, these taxa reflect their relatedness. shape and ornamentation (Fig. 1), with the exception of Marticorena (1963) described the pollen of C. colocyn- PI 482261 which had a rugulate ornamentation (Fig. 2A). this as being approximately 52 to 55 mM ´ 56 to 64 mM in Jeffrey (1964) described pollen of Benincaseae (including size, subprolate in shape, and with a reticulate ornamenta- Citrullus and Acanthosicyos) as 3-colporate, usually reticu- tion. Pollen of C. lanatus was described as oblate-spheroidal late, and rarely spinulose. All Citrullus spp. examined in the with a reticulate exine and being of approximately the present study were 3-colporate and, except as noted previ- same size as pollen of C. colocynthis. Pollen of C. naudinianus ously, reticulate. We found pollen of C. naudinianus to be (as A. naudinianus) was reported as being of the same form, similar in size, shape, and ornamentation to other Citrullus size, and ornamentation as C. lanatus, although no pictures spp. (Fig. 1). Statistically significant differences in size were were provided of any of these. Wen’ge et al. (2003) noted detected among the pollen of the Citrullus spp. examined that the shape of pollen of diploid watermelon cultivars (Table 1). However, the significance of these differences, (C. lanatus) was suboblate and that pollen grain size varied if any, remains to be determined. Intraspecific variation in among (four) cultivars (polar axis: 54.5–63.8 mM, equato- pollen size in Citrullus lanatus has been documented (Wen’ge rial axis: 44.5–47.4 mM) with an average polar/equatorial et al., 2003). The full extent of the variation in pollen size (P/E) ratio of 1.28, whereas the pollen of polyploid cultivars

Fig. 1. Scanning electron microscopy photographs of pollen grains of (A) C. lanatus ‘Sugar Baby’ (PI 665007), (B) C. mucosospermus/PI 306782, (C) C. rehmii/PI 670011, (D) C. ecirrhosus/PI 673135, (E) C. colocynthis/PI 652554, and (F) C. naudinianus/GRIF 14032.

crop science, vol. 57, march–april 2017 www.crops.org 859 accessions or taxa was observed (Fig. 1) in the present study. Environmental effects may also be a factor. Pollen of all Citrullus taxa was distinct from four related genera (Fig. 3). Our initial observation of the pollen of PI 482261, a C. amarus originating from Zimbabwe, revealed a rugulate ornamentation (Fig. 2A), quite distinct from the reticulate ornamentation present on the pollen grains of the other Citrullus spp. examined. A close relationship but distinct division between C. amarus and the cultivated sweet watermelon has been demonstrated (Mujaju et al., 2010, 2011; Chomicki and Renner, 2015). The unique morphology of the pollen of C. amarus PI 482261 prompted us to examine additional specimens of C. amarus from that same Fig. 2. Pollen ornamentation of C. amarus accessions (A) PI geographic region. Among the 15 additional samples from 482261 (mutant) and (B) PI 482309 (normal). Zimbabwe that were examined, only PI 482312 was found to have rugulate ornamentation. The ornamentation of the was spheroidal. Perveen and Qaiser (2008) reported the remaining 14 samples was reticulate (Fig. 2B), as determined pollen of C. colocynthis to be oblate-spheroidal in shape and via both electron and light microscopy. The exine of both slightly smaller (~47 × 54 mM) than that reported by Mar- PI 482261 and 482312 differed slightly in appearance(Fig. 4) ticorena (1963) with a coarsely reticulate tectum. Levi et al. and bore a resemblance to the type-4 mutants described in (2010) reported pollen of cultivated C. lanatus ‘Charleston Arabidopsis thaliana (L.) Heynh. (Suzuki et al., 2008). Gray’ to be 49 (P) ´ 33 mM (E), or a P/E of 1.64. Pollen of To examine the possibility of an underlying genetic C. colocynthis was slightly smaller at 54 (P) ´ 29 mM (E) for basis for the rugulate ornamentation, reciprocal crosses a P/E of 1.86, whereas pollen of a C. lanatus (C. amarus) was were made between PI 482261 (mutant) and PI 482309

64 (P) ´ 39 mM (E) for a P/E of 1.64. (normal). An examination of the pollen from six F1 plants In general, our data support earlier observations of of both crosses using light microscopy (Fig. 5) revealed pollen of Citrullus spp. All specimens were 3-colporate only the reticulate exine of the normal (PI 482309)

and reticulate (or in one instance, rugulate). However, the parent. A single F1 plant (PI 482261 ´ PI 482309) was pollen grains observed in the present study were prolate, selected, selfed, and used as a male parent in backcrosses

having a P/E between 1.33 and 2.0 (Erdtman, 1943) and to PI 482309. Among the 48 F2 plants examined, 10 pro- being more elongate than reported by Marticorena (1963) duced rugulate pollen and 38 reticulate pollen (c2 = 0.44, and Wen’ge et al. (2003) (Table 1, Fig. 1). The methods used p = 0.51). Among the 48 BC progeny, 22 were rugulate and to prepare the pollen reported in these earlier studies are 26 were reticulate (c2 = 0.33, p = 0.56). We acknowledge unclear. The differences in pollen size and shape reported the small size of the populations used; however, the data sug- here may be due to differences in the methods used in these gest a genetic (recessive) basis for this mutation of the pollen earlier studies to fix or observe the pollen grains before or exine ornamentation in this citron-type melon. Suzuki et during their microscopic examination, or due to genetic al. (2008) noted a single recessive gene as the basis for some effects. Variability in pollen size both within and among exine mutants in A. thaliana, whereas the number of genes

Table 1. Shape and mean dimensions (mM) of pollen (n = 10) of seven Citrullus spp. Taxon Accession no. Shape† Polar axis (P) Equatorial axis (E) P/E Ornamentation —————————————— mM —————————————— C. colocynthis 652554 Tr/Pr 76.8c‡ 44.8c 1.7b Reticulate C. ecirrhosus 673135 Tr/Pr 73.5d 46.5b 1.6c Reticulate C. amarus§ 482261 Tr/Pr 85.4a 44.7bc 1.9a Rugulate C. amarus§ 482309 Tr/Pr 83.5a 43.2cd 1.9a Reticulate C. mucosospermus¶ 306782 Tr/Pr 75.5cd 49.1a 1.6c Reticulate C. lanatus# 665007 Tr/Pr 64.5f 41.4d 1.6c Reticulate C. rehmii 670011 Tr/Pr 70.0e 38.9e 1.8b Reticulate C. naudinianus GRIF 14032 Tr/Pr 81.2b 43.1cd 1.9a Reticulate † Tr, tricolpate; Pr, prolate.

‡ Means followed by the same letter are not significantly different.

§ Citron.

¶ Egusi.

# Cultivated dessert watermelon.

860 www.crops.org crop science, vol. 57, march–april 2017 Fig. 3. Scanning electron microscopy photographs of pollen grains of (A) Lagenaria siceraria/PI 381824, (B) Luffa acutangula/GRIF 5611, (C) Cucurbita okeechobeensis ssp. martinezii/PI 512102, and (D) Cucumis spp./PI 618817. controlling other mutants was uncertain due to difficulties The C value reported here for PI 665007 (C. lanatus culti- in scoring the pollen. Exine pattern formation in A. thali- var Sugar Baby) and the previously published value for C. ana involved several physiological processes (Ariizumi and vulgaris. Arumuganathan and Earle (1991b) are substantiated Toriyama, 2007). The ecological significance of the pollen per the genome sequence of C. lanatus (Guo et al., 2013). exine mutations described here, if any, is unknown. We The C values for C. amarus and C. mucosospermus are nota- propose the gene designation rp (rugulate pollen) for the bly higher than the C values of the other taxa examined. A mutant allele present in PI 482261. The genetic basis for the broader survey of additional plant materials may shed light mutant allele present in PI 482312 awaits determination. on the basis for, and the extent of, these differences. Arumuganathan and Earle (1991b) reported the DNA Citrullus naudinianus (as Acanthosicyos naudinianus) tol- content of Citrullus vulgaris (C. lanatus) as 0.88 to 0.90 pg per erates a saline subsoil, has wide ecological adaptation, 2C nuclei. This was similar to several other members of the and produces edible fruit that store well (Bosch, 2004). family including Cucumis melo L. (0.94–1.04), Cucumis sativus Given its native habitat, it is likely drought tolerant. Citrul- L. (0.76), and Cucurbita pepo L. (1.04–1.08), as reported by lus naudinianus has been recognized as a candidate for the these authors. However, nuclear DNA contents (C values) development of a high-yielding, dry-country crop for food for other Citrullus spp. have not been previously reported. and oil (Bosch, 2004). The extent to which C. naudinianus Our analysis revealed pg per 2C nuclei (±SD) values of 1.04 might play a role in the improvement of the cultivated (0.008), 1.03 (0.045), 0.82 (0.007), 0.87 (0.009), 0.80 (0.018), C. lanatus, or vice versa, remains to be determined. We 0.89 (0.006), and 0.87 (0.022) for C. amarus (PI 482261), were unable to locate reports in the literature on the C. mucosospermus (PI 306782), C. colocynthis (PI 652554), evaluation of this taxon for its agronomic or horticultural C. ecirrhosus (PI 673135), C. lanatus (PI 665007), C. naudini- traits, other than general observations on its adaptability anus (GRIF 14032), and C. rehmii (PI 670011), respectively. and the edibility of its fruit (which are sometimes bitter).

crop science, vol. 57, march–april 2017 www.crops.org 861 Fig. 6. (A) Storage root of a C. colocynthis (PI 652554) ´ C.

naudinianus (GRIF 14032) F1 hybrid. (B) Tendrils of GRIF 14032 (upper), PI 652554 (lower), and eir F1 hybrid (center). (C) Mature leaves of PI 652554 (left), GRIF 14032 (right), and their F1 hybrid (center).

or more deeply lobed than either of the parents. Hybrid Fig. 4. Close-up of pollen ornamentation of C. amarus (A) PI plants produced a storage root, rooted readily at the 482261 and (B) PI 482312. nodes, and exhibited an angular growth pattern of the The literature appears to be lacking in information on its stem—characteristics of C. naudinianus (Fig. 6). Recip- disease and/or insect resistance characteristics. rocal crosses (A. naudinianus ´ C. colocynthis) were also Hybridizations between C. colocynthis (PI 652554) successfully made. All hybrid plants that have produced and C. naudinianus (GRIF 14032) were attempted. Most male flowers to date (three) are sterile and produce only (75–95%/fruit) of the “seeds” that developed did not con- shrunken and/or partially developed pollen grains. tain an embryo and consisted of only an empty seed coat. Six attempts to cross C. lanatus cultivar Sugar Baby Approximately 10% of the apparently viable seed germi- with C. naudinianus (GRIF 14032) were unsuccessful. nated in vitro. The hybrid plants possessed morphological The lack of available male flowers on GRIF 14032 and its characteristics that were generally intermediate to those of failure to produce female flowers in 2015 precluded addi- the parents, including leaf size and shape and tendril shape tional attempts. Genotypic effects on crossability among (Fig. 6). The leaves of the hybrid plants were frequently as Citrullus spp. have been demonstrated (Levi et al., 2011), and the possibility of successful C. lanatus ´ C. naudinianus (or reciprocal) hybridizations cannot be totally discounted, assuming a suitable parent pair could be identified. Indeed, Shimotsuma (1963) reported on the cross-compatibility of C. naudinianus (A. naudinianus) with C. ecirrhosus, C. colocynthis, and C. vulgaris (C. lanatus), although no mention was made of the germinability of the seed or the viability of the progeny from those hybridizations. In our hands, Citrullus naudinianus was also successfully Fig. 5. Pollen of (A) PI 482312 (mutant) and (B) PI 482312 ´ PI hybridized with C. rehmii. Citrullus rehmii is an herbaceous 482309 (normal) F1, as observed via light microscopy.

862 www.crops.org crop science, vol. 57, march–april 2017 annual native to the Namib Desert. This monoecious spe- progeny can be obtained. To date, all C. rehmii ´ C. nau- cies forms a tap root and is readily recognized by the unique dinianus hybrids have been male sterile (Fig. 7). texture and mottling of its fruit (De Winter, 1990). We In summary, the palynological data support the close observed that the percentage (and number) of fully devel- relationship among all Citrullus spp. including C. nau- oped seed/fruit was higher in C. rehmii ´ C. naudinianus dinianus, a taxon morphologically distinct from all other crosses than in C. colocynthis ´ C. naudinianus crosses (data members of this genus (Fig. 8). Two genebank accessions not shown). Crosses of C. naudinianus ´ C. rehmii also of C. amarus with a mutated (rugulate) exine pattern were resulted in fully developed viable seed. De Winter (1990) identified. The C values for all taxa examined were deter- had previously noted that C. rehmii could be hybridized mined. Both C. colocynthis and C. rehmii were successfully with C. lanatus, C. colocynthis, and C. ecirrhosus and that hybridized with C. naudininanus, and the crosses produced hybrid progeny of all crosses were fertile and intermediate viable and (highly) vigorous (though male sterile) progeny. in character. As C. rehmii is more closely related to C. lanatus than is C. colocynthis (Chomicki and Renner, 2015), that Acknowledgments is, perhaps, not unexpected. Given that C. colocynthis and We gratefully acknowledge the technical support provided by C. rehmii can be hybridized with C. lanatus (De Winter, Mr. Chris Tatum, University of Georgia, Griffin, GA. Mention 1990; Levi et al., 2011), either of these taxa may have poten- of trade names or commercial products in this publication is tial as a means to facilitate the transfer of genetic material solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA; from C. naudinianus into C. lanatus, assuming fertile USDA is an equal opportunity provider and employer.

Fig. 7. Shrunken pollen grains typical of C. rehmii (PI 670011) ´ C. naudinianus (GRIF 14032) F1 hybrids (left) and those of C. colocynthis (PI 652554) ´ C. naudinianus (GRIF 14032) F1 hybrids (right).

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