Art15.indd Journal of Applied Botany and Food Quality 82, 83 - 89 (2008) Palacký University in Olomouc, Faculty of Science, Department of Botany, Olomouc, Czech Republic Embryo rescue of cucumber (Cucumis sativus), muskmelon (C. melo) and some wild Cucumis species (C. anguria, C. zeyheri, and C. metuliferus) Skálová D.*, Navrátilová B., Lebeda A. (Received September 19, 2007) Summary Cucumis sativus is one of the most economically important crops of the Cucurbitaceae. Recent cucumber cultivars are susceptible to some serious diseases and pests, including downy mildew, powdery mildew, nematodes, and spider mites. Sources of resistance to these pathogens and pests were identified in some accessions of wild Cucumis spe- cies. One possible way of introducing these resistances into cucumber germplasm is interspecific hybridization. However, C. sativus is sexually incompatible with nearly all other Cucumis species, because of substantially different chromosome numbers, n = 7 in C. sativus versus n = 12 in C. melo and most wild Cucumis species. Overcoming this obstacle can be accomplished through the use of embryo rescue and/or ovule culture. Results of experiments using these methods, especially of embryo rescue of cucumber and selected wild Cucumis species after intra- and interspecific hybridization, are summarized in this paper. Various culture media and selected genotypes were tested in our experiments. Successful regeneration of mature embryos of some Cucumis spp. was observed on all types of media, and callus or sporadic plant formation from immature embryos and seeds occurred on media with coconut water and gibberellic acid. Abbreviations BA – benzyladenin; IBA – indole-3-butyric acid; MS – Murashige and Skoog medium (1962). Introduction The Cucurbitaceae family consist of approximately 118 genera and 825 species almost equally divided between the New and Old World, with an emphasis on tropical regions. One of the most important genera is Cucumis (JEFFREY, 2001), which has two subgenera. Subgenus Cucumis considered to be of Asiatic origin (n = 7), and subgenus Melo Miller of African origin (n = 12). Subgenus Cucumis includes Cucumis sativus L. and C. hystrix Chakrav. Although Cucumis hystrix resembles C. sativus morphologically and biochemically, its base chromosome number (n = 12) is the same as C. melo, which is included with wild Cucumis species in subgenus Melo (LEBEDA et al., 2007). Cucumber cultivars are susceptible to some serious diseases and pests. Genes for resistance to several pathogens and pests that are not known to occur in cucumber have been found in accessions of wild Cucumis species (LEPPIK, 1966; CHEN and ADELBERG, 2000; CHEN et al., 2003; LEBEDA et al., 2007). Accessions of Cucumis anguria L., C. metuliferus E. Mey ex Naud. and C. zeyheri Sond. have high levels of resistance to root-knot nematode (den NIJS and CUSTERS, 1990; WEHNER et al., 1990). Very high levels of resistance to Tetranychus urticae were found in some accessions of C. zeyheri (LEBEDA, 1996). Cucumis metuliferus also displays resistance to Squash mosaic virus (SqMV) and Water- melon mosaic virus (WMV) (MCCARTHY et al., 2001). Cucumis melo line MR-1 and some other accessions have some resistance to cu- cumber downy mildew (Pseudoperonospora cubensis) (LEBEDA et al., 1996, 1999, 2007). One possible method of introducing these resis- tance genes to cucumber cultivars is via interspecific hybridization (LEBEDA et al., 2007). To this end, it is necessary to use unconventional techniques, including various methods of in vitro culture, due to hybridization barriers and embryo abortion in the early globular stage of embryo development (ONDŘEJ et al., 2001). One approach to overcome this crossability barrier, which is primarily caused by different chromosome numbers, is to use embryo culture, a method of embryo rescue (CHEN and ADELBERG, 2000; LEBEDA et al., 1996; SKÁLOVÁ et al., 2004). The composition of the culture media has the major influence on the successful regeneration of isolated and culti- vated embryos (SKÁLOVÁ et al., 2004). Successful embryo rescue techniques for the genus Cucumis is con- sidered important for future efforts toward interspecific hybridization (LEBEDA et al., 2007; SKÁLOVÁ et al., 2004). The main purpose of this paper is to report our recent results in the research of Cucumis spp. embryo rescue. Selected wild species and new types of media were tested for supporting regeneration, especially of immature embryos. Materials and methods Plant material Selected Cucumis species were used for embryo rescue experiments (Tab. 1). The plant material originated from the vegetable germplasm collection of the Research Institute of Crop Production (Prague), Gene Bank Department, Olomouc Workplace, Czech Republic (Web site: , EVIGEZ) and the USDA-ARS North Central Regional Plant Introduction Station, Iowa State University, Ames, IA. The plants were cultivated in a glasshouse at the Department of Botany, Palacký University in Olomouc, Czech Republic (25°C / 15°C day/night; with daily watering and weekly fertilization by Kristalon Start (Hydro Agri, Rotterdam, Netherlands), 10 ml of fertilizer (19:6:20 N:P:K) /10 l of H 2 O), in organic substrate (Florcom SP, BBcom s.r.o, Letohrad, Czech Republic), and without pest control. Embryo culture Fruits of selected Cucumis species (Tab. 1) were harvested after three days or one, two and three weeks, and in the case of Cucumis sativus and C. anguria, also after six weeks post hand self-pollination. For cross-pollination, C. sativus was used as a maternal parent and other Cucumis species (Tab. 1) as paternal parents, and the fruits were harvested two weeks after hand cross-pollination. They were surface sterilized with 70% ethanol (fruits were rinsed and then flamed) and seeds (S) were dissected. Immature seeds (originating from the 3 day fruits after self-pollination and from 2 week fruits after cross-polli- nation) were cultured intact. Zygotic embryos (E) were excised from the seeds of 1, 2, 3 and 6 week fruits while viewed using a stereoscopic binocular microscope under sterile conditions. Embryos or seeds were cultivated on various media (Tab. 2) in test tubes for six weeks at 25oC in the dark (5 ml of medium per tube). One of these was OK-medium, which acted as a control, and the others, ON-, CW-, and GA-medium, were supplemented with components which are thought to have a positive influence on embryogenesis: casein hydrolysate, coconut water, and gibberellic acid. MS-medium (MURASHIGE and SKOOG, 1962) was used as a basis for macroelements, 84 Skálová D., Navrátilová B., Lebeda A. microelements and vitamins. Other components were supplemented depending on the type of medium (ascorbic acid, casein hydrolysate, α- glutamin, IBA, BA, sucrose, and agar), and the pH was adjusted to 5.8 before autoclaving. Solutions with coconut water and gibberellic acid were filter-sterilized (Steritope, Millipore, Billerica, MA) and added to the autoclaved part of the medium. After germination, the embryos or seeds were transferred to a culture room (22±2oC and 16h-day/8h-night photoperiod, 32 - 36 µmol · m-2 · s-1) and then the type and frequency (%) of regeneration of embryos of various ages and seeds on the media were evaluated and compared. We describe three types of regeneration in our experiments – 1. callus formation (C; no organogenesis was observed from isolated embryos and seeds; only callus without stems, roots or leaves); 2. abnormal plant formation (AP; indirect organogenesis from isolated embryos and seeds was observed, such as the formation of stems, roots or leaves on callus; vitrificated plants also occurred – callus formation on developed stems, roots or leaves) and 3. normal plant formation (P; direct organogenesis from isolated embryos and seeds was observed, with the development of entire plants) (Fig. 1). The experiments were repeated. The frequency of regeneration was expressed in percent (average percentages were presented). In graphs, Y-error bars were used to represent the standard deviation of means. Results and discussion Embryo rescue after hand self-pollination in Cucumis species The most important aspects to consider regarding embryo development in cultures are the age of embryos (seeds), the genotype, a suitable Fig. 1: Various types of regeneration from isolated embryos or seeds of Cucumis species (A – normal plant formation; B – abnormal plant formation; C – callus formation). Tab. 1: Cucumis species used for embryo rescue. Cucumis species Abbreviation Accession number C. sativus (line SM-6514) CS CZ 09H3900768 C. anguria var. longipes CA PI 249896 C. zeyheri CZ PI 364473 C. melo (line MR-1) CM1 PI 124111 C. melo var. Charentais CM2 PI 261778 C. metuliferus CME PI 292190 Tab. 2: Composition of culture media. Culture Basic Other components medium medium OK MS 20mg/l ascorbic acid, 0.01mg/l IBA, 0.01mg/l BA, 20mg/l sucrose, 8g/l agar (control medium) ON MS 1g/l casein hydrolysate, 0.01mg/l IBA, 0.01mg/l BA, 20g/l sucrose, 6g/l agar CW MS 5% coconut water, 200mg/l α-glutamin, 0.01mg/l IBA, 0.01mg/l BA, 60g/l sucrose, 6g/l agar GA MS 0.3mg/l gibberellic acid, 0.01mg/l IBA, 0.01mg/l BA, 20g/l sucrose, 8g/l agar Explanatory notes: MS = Murashige and Skoog (1962); OK, ON, CW, GA = abbreviations for MS-media supplemented with different type of additions sup- porting embryogenesis; IBA = indole-3-butyric acid; BA = benzyladenin. A C B Embryo rescue of cucumber 85 Tab. 3: Frequency (%) of successful regeneration of Cucumis genotypes on various culture media. Cucumis spp. CS CA CZ CM1 CM2 CME Age Medium 3 days OK 22 35 6 6 0 20 ON 61 7 8 11 5 30 CW 72 27 12 6 10 30 GA 72 – 4 17 0 23 1 week OK 56 10 10 60 40 10 ON 50 20 10 25 0 10 CW 50 20 20 60 20 25 GA 60 - 60 80 40 28 2 weeks OK 20 50 55 89 30 0 ON 78 20 40 89 20 5 CW 78 50 70 100 23 20 GA 40 40 40 100 30 15 3 weeks OK 100 90 80 50 43 100 ON 86 80 90 45 100 100 CW 86 80 75 55 100 50 GA – – 60 40 83 75 6 weeks OK 100 100 – – – – ON 100 100 – – – – CW 100 100 – – – – GA – – – – – – Explanatory notes: CS, CA, CZ, CM1, CM2, CME – selected genotypes of Cucumis (Tab. 1); OK, ON, CW, GA – various types of media (Tab. 2); – - variant was not tested. culture medium, the concentration of sucrose and growth regulators and the influence of light (ONDŘEJ and NAVRÁTILOVÁ, 1999). Our recent experiments focused on the selection of the most suitable geno- types and media for embryo rescue in the genus Cucumis. Embryos and seeds isolated from fruits in different time periods after hand self-pollination were cultivated on various types of media, and the type and frequency (%) of regeneration was compared (Tabs. 3 and 4; Fig. 4). For cucumber (C. sativus), the in vitro response of one genotype, SM 6514, was studied. We observed normal development of mature embryos (6 week) to entire plants on the OK- and ON-media (100% regeneration; Fig. 1). On the other hand, coconut water in CW-medium often supported indirect regeneration with callus development on mature isolated embryos (Fig. 2). A similar effect of coconut water was noted by ONDŘEJ et al. (2000). Callus formation and abnormal plant formation were noted during the development of the 1, 2 and 3 week embryos (especially on CW-medium; Fig. 1). The 3 day seeds developed to plants on ON-medium (61%), and callus development and abnormal plant development were recorded on GA-medium (72%). Regeneration of 3 and 6 week embryos of C. anguria resembled results from C. sativus. The most suitable media for normal plant develop- ment were OK (90%; 100%) and ON (80%; 100%), and abnormal plant formation was again noted on the CW-medium. Immature embryos and seeds were regenerated to entire plants on OK-medium (Fig. 2). For 3 week embryos of C. zeyheri, OK- and ON-media (80%; 90%) were suitable, but for the 2 week embryos it was CW-medium (70%). ONDŘEJ et al. (2000) also found these media to be suitable for C. zeyheri. NUÑEZ-PALENIUS et al. (2006) reported a beneficial effect of coconut water on embryo resuce of Cucumis melo, and GORALSKI and PRZYWARA (1998) observed its positive effects on regeneration of immature embryos generally. Regeneration of immature 1 week embryos and the 3 day seeds was the most efficient on GA-medium (60%). The positive influence of GA 3 (but at a different concentration) was recorded in previous experiments (ONDŘEJ et al., 2002). The most important species for interspecific hybridization of cucumber is considered to be muskmelon (C. melo (line MR-1)). We observed 100% regeneration of its 2 week embryos on CW- and GA-media; however, the type of regeneration was only callus or abnormal plants (Fig. 1). Normal plants were obtained from embryos cultivated on OK- and ON-media (89%). ONDŘEJ et al. (2000) reported that the most suitable medium for C. melo (line MR-1) was generally the OK- medium. Gibberellic acid (GA-medium) was suitable for the 3 day seeds and 1 week embryos (60%; 80%). Embryos and seeds of a second genotype of muskmelon, C. melo var. Charentais, regenerated less frequently than did line MR-1. The most suitable media for the 3 week embroys were ON- and CW- (only callus formation; 100%). Gibberellic acid and coconut water were suitable for all immature embryos (2 and 1 week) and seeds (3 day) (callus formation predominated) (Fig. 2). The most suitable medium for C. metuliferus embryos was OK- medium, but lower reduced concentrations of sucrose and growth regulators (ONDŘEJ et al., 2000). The 3 week embryos of C. metuliferus expressed the best results on OK- and ON-media (100%). No re- generation of plants was observed on media with coconut water and gibberellic acid. Nevertheless, these media were the best for the cultivation of the 2 week, 1 week and the 3 day seeds (mostly callus formation, rarely abnormal plant production was observed). It is clear from these experiments that each species responds differently to these in vitro culture conditions. Our results confirm the fact that the successful cultivation (= obtaining the entire plants through direct organogenesis) of immature embryos and seeds is more complicated than is regeneration from mature embryos and seeds (PREŤOVÁ, 1995). 86 Skálová D., Navrátilová B., Lebeda A. Fig. 2: Regeneration of embryos isolated at different time after a hand self-pollination from various Cucumis genotypes (A – 1 week C. sativus embryo on CW- medium; B – 2 week C. anguria embryo on OK-medium; C – 2 week C. melo (line MR-1) embryo on CW-medium; D – 2 week C. melo (var. Charentais) embryo on GA-medium). Tab. 4: Types of regeneration of Cucumis genotypes on various culture media. Cucumis species CS CA CZ CM1 CM2 CME Age Medium 3 days OK P, AP P P, AP C 0 C ON P AP AP C C C CW AP AP P C C C GA C, AP – P C 0 C 1 week OK P P P, AP P C C ON P AP AP AP C, AP C CW AP AP P C, AP C, AP C GA AP – P AP C C 2 weeks OK P P P P C C ON P P P P C C CW AP AP P, AP AP AP AP, C GA AP – P, AP AP AP AP, C 3 weeks OK P P P P C P ON P P P P AP P CW P, AP P, AP P, AP AP AP C GA – – – AP C C 6 weeks OK P P – – – – ON P P – – – – CW P, AP P, AP – – – – GA – – – – – – Explanatory notes: CS, CA, CZ, CM1, CM2, CME – selected genotypes of Cucumis (Tab. 1); OK, ON, CW, GA – various types of media (Tab. 2); C – callus formation, AP – abnormal plant formation, P – normal plant formation; – - variant was not tested. A C B D Embryo rescue of cucumber 87 small roots or a shoot meristem, however callus formation pre- dominated. Two embryos developed into entire flowering plants and their hybrid origin was confirmed using isozyme analyses and flow cytometry. Only callus formation was observed in our experiments with classic hybridization of cucumber with other Cucumis species. With respect to the evaluation of the success of cross-pollination, the highest frequency of fruits was obtained with C. metuliferus as an paternal parent (74%; Fig. 5). On the other hand, the worst partner for cucumber was C. anguria (19 % successful pollination; Fig. 5). Only callus formation was observed after hybridization with C. melo (line MR-1) or C. metuliferus. The frequency of regeneration was very low (1.5% for C. melo and 1.7% for C. metuliferus). ONDŘEJ et al. (2001) had similar results with interspecific hybridization in the genus Cucumis. As suitable media for obtained hybrid embryos were Embryo rescue after hand cross-pollination in Cucumis species Cucumis sativus as a maternal parent and C. melo and selected wild Cucumis species (Tab. 1) as paternal parents were used in interspecific hybridization. Embryos and seeds isolated from fruits after two weeks of hand cross-pollination were cultivated on several types of media. Because of the different embryos responses on various media that we observed from previous hand self-pollination, we focused on frequency of successful pollination (= formation of fruits; Fig. 5) and on the type and frequency of regeneration after hand cross-pollination with various interspecific partners (Tab. 5). The regeneration of embryos after interspecific hybridization of Cucumis has been infrequently accomplished. In total, the regeneration of seven embryos from interspecific crosses of C. sativus × C. melo was observed (LEBEDA et al., 1996, 1999). Five embryos developed Fig. 3: Regeneration of embryos isolated two weeks after a hand cross-pollination of C. sativus with other Cucumis species (A – fruit developing from C. sativus × C. melo; B – fruit developing from C. sativus × C. zeyheri; C, D – callus formation on embryos from C. sativus × C. melo on GA-medium). Tab. 5: Results of interspecific hybridization of C. sativus with Cucumis species. Partner No. of No. of No. of No. of No. and type of Successful pollinations obtained fruits isolated seeds isolated embryos regener. media CM1 27 13 260 260 8 (C) OK (3C), CW (2C), GA (3C) CM2 18 8 120 160 0 - CA 11 2 80 0 0 - CZ 16 7 280 0 0 - CME 8 6 240 0 4 (C) CW (2C), GA (2C) Explanatory notes: Partner for IH – partner for interspecific hybridization with C. sativus; CM1, CM2, CA, CZ, CME – selected genotypes of Cucumis (Tab. 1); OK, ON, CW, GA – various types of media (Tab. 2); C – callus formation. A C B D 88 Skálová D., Navrátilová B., Lebeda A. 15 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 OK ON CW GA M e d iu m Rege neration (%) CME CM2 CM1 CZ CA CS Fig. 4: Frequency (%) of regeneration 3 day seeds of tested Cucumis species (CS, CA, CZ, CM1, CM2, CME – selected genotypes of Cucumis species (Tab. 1); OK, ON, CW, GA – various types of media (Tab. 2); Y-error bars represent standard deviations). 0 10 20 30 40 50 60 70 80 90 100 CM1 CM2 CA CZ CME Partner for IH R eg en er a ti o n (% ) Fig. 5: Frequency (%) of successful hand cross-pollination (formation of fruits) C. sativus with Cucumis species (CS, CA, CZ, CM1, CM2, CME – selected genotypes of Cucumis species (Tab. 1); IH partner – partner for interspecific hybridization with C. sativus; Y-error bars represent standard deviations). appeared CW- and GA-. There was observed a positive influence of coconut water and gibberellic acid again on immature embryos (GORALSKI and PRZYWARA, 1998; ONDŘEJ et al., 2002; NUÑEZ- PALENIUS et al., 2006). However, no direct or indirect organogenesis was noted from these hybridizations. It is necessary to use other strategies to obtain hybrids from crossing C. sativus with other Cucumis species. For example the polyploidization of cucumber maternal plant was used and entire plants were developed after cross C. sativus × C. melo (SKÁLOVÁ et al., 2006). Conclusions From this study, it is evident that methods of embryo rescue in the genus Cucumis were successful. Direct organogenesis was observed in selected Cucumis species cultivated on media supporting embryo- genesis. An important hybridization partner for cucumber, C. melo line MR-1, regenerated with 10 % frequency but only formed callus. Among wild Cucumis species, immature C. metuliferus embryos (3 day), showed the highest frequency of regeneration (26%). These two genotypes were characterized by callus formation from inter- specific hybridization with cucumber. The addition of coconut water (CW-medium) and gibberellic acid (GA-medium) to culture media had positive effects on the regeneration of immature embryos (27%; 29%). These same media supported callus formation, the only degree of regeneration, in the case of interspecific hybrids between C. sativus and C. melo or C. metuliferus. Future work will concentrate on opti- mizing methods to increase the frequency of regeneration and make it possible to recover entire plants from the interspecific hybridization of cucumber with muskmelon. Acknowledgements Authors thanks to Dr. M.P. Widrlechner (USDA-ARS NCRPIS, Iowa State University, Ames Iowa, USA) and Dr. H.S. 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North Carolina State University, Raleigh (NC, USA), 2006, 51-59. WEHNER, T.C., CADE, R.M., LOCY, R.D., 1990: Cell, tissue and organ culture techniques for genetic improvement of Cucurbits. In: Bates, D. et al. (eds.), Biology and Utilization of the Cucurbitaceae, Cornell University Press, Ithaca, New York, 367-381. Address of the authors: RNDr. Dagmar Skálová, Ph.D.*, RNDr. Božena Navrátilová, Ph.D. and Prof. Ing. Aleš Lebeda, DrSc.; Department of Botany, Faculty of Science, Palacký University in Olomouc, Šlechtitelu 11, 783 71 Olomouc – Holice, Czech Republic. *corresponding author (dagmar.skalova@upol.cz)