CT. A synthetic amphidiploid was developed through organogenesis in tissue culturefrom an interspecific F1hybrid betweenCucumis sativus L. andC. hystrix Chakr. The resultsfrom flow cytometry indicated that, on average, 7.3% of the regenerants with unique morphology were chromosome-doubled F1hybrids. The 2C DNA content of the original F1hybrid was1.17 pg, and the 4C DNA content was 2.35 pg. Frequency of chromosome doubling between thetwo genotypes used in this study varied considerably

Adelberg, Jeffrey

Chen, Jinfeng

Rhodes, Billy

Skorupska, Halina

Staub, Jack E

English

Clemson University: TigerPrints

Clemson UniversityTigerPrintsPublications Plant and Environmental Sciences1998A New Synthetic Amphidiploid in Cucumis from aC. sativus x C. hystrix F1 Interspecific HybridJeffrey AdelbergClemson University, jadlbrg@clemson.eduJinfeng ChenClemson UniversityJack E. StaubUSDAHalina SkorupskaClemson UniversityBilly RhodesClemson UniversityFollow this and additional works at: https://tigerprints.clemson.edu/ag_pubsPart of the Agriculture CommonsThis is brought to you for free and open access by the Plant and Environmental Sciences at TigerPrints. It has been accepted for inclusion inPublications by an authorized administrator of TigerPrints. For more information, please contact kokeefe@clemson.edu.Recommended CitationPlease use publisher's recommended citation.A New Synthetic Amphidiploid in Cucumis from aC. sativus x C. hystrix F1 Interspecific HybridJinfeng Chen and Jeffrey W. AdelbergDepartment of Horticulture, Clemson University, Clemson, SC 29634Jack E. StaubUSDA—ARS, Department of Horticulture, University of Wisconsin, Madison 53706Halina T. SkorupskaDepartment of Agronomy, Clemson University, Clemson, SC 29634Billy B. RhodesDepartment of Horticulture, Clemson University, Clemson, SC 29634ADDITIONAL INDEX WORDS. amphidiploid, chromosome doubling, interspecific hybridization,Cucumis, organogenesisABSTRACT. A synthetic amphidiploid was developed through organogenesis in tissue culturefrom an interspecific F 1 hybrid between Cucumis sativus L. and C. hystrix Chakr. The resultsfrom flow cytometry indicated that, on average, 7.3% of the regenerants with unique morphol-ogy were chromosome-doubled F1 hybrids. The 2C DNA content of the original F1 hybrid was1.17 pg, and the 4C DNA content was 2.35 pg. Frequency of chromosome doubling between thetwo genotypes used in this study varied considerably.Jnterspecific hybridization is often attempted for as a means to gene introgres-sion between taxa. These efforts should bespecially encouraged in Cucurnis crops since theirlimited diversity and poor resistance of pest (Staubet al., 1987). An interspecific cross was successfullymade between Cucwnissativus and C. htstrix (Chen etal., 1997), and offered the possibility to introduce thedesirable characters such as the tolerance to hightemperature and low light intensity from C. hystrix tocultivated C. sativus (Chen, J . F., 1995-1998, personalobservation). However, reciprocal crossing of thoseF1 plants to either parent and self-crossing indi -cated that the hybrid was both male- and female-sterile. The F1 hybrid has an odd number of chro-mosomes in diploid complement of somatic cells(2n = 19 chromosomes including n = 7 from C.sativus and n = 12 from C. hystrix).The problem of interspecific hybrid sterilityhas been reported in many crops (Lu and Bridgen,1997). Reasons for the sterility may vary, but inmost cases, sterility is associated with meioticThis work was conducted while J.F. Chen was supported by BARDgrant US-2809-96R. Flow cytornetry was performed with theableassis-ta nce of J . Whitesides, Clemson Univ. Multiuser Flow Cytometry Labo-r1 tory. J. F. Chen is the corresponding author.abnormalities. Many experiments indicated thatthe fertility problem could be overcome by dou-bling the number of chromosomes. If we coulddouble the 19 chromosomes to 38 chromosomes,each chromosome has a homologous partner forpairing in meiosis, viable pollen grains and eggcells would then likely be produced, and thus,fertility would be restored.Chemical treatment, in vitro callus culture(somaclonal variation), and meiotic induction aremajor ways to double chromosome (Sybenga,1992). Coichicine is one of the most common usedchemical agent to double chromosome. However,chromosome doubling of C. sutivus x C. hystrixthrough various colchicine treatments has beentried previously without success (Chen et al., 1997).Spontaneous chromosome doubling in tissue cul-ture through organogenesis of diploid plants hasbeen reported in potato (Karp et al., 1990), Daucuscarota (Mok et al., 1976), Kallstroemia pubesct'ns(Sengupta et al., 1987), Cucuinis melo (Ezura et al.,1992; Adelberg, 1993), and C. sativus. Here wereport a chromosome doubling through tissueculture for the development of a new amphidip-bid in Cucuinis.336	 Cii i ni I	 I ii '98*hkAldIMaterials and methodsCucuinis hystrix and commercial C. sativusfrom North and South China were used in thisexperiment. Seeds of C. hystrix were collected inSouth China (Chen et al., 1994). North Chinesecucumber 'Beijing Jietou' was originally obtainedfrom C.Z. Qi, of the Vegetable Research Institute,Chinese Agricultural Academy of Sciences, Beijing,and the seeds were increased at the University ofWisconsin—Madison in 1997. The South Chinesecucumber 'Er Zhaozi' was obtained from Z.B.Gong, of the Chengdu Seed Company no. 2,Chengdu city, Sichuan of China. C. hystrix wastransplanted in field between June and October,and C. scitivus was grown in greenhouse betweenJuly and November 1997 at Clemson University.The interspecific crossing was made between Sep-tember and October 1997 using the proceduredescribed by Chen et al. (1997).The hybrid fruit were harvested 30 days afterpollination. The surface of the fruit was sterilizedwith 70% ethanol and seeds were extracted. Em-bryos were aseptically excised under a stereo-scopic binocular microscope, and cultured on MS(Murashige and Skoog, 1962) hormone free me-dium containing 30 g . L 1 sucrose, 8 to 12 g. L 1 agarat pH 5.6. Six embryos were placed in glass jars (5x 8 cm) containing 40 mL of MS medium. Theembryos were incubated at24 C under fluorescentlighting an 16 h light (100pmol•m-2 s 1 ): 8 h dark pho-toperiod for 60 days tillplantlet formed. After 90days, the edges of the leaftissues of the plantlets werecut off and sliced into 0.5cm explants. The explantswere placed abaxial side upon a MS medium contain-ing 2.4-D 1.0 mg-L-1 , BA 0.5mg.L 1 , ABA 0.4 mg•L 1 , andAgNO3 10 mg-L- 1 . After 4weeks incubation underlights, they were transferredto MS hormone-free me-dium. After 3 to 4 weeks, theCuLRIIII CF\I '98shoots were excised for callus, and transferred tofresh MS medium with 1.0 mg-L- 1 IBA to formroots. Once small roots had developed, the plant-lets were gently removed from the agar, rinsedwith water, and placed into moist artificial soil.The plants were acclimatized under a plastic doomfor I week and then cultured in the greenhouse.Newly expanded leaf tissues of primary re-generants from tissue culture were cut into smallpieces in MgSO4 buffer. After washing with lxPBS buffer, the suspension of nuclei was filteredthrough one layer of miracloth with pore size 15(m. The nuclei were adjusted to the concentrationof 2 x 105 mL before stain with propidium iodide(PT) in a solution containing RNase. Nuclei wereanalyzed with the flow cytometer Epics 751(Coulter Corporation). Excitation of PT was pro-vided by the 488 nm line (400 mW) of an argonlaser (model 1-90, Coherent) and the red fluores-cence emitted by PT was collected through a 635nmband pass filter. Chicken red blood cells (CRBC)with DNA content of 1.88 pg were used as aninternal standard. Each sample was analyzed bothwith and without the standard to detect ploidylevel. The data were presented as histograms, andFigure 1. Interspecific hybrid F 1 plants regeneratedfrom tissue culture. (left) Chromosome doubled F1plant. (right) Normal sterile F 1 plant.-3371250020001500>.0CwCrLI.1000500— 0) N LU U) - C) N U) U) - C) N U) C') — 0) N LI) C') ,- 0) N LI) C') ,- 0) N LI) C') - 0).— C'J CO	 U) (0 N- 0) co 0) 0) ,- C'J (%J C')	 II) (I) (0 N 0) 0) 00 ,- C\J C')c, CM C\J C\J C\i C'J NJChannel numberFigure 2. Histogram of number of nuclei per channel,a function of relative fluorescence intensity resultingfrom flow cytometry of nuclei stained withpropidium iodide.the nuclear DNA content was calculated by (posi-tion of plant nuclear peak/position of CRBCnuclear peak) x 1.88.Results and discussionA total of 854 plants were obtained throughorganogenesis in our experiment. Among those,there was a group of 62 plants (7.3%) that distin-guished themselves from the rest with uniquecharacters in morphology, such as the curve on theedge of the leaves, and short stout internodes (Fig.1). From the 62 plants, 10 were sampled to flowcytometer analysis. The results from flow cytom-etry indicated those plants were chromosome-doubled F1 hybrid (Fig. 2). The peak position ofG0/G 1 nuclei was used to calculate 2C nuclearDNA content. Based on the DNA content of inter-nal standard CRBC 1.88 pg, the DNA content ofthe original F1 hybrid was 1.17 pg, while that in thechromosome-doubled F1 hybrid was 2.35 pg.Considerable differences in the percentage ofchromosome-doubled plants were observedamong the C. sativus genotypes. In the hybridwhere the 'Beijing Jietou' used as seed parent, 40chromosome- doubled plants were obtained from761 regenerants (5.3%), while in the case of 'ErZhaozi', 22 chromosome doubled plants wereobtained from 93 regenerants (23.7%). This obser-vation is in agreement with our previous experi-ment with C. melo (Adelberg and Chen, 1998).Allopolyploid have been very important inthe evolution of many plant families (Simmonds,1976), and several important crops are allopolyp-bid. In Cucurbitaceae, amphidiploidy was reportedfrom C. maxima xC. moschata (Pearson etal., 1951), andC. ançliria x C. dipsaceus (Yadava et al., 1986). Therewere no successful efforts on cucumber and melon,the two most commercially important Cucwnis spp.The amphidiploid developed in our experiment cre-ated a new genetic material and a potential agricul-tural product that has a new combination of ge-nomes, which has not previously existed. Withthis success, the enhancement of the current cu-cumber germplasm by backcrossing is possible.The characterization, fertility, and chromosomebehavior in meiosis of this amphidiploid will be33S	 (	 IuIcarried out in our follow-up experiments.Literature citedAdelberg, J.W., B.B. Rhodes, and H.T. Skorupska. 1993.Generating tetraploid melons in tissue culture. ActaHort. 336:373-380.Adelberg, J.W. and J.F. Chen. 1998. Environmental andgenetic factors affect frequency of tetraploid regener-ants from immature cotyledon of melon. HortScience33:533 (abstr.).Chen, J.F. and J.E. Staub. 1997. Attempts at coichicinedoubling of an interspecific hybrid of Cucumis sat ivusL. x C. Iiyst rix Chakr. by direct embryo culture. Cucur-bit Genet. Coop. Rpt. 20:24-26.Chen,J.F.,J.E. Staub, Y. Tashiro, S. Isshiki, and S. Miyazaki.1997. Successful interspecific hybridization betweenCucumis sativus L. and C. hystrix Chakr. Euphytica96:413-419.Chen, J.F., S.L. Zhang, and X.G. Zhang, 1994. Thexishuangbanna gourd, a traditionally cultivated plantof the Hani people, Xishuangbanna, Yunnan, China.Cucurbit Genet. Coop. Rpt. 17:18-20.Ezura, H., H. Amagai, K. Yoshioka, and K. Oosawa. 1992.Highly frequent appearance of tetraploidy in regener-ated plants, a universal phenomenon in tissue cultureof melon (Cucumis melo L.). Plant Sci. 85:209-213.Karp, A. 1990. p.379-399. In: Y.P.S. Bajaj (ed.). Biotechnol-ogy in agriculture and forestry. vol. 11. Somaclonalvariation in crop plants. I. Springer-Verlag, New York.Kim, S., J . Chang, H. Cha, and K. Lee. 1988. Callus growthand plant regeneration from diverse cultivars of cu-cumber (Cucuinis safivus L. ). Plant Cell Tissue OrganCult. 12:67-74.Lu, Ch.S. and M.P. Bridgen. 1997. Chromosome doublingand fertility study of Alsf roernerin auren x A. cariophy!!aea.Euphytica 94:75-81.Mok, M.C., W.H. Gabelman, and F. Skoog. 1976. Caro-tenoid synthesis in tissue cultures of Daucus Ca rota L. J.Amer. Soc. Hort. Sci. 101:442-449.Murashige, T. and F.A. Skoog. 1962. A revised mediumfor rapid growth and bioassays with tobacco tissuecultures. Physiol. Plant 15:473-497.Pearson, O.H., R. Hopp, and G.W. Bohn. 1951. Notes onspecies crosses in Cucurbifa. Proc. Amer. Soc. Hon. Sci.57:310.Sengupta,J., G.C. Mitra, and A.K. Sharma. 1987. Study ofchromosomes in two callus lines and in regeneratedplants of Kalistroernia pubescens (C. Don) Dandy.Cytologia 52:767-770.Simmonds, N.W. 1976. Evolution of crop plants. Long-man, London.Staub, J.E., L. Fredrich, and T.L. Marty. 1987. Electro-phoretic variation in crosscompatible wild diploidspecies of Cucurnis. Can. J . Bot. 65:792-798.Sybenga. 1992. Cytogenetics in plant breeding. Springer-Verlag, Berlin.Yadava, K. S., A.K. Singh, R.P. Roy, and U.C. Jha. 1986.Cytogenetics in Cucumis L. VI, synthetic amphidip-bids. Nucleus 29:58-62.hw^^^Fj

A New Synthetic Amphidiploid in Cucumis from a C. sativus x C. hystrix F1 Interspecific Hybrid

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A New Synthetic Amphidiploid in Cucumis from a C. sativus x C. hystrix F1 Interspecific Hybrid

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