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

Jinfeng Chen and Jeffrey W. Adelberg

Department of Horticulture, Clemson University, Clemson, SC 29634

Jack E. Staub

USDA­ARS, Department of Horticulture, University of Wisconsin, Madison 53706

Halina T. Skorupska

Department of Agronomy, Clemson University, Clemson, SC 29634

Billy B. Rhodes

Department of Horticulture, Clemson University, Clemson, SC 29634

Additional index words. amphidiploid, chromosome doubling, interspecific hybridization, Cucumis, organogenesis

Abstract. A synthetic amphidiploid was developed through organogenesis in tissue culture from an interspecific F1 hybrid between Cucumis sativus L. and C. hystrix Chakr. The results from flow cytometry indicated that, on average, 7.3% of the regenerants with unique morphology were chromosome-doubled F1 hybrids. The 2C DNA content of the original F1 hybrid was 1.17 pg, and the 4C DNA content was 2.35 pg. Frequency of chromosome doubling between the two genotypes used in this study varied considerably.

 

Interspecific hybridization is often attempted for as a means to gene introgression between taxa. These efforts should be specially encouraged in Cucumis crops since their limited diversity and poor resistance of pest (Staub et al., 1987). An interspecific cross was successfully made between Cucumis sativus and C. hystrix (Chen et al., 1997), and offered the possibility to introduce the desirable characters such as the tolerance to high temperature and low light intensity from C. hystrix to cultivated C. sativus (Chen, J. F., 1995­1998, personal observation). However, reciprocal crossing of those F1 plants to either parent and self-crossing indicated that the hybrid was both male- and female-sterile. The F1 hybrid has an odd number of chromosomes 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 sterility has been reported in many crops (Lu and Bridgen, 1997). Reasons for the sterility may vary, but in most cases, sterility is associated with meiotic

abnormalities. Many experiments indicated that the fertility problem could be overcome by doubling the number of chromosomes. If we could double the 19 chromosomes to 38 chromosomes, each chromosome has a homologous partner for pairing in meiosis, viable pollen grains and egg cells would then likely be produced, and thus, fertility would be restored.

Chemical treatment, in vitro callus culture (somaclonal variation), and meiotic induction are major ways to double chromosome (Sybenga, 1992). Colchicine is one of the most common used chemical agent to double chromosome. However, chromosome doubling of C. sativus x C. hystrix through various colchicine treatments has been tried previously without success (Chen et al., 1997). Spontaneous chromosome doubling in tissue culture through organogenesis of diploid plants has been reported in potato (Karp et al., 1990), Daucus carota (Mok et al., 1976), Kallstroemia pubescens (Sengupta et al., 1987), Cucumis melo (Ezura et al., 1992; Adelberg, 1993), and C. sativus. Here we report a chromosome doubling through tissue culture for the development of a new amphidiploid in Cucumis.

This work was conducted while J.F. Chen was supported by BARD grant US-2809-96R. Flow cytometry was performed with the able assistance of J. Whitesides, Clemson Univ. Multiuser Flow Cytometry Laboratory. J.F. Chen is the corresponding author.

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Materials and methods

Cucumis hystrix and commercial C. sativus from North and South China were used in this experiment. Seeds of C. hystrix were collected in South China (Chen et al., 1994). North Chinese cucumber 'Beijing Jietou' was originally obtained from C.Z. Qi, of the Vegetable Research Institute, Chinese Agricultural Academy of Sciences, Beijing, and the seeds were increased at the University of Wisconsin­Madison in 1997. The South Chinese cucumber 'Er Zhaozi' was obtained from Z.B. Gong, of the Chengdu Seed Company no. 2, Chengdu city, Sichuan of China. C. hystrix was transplanted in field between June and October, and C. sativus was grown in greenhouse between July and November 1997 at Clemson University. The interspecific crossing was made between September and October 1997 using the procedure described by Chen et al. (1997).

The hybrid fruit were harvested 30 days after pollination. The surface of the fruit was sterilized with 70% ethanol and seeds were extracted. Embryos were aseptically excised under a stereoscopic binocular microscope, and cultured on MS (Murashige and Skoog, 1962) hormone free medium containing 30 g·L­1 sucrose, 8 to 12 g·L­1 agar at pH 5.6. Six embryos were placed in glass jars (5 ¥ 8 cm) containing 40 mL of MS medium. The embryos were incubated at 24 oC under fluorescent lighting an 16 h light (100 µmol·m­2·s­1 ): 8 h dark photoperiod for 60 days till plantlet formed. After 90 days, the edges of the leaf tissues of the plantlets were cut off and sliced into 0.5 cm explants. The explants were placed abaxial side up on a MS medium containing 2.4-D 1.0 mg·L­1, BA 0.5 mg·L­1, ABA 0.4 mg·L­1, and AgNO3 10 mg·L­1. After 4 weeks incubation under lights, they were transferred to MS hormone-free medium. After 3 to 4 weeks, the

shoots were excised for callus, and transferred to fresh MS medium with 1.0 mg·L­1 IBA to form roots. Once small roots had developed, the plantlets were gently removed from the agar, rinsed with water, and placed into moist artificial soil. The plants were acclimatized under a plastic doom for 1 week and then cultured in the greenhouse.

Newly expanded leaf tissues of primary regenerants from tissue culture were cut into small pieces in MgSO4 buffer. After washing with 1¥ PBS buffer, the suspension of nuclei was filtered through one layer of miracloth with pore size 15 (m. The nuclei were adjusted to the concentration of 2 ¥ 105 mL before stain with propidium iodide (PI) in a solution containing RNase. Nuclei were analyzed with the flow cytometer Epics 751 (Coulter Corporation). Excitation of PI was provided by the 488 nm line (400 mW) of an argon laser (model I-90, Coherent) and the red fluorescence emitted by PI was collected through a 635 nm band pass filter. Chicken red blood cells (CRBC) with DNA content of 1.88 pg were used as an internal standard. Each sample was analyzed both with and without the standard to detect ploidy level. The data were presented as histograms, and

Figure 1. Interspecific hybrid F1 plants regenerated from tissue culture. (left) Chromosome doubled F1 plant. (right) Normal sterile F1 plant.

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Figure 2. Histogram of number of nuclei per channel, a function of relative fluorescence intensity resulting from flow cytometry of nuclei stained with propidium iodide.

chromosome-doubled plants were observed among the C. sativus genotypes. In the hybrid where the 'Beijing Jietou' used as seed parent, 40 chromosome- doubled plants were obtained from 761 regenerants (5.3%), while in the case of 'Er Zhaozi', 22 chromosome doubled plants were obtained from 93 regenerants (23.7%). This observation is in agreement with our previous experiment with C. melo (Adelberg and Chen, 1998).

Allopolyploid have been very important in the evolution of many plant families (Simmonds, 1976), and several important crops are allopolyploid. In Cucurbitaceae, amphidiploidy was reported from C. maxima x C. moschata (Pearson et al., 1951), and C. anguria x C. dipsaceus (Yadava et al., 1986). There were no successful efforts on cucumber and melon, the two most commercially important Cucumis spp. The amphidiploid developed in our experiment created a new genetic material and a potential agricultural product that has a new combination of genomes, which has not previously existed. With this success, the enhancement of the current cucumber germplasm by backcrossing is possible. The characterization, fertility, and chromosome behavior in meiosis of this amphidiploid will be carried out in our follow-up experiments.

the nuclear DNA content was calculated by (position of plant nuclear peak/position of CRBC nuclear peak) ¥ 1.88.

Results and discussion

A total of 854 plants were obtained through organogenesis in our experiment. Among those, there was a group of 62 plants (7.3%) that distinguished themselves from the rest with unique characters in morphology, such as the curve on the edge of the leaves, and short stout internodes (Fig. 1). From the 62 plants, 10 were sampled to flow cytometer analysis. The results from flow cytometry indicated those plants were chromosome-doubled F1 hybrid (Fig. 2). The peak position of G0/G1 nuclei was used to calculate 2C nuclear DNA content. Based on the DNA content of internal standard CRBC 1.88 pg, the DNA content of the original F1 hybrid was 1.17 pg, while that in the chromosome-doubled F1 hybrid was 2.35 pg.

Considerable differences in the percentage of

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and plant regeneration from diverse cultivars of cucumber (Cucumis sativus L. ). Plant Cell Tissue Organ Cult. 12:67­74.

Lu, Ch.S. and M.P. Bridgen. 1997. Chromosome doubling and fertility study of Alstroemeria aurea x A. caryophyllaea. Euphytica 94:75­81.

Mok, M.C., W.H. Gabelman, and F. Skoog. 1976. Carotenoid synthesis in tissue cultures of Daucus carota L. J. Amer. Soc. Hort. Sci. 101:442­449.

Murashige, T. and F.A. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473­497.

Pearson, O.H., R. Hopp, and G.W. Bohn. 1951. Notes on species crosses in Cucurbita. Proc. Amer. Soc. Hort. Sci. 57:310.

Sengupta, J., G.C. Mitra, and A.K. Sharma. 1987. Study of chromosomes in two callus lines and in regenerated plants of Kallstroemia pubescens (G. Don) Dandy. Cytologia 52:767­770.

Simmonds, N.W. 1976. Evolution of crop plants. Longman, London.

Staub, J.E., L. Fredrich, and T.L. Marty. 1987. Electrophoretic variation in crosscompatible wild diploid species of Cucumis. Can. J. Bot. 65:792­798.

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 amphidiploids. Nucleus 29:58­62.

Literature cited

Adelberg, J.W., B.B. Rhodes, and H.T. Skorupska. 1993. Generating tetraploid melons in tissue culture. Acta Hort. 336:373­380.

Adelberg, J.W. and J.F. Chen. 1998. Environmental and genetic factors affect frequency of tetraploid regenerants from immature cotyledon of melon. HortScience 33:533 (abstr.).

Chen, J.F. and J.E. Staub. 1997. Attempts at colchicine doubling of an interspecific hybrid of Cucumis sativus L. x C. hystrix Chakr. by direct embryo culture. Cucurbit Genet. Coop. Rpt. 20:24­26.

Chen, J.F., J.E. Staub, Y. Tashiro, S. Isshiki, and S. Miyazaki. 1997. Successful interspecific hybridization between Cucumis sativus L. and C. hystrix Chakr. Euphytica 96:413­419.

Chen, J.F., S.L. Zhang, and X.G. Zhang, 1994. The xishuangbanna gourd, a traditionally cultivated plant of the Hani people, Xishuangbanna, Yunnan, China. Cucurbit Genet. Coop. Rpt. 17:18­20.

Ezura, H., H. Amagai, K. Yoshioka, and K. Oosawa. 1992. Highly frequent appearance of tetraploidy in regenerated plants, a universal phenomenon in tissue culture of melon (Cucumis melo L.). Plant Sci. 85:209­213.

Karp, A. 1990. p. 379­399. In: Y.P.S. Bajaj (ed.). Biotechnology in agriculture and forestry. vol. 11. Somaclonal variation in crop plants. I. Springer-Verlag, New York.

Kim, S., J. Chang, H. Cha, and K. Lee. 1988. Callus growth

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