, Volume 189, Issue 3, pp 433–443 | Cite as

Creation of novel interspecific-interploid Hylocereus hybrids (Cactaceae) via embryo rescue

  • Aroldo Cisneros
  • Reinerio Benega Garcia
  • Noemi Tel-ZurEmail author


In this study we developed a technique for rescuing embryos at a very early developmental stage following interspecific-interploid crosses between Hylocereus sp. Controlled hand pollinations were performed between the tetraploid H. megalanthus (Vaup.) Bauer as the female parent and either diploid H. monacanthus (Lem.) Britton et Rose or H. undatus (Haw.) Britton et Rose as the male parent. The fertilized ovules were excised from ovaries 10 or 30 days after pollination (DAP). Pollinated ovules containing the funiculi and placental tissue and immature embryos were placed on half-strength basal Murashige and Skoog (MS) medium containing 680 μM glutamine, 0.54 μM α-naphthaleneacetic acid, and 0.45 μM thidiazuron and supplemented with 0.00, 0.09, 0.17 or 0.26 M sucrose concentrations. The best ovule response was recorded at 30 DAP, and the most prolific callus formation was observed at 10 DAP. Callus formation was observed in most of the treatments using whole ovules but not in the isolated immature embryos. The calli were mucilaginous or compact, transparent and friable, but they did not form embryogenic structures. Embryo development was significantly affected by the sucrose concentration, and the best results were obtained with 0.17 M sucrose. More than 70 % of the obtained hybrids were successfully hardened off and transplanted in soil where they grew normally. Ploidy level analyses of 77 putative hybrids exposed diploid, triploid, tetraploid, and higher than tetraploid levels. Among those hybrids studied, 22 progenies were randomly chosen for amplified fragment length polymorphism analysis, and all were identified as genuine hybrids. The technology described here is an additional stage in the breeding program for Hylocereus species resulting in novel, interspecific hybrids obtained using the embryo rescue technique.


Amplified fragment length polymorphism (AFLP) Flow cytometry α-Naphthaleneacetic acid Ovule culture Thidiazuron Vine cacti 



Amplified fragment length polymorphism


Ben-Gurion University of the Negev


Days after pollination


Immature embryos


Murashige and Skoog


α-Naphthaleneacetic acid


Nuclei isolation buffer




Whole ovules



The authors gratefully acknowledge the partial support of this work by a Zin Fellowship from the Kreitman School for Advanced Graduate Studies and by a doctoral fellowship granted to A. Cisneros by the Albert Katz International School for Desert Studies (BGU). We also thank Prof. G. Grafi for instructive discussions and valuable comments on the manuscript. We want to extend our gratitude to Mr. J. Mouyal and to the late Dr. B. Schneider for their valuable assistance.


  1. Ahmad F, Slinkard AE (2003) Limitations to bridge-species facilitated alien gene transfers in chickpea: pre-fertilization events. J Genet Breed 57:69–74Google Scholar
  2. Barthlott W, Hunt DR (1993) Cactaceae. In: Kubitzki K (ed) The families and the genera of vascular plants, vol 2. Springer-Verlag, Berlin, pp 161–196Google Scholar
  3. Chen Y, Kenaschuk EO, Procunier JD (1998) Plant regeneration from anther culture in Canadian cultivars of flax (Linum usitatissimum L.). Euphytica 102:183–189CrossRefGoogle Scholar
  4. Cisneros A, Tel-Zur N (2010) Embryo rescue and plant regeneration following interspecific crosses in the genus Hylocereus (Cactaceae). Euphytica 174:73–82CrossRefGoogle Scholar
  5. Cisneros A, Benega GR, Tel-Zur N (2011) Ovule morphology, embryogenesis and seed development in three Hylocereus species (Cactaceae). Flora 206(12):1076–1084CrossRefGoogle Scholar
  6. Clarke HJ, Wilson JG, Kuo I, Lülsdorf MM, Mallikarjuna N, Kuo J, Siddique KHM (2006) Embryo rescue and plant regeneration in vitro of selfed chickpea (Cicer arietinum L.) and its wild annual relatives. Plant Cell Tissue Organ 85:197–204CrossRefGoogle Scholar
  7. Dag A, Mizrahi Y (2005) Effect of pollination method on fruit set and fruit characteristics in the vine cactus Selenicereus megalanthus (‘‘yellow pitaya’’). J Hortic Sci Biotechnol 80:618–622Google Scholar
  8. Doležel J, Binarová P, Lucretti S (1989) Analysis of nuclear DNA content in plant cells by flow cytometry. Biol Plant 31:113–120CrossRefGoogle Scholar
  9. Fratini R, Ruiz ML (2006) Interspecific hybridization in the genus Lens applying in vitro embryo rescue. Euphytica 150:271–280CrossRefGoogle Scholar
  10. Honjo R, Ikeda T, Taji A, Uragami A (2009) Effects of various sucrose concentration and ancymidol on shoot elongation and water status of Asparagus in vitro. Acta Hortic 829:299–303Google Scholar
  11. Hunt D (2006) The new cactus lexicon. DH Books, Milborne PortGoogle Scholar
  12. Ikeda T, Iwaya-Inoue M, Fukuyama T, Nonami H (2000) Trehalose changes hydraulic conductance of tissue-cultured soybean embryos. Plant Biotechnol 17:119–125CrossRefGoogle Scholar
  13. Joshi M, Sujatha K, Hazra S (2008) Effect of TDZ and 2,4-D on peanut somatic embryogenesis and in vitro bud development. Plant Cell Tissue Organan Cult 94:85–90CrossRefGoogle Scholar
  14. Kaushal P, Malaviya DR, Roy AK, Kumar B, Tiwari A (2005) Trifolium alexandrinum × T. resupinatum—interspecific hybrids developed through embryo rescue. Plant Cell Tissue Organ 83:37–144CrossRefGoogle Scholar
  15. Leng P, Yamamura H (2006) Fruit set and embryo rescue in crosses using parthenocarpic ‘Mopanshi’ persimmon. Sci Hortic Amst 107:332–336CrossRefGoogle Scholar
  16. Li H, Devaux P (2003) High frequency regeneration of barley doubled haploid plants from isolated microspore culture. Plant Sci 164:379–386CrossRefGoogle Scholar
  17. Lichtenzveig J, Abbo S, Nerd A, Tel-Zur N, Mizhrahi Y (2000) Cytology and mating systems in the climbing cacti Hylocereus and Selenicereus. Am J Bot 87:1058–1065PubMedCrossRefGoogle Scholar
  18. Liu GS, Qi DM, Zhang WD, Liu JS, Li HJ (2004) Highly efficient embryo germination in vitro shortens the breeding cycle in Leymus chinensis. In Vitro Cell Dev Plant 40:321–324CrossRefGoogle Scholar
  19. Merten S (2003) A review of Hylocereus production in the United States. J PACD 5:98–105Google Scholar
  20. Mizrahi Y, Nerd A (1999) Climbing and columnar cacti. In: Janick J (ed) New arid land fruit crops. Perspective in new crops and new uses. ASHS Press, Alexandria, pp 358–366Google Scholar
  21. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissues cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  22. Nuñez-Palenius HG, Klee HJ, Cantliffe DJ (2006) Embryo-rescue culture of the ‘Galia’ muskmelon (Cucumis melo L. var. reticulatus Ser.) male parental line. Plant Cell Tissue Organ 85:345–352CrossRefGoogle Scholar
  23. Okawai Y, Ikeda T, Taji A (2009) Growth and water status of tissue-cultured strawberry plants as affected by salt stress and different sugar concentration in media. Acta Hortic 829:417–421Google Scholar
  24. Reed SM (2005) Embryo rescue. In: Trigiano RN, Gray DJ (eds) Plant development and biotechnology. CRC Press, Boca Raton, pp 235–240Google Scholar
  25. Sage TL, Strumas F, Cole WW, Barret SCH (1999) Differential ovule development following self and cross pollination: the basis of self-sterility in Narcissus triandrus (Amaryllidaceae). Am J Bot 86:855–870PubMedCrossRefGoogle Scholar
  26. Tel-Zur N, Abbo S, Bar-Zvi D, Mizrahi Y (2003) Chromosome doubling in vine cacti hybrids. J Hered 94:329–333PubMedCrossRefGoogle Scholar
  27. Tel-Zur N, Abbo S, Bar-Zvi D, Mizhrahi Y (2004) Genetic relationships among Hylocereus and Selenicereus vine cacti (Cactaceae): evidence from hybridization and cytological studies. Ann Bot 94:527–534PubMedCrossRefGoogle Scholar
  28. Tel-Zur N, Abbo S, Mizrahi Y (2005) Cytogenetics of semi-fertile triploid and aneuploid intergeneric vine cacti hybrids. J Hered 6:24–131Google Scholar
  29. Tikhenko N, Rutten T, Voylokov A, Houben A (2008) Analysis of hybrid lethality in F1 wheat–rye hybrid embryos. Euphytica 159:367–375CrossRefGoogle Scholar
  30. Tonguç M, Griffiths PD (2004) Development of black rot resistant interspecific hybrids between Brassica oleracea L. cultivars and Brassica accession A 19182, using embryo rescue. Euphytica 136:313–318CrossRefGoogle Scholar
  31. Valiente-Banuet A, Gally RS, Arizmendi MC, Casas A (2007) Pollination biology of the hemiepiphytic cactus Hylocereus undatus in the Tehuacán Valley, Mexico. J Arid Environ 68:1–8CrossRefGoogle Scholar
  32. Wang J, Huang L, Bao M, Liu G (2009) Production of interspecific hybrids between Lilium longiflorum and L. lophophorum var linearifolium via ovule culture at early stage. Euphytica 167:45–55CrossRefGoogle Scholar
  33. Weiss J, Nerd A, Mizrahi Y (1994) Flowering behavior and pollination requirements in climbing cacti with fruit crop potential. HortScience 29:1487–1492Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Aroldo Cisneros
    • 1
  • Reinerio Benega Garcia
    • 1
  • Noemi Tel-Zur
    • 1
    Email author
  1. 1.French Associates Institute for Agriculture and Biotechnology of Drylands, The Jacob Blaustein Institutes for Desert Research (BIDR)Ben-Gurion University of the Negev (BGU)Sede-BoqerIsrael

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