doi: 10.15389/agrobiology.2017.1.143eng

UDC 635.342:631.527.5:581.4


convar. capitata var. alba DC) BASED ON DOUBLED HAPLOIDS

V.F. Pivovarov, L.L. Bondareva, N.A. Shmykova, D.V. Shumilina,
A.I. Mineikina

All-Russian Research Institute of Breeding and Seed Production of Vegetable Crops, Federal Agency of Scientific Organizations, 14, ul. Selektsionnaya, pos. VNIISSOK, Odintsovskii Region, Moscow Province, 143080 Russia,

Received June 7, 2016


Presently, cabbage breeding is mainly focused on F1 hybrids necessitating constant parental lines to be obtained. Doubled haploid (DH) technology based on isolated microspore in vitro culture is widely used to produce pure lines of brassica crops. This method allows us to rapidly develop homozygous lines, in contrast to time-consuming traditional breeding for heterosis in cross-pollinating crops which takes 7 to 10 years for annuals and 14 to 20 years for biennial plants. One of the objectives of DH technology is to provide the all possible number of doubled haploid plants that allows more fully encompass the spectrum of genetic recombination, including the recessive locus. The aim of our study was to evaluate economically important traits in white cabbage (Brassica oleracea L. convar. capitata var. alba DC) constant doubled haploid lines of late ripening and to improve the technology for producing DH based F1 hybrids. Eleven breeding lines of late ripening cabbage were used to obtain doubled haploid lines from microspore in vitro culture. Of the obtained lines, twelve doubled haploid genotypes were selected for further use based on evaluation of ploidy and combining ability. Seed progeny was reproduced by hybridization of regenerated plants in a climatic chamber (2014-2015). We used the schemes of creating self-incompatible lines and two-line-based hybrids. In the field trials (Moscow region, 2014-2015), the doubled haploids and their hybrid combinations were compared to the standard (Severyanka F1) for the main valuable characteristics (i.e. the content of dry matter, nitrates, and vitamin C). The field resistance to Fusarium wilt, alternariosis, and pest damage were determined at cabbagehead technical maturity. The resistance to clubroot was assessed under artificial infection. There was a direct relationship of the average number of chromosomes to the number of chloroplasts in the stomata guard cells and the length of guard cells. The frequency of spontaneous doubling of the chromosomes numbers varied from 50.0 % to 87.5 % in different geotypes. A total of 11 to 73 % produced lines were high self-incompatible. Their geitonogamic pollination in the topcrosses resulted in 42 hybrid combinations. The model of F1 hybrid most fully responding to consumer market demands was developed. Ten promising hybrid combinations which matched the model parameters in two-year field testing were recommended for variety testing. Hybrids were characterized by uniformity, high biochemical quality, the resistance to major diseases and pests and the yield of 104.60±8.27 t/ha. The dry matter content reached to 10.5 %, the sugar content was about 4.21-5.10 %, and ascorbic acid level ranged from 21.12 to 38.70 mg%. Both the highest level of ascorbic acid (92.0 mg%) and the smallest nitrate accumulation (33 mg/kg) were characteristic of one hybrid combination.

Keywords: white cabbage, Brassica oleracea L., doubled haploid lines, heterosis F1 hybrids, in vitro isolated microspore culture, self-incompatibility, DH-technology, spontaneous doubling, ploidy.


Full article (Rus)

Full text (Eng)



  1. Krasheninnik N.V. Gavrish, 2010, 2: 16-19 (in Russ.).
  2. Nakanishi T. An effective time for CO2 gas treatment in overcoming self-incompatible cabbage in Brassica. Plant Cell Physiol., 1973, 14: 837-873.
  3. Pearson O.H. Breeding plants of the cabbage group. Bull. Calif. Agric. Exp. Stn., 1932, 532: 3-22.
  4. Kryuchkov A.B. Izvestiya TSKHA, 1972, 1: 124-131 (in Russ.).
  5. Pivovarov V.F., Startsev V.I. Kapusta, ee vidyiraznovidnosti (raznoobrazieisposobyvyrashchivaniya) [Cabbage: multiplicity of species, cultivars and their cultivation]. Moscow, 2006 (in Russ.).
  6. Bondareva L.L. Nauchnoe obosnovanie i razrabotka sistemy metodov selektsii i semenovodstva kapustnykh kul'tur. Avtoreferat doktorskoi dissertatsii [Fundamentals and developed techniques for Brassica breeding and seed production. DSci Thesis]. Moscow, 2009 (in Russ.).
  7. Zhang W., Fu Q., Dai X.G., Bao M.Z. The culture of isolated microspores of ornamental kale (Brassica oleracea var. acephala) and the importance of genotype to embryo regeneration. Scientia Horticulturae, 2008, 117(1): 69-72 CrossRef
  8. Dunwell J.M. Haploids in flowering plants: origins and exploitation. Plant Biotechnol. J., 2010, 8: 377-424 CrossRef
  9. Winarto B., Teixeira da Silva J.A. Microspore culture protocol for Indonesian Brassica oleracea. Plant Cell Tiss. Organ Cult., 2011, 107: 305-315 CrossRef
  10. Kim J., Lee S.S. Identification of monogenic dominant male sterility and its suppressor gene from an induced mutation using a broccoli (Brassica oleracea var. italica) microspore culture. Hortic. Environ. Biotechnol., 2012, 53(3): 237-241 CrossRef
  11. Yuan S.X., Su Y.B., Liu Y.V., Fang Z.Y., Yang L.M., Zhuang M., Zhang Y.Y., Sun P.T. Effects of pH, MES, arabinogalactan-proteins on microspore cultures in white cabbage. Plant Cell Tiss. Organ Cult., 2012, 110: 69-76 CrossRef
  12. Gu H., Zhao Z., Sheng X., Yu H., Wang J. Efficient doubled haploid production in microspore culture of loose-curd cauliflower (Brassica oleracea var. botrytis). Euphytica, 2014, 195: 467-475 CrossRef
  13. Shmykova N.A., Shumilina D.V., Suprunova T.P. Vavilovskii zhurnal genetiki i selektsii, 2015, 19(1): 111-120 (in Russ.).
  14. Devaux P., Pickering R. Haploids in the improvement of Poaceae. In: Biotechnology in Agriculture and Forestry. V. 56. C.E. Palmer, W.A. Keller, K.J. Kasha (eds.). Springer, Berlin-Heidelberg, 2005: 215-242 CrossRef
  15. Kasha K.J., Maluszynski M. Production of doubled haploids in crop plants. An introduction. In: Doubled haploid production in crop plants: a manual. M. Maluszynski, K.J. Kasha, B.P. Forster, I. Szarejko (eds). Springer Netherlands, 2003: 1-4 CrossRef
  16. Shmykova N.A., Suprunova T.P., Pivovarov V.F. Biotechnologies and molecular methods in vegetable crop breeding (to 95th Anniversary of VNIISSOK). Agricultural Biology, 2015, 50(5): 561-570 CrossRef (in Engl.).
  17. Monakhos S.G. Kartofel' i ovoshchi, 2014, 9: 34-35 (in Russ.).
  18. Reznikova S.A. Tsitologiya i fiziologiya razvivayushchegosya pyl'nika [Cytology and physiology on developing anther]. Moscow, 1984 (in Russ.).
  19. Sari N., Abak K., Pitrat M. Comparison of ploidy level screening methods in watermelon: Citrullus lanatus (Thunb.) Matsum. and Nakai. Scientia Horticulturae, 1999, 82: 265-277.
  20. Qin X., Rotino G.L. Chloroplast number in guard cells as ploidy indicator of in vitro-grown androgenic pepper plantlets. Plant Cell Tiss. Organ Cult., 1995, 41(2): 145-149.
  21. Hamaoka Y., Fujita Y., Iwai S. Nimber of chloroplasts in haploids and diploids produced via anther culture in Brassica campestris. Plant Tissue Culture Letters, 1991, 8(2): 67-72.
  22. Galbraith D.W., Lambert G.M., Macas J., Dolezel J. Analysis of nuclear DNA content and ploidy in higher plants. Current Protocols in Cytometry, 2001, 2(7.6): 7.6.1-7.6.227 CrossRef
  23. Hcini K., Walker D.J., Gonzales E., Frayssinet N., Correal E., Bou
    zid S. Estimation of nuclear DNA content and determination of ploidy level in Tunisian populations of Atriplex halimus L. by flow cytometry. In: Flow cytometry — recent perspectives. I. Schmid (ed.). InTech, Rijeka, Croatia, 2012: 103-118 CrossRef
  24. Winarto B., Mattjik N.A., Purwito A., Marwoto B. Ploidy screening of anthurium (Anthurium andreanum Linden ex André) regenerants derived from anther culture. Scientia Horticulturae, 2010, 127(1): 86-90 CrossRef
  25. Friedt W., Zarhloul K. Haploids in the improvements of Crucifers. In: Biotechnology in Agriculture and Forestry. V. 56. C.E. Palmer, W.A. Keller, K.J. Kasha (eds.). Springer, Berlin-Heidelberg, 2005: 191-213 CrossRef
  26. Smykalova I. Vetrovcova M., Klima M., Machackova M., Griga M. Efficiency of microspore culture for doubled haploid production in the breeding project «Czech Winter Rape». Czech J. Genet. Plant Breed., 2006, 42: 58-71.
  27. Ferrie A.M.R., Caswell K.L. Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tiss. Organ Cult., 2011, 104: 301-309 CrossRef
  28. Takahata Y., Takahashi Y., Tsuwamoto R. Microspore culture and doubled haploid. In: Biotechnology of Crucifers. S.K. Gupta (ed.). Springer, 2013: 45-62 CrossRef
  29. Lichter R. Induction of haploid plants from isolated pollen of Brassica napus. Z. Pflanzenphysiol., 1982, 105: 427-434.
  30. Gamborg O.L., Miller R.A., Ojima K. Nutrients requirements of suspension cultures of soybean root cells. Exp. Cell Res., 1968, 50: 151-158 CrossRef
  31. Murashige T., Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 1962, 15: 473-497 CrossRef
  32. Shumilina D.V., Shmykova N.A., Bondareva L.L., Suprunova T.P. Izvestiya RAN, 2015, 4: 368-375 CrossRef (in Russ.).
  33. Pukhal'skii V.A., Solov'ev A.A., Badaeva E.D., Yurtsev V.N. Praktikum po tsitologii i tsitogenetike rastenii [Plant cytology and cytogenetics — practical works]. Moscow, 2007 (in Russ.).
  34. Monakhos S.G., Nguen M.L., Bezbozhnaya A.V., Monakhos G.F. A relationship between ploidy level and the number of chloroplasts in stomatalguard cells indiploid and amphidiploid Brassica species. Agricultural Biology, 2014, 5: 44-54 CrossRef (in Engl.).
  35. Monakhos G.F., Monakhos S.G. Kapusta pekinskaya Brassica rapa L. Em. Metzg. ssp. pekinensis (Lour.) Hanelt. Biologicheskie osobennosti, genetika, selektsiya i semenovodstvo [Brassica rapa L. Em. Metzg. ssp. pekinensis (Lour.) Hanelt. — biology, genetics, breeding and seed production]. Moscow, 2009 (in Russ.).
  36. Dospekhov B.A. Metodika opytnogo dela [Experimental techniques]. Moscow, 1985 (in Russ.).
  37. Sapozhnikova E.V., Dorofeeva L.S. Konservnaya i ovoshchesushil'naya promyshlennost', 1966, 5: 29 (in Russ.).
  38. Kvasnikov B.V., Antonov Yu.P. Zashchita rastenii, 1972, 9: 16 (in Russ.).
  39. Hansen M. Production of homogeneous varieties in Brassica. Modern plant Breeding. Proc. NJF Congress: Agriculture and Society. Norway, Aas (28 Jun-1 Jul 1999). Nordisk Jordbrugsforskning (Denmark), 1999, 81(3): 148-153.
  40. Rudolf K., Bohanec B., Hansen M. Microspore culture of white cabbage, Brassica oleracea var. capitata L.: Genetic improvement of nonresponsive cultivars and effect of genome doubling agents. Plant Breeding, 1999, 118: 237-241 CrossRef
  41. Baidina A.V., Monakhos S.G. Selektsiya kapusty na baze udvoennykh gaploidov. Kartofel'iovoshchi, 2015, 11: 39-40 (in Russ.).
  42. Dias S.J.C. Protocol for broccoli microspore culture. In: Doubled haploid production in crop plants: a manual. M. Maluszynski, K.J. Kasha, B.P. Forster, I. Szarejko (eds). Springer Netherlands, 2003: 195-204.