Article | 06. 2014 Vol. 32, Issue. 3
Microspore-derived Embryo Formation and Morphological Changes during the Isolated Microspore Culture of Radish (Raphanus sativus L.)



Nature Resource Institute, Mokpo National University1
Department of Horticultural Science, Mokpo National University2
Department of Bioresource Engineering, Sejong University3




2014.06. 382:389


PDF XML




Raphanus sativus L. cv. Taebaek, a efficiently microspore-derived embryo (MDE)-forming cultivar, and ‘Chungwoon’, a non-MDE-forming cultivar were selected as donor plants for isolated microspore culture. Radish flower bud of 2.0 (small, S), 4.0 (medium, M), and 6.0 (large, L) ± 0.5 mm in length were isolated to determine the temporal relationship between flower bud size and MED yield. Anatomical observations revealed no difference in the structure of the flower buds between the two cultivars. In both cultivars, the stigmas were much longer than the floral leaf in M-sized flower buds. The MDE yields for ‘Taebaek’ per petri dish were 6.6 and 1.3 for M- and L-sized of flower buds, respectively, but MDE formation was not induced in the S flower buds. On the other hand, ‘Chungwoon’ failed to form MDEs in all flower buds. The microspore density of ‘Taebaek’ was 1.3 times more than that of ‘Chungwoon’ for M sized flower buds. Of the M-sized buds from ‘Taebaek’ and ‘Chungwoon’, 92.1 and 81.6%, respectively, were in the late uninucleate microspore stage, which is characterized by the highest frequency of MDE formation. Anatomical observations of MDE formation revealed that the microspores were able to divide to form a primordium from which cell division took place continuously in the ‘Teabeak’ cultivar. However, the microspores of ‘Chungwoon’ failed to progress beyond the primodium stage, resulting in lack of MDE formation. By contrast, after the formation of the primordium, various developmental stages of embyos from microspore were observed in the ‘Taebaek’ cultivar. These results can be used to determine MDE forming potentials of radish cultivars.



1. Biddington, N.L. 1992. The influence of ethylene in plant tissue culture. Plant Growth Regulat. 11:173-187.  

2. Binarova, P., G. Hause, V. Genklova, J.H.G. Bordewener, and M.M.L. Campagne. 1997. A short severe heat shock is required to induce embryogenesis in late bicellular pollen of Brassica napus L. Sexual Plant Reproduction 10:200-208.  

3. Burnett, L., S. Yarow, and B. Huang. 1992. Embryogenesis and plant regeneration from isolated microspores of Brassica rapa L. ssp. oleifera. Plant Cell Rep. 11:215-218.  

4. Cho, M.S. and F.J. Zapata. 1990. Plant regeneration from isolated microspore of Indica rice. Plant Cell Physiol. 31:881-885.  

5. Chun, C. and H. Na. 2011a. Microspore-derived embryo formation in response to cold pretreatment, washing medium, and medium composition of radish (Raphanus sativus L.). Kor. J. Hort. Sci. Technol. 29:494-499.  

6. Chun, C., H. Park, and H. Na. 2011b. Microspore-derived embryo formation in radish (Raphanus sativus L.) according the nutritional and environmental conditions. Hort. Environ. Biotechnol. 52: 530-535.  

7. Chuong, P.V., C. Deslauriers, L.S. Kott, and W.D. Beversdorf. 1988. Effects of donor genotype and bud sampling on microspore culture of Brassica napus. Canadian J. Bot. 66:1653-1657.  

8. Cloutier, C., M Cappadocia, and B.S. Landry. 1995. Study of microspore-culture responsiveness in oilseed rape (Brassica napus L.) by comparative mapping of a F2 population and two microspore-derived populations. Theoretical Appl. Genet. 91:841-847.  

9. Curtis, I.S. 2007. Radish, p. 381-389. In: E.C.V. Pua and M.R. Davey (des.). Biotechnology in agriculture and forestry. Springer, Dordrecht.  

10. Curtis, I.S. 2011. Genetic engineering of radish: Current achievements and future goals. Plant Cell Rep. 30:733-744.  

11. da Silva Dias, J.C. 1999. Effect of activated charcoal on Brassica oleracea microspore culture embryogenesis. Euphytica 108:65-69.  

12. Forster, B.P., E. Heberle-Bors, K.J. Kasha, and A. Touraev. 2007. The resurgence of haploids in higher plants. Trends Plant Sci. 12:368-375.  

13. Gallie, D.R. and T.E. Young. The ethylene biosynthetic and perception machinery is differentially expressed during endosperm and embryo development in maize. Mol. Gen. Genomics 271: 267-281.  

14. Gamborg, O.L., R.A. Miller, and K. Ojima. 1968. Nutrient requirements of suspension culture of soybean root cells. Exp. Cell Res. 50:151-158.  

15. Gu, H.H., P. Hagberg, and W.J. Zhou. 2004. Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.). Plant Growth Regulat. 42:137-143.  

16. Hansen, M. and K. Svinnset. 1993. Microspore culture of swede (Brassica napus ssp. rapifera) and the effects of fresh and conditioned media. Plant Cell Rep. 12:496-500.  

17. Heberle-Bors, E. 1989. Isolated pollen culture in tobacco: Plant reproductive development in a nutshell. Sexual Plant Reproduction 2:1-10.   

18. Lichter, R. 1982. Induction of haploid plants from isolated pollen of Brassica napus Z. Pflanzenphysiol. 105:427-434.   

19. Polsoni, L., L.S. Kott, and W.D. Beversdorf. 1988. Large-scale microspore culture technique for mutation-selection studies in Brassica napus. Canadian J. Bot. 66:1681-1685.  

20. Raina, S.K. and S.T. Irfan. 1998. High-frequency embryogenesis and plantlet regeneration from isolated microspores of Indica rice. Plant Cell Rep. 17:957-962.  

21. Seguí-Simarro, J.M. and F. Nuez. 2008. How microspores transform into haploid embryos: Changes associated with embryogenesis induction and microspore-derived embryogenesis. Physiol. Plant. 134:1-12.  

22. Senratna, T., L. Kott, W.D. Beversdorf, and B.D. Mckersie. 1991. Desiccation of microspore derived embryos of oilseed rape (Brassica napus L.). Plant Cell Rep. 10:342-344.   

23. Smykal, P. 2000. Pollen embryogenesis-The stress mediated switch from gametophytic to sporophytic development. Current status and future prospects. Biol. Plant. 43:481-489.  

24. Takahata, Y., D.C.W. Brown, and W.A. Keller. 1991. Effect of donor plant age and inflorescence age on microspore culture of Brassica napus L. Euphytica 58:51-55.  

25. Takahata, Y., H. Komatsu, and N. Kaizuma. 1996. Microspore culture of radish (Raphanus sativus L.): Influence of genotype and culture conditions on embryogenesis. Plant Cell Rep. 16:163-166.  

26. Takahashi, Y., S. Yokoi, and Y. Takahata. 2011. Improvement of microspore culture method for multiple samples in Brassica. Breeding Sci. 61:96-98.  

27. Tarnowski, B.I., F.G. Spinale, and J.H. Nicholson. 1991. DAPI as a useful stain for nuclear quantitation. Biotechnol. Histochem. 66:297-302.  

28. Telmer, C.A., D.H. Simmonds, and W. Newcomb. 1992. Determination of developmental stage to obtain high frequencies of embryogenic microspores in Brassica napus. Physiol. Plant. 84:417-423.  

29. Touraev, A., A. Ilham, O. Vicente, and E. Heberle-Bors. 1996a. Stress-induced microspore embryogenesis in tobacco: An optimized system for molecular studies. Plant Cell Rep. 15:561-565.  

30. Touraev, A., A. Indrianto, I. Wratschko, O. Vicente, and E. Heberle- Bos. 1996b. Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperature. Sexual Plant Reproduction 9:209-215.  

31. Touraev, A., M. Pfosser, O. Vicente, and E. Heberle-Bors. 1996c. Stress as the major signal controlling the developmental fate of tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis. Planta 200:144-152.  

32. Touraev, A., M. Pfosser, and E. Heberle-Bors. 2001. The microspore: A haploid multipurpose cell. Advances Botanical Res. 35:53-109.  

33. Weber, S., F. Unker, and W. Friedt. 2005. Improved doubled haploid production protocol for Brassica napus using microsproe colchicine treatment in vitro and ploidy determination by flow cytometry. Plant Breeding 124:511-513.  

34. Wu, L.M., Y.M. Wei, and Y.L. Zheng. 2006. Effect of silver nitrate on the tissue culture of immature wheat embryos. Russian J. Plant Physiol. 53:530-534.  

35. Xie, J., M. Gao, Q. Cai, X. Cheng, Y. Shen, and Z. Liang. 1995. Improved isolated microspore culture efficiency in medium with maltose and optimized growth regulator ombination in Japonica rice (Oryza sativa). Plant Cell. 42:245-250.  

36. Xie, J.H., M.W. Gao, Z.Q. Liang, Q.Y. Shu, X.Y. Cheng, and Q.Z. Xue. 1997. The effect of cool-pretreatment on the isolated microspore culture and the free amino acid change of anthers in Japonica rice (Oryza sativa L). J. Plant Physiol. 151:79-82.  

37. Yeung, E.C., M.H. Rahman, and T.A. Thorpe. 1996. Comparative development of zygotic and microspore-derived embryos in Brassica napus L. cv. Topas. I. histodifferentiation. Int. J. Plant Sci. 157:27-39.  

38. Yeung, E.C. 2002. The canola microspore-derived embryo as a model system to study developmental processes in plants. J. Plant Biol. 45:119-133.  

39. Žárský, V., D. Garrido, L. Říhová, J. Tupý, O. Vicente, and E. Heberle-Bors. 1992. Derepression of the cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis. Sexual Plant Reproduction 5:189-194.  

40. Žárský, V., D. Garrido, N. Eller, J. Tupy, O. Vicente, F. Schoffl, and E Heberle-Bors. 1995. The expression of a small heat shock gene is activated during induction of tobacco pollen embryogenesis by starvation. Plant Cell Environ. 18:139-147.