Article | . 2018 Vol. 36, Issue. 2
IIdentification and Variation of Major Aliphatic Glucosinolates in Doubled Haploid Lines of Radish (Raphanus sativus L.)

Department of Integrative Plant Science, Chung-Ang University1
Department of Horticultural Science, Mokpo National University2

2018.. 202:311


The aim of this study was to determine the concentration of glucoraphenin (GRE) and glucoraphasatin (GRH) from 41 doubled haploid (DH) lines of radish (Raphanus sativus L.) cultivar obtained by isolated microspore culture. Microspores from flower buds (4.0 ± 0.5 mm in length) were cultured in Nitsch and Nitsch medium, and microspore-derived embryos were regenerated into complete plantlets in Murashige and Skoog medium. The average embryo yield was 5.0 ± 2.9 per petri dish. Seeds obtained by bud pollination from the DH lines were planted and leaves of 4-week old seedlings were used for analysis of glucosinolate (GSL) content. In order to determine the GSL content, GRE from seeds and GRH from the roots were extracted and purified to 96.2 and 71.0%, respectively. The amount of GRH markedly varied among the DH lines, ranging from 2.6 to 31.5 mg per gram dry weight. GRH contents were reduced by as much as 14.1% or increased by up to 171.2% compared with the donor plant. There was as much as a 6.1-fold difference in the amount of GRE between the DH lines. Among the 41 DH lines tested, the sum of GRE and GRH contents were significantly reduced in 14 DH lines compared to the donor plant (p = 0.95), whereas the contents were increased in only three DH lines. Our results will be useful for breeding radish genotypes with low and high content of GSL, and the DH lines developed in this study will be useful germplasm for elucidating the regulation of the biosynthesis of aliphatic GSLs in radish plants.

1. Babbar SB, Agarwal PK, Sahay S, Bhojwani SS (2004) Isolated microspore culture of Brassica: an experimental tool for developmental studies and crop improvement. Indian J Biotechnol 3:185-202   

2. Baillie AM, Epp DJ, Hutcheson D, Keller WA (1992) In vitro culture of isolated microspores and regeneration of plants in Brassica campestris. Plant Cell Rep 11:234-237.  

3. Barillari J, Cervellati R, Paolini M, Tatibouet A, Rollin P, Iori R (2005) Isolation of 4-methylthio-3-butenyl glucosinolate from Raphanus sativus sprouts (kaiware daikon) and its redox properties. J Agric Food Chem 53:9890-9896.  

4. Barillari J, Iori R, Broccoli M, Pozzetti L, Canistro D, Sapone A, Bonamassa B, Biagi GL, Paolini M (2007) Glucoraphasatin and glucoraphenin, a redox pair of glucosinolates of brassicaceae, differently affect metabolizing enzymes in rats. J Agric Food Chem 55:5505-5511.  

5. Barro F, Fernández-Escobar J, De la Vega M, Martín A (2003) Modification of glucosinolate and erucic acid contents in doubled haploid lines of Brassica carinata by UV treatment of isolated microspores. Euphytica 129:1-6.  

6. Bhatia R, Dey SS, Sood S, Sharma K, Parkash C, Kumar R (2017) Efficient microspore embryogenesis in cauliflower (Brassica oleracea var. botrytis L.) for development of plants with different ploidy level and their use in breeding programme. Sci Hortic 216:83-92.  

7. Chun CH, Na HY (2011) Microspore-derived embryo formation in response to cold pretreatment, washing medium, and medium composition of radish (Raphanus sativus L.). Korean J Hortic Sci Technol 29:494-499  

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

9. Clarke DB (2010) Glucosinolates, structures and analysis in food. Anal Methods 2:310-325.  

10. Curtis IS (2003) The noble radish: past, present and future. Trends Plant Sci 8:305-307.  

11. Curtis IS (2011) Genetic engineering of radish: current achievements and future goals. Plant Cell Rep 30:733-744. s00299-010-0978-6  

12. Custers J (2003) Microspore culture in rapeseed (Brassica napus L.). In M Maluszynski, KJ Kasha, BP Forster, I Szarejko, eds. Doubled Haploid Production in Crop Plants. Springer, Dordrecht, The Netherlands, pp 185-193.  

13. Da Silva Dias JC (2003) Protocol for broccoli microspore culture. In M Maluszynski, KJ Kasha, BP Forster, I Szarejko, eds. Doubled Haploid Production in Crop Plants. Springer, Dordrecht, The Netherlands, pp 195-204.  

14. Dawson GW, Hick AJ, Bennett RN, Donald A, Pickett JA, Wallsgrove RM (1993) Synthesis of glucosinolate precursors and investigations into the biosynthesis of phenylalkyl- and methylthioalkylglucosinolates, J Biol Chem 268:27154-27159  

15. Fahey JW, Zalcmann AT, Talalay P (2001) The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56:5-51.  

16. Ferrie A (2003) Microspore culture of Brassica species. In M Maluszynski, KJ Kasha, BP Forster, I Szarejko, eds. Doubled Haploid Production in Crop Plants. Springer, Dordrecht, The Netherlands, pp 205-215.  

17. Ferrie A, Caswell K (2011) Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tissue Organ Cult 104:301-309.  

18. Ferrie AM, Möllers C (2011) Haploids and doubled haploids in Brassica spp. for genetic and genomic research. Plant Cell Tissue Organ Cult 104:375-386.  

19. Forster BP, Heberle-Bors E, Kasha KJ, Touraev A (2007) The resurgence of haploids in higher plants. Trends Plant Sci 12:368-375.  

20. Grubb CD, Abel S (2006) Glucosinolate metabolism and its control. Trends Plant Sci 11:89-100.  

21. Gu H, Zhou W, Hagberg P (2003) High frequency spontaneous production of doubled haploid plants in microspore cultures of Brassica rapa ssp. chinensis. Euphytica 134:239-245.  

22. Han N, Kim SU, Park HY, Na H (2014) Microspore-derived embryo formation and morphological changes during the isolated microspore culture of radish (Raphanus sativus L.). Korean J Hortic Sci Technol 32:382-389. 2014.13170  

23. Han N, Su’udi M, Kim J (2015) The major aliphatic glucosinolate content in Korean radish during vegetative and reproductive growth. Hortic Environ Biotechnol 56:152-158.  

24. Heber D (2004) Vegetables, fruits and phytoestrogens in the prevention of diseases. J Postgrad Med 50:145-149  

25. Higdon JV, Delage B, Williams DE, Dashwood RH (2007) Cruciferous vegetables and human cancer risk: epidemiologic evidence and mechanistic basis. Pharmacol Res 55:224-236.  

26. Ito H, Horie H (2008) A chromatographic method for separating and identifying intact 4-methylthio-3-butenyl glucosinolate in Japanese radish (Raphanus sativus L.). Jpn Agric Res Q 42:109-114.  

27. Jo JS, Bhandari SR, Kang GH, Lee JG (2016) Comparative analysis of individual glucosinolates, phytochemicals and antioxidant activities in broccoli breeding lines. Hortic Environ Biotechnol 57:392-403  

28. Kakizaki T, Kitashiba H, Zou Z, Li F, Fukino N, Ohara T, Nishio T, Ishida M (2017) A 2-oxoglutarate-dependent dioxygenase mediates the biosynthesis of glucoraphasatin in radish. Plant Physiol 173:1583-1593.  

29. Kuang P, Liang H, Yuan Q (2010) Isolation and purification of glucoraphenin from radish seeds by low-pressure column chromatography and nanofiltration. Sep Sci Technol 46:179-184.  

30. Lichter R (1982) Induction of haploid plants from isolated pollen of Brassica napus. Z Pflanzenphysiol 105:427-434. S0044-328X(82)80040-8  

31. Lionneton E, Beuret W, Delaitre C, Ochatt S, Rancillac M (2001) Improved microspore culture and doubled-haploid plant regeneration in the brown condiment mustard (Brassica juncea). Plant Cell Rep 20:126-130.  

32. Montaut S, Barillari J, Iori R, Rollin P (2010) Glucoraphasatin: chemistry, occurrence, and biological properties. Phytochemistry 71:6-12.  

33. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473-497.  

34. Saavedra MJ, Borges A, Dias C, Aires A, Bennett RN, Rosa ES, Simoes M (2010) Antimicrobial activity of phenolics and glucosinolate hydrolysis products and their synergy with streptomycin against pathogenic bacteria. Med Chem 6:174-183. doi. org/10.2174/ 1573406411006030174  

35. Seguí‐Simarro JM, Nuez F (2008) How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore‐derived embryogenesis. Physiol Plant 134:1-12.  

36. Smýkal P (2000) Pollen embryogenesis-the stress mediated switch from gametophytic to sporophytic development. Current status and future prospects. Biol Plant 43:481-489.  

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

38. Zeng A, Song L, Cui, Y, Yan J (2017) Reduced ascorbate and reduced glutathione improve embryogenesis in broccoli microspore culture. S Afr J Bot 109:275-280.