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Production of plants resistant to Alternaria carthami via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

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The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamus tinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 105 spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min−1 mg−1 protein) from organogenesis and 47% (0.23 μmol min−1 mg−1 protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg−1 protein) and 19.5% (145 U mg−1 protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.

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Fig. 1
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Thidiazuron, N-phenyl-N′-1,2,3-thiadiazol-5-yl-urea

GA3 :

Gibberellic acid


α-naphthaleneacetic acid

AgNO3 :

Silver nitrate


Potato dextrose agar


Plant growth regulators


Thiamine hydrochloride


Fungal culture filtrate


Murashige and Skoog germination medium (Becwar et al. 1990)






Superoxide dismutase


Nimbkar Agricultural Research Institute


  1. Ahmed KS, Mesterhazy A, Sagi F (1991) In vitro techniques for selecting wheat (Triticum aestivum L.) for Fusarium resistance I: double layer culture technique. Euphytica 57:251–257

  2. Amin Gh (1991) Popular medicinal plants of Iran. Health Ministry Press, Tehran, p 118

  3. Anderson MD, Prasad TK, Stewart CR (1995) Changes in isozymes profiles of catalase, peroxidase, and glutathione reductase during acclimation to chilling in mesocotyls of maize seedlings. Plant Physiol 109:1247–1257

  4. Becwar MR, Nagmani R, Wann SR (1990) Initiation of embryogenic cultures and somatic embryo development in loblolly pine (Pinus taeda). Can J For Res 20:810–817

  5. Bergman JW, Riveland CR, Flynn CR, Carison GR, Wichman DM (2001) Registration of safflower. Crop Sci 41:1640

  6. Biondi S, Mirza J, Mittempergher L, Bagni N (1991) Selection of elm cell cultures variants resistant to Ophiostoma ulmi culture filtrate. J Plant Physiol 137:631–634

  7. Bradford MN (1976) A rapid and sensitive method for the quantitation of microgram quantity of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  8. Chaoui A, Mazhoudi S, Ghorbal MH, Ferjani EE (1997) Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzyme activities in bean (Phaseolus vulgaris L.). Plant Sci 127:139–147

  9. Chowdhury S (1944) An Alternaria disease of safflower. Ind Bot Soc 23:59–65

  10. Cyberlipid Center (2007) Fats and oils, minor vegetable oils, 21st August, http://www.cyberlipid.org/glycer/glyc0053.htm

  11. Daub ME (1986) Tissue culture and the selection of resistance to pathogens. Ann Rev Phytopathol 24:159–186

  12. Egley GH, Paul RN Jr, Vaughn KC, Duke SO (1983) Role of peroxidase in the development of water-impermeable seed coats in Sida spinosa L. Planta 157:224–232

  13. Ganesan M, Jayabalan N (2006) Isolation of disease-tolerant cotton (Gossypium hirsutum L. cv. SVPR 2) plants by screening somatic embryos with fungal culture filtrate. Plant Cell Tissue Organ Cult 87:273–284

  14. Gentile A, Tribulato E, Deng ZN, Galun E, Fluh R, Vardi A (1993) Nucellar callus of `Femminello’ lemon, selected for tolerance to Phoma tracheiphila toxin, shows enhanced release of chitinase and glucanase into the culture medium. Theor Appl Genet 86:527–532

  15. Hatta R, Ito K, Hosaki Y, Tanaka T, Tanaka A, Yamamoto M, Akimitsu K, Tsuge T (2002) A conditionally dispensable chromosome controls host-specific pathogenicity in the fungal plant pathogen Alternaria alternate. Genetics 161:59–70

  16. Hayashi N, Tamnabe T, Tsuge S, Nishimura K, Kohmoto K (1990) Determinations of host selective toxin production during spore germination of Alternaria alternata by high-performance liquid chromatography. Phytopathol 80:1088–1091

  17. Hernandez JA, Jiminez A, Mullineaux PM, Sevilla F (2000) Tolerance of pea (Pisum sativum L.) to long-term stress is associated with induction of antioxidant defenses. Plant Cell Environ 23:853–862

  18. Jayasankar S, Litz RE (1998) Characterization of resistance in mango embryogenic cultures selected for resistance to Colletotrichum gloeosporioides culture filtrate and phytotoxin. Theor Appl Genet 96:823–831

  19. Jayasankar S, Li Z, Gray DJ (2000) In-vitro selection of Vitis vinifera ‘Chardonnay’ with Elsinoe ampelina culture filtrate is accompanied by fungal resistance and enhanced secretion of chitinase. Planta 211:200–208

  20. Kneusel RE, Schiltzg E, Matern U (1994) Molecular characterization and cloning of an esterase which inactivates the macrolide toxin brefeldin A. J Biol Chem 269:3449–3456

  21. Lynch DR, Coleman MC, Lyon GD (1991) Effect of Alternaria solani culture filtrate on adventitious shoot regeneration in potato. Plant Cell Rep 9:607–610

  22. Maekawa N, Yamamoto S, Nishimura K, Kohmoto M (1984) Studies on host-specific AF-toxins produced by Alternaria strawberry pathotype causing Alternaria black spot of strawberry. I. Production of host-specific toxins and their biological activity. Ann Phytopathol Soc Jpn 50:600–609

  23. Maier FJ, Oettler G (1992) Selection for the Fusarium toxin deoxynivalenol in callus cultures of triticale. Third European Fusarium seminar. September 22–24. IHAR Radzikow, pp 43–49

  24. Mortensen K, Bergman JW, Burns EE (1983) Importance of Alternaria carthami and A. alternaria in causing leaf spot diseases in safflower. Plant Disease 67:1187–1190

  25. NARI (2005) Safflower improvement programme at the Nimbkar Agricultural Research Institute, Maharashtra, India. http://nariphaltan.virtualave.net/safflower.htm

  26. Olson PD, Varner JE (1993) Hydrogen peroxide and lignification. Plant J 4:887–892

  27. Petrie GA (1974) Fungi associated with seeds rape, flax and safflower in Western Canada, 1968–73. Can Plant Dis Surv 54:155–165

  28. Piacentini MP, Fraternale D, Piatti E, Ricci D, Vetrano F, Dacha M, Accorsi A (2001) Senescence delay and change of antioxidant enzyme levels in Cucumis sativus L. etiolated seedlings by ELF magnetic fields. Plant Sci 161:45–53

  29. Rao MV, Paliyath G, Ormrod DP (1996) Ultraviolet-B- and ozone-induced biochemical changes in antioxidant enzymes of Arabodopsis thaliana. Plant Physiol 110:125–136

  30. Smith VH, Jimmerson J (2005) safflower. Briefing 58:1–4

  31. Song HS, Lim SM, Widholm JM (1994) Selection and regeneration of soybeans resistant to the pathotoxic culture filtrates of Septoria glycines. Phytopathol 84:948–951

  32. Spychalla JP, Desborough SL (1990) Superoxide dismutase, catalase and alpha tocopherol content of stored potato tubers. Plant Physiol 94:1214–1218

  33. Tanaka A, Shiotani H, Yamamoto M, Tsuge T (1999) Insertional mutagenesis and cloning of the genes required for biosynthesis of the host-specific AK-toxin in the Japanese pear pathotype of Alternaria alternata. Mol Plant Microbe Interact 12:691–702

  34. Thanutong P, Furusawa I, Yamamota M (1983) Resistant tobacco plants from protoplast-derived calluses selected for their resistance to Pseudomonas and Alternaria toxins. Theor Appl Genet 66:209–215

  35. Venkatachalam P, Jayabalan N (1996) In vitro screening of groundnut (Arachis hypogaea L.) cell lines and regeneration of plants resistant to pathotoxic culture filtrate of Cercosporidium personatum. Plant Tissue Cult 6:7382

  36. Vijaya Kumar J, Ranjitha Kumari BD (2005) Effect of phytohormones on multiple shoot induction in cv. NARI-6 of safflower (Carthamus tinctorius L.). J Plant Biotechnol 7:149–153

  37. Weiss EA (1971) Castor, sesame and safflower. Leonard Hill, London

  38. Woodbury W, Spencer AK, Stahman MA (1971) An improved procedure for using ferricyanide for detecting catalase isozymes. Anal Biochem 44:301–305

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We are grateful to Prasad RD, Directorate of Oilseeds Research, Rajendranagar, Hyderabad, India for providing Alternaria carthami culture. We thank Nimbhkar Agricultural Research Institute (NARI), Maharashtra, India for providing seeds for this study.

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Correspondence to B. D. Ranjitha Kumari.

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Vijaya Kumar, J., Ranjitha Kumari, B.D., Sujatha, G. et al. Production of plants resistant to Alternaria carthami via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates. Plant Cell Tiss Organ Cult 93, 85–96 (2008). https://doi.org/10.1007/s11240-008-9346-4

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  • Carthamustinctorius L.
  • Medicinal plant
  • Embryogenic calli
  • Organogenesis
  • Somatic embryogenesis
  • Plant regeneration
  • Alternaria leaf spot
  • Disease resistance
  • Antioxidant enzymes