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Research Article

volume 39 issue 2 (june 2019) : 96-101,   Doi: 10.18805/ag.D-4714

Sterilization procedure and callus regeneration in black turmeric (Curcuma caesia)

A
A S Abubakar
R
R N Pudake
1School of Agriculture, Lovely Professional University, Phagwara, Punjab, India
Cite article:- Abubakar S A, Pudake N R (2019). Sterilization procedure and callus regeneration in black turmeric (Curcuma caesia). Agricultural Science Digest. 39(2): 96-101. doi: 10.18805/ag.D-4714.

ABSTRACT

Sterilization procedure, media composition, explants selection and control of physical environment are critical for successful cultures and callus induction with surface sterilization being very challenging in most plants. Five different sterilization methods were evaluated to come up with the best for subsequent use to establish an in vitro regeneration method for the induction of callus in Curcuma caesia using excised leaf and rhizome explants. Murashige and Skoog (MS) media supplemented with various concentration of 2,4-Dichlorophenoxy acetic acid (2,4-D)/Indole-3-acetic acid (IAA) (0.5- 5.0mg/L), singly or in combination with Benzyl aminopurine (BAP)/Kinetin (KIN) (0.1-5.0mg/L), 0.3% sucrose and 0.08% agar were used. The result of the sterilization procedures showed 15% NaHClO3 (5min) + 70% Ethanol (30s) + 0.1% HgCl2 (5min) to be the most effective in controlling contamination in C. caesia among all the treatments tested. The response to callus induction was found to depend on the type of explants used and growth regulators combination. Leaf explants gave the highest percentage of callus induction. Highest percentage of callus induction (66.70%) was obtained in the growth regulator combination of 2, 4-D (0.5mg/L) + BAP (0.1mg/L) and least (14.29%) in IAA (2.0mg/L) + BAP (0.5mg/L). Equal and higher concentration of 2, 4-D + BAP of 5.0mg/L each also provided better result (40.00%). No callus was obtained in all the single concentration of 2, 4-D used.

REFERENCES

  1. Abubakar, A. S. and Pudake, R. M. (2014). Curcuma caesia: A wonder herb with medicinal properties (review). Indian J. Scholarly Res., 3(6): 1-4
  2. Abubakar, A. S., Yahaya, S. U., Shaibu, A.S., Ibrahim, H., Ibrahim1, A.K., Lawan, Z.M. and Isa, A.M. (2018). In vitro propagation of sweet potato (Ipomoea batatas (L.) Lam.) cultivars. Agric. Sci. Digest., 38(1): 17-21
  3. Afolabi, A. S., Oyebanji, O. B., Nweke, O., Odebunmi, O., Galadima, N. B., Idris, M. S., et. al., (2009) Simple, effective and economical explant-surface sterilization protocol for cowpea, rice and sorghum seeds. Afr. J. Biotec., 8(20): 5395-5399.
  4. Behera, K. K., Pani, D. and Sahoo, S. (2010). Effect of plant growth regulator on in vitro multiplication of turmeric Curcuma longa L. cv. Ranga). Inter. J. Bio. & Tech. 1(1):16-23.
  5. Cousins, M. M. (2008). Development of in vitro protocols to enhance secondary metabolite production from turmeric (Curcuma longa L.). A thesis presented to the graduate school of Clemson Univ. in partial fulfillment of the requirements for the degree Masters of Science plant and environmental sci.
  6. Ghosh, A., Chatterjee, P., and Ghosh, P. (2013). A protocol for rapid propagation of genetically true to type Indian turmeric (Curcuma longa L.) through in vitro culture technique. Adv. in App.Sci. Res., 4(3):39-45
  7. Gopal, J., Minocha, J. L., and Dhaliwal, H. S. (1998). Microtuberization in potato (Solanum tuberosum L.) Plant Cell Reports, 17(10), 794-798.
  8. Hashemy, T., Maki, H., Yamada, Y., Kaneko, T. S. and Syono, K. (2009). Effects of light and cytokinin on in vitro micropropagation and microrhizome production in turmeric (Curcuma longa L.) Plant Bio. & Tech., 26: 237–242.
  9. Jala, A. (2013). The effect of the 2, 4-Dichlorophenoxy acetic acid, benzyl adenine and paclobutrazol, on vegetative tissue-derived somatic embryogenesis in turmeric (Curcuma var. Chattip). Inter. Transaction J Eng., Management, & App. Sci. & Tech., 4(2): 105-110.
  10. Jamil, M., Kim, J. K., Akram, Z., Ajmul, S. and Raha, E. S. (2007). Regeneration of ginger plant from callus through organogenesis and effect of CO2 enrichment on the differentiation of regenerative plant. Biotechnology, 6(1): 101-104 
  11. Leifert, C., Morris, C., and Waites, W. M. (1994). Ecology of microbial saprophytes and Pathogens in field grown and tissue cultured plants. CRC Critical Reviews Plant Sci., 13:139-183
  12. Mannangatti, K. and Narayanasamy, M. (2008). Anti-fungal protein from Curcuma caesia Roxb. J. Biotechnol., S136–90.
  13. Miachir, J. I., Moretti Romani, V. L., Amaral, A. F. de C., Mello, M. O., Crocomo, O. J. and Melo, M. (2004). Micropropagation and callogenesis of Curcuma zedoaria, Roscoe. Sci. Agric. (Piracicaba, Braz.), 61(4): 427-432
  14. Nakagarwara, S., Goto, T, Nara, M., Ozawa, Y., Hotta, K., Arata, Y. (1998) Spectroscopic characterisation and the pH dependence of bacterial activity of the aqueous chlorine solution. Anal. Sci., 14: 691-698.
  15. Nayak, S. and Naik, P. K. (2006). Factors effecting in vitro microrhizome formation and growth in Curcuma longa l. and improved field performance of micropropagated plants. Science Asia, 32: 31-37.
  16. Paliwal, P., Pancholi, S.S and Patel, R.K. (2011). Pharmacognostic parameters for evaluation of the rhizomes of Curcuma caesia. J. Adv. Pharm. Tech. Res., 2(1): 56–61.
  17. Pandey, A.K. and Chowdhary, A.R. (2003). Volatile constituents of rhizome oil of Curcuma caesia Roxb. from central India. Flavour Fragr. J., 18(5): 463–5.
  18. Parthasarathy, V. A. and Sasikumar, B. (2006). Biotechnology of Curcuma, CAB Reviews: Perspectives in Agric, Vet. Sci., Nut. & Nat. Resources, 1(20): 1-9.
  19. Prakash, S., Elangomathavan, R., Seshadri, S., Kathiravan, K. and Ignacimuthu, S, (2004) Efficient regeneration of Curcuma amada Roxb plantlets from rhizome and leaf sheath explants. Plant Cell Tiss. Org. Cult., 78: 159-165.
  20. Prasad, S. and Aggarwal, B.B. (2011). The Golden Spice From Traditional Medicine to Modern Medicine. Taylor and Francis Group, LLC. Bookshelf ID: NBK92752PMID
  21. Rahman, M. M., Amin, M. N., Ahamed, T., Ahmad, S., Habib, A., Ahmed, R., Ahmed, M. B. and Ali, M.R. (2005). In vitro rapid propagation of black thorn (Kaempferia galanga L.): A rare medicinal and aromatic plant of Bangladesh. J. Biol. Sci., 5(3): 300-304.
  22. Saensouk, P. (2011). Callus induction and plant regeneration from leaf explants of Cornukaempferia aurantiflora, Mood & Larsen. Pak. J. Bot., 43(5): 2415-2418.
  23. Sarma, I., Deka, A. C., Sarma S. and Sarma, T. C. (2011). High frequency clonal propagation and microrhizome induction of Curcuma longa L. (cv lakadong)- A rich source of curcumin of North East India. The Bioscan, an Inter Quaterly J. Life Sci., 6(1): 11-18.
  24. Shirgurkar, M.V., John, C.K. and Nadgauda, R.S. (2001). Factors affecting in vitro microrhizome production in turmeric. Plant Cell Tiss. Org. Cult., 64; 5–11.
  25. Srirat, P., Sirisansaneeyakul, S., Parakulsuksatid, P., Prammanee, S. and Vanichsriratana, W. (2008). In vitro shoot propagation of Curcuma longa L. from rhizome bud explants. The 3rd Inter. Conf. on Fermentation Tech. for Value added Agric. Products. pp13.
  26. Srivastava, N., Kamal, B., Sharma, V. Negi, Y. K., Dobriyal, A.K., Gupta, S. and Jadon, V. S. (2010). Standardization of sterilization protocol for micropropagation of Aconitum heterophyllum- An endangered medicinal herb. Academic Arena, 2(6): 37-42.
  27. Sultana, A., Hassan, L., Ahmad, S. D., Shah, A.H., Batool, F., Islam, M.A., Rahman, R. and Moonmoon, S. (2009). In vitro regeneration of ginger using leaf, shoot tip and root explants. Pak. J. Bot., 41(4): 1667-1676.
  28. Sundram, T. C.M., Annuar, M. S. and Khalid, N. (2012).Optimization of culture condition for callus induction from shoot buds for establishment of rapid growing cell suspension cultures of Mango ginger (Curcuma mangga). Australian J. Crop Sci., 6(7): 1139-1146. 
  29. Taha, H. S., Abbas, M. S., Aly, U. I. and Gaber, E. I. (2013). New aspects for callus production, regeneration and molecular characterization of ginger (Zingiber officinale Rosc.). Med. Aromat. Plants 2(6): 1-8.
  30. Tuan, V. C., Hoang, V. D., and Loc, N. H. (2011). Cell suspension culture of Zedoary (Curcuma zedoaria Roscoe). VNU J. Nat. Sci. & Tech., 27: 64-70.
  31. VSN International: GenStat for Windows 17th edition. VSN International, Hemel Hempstead, UK.
  32. Zhang, S., Liu, N., Sheng, A., Ma, G. and Wu, G. (2011). Direct and callus-mediated regeneration of Curcuma soloensis Valeton (Zingiberaceae) and ex vitro performance of regenerated plants. Scientia Horticulturae, 130(4): 899-905.
  33. Zuraida, A. R., Izzati, K. F. L., Nazreena, O. A., Radziah, C. M. Z. C., Asyikin, S. G. S. N. and Sreeramanan S. (2014) In vitro regeneration of Curcuma caesia plantlets from basal part and via somatic embryogenesis. Adv. in Bioscience and Biotech., 5: 363-372. 
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    Research Article

    volume 39 issue 2 (june 2019) : 96-101,   Doi: 10.18805/ag.D-4714

    Sterilization procedure and callus regeneration in black turmeric (Curcuma caesia)

    A
    A S Abubakar
    R
    R N Pudake
    1School of Agriculture, Lovely Professional University, Phagwara, Punjab, India
    Cite article:- Abubakar S A, Pudake N R (2019). Sterilization procedure and callus regeneration in black turmeric (Curcuma caesia). Agricultural Science Digest. 39(2): 96-101. doi: 10.18805/ag.D-4714.

    ABSTRACT

    Sterilization procedure, media composition, explants selection and control of physical environment are critical for successful cultures and callus induction with surface sterilization being very challenging in most plants. Five different sterilization methods were evaluated to come up with the best for subsequent use to establish an in vitro regeneration method for the induction of callus in Curcuma caesia using excised leaf and rhizome explants. Murashige and Skoog (MS) media supplemented with various concentration of 2,4-Dichlorophenoxy acetic acid (2,4-D)/Indole-3-acetic acid (IAA) (0.5- 5.0mg/L), singly or in combination with Benzyl aminopurine (BAP)/Kinetin (KIN) (0.1-5.0mg/L), 0.3% sucrose and 0.08% agar were used. The result of the sterilization procedures showed 15% NaHClO3 (5min) + 70% Ethanol (30s) + 0.1% HgCl2 (5min) to be the most effective in controlling contamination in C. caesia among all the treatments tested. The response to callus induction was found to depend on the type of explants used and growth regulators combination. Leaf explants gave the highest percentage of callus induction. Highest percentage of callus induction (66.70%) was obtained in the growth regulator combination of 2, 4-D (0.5mg/L) + BAP (0.1mg/L) and least (14.29%) in IAA (2.0mg/L) + BAP (0.5mg/L). Equal and higher concentration of 2, 4-D + BAP of 5.0mg/L each also provided better result (40.00%). No callus was obtained in all the single concentration of 2, 4-D used.

    REFERENCES

    1. Abubakar, A. S. and Pudake, R. M. (2014). Curcuma caesia: A wonder herb with medicinal properties (review). Indian J. Scholarly Res., 3(6): 1-4
    2. Abubakar, A. S., Yahaya, S. U., Shaibu, A.S., Ibrahim, H., Ibrahim1, A.K., Lawan, Z.M. and Isa, A.M. (2018). In vitro propagation of sweet potato (Ipomoea batatas (L.) Lam.) cultivars. Agric. Sci. Digest., 38(1): 17-21
    3. Afolabi, A. S., Oyebanji, O. B., Nweke, O., Odebunmi, O., Galadima, N. B., Idris, M. S., et. al., (2009) Simple, effective and economical explant-surface sterilization protocol for cowpea, rice and sorghum seeds. Afr. J. Biotec., 8(20): 5395-5399.
    4. Behera, K. K., Pani, D. and Sahoo, S. (2010). Effect of plant growth regulator on in vitro multiplication of turmeric Curcuma longa L. cv. Ranga). Inter. J. Bio. & Tech. 1(1):16-23.
    5. Cousins, M. M. (2008). Development of in vitro protocols to enhance secondary metabolite production from turmeric (Curcuma longa L.). A thesis presented to the graduate school of Clemson Univ. in partial fulfillment of the requirements for the degree Masters of Science plant and environmental sci.
    6. Ghosh, A., Chatterjee, P., and Ghosh, P. (2013). A protocol for rapid propagation of genetically true to type Indian turmeric (Curcuma longa L.) through in vitro culture technique. Adv. in App.Sci. Res., 4(3):39-45
    7. Gopal, J., Minocha, J. L., and Dhaliwal, H. S. (1998). Microtuberization in potato (Solanum tuberosum L.) Plant Cell Reports, 17(10), 794-798.
    8. Hashemy, T., Maki, H., Yamada, Y., Kaneko, T. S. and Syono, K. (2009). Effects of light and cytokinin on in vitro micropropagation and microrhizome production in turmeric (Curcuma longa L.) Plant Bio. & Tech., 26: 237–242.
    9. Jala, A. (2013). The effect of the 2, 4-Dichlorophenoxy acetic acid, benzyl adenine and paclobutrazol, on vegetative tissue-derived somatic embryogenesis in turmeric (Curcuma var. Chattip). Inter. Transaction J Eng., Management, & App. Sci. & Tech., 4(2): 105-110.
    10. Jamil, M., Kim, J. K., Akram, Z., Ajmul, S. and Raha, E. S. (2007). Regeneration of ginger plant from callus through organogenesis and effect of CO2 enrichment on the differentiation of regenerative plant. Biotechnology, 6(1): 101-104 
    11. Leifert, C., Morris, C., and Waites, W. M. (1994). Ecology of microbial saprophytes and Pathogens in field grown and tissue cultured plants. CRC Critical Reviews Plant Sci., 13:139-183
    12. Mannangatti, K. and Narayanasamy, M. (2008). Anti-fungal protein from Curcuma caesia Roxb. J. Biotechnol., S136–90.
    13. Miachir, J. I., Moretti Romani, V. L., Amaral, A. F. de C., Mello, M. O., Crocomo, O. J. and Melo, M. (2004). Micropropagation and callogenesis of Curcuma zedoaria, Roscoe. Sci. Agric. (Piracicaba, Braz.), 61(4): 427-432
    14. Nakagarwara, S., Goto, T, Nara, M., Ozawa, Y., Hotta, K., Arata, Y. (1998) Spectroscopic characterisation and the pH dependence of bacterial activity of the aqueous chlorine solution. Anal. Sci., 14: 691-698.
    15. Nayak, S. and Naik, P. K. (2006). Factors effecting in vitro microrhizome formation and growth in Curcuma longa l. and improved field performance of micropropagated plants. Science Asia, 32: 31-37.
    16. Paliwal, P., Pancholi, S.S and Patel, R.K. (2011). Pharmacognostic parameters for evaluation of the rhizomes of Curcuma caesia. J. Adv. Pharm. Tech. Res., 2(1): 56–61.
    17. Pandey, A.K. and Chowdhary, A.R. (2003). Volatile constituents of rhizome oil of Curcuma caesia Roxb. from central India. Flavour Fragr. J., 18(5): 463–5.
    18. Parthasarathy, V. A. and Sasikumar, B. (2006). Biotechnology of Curcuma, CAB Reviews: Perspectives in Agric, Vet. Sci., Nut. & Nat. Resources, 1(20): 1-9.
    19. Prakash, S., Elangomathavan, R., Seshadri, S., Kathiravan, K. and Ignacimuthu, S, (2004) Efficient regeneration of Curcuma amada Roxb plantlets from rhizome and leaf sheath explants. Plant Cell Tiss. Org. Cult., 78: 159-165.
    20. Prasad, S. and Aggarwal, B.B. (2011). The Golden Spice From Traditional Medicine to Modern Medicine. Taylor and Francis Group, LLC. Bookshelf ID: NBK92752PMID
    21. Rahman, M. M., Amin, M. N., Ahamed, T., Ahmad, S., Habib, A., Ahmed, R., Ahmed, M. B. and Ali, M.R. (2005). In vitro rapid propagation of black thorn (Kaempferia galanga L.): A rare medicinal and aromatic plant of Bangladesh. J. Biol. Sci., 5(3): 300-304.
    22. Saensouk, P. (2011). Callus induction and plant regeneration from leaf explants of Cornukaempferia aurantiflora, Mood & Larsen. Pak. J. Bot., 43(5): 2415-2418.
    23. Sarma, I., Deka, A. C., Sarma S. and Sarma, T. C. (2011). High frequency clonal propagation and microrhizome induction of Curcuma longa L. (cv lakadong)- A rich source of curcumin of North East India. The Bioscan, an Inter Quaterly J. Life Sci., 6(1): 11-18.
    24. Shirgurkar, M.V., John, C.K. and Nadgauda, R.S. (2001). Factors affecting in vitro microrhizome production in turmeric. Plant Cell Tiss. Org. Cult., 64; 5–11.
    25. Srirat, P., Sirisansaneeyakul, S., Parakulsuksatid, P., Prammanee, S. and Vanichsriratana, W. (2008). In vitro shoot propagation of Curcuma longa L. from rhizome bud explants. The 3rd Inter. Conf. on Fermentation Tech. for Value added Agric. Products. pp13.
    26. Srivastava, N., Kamal, B., Sharma, V. Negi, Y. K., Dobriyal, A.K., Gupta, S. and Jadon, V. S. (2010). Standardization of sterilization protocol for micropropagation of Aconitum heterophyllum- An endangered medicinal herb. Academic Arena, 2(6): 37-42.
    27. Sultana, A., Hassan, L., Ahmad, S. D., Shah, A.H., Batool, F., Islam, M.A., Rahman, R. and Moonmoon, S. (2009). In vitro regeneration of ginger using leaf, shoot tip and root explants. Pak. J. Bot., 41(4): 1667-1676.
    28. Sundram, T. C.M., Annuar, M. S. and Khalid, N. (2012).Optimization of culture condition for callus induction from shoot buds for establishment of rapid growing cell suspension cultures of Mango ginger (Curcuma mangga). Australian J. Crop Sci., 6(7): 1139-1146. 
    29. Taha, H. S., Abbas, M. S., Aly, U. I. and Gaber, E. I. (2013). New aspects for callus production, regeneration and molecular characterization of ginger (Zingiber officinale Rosc.). Med. Aromat. Plants 2(6): 1-8.
    30. Tuan, V. C., Hoang, V. D., and Loc, N. H. (2011). Cell suspension culture of Zedoary (Curcuma zedoaria Roscoe). VNU J. Nat. Sci. & Tech., 27: 64-70.
    31. VSN International: GenStat for Windows 17th edition. VSN International, Hemel Hempstead, UK.
    32. Zhang, S., Liu, N., Sheng, A., Ma, G. and Wu, G. (2011). Direct and callus-mediated regeneration of Curcuma soloensis Valeton (Zingiberaceae) and ex vitro performance of regenerated plants. Scientia Horticulturae, 130(4): 899-905.
    33. Zuraida, A. R., Izzati, K. F. L., Nazreena, O. A., Radziah, C. M. Z. C., Asyikin, S. G. S. N. and Sreeramanan S. (2014) In vitro regeneration of Curcuma caesia plantlets from basal part and via somatic embryogenesis. Adv. in Bioscience and Biotech., 5: 363-372. 
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