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volume 37 issue 4 (august 2014) : 345-352,   Doi: 10.5958/0976-0571.2014.00642.0

ASSESSMENT OF SOMACLONAL VARIATION AND STABILITY IN IN VITRO REGENERATED GRASSPEA PLANTS USING SDS-PAGE

S
Surendra Barpete
*
*
N
N.C. Sharma
S
Shiv Kumar1
1Department of Biochemistry and Genetics, Barkatullah University, Bhopal–462 001, India
Cite article:- Barpete Surendra, *, Sharma N.C., Kumar1 Shiv (2025). ASSESSMENT OF SOMACLONAL VARIATION AND STABILITY IN IN VITRO REGENERATED GRASSPEA PLANTS USING SDS-PAGE. Legume Research. 37(4): 345-352. doi: 10.5958/0976-0571.2014.00642.0.

ABSTRACT

Tissue culture may be one of the possible sources of variation for crop improvement. To assess variation and stability in regenerated plants, shoots were regenerated from the callus derived from axillary explants of 11 grasspea genotypes, and their shoot protein profiles were compared with those of seed cultured plants. The highest response of callus induction (87%) was observed when 2.0 mg/l2,4-dichlorophenoxyacetic acid(2,4-D) and 0.25 mg/l BAP were supplemented in Murashige and Skoog’s (MS) medium. a-naphthaleneacetic acid(NAA) and 6-benzylaminopurine(BAP) showed variable redifferentiation response along with callus formation. The MS medium supplemented with 0.5 mg/l BAP showed better multiplication and elongation of shoots.Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a unique protein band of 43 kDa in both tissue and seed cultured plants of Pusa 24. Polypeptide banding pattern of regenerated plants from 11 grasspea genotypes did not deviate from the banding pattern of parental seed protein. Similarity coefficient values ranged from 0.37 to 0.85 with a mean of 0.43 among the 55 genotypic combinations. Comparison of protein bands between calli raised regenerated shoots and parental seeds revealed the absence of somaclonal variation in regenerated plants, suggesting that the regeneration protocol used in the present study can be used for genomics enabled improvement in grasspea without the risk of additional variation or instability.

REFERENCES

  1. Bapat, S.A., Rawal, S.K., Mascarenhas, A.F. (1992).Isozyme profiles during ontogeny of somatic embryos in wheat (Triticum aestivum). Plant Sci. 82:235-242.
  2. Barik, D.P., Mohapatra, U., Chand, P.K.(2005).High frequency in-vitro regeneration of Lathyrussativus L. Biol. Planta. 49:637-639.
  3. Barik, D.P., Naik, S.K., Mohapatra, U., Chand, P.K. (2004). High frequency plant regeneration by in-vitro shoot proliferation in cotyledonary node explants of grasspea (LathyrussativusL.). In vitro Cell Dev. Biol. Plant 40:467-470.
  4. Barpete, S., Sharma, N.C., Parmar, D., Dhingra, M. (2009).In vitro induction of multiple shoots and plant regeneration of Khesari Dal (Lathyrussativus L.) using cotyledonary node explant.Indian Appl. Pure Biol.24:81-86.
  5. Barpete, S., Sharma, N.C., Parmar, D., Dhingra, M. (2008).In-Vitro Regeneration of Lathyrussativus L.Nat. Life Sci.05:235-238.
  6. Bekheet, S.A., Taha, H.S., Matter, M.A. (2007).In vitro regeneration of sugar beet propagules and molecular analysis of the regenerants. Arab J.Biotechnol.10:321-332.
  7. Celebi, A., Acik, L., Aytac, Z. (2009). Biosystematics studies among EbenusL. species based on morphological, RAPD- PCR and seed protein analyses in Turkey. Pakistan J. Bot.41:2477-2486.
  8. Gharyal, P.K., Maheshwari, S.C. (1980). Plantlet formation from callus cultures of a legume, Lathyrussativus cv. L.S.D.-3.Pflanzen Physiol.100:359-362.
  9. Girma, D., Korbu, L.J. (2012). Genetic improvement of grass pea (Lathyrussativus) in Ethiopia: an unfulfilled promise. Plant Breeding 131:231-236. doi:10.1111/j.1439- 523.2011.01935.x.
  10. Girma, D. (2010). Ethiopian Grass Pea (Lathyrussativus L.) Started the Genomics Era.Lambert Academic Publishing, Koeln, Germany.
  11. Hameed, A., Shah, T.M., Atta, B.M., Iqbal, N., Haq, M.A., Ali, H. (2009).Comparative seed storage protein profiling of Kabuli chickpea genotypes. Pakistan J. Bot.41:703-710.
  12. Hanbury, C.D., White, C.L., Mullan, B.P., Siddique, K.H.M. (2000). A review of the potential of Lathyrussativus L and L. Cicera L seed for use as animal feed.Anim. Feed Sci. Tech.87:1-27.
  13. Jaccard, P. (1908).Nouvellesrecherchessur la distribution florale.Bull. Soc.Vaudoise Sci. Nat.44:223-270.
  14. Kendir, H., Sahin, D.N., Khawar, K.M., Ozcan, S. (2009).In-vitro plant regeneration from Turkish Grasspea (Lathyrussativus L.) using immature zygotic embryo explant.Agricult. Environ.Biotechn. 23:1177-1180.
  15. Kiarostami, K.H., Ebrahimzadeh, H. (2001).Changes of proteins and oxidative enzymes in seeds in vitro regeneration plants of three Iranian cultivars of wheat (Triticumaestivusm L.).Pakistan J. Bot. 33:257-266.
  16. Kumar, S., Bejiga, G., Ahmed, S., Nakkoul, H., Sarkar, A. (2011).Genetic Improvement of grasspea for low neurotoxin (â-ODAP) content.Food Chem.Toxicol.49:589-600.
  17. Kumar, S., Reddy, T.P., Reddy, G.M.(1983). Plantlet regeneration from different callus culture of Pigeon pea (CajanuscajanL.). Plant Sci.Lett.32:271-278.
  18. Lammeli, U.K. (1970).Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227:680–685.
  19. Malik, K.A., Ali-Khan, S.T., Saxena, P.K. (1993).High frequency organogenesis from direct seed culture in Lathyrus. Ann. Bot.72:629-637.
  20. Murashige, T., Skoog, F. (1962).A revised medium for rapid growth and bioassays with tobacco tissue culture.Plant Physiol.15:473-497.
  21. Przybylska, J., Przybylsk, Z.Z., Kranjewski, P. (2000). Diversity of seed globulins in Lathyrussativus L. and some related species. Genet.Resour.Crop Evol.47:239-246.
  22. Rao, S.L.N. (1978).A sensitive and specific colorimetric method for the determination of alpha, beta-ODAP, the Lathyrussativus neurotoxin. Ann.Biochem.86:386-395.
  23. Reinert, J., Bajaj, Y.P.S. (1977).Applied and Fundamental Aspects of Plant Cell, Tissue and Oragan Culture.Springer- Verlag Berlin, Heidelberg, New York.
  24. Rohlf, F.J. (1998). NTSYS-pc: Numerical Taxonomy and Multivariate Analysis System. Version 2.1 Exeter Publications, NY.
  25. Roy, P.K., Singh, B., Mehta, S.L., Barat, G.K., Gupta, N., Kirti, P.B., Chopra, V.L. (1991).Plant regeneration from leaf disc of Lathyrussativus.Indian Exp.Biol.29:327-330.
  26. Sammour, H.R., Mustafa, A.E.Z., Badr, S., Tahr, W. (2007).Genetic variability of some quality traits in LathyrusSpp. Germplasm.ActaAgricult.Slovenica90:33-43.
  27. Santha, I.M., Mehta, S.L. (2001).Development of low ODAP somaclones of Lathyrussativus. Lathyrus Lathyrism Newslett. 2:42.
  28. Sinha, R.R., Das, K., Sen, S.K. (1982).Plant regeneration from stem-derived callus of the seed legume LathyrussativusL. Plant Cell Tissue Organ Cult.2:67-76.
  29. Sneath, P.H.A., Sokal, R.R. (1973). Numerical taxonomy: The principles and practice of numerical classification. - Freeman and Company, San Francisco.
  30. Sujatha, M., Kumar, D.V. (2007).In vitro bud regeneration of Carthamustinctoriusand wild Carthamus species from leaf explants and axillary buds.BiolPlanta.51:782-786.
  31. Van-Dorrestein, B., Baum, M., Abd-El-Moneim, A.M. (1998).Use of somaclonal variation in Lathyrussativus (grass pea) to select variants with low ODAP concentration. In: Proceedings of the 3rd European Conference on Grain Legumes, Valladolid, Spain, p. 364.
  32. Zaher, E.A., Mustafa, M.A. (2007).Genetic variation among Egyptian cultivars of Viciafeba L.Pakistan J BiolSci10:4204-4210.
  33. Zambre, M., Chowdharey, B., Kuo, Y.H., Montagu, M.V., Angenon, G., Lambein, F. (2002). Prolific regeneration of fertile plants from green nodular callus induced from meristematic tissue in LathyrussativusL. (grasspea). Plant Sci.163:1107– 1112.
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    volume 37 issue 4 (august 2014) : 345-352,   Doi: 10.5958/0976-0571.2014.00642.0

    ASSESSMENT OF SOMACLONAL VARIATION AND STABILITY IN IN VITRO REGENERATED GRASSPEA PLANTS USING SDS-PAGE

    S
    Surendra Barpete
    *
    *
    N
    N.C. Sharma
    S
    Shiv Kumar1
    1Department of Biochemistry and Genetics, Barkatullah University, Bhopal–462 001, India
    Cite article:- Barpete Surendra, *, Sharma N.C., Kumar1 Shiv (2025). ASSESSMENT OF SOMACLONAL VARIATION AND STABILITY IN IN VITRO REGENERATED GRASSPEA PLANTS USING SDS-PAGE. Legume Research. 37(4): 345-352. doi: 10.5958/0976-0571.2014.00642.0.

    ABSTRACT

    Tissue culture may be one of the possible sources of variation for crop improvement. To assess variation and stability in regenerated plants, shoots were regenerated from the callus derived from axillary explants of 11 grasspea genotypes, and their shoot protein profiles were compared with those of seed cultured plants. The highest response of callus induction (87%) was observed when 2.0 mg/l2,4-dichlorophenoxyacetic acid(2,4-D) and 0.25 mg/l BAP were supplemented in Murashige and Skoog’s (MS) medium. a-naphthaleneacetic acid(NAA) and 6-benzylaminopurine(BAP) showed variable redifferentiation response along with callus formation. The MS medium supplemented with 0.5 mg/l BAP showed better multiplication and elongation of shoots.Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a unique protein band of 43 kDa in both tissue and seed cultured plants of Pusa 24. Polypeptide banding pattern of regenerated plants from 11 grasspea genotypes did not deviate from the banding pattern of parental seed protein. Similarity coefficient values ranged from 0.37 to 0.85 with a mean of 0.43 among the 55 genotypic combinations. Comparison of protein bands between calli raised regenerated shoots and parental seeds revealed the absence of somaclonal variation in regenerated plants, suggesting that the regeneration protocol used in the present study can be used for genomics enabled improvement in grasspea without the risk of additional variation or instability.

    REFERENCES

    1. Bapat, S.A., Rawal, S.K., Mascarenhas, A.F. (1992).Isozyme profiles during ontogeny of somatic embryos in wheat (Triticum aestivum). Plant Sci. 82:235-242.
    2. Barik, D.P., Mohapatra, U., Chand, P.K.(2005).High frequency in-vitro regeneration of Lathyrussativus L. Biol. Planta. 49:637-639.
    3. Barik, D.P., Naik, S.K., Mohapatra, U., Chand, P.K. (2004). High frequency plant regeneration by in-vitro shoot proliferation in cotyledonary node explants of grasspea (LathyrussativusL.). In vitro Cell Dev. Biol. Plant 40:467-470.
    4. Barpete, S., Sharma, N.C., Parmar, D., Dhingra, M. (2009).In vitro induction of multiple shoots and plant regeneration of Khesari Dal (Lathyrussativus L.) using cotyledonary node explant.Indian Appl. Pure Biol.24:81-86.
    5. Barpete, S., Sharma, N.C., Parmar, D., Dhingra, M. (2008).In-Vitro Regeneration of Lathyrussativus L.Nat. Life Sci.05:235-238.
    6. Bekheet, S.A., Taha, H.S., Matter, M.A. (2007).In vitro regeneration of sugar beet propagules and molecular analysis of the regenerants. Arab J.Biotechnol.10:321-332.
    7. Celebi, A., Acik, L., Aytac, Z. (2009). Biosystematics studies among EbenusL. species based on morphological, RAPD- PCR and seed protein analyses in Turkey. Pakistan J. Bot.41:2477-2486.
    8. Gharyal, P.K., Maheshwari, S.C. (1980). Plantlet formation from callus cultures of a legume, Lathyrussativus cv. L.S.D.-3.Pflanzen Physiol.100:359-362.
    9. Girma, D., Korbu, L.J. (2012). Genetic improvement of grass pea (Lathyrussativus) in Ethiopia: an unfulfilled promise. Plant Breeding 131:231-236. doi:10.1111/j.1439- 523.2011.01935.x.
    10. Girma, D. (2010). Ethiopian Grass Pea (Lathyrussativus L.) Started the Genomics Era.Lambert Academic Publishing, Koeln, Germany.
    11. Hameed, A., Shah, T.M., Atta, B.M., Iqbal, N., Haq, M.A., Ali, H. (2009).Comparative seed storage protein profiling of Kabuli chickpea genotypes. Pakistan J. Bot.41:703-710.
    12. Hanbury, C.D., White, C.L., Mullan, B.P., Siddique, K.H.M. (2000). A review of the potential of Lathyrussativus L and L. Cicera L seed for use as animal feed.Anim. Feed Sci. Tech.87:1-27.
    13. Jaccard, P. (1908).Nouvellesrecherchessur la distribution florale.Bull. Soc.Vaudoise Sci. Nat.44:223-270.
    14. Kendir, H., Sahin, D.N., Khawar, K.M., Ozcan, S. (2009).In-vitro plant regeneration from Turkish Grasspea (Lathyrussativus L.) using immature zygotic embryo explant.Agricult. Environ.Biotechn. 23:1177-1180.
    15. Kiarostami, K.H., Ebrahimzadeh, H. (2001).Changes of proteins and oxidative enzymes in seeds in vitro regeneration plants of three Iranian cultivars of wheat (Triticumaestivusm L.).Pakistan J. Bot. 33:257-266.
    16. Kumar, S., Bejiga, G., Ahmed, S., Nakkoul, H., Sarkar, A. (2011).Genetic Improvement of grasspea for low neurotoxin (â-ODAP) content.Food Chem.Toxicol.49:589-600.
    17. Kumar, S., Reddy, T.P., Reddy, G.M.(1983). Plantlet regeneration from different callus culture of Pigeon pea (CajanuscajanL.). Plant Sci.Lett.32:271-278.
    18. Lammeli, U.K. (1970).Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227:680–685.
    19. Malik, K.A., Ali-Khan, S.T., Saxena, P.K. (1993).High frequency organogenesis from direct seed culture in Lathyrus. Ann. Bot.72:629-637.
    20. Murashige, T., Skoog, F. (1962).A revised medium for rapid growth and bioassays with tobacco tissue culture.Plant Physiol.15:473-497.
    21. Przybylska, J., Przybylsk, Z.Z., Kranjewski, P. (2000). Diversity of seed globulins in Lathyrussativus L. and some related species. Genet.Resour.Crop Evol.47:239-246.
    22. Rao, S.L.N. (1978).A sensitive and specific colorimetric method for the determination of alpha, beta-ODAP, the Lathyrussativus neurotoxin. Ann.Biochem.86:386-395.
    23. Reinert, J., Bajaj, Y.P.S. (1977).Applied and Fundamental Aspects of Plant Cell, Tissue and Oragan Culture.Springer- Verlag Berlin, Heidelberg, New York.
    24. Rohlf, F.J. (1998). NTSYS-pc: Numerical Taxonomy and Multivariate Analysis System. Version 2.1 Exeter Publications, NY.
    25. Roy, P.K., Singh, B., Mehta, S.L., Barat, G.K., Gupta, N., Kirti, P.B., Chopra, V.L. (1991).Plant regeneration from leaf disc of Lathyrussativus.Indian Exp.Biol.29:327-330.
    26. Sammour, H.R., Mustafa, A.E.Z., Badr, S., Tahr, W. (2007).Genetic variability of some quality traits in LathyrusSpp. Germplasm.ActaAgricult.Slovenica90:33-43.
    27. Santha, I.M., Mehta, S.L. (2001).Development of low ODAP somaclones of Lathyrussativus. Lathyrus Lathyrism Newslett. 2:42.
    28. Sinha, R.R., Das, K., Sen, S.K. (1982).Plant regeneration from stem-derived callus of the seed legume LathyrussativusL. Plant Cell Tissue Organ Cult.2:67-76.
    29. Sneath, P.H.A., Sokal, R.R. (1973). Numerical taxonomy: The principles and practice of numerical classification. - Freeman and Company, San Francisco.
    30. Sujatha, M., Kumar, D.V. (2007).In vitro bud regeneration of Carthamustinctoriusand wild Carthamus species from leaf explants and axillary buds.BiolPlanta.51:782-786.
    31. Van-Dorrestein, B., Baum, M., Abd-El-Moneim, A.M. (1998).Use of somaclonal variation in Lathyrussativus (grass pea) to select variants with low ODAP concentration. In: Proceedings of the 3rd European Conference on Grain Legumes, Valladolid, Spain, p. 364.
    32. Zaher, E.A., Mustafa, M.A. (2007).Genetic variation among Egyptian cultivars of Viciafeba L.Pakistan J BiolSci10:4204-4210.
    33. Zambre, M., Chowdharey, B., Kuo, Y.H., Montagu, M.V., Angenon, G., Lambein, F. (2002). Prolific regeneration of fertile plants from green nodular callus induced from meristematic tissue in LathyrussativusL. (grasspea). Plant Sci.163:1107– 1112.
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