Abstract
An optimized regeneration method for commercial winter rape of the Bn1 line was proposed. Hypocotyl fragments of 6-day-old seedlings were used as explants. Regeneration occurred via organogenesis on an MS medium supplemented with 3 mg/L of 6-benzylaminopurine and 2 mg/L of 2-isopentyladenine. All obtained regenerated plants successfully formed roots on hormone-free media and were adapted to the soil conditions. The vernalization conditions were specified to promote budding and flowering at the level of 83.93 ± 5.33%. The PCR analysis using the ISSR 2 and ISSR 15 markers has shown that no somaclonal variations in Bn1 winter rape occur under the protocols based on the proposed methodology. Thus, the developed methodology is efficient and economically profitable for obtaining biotechnology-based winter rape plants.
Similar content being viewed by others
REFERENCES
Maheshwari, P., Selvaraj, G., and Kovalchuk, I., Optimization of Brassica napus (canola) explant regeneration for genetic transformation, New Biotechnol., 2011, vol. 29, no 1, pp. 144–155. https://doi.org/10.1016/j.nbt.2011.06.014
Hoang, T.G. and Raldugina, G.N., Regeneration of transgenic plants expressing the GFP gene from rape cotyledonary and leaf explants: effects of the genotype and ABA, Russ. J. Plant Physiol., 2012, vol. 59, no. 3, pp. 406–412. https://doi.org/10.1134/S1021443712030089
Hussain, S., Rasheed, A., Latif, M., Mahmood, T., and Saqlan Naqvi, S.M., Canola (Brassica napus L.) regeneration and transformation via hypocotyl and hypocotyl derived calli, Sarhad J. Agric., 2014, vol. 30, no. 2, pp. 165–172.
Bhalla, P.L. and Singh, M.B., Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea,Nat. Prot., 2008, vol. 3, no. 2, pp. 181–189. https://doi.org/10.1038/nprot.2007.527
Mashayekhi, M., Shakib, A.M., Ahmad-Raji, M., and Ghasemi Bezdi, K., Gene transformation potential of commercial canola (Brassica napus L.) cultivars using cotyledon and hypocotyl explants, Afr. J. Biotechnol., 2008, vol. 7, no. 24, pp. 4459–4463.
Rahnama, H. and Sheykhhasan, M., Transformation and light inducible expression of cry1Ab gene in oilseed rape (Brassica napus L.), J. Sci., 2016, vol. 27, no. 4, pp. 313–319.
Bates, R., Craze, M., and Wallington, E.J., Agrobacterium-mediated transformation of oilseed rape (Brassica napus), Curr. Prot. Plant Biol., 2017, vol. 2, pp. 287–298. https://doi.org/10.1002/cppb.20060
Ikeuchi, M., Ogawa, Y., Iwase, A., and Sugimoto, K., Plant regeneration: cellular origins and molecular mechanisms, Development, 2016, vol. 143, no. 9, pp. 1442–1451. https://doi.org/10.1242/dev.134668
Lone, J.A., Gupta, S.K., Wani, S.H., Bhat, M.A., and Lone, R.A., In vitro regeneration studies in Brassica napus with response to callus induction frequency and regeneration frequency, Int. J. Agric., Environ. Biotechnol., 2016, vol. 9, no. 5, pp. 755–761. https://doi.org/10.5958/2230-732X.2016.00098.X
Akter, S., Mollika, S.R., Sarker, R.H., and Hoque, M.I., Agrobacterium-mediated genetic transformation of two varieties of Brassica juncea (L.) using marker genes, Plant Tiss. Cult. Biotechnol., 2016, vol. 26, no. 1, pp. 55–65. https://doi.org/10.3329/bjar.v34i2.5802
Liu, X.X., Lang, S.R., Su, L.Q., Liu, X., and Wang, X.F., Improved Agrobacterium-mediated transformation and high efficiency of root formation from hypocotyl meristem of spring Brassica napus “Precocity” cultivar, Genet. Mol. Res., 2015, vol. 14, no. 4, pp. 16 840–16 855. https://doi.org/10.4238/2015
Hocheva, E.A., Sakhno, L.O., and Kuchuk, M.V., Method for producing transformed rape plants by method of agrobacterial transformation, UA Patent no. u200811768 model 39 205, 2009, Bull. 3.
Ravanfar, S.A., Orbovic, V., Moradpour, M., Abdul Aziz, M., Karan, R., Wallace, S., and Parajuli, S., Improvement of tissue culture, genetic transformation, and applications of biotechnology to Brassica,Biotechnol. Genet. Eng. Rev., 2017, vol. 33, no. 1, pp. 1–25. https://doi.org/10.1080/02648725.2017.1309821
Bairu, M.W., Aremu, A.O, and Staden, J., Somaclonal variation in plants: causes and detection methods, Plant Growth Regul., 2010, vol. 63, no. 2, pp. 147–173. https://doi.org/10.1007/s10725-010-9554-x
Reddy, M.P., Sarla, N., and Siddiq, E.A., Inter simple sequence repeat (ISSR) polymorphism and its application in plant breeding, Euphytica, 2002, vol. 128, pp. 9–17. https://doi.org/10.1023/A:1020691618797
Murashige, T. and Skoog, F., A revised medium for rapid growth and bio assays with tobacco tissue cultures, Physiol. Plant., 1962, vol. 15, pp. 473–497.
Rogers, S.O. and Bendich, A.J., Extraction of total cellular DNA from plants, algae and fungi, Plant Mol. Biol. Man., 1994, pp. 183–190. https://doi.org/10.1007/978-94-011-0511-8_12
Godwin, I., Aitken, E., and Smith, L., Application of inter simple sequence repeat (ISSR) markers to plant genetics, Electrophoresis, 1997, vol. 18, no. 9, pp. 1524–1528. https://doi.org/10.1002/elps.1150180906
Mahjooba, B., Zarinib, H.N., Hashemia, S.H., and Shamasbia, F.V., Comparison of ISSR, IRAP and REMAP markers for assessing genetic diversity in different species of Brassica sp., Russ. J. Genet., 2016, vol. 52, no. 12, pp. 1272–81. https://doi.org/10.1134/s1022795416120073
Debergh, P.C. and Zimmerman, R.H., Micropropagation: Technology and Application, Dordrecht: Kluwer Academic, 1991.
Savelieva, E.M. and Tarakanov, I.G., Control of flowering in canola plants with various response to photoperiodic and low-temperature induction, Izv. Timiryaz. Agricult. Acad., 2014, vol. 2, pp. 57–68.
Filek, M., Koscielniak, J., Macháčková, I., and Krekule, J., Generative development of winter rape (Brassica napus L.)—the role of vernalization, Int. J. Plant Dev. Biol., 2007, vol. 1, no. 1, pp. 57–63.
Waalen, W.M., Stavang, J.A., Olsen, J.E., and Rognli, O.A., The relationship between vernalization saturation and the maintenance of freezing tolerance in winter rapeseed, Environ. Exp. Bot., 2014, vol. 106, pp. 164–173. https://doi.org/10.1016/j.envexpbot.2014.02.012
Sakhno, L.A., Gocheva, E.A., Komarnitskii, I.K., and Kuchuk, N.V., Stable expression of the promoterless bar gene in transformed rapeseed plants, Cytol. Genet., 2008, vol. 42, no. 1, pp. 21–28.https://doi.org/10.1007/s11956-008-1003-7
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.
Additional information
Translated by N. Tarasyuk
About this article
Cite this article
Hnatyuk, I.S., Varchenko, O.I., Kuchuk, M.V. et al. Development of an Effective In Vitro Regeneration System for Ukrainian Breeding Winter Rape Brassica napus L.. Cytol. Genet. 54, 341–346 (2020). https://doi.org/10.3103/S0095452720040039
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.3103/S0095452720040039