Abstract
An in vitro plant generation and genetic transformation protocol was established in sesame (Sesamum indicum L. cv Rama) through biolistic particle gun bombardment. 5-day-old apical, meristematic tissues of in vitro-germinating seedlings were used as explants. 10–15 Multiple shoots were generated from each explant using Murashige and Skoog basal medium containing 18.0 µM benzylamino purine and 5.37 µM naphthalene acetic acid. Four independent sets of transformation were carried out and each set consisted of three independent experiments each comprising three replications with 30 explants per replication. A synthetically designed bialaphos resistance gene (bar) was used for transformation. The positive transformants containing the bar gene were selected in growth medium containing 2.5 mg/L bialaphos. Green shoots recovered from bombarded explants were subjected to root development on Murashige and Skoog basal medium containing 5.37 µM naphthalene acetic acid. The rooted shoots were established in soil and grown to maturity in greenhouse. Polymerase chain reaction (PCR), Southern and reverse-transcription PCR, real-time quantitative PCR, western blot and enzymatic assay of four putative transformants from independent sets provided evidence for full-length gene integration as well as high level expression of the transgene. Analysis of the T1 plants revealed a stable inheritance of the transgene through the progenies. This is the first report of biolistic mediated stable transformation of sesame and should pave the way for future genetic engineering strategies to be employed for improvement of this very important oil-seed crop.
Similar content being viewed by others
References
Agrawal PK, Kohli A, Twyman RM, Christou P (2005) Transformation of plants with multiple cassettes generates simple transgene integration patterns and high expression levels. Mol Breed 16:247–260
Al-Shafeay AF, Ibrahim AS, Nesiem MR, Tawfik MS (2011) Establishment of regeneration and transformation system in Egyptian sesame (Sesamum indicum L.) cv Sohag1. GM Crops 2:182–192
Aragao FJL, Sarokin L, Vianna GR, Rech EL (2000) Selection of transgenic meristematic cells utilizing a herbicidal molecule results in the recovery of fertile transgenic soybean (Glycine max L. Merril) plants at high frequency. Theor Appl Genet 101:1–6
Ashri A (1989) Sesame. In: Robbelen G, Downey RK, Ashri A (eds) Oil crops of the world: their breeding and utilization. McGraw Hill, New York, pp 375–387
Barampuram S, Zhang ZJ (2011) Recent advances in plant transformation. Birchler JA (ed) Plant chromosome engineering: methods and protocols. Methods Mol Biol 701:1–32. doi:10.1007/978-1-61737-957-4_1
Bhaskaran P, Jayabalan N (2006) In vitro mass propagation and diverse callus orientation on Sesamum indicum L.—an important oil plant. J Agric Tech 2:259–269
Bhattacharyya J, Chakraborty A, Roy S, Pradhan S, Mitra J, Chakraborty M, Manna A, Sikdar N, Chakraborty S, Sen SK (2015) Genetic transformation of cultivated jute (Corchorus capsularis L.) by particle bombardment using apical meristem tissue and development of stable transgenic plant. Plant Cell Tiss Organ Cult 121:311–324
Biabani AR, Pakniyat H (2008) Evaluation of seed yield-related characters in sesame (Sesamum indicum L.) using factor and path analysis. Pak J Biol Sci 11(8):1157–1160
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Breitler JC, Labeyrie A, Meynard D, Legavre T, Guiderdoni E (2002) Efficient microprojectile bombardment mediated transformation of rice using gene cassettes. Theor Appl Genet 104:709–719
Campbell BT, Baeziger PS, Mitra A, Sato S, Clemente T (2000) Inheritance of multiple genes in wheat. Crop Sci 40:1133–1141
Cao J, Duan X, McElroy D, Wu R (1992) Regeneration of herbicide resistant transgenic rice plants following microprojectile mediated transformation of suspension culture cells. Plant Cell Rep 11:586–591
Chakraborti P, Ghosh A (2009) Variation in callus induction and root-shoot bud formation depend on seed coat of sesame genotypes. Res J Bot 5:14–19
Chattopadhyaya B, Banerjee J, Basu A, Sen SK, Maiti MK (2010) Shoot induction and regeneration using internodal transverse thin cell layer culture in Sesamum indicum L. Plant Biotechnol Rep 4:173–178
Chowdhury S, Basu A, Ray Chaudhuri T, Kundu S (2014a) In-vitro characterization of the behaviour of Macrophomina phaseolina (Tassi) Goid at the rhizosphere and during early infection of roots of resistant and susceptible varieties of sesame. Eur J Plant Pathol 138:361–375
Chowdhury S, Basu A, Kundu S (2014b) A new high-frequency Agrobacterium-mediated transformation technique for Sesamum indicum L. using de-embryonated cotyledon as explant. Protoplasma. doi:10.1007/s00709-014-0625-0)
Christou P (1995) Strategies for variety-independent genetic transformation of important cereals, legumes and woody species utilizing particle bombardment. Euphytica 85:13–27
Christou P, McCabe DE, Martinell BJ, Swain WF (1990) Soybean genetic engineering—commercial production of transgenic plants. Trends Biotechnol 8:145–151
Christou P, Ford TL, Kofron M (1991) Production of transgenic rice (Oryzae sativa L.) plant from agronomically important Indica and Japonica varieties via electrically discharged particle acceleration of exogenous DNA into immature zygotic embryo. Biotechnology 9:957–962
Datta SK, Peterhans A, Datta K, Potrykus I (1990) Genetically engineered fertile Indica rice recovered from protoplasts. Nat Biotechnol 8:736–740
De Block M, Botterman J, Vandewiele M, Dockx J, Thoen C, Gossele V, Movva NR, Thompson C, Van Montagu M, Leemans J (1987) Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J 6:2513–2518
DeBuck S, Van Montagu M, Depicker A (2001) Transgene silencing of invertedly repeated transgenes is released upon deletion of one of the transgenes involved. Plant Mol Biol 46:433–445
DeBuck S, Windels P, De-Loose M, Depicker A (2004) Single-copy T-DNAs integrated at different positions in the Arabidopsis genome display uniform and comparable beta-glucuronidase accumulation levels. Cell Mol Life Sci 61:2632–2645
Devi PB, Sticklen MB (2003) In vitro culture and genetic transformation of sorghum by microprojectile bombardment. Plant Biosyst 137:249–254
Doyle J, Doyle J (1990) Isolation of plant DNA from fresh tissue. BRL FOCUS 12:13–15
FAOSTAT (2008) http://faostat.fao.org
George L, Bapat VA, Rao PS (1987) In vitro multiplication of sesame (Sesamum indicum L. CV. PT) through tissue culture. Ann Bot 60:17–22
George L, Bapat VA, Rao PS (1989) Plant regeneration in vitro in different cultivars of sesame (Sesamum indicum L.). Proc Indian Acad Sci (Plant Sci.) 99(2):135–137
Ghosh M, Saha T, Nayak P, Sen SK (2002) Genetic transformation by particle bombardment of cultivated jute, Corchorus capsularis L. Plant Cell Rep 20:936–942
Gordon-Kamm WJ, Spencer TM, Mangano ML et al (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2:603–618
Grantham R, Perrin P, Mouchiroud D (1986) Pattern of codon usage of different kinds of species. Oxford Surv Evol Biol 3:48–81
Jin UH, Chun JA, Han MO, Lee JW, Yi YB, Lee SW, Chung CH (2005) Sesame hairy root cultures for extra-cellular production of a recombinant fungal phytase. Prog Biochem 40:3754–3762
Kamal-Eldin A, Appleqvist LǺ (1994) Variation in the compositions of sterols, tocopherols and lignans in seed oils from four Sesamum species. J Am Oil Chem Soc 71:149–156
Lowe BA, Prakash NS, Melissa W, Mann MT, Spencer TM, Boddupalli RS (2009) Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA. Transgenic Res 18:831–840. doi:10.1007/s11248-009-9265-0
Mary RJ, Jayabalan N (1997) Influence of growth regulators on somatic embryogenesis in sesame. Plant Cell Tissue Organ Cult 49:67–70
Matzke AJM, Matzke MA (1998) Position effects and epigenetic silencing of plant transgenes. Curr Opin Plant Biol 1:142–148
Mondal N, Bhat VK, Srivastava SP (2010) Variation in fatty acid composition in Indian germplasm of sesame. J Am Oil Chem Soc 87:1263–1269
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Phys Plant 15:437–497
Murray EE, Lotzer J, Eberle M (1989) Codon use in plant genes. Nucl Acids Res 17:477–498
Ogasawara T, Chiba K, Tada M (1993) Production and high yield of napthoquinone by a hairy root culture of Sesamum indicum. Phytochemistry 33:1095–1098
Ram R, Catlin D, Romero J, Cowley C (1990) Sesame: new approaches for crop improvement. In: Janick J, Simon JE (eds) Advances in new crops. Timber Press, Portland, pp 225–228
Rathore KS, Chowdhury VK, Hodges TK (1993) Use of bar as a selectable marker gene and for the production of herbicide resistant rice plants from protoplast. Plant Mol Biol 21:871–884
Schmidt MA, Lafayette PR, Artelt BA, Parrott WA (2008) A comparison of strategies for transformation with multiple genes via microprojectile-mediated bombardment. In Vitro Cell Dev Biol Plant 44:162–168
Seo HY, Kim YJ, Park TI, Kim HS, Yun SJ, Park KH, Oh MK, ChoiMY PaikCH, Lee YS, Choi YE (2007) High-frequency plant regeneration via adventitious shoot formation from de-embryonated cotyledon explants of Sesamum indicum L. In Vitro Cell Dev Biol Plant 43:209–214
Shashidhara N, Ravikumar H, Ashoka N, Santosh DT, Pawar P, Lokesha R, Janagoudar BS (2011) Callus induction and sub-culturing in sesame (Sesamum indicum L.): a basic strategy. Int J Agric Env Biotechnol 42:153–156
Shaw WV (1975) Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol 43:737–755
Silme RS, Cagirgan MI (2010) Screening for resistance to Fusarium wilt in induced mutants and world collection of sesame under intensive management. Turk J Field Crops 15:89–93
Sticklen MB, Oraby HF (2005) Shoot apical meristem: a sustainable explant for genetic transformation of cereal crops. In Vitro Cell Dev Biol Plant 41:187–200
Taskin K, Ercan A, Turgut K (1999) Agrobacterium tumefaciens-mediated transformation of sesame (Sesamum indicum L.). Tr J Bot 23:291–295
Tiwari S, Kumar S, Gontia I (2011) Biotechnological approaches for sesame (Sesamum indicum L.) and Niger (Guizotia abyssinica L.f. Cass.). As Pac J Mol Biol Biotechnol 19(1):2–9
Verwoerd TC, Dekker BM, Hoekema A (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nucl Acids Res 17:2362
Wang Y, Zhu W, Levy DE (2006) Nuclear and cytoplasmic mRNA quantification by SYBR green based real-time RT-PCR. Methods 39:356–362
Were BA, Gudu S, Onkware AO, Carlson AS, Welander M (2006) In vitro regeneration of sesame (Sesamum indicum L.) from seedling cotyledon and hypocotyl explants. Plant Cell Tissue Organ Cult 85:235–239
Wilmink A, Dons JJM (1993) Selective agents and marker genes for use in transformation of monocotyledonous plant. Plant Mol Biol Rep 11:165–185
Xu ZQ, Jia JF, Hu ZD (1997) Somatic embryogenesis in Sesamum indicum L. cv. Nigrum. J. Plant Physiol 150:755–758
Yadav M, Chaudhary D, Sainger M, Jaiwal PK (2010) Agrobacterium tumefaciens mediated genetic transformation of sesame (Sesamum indicum L.). Plant Cell Tissue Organ Cult 103:377–386
Younghee K (2007) Effects of BA, NAA, 2, 4-D and AgNO3 treatments on the callus induction and shoot regeneration from hypocotyl and cotyledon of sesame (Sesamum indicum L.). J Korean Soc Hort Sci 142:70–74
Zhang H, Miao H, Wang L, Qu L, Liu H, Wang Q, Yue M (2013) Genome sequencing of important oilseed crop Sesamum indicum L. Genome Biol 14:401
Zhong H, Sun B, Warkentin D, Zhang S, Wu R, Wu T, Sticklen MB (1996) The competence of maize shoot meristems for integrative transformation and inherited expression of transgenes. Plant Physiol 110:1097–1107
Acknowledgments
The authors extend their sincere appreciation for Shila Bhattacharjee for her extensive assistance in the tissue culture. The authors are also thankful to Sudarshan Maity, Uttam Dogra and Subhash Ghosh for maintenance and rearing of transgenic sesame plants under containment conditions. Grant support to the laboratory from Indian Council of Agricultural Research (ICAR) is thankfully acknowledged. Finally, the authors express their sincere gratitude to the distinguished editor and the anonymous reviewers, whose incisive comments were of immense help for improvement of the quality of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Bhattacharyya, J., Chakraborty, A., Mitra, J. et al. Genetic transformation of cultivated sesame (Sesamum indicum L. cv Rama) through particle bombardment using 5-day-old apical, meristematic tissues of germinating seedlings. Plant Cell Tiss Organ Cult 123, 455–466 (2015). https://doi.org/10.1007/s11240-015-0848-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-015-0848-6