Agrobacterium tumefaciens-mediated genetic transformation of sesame (Sesamum indicum L.)

Abstract

Sesame (Sesamum indicum) is an important oil seed crop that has not yet been transformed genetically. We report herein for the first time successful recovery of fertile transgenic plants of sesame from cotyledon explants inoculated with Agrobacterium tumefaciens carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a b-glucuronidase (GUS) gene (uidA) interrupted with an intron. Green shoots recovered from A. tumefaciens-infected explants on selection medium [Murashige and Skoog (MS) basal medium containing 25.0 lM benzyladenine (BA), 25.0 mg l -1 kanamycin and 400.0 mg l -1 cefotaxime] were rooted on MS basal medium containing 2.0 lM indole-3-butyric acid and 5.0 mg l -1 kanamycin. The rooted shoots were established in soil and grown to maturity to collect seeds. The presence, integration and expression of transgenes in putative T 0 plants were confirmed by polymerase chain reaction (PCR), Southern blot hybridization and GUS histochemical assay, respectively. GUS activity was detected in vegetative and reproductive parts of T 0 and T 1 plants. Presence of thiol compounds in coculture medium and kanamycin selection at shoot regeneration and at rooting stages were found to be critical for transformation. The transgenes were inherited in Mendelian fashion in T 1 progeny as detected by PCR. RT-PCR analysis of T 1 plants confirmed the presence of transcripts of uidA gene. The transformation frequency was 1.01%, and 22-24 weeks were required from seed to seed generation time. This protocol can be used to transfer new traits in sesame for quantitative and qualitative improvement.

Characterization of inhibitor(s) of β-glucuronidase enzyme activity in GUS-transgenic wheat

“…It is used to indicate transient and stable transformations (Serres et al 1992), and to estimate the relative contribution and strength of promoter elements in the transcriptional activation. Most recently, GUS histochemical assay was utilized in the production of markerfree Petunia hybrida and tomato (Khan et al , 2011, and in the production of a more efficient Agrobacterium tumefaciens-mediated transformation protocol in melon (Ntui et al 2010), in Lilium (Azadi et al 2010), in Petunia hybrida (Thirukkumaran et al 2009), in pineapple (Gangopadhyay et al 2009), in Piper colubrinum (Mani and Manjula 2011), in sesame (Yadav et al 2010), in…”

Metabolic engineering of fatty acid biosynthetic pathway in sesame (Sesamum indicum L.): assembling tools to develop nutritionally desirable sesame seed oil

“…In light of the recent findings, many genes controlling the TAG biosynthetic pathway were identified, though the detail of FAD3 gene is yet to reveal. From the past few years reports of sesame transformation (Yadav et al 2010;Al-Shafeay et al 2011;Chowdhury et al 2014) were well documented. Although, a detailed knowledge of the FA biosynthetic pathway involved in lipid synthesis for sesame is not documented, but FA profile suggested that, other related vegetable oil plant FA synthesis definitely carry the similarity with sesame.…”

Sesame

“…Tissue culture and regeneration through in vitro culturing can speed up breeding process by producing a number of stable regenerants via callus or somatic embryogenesis in a short span of time. In sesame in vitro culturing, cotyledon (Yadav et al 2010 ) hypocotyl and shoot tips (Baskaran and Jayabalan 2006 ) have been reported to be more responsive to callus induction and plant regeneration. Appropriate concentrations of plant growth regulators and their combinations are very important to achieve successful plant regeneration from cultured cells and tissues, and were optimized in different studies.…”

“…Appropriate concentrations of plant growth regulators and their combinations are very important to achieve successful plant regeneration from cultured cells and tissues, and were optimized in different studies. Application of BAP (benzylaminopurine) in the nutrient media was reported essential and the most effective cytokinin for shoot induction and plant regeneration in S. indicum (Yadav et al 2010 ) . Baskaran and Jayabalan ( 2006 ) studied the effects of plant growth regulators on callus induction in hypocotyls and cotyledon explants of sesame, and reported callus induction on media containing 2.2-22.6 m M 2, 4-D and 2.6-26.8-m M NAA ( a -naphthalene acetic acid), increased shoot proliferation on BAP and Kn (kinetin), whereas rooting took place on NAA (8.0 m M).…”

“…A signifi cant interaction between hormonal concentration and macronutrients for plant regeneration was recorded, and application of 20-m M TDZ along with 2.5-m M IAA was found the best for successful plant regeneration. Yadav et al ( 2010 ) optimized an A. tumefaciens -mediated transformation protocol to generate fertile transgenic sesame plants. In this method, cotyledon explants were used for plant regeneration via multiple shoot organogenesis.…”