An efficient plant regeneration and Agrobacterium-mediated genetic transformation of Tagetes erecta

Abstract

Tagetes erecta, L. an asteraceous plant of industrial and medicinal value, contains important compounds like pyrethrins, thiophenes and lutein, possessing immense potential for insecticidal, nematicidal and nutraceutical activities. Considering the importance and demand for these natural compounds, genetic manipulation of this crop for better productivity of secondary metabolites holds great significance. A rapid and reproducible direct regeneration and genetic transformation system is the prerequisite for genetic manipulation of any crop. This paper elucidates the establishment of an efficient direct regeneration and transformation protocol of T. erecta using Agrobacterium tumefaciens. Investigation of the effects of different types of explants (Hypocotyls, cotyledonary leaves, rachis and leaf sections) and different BAP and GA3 combinations on the regeneration frequency of T. erecta suggested that the best regeneration frequency (66 %) with an average of 5.08 ± 0.09 shoot buds/explant was observed from hypocotyl explants cultured on media containing 1.5 mg/l BAP and 5 mg/l GA3. The transformation protocol was established using A. tumefaciens strain LBA4404, containing the binary vector pBI121, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Various parameters like optimization of kanamycin concentration (200 mg/l) for selection, standardization of cocultivation time (45 min) and acetosyringone concentration (150 μM) for obtaining higher transformation frequency were established using hypocotyl explants. The selected putative transgenic shoots were subsequently rooted on the Murashige and Skoog medium and transferred to the green house successfully. The plants were characterised by analysing the gus expression, amplification of 600 bp npt II fragment and Southern blot hybridization using the PCR-amplified gusA fragment as probe. The standardised protocol established during the study will open new vistas for genetic manipulation and introduction of desired genes for genetic improvement of T. erecta.

Establishment of a High-efficiency Genetic Transformation System of Cucumber (Cucumis sativus) using Csexpansin 10 (CsEXP10) Gene

“…Acetosyringone has been used to enhance Agrobacteriummediated genetic transformation (Mishra et al, 2013;Manickavasangam et al, 2013;Gupta and Rahman, 2015). In this invesitgation, we observed the effect of different concentrations of acetosyringone in the A. tumefaciens inoculum solution on the transformation efficiency of the cucumber cotyledonary node explants (Table 3).…”

Agrobacterium tumefaciens mediated genetic transformation of Ocimum gratissimum: A medicinally important crop

“…(600). A. tumefaciens cells were harvested by centrifugation at 5000 rpm Gupta and Rahman (2014). The explants were exposed to Agrobacterium inoculum having O.D.…”

“…and infection time A. tumefaciens strain LBA4404 containing the binary vector pBI121 ( Fig. 1) was used to infect the explants for optimization of various parameters crucial for ATMT as reported by Gupta and Rahman (2014). Single colony of A. tumefaciens was picked up with a sterile loop and was grown in Yeast extract broth (YEB) media in the presence of 50 mg/L kanamycin, 25 L kanamycin, 25 mg/L rifampicin and 250 mg/L of streptomycin at 28 • C for overnight at 200 rpm on incubator shaker to obtain the desired O.D.…”

<i>In vitro</i> regeneration and <i>Agrobacterium</i>-mediated transformation of male- sterile marigold (<i>Tagetes erecta</i> L.)

“…After the inoculation of A. tumefaciens strain LBA4404 harboring pIG121-Hm (Ohta et al 1990), the leaf explants were cultured on SIM and then the regenerated shoots were stained with 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) for visualization of the transgenic cells (Jefferson et al 1987). The male-sterile marigold used in our study is more sensitive to kanamycin than those used in previous studies (50-400 mg/L kanamycin has been used for selection in the previous studies; Gupta and Rahman 2015;Kim et al 2007;Vanegas et al 2006). In our case, most of the regenerated shoots showed necrosis after 3 to 4 weeks of cultivation on SIM even with low kanamycin concentration (20 mg/L).…”

“…They used explants of four different tissues, such as leaf primordium and shoot tips, as recipients of A. tumefaciens and performed histochemical detection of β-glucuronidase (GUS) activity in explants, 3 days after inoculation with Agrobacterium. Recently, Gupta and Rahman (2015) reported stable Agrobacteriummediated transformation of T. erecta and histochemical detection of GUS expression in transgenic leaves. All previous studies on transgenic marigold used different parts of seedlings and subsequent young plantlets, such as cotyledons and young leaves, as explants for Agrobacterium inoculation.…”