Effects of explant age, germination medium, pre-culture parameters, inoculation medium, pH, washing medium, and selection regime on Agrobacterium-mediated transformation of tomato

Abstract

An efficient protocol was developed for Agrobacterium tumefaciens-mediated transformation of tomato (Solanum lycopersicum) cultivars using cotyledon explants. The transformation frequency was assessed in response to several different factors, including seed germination medium, seedling age, pre-culture duration, pre-culture and cocultivation media, inoculation medium, medium pH, washing medium, and kanamycin concentration in initial selection medium. Cotyledons excised from 6-d-old seedlings germinated on half-strength Murashige and Skoog's (MS) basal medium containing 8.9 μM benzyladenine (BA) produced the most suitable explant material. Six days of explant pre-culture and 5 min inoculation with Agrobacterium culture in MS medium, containing 8.9 μM BA, 9.3 μM kinetin, and 0.4 mg l −1 thiamine at pH 5.0, significantly improved the transformation frequency. The addition of a tobacco feeder cell layer, however, did not lead to any significant improvement in the transformation rate. Kanamycin at 20 mg l −1 in the selection medium for the initial 10 d resulted in the highest transformation frequency. Combining the best conditions for each parameter resulted in an overall transformation efficiency of 44.3 %. Gene transfer was confirmed through PCR and Southern blot analyses. Mendelian inheritance ratios were found in 71.5 % of the independent transgenic lines from self-fertilized T 1 progeny. The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh (H-86), Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivars.

Genetic Improvement of Tomato (Solanum lycopersicum) with AtDREB1A Gene for Cold Stress Tolerance using Optimized Agrobacterium-mediated Transformation System

“…The pre-culture period increases the regeneration percentage by eliminating the hypersensitive response of explants induced by Agrobacterium in co-cultivation stage (Arcos-Ortega et al, 2010). The pre-culture period is an important factor that significantly affects the TE in tomato (Rai et al, 2012). TE is closely linked with cell division or dedifferentiation (Arias et al, 2006).…”

“…These differences might be due to difference in Agrobacterium strain, type and age of explants and genotypes. Rai et al (2012) optimized an efficient Agrobacterium-mediated transformation protocol for tomato cultivars using cotyledons explants and reported that six days pre-culturing period significantly improved the TE upto 44.3% in tomato. Similarly, Duan et al (2010) conducted a study on tomato cv.…”

Screening and kinetic studies of catharanthine and ajmalicine accumulation and their correlation with growth biomass inCatharanthus roseushairy roots

“…Putative transformants were obtained by co-culturing the explants with A. rhizogenes for 0-3 d. After 12 d of culture (Table 1), the hairy root started appearing from wounded sites of explants co-cultured for 3 d with both the bacterial strains ( Figure 1A and B), while in other explants co-cultured for 0 and 2 d, the hairy root appeared after more than 12 d. The increase in the co-culture period ensures the rapid transformation of cells infected by the bacteria, which allowed the induction of hairy roots within less time (Rai et al 2012). Both the A. rhizogenes strains used in our study showed a good transformation frequency (Table 1).…”

TRANSFORMASI GEN PADA NILAM UNTUK KETAHANAN TERHADAP PENYAKIT UTAMA MENGGUNAKAN Agrobacterium tumefaciens

“…Pada eksplan tanaman tomat yang tidak diinduksi, transformasi gen terjadi hanya 0,45% sedangkan bila diinduksi selama 2 hari, efisiensi transformasi meningkat menjadi 6,32% (Rai et al 2012). Eksplan yang diinduksi dapat mengaktifkan pembelahan sel sehingga dapat meningkatkan transformasi.…”