Abstract
Many researchers have developed various methods for in-planta or floral dip transformation of Arabidopsis thaliana, one of the simple protocol and widely used to produce transgenic Arabidopsis. As the efficiency and ease of getting a transformant is very much time consuming effort and less number of the transformants people get, we have developed a little modified transformation protocol to avoid the disparities. Four types of inoculums (inoculum1, inoculum2, inoculum3 and inoculum4) were used to check the transformation efficiency out of which Inoculum3 showed the highest rate of transformation among the four types. 0.07% Twin-20 also acts in same manner as silwet L-77 to increase the rate of transformation efficiency and glucose instead of sucrose can be used in inoculum to transform Arabidopsis. After vacuum infiltration keeping the Agrobacterium infected plants for 7-8 hrs horizontally in low light at 280C temperature condition, considered best to get an increased number of transformed seeds. Modified protocol produced ~12-14% increase in transformants. Selection pots (kanamycin supplemented soil filled pots) in place of selection plates (Kanamycin supplemented Murashige and Skoog agar plates) proved beneficial as no MS medium and no aseptic condition is required for selection of transformed plants. This increase in transformation efficiency consequently increased the percentage of homozygous and single copied stable transgenic lines.
Highlights
Efficient study of plant gene function has been permissible through transgenic approaches
Four types of inoculums were used to check the transformation efficiency out of which Inoculum-3 showed the highest rate of transformation among the four types. 0.07% Twin-20 acts in same manner as silwet L-77 to increase the rate of transformation efficiency and glucose instead of sucrose can be used in inoculum to transform Arabidopsis
Out of four types of inoculums, inoculum-3 gave best result to produce increased number of transformants. 12% - 14% (18 ± 4 antibiotic resistant plants from 100 seeds) increase in the number of transformants on kanamycin plate was observed than the standard inoculums used (Figure 2(c)). 0.075% silwet L-77 was considered best to get increased number of tranformants. ~ 150 seeds were taken from each type for screening on kanamycin plates
Summary
Efficient study of plant gene function has been permissible through transgenic approaches. This account of transformation approach was frequently named as the ‘‘Agrobacterium vacuum infiltration method’’ [3] It involves uprooting of flowering Arabidopsis plants, vacuum infiltration of the plants using an Agrobacterium cell suspension, re-planting, harvesting of seed several weeks later, and screening for primary transformants on medium containing the appropriate selective agent (usually an antibiotic or a herbicide). We provide some modifications in steps of floral-dip method of Arabidopsis that eradicates the need to screen on sterile conditions as only agro-peat soil treated with kanamycin is a suitable alternative to an agar substrate during the seed selection process In another development, we provide a description of modified inoculum preparation for infiltration that supports direct dipping and plant transformation, thereby enhancing the chance of more transformant production having homozygous and single copy of integrated target gene
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