Effective Agrobacteriummediated transformation of pineapple with CYP1A1 by kanamycin selection technique

Abstract

Initial calli was induced from leaf base of aseptic shoots. These calli were co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a plant expression vector pUHA1 containing a human cytochrome P4501A1 (CYP1A1) gene for 3 days. Then, the infected calli were transferred to differentiation medium. Adventitious buds were generated after about 10 day's incubation. The generated shoots were cultured on a medium containing 30 to 50 mg L-1 Km for screening. The selected Km-resistant shoots were subsequently transferred to rooting medium for rooting. We derived 95 Kmresistant plants from four infection groups in total. The transformation frequency was 0.12 to 2.69%. Further analysis by polymerase chain reaction (PCR) and Southern blotting confirmed that the positive frequency of Km-resistant plants was 53.58%. We also confirmed that these protocols below were essential for A. tumefaciens-mediated genetic transformation of pineapple calli: taking agar as medium gelling agent, adding AS to co-culture medium, increasing selection times and gradually increasing the concentration of Km in the selection medium.

An Efficient Agrobacterium Mediated Transformation of Pineapple with GFP-Tagged Protein Allows Easy, Non-Destructive Screening of Transgenic Pineapple Plants

“…Successful and efficient transformation systems are also dependent on the efficiency and success of regeneration [18]. We have developed an efficient and cost-effective regeneration system for pineapple [19], which we used in the present study to obtain transformed plants.…”

“…There is some consensus that NPT-II may not be suitable for the transformation of legume crops and monocotyledons [18]. In our analysis, we used hygromycin resistance to select putative transformants during the regeneration process, similarly to previous related studies [7].…”

“…In our analysis, we used hygromycin resistance to select putative transformants during the regeneration process, similarly to previous related studies [7]. In addition to hygromycin, Basta (herbicide tolerance) and kanamycin have also been used for pineapple transformant selection in previous work [5,18].…”

AbhemCencoding porphobilinogen deaminase plays an important role in chlorophyll biosynthesis and function in albinoAnanas comosusvar.bracteatusleaves

“…Nickel agarose affinity chromatography was performed after ultrasound chromatography to purify the protein. The recombinant protein was expressed in E. coli Rosetta-gami (DE3) following the protocol of Ma et al (2012), and its enzymatic activity was detected using the method of Lee et al (1992).…”