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 Km-resistant 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. Key words: Ananas comosus, CYP1A1 gene, genetic transformation, Agrobacterium tumefaciens.
Highlights
Pineapple (Ananas comosus) is the number one agricultural commodity in certain parts of the world (Gangopadhyay et al, 2009), and the third most important fruit crop in the tropics and subtropics, after banana and citrus (Rohrbach et al, 2002)
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
Further analysis by polymerase chain reaction (PCR) and Southern blotting confirmed that the positive frequency of Km-resistant plants was 53.58%
Summary
Jun Ma1,2, Ye-hua He1*, Cheng-hou Wu1, He-ping Liu, Zhong-yi Hu1 and Guang-ming Sun. 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. The infected calli were transferred to differentiation medium. 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 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
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