Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration

Abstract

A reproducible and efficient transformation method was developed for the banana cv. Rasthali (AAB) via Agrobacterium-mediated genetic transformation of suckers. Three-month-old banana suckers were used as explant and three Agrobacterium tumefaciens strains (EHA105, EHA101, and LBA4404) harboring the binary vector pCAMBIA1301 were used in the co-cultivation. The banana suckers were sonicated and vacuum infiltered with each of the three A. tumefaciens strains and co-cultivated in the medium containing different concentrations of acetosyringone for 3 days. The transformed shoots were selected in 30 mg/l hygromycin-containing selection medium and rooted in rooting medium containing 1 mg/l IBA and 30 mg/l hygromycin. The presence and integration of the hpt II and gus genes into the banana genome were confirmed by GUS histochemical assay, polymerase chain reaction, and southern hybridization. Among the different combinations tested, high transformation efficiency (39.4 ± 0.5% GUS positive shoots) was obtained when suckers were sonicated and vacuum infiltered for 6 min with A. tumefaciens EHA105 in presence of 50 μM acetosyringone followed by co-cultivation in 50 μM acetosyringone-containing medium for 3 days. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into banana has been developed and that this transformation system could be useful for future studies on transferring economically important genes into banana.

Transformasi Genetik Pisang Ambon dengan Gen Kitinase dari Padi

“…Transformasi genetik pisang telah dilakukan oleh Sreeramanan et al (2006b) Sreeramanan et al (2009) menunjukkan bahwa efisiensi transformasi tertinggi pada transformasi genetik pisang diperoleh dengan menggunakan higromisin 20 mg/l, sedangkan waktu kokultivasi terbaik adalah 3 hari (Subramanyam et al, 2011).…”

“…Hasil yang sama diperoleh dari hasil penelitian Sreeramanan et al (2009) yang melakukan transformasi pisang dengan gen chitinase. Selanjutnya Subramanyam et al (2011) menggunakan higromisin 30 mg/l untuk menyeleksi pisang cv. Rasthali (AAB) yang ditransformasi dengan A. tumefaciens strain EHA 105.…”

Recent Advances in Fruit Species Transformation

“…Today, application of these modifications solely or in combination with other approaches has made it possible to transfer foreign DNA via Agrobacterium even to monocots (Hiei et al, 1994;Ishida et al, 1996;Hensel et al, 2011) which were initially transformed with direct gene transfer methods since Agrobacterium is not a natural host. Following its first successful usage in soybean and Ohio buckeye (Trick & Finer, 1997), SAAT was also applied recently to fruit species including orange (Oliveira et al, 2009); banana (Subramanyam et al, 2011) and grapevine . In the last paper the developed efficient methodology that combined SAAT with vacuum infiltration allowed to obtain reporter gene expression in different newly formed organs such as stems, petioles and leaves.…”

“…Almond (Costa et al, 2006); apple (Smolka et al 2009;Lau & Korban, 2010;Vanblaere et al, 2011); banana (Subramanyam et al, 2011); fig (Yancheva et al, 2005); kiwifruit (Tian et al, 2011); peach (Padilla et al, 2006); strawberry (Mercado et al, 2010); peanut (Asif et al, 2011); watermelon (Huang et al, 2011) and pear (Sun et al, 2011) are some examples of transformed cultivars for some fruit species that the transformed tissues were regenerated via organogenesis. Since organogenesis protocols are developed for many different fruit species, it is easier to adapt the regeneration system into genetic transformation methods (Frary & Eck, 2005).…”

Plant biotechnology in support of the Millennium Goals

“…The problem of expressing pharmacologically active peptides in plants is addressed in the study by Luchakivskaya et al (2011) and Soria-Guerra et al (2011). Finally, Subramanyam et al (2011) and Basnayake et al (2011) present progress towards the transformation of banana and diverse sugarcane cultivars, respectively.…”