Improvement of the plastid transformation protocol by modifying tissue treatment at pre- and post-bombardment in tobacco

Abstract

Plastid transformation in higher plants has been established in tobacco using particle bombardment, and the protocol has been used as a model in other plant species; however, as target materials, tobacco leaves may be partially exposed to unexpected abiotic stresses-drought stress during post-bombardment culture and damage due to cutting before selection culture. Plastid transformation e ciency may be increased by modi cation of leaf treatment to lessen such damage. In the modi ed protocol, tobacco leaves were cut into pieces (5×5 mm) and placed on the culture medium plate (approximately y pieces per plate) for one day before bombardment. A er bombardment, these pieces were transferred onto the non-selection medium and cultured for three days; they were then transferred onto spectinomycin selection culture medium. A transformation vector containing an aminoglycoside 3′-adenylyltransferase gene and a green uorescence protein gene were used for plastid transformation. Approximately four independent plastid transformants per bombarded plate were obtained on average using the modi ed protocol; the transformation e ciency was 1.6 times higher than that in a control experiment using the standard protocol. Modi ed leaf treatment improved the e ciency and stability of plastid transformation. is nding should aid plastid transformant production in tobacco and other plant species.

Efficient plastid transformation in tobacco using small gold particles (0.07^|^#8211;0.3^|^#8201;^|^micro;m)

“…Kausch et al (1995) reported that DNA/gold particles formed extremely loose aggregates and were finely dispersed compared with tungsten particles. In our previous report, the DNA/gold solution with 0.6-µm gold particles was easily suspended by pipetting and low vortexing (Okuzaki and Tabei 2012). However, in our examination of DNA coating of small gold particles, the particles with aggregated DNA, and the aggregate could not be resuspended by pipetting and vortexing (data not shown).…”

“…The DNA/gold mixtures (10 µl) were applied onto a macrocarrier, dried, and bombarded on a sample plate containing 50 pieces of tobacco leaf (5×5 mm) as described by Okuzaki and Tabei (2012). Two shots per plate were performed using a 1100-psi (7.6 MPa) or 1350-psi (9.3 MPa) rupture disc under partial vacuum (711 mmHg) in a PDS-1000/He system (Bio-Rad Laboratories).…”

“…The transformation vector pNtag (Okuzaki and Tabei 2012), which contains the spectinomycin-resistance selectable marker aadA and the visual marker GFP, was extracted using the Qiagen HiSpeed Plasmid Maxi Kit (Qiagen KK, Tokyo, Japan) and used to coat the gold particles as follows. A stock solution of each gold particle (40 mg ml −1 ) was sonicated (40 kHz) for 1-2 min before coating by floating the 1.5-ml tubes on water in an ultrasonic cleaner (USD-1R; AS ONE, Osaka, Japan).…”

“…Preparation of tobacco (N. tabacum L. cv. Petit Havana SR1) and spectinomycin (500 mg l −1 ) selection culturing were performed according to the modified tobacco plastid transformation protocol (Okuzaki and Tabei 2012).…”

“…Therefore, we produced a series of small gold particles and evaluated whether these particles were suitable for plastid transformation. In order to evaluate the practical utility of small gold particles, we applied them to our improved tobacco plastid transformation system (Okuzaki and Tabei 2012). We also assessed the appropriate acceleration pressure for the small gold particles.…”