Standardization of Protocol for Agrobacteriummediated Transformation in Potato (Solanum tuberosum L.)

Abstract

The experiment was conducted at the Laboratory of Biotechnology, Biotechnology Division, Bangladesh Agricultural Research Institute, Gazipur- 1701 during July 2007 to June 2008. An efficient and reproducible protocol for the production of transgenic potato plants was developed by inoculating internode explants of potato with Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pBI121 having one reporter gene (gus) and selectable marker gene (nptII) resistant to Kanamycin. The transformation experiment was done by optimizing two important parameters names infection time and cocultivation period. Most of the explants produced shoots within 21 days on 5 mg/l Zeatin riboside (ZR) and 50 mg/l Kanamycin supplemented MS medium without introducing callus. The infected explants produced 8.27 and 6.42 shoots in Asterix and Diamant varieties, respectively within 21 days. Transgenes were confirmed by molecular analysis. DNA from well established rooted plants confirmed nptII positive through PCR analysis. The transformation rates were 28.97 and 24.37% in Asterix and Diamant, respectively. Putative transformed plants of Diamant and Asterix varieties produced roots in ½MS medium supplemented with 50/mg Cefotaxim, 50 mg/l Kanamycin and 0.5 mg/l IBA. DOI: http://dx.doi.org/10.3329/bjar.v37i2.11220 Bangladesh J. Agril. Res. 37(2): 185-194, June 2012

Design of expression cassettes using the Cry1Ba1 gene for potato (Solanum tuberosum L.) varieties

“…Employing a regeneration medium containing 50 mg L -1 of kanamycin as a selection agent has proven useful for the preliminary selection of possible transformants when using potato explants; they avoid selection pressure and loss of possible transformed explants by oxidation, chlorosis or tissue necrosis when higher concentrations of the antibiotic are used. This is demonstrated by some authors that have used concentrations of 100 mg L -1 (Andersson et al, 2003;Khan et al, 2006;López and Chaparro, 2007;Meiyalaghan et al, 2011;Molla et al, 2012;Torres et al, 2012).…”

“…The explants were placed in contact with the bacterial suspension for 8 min, using as a control a group of 40 explants not subjected to cocultivation and transferred directly to MS solid culture medium without the addition of hormones. We transferred the co-cultivated explants to Petri dishes with solid coculture medium and incubated them in the dark at a temperature of 20 ± 3°C for 18 h. Then, we transferred the explants to a regeneration medium supplemented with 250 mg L -1 of cefotaxime for 7 d, and transferred to the same medium supplemented with 50 mg L -1 of kanamycin as the selection concentration that has been used for explants of Pastusa Suprema (López and Chaparro-Giraldo, 2007;Torres et al, 2012) and other varieties (Andersson et al, 2003;Khan et al, 2006;Meiyalaghan et al, 2011;Molla et al, 2012).…”

Functional Analysis of StPHT1;7, a Solanum tuberosum L. Phosphate Transporter Gene, in Growth and Drought Tolerance

“…The pBIN19 expression vector of the interference fragment was constructed via the LR reaction and was named pBIN19-StPHT1;7i after successful sequencing; it was introduced into the LBA4404 Agrobacterium (Mei5, Beijing, China) using the freeze-thaw method. The transformation of transgenic Agrobacterium into Desiree leaves was conducted following the methods described by Molla et al [ 52 ] with minor modifications. Our transformation media, which differed from that of Molla et al [ 52 ], is described in Table 2 .…”

Construction of Potato DM1-3-516-R44 (DM) Transgenic System Based on Agrobacterium Transformation

“…Similar to callus induction medium selection, hormone selection also played a vital role in shoots induction. We also selected 4 hormones to determine the ratio of hormones (Supplement table [4][5][6] [23, [26][27][28]. Large difference in induction rate led to different concentration ratios.…”

“…Infection concentration and co-cultivation time were the transformation of the factors influence based on agrobacterium infection. Some researchers also analyzed and adjusted the agrobacterium concentration, co-culture time and infection method, and finally obtained a better way [18,[27][28][29][30].…”