Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.)

Abstract

Transient expression studies using blueberry leaf explants and monitored by beta-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 microM for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 microM AS. Explants were then placed on modified WPM supplemented with 1.0 mg l(-1) thidiazuron, 0.5 mg l(-1) alpha-naphthaleneacetic, 10 mg l(-1) kanamycin (Km), and 250 mg l(-1) cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 microE m(-2) s(-1) at 25 degrees C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.

<i>Agrobacterium tumefaciens</i>-Mediated Transformation of Wild Tobacco Species <i>Nicotiana debneyi</i>, <i>Nicotiana clevelandii</i>, and <i>Nicotiana glutinosa</i>

“…The pBISN1 containing a potato ST-LS1 intron interrupted gusA to inhibit GUS expression in Agrobacterium cells was routinely used to optimize our transformation protocols for different plant species [18]- [22]. In this study, transient GUS expression assay was conducted after 4-d co-cultivation.…”

Improvement of cold tolerance of the half-high bush Northland blueberry by transformation with the LEA gene from Tamarix androssowii

“…(Graham et al 1996;Cao et al 1998Cao et al , 2003Song and Sink 2004). In this study, we have transformed the blueberry cultivar Northland with the LEA gene to enhance its cold tolerance.…”

“…When the Km concentration was higher than 30 mg l -1 , shoot growth was completely inhibited and all explants turned dark brown within 40 days (Table 3). Compared to Aurora, Bluecrop, Brigitta and Legacy high bush blueberries for example, where 10 mg l -1 km is employed (Song and Sink 2004), half-high bush Northland blueberry leaf tissues are less sensitive to Km. Thus, for stable transformation studies, the final selection medium used in transgenic studies was MWPMZ supplemented with 250 mg l -1 cefotaxime and 30 mg l -1 km.…”

Biochemical characterization of somatic embryogenesis and genetic transformation studies in Terminalia chebula Retz.: An immensely valuable medicinal tree

“…Calli was incubated in PIM II bacterial suspension containing 100 µM acetosyringone for 10 min was found suitable for transformation in T.chebula. In earlier report a similar 100 µM acetosyringone in bacterial suspension was improved transformation frequency in Vaccinium corymbosum (Song and Sink, 2004). However, bacterial suspension containing different concentrations of acetosyringone 10-25 mM in case of Hevea brasiliensis and 50µM in case of Chamaecyparis obtusa was used for explants cocultivation (Jayashree et al, 2003;Andrade et al, 2017).…”

“…As the concentration of the antibiotic increased from 100 to 400 mg/l, the degree of inhibition of bacterial growth was evident and 350 mg/l was found optimal. Cefotaxime at the concentration of 250 mg/l was used in shoot regeneration medium to control bacteria in Vaccinium corymbosum (Song and Sink, 2004). Antibiotics such as cefotaxime, carbenecillin, timentin, augmentin, triacillin, etc., were used to control bacterial growth in transformation experiments in tree species (ref : Table II).…”