An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.)

Abstract

An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agarsolidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previouslydescribed procedures. Transgenic plant recovery was increased 3.5-6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 lM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera 'Merlot', 'Shiraz' and 'Thompson Seedless' as well as Vitis hybrid 'Seyval Blanc'.

Use of the VvMybA1 gene for non-destructive quantification of promoter activity via color histogram analysis in grapevine (Vitis vinifera) and tobacco

“…Cefotaxime and carbenicillin were obtained from Caisson Laboratories, Inc. (North Logan, UT, USA). Initiation and maintenance of somatic embryos (SE) from young leaves of grapevine (V. vinifera 'Thompson Seedless') were carried out as previously described (Li et al 2008). SE at a mid-cotyledonary stage of development were used in transformation experiments.…”

“…Rooted transgenic tobacco plants were transplanted into ProMix BX in 8-in pots and maintained in the greenhouse for observation and tissue collection. Gene insertion of grapevine SE (V. vinifera 'Thompson Seedless') was carried out using previously described procedures (Li et al 2008). Specifically, SE were submerged in Agroabcterium suspension for 10 min and then transferred onto filter papers pre-soaked with X2 liquid medium in a 15 9 100 mm Petri dish.…”

“…Cultures were incubated in the dark for 72 h. SE were then moved to semi-solid X6 medium supplemented with 100 mgl -1 each of cefotaxim and carbenicillin (Haarlem, The Netherlands) and maintained in the dark at 25°C for 7 d before being used in color histogram analysis of transient expression. Medium composition and preparation were described previously (Li et al 2008). …”

Genetic Transformation Strategies in Fruit Crops

“…These efforts did not report major morphological or anatomical differences in the generated E and PE masses when compared to the above described procedures using solid media (Jayasankar et al, www.intechopen.com 2003). Recently, Li et al (2008) improved this fundamental methodology by introduction of a flask-based calli processing before starting the second round of sub-culturing in solid X6 medium. This additional step consisted of four consecutive sub-cultures using agitated flasks containing DMcck; i.e.…”

“…Alternatively, convenient procedures to introduce material from the field have been reached by proper cultivation of grapevine sterile buds in solid C2D4B medium (Araya et al, 2008;Gray, 1995) by one to four months and then culturing the processed tissues into NB2 solid medium. The use of induction media based on modified MS or Nitsch (described by Li et al, 2008), established the basis for additional improvements in grapevine SE. This time, authors were focused on the yield of the system.…”