Factors influencing successful Agrobacterium-mediated genetic transformation of wheat

Abstract

The development of a robust Agrobacteriummediated transformation protocol for a recalcitrant species like bread wheat requires the identification and optimisation of the factors affecting T-DNA delivery and plant regeneration. We have used immature embryos from range of wheat varieties and the Agrobacterium strain AGL1 harbouring the pGreen-based plasmid pAL156, which contains a T-DNA incorporating the bar gene and a modified uidA (b-glucuronidase) gene, to investigate and optimise major T-DNA delivery and tissue culture variables. Factors that produced significant differences in T-DNA delivery and regeneration included embryo size, duration of pre-culture, inoculation and co-cultivation, and the presence of acetosyringone and Silwet-L77 in the media. We fully describe a protocol that allowed efficient T-DNA delivery and gave rise to 44 morphologically normal, and fully fertile, stable transgenic plants in two wheat varieties. The transformation frequency ranged from 0.3% to 3.3%. Marker-gene expression and molecular analysis demonstrated that transgenes were integrated into the wheat genome and subsequently transmitted into progeny at Mendelian ratios.

Virus-Induced Gene Silencing Identifies an Important Role of the TaRSR1 Transcription Factor in Starch Synthesis in Bread Wheat

“…The euAPETALA2 (euAP2) group of the APETALA2/ethylene-responsive element binding protein (AP2/EREBP) family is characterized by the APETALA2 (AP2) domain, and some members of this group function in the boundaries of floral organ identity, floral meristem identity, or the control of floral organ number [20,21,22,23]. In our previous study, transcription levels of Rice Starch Regulator 1 ( RSR1 ), an euAP2 group transcription factor, was significantly and negatively associated with the transcription levels of 11 starch synthesis-related enzyme genes during the wheat grain-filling period, suggesting that TaRSR1 may negatively regulate the expression of some starch synthesis-related enzyme genes in wheat grains [7].…”

“…In bread wheat, particle bombardment and co-cultivation with Agrobacterium tumefaciens have been used to explore the function of candidate genes. However, the disadvantages of these two methods include multiple copy insertions, cultivar-specificity, time consumption, and low transformation efficiency; this species may be the last cereal to be genetically transformed [23,24]. The barley stripe mosaic virus–virus induced gene silencing (BSMV-VIGS) was recently developed as an attractive tool for the rapid generation of gene knockdown phenotypes in plants.…”

Identification and Comparative Analysis of microRNA in Wheat (Triticum aestivum L.) Callus Derived from Mature and Immature Embryos during In vitro Culture

“…Feasible and efficient tissue culture plays an important role in plant genetic engineering (Wu et al, 2003). Common wheat ( Triticum aestivum L.; 2n = 6x = 42; AABBDD) is an important staple crop cultivated worldwide.…”

Gene Targeting Without DSB Induction Is Inefficient in Barley

“…These plants were self-pollinated and the progeny (T1) analyzed in more detail. To determine the number of independent transgene inserts in the genome, inheritance of PPT resistance in the segregating progeny was analyzed with the leaf painting assay (Wu et al, 2003). Sixteen out of 23 GP-RAD51-1 siblings were PPT resistant closely matching the expected ratio of 3:1 for Mendelian inheritance of a single gene.…”

“…All tissue culture media were as described (Jacobsen et al, 2006) except that casein hydrolysate (Hydrolysate N-Z-Amine-A) was omitted. The leaf painting assay (Wu et al, 2003) was used for segregation analyses of greenhouse grown plants as described (Jones and Sparks, 2008). …”