Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment

Abstract

Two groups of linear gene constructs (gus and bar, and 1Ax1 and bar) lacking vector backbone sequences were independently transferred into the elite wheat (Triticum aestivum L.) variety EM12, and genetically stable transgenic plants with low copy number transgene integration were recovered. Co-transformation experiments were carried out in parallel using either circular whole plasmid(s) or linear gene cassettes which were purified from the same plasmid by restrictive digestion, each cassette consisting of a promoter, an open reading frame, and a terminator. Six transgenic wheat lines transformed with 1Ax1 plus bar gene cassettes, five lines with gus plus bar gene cassettes, three lines with p1Ax1 plus pAHC20, and two lines with pAHC25 were regenerated with transformation frequencies of 0.6, 0.5, 0.3, and 0.2%, respectively. Southern blotting analysis showed that there were 1-4 hybridizing bands in transgenic lines carrying gene cassettes, of which most lines displayed single-copy transgene insertion. Expression analyses showed that 50.5% of the T1 lines carrying gus plus bar gene cassettes have the expression signals of two genes. SDS-PAGE analysis of the T1 generation revealed that 71% of herbicide-resistant plants carrying 1Ax1 plus bar gene cassettes expressed the high molecular weight subunit 1Ax1 in the endosperm. Gene cassettes were transmitted and segregated in the subsequent generations, in simple Mendelian ratios. In addition, reverse transcription-polymerase chain reaction (RT-PCR) results confirmed that 1Ax1 gene cassettes were expressed specifically in the endosperm of the transgenic wheat plant. It is proposed that gene transfer using multiple gene cassettes offers an efficient and rapid method to obtain the single-copy transgenic wheat.

Relaxed chromatin induced by histone deacetylase inhibitors improves the oligonucleotide-directed gene editing in plant cells

“…The original pAHC25 plasmid (Christensen and Quail 1996;Yao et al 2006) was modified by removing the GUS gene using SacI and SmaI restriction enzymes, and the transformation plasmid pAHC25::mGFP was constructed by inserting the mGFP gene between the SacI and SmaI restriction sites. Transgenic maize cells were produced using a biolistic transformation method by bombardment of the …”

Methods to Transfer Foreign Genes to Plants

“…Since that time, rapid developments in transformation technology have resulted in the genetic modification of many plant species. Methods for introducing diverse genes into plant cells include Agrobacterium tumefaciens-mediated transformation (De la Riva, 1998;Hooykaas & Schilperoort, 1992;Sun et al, 2006;Tepfer, 1990;Zupan & Zambryski, 1995), recently reclassified as Rhizobium radiobacter, direct gene transfer into protoplasts (Gad et al, 1990;Karesch et al, 1991;Negrutiu et al, 1990;Neuhaus & Spangenberg, 1990), and particle bombardment (Birch & Franks, 1991;Christou, 1992;Seki et al, 1991;Takeuchi et al, 1992Takeuchi et al, , 1995Yao et al, 2006).…”

“…Particle bombardment is a technique used to introduce foreign DNA into plant cells (Birch & Franks, 1991;Christou, 1992Christou, , 1995Gan, 1989;Takeuchi et al, 1992;Yao et al, 2006) (Figure 1 ) . G o l d o r t u n g s t e n p a r t i c l e s ( 1 -2 µ m ) a r e c o a t e d w i t h t h e D N A t o b e u s e d f o r transformation.…”

Advances in genetic engineering of marine algae

“…The direct DNA-transfer methods such as glass beads and biolistic bombardment usually lead to a large number of integrated gene copies in the receptor algal genome, which may increase silencing effects. Single or low number copy transgenic lines can be obtained by transformation cassettes and control the amount of cassette DNA when using the biolistic method (Lowe et al, 2009, Yao et al, 2006.…”