Expression of Engineered Nuclear Male Sterility in Brassica napus (Genetics, Morphology, Cytology, and Sensitivity to Temperature)

Abstract

A dominant genetic male sterility trait obtained through transformation in rapeseed (Brassica napus) was studied in the progenies of 11 transformed plants. l h e gene conferring the male sterility consists of a ribonuclease gene under the control of a tapetumspecific promoter. Two ribonuclease genes, RNase T1 and barnase, were used. The chimaeric ribonuclease gene was linked to the bialophos-resistance gene, which confers resistance to the herbicide phosphinotricine (PPT). l h e resistance to the herbicide was used as a dominant marker for the male sterility trait. l h e study presented here concerns three aspects of this engineered male sterility: genetics correlated with the segregation of the 1-DNA in the progenies; expression of the male sterility in relation to the morphology and cytology of the androecium; and stability of the engineered male sterility under different culture conditions. Correct segregation, 50% male-sterile, PPT-resistant plants, and 50% male-fertile, susceptible plants were observed in the progeny of seven transformants. l h e most prominent morphological change in the male-sterile flowers was a noticeable reduction i n the length of the stamen filament. l h e first disturbances of microsporogenesis were observed from the free microspore stage and were followed by a simultaneous degeneration of microspore and tapetal cell content. At anthesis, the sterile anthers contained only empty exines. In some cases, reversion to fertility of male-sterile plants has been observed. Both ribonuclease genes are susceptible to instability. lnstability of the RNase TI-male sterility trait increased at temperatures higher than 25°C. Our results do not allow us to confirm this observation for the barnase male-sterile plants. However, the male-sterile plants of the progeny of two independent RNase T I transformants were stably male sterile under all conditions studied.

The gene responsible for borate cross-linking of pectin Rhamnogalacturonan-II is required for plant reproductive tissue development and fertilization

“…The slides were rinsed with PBS and immersed in methanol containing 0.3% (vol͞vol) H 2 O 2 for 10 min to inactivate endogenous peroxidases. The slides were then incubated for 1 h at room temperature in a 1% (vol͞vol) solution of anti-RG-II polyclonal antibody (29), washed with 0.1% Tween 20 in PBS, and agitated for 1 h in PBS containing 1,000-fold-diluted horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Sigma, St. Louis, MO). Proteins were visualized by incubating the slides in PBS containing 0.03% (vol͞vol) 3,3-diaminobenzidine and 0.003% (vol͞vol) H 2 O 2 .…”

Genomic stability and stability of expression in genetically modified plants

“…Therefore, a complete T-DNA sequence might be expected in the genome after integration (for review see Zupan & Zambryski 1995). However, in 20-30% of the transgenic plants investigated, sequences that extend beyond 25 base pair borders of the plasmid have been found (Denis et al 1993;Martineau et cil 1994;Denis 1994). The right border of the T-DNA is generally retained, while the left border quite often is not faithfully utilized.…”

“…The right border of the T-DNA is generally retained, while the left border quite often is not faithfully utilized. In some cases even integration of the almost complete Ti plasmid has been found (Denis et al 1993;Denis 1994). These observations show that in Agrobacterium read-through of T-DNA molecules beyond the border is possible.…”

Cytological mechanism of pollen abortion resulting from allelic interaction of F1 pollen sterility locus in rice (Oryza sativa L.)

“…So far six F 1 pollen sterility loci had been identified, and three of them, S-a, S-b, and S-c loci, had been mapped on chromosomes by using of RFLP and SSR makers (Zhuang et al, 1999;Zhuang, Fu & Zhang, 2002;Li et al, 2002). Pollen abortion caused by the male-sterility-inducing ogu cytoplasm, chemeric genes for the expression of a modified b-1,3-glucanase, the ribonuclease genes and callose synthases genes have also been reported (Gourret, Delourme & Renard, 1992;Worrall et al, 1992;Denis et al, 1993;Enns et al, 2005).…”