Method for obtaining improved fertility restorer lines for transgenic male sterile crop plants and a DNA construct for use in said method

A method to obtain efficient fertility restorer lines in crop plants for hybrid seed production, said method comprising:

• i) regenerating transformed crop plants from plant cells transformed with a DNA construct containing two different coding sequences of a barstar fertility restorer gene both of which encode the same protein product, the first of said sequences being a naturally occurring wild type gene sequence and the second of said sequences being a modified sequence generated by modification of the wild type sequence using codon degeneracy for expression in dicotyledonous crop plants to avoid homology between the modified sequence and the wild type sequence at the DNA and mRNA levels so as to reduce susceptibility to homology-based post-transcriptional gene silencing, said DNA construct comprising:
• a) a first transcription unit comprising the wild type sequence of the barstar restorer gene under transcriptional control of a first tapetum-specific promoter, which promoter is used to express a male sterility gene in corresponding male sterile plants, wherein said restorer gene sequence is fused to a transcription termination signal, including a polyadenylation signal,
• b) a second transcription unit comprising a modified sequence of the barstar restorer gene under transcriptional control of a second tapetum-specific promoter and fused to a transcription termination signal including a polyadenylation signal, and
• c) a third transcription unit comprising a selectable marker gene under transcriptional control of a strong constitutive promoter and fused to a transcription termination signal including a polyadenylation signal;
• wherein the first and second tapetum-specific promoters have overlapping temporal expression patterns in tapetal tissues of said crop plants;
• wherein said first tapetum-specific promoter is used to express a male sterility gene in a corresponding male sterile plant;
• and
• wherein expression from said second tapetum-specific promoter begins earlier than said first tapetum-specific promoter,
• (ii) identifying transformed crop plants having a single copy of the DNA construct,
• (iii) crossing the above single copy plants with male sterile barnase lines,
• (iv) subjecting the F1 progeny obtained from crosses between barnase and barstar lines to molecular analysis to identity fertility restored plants, and
• (v) testing pollen viability of fertility restored plants to determine extent of restoration.