The canola lines MS1 and RF1 were developed using genetic
engineering techniques to provide a pollination control system for
the production of hybrid oilseed rape (MS1xRF1) expressing male
sterility and tolerance to glufosinate ammonium. The novel
hybridization system involves the use of two parental lines, a male
sterile line MS1 and a fertility restorer line RF1. The transgenic
MS1 plants do not produce viable pollen grains and cannot
self-pollinate. In order to completely restore fertility in the
hybrid progeny, line MS1 must be pollinated by a modified plant
containing a fertility restorer gene, such as line RF1. The
resultant F1 hybrid seed, derived from the cross between MS1 x RF1,
generates hybrid plants that produce pollen and are completely
fertile.
The male-sterile trait was introduced in MS1 by inserting the
barnase gene, isolated from Bacillus amyloliquefaciens, a common
soil bacterium that is frequently used as a source for industrial
enzymes. The barnase gene encodes for a ribonuclease enzyme (RNAse)
that is expressed only in the tapetum cells of the pollen sac
during anther development. The RNAse affects RNA production,
disrupting normal cell functioning and arresting early anther
development, thus leading to male sterility.
The transgenic line RF1 was produced by genetically engineering
plants to restore fertility in the hybrid line. Transgenic RF1
plants contain the barstar gene, also isolated from Bacillus
amyloliquefaciens. The barstar gene codes for a ribonuclease
inhibitor (barstar enzyme) expressed only in the tapetum cells of
the pollen sac during anther development. The ribonuclease
inhibitor (barstar enzyme) specifically inhibits barnase RNAse
expressed by the MS1 line. Together, the RNAse and the ribonuclease
inhibitor form a very stable one-to-one complex, in which the RNAse
is inactivated. As a result, when pollen from the restorer line RF1
is crossed to the male sterile line MS1, the resultant progeny
express the RNAse inhibitor in the tapetum cells of the anthers,
allowing hybrid plants to develop normal anthers and restoring
fertility.
Both transgenic lines MS1 and RF1 were also engineered to express
tolerance to glufosinate ammonium, the active ingredient in
phosphinothricin herbicides (Basta®, Rely®, Finale®, and Liberty®).
Glufosinate chemically resembles the amino acid glutamate and acts
to inhibit an enzyme, called glutamine synthetase, which is
involved in the synthesis of glutamine. Essentially, glufosinate
acts enough like glutamate, the molecule used by glutamine
synthetase to make glutamine, that it blocks the enzyme's usual
activity. Glutamine synthetase is also involved in ammonia
detoxification. The action of glufosinate results in reduced
glutamine levels and a corresponding increase in concentrations of
ammonia in plant tissues, leading to cell membrane disruption and
cessation of photosynthesis resulting in plant withering and
death.
Glufosinate tolerance in these canola lines was the result of
introducing a gene encoding the enzyme
phosphinothricin-N-acetyltransferase (PAT) isolated from the common
aerobic soil actinomycete, Streptomyces hygroscopicus. The PAT
enzyme catalyzes the acetylation of phosphinothricin, detoxifying
it into an inactive compound. The PAT enzyme is not known to have
any toxic properties.
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