Development of Radicchio Rosso lines RM3-3, RM3-4, and RM3-6.  To develop a male  sterile chicory plant, a ribonuclease encoded by barnase gene, has been engineered into  chicory.  The ribonuclease, which is expressed only in the tapetum cells of the pollen sac  during anther development, blocks pollen development, thus producing a male sterile  plant.  To direct the expression of barnase to pollen cells, a pollen specific promoter,  pTA 29 (Seurinck <i>et al</i>., 1990) was used to direct the synthesis of ribonuclease enzyme.<br>Expression of barnase in pollen cells apparently results in degradation of host RNAs and blocking development of mature viable pollen.  <i>Barnase</i> gene encodes for an extracellular ribonuclease protein in <i>Bacillus amyloliquefaciens</i>, a common soil microorganism.  <i>Bacillus amyloliquefaciens</i> also contains an intracellular protein called barstar which specifically inhibits barnase by combining with it in a one-to-one complex (Hartely, 1989).  Thus, barstar is produced intracellularly by the same organism that secretes barnase (Hartely and Smeaton, 1973).  <br> <br>The second gene (<i>bar</i>) inserted into the Radicchio Rosso lines RM3-3, RM3-4, and RM3-6 genome encodes for phosphinothricin acetyltransferase (PAT) enzyme which inactivates phosphinothricin, the active component in the herbicide glufosinate.  The <i>bar</i> gene is produced in Radicchio Rosso lines RM3-3, RM3-4, and RM3-6 under the direction of <i>Arabidospsis thaliana</i>  PssuAra promoter and nopaline synthase termination/polyadenylation sequences.  Although Radicchio Rosso lines RM3-3, RM3-4, and RM3-6 are tolerant to the herbicide, hybrid progeny (the seeds that would be available to the farmers) developed from these lines may or may not be tolerant to the herbicide.  If the female parent is not herbicide tolerant, then only half of the progeny hybrid chicory would carry the tolerance gene.  This would mean that farmers could not apply the herbicide because 50% of the plants would die.  However, if the female parent in the hybrid selection scheme was glufosinate tolerant, all the progeny would herbicide tolerant and farmers could apply the herbicide (assuming the herbicide is registered by EPA for use on chicory).