title: Keel petal incision: a simple and efficient method for genetic crossing in Medicago truncatula.
abstract: BACKGROUND
  Genetic crossing is an essential tool in both forward and reverse genetic approaches to understand the biological functions of genes. For Medicago truncatula (barrel medic) various crossing techniques have been used which differ in the methods used to dissect the female parent's unopened flower bud to remove immature anthers for prevention of self-pollination. Previously described methods including front, side or back incision methods may damage the flower bud, impeding successful fertilization and/or seed development because they may allow pollen to dislodge and floral organs to desiccate after crossing, all of which diminish the success rates of crossing.
  
  
  RESULTS
  We report the keel petal incision method for genetic crossing in M. truncatula ecotype R108 and demonstrate successful crosses with two other M. truncatula ecotypes, A17 and A20. In the method presented here, an incision is made along the central line of the keel petal from the bottom 1/3rd of the female parent's flower bud to its distal end. This allows easy removal of anthers from the flower bud and access for cross-pollination. After pollination, the stigma and the deposited pollen from the male donor are covered by the keel petal, wing petals and standard petal, forming a natural pouch. The pouch prevents dislodging of deposited pollen from the stigma and protects the internal floral organs from drying out, without using cling-film or water-containing chambers to maintain a humid environment. The keel petal incision method showed an approximate 80% success rate in the M. truncatula R108 ecotype and also in other ecotypes including Jemalong A17 and A20.
  
  
  CONCLUSIONS
  Our keel petal incision protocol shows marked improvement over existing methods with respect to the ease of crossing and the percentage of successful crosses. Developed for the M. truncatula R108 ecotype, the protocol has been demonstrated with A17 and A20 ecotypes and is expected to work with other ecotypes. Investigators of varying experience have achieved genetic crosses in M. truncatula using this method.
authors: Department of Biological Sciences, University of North Texas, 1155 Union Circle #305220, Denton, Texas 76203, USA.
authors: Vijaykumar
authors: V
authors: Veerappan
authors: Department of Biological Sciences, University of North Texas, 1155 Union Circle #305220, Denton, Texas 76203, USA.
authors: Khem
authors: K
authors: Kadel
authors: Department of Biological Sciences, University of North Texas, 1155 Union Circle #305220, Denton, Texas 76203, USA.
authors: Naudin
authors: N
authors: Alexis
authors: Department of Biological Sciences, University of North Texas, 1155 Union Circle #305220, Denton, Texas 76203, USA.
authors: Ashley
authors: A
authors: Scott
authors: Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA.
authors: Igor
authors: I
authors: Kryvoruchko
authors: Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA.
authors: Senjuti
authors: S
authors: Sinharoy
authors: Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA.
authors: Mark
authors: M
authors: Taylor
authors: Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA.
authors: Michael
authors: M
authors: Udvardi
authors: Department of Biological Sciences, University of North Texas, 1155 Union Circle #305220, Denton, Texas 76203, USA.
authors: Rebecca
authors: R
authors: Dickstein
keywords: Artificial hybridization; Barrel medic; Genetic crossing; Keel petal; Legume; Medicago truncatula