Transformation of recalcitrant maize elite inbreds using in vitro shoot meristematic cultures induced from germinated seedlings
Two new methods of transformation for recalcitrant maize elite inbreds (B73 and a Pioneer Hi-Bred inbred) were successfully developed using shoot meristematic cultures (SMCs) derived from germinated seedlings. One of the methods – the sector proliferation method – involved in vitro induction and proliferation of SMCs from transgenic sectors. These transgenic sectors derived from the bombardment of shoot apical meristems in immature embryos. Using this method, transgenic T1 and T2 progeny were obtained from the Pioneer Hi-Bred maize inbred, PHTE4. The other method – the SMC method – involved direct bombardment of SMCs. Using the second method, transgenic T1 and T2 progeny were produced from the publicly held maize inbred B73. Cellular and molecular analyses showed that SMCs were mainly induced from the nodal regions within the elongating in vitro stem tissues. The induced SMCs, characterized by large numbers of cells expressing KN1, have the potential to produce multiple adventitious shoot meristems. The use of induction and maintenance media containing higher levels of Cu2+ or Zn2+, not needed in earlier investigations on sweet corn, was found to be critical for the successful in vitro culture and transformation of some maize inbreds.
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