Establishment of transformation systems using mature seeds of maize inbred lines

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Martínez Nicolás, Juan
Major Professor
Kan Wang Michael Lee Philip W. Becraft
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Maize is one of the most important crops for plant biologists not only for its tremendous values for agriculture but also for its wealthy genetic information for biology. Recent years, the advent of biotechnology has further expanded the possibility of this crop. One of the most important tools for crop improvement and basic biological studies is genetic transformation. While plant genetic transformation using either Agrobacterium tumefaciens or biolistic gun as delivery systems has been available for more than 20 years, genetic transformation of many plant species, especially cereal crops, is still a challenging task for most laboratories.;The efficiency in genetic transformation depends upon the establishment of a robust and reproducible plant tissue culture system as well as an efficient delivery system for transformation. For current maize transformation system, immature embryos are routinely used as starting material for Agrobacterium infection or particle bombardment. The major problem for using the immature embryos is that it requires maize plants to be grown in the greenhouse year-round to meet the research demands. This practice requires large greenhouse spaces, quality growth conditions and experienced supporting staff.;One other problem with the current maize transformation system is that only Hi II, a hybrid germplasm bred specifically for tissue culture purposes, is amendable for transformation. For the maize community, inbred lines are the most desirable target for transformation due to their genetic background and agronomic importance in crop improvement.;This research program aimed at developing a transformation system using maize mature seeds as starting material for inbred lines B73, B104, H99, Mo17 and W22.;The first challenge in this study was to obtain sterilized mature embryo material for callus initiation. We have established an efficient and optimized seed sterilization protocol for maize seeds harvested from field-grown plants which have an increased amount of seed-borne pathogens. Our protocol ensures 98-100% sterility of plant material without noticeable compromise of the vigor and callus initiation.;Two different approaches: shoot meristematic cultures (SMC) and somatic embryogenic cultures (SEC) have been evaluated to obtain transformation-target tissue. Although SMC of different inbred lines could be obtained in a non-genotype dependent fashion, and GUS expression was detected when performing genetic transformation using Agrobacterium-mediated and the biolistic gun, no plants were recovered using this tissue culture system. On the other hand SEC was observed in all inbred lines at different levels, however just H99 responded with high frequency on callus induction medium (71%). Bombardment of SEC of inbred line H99 resulted in fertile transgenic plants. Analysis of progeny indicated that both gus and bar transgenes have been transmitted to the next generation. Bialaphos resistant callus frequency (BRCF) ranged from 24.1% to 74% with this system.

Tue Jan 01 00:00:00 UTC 2008