The transformation protocol hereby reported is quite efficient. The frequency of transformation of immature embryos, which was constantly over 50 % and as high as 95.0 % in the best case, was higher than the ones previously reported by roughly ten times. Therefore, one of the issues of wheat, which was a low efficiency of transformation methods, appeared to be resolved now.

Many factors needed to be examined and adjusted in order to achieve this much of improvement. The list of key factors per se in wheat transformation, including choice of genotype, quality of immature embryos, media composition, strain of

A. tumefaciens, pre-treatment of embryos and handling of tissues, was not much different from those studied in rice and maize, but the details and specific parameters for the factors were quite different. For example, both centrifuging and heating as pre-treatments were effective in rice and maize, but only centrifuging was effective in wheat. With respect to the handling of tissues, the process of the excision of embryo axes two days after the co-cultivation was not included in the protocols for transformation of other cereals.

The composition of the medium for co-cultivation of embryos and A. tumefaciens was a factor not well optimized before the present study, and a lot of experiments were needed. A combination good for wheat transformation was found by primarily looking after conditions suitable for stronger transient expression of the GUS gene in and the callus induction from the immature embryos after the co-cultivation and was further optimized.

It should also be noted that the windows of optimal ranges of parameters for the key factors in wheat seemed to be narrower than those in rice and maize. We experienced that, although the frequency of transformation in wheat was higher than that reported for maize when all the factors were optimal, the frequency dropped more drastically than maize when one or more of the factors became suboptimal.

The fact that too many factors with narrow optimal windows were involved in wheat transformation may explain both low frequency of transformation and low reproducibility of a protocol at a different laboratory before the present study. In addition, critical importance of the use of healthy immature embryos harvested at the right stage from wheat plants vigorously growing in a well-conditioned greenhouse, like any other cereal species, may explain them further. A greenhouse could be different in many ways from another no matter how similar they are and no matter how well the conditions are controlled. Therefore, a certain adjustment of the protocol by trials and errors may be inevitable for immature embryos from other greenhouses.

Commonly recognized advantage of Agrobacterium-mediated transformation, such as low copies of transgenes in the transformants and the stable Mendelian inheritance of the expression of transgenes, were well demonstrated in wheat. Thus, wheat has finally joined the list of cereals that can be transformed efficiently by

A. tumefaciens. The next challenge for the present protocol is how reproducible it is at other laboratories. Chances are good because the many factors were well optimized now and the very high frequency of transformation was recorded in the present study. Finding of Fielder as a suitable genotype is another positive factor. In fact, the protocol recommended by the present study has already been tested successfully in more than ten leading laboratories in the world, and in this process, hands-on guidance provided by scientists experienced in wheat tissue culture was very helpful (personal communication). It is also likely that efforts will be made to develop methods for transformation of genotypes other than Bobwhite and Fielder, making use of the current protocol as a starting point. Therefore, the protocol must be very useful in basic and applied study of molecular biology, genomics, biotechnology and breeding in wheat.

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