A) Wheat x maize crosses. When hexaploid wheat is pollinated with maize, fertilization typically occurs in about 30% of florets. Fertilization has been recorded in 25 of the 26 wheat X maize genotype combinations so far studied (the one failure was in a cross where only 11 florets were examined) but, curiously, double fertilization is relatively rare and most florets in which fertilization has occurred contain an embryo but lack an endosperm, the polar nuclei remaining unfertilized. For example, when 343 florets of 'Chinese Spring' wheat fixed 48h after pollination with 'Seneca 60' maize were examined 80 contained only an embryo, 8 contained only an endosperm, while 12 had both an embryo and an endosperm, giving an overall total of 100/343 (29.2%).
Proof of successful hybridization was obtained from zygotes at metaphase since these contained the expected F1 combination of 21 wheat chromosomes and 10 very much smaller maize chromosomes. However, the centromeres of the maize chromosomes are usually very indistinct and show little affinity for spindle microtubules. As a result they are rapidly eliminated and the developing embryos have only a haploid complement of wheat chromosomes. Wheat x maize crosses are potentially an attractive system for wheat haploid production via chromosome elimination since maize is relatively insensitive to the action of dominant alleles of the so-called crossability genes in wheat (Kr1 and Kr2) which normally reduce the frequency of fertilization in crosses between wheat and alien species such as rye or Hordeum bulbosum.
Unfortunately embryos allowed to develop on plants almost always abort before they reach a stage where they are viable in embryo culture, and only 1, which developed into a haploid wheat plant, was recovered from 2,440 florets. A sample of this size would be expected to contain 592 embryos (calculated from data on the frequency of egg-cell fertilization in florets fixed for cytological analysis 48h after pollination).
However, we have recently succeeded in recovering haploid wheat plants from wheat x maize crosses at a much higher frequency by using an alternative culture method which will allow embryos to develop in the absence of any detectable endosperm. Spikelets containing cross-pollinated florets are removed from plants 2 days after pollination, surface sterilized, and placed on Murashige and Skoog medium containing 0.1mg 1-1 2,4-D. After 3 weeks incubation at 20 C the florets are dissected and the embryos are transferred to embryo culture medium. A total of 47 embryos were recovered from 706 florets and of these, 31 developed into haploid plants. This was estimated to be 17.0% of the number of embryos that would have been present 48h after pollination, making this method 100 times more efficient for recovering plants than allowing embryo development in vivo.
B) Barley x maize crosses. During 1987 we also found that 'Sultan' barley x 'Seneca 60' maize crosses result in fertilization. In 100 florets fixed 48h after pollination 4 contained only an embryo, 8 contained only an endosperm, and 16 had both an embryo and an endosperm. Thus the overall frequency of fertilization (28/100) was similar to that found in wheat x maize crosses, but the frequency of double fertilization was higher so that most embryos were accompanied by an endosperm, albeit one that was highly abnormal. Again proof of hybridization was obtained from zygotes fixed 48h after pollination since these contained the expected F1 combination of 7 barley chromosomes and 10 much smaller maize chromosomes. Interestingly, maize chromosomes showed well-defined centromeres in zygotes and although they were subsequently eliminated, at least some persisted for at least 4 cell cycles. So far no plants have been recovered from barley x maize crosses but they have not yet been tried in spikelet culture.
C) Potential uses of these crosses. Wheat x maize crosses have potential for wheat haploid production, particularly for the many wheats which carry dominant alleles of the Kr genes, but our main interest is to transfer maize DNA into wheat by sexual hybridization. All of the wheat x maize crosses investigated so far have rapidly lost the maize chromosomes but since we know that zygotes contain a complete haploid complement of maize chromosomes it might still be possible to transfer DNA by inducing intergenomic translocations prior to elimination. At present we are trying to do this using X-ray irradiation. An obvious goal for this work is the transfer of maize transposable elements into wheat, with a view to developing a stock which could be used for 'transposon tagging' experiments. This might also be feasible for barley if plants can be recovered using the spikelet culture method.
D.A. Laurie and M.D. Bennett
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