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Plant Materials: Corn Polyploids

Tetraploid stocks of common inbred lines of maize are being produced by nitrous oxide gas treatment shortly after self-fertilization. The self-pollinated plants are placed in an iron chamber and are exposed to pressurized nitrous oxide. Because of the gaseous nature of the treatment. good penetration into the internal tissues can be achieved, which is not possible with colchicine. Nitrous oxide is a mitotic inhibitor and blocks the first developmental division to create a tetraploid. Autotetraploids of B73, W22, Oh43 and A188 have been produced by this method and trials with several other common inbreds are underway.

Plants of diploid inbred lines are being readied for nitrous oxide gas treatment. The plants are self pollinated on day one. At about 30 hours following pollination, the tassel is removed and the plant is placed in the chamber for 20 hours. At the conclusion of the treatment, the plants are removed and the ears allowed to mature. Seedlings grown from the treated ear are subjected to root tip chromosome counts and the tetraploid inbreds are selected, grown and self pollinated. To date the project has produced tetraploids of inbred lines Oh43, A188, H99, W22, B73, stock 6, and Mo17. Others have been treated and await screening.

Monoploids of the same material can be created by crossing the progenitor diploid inbred lines by a line referred to as “stock 6” that carries an anthocyanin pigment marker, R-scm2 . This allele of the rregulatory gene conditions color in the embryo of the kernel as well as the aleurone layer of the endosperm. When the inbred lines are crossed as female by this specific male parent, monoploids can be recognized as those kernels that lack pigment in the embryo, but have it in the endosperm. Stock 6 typically induces maternal monoploids in this type of cross at a frequency of 2-4%.

Nitrous oxide chamber. Self-pollinated plants are placed in the chamber to double the chromosome number just following fertilization.

Triploids of the same inbred lines can be produced by generating diploid sperm by trifluralin treatment of developing tassels. Trifluralin blocks chromosome separation and if applied at the time of second pollen mitosis, a single diploid sperm is produced. This diploid sperm is capable of “single fertilization”, as opposed to the typical double fertilization event in angiosperms. A subsequent pollination with untreated pollen will fertilize the remaining nucleus (egg or central cell). If the second pollen parent carries the R-scm2 marker, then the triploids generated can be recognized by the absence of pigment in the embryo.

Ears of inbred Oh43 at the diploid and tetraploid levels.

Hexaploid plants potentially could be generated in the same manner using the tetraploids as the starting material. In this case, tetraploid sperm would be generated by trifluralin and used onto tetraploid females that would produce diploid eggs. Hexaploid maize exhibits reduced vigor, but if they shed pollen, as previously produced hexaploids are known to do, they can be backcrossed to tetraploid stocks to produce pentaploids. Thus through this series of steps, one can potentially generate a 1x-6x series with an identical genotype.

Ears of inbred A188 at the diploid and tetraploid levels.

From our initial comparisons of diploid, triploid and tetraploid inbreds, it is clear that plant vigor declines with increasing ploidy when comparing strict inbred materials. A comparison with hybrids in the ploidy series is underway.

A ploidy series of inbred Oh43 is depicted at the monoploid, diploid and tetraploid levels. The tetraploid inbred lines were generated by nitrous oxide gas treatment shortly after fertilization to form zygotes with a doubled chromosome number.
A ploidy series of inbred Oh43 at the diploid, tetraploid and hexaploid levels. The hexaploid was induced by trifluralin treatment of pollen of a tetraploid before self pollination. The resulting progeny were screened by root tip chromosome counts. Photo by Hong Yao. The person in the photo is Weichang Yu.

Corn lines now available are tetraploid derivatives of inbreds W22, Oh43, B73 and A188. These lines and their direct diploid progenitors have been sent to the Maize Genetics Cooperation Center.

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