Institute of Radiation Breeding

Induction of bicellular pollen and dihaploidization of tetraploid maize
--Kato, A

Recently, antimicrotubule agents have been used in chromosome doubling in maize anther culture. These chemicals were originally developed as herbicides. Compared with colchicine, they are very cheap and effective. Trifluralin is one of them and I examined the effect of trifluralin on in vivo maize microsporogenesis and succeeded in the induction of restituted bicellular pollen.

I sprayed 0.05-0.2% of a Trefanocide (44.5% trifluralin emulsion) solution (with the addition of 0.1% of spreading agent Alsoap) on the tassels of a diploid inbred line Oh43 at 8-10 days before flowering. At that time microspores in the anthers were at the monocellular to bicellular stages. Microscopic observation revealed the presence of restituted bicellular pollen grains (Fig. 1) mixed in the normal tricellular pollen grains in the 0.2% treatment. The sperm cells in the bicellular pollen grains were diploid since they presumably originated from the nondisjunction of the chromosomes at the second pollen mitosis.

The ears pollinated with the treated pollen exhibited an increased number of shriveled kernels which resembled a 2n x 4n cross in maize (Fig. 2). In the 0.2% treatment, 46% of the kernels were shriveled (Table 1). Thirty-seven percent of the shriveled kernels had a small embryo. I carefully planted the shriveled kernels in moist vermiculite but none of them germinated.

Table 1. Kernel development on the ears pollinated by trifluralin-treated pollen.
Trefanocide concentration (%) No. of ears pollinated No. of plump kernels No. of shriveled kernels
0.0 3 977 (99.4) 6 (0.6)
0.05 2 710 (98.6) 10 (1.4)
0.1 2 519 (86.2) 83 (13.8)
0.2 7 694 (54.0) 590 (46.0)

The restituted bicellular pollen has only one sperm cell. If the sperm cell fertilizes only polar nuclei and if the egg cell is not fertilized, the ovule may develop into a haploid kernel. I determined whether the bicellular pollen produced by diploid plants induced dihaploids on tetraploid maize ears. I pollinated the ears of a tetraploid maize line Q28-1 with trifluralin-treated Oh43 pollen (0.3% Trefanocide solution). I obtained 117 plump kernels from the seven ears. Ploidy levels of the 85 seedlings among them were determined: 65 seedlings were tetraploid, 12 were triploid and eight were diploid. The tetraploid cases may have originated from the union of a diploid sperm cell and diploid egg cell and the triploid cases from the union of a haploid sperm cell and diploid egg cell. In both cases polar nuclei must have been fertilized by a diploid sperm cell, because the union of polar nuclei (2n+2n) of tetraploid maize and a haploid sperm cell (n) should result in the development of shriveled kernels. The eight diploid cases were attributed to dihaploidization and the rate was 9.4%.

Figure 1. Restituted bicellular pollen grain induced by trifluralin treatment (S - sperm cell, V - vegetative nucleus).

Figure 2. Control ear (left) and ear pollinated with trifluralin (0.2% Trefanocide solution)-treated pollen (right).

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