Doubled Haploid Broccoli (Brassica olearacea var. italica) Plants from Anther Culture.

Broccoli (Brassica olearecea var. italica) is a cole crop grown for its floral heads and stalks. It is rich in bioactive chemicals good for human health. Broccoli has been consumed as a vegetable since Roman times, but its production and consumption have increased significantly over the past few decades. Breeders try to develop new broccoli varieties with high yield, improved quality, and resistance to biotic and abiotic stresses. Almost all new broccoli varieties are F1 hybrids. Development of inbred broccoli lines that can be used as parents in hybrid production is a time-consuming and difficult process. Haploidization techniques can be utilized as a valuable support in broccoli breeding programs to speed up the production of genetically pure genotypes. Haploid plants of broccoli can be produced from immature male gametophytes via anther and microspore cultures with similar success rates. The most important parameters affecting the success of haploidization in broccoli are the genetic background (genotype) and the developmental stage of the microspores. Broccoli genotypes differ in their responses to androgenesis induction. The highest androgenesis response could be induced from microspores in late uninucleate and early binucleate stages. Recovery of diploid broccoli plants from haploids is possible via spontaneous and induced doubling. Doubled haploid (DH) broccoli lines are considered to be fully homozygous. Therefore, the production of DH lines is an alternative way to obtain pure inbred lines that can be utilized as parents in the development of new F1 hybrid varieties showing high levels of heterosis, high-quality heads, and uniform harvestable crop. We are using an anther culture-based haploid plant production system to develop DH broccoli lines in our broccoli breeding program. DH broccoli lines are produced from different genetic backgrounds within a year and handed to broccoli breeders.

In Vitro Gynogenesis in Leek (Allium ampeloprasum L.).

Leek (A. ampeloprasum L.) is an economically important vegetable crop from Alliaceae family. It is a non-bulb forming biennial species grown for its pseudostem and leaves. Leek is a tetraploid with one of the largest genomes known among cultivated plant species. It has enormous economic importance all around the world for many purposes such as vegetable, medicinal herb, and food seasoning. Production and consumption of leek is in rise all around the world and breeders are trying to develop new F1 hybrid varieties with desired agronomical traits. Although self-compatible, leek shows high tendency toward outcrossing and display severe inbreeding depression when selfed with its own pollen. Therefore, inbred development through classical breeding techniques is very difficult in this crop. Traditional leek genotypes are highly heterozygous, open pollinated varieties. There is a high demand for F1 hybrid varieties with resistance to biotic and abiotic stresses and high-quality plants. Our group is trying to incorporate gynogenesis-based doubled haploid technology to leek improvement programs. Over the years, many experiments were carried out to determine the gynogenic potential of donor leek genotypes of different genetic backgrounds in different induction media. Here, we report a protocol allowing production of green gynogenic leek plants via single step culture of unopened flower buds. Ploidy levels of gynogenic regenerants are determined by flow cytometry analysis. A majority of the gynogenic leek regenerants produced survived well in vivo.

Doubled Haploid Onion (Allium cepa L.) Production Via In Vitro Gynogenesis.

Onion (A. cepa L.) is an outcrossing biennial species with a very large genome. Development of genetically uniform (inbred) lines highly desired by onion breeders is a difficult process due to high level of heterozygosity. Inbred onion development may take up to five generations (~10 years) by classical selfing technique. Onion shows severe inbreeding depression, which further complicates production of lines with stabilized important agronomic traits. When applied successfully, haploidization technology can be useful in the development of fully homozygous onion lines in 2 years. Although production of haploid and doubled haploid (DH) onions via gynogenesis was reported more than three decades ago, successful production and utilization of DHs in onion breeding is still far behind of expectations of breeders. The main obstacles in front of the success include high variation in the response of donor materials to gynogenesis induction and difficulties faced in the process of obtaining DHs from haploid plants. We use a DH production procedure enabling us to develop DH plants from a wide range of onion donor materials. This procedure is based on production of haploid plants via single step culture of unopened flower buds, detection of haploid plants among gynogenic regenerants, and converting these plants to fecund DHs using a combination of ploidy manipulation techniques. The bulbs of DHs are produced in about 1 year after the initiation of induction cultures and selfed seeds are produced from fecund DH plants when they flower in the second year.