Şeker pancarında (Beta vulgaris) ginogenezis indüksiyonu ve katlanmış haploid bitki üretimi

The first chapter contains a brief introduction and a general literature review. Sugar was first extracted from sugar beet in 1747, with an approximate rate of 1.6% extractable sugar. During more than two centuries, researchers' attempts increased the sugar amount up to 20%. Majority of those successes were because of conventional breeding methods. In the early 19th century, a spontaneously induced haploid plant was discovered. The doubled haploid technique provides researchers with a complete homozygous plant, which is of high value for breeders, and let them shorten breeding duration for biennial plants from about ten years to two years. However, for sugar beet, from 1945 doubled haploid sugar beet technique was employed towards breeding sugar beets with other beneficial traits other than sugar concentration, i.e. resistance or tolerance to biotic or abiotic stresses. Several doubled haploid methods were examined, categorized in vivo and in vitro methods. Androgenesis, the most favorable methods mostly resulted in callus or diploid plants instead of haploid or doubled haploid plants. Except for gynogenesis, none of the applied methods was promising. Therefore, the only option was gynogenesis to be employed in sugar beet doubled haploid breeding programs. Sugar beet is an inter-breeding allogamous plant. Thus, there is a considerable variation among different varieties, cultivars, and genotypes. As a result, the available methods reported in the scientific literature sometimes cannot be successfully implemented for other genotypes. Sugar beet is an economically important crop for the countries in the temperate region of the northern hemisphere, including Turkey, which is a sugar beet seed importer country from western countries, e.g. Germany, the Netherland, Denmark, or Sweden. To produce sugar beet sustainably in Turkey, the country needs to breed and produce the required seeds and locally adaptable genotypes. Therefore, this project was launched to be a bridge between two parts of a long-term breeding program, started from more than a decade ago and is continued. The aims of these experiments reported in four chapters in the present thesis were haploid sugar beet induction through in vitro culture of unfertilized ovules (Chapter II); ameliorating hyperhydricity of the gynogenic explants (Chapter III); Doubled haploid sugar beet induction (Chapter IV); Increasing the propagation rate of the doubled haploid explants (Chapter V). The summaries of the chapters are provided in the following paragraphs. The second chapter describes a protocol studying the effects of an interaction between cold pretreatment of six genotypes of sugar beet inflorescences at 4 °C for one week or more and 6-benzylaminopurine (BAP) concentrations (1 or 2 mg L‒1) to increase the response rate of haploid embryo induction.. Ovules were removed from the unfertilized flowers and cultured on in vitro media. The interaction of three variables was examined, i.e. genotype, cold pretreatment, and hormonal treatment. In comparison with freshly cultured ovules, cold pretreatment for one week almost doubled the mean of haploid plantlet induction rate. Supplementing BAP at 2 mg L‒1 nearly doubled the induction rate of the cultured ovules, followed by 1 mg L‒1 BAP in comparison with the hormone-free medium. Interaction of 2 mg L‒1 BAP with one-week cold pretreatment induced the highest gynogenesis rate, but the hormonal treatment resulted in hyperhydricity. There was a considerable variation among the genotypes in their responses to the treatments. Genotype and cold pretreatment also showed a significant interaction. Cold pretreatment for longer than one week, i.e. 2–5 weeks resulted in similar or lower amounts of gynogenesis in comparison with the control (freshly cultured ovules). Ovules of one of the genotypes (SG3) treated with one-week cold pretreatment and 1 mg L−1 BAP produced the highest percentage of regenerants. BAP at 1 or 2 mg L−1 increased the gynogenesis rates 1.7 and 2 fold, respectively. Cold pretreatment increased the haploid embryo induction from a mean of 6.49% to 11.3% after one-week cold pretreatment. The third chapter describes a study involving the effects of media with different concentrations of BAP and/or kinetin (Kin) as hormonal treatments, sucrose, and a solidifying agent (Phytagel) on haploid sugar beet explants' proliferation and hyperhydricity. After inducing haploid embryos and initial propagation of the explants, they were treated with ten different concentrations of the above-mentioned chemicals over six weeks. After applying the treatments, the mean of proliferation and the mean of hyperhydricity of the explants were compared. It was observed that Kin with a reasonable amount of proliferation and minimum rate of hyperhydricity performed better than BAP in different concentrations and combinations. Highest proliferation with the least hyperhydricity was obtained with 0.2 mg L−1 Kin, 10 g L−1 sucrose, and 6.5 mg L−1 Phytagel. The variables were negatively correlated (τb = −.648, n = 36, p < .001). The fourth chapter describes a detailed study of a highly efficient protocol to multiply the number of haploid plants in sugar beet and subsequent chromosome doubling. In this chapter, the interactions between cold pretreatment, seven genotypes of sugar beet, and Kin to improve haploid embryo induction were studied. In addition, the effects of the color of ovules, flower bud position, and comma‑form ovule on haploid embryo induction were investigated. Cold pretreatment for one-week, Kin supplementation, and genotype were influential in stimulating the ovules. Moreover, the main effects of flower bud position, ovule color, and comma-form ovule on gynogenic response were significant. Two-way and three-way interactions of the variables were also statistically significant. Kin at 0.05 or 0.5 significantly induced more gynogenic embryo induction in comparison with the control. The difference in gynogenic embryo induction between the most and the least responsive genotypes was about 4-fold. The effect of interaction between cold pretreatment and Kin was most prominent when 0.05 mg L−1 was used. However, for freshly cultured ovules, Kin was not statistically significant. The hormonal treatments' effects on the genotypes were different, e.g. a genotype (SG5) was highly benefitted from 0.5 mg L−1 Kin and reached as much as 24% of induction, whereas another genotype (SG4) was not responsive to any of the hormonal treatments. When the effect of ovule position on inflorescence was studied, it was observed that the ovules removed from the flowers grew on the lower part of the inflorescence were more responsive and produced more gynogenic embryos than the ones removed from the upper part of the inflorescence. The ovules turned brown after one month produced higher percentages of the gynogenic embryo as compared with the ovules remained white. Colchicine at 5 g L–l for 5 min was used to double the chromosome number of the haploid plantlets because the treatment over 3 or 7 min resulted in lower amounts of doubled haploid plants. However, the genotype responses to the doubling treatments were not significantly different. In the fifth chapter, with an aim of increasing the number of doubled haploid explants, the effects of five levels of proline (0.0, 0.1, 0.2, 0.3 or 0.4 mM) on the explants' proliferation, propagation, and shoot length were compared. The amino acid was supplemented to the most productive medium that is described in chapter III which contained 0.2 mg L−1 Kin, 10 g L−1 sucrose, and 6.5 mg L−1 Phytagel (i.e., treatment HT9). Proline at 0.3 mM induced the highest amount of proliferation and propagation, while proline-free medium resulted in the lowest amount of proliferation, and induced one of the lowest amounts of propagation. Proline at 0.3 mM induced the shortest shoots, whereas 0.1 mM proline induced the longest shoots. The proliferated explants were suitable for propagation (τb = 0.822, SE = 0.027, n = 75, p < 0.001). However, both proliferation and propagation showed negative correlation (τb = –0.565 and –0.601, SE = 0.061 and 0.054, respectively, n = 75, p < 0.001). For the first time, our results show beneficial effects of proline on in vitro proliferation and propagation of sugar beet. The six chapter covers the main conclusions. In summary, it can be noted that haploid plantlet induction rate can be improved by cold pretreatment of inflorescences for one week at 4 °C. Moreover, BAP supplementation may induce more gynogenesis. However, the higher level of BAP may lead to higher abnormal development of emerged structures, e.g. hyperhydricity and necrosis. The technique appears highly genotype-dependent. However, Kin seemed a better alternative than BAP in inducing non-hyperhydric plantlets. Ovule color and the position of the flower bud on the inflorescence showed influential in gynogenesis, which was statistically significant. Proline at 0.4 mM might be deleterious to in vitro growth of sugar beet. Proline at 0.3 mM induced more proliferation. Although proline at 0.1 mM was less favorable, it yielded better proliferation and propagation rates in comparison with the proline-free medium. The longest shoots were produced by 0.1 mM proline, while the shortest ones grew on the medium with 0.3 mM proline.